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Actin assembly and disassembly factors regulate BCR organization and signaling Bolger-Munro, Madison 2020

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Actin assembly and disassembly factors regulate BCR organization and signaling   by  Madison Bolger-Munro  B.Sc., The University of British Columbia, 2013  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   April 2020  © Madison Bolger-Munro, 2020    ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, the dissertation entitled: Actin assembly and disassembly factors regulate BCR organization and signaling   submitted by Madison Bolger-Munro in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Immunology  Examining Committee: Michael R Gold, Microbiology and Immunology  Supervisor  Kurt Haas, Cell and Developmental Biology Supervisory Committee Member  Geoffrey Wasteneys, Botany University Examiner Timothy O’Connor, Cell and Developmental Biology University Examiner  Additional Supervisory Committee Members: Ninan Abraham, Microbiology and Immunology  Supervisory Committee Member Kenneth Harder, Microbiology and Immunology Supervisory Committee Member    iii Abstract The immune synapse is a contact-dependent cellular communication platform that is important for activating, amplifying and executing immune responses. A B cell immune synapse is formed when B cells encounter antigens (Ags) on the surface of an Ag-presenting cell (APC). The binding of the B cell receptor (BCR) to these Ags triggers dynamic, multi-scale reorganization of the BCR and associated signal transduction machinery. Initially, BCRs are gathered into microclusters that are dispersed throughout the B cell-APC contact site. Microclusters are then moved centripetally and coalesce into the central supramolecular activation cluster (cSMAC) of an immune synapse. Although the formation of BCR-Ag microclusters recruits key signaling enzymes and increases BCR signaling efficiency, the mechanisms controlling BCR organization during immune synapse formation are not fully understood. Importantly, how the spatial patterning of the BCR promotes the translation of extracellular information (recognition of membrane-associated Ags) into functional outcomes (B cell activation) is not clear. I showed that the coalescence of BCR microclusters is dependent on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, I showed that this dynamic spatial reorganization of BCR microclusters is critical for amplifying proximal BCR signaling reactions and enhancing the ability of membrane-associated Ags to induce transcriptional responses and proliferation. My finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound Ag, but not for soluble Ags, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound Ags. I also demonstrated that the disassembly of actin networks mediated by the actin severing protein, cofilin, and its regulators is important for cytoskeletal remodeling during immune synapse formation. In addition, I showed that actin dynamics mediated by cofilin activity is important for amplifying BCR signaling in response to membrane-bound Ags. Thus, by controlling the dynamic spatial reorganization of BCR-Ag microclusters, the actin cytoskeleton amplifies APC-induced BCR signaling and B cell activation responses.        iv Lay Summary B-lymphocytes are white blood cells that protect the body against pathogenic infection. B cells make antibodies that neutralize and eliminate invading pathogens and are important for the maintenance of health. The activation of B cells must be tightly controlled as inappropriate activation could lead to the development of autoimmune diseases. B cells become activated when a receptor on their surface, called the B cell receptor, binds to foreign molecules (also called antigens) that are presented to B cells on the surface of other immune cells in the body. This study investigates the role of the actin cytoskeleton in organizing the B cell receptor at the interface between the B cell and the antigen-presenting cell. Using specialized microscopy approaches, I showed that the actin-dependent spatial organization of B cell receptors amplifies intracellular signaling and promotes B cell activation. This study provides new insights into how B cell activation is controlled.     v  Preface Status of data chapters and my contributions to them  The material contained in this thesis is derived from the following publications or manuscripts:   A version of Chapters 3 and 4 has been published. Bolger-Munro M, Choi K, Scurll JM, Abraham L, Chappell RS, Sheen D, Dang-Lawson M, Wu X, Priatel JJ, Coombs D, Hammer JA, Gold MR. Arp2/3 complex-driven spatial patterning of the BCR enhances immune synapse formation, BCR signaling and B cell activation. eLife. 2019 Jun 3;8:e44574. doi: 10.7554/eLife.44574. PMID: 31157616; PMCID: PMC6591008. I performed and analyzed all the experiments in these chapters. Experiments in Figures 4.4, 4.7, and 4.10 were performed with the assistance of Kate Choi. Kate Choi also provided and comments on the manuscript. Figure 4.8 was performed with the assistance of Dr. Libin Abraham. May Dang-Lawson and Duke Sheen helped generate plasmid constructs used in this study. Josh Scurll, Rhys Chappell wrote image analysis scripts. Dr. John Priatel provided Nur77-GFP mice and comments on the manuscript. Dr. Daniel Coombs provided advice on image analysis and comments on the manuscript. Drs. Xufeng Wu and John Hammer provided critical support for experimental set-up and data acquisition on super-resolution microscopes as well as comments on the manuscript. Additional microscopy support was provided by the UBC Life Sciences Institute Imaging Facility and by Drs. Harshad Vishwasrao and Hari Shroff from the NIH Advanced Imaging and Microscopy Resource. Michael Gold and I conceptualized the project and wrote the published paper.        vi Chapter 5 is being prepared for submission in June 2020.  Figure 5.1: Yi Tian Liu, Faith Cheung, and Kate Choi performed these experiments and I analyzed the data. Figure 5.2: I performed these experiments with Faith Cheung. Figure 5.3: I performed these experiments with Yi Tian Liu, Faith Cheung, and Nikola Deretic and I analyzed the data. I performed the experiments in Figures 5.4 and 5.5. Figures 5.6-5.9: I performed these experiments with Yi Tian Liu and Faith Cheung and I analyzed the data. Michael Gold and I conceptualized the project and will write the manuscript based on my thesis chapter.   Animal studies were conducted in the Modified Barrier Facility at the University of British Columbia. All of the animals were handled according to protocols approved by the University of British Columbia Animal Care Committee (mouse breeding license #A18-0334; animal use licenses #A15-0162 and A19-0177).    vii Table of Contents  Abstract ......................................................................................................................................... iii	Lay Summary ............................................................................................................................... iv	Preface .............................................................................................................................................v	Table of Contents ........................................................................................................................ vii	List of Tables ................................................................................................................................ xv	List of Figures ............................................................................................................................. xvi	List of Videos ................................................................................................................................ xx	List of Abbreviations ................................................................................................................ xxii	Acknowledgements .................................................................................................................. xxvi	Chapter 1: Introduction ................................................................................................................1	1.1	 The B cell immune response .............................................................................................. 1	1.1.1	 B cells in health and disease ....................................................................................... 1	1.1.2	 Structure and function of the BCR .............................................................................. 4	1.1.3	 Tonic BCR signaling: regulation by actin-dependent membrane compartmentalization .............................................................................................................. 9	1.1.4	 BCR triggering by Ag ............................................................................................... 12	1.2	 Ag presentation to B cells ................................................................................................ 13	1.2.1	 Sites of Ag encounter ................................................................................................ 13	1.2.2	 Ag capture and presentation to B cells ..................................................................... 14	1.3	 The B cell immune synapse ............................................................................................. 17	1.3.1	 Immune synapses ...................................................................................................... 17	  viii 1.3.2	 Cytoskeletal reorganization drives immune synapse formation ............................... 19	1.3.3	 Ag extraction, processing, and presentation to T cells ............................................. 24	1.4	 The actin cytoskeleton ..................................................................................................... 25	1.4.1	 Actin assembly .......................................................................................................... 26	1.4.2	 Actin nucleators ........................................................................................................ 27	1.4.3	 NPFs activate the Arp2/3 complex ........................................................................... 28	1.4.4	 Assembly of branched actin networks by the Arp2/3 complex ................................ 31	1.4.5	 Disassembly of branched actin networks .................................................................. 35	1.5	 Regulation of the Arp2/3 complex-nucleated cytoskeleton at the B cell  immune synapse ........................................................................................................................ 37	1.6	 Hypothesis and specific aims ........................................................................................... 41	Chapter 2: Methods .....................................................................................................................42	2.1	 Cell isolation and culture ................................................................................................. 42	2.1.1	 Primary B cell isolation and culture .......................................................................... 42	2.1.2	 B cell lines and cell culture ....................................................................................... 42	2.1.3	 APC cell lines and cell culture .................................................................................. 43	2.2	 Transfections .................................................................................................................... 43	2.2.1	 Transient transfection of B cell lines ........................................................................ 43	2.2.2	 Transient transfection of APCs ................................................................................. 45	2.3	 Cytoskeletal inhibitors ..................................................................................................... 47	2.4	 Immunoblotting................................................................................................................ 47	2.5	 B cell spreading on immobilized anti-Ig .......................................................................... 48	2.6	 B cell interactions with APCs .......................................................................................... 49	  ix 2.6.1	 Preparation of APCs ................................................................................................. 49	2.6.2	 Interaction of primary murine B cells with APCs .................................................... 49	2.6.3	 Interaction of B cell lines with APCs ....................................................................... 49	2.7	 Cell staining for fluorescence microscopy ....................................................................... 50	2.7.1	 Immunostaining for fluorescence microscopy .......................................................... 50	2.7.2	 Immunostaining for STED microscopy .................................................................... 52	2.7.3	 Single-particle tracking (SPT) .................................................................................. 52	2.8	 Microscopy ...................................................................................................................... 53	2.8.1	 Confocal microscopy ................................................................................................ 53	2.8.2	 TIRF microscopy (TIRFM) ...................................................................................... 53	2.8.3	 High-resolution live-cell imaging of B cells spreading on anti-Ig-coated  coverslips .............................................................................................................................. 53	2.8.4	 High-resolution live-cell imaging of B cell-APC interactions ................................. 54	2.8.5	 STED microscopy ..................................................................................................... 54	2.8.6	 Fluorescence recovery after photobleaching (FRAP) ............................................... 54	2.9	 Image analysis .................................................................................................................. 56	2.9.1	 Quantification of cell spreading area and actin organization .................................... 56	2.9.2	 Identification of Ag clusters, quantification of fluorescence intensity, and cSMAC formation using ImageJ ......................................................................................................... 57	2.9.3	 Identification of Ag clusters and quantification of fluorescence intensity using a custom MATLAB program ................................................................................................... 57	2.9.4	 Quantification of fluorescence overlap ..................................................................... 58	  x 2.9.5	 Quantification of the distance between each microcluster and the center of the immune synapse .................................................................................................................... 58	2.9.6	 SPT ............................................................................................................................ 60	2.10	 Flow cytometry .............................................................................................................. 60	2.10.1	 Staining for surface BCR levels for flow cytometry .............................................. 60	2.10.2	 Staining for intracellular actin filaments by flow cytometry .................................. 60	2.10.3	 Quantifying BCR-induced transcriptional responses using flow cytometry .......... 61	2.10.4	 Staining for upregulation of surface activation markers using flow cytometry ...... 61	2.10.5	 Quantifying BCR-induced proliferation of B cells using flow cytometry .............. 61	2.10.6	 Flow cytometry ....................................................................................................... 62	2.11	 Statistical analysis .......................................................................................................... 62	Chapter 3: Actin networks nucleated by the Arp2/3 complex control BCR organization at the immune synapse .....................................................................................................................65	3.1	 Introduction ...................................................................................................................... 65	3.1.1	 Reorganization of the actin cytoskeleton drives immune synapse formation ........... 65	3.1.2	 Actin nucleation at the B cell immune synapse ........................................................ 66	3.1.3	 The activation of the Arp2/3 complex ...................................................................... 67	3.1.4	 Systems to study B cell immune synapse formation and function ........................... 67	3.1.5	 Rationale and hypothesis .......................................................................................... 70	3.2	 Results .............................................................................................................................. 71	3.2.1	 B cells generate dynamic actin-rich membrane protrusions that scan Ag presenting surfaces for Ag ...................................................................................................................... 71	3.2.2	 The Arp2/3 complex is important for centralization of BCR-Ag microclusters ...... 74	  xi 3.2.3	 The Arp2/3 complex is important for BCR-induced actin reorganization and dynamics at the immune synapse .......................................................................................... 88	3.2.4	 The Arp2/3 complex-dependent actin structures surround BCR microclusters and drive their centralization ....................................................................................................... 94	3.3	 Discussion ...................................................................................................................... 101	3.3.1	 Summary of findings .............................................................................................. 101	3.3.2	 Ag probing behaviour at the B cell immune synapse ............................................. 102	3.3.3	 Actin retrograde flow is required for cSMAC formation ....................................... 103	3.3.4	 The three-step model of microcluster transport ...................................................... 106	3.3.5	 NPFs direct Arp2/3 complex-dependent actin assembly ........................................ 110	3.3.6	 Perspectives ............................................................................................................. 111	Chapter 4: Arp2/3 complex activity amplifies APC-induced BCR signaling and enhances B cell activation ..............................................................................................................................113	4.1	 Introduction .................................................................................................................... 113	4.1.1	 BCR signaling induces the formation of microsignalosomes ................................. 113	4.1.2	 BCR-CD19 interactions amplify BCR signaling in response to  membrane-bound Ags ......................................................................................................... 114	4.1.3	 Rationale and hypothesis ........................................................................................ 115	4.2	 Results ............................................................................................................................ 117	4.2.1	 Arp2/3 complex activity amplifies BCR signaling at the immune synapse ........... 117	4.2.2	 Inhibiting the Arp2/3 complex increases tonic BCR signaling as well as BCR and CD19 diffusion ................................................................................................................... 123	4.2.3	 Arp2/3 complex activity is important for BCR-CD19 interactions ........................ 130	  xii 4.2.4	 Arp2/3 complex activity is important for BCR-induced B cell activation  responses ............................................................................................................................. 134	4.3	 Discussion ...................................................................................................................... 146	4.3.1	 Summary of findings .............................................................................................. 146	4.3.2	 Arp2/3 complex-dependent actin structures may stabilize BCR  microsignalosomes .............................................................................................................. 147	4.3.3	 Arp2/3 complex-dependent BCR clustering and spatial organization may amplify BCR signaling ..................................................................................................................... 150	4.3.4	 The Arp2/3 complex stabilizes BCR-CD19 interactions ........................................ 152	4.3.5	 Mechanosensitivity of BCR signaling .................................................................... 153	4.3.6	 Arp2/3 complex-dependent actin dynamics control Ag affinity and density thresholds ............................................................................................................................ 154	4.3.7	 Functions of the B cell cSMAC .............................................................................. 155	4.3.8	 Is the Arp2/3 complex-nucleated actin network required for the humoral immune response? ............................................................................................................................. 156	4.3.9	 Perspectives ............................................................................................................. 157	Chapter 5: The role of actin disassembly factors in the B cell spreading response, BCR microcluster organization and BCR signaling ........................................................................162	5.1	 Introduction .................................................................................................................... 162	5.2	 Results ............................................................................................................................ 166	5.2.1	 Wdr1 and LIMK regulate cofilin activity in B cells ............................................... 166	5.2.2	 The Wdr1-LIMK-cofilin pathway is important for B cell spreading on  immobilized anti-Ig ............................................................................................................. 170	  xiii 5.2.3	 Cofilin-mediated actin disassembly is important for actin organization at the immune synapse .................................................................................................................. 176	5.2.4	 Cofilin is important for APC-induced BCR signaling and cSMAC formation ...... 178	5.2.5	 The cofilin regulatory proteins Wdr1 and LIMK are important for BCR-Ag microcluster organization and proximal BCR signaling ..................................................... 182	5.3	 Discussion ...................................................................................................................... 188	5.3.1	 Summary of findings .............................................................................................. 188	5.3.2	 Regulation of cofilin by phosphorylation ............................................................... 188	5.3.3	 Actin disassembly factors control actin organization and dynamics in B cells ...... 189	5.3.4	 Actin disassembly factors are important for immune synapse  formation in B cells ............................................................................................................. 192 5.3.5	 Perspectives ............................................................................................................. 193	Chapter 6: Overall discussion ...................................................................................................196	6.1	 Summary of main findings............................................................................................. 196	6.2	 Contributions of the Arp2/3 complex-dependent actin dynamics to the three-step model of BCR-Ag microcluster centralization .................................................................................. 197	6.2.1	 The Arp2/3 complex is important for actin retrograde flow at the cell periphery .. 197	6.2.2	 Actomyosin structures at the immune synapse ....................................................... 197	6.2.3	 Arp2/3 complex-dependent regulation of microtubules ......................................... 200	6.2.4	 An updated view of the three-step model for microcluster centralization and cSMAC formation ............................................................................................................................. 201	6.3	 Actin dynamics may control Ag density and affinity thresholds for  membrane-bound Ags ............................................................................................................. 201	  xiv 6.4	 The Arp2/3 complex in other B cell subsets .................................................................. 206	6.4.1	 Immune synapse formation and central tolerance .................................................. 206	6.4.2	 Germinal center B cells ........................................................................................... 207	6.5	 Conclusions .................................................................................................................... 209	Bibliography ...............................................................................................................................211	   xv List of Tables  Table 1 Plasmid constructs used to express proteins in B cells .................................................... 44	Table 2 Primary antibodies used for immunoblotting .................................................................. 48	Table 3 Primary antibodies used for immunostaining .................................................................. 50	Table 4 Secondary antibodies used for immunostaining .............................................................. 51	Table 5 Fluorophore-conjugated phalloidin probes for visualization of actin filaments .............. 51	Table 6 Microscopy systems used ................................................................................................ 55	   xvi List of Figures Figure 1.1 BCR signaling pathways ............................................................................................... 8	Figure 1.2 Ag capture by follicular dendritic cells, subcapsular macrophages, and dendritic cells....................................................................................................................................................... 16	Figure 1.3 B cell immune synapse formation ............................................................................... 23	Figure 1.4 Functions of NPFs ....................................................................................................... 30	Figure 1.5 Regulation of branched actin networks ....................................................................... 34	Figure 2.1 Plasmid map for mHEL-HaloTag ............................................................................... 46	Figure 2.2 Schematic of kymograph analysis ............................................................................... 56	Figure 2.3 Quantification of the distance between each microcluster and the center of the immune synapse ............................................................................................................................ 59	Figure 2.4 Example of gating strategy for analysis of Nur77GFP expression ................................ 63	Figure 2.5 Example of gating strategy for analysis of CD69 and CD86 upregulation and blast cell formation ....................................................................................................................................... 64	Figure 3.1 B cells generate actin-based protrusions in response to APC-bound Ags ................... 73	Figure 3.2 siRNA-mediated knockdown of Arp3 in A20 B cells ................................................. 75	Figure 3.3 CK-666 treatment does not decrease B cell viability .................................................. 76	Figure 3.4 CK-666 does not alter actin organization or dynamics in the COS-7 APCs ............... 77	Figure 3.5 Arp2/3 complex function is important for centralization of BCR-Ag microclusters in A20 D1.3 B cells ........................................................................................................................... 81	Figure 3.6 Arp2/3 complex function is important for centralization of BCR-Ag microclusters in ex vivo murine B cells .................................................................................................................. 82	  xvii Figure 3.7 The Arp2/3 complex controls the distribution of BCR-Ag microclusters at the B cell immune synapse ............................................................................................................................ 84	Figure 3.8 The Arp2/3 complex is important for the centripetal movement of BCR-Ag microclusters towards the cSMAC ............................................................................................... 87	Figure 3.9 Actin and myosin structures in A20 B cells spreading on immobilized anti-IgG ....... 89	Figure 3.10 The Arp2/3 complex is important for BCR-induced actin reorganization ................ 92	Figure 3.11 The Arp2/3 complex is important for BCR-induced actin reorganization and dynamics ....................................................................................................................................... 93	Figure 3.12 The Arp2/3 complex is important for actin and BCR microcluster dynamics at the B cell-APC immune synapse ............................................................................................................ 97	Figure 3.13 Arp2/3 complex-dependent actin structures encage BCR-Ag microclusters on membrane protrusions ................................................................................................................... 98	Figure 3.14 Arp2/3 complex activity does not regulate B cell-APC contact area ........................ 99	Figure 3.15 Inhibition of myosin II does not affect cSMAC formation ..................................... 100	Figure 3.16 The three-step model of Ag receptor centralization ................................................ 108	Figure 4.1 Readouts of BCR signaling and B cell activation ..................................................... 116	Figure 4.2 Arp2/3 complex activity amplifies proximal BCR signaling .................................... 119	Figure 4.3 The Arp2/3 complex amplifies proximal BCR signaling in B cells from MD4  mice ............................................................................................................................................. 120	Figure 4.4 Arp2/3 complex activity increases proximal BCR signaling in response to APC-bound Ag but is dispensable for signaling in response to soluble Ags .................................................. 122	Figure 4.5 The Arp2/3 complex amplifies Syk phosphorylation ................................................ 125	Figure 4.6 The Arp2/3 complex amplifies Syk phosphorylation in MD4 mice ......................... 126	  xviii Figure 4.7 The Arp2/3 complex regulates tonic signaling in resting B cells .............................. 128	Figure 4.8 The Arp2/3 complex regulates the lateral mobility of BCRs and CD19 in resting B cells ............................................................................................................................................. 129	Figure 4.9 Arp2/3 complex activity increases CD19 phosphorylation in response to APC-bound Ags .............................................................................................................................................. 133	Figure 4.10 Arp2/3 complex activity is required for inducing transcriptional responses and cell cycle entry. .................................................................................................................................. 137	Figure 4.11 Depleting the Arp2/3 complex in APCs does not affect B cell activation responses..................................................................................................................................................... 140	Figure 4.12 Arp2/3 complex activity is required in the first 5 min of APC contact ................... 142	Figure 4.13 Inhibition of myosin does not affect upregulation of B cell activation markers ..... 144	Figure 4.14 Arp2/3 complex activity is required for B cell proliferation in response to APC-bound Ags ................................................................................................................................... 145	Figure 4.15 The actin cytoskeleton regulates BCR signaling ..................................................... 148	Figure 5.1 Wdr1 and LIMK regulate cofilin activity in B cells .................................................. 168	Figure 5.2 Wdr1 siRNA knockdown increases cellular filamentous actin levels ....................... 169	Figure 5.3 The Wdr1-LIMK-cofilin network regulates B cell spreading on immobilized anti-Ig..................................................................................................................................................... 173	Figure 5.4 The Wdr1-LIMK-cofilin network is important for BCR-induced actin dynamics ... 174	Figure 5.5 The Wdr1-LIMK-cofilin network shapes the actin architecture at the Ag contact site..................................................................................................................................................... 178	Figure 5.6 Cofilin is important for BCR-Ag microcluster organization and amplification of proximal BCR signaling ............................................................................................................. 181	  xix Figure 5.7 Wdr1 is important for BCR-Ag microcluster organization and amplification of proximal BCR signaling in A20 D1.3 B cells ............................................................................. 184	Figure 5.8 LIMK activity is important for BCR-Ag microcluster organization and for amplification of proximal BCR signaling in A20 D1.3 B cells .................................................. 186	Figure 5.9 LIMK activity is important for BCR-Ag microcluster organization and amplifying proximal BCR signaling in B cells from MD4 mice .................................................................. 187	Figure 6.1 Actin dynamics may control Ag density and affinity thresholds for membrane-bound Ags .............................................................................................................................................. 204	     xx List of Videos Video 1 Dynamic actin-based protrusions and BCR-Ag microclusters at the B cell-APC contact site	Video 2 Actin dynamics in B cells spreading on immobilized anti-IgG antibodies	Video 3 BCR-Ag microclusters coalesce into a cSMAC in B cells expressing control siRNA	Video 4 Impaired cSMAC formation in B cells expressing Arp3 siRNA	Video 5 BCR-Ag microclusters coalesce into a cSMAC in CK-689-treated B cells	Video 6 Impaired cSMAC formation in CK-666-treated B cells	Video 7 Peripheral actin dynamics in CK-689-treated B cells plated on immobilized  anti-IgG	Video 8 Impaired peripheral actin dynamics in CK-666-treated B cells plated on immobilized anti-IgG	Video 9 Actin and BCR-Ag microcluster dynamics in CK-689-treated B cells interacting with APCs	Video 10 Impaired actin and BCR-Ag microcluster dynamics in CK-666-treated B cells interacting with APCs	Video 11 Peripheral actin dynamics in control siRNA-expressing B cells plated on immobilized anti-IgG	Video 12 Peripheral actin dynamics in cofilin siRNA-expressing B cells plated on immobilized anti-IgG	Video 13 Peripheral actin dynamics in Wdr1 siRNA-expressing B cells plated on immobilized anti-IgG	   xxi Video 14 Peripheral actin dynamics in DMSO-treated B cells plated on immobilized  anti-IgG	Video 15 Peripheral actin dynamics in LIMKi3-treated B cells plated on immobilized anti-IgG	    xxii List of Abbreviations ABC-DLBCL: activated B cell-like subtype of diffuse large B cell lymphoma ADF: actin-depolymerizing factor AIP1: actin-interacting protein 1  Ag: antigen  APC: antigen-presenting cell Arp2/3: actin-related protein 2/3  B-CLL: B cell-type chronic lymphocytic leukemia BCR: B cell receptor BLNK: B cell linker protein Btk: Bruton’s tyrosine kinase BSA: bovine serum albumin  CaMK: Ca2+/calmodulin‐dependent protein kinase CAP: cyclase-associated protein CR1: complement receptor 1 CR2: complement receptor 2 CR3: complement receptor 3 cSMAC: central supramolecular activating cluster CTL: cytotoxic T lymphocyte DAG: diacylglycerol DAM: dissociation-activation model DLBCL: diffuse large B-cell lymphoma DMEM: Dulbecco’s Modified Eagle Medium dSMAC: distal supramolecular activating cluster EGF: epidermal growth factor EM- CCD: electron multiplier-charge-coupled device  Ena/VASP: Enabled/vasodilator-stimulated phosphoprotein ERM: ezrin-radixin-moesin FACS: fluorescence-activated cell sorting  FCS: fetal calf serum   xxiii FOXO1: Forkhead box O1  FRAP: fluorescence recovery after photobleaching FRET: fluorescence resonance energy transfer GEF: guanine nucleotide exchange factor  GFP: green fluorescent protein GM: geometric mean  GM-CSF: granulocyte macrophage colony-stimulating factor GMF: glia maturation factor HEL: hen egg lysozyme HS1: hematopoietic cell-specific Lyn substrate 1 Ig: immunoglobulin  IP3: inositol trisphosphate  ITAM: immunoreceptor tyrosine-based activation motif ITIM: immunoreceptor tyrosine-based inhibition motif ISIM: instant structured illumination microscopy JMY: junction mediating and regulatory protein LAT: linker for activation of T cells LIMK: LIM domain kinase LoG: Laplacian of Gaussian MHC: major histocompatibility complex mHBS: modified HEPES-buffered saline mHEL: membrane-bound HEL mIg: membrane-bound immunoglobulin MTOC: microtubule organizing center mTOR: mammalian target of rapamycin NFAT: nuclear factor of activated T cells NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells NK cell: natural killer cell NP: hydroxy-3-nitrophenyl acetyl NP-Ficoll: NP-conjugated to Ficoll   xxiv NPF: nucleation promoting factor NP-KLH: NP conjugated to keyhole limpet hemocyanin  NRK: Nck-interacting kinase N-WASp: neural Wiskott-Aldrich Syndrome protein PAK: p21-activated kinase pAkt: phosphorylated Akt PBS: phosphate-buffered saline pCD19: phosphorylated CD19 pCD79: phosphorylated CD79 p-cofilin: phosphorylated cofilin pERK: phosphorylated ERK PFA: paraformaldehyde PH domain: pleckstrin homology domain PI3K: phosphoinositide 3-kinase  PIP2: phosphatidylinositol 4,5-bisphosphate  PIP3: phosphatidylinositol 3,4,5-trisphosphate  PKC: protein kinase C PLC: phospholipase C pMHC: peptide-bound major histocompatibility complex protein  pSyk: phosphorylated Syk pnBB: (S)-nitro-blebbistatin pSMAC: peripheral supramolecular activating cluster RFP: red fluorescent protein RPMI: Roswell Park Memorial Institute ROCK: Rho-associated protein kinase ROI: region of interest SEM: standard error of the mean  SIM: structured illumination microscopy SH2 domain: Src homology 2 domain  SSH: Slingshot   xxv SHP-1: SH2 domain-containing phosphatase-1 SHIP-1: SH2 domain containing inositol polyphosphate 5-phosphatase 1  SPT: Single particle tracking STED: stimulated emission depletion  Syk: spleen tyrosine kinase TCR: T cell receptor TESK: testicular protein kinases (TESKs)  TIRFM: total internal reflection microscopy TIRF-SIM: total internal reflection-structured illumination TLR: Toll-like receptor WAS: Wiskott-Aldrich Syndrome WASp: Wiskott-Aldrich Syndrome protein WASH: WASp and Scar homolog WAVE: WASp-family verprolin-homologous protein Wdr1: WD-repeat protein 1 WH2: WASp homology 2 WHAMM: WASP homolog associated with actin, membranes, and microtubules WIP: Wiskott-Aldrich Syndrome protein interacting protein WRC: WAVE regulatory complex Y: Tyrosine        xxvi Acknowledgements I would first like to thank my supervisor, Dr. Michael Gold. Thank you for your patience and trust. You have taught me so much about scientific thought, mentorship, communication, and grammar (amongst many, many other things). Thank you for allowing me to fail, learn from my mistakes, and then always supporting me to keep going. I am also so grateful for the opportunities I have been given while in your lab. Thank you for trusting me to pursue scientific questions in other labs and share our work with the research community. Your enthusiasm for science and mentoring is truly inspiring, and I hope to one day accumulate half of your encyclopedic knowledge. I would also like to thank my committee members, Drs. Ninan Abraham, Kurt Haas, and Ken Harder for always pushing for their consistent support.  I would also like to thank the investigators that invited me into their labs to learn new techniques. Dr. John Hammer opened his lab to me so I could use super-resolution imaging techniques to investigate the interactions between B cells and antigen-presenting cells. This was a major contribution to my eLife publication. Dr. Xufeng Wu opened her home to me to support this study and provided me with so much support and guidance. I was also granted the incredible opportunity to spend eight months with Drs. Paolo Pierobon and Ana-Maria Lennon-Duménil to pursue questions and learn techniques to further explore the interactions between B cells and APCs from a biophysics perspective. This visit would not have been possible without the support of the Friedman Award for Scholars in Health. I am incredibly humbled and grateful for this opportunity. Merci to all of the Lemmons: Hélène Moreau, Zahraa Alraies, Maria-Graciela Delgado, Claudia Rivera, Doriane Sanséau, Odile Malbec, Aleksandra Chikina, and especially Judith Pineau. My time in your lab was terrific, and I felt truly supported by every one of you. I have also had the wonderful opportunity to collaborate with Dr. Dan Coombs and his group in the Department of Mathematics at UBC. Thank you Dan for many great discussions and especially to Josh Scurll for helping me so much with image analysis and making complex ideas understandable!    I would not be the scientist that I am now without the support of past and present members of the Gold lab. Thank you for questioning me, answering me, putting up with me, cleaning up after me, and inspiring me. Kate Choi and May Dang-Lawson have helped me in more ways than I can begin to understand. Thank you for being patient with me and teaching me   xxvii every day. There are not enough thank yous for all the help I have received from you both. Without Kate, many of the experiments I attempted would not have been possible. I am also very grateful to my friend and colleague, Caitlin Pritchard, for never making me feel guilty about my candy consumption. Jia Wang, Spencer Freeman, and Sonja Christian have been and continue to be exceptional role models to me. Libin Abraham, your support and generosity have been so critical to my success in graduate school. Thank you for your relentless passion for science and obsession with rigor and thoroughness. I have learned so much from you. I would also like to thank Amanda Krystal, Lydia Yeo, Duke Sheen, Anika Krishnan, Jason Liu, Faith Cheung, and Nikola Deretic. I am inspired by and proud of all that you have accomplished in the lab in such a short time. Watching the future generation of scientists become enthusiastic about research and discovery has kept me motivated and excited throughout my Ph.D. Thank you.  The Life Sciences Institute is full of fantastic people and scientists. The Matsuuchi lab, especially Farnaz Pournia, have been great neighbors, friends, and colleagues. The Abraham lab, especially Abdalla Sheikh and Etienne Melese, are always coming to the office for snacks and interesting discussions. I have really enjoyed being a part of the TriLab group and very much appreciate the space to explore new scientific interests each week at journal club. These weekly meetings have been very important to my Ph.D. journey, so thank you, Drs. Linda Matsuuchi, Ninan Abraham, and Michael Gold for making this group and meeting so enjoyable! I would also like to acknowledge the Department of Microbiology and Immunology. It has been so wonderful to be part of such a supportive and exciting community.  I have been fortunate enough to be surrounded by amazingly supportive friends that also inspire me and continually push me to do my best in whatever I undertake. Thank you to Michaela Eaton-Kent and Kirnjot Mehat, for always, always believing in me. Rachel Hawtin, Rebecca Wheeler, Alanna Joncas, and Mackenzie Deane have been there for me for 25+ years and will be for 100 more. These women inspire me more than I can articulate.  Finally, I would like to thank my family. My grandparents have provided me with unwavering support and love my whole life, as well as a deep appreciation for the merits of hard work. To my parents, Lesley Bolger-Munro and David Munro, thank you for always believing in me and sacrificing so much for me. Because of you, I know I can do anything. I love you. And Connor Morgan-Lang, thank you for being fresh tracks on a bluebird day!    1 Chapter 1: Introduction  1.1 The B cell immune response  1.1.1 B cells in health and disease B lymphocytes play a critical role in health and disease by producing antibodies and cytokines and by acting as antigen (Ag)-presenting cells (APCs) for T cells. The activation of mature B cells, as well as their subsequent differentiation into antibody-secreting cells and memory B cells, depends on the binding of Ag to the B cell receptor (BCR). Upon Ag binding, BCR-dependent signaling cascades are initiated, which culminate in the induction of transcriptional responses and metabolic changes that drive cell cycle entry, proliferation, and differentiation. Dysregulation of BCR signaling and B cell activation can cause immunodeficiencies, autoimmunity, and B cell malignancies. Hence, the ability to therapeutically modulate B cell activation and optimize vaccine strategies has significant health implications. Activated B cells that have differentiated into plasma cells secrete antibodies with the same Ag specificity as the BCRs on the parental mature B cell that underwent Ag-induced activation. Antibodies can provide immune protection by neutralizing pathogens and toxins by blocking their ability to bind attachment or entry receptors. Antibody binding can also aid in the elimination of the pathogen or toxin by promoting phagocytosis or by activating lytic immune responses such as complement-mediated lysis or antibody-dependent cellular cytotoxicity. In addition to initiating signaling reactions in response to Ag binding, the other major function of the BCR is receptor-mediated endocytosis of the Ag. The subsequent trafficking of the Ag through the endolysosome pathway allows Ag to be processed into peptides and loaded onto major histocompatibility complex (MHC) II proteins for presentation to CD4+ T cells (Yuseff et al., 2013). Activated B cells also express T cell co-stimulatory molecules such as CD80 and CD86, which provide essential second signals for T cell activation. T cells activated by these peptide-MHC II complexes can then provide the B cell with “T cell help” by expressing the B cell co-stimulatory protein CD40 ligand on their surface and by secreting B cell-activating cytokines such as IL-4 and IL-21 (Crotty, 2011). T cell help drives Ig class switching as well as   2 the germinal center response, where B cells undergo somatic hypermutation of their Ig heavy and light chain V regions, resulting in affinity maturation of the antibodies that they will produce (Crotty, 2011; Silva and Klein, 2015). The germinal center reaction is critical for the development of high-affinity and long-lived antibody responses as well as the differentiation of memory B cells (Silva and Klein, 2015). Thus, B cell activation is critical for providing both early and long-lasting antibody-based immunity.  Activated B cells can also play an important role in modulating inflammatory responses and immune responses by presenting Ags to T cells and secreting cytokines (Barnas et al., 2019; Getahun and Cambier, 2019; Lund, 2008; Shen and Fillatreau, 2015). The APC and cytokine-producing function of B cells are particularly important in autoimmune diseases such as type 1 diabetes (Silveira and Grey, 2006), systemic lupus erythematosus (Jacob and Stohl, 2010), rheumatoid arthritis (Fillatreau, 2018), and multiple sclerosis (Fillatreau, 2018). The repertoire of cytokines secreted by B cells depends on the context in which the B cell encounters Ag, for example the presence of Toll-like receptor (TLR) ligands and cytokines (Fillatreau, 2018; Lund, 2008). B cells are a major source of the pro-inflammatory cytokine IL-6, which drives differentiation of pro-inflammatory effector T cell subsets (Jones et al., 2018; Matsushita, 2019). Elevated levels of IL-6 are hallmarks of rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis (Jones et al., 2018). B cells also produce the pro-inflammatory cytokine granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF enhances inflammatory responses by myeloid cells and significantly contributes to the pathogenesis of multiple sclerosis (Li et al., 2015, 2018). Conversely, IL-10-producing B cells are critical for the downregulating inflammatory responses in mice and humans (Kalampokis et al., 2013).  Because B cells secrete antibodies and have powerful modulatory effects on other immune cells via their ability to present Ags to T cells and secrete cytokines, the activation of B cells must be tightly regulated. Defects in BCR signaling can result in immunodeficiency diseases such as common variable immune deficiency (Ahn and Cunningham-Rundles, 2009; Durandy et al., 2013; Liadaki et al., 2013; Smith and Cunningham-Rundles, 2019) and X-linked agammaglobulinemia (Durandy et al., 2013; Fried and Bonilla, 2009; Liadaki et al., 2013; Smith and Cunningham-Rundles, 2019). Conversely, excessive, unregulated BCR signaling can lead to autoimmune diseases such as systemic lupus erythematosus (Rawlings et al., 2017; Yanaba et al.,   3 2008) or to B cell malignancies such as B cell-type chronic lymphocytic leukemia (B-CLL) (Bosch and Dalla-Favera, 2019) and the activated B cell-like subtype of diffuse large B cell lymphoma (ABC-DLBCL) (Miao et al., 2019).  Depletion of B cells is currently used as a treatment for a variety of autoimmune diseases and B cell malignancies. Direct depletion of B cells mediated by rituximab, which binds to CD20, a transmembrane protein expressed on normal and pathogenic B cells is now the standard of treatment for B cell malignancies, including DLBCL and CLL (Salles et al., 2017). Anti-CD20 is also used in the treatment of rheumatoid arthritis and multiple sclerosis (Barnas et al., 2019; Li et al., 2018). Indirect depletion of B cells via inhibiting pro-survival signaling pathways with belimumab, which neutralizes the B cell-activating factor BAFF, is currently used for the treatment of systemic lupus erythematosus (Barnas et al., 2019). Because some B cell malignancies are driven by constitutive BCR signaling, components of the BCR signaling pathway are major targets for the development of therapeutics. In particular, Ibrutinib, a small molecule inhibitor of Bruton’s tyrosine kinase (Btk), an essential component of multiple B cell signaling pathways, is used to treat B cell malignancies such as CLL and mantle cell lymphoma (Burger and Wiestner, 2018; Puri et al., 2013). Moreover, spleen tyrosine kinase (Syk), which is required for almost all signaling events downstream of the BCR, has emerged as an important target for the treatment of B cell malignancies and B cell-mediated autoimmune diseases such as rheumatoid arthritis and lupus (Deng et al., 2016; Puri et al., 2013). Precise control of B cell activation is critical for human health and disease. As described below, B cells are often activated in vivo by APCs that capture and concentrate Ags. The primary goal of my thesis is to understand how the actin cytoskeleton contributes to the regulation of B cell activation in response to Ags that are displayed on the surface of APCs. A more thorough understanding of this process could suggest novel therapeutic targets for treating immunodeficiency diseases, autoimmune diseases, and B cell malignancies.    4 1.1.2 Structure and function of the BCR Like many other activating receptors on immune cells, the BCR is comprised of two subunits: an Ag- or ligand-binding subunit and a signaling subunit (Abraham et al., 2016; Reth, 1992). The Ag-binding component of the BCR is a membrane-bound immunoglobulin molecule (mIg) that has the identical V regions and Ag-binding site as secreted antibodies that would be made by plasma cells derived from that B cell. The ability of B cells to produce membrane-bound and secreted antibodies with the same Ag-specificity is due to alternative splicing of secreted (S) and membrane (M) exons in the Ig heavy chain mRNA. The M exons encode a C-terminal transmembrane domain. The Ag-binding subunit of the BCR is non-covalently associated with the signaling subunit, which is comprised of CD79a (Igα) and CD79b (Igβ) polypeptides (Abraham et al., 2016; Hombach et al., 1990). Both CD79a and CD79b contain immunoreceptor tyrosine-based activation motifs (ITAMs) in their cytoplasmic domains, which initiate intracellular signaling by recruiting and activating tyrosine kinases (Abraham et al., 2016; Dal Porto et al., 2004; Gold et al., 1990, 1991). The BCR has essential functions in B cell development, survival, and responses to Ag. The formation of a properly folded Ig heavy chain that associates with a surrogate Ig light chain and with CD79a/b serves as an important checkpoint in B cell development (Abraham et al., 2016). Cell surface expression of this pre-BCR, which consists of an Ig heavy chain, the Vpre-B and λ5 surrogate light chains, and CD79a/b, results in signaling that is required for B cell survival and differentiation (Abraham et al., 2016; Herzog et al., 2009; Nemazee, 2017; Übelhart et al., 2015). It is not known whether this signaling is initiated by extracellular ligands found on bone marrow stromal cells or via a ligand-independent mechanism, for example spontaneous pre-BCR clustering (Bankovich et al., 2007; Gauthier et al., 2002). Once this checkpoint has been passed, the rearrangement and expression of the Ig light chain genes is induced by signals that are dependent on the cell surface expression of the pre-BCR. Once the BCR with conventional Ig light chains is expressed, self-reactive B cells that bind Ag can undergo receptor editing, where rearrangements of the Ig genes can generate a new light chain to pair with the existing heavy chain (Nemazee, 2017). B cells that remain self-reactive after receptor editing are deleted by apoptosis. Alternatively, autoreactive B cells can be rendered anergic, a state in which B cells are unresponsive to Ags (Yarkoni et al., 2010). The deletion or silencing of autoreactive   5 B cells in the early stages of B cell development is referred to as central tolerance. Autoreactive B cells that escape central tolerance and react strongly to autoantigens in peripheral tissues can be removed by clonal deletion or silenced by induction of anergy, in a process known as peripheral tolerance (Burnett et al., 2019; Goodnow et al., 1988; Tan et al., 2019). These checkpoints are critical for preventing autoimmunity (Cashman et al., 2019; Meffre and O’Connor, 2019). The deletion or anergy induction that mediates central and peripheral tolerance requires signaling by the BCR (Nemazee, 2017; Yarkoni et al., 2010). Autoreactive B cells with mutations that impair BCR signaling can escape negative selection (Cyster et al., 1996; Nemazee, 2017). For example, patients with Wiskott-Aldrich Syndrome (WAS), which is caused by loss-of-function mutations in the actin regulator, WAS protein (WASp), have aberrant BCR and TCR signaling (Candotti, 2018). These patients also have an increased prevalence of autoimmunity and higher serum titers of autoreactive antibodies (Candotti, 2018). The altered BCR signaling in immature B cells is thought to allow the escape of autoreactive immature B cells or the positive selection of autoreactive transitional B cells in these patients (Cashman et al., 2019; Castiello et al., 2014). In response to Ag, the BCR has two functions: 1) initiating intracellular signaling and 2) eliciting T cell help by internalizing Ags for processing and presentation on MHC II proteins (Abraham et al., 2016). Ag binding induces BCR clustering and ITAM phosphorylation, resulting in the initiation of the BCR signaling cascade, which is described in detail below. The combined effects of activating this signaling network are changes in the B cell cytoskeleton, metabolism, and gene expression patterns, leading to cell cycle entry. Full activation of the B cell, including proliferation and differentiation, requires a second signal such as T cell help or signals initiated by the binding of microbial-associated molecular patterns to Toll-like receptors (TLRs).   1.1.3 BCR signaling pathways   Ag binding to the BCR induces phosphorylation of ITAMs in the cytoplasmic tail of CD79a and CD79b by Src-family kinases such as Lyn and by Syk (Abraham et al., 2016; Dal Porto et al., 2004; Engels and Wienands, 2011; Packard and Cambier, 2013). Phosphorylation of the tyrosine residues in the ITAMs of the BCR creates a binding site for Src homology family 2   6 (SH2) domain-containing proteins. Doubly phosphorylated ITAMs recruit Syk, which is phosphorylated by nearby Src-family kinases. Activated Syk can then phosphorylate nearby ITAMs in other BCRs as well as downstream components of the BCR signaling cascade. Syk-dependent phosphorylation of scaffolding proteins such as B cell linker protein (BLNK/SLP-65) and the co-receptor CD19 promotes the assembly of BCR-associated microsignalosomes, which contain key signaling proteins such as phosphoinositide 3-kinase (PI3K), Btk, phospholipase C gamma 2 (PLCγ2), and Vav (Kurosaki, 2002). Activation of these signaling proteins, and hence microsignalosome formation, is essential for B cell activation. Indeed, loss-of-function mutations in Syk, Btk, PLCγ2, BLNK, and Vav result in dramatic impairment of B cell development and function (Cornall et al., 2000; Doody, 2000; Doody et al., 2001; Foucault et al., 2005; Fu et al., 1998; Hashimoto et al., 2000; Inabe et al., 2002; Ishiai et al., 1999; Khan et al., 1995; Liu et al., 2011; Pappu et al., 1999; Rawlings, 1999; Takata and Kurosaki, 1996; Tarakhovsky et al., 1995; Tedford et al., 2001; Turner et al., 1997b, 1997a; Wang et al., 2000; Weber et al., 2008; Wienands et al., 1998; Zeng et al., 2000; Zhang et al., 1995).  BCR-dependent phosphorylation of CD19, as well as other adaptor proteins such as BCAP (Okada et al., 2000), recruits PI3K to the plasma membrane where it phosphorylates phosphatidylinositol 4,5-bisphosphate (PIP2), generating the second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3) (Abraham et al., 2016; Dal Porto et al., 2004; Engels and Wienands, 2011; Packard and Cambier, 2013). PIP3 promotes the recruitment and activation of pleckstrin homology (PH) domain-containing proteins, in particular, the pro-survival kinase Akt. Akt can then phosphorylate and activate the nutrient sensor mTOR, which regulates B cell growth, metabolism, and proliferation (Limon and Fruman, 2012). Changes in the metabolic activity of B cells upon Ag encounter are critical for the germinal center response, B cell proliferation, and antibody production (Akkaya et al., 2018; Egawa and Bhattacharya, 2019; Jellusova, 2018, 2020). Akt also phosphorylates the Forkhead box O1 (FOXO1) transcription factor, which controls the expression of genes that suppress the proliferation and survival of B cells (Szydłowski et al., 2014). Akt-dependent phosphorylation of FOXO1 mediates its export from the nucleus allowing for B cell survival and proliferation. BLNK-dependent co-localization of PLCγ2 and Btk, followed by the phosphorylation of PLCγ2 by Btk, results in the activation of PLCγ2, which cleaves PIP2 into the second messengers inositol   7 trisphosphate (IP3) and diacylglycerol (DAG). IP3 initiates Ca2+ release, whereas DAG activates the Ras and Rap GTPases as well as protein kinase C (PKC) family members. A cumulative effect of activating these pathways is the activation of the transcription factors NFAT and NF-κB, which initiate changes in gene expression that precede and promote B cell activation responses such as cell cycle entry and proliferation (Crabtree and Olson, 2002; Healy et al., 1997; Schulze-Luehrmann and Ghosh, 2006). As well, the active GTP-bound forms of the Rap, Rac, and Cdc42 GTPases control BCR-induced changes in the cytoskeleton, including the remodeling of the submembrane actin network (Arana et al., 2008a; Burbage et al., 2015; Lin et al., 2008; Tybulewicz and Henderson, 2009). The actin cytoskeleton controls the spatial organization of BCRs on the cell surface. As discussed in section 1.7, this has an important role in amplifying BCR signaling in response to membrane-bound Ags. To prevent excessive or inappropriate B cell activation, BCR signaling must be tightly regulated (Bounab et al., 2013). Upon BCR crosslinking, Lyn phosphorylates the three immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in the cytoplasmic tail of the inhibitory co-receptor CD22. Phosphorylation of these ITIMs recruits SH2 domain-containing phosphatase-1 (SHP-1) as well as the inositol phosphatase SHIP-1 via their SH2 domains. SHP-1 dephosphorylates the CD79a/b ITAMs as well as the critical tyrosine residues in Syk and BLNK (Adachi et al., 2001; Mizuno et al., 2000). SHIP-1 opposes the PI3K pathway by dephosphorylating PIP3. Loss of Lyn, SHP-1, SHIP-1, or CD22 results in increased BCR signaling and the development of lupus-like autoimmune disease in mice (Bounab et al., 2013; Cornall et al., 1998; Khalil et al., 2012; Leung et al., 2013; O’Keefe et al., 1996; Pao et al., 2007).       8   Figure 1.1 BCR signaling pathways  Ag binding induces BCR clustering and the recruitment of signaling enzymes to form the BCR microsignalosome, a complex that initiates downstream signaling reactions. Src-family kinases, such as Lyn, phosphorylate the ITAMs of the CD79a/b subunit. This creates binding sites for the Src homology 2 (SH2) domains in other proteins such Syk. Syk initiates critical signaling pathways that collectively lead to cytoskeletal reorganization and changes in gene expression. The interaction of BCRs with CD19 amplifies BCR signaling and plays an important role in BCR-induced activation of phosphoinositide 3-kinase (PI3K), which consists of a p85 subunit and a p110 catalytic subunit. Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the lipid second messenger generated by PI3K, promotes the membrane recruitment and activation of multiple proteins that contain pleckstrin homology (PH) domains, in particular, the Akt pro-survival kinase. See text for more detail. Adapted from Abraham et al., 2016.        9 1.1.3 Tonic BCR signaling: regulation by actin-dependent membrane compartmentalization The survival of mature circulating B cells requires signals from the BCR (Lam et al., 1997). Mature B cells lacking either the mIg component of the BCR or the CD79a/b ITAMs have a dramatically shorter half-life in the periphery: only 3-6 days compared to the typical 40 days (Kraus et al., 2004; Lam et al., 1997; Monroe, 2006). In contrast, replacing the mIg with mutant forms that cannot bind to Ag does not impair B cell development or decrease cell survival. This suggests that B cell survival depends on Ag-independent signals that remain below the signaling threshold required for B cell activation.  Unrestricted Ag-independent BCR signaling and subsequent B cell activation can result in autoimmunity or B cell malignancies (Puri et al., 2013). Thus, the low-level constitutive or “tonic” signals required for B cell survival must be strictly regulated. The magnitude of BCR signaling depends on a delicate balance between activating kinase and inhibitory phosphatase reactions (Abraham et al., 2016; Bounab et al., 2013; Kurosaki and Hikida, 2009). Global inhibition of tyrosine phosphatases by vanadate compounds results in robust Ag-independent BCR signaling (Wienands et al., 1996). As well, mice lacking important phosphatases such as SHP-1 and SHIP-1 have a lower threshold for activation and develop autoimmunity (Bounab et al., 2013). The balance between the activation and inhibition of BCR signaling pathways is also controlled by regulating the access of kinases and phosphatases to their substrates. In this regard, the plasma membrane acts as a signaling platform, and the recruitment of signaling enzymes (e.g. Syk, Akt, PKC enzymes, GTPases) to the plasma membrane (e.g. via SH2 and SH3 domains, lipid-binding domains, reversible lipid modifications) is often required for their activation and function (Gold, 2002). Moreover, the segregation of signaling components into distinct plasma membrane domains provides another important mechanism for controlling receptor signaling (Garcia-Parajo et al., 2014; Mattila et al., 2016).  By controlling the compartmentalization of the plasma membrane, the submembrane actin cytoskeleton plays a significant role in restricting tonic BCR signaling. The submembrane actin cytoskeleton is a dense meshwork of actin filaments that is organized by over 100 actin-binding proteins and is closely tethered to the plasma membrane via interactions with membrane proteins and lipids (Chugh and Paluch, 2018). Because the submembrane actin network is in   10 close proximity to the plasma membrane, actin filaments act as a fence or barrier that restricts the lateral diffusion of membrane components. The diameter of these actin-defined membrane compartments is estimated to be approximately 40-300 nm (Kusumi et al., 2012). In B cells, the submembrane actin cytoskeleton restricts the diffusion of BCRs and other transmembrane components of the BCR signaling network, including the co-receptor CD19 (Treanor et al., 2010; Freeman et al., 2015; Mattila et al., 2013). Single-particle tracking (SPT) studies demonstrated that the removal of actin-based diffusion barriers, or the dissociation of the submembrane actin cytoskeleton from the plasma membrane, increases the lateral mobility of BCRs, leading to spontaneous Ag-independent signaling (Abraham et al., 2017; Freeman et al., 2015; Treanor et al., 2010, 2011). This signaling response is dependent on BCR-BCR and BCR-CD19 interactions (Treanor et al., 2010; Mattila et al., 2013). These findings are consistent with the collision-coupling model of receptor signaling, which postulates that lateral diffusion of membrane proteins is required for promoting protein-protein interactions that initiate and potentiate signal transduction (Treanor, 2012; Mattila et al., 2016). Thus, in resting B cells, the actin cytoskeleton limits BCR-BCR and BCR-CD19 interactions in order to restrict Ag-independent BCR signaling to a level that supports cell survival but which is below the threshold of signaling required for B cell activation.  The segregation of receptors from the adaptor proteins that act as platforms for the assembly of signaling complexes may be a conserved principle for regulating receptor signaling. Before the recent development of super-resolution imaging techniques, our understanding of the organization of surface proteins was hampered by the diffraction limit of visible light (~200-250 nm). With resolution limits approaching 10-20 nm, super-resolution microscopy has revealed that many proteins on the plasma membrane, including BCRs, are organized into pre-formed clusters with diameters of approximately 20-200 nm (Gold and Reth, 2019). Given the 40-300 nm of actin-bounded membrane nanocompartments, it is likely that only one nanocluster occupies a given nanocompartment. Thus, a key function of the submembrane actin cytoskeleton could be to limit the interaction of nanoclusters.  In mast cells, FcεRI nanoclusters are separated from clusters of the adaptor protein LAT in resting cells (Wilson et al., 2001) but come together upon receptor ligation. Similarly, in T cells, T cell receptors (TCRs) are segregated from LAT in the resting state (Lillemeier et al.,   11 2010) but concatenate (i.e. contact each other rather than coalesce) into microclusters with spatially segregated domains upon binding of peptide-MHC complexes to TCRs (Yi et al., 2019). Because the actin cytoskeleton is in close proximity to plasma membrane components and is highly dynamic, it is emerging as an essential regulator of receptor compartmentalization, allowing for the precise fine-tuning of receptor signaling. Thus, cytoskeletal control of the organization of distinct clusters of signaling molecules on the plasma membrane could be a key determinant of receptor signaling output in lymphocytes. Remodeling of the actin cytoskeleton permits and supports interactions between receptor nanoclusters, which is required to initiate and sustain signaling (Mattila et al., 2016).  Because BCR signaling requires BCR-BCR and BCR-CD19 interactions, the actin cytoskeleton plays an important role in limiting the amount of BCR signaling in the absence of Ag by restricting the lateral mobility of BCRs within the plasma membrane. In addition to limiting BCR-BCR interactions, the actin cytoskeleton may also limit the interactions of the BCR with CD19, which may have a role analogous to that of LAT in TCR activation. CD19 phosphorylation is mediated by BCR-associated kinases such as Syk and is required for the activation of the PI3K and Vav (Buhl et al., 1997; O’Rourke et al., 1998; Tuveson et al., 1993). How the submembrane actin cytoskeleton controls the relative organization of BCR and CD19 nanoclusters so as to allow the tonic low-level BCR signaling required for B cell survival, while preventing B cell activation, is not fully understood. The compartmentalization of BCRs into nanoclusters could allow for low-level tonic signaling by allowing BCR-BCR collisions within the nanocluster. As well, the actin barriers that separate nanoclusters are dynamic and frequently turned over, which could allow limited interactions between adjacent BCR nanoclusters, and between BCR and CD19 nanoclusters, which would contribute to tonic BCR signaling. As described below in section 1.1.4, a critical early event in Ag-stimulated BCR signaling is the localized disassembly of actin-based diffusion barriers. This allows more extensive BCR-BCR and BCR-CD19 interactions that result in greatly increased BCR signaling.       12 1.1.4 BCR triggering by Ag How extracellular Ag signals are translated across the cell membrane to initiate BCR signaling is not fully understood. Both changes in the spatial organization of BCRs and structural changes within the BCR could be involved. In the dissociation-activation model (DAM) of BCR triggering put forward by Reth et al. (Maity et al., 2015; Yang and Reth, 2010a, 2010b), most BCRs on resting B cells exist as autoinhibited oligomers. In these oligomers, the CD79a/b domains are inaccessible to kinases because of their tight association with each other. Upon binding of Ags to the Ag-binding site in the mIg subunit, BCRs within the oligomer move away from each other, presumably due to conformational changes in the mIg extracellular domains. This allows cytoplasmic Syk to bind to the newly exposed ITAMs and become activated. Evidence supporting this model was provided by proximity ligation assays showing that BCR oligomers dissociate after Ag binding and only then bind Syk (Kläsener et al., 2014). Further dissociation of BCR oligomers results from “inside out signaling” in which Syk phosphorylates the ITAMs of nearby BCR oligomers, including ones that are not bound to Ag, allowing more Syk binding (Kläsener et al., 2014). BCR-activated Src-family kinases could also contribute to signal spreading via the trans-phosphorylation of ITAMs in nearby unbound BCRs, allowing the further recruitment and activation of Syk (Gold, 2002). This creates a positive feedback loop that amplifies BCR signaling (Mukherjee et al., 2013).  In the conformational change model put forth by Pierce et al., Ag binding to the BCR results in conformational changes that induce BCR oligomerization and signaling (Tolar and Pierce, 2010; Tolar et al., 2005, 2009). Using a FRET-based approach, these authors demonstrated that in resting B cells, BCRs exist as monomers (Tolar et al., 2005). Ag-induced oligomerization of BCRs requires the Cµ4 domains, which undergo conformational change only upon encounter of membrane-bound Ag (Tolar et al., 2009). These authors speculate that forces transmitted through the BCR cause changes that expose the Cµ4 oligomerization surface, allowing for BCR clustering. However, with the development of super-resolution imaging tools, it has become increasingly clear that many of the BCRs on the surface of resting B cells form nanoclusters of diverse sizes. Recently, the conformational change model has been revisited. Again, using a FRET-based system, conformational changes in the heavy chain of mIg were observed, as well as changes in the spatial relationship between the mIg and CD79b (Shen et al.,   13 2019). These authors found that IgM and IgG isoforms undergo distinct conformational changes and that the extent of the conformational change correlates with the magnitude of BCR signaling and whether or not B cell activation ensues. After the initial activation of BCR monomers or oligomers, BCR signaling could then be further amplified by the increased BCR mobility and by BCR clustering that is facilitated by BCR signaling pathways that promote the breakdown of the submembrane actin network (see section 1.3.2 below). According to the collision-coupling model, the release of BCRs from actin-based diffusion barriers increases the frequency of BCR-BCR and BCR-CD19 interactions that are important for BCR signaling (Treanor, 2012). SPT and super-resolution microscopy studies support this model. The breakdown of actin-based diffusion barriers increases BCR mobility (Batista et al., 2010; Freeman et al., 2015; Mattila et al., 2013; Treanor et al., 2010) and may allow receptors and signaling proteins that are separated by the membrane compartmentalization on resting B cells to interact and initiate BCR signaling cascades. In this way the increased actin turnover initiated by Ag-induced BCR signaling may permit the concatenation of BCR nanoclusters as well as their interactions with clusters of key adaptor proteins such as CD19 (Gold and Reth, 2019).  1.2 Ag presentation to B cells   1.2.1 Sites of Ag encounter  B cells must protect against a vast range of pathogenic challenges. To meet this challenge, each B cell has a unique Ag binding specificity. All of the BCRs on a single cell contain the same Ag-binding site, which has significant affinity for one Ag or for related Ags that share a similar 3-dimensional epitope. BCRs can bind to soluble Ags, Ag-bearing particles (i.e. viruses), and Ags on the surface of other cells, as well as single cell or multicellular pathogens. To promote the recognition of Ag by the rare B cells whose BCR has affinity for that Ag, Ags are concentrated in secondary lymphoid organs such as lymph nodes and the spleen. These organs contain B cell follicles that are highly enriched in circulating follicular B cells as well as APCs that can present Ags to B cells.   14 Ags are brought to secondary lymphoid organs either via the blood or lymph or are delivered from the tissues by dendritic cells. The mechanism of Ag delivery to these sites depends on the size of the Ag, whether or not it has been opsonized by complement or pre-existing antibodies, and the type of organ (Cyster, 2010). Blood-borne Ags are delivered to the spleen, where they are captured by dendritic cells and macrophages (Heesters et al., 2016). Ags in the lymphatic fluid enter draining lymph nodes. How Ags that are carried by the lymph enter the B cell follicle depends on the molecular weight of the Ag. Low molecular weight (<70 kDa) soluble Ags can penetrate into the B cell follicle via pores in the subcapsular sinus and can flow into follicles via the conduit network. These Ags can then either bind directly to cognate (i.e. Ag-specific) B cells (Pape et al., 2007) or be captured by and concentrated on follicular dendritic cells (Clark, 1962; Pape et al., 2007). Larger Ags such as bacteria, viruses, and immune complexes do not enter the B cell follicle directly but can be captured by a specialized population of macrophages that line the subcapsular sinus (Carrasco and Batista, 2007; Gonzalez et al., 2010; Junt et al., 2007; Phan et al., 2009). The position of the subcapsular sinus macrophages allows them to simultaneously sample the incoming lymphatic fluid and maintain contact with the B cell follicle. Transport of the Ag along the membrane of a subcapsular sinus macrophage, or alternatively via transcytosis of the Ag across the macrophage, exposes the captured Ag to B cells within the follicle. There, the Ag can be recognized by cognate B cells or shuttled to follicular dendritic cells by non-cognate B cells (Phan et al., 2009). Dendritic cells can also bind Ags either in the peripheral tissues or within the subcapsular sinus, transport them into the lymphoid follicle, and retain them on the surface in an intact form.   1.2.2 Ag capture and presentation to B cells  B cells are most efficiently activated by Ags that are presented on the surface of APCs such as subcapsular sinus macrophages, follicular dendritic cells, and dendritic cells (Batista and Harwood, 2009). These B cell APCs capture Ags via a wide range of surface receptors including, pattern recognition receptors, carbohydrate-binding scavenging receptors, Fc receptors, and complement receptors. Long-term retention of Ag the surface of APCs concentrates Ags, allowing rare cognate B cells to detect them (Heesters et al., 2013; Unanue et al., 1969). These APCs express the integrin ligands VCAM-1 and ICAM-1, which allow B cells to adhere to them   15 (Heesters et al., 2016). In addition, follicular dendritic cells secrete the chemokine CXCL13, which is a potent chemoattractant for B cells (Heesters et al., 2014). Subcapsular sinus macrophages capture complement-opsonized Ags via complement receptor 3 (CR3; also known as MAC-1, the αMβ2 integrin, and CD11b/CD18) and use FcγRIIB to capture Ags that have been opsonized by pre-existing circulating antibodies (i.e. immune complexes). Subcapsular sinus macrophages can also capture carbohydrate Ags and glycoproteins via the C-type lectin CD209 (DC-SIGN). Subcapsular sinus macrophages can retain Ag on their surface for at least 72 hr after Ag exposure (Unanue et al., 1969). Recently, it has been demonstrated that under inflammatory conditions the ring of subcapsular sinus macrophages around the follicle become highly disorganized and these macrophages enter into the B cell follicle (Gaya et al., 2015). This likely enhances B cell responses to primary infection by allowing the entry of Ag-carrying APCs directly into the B cell follicle and by increasing Ag shuttling to follicular dendritic cells (Gaya et al., 2015). Dendritic cells are the most potent APCs for T cells but can also present intact, unprocessed Ags to B cells. Both resident and migratory dendritic cells accumulate near the high endothelial venules, the sites where B cells first enter secondary lymphoid organs. In vivo microscopy studies have shown that B cells survey these dendritic cells and that Ags captured by these cells can activate B cells (Qi et al., 2006). These dendritic cells can also capture Ags via FcγRIIB or CD209. Although dendritic cells can act as APCs for B cells, follicular dendritic cells are thought to be the most important APC for eliciting high-affinity antibody responses (Batista and Harwood, 2009). In contrast to conventional dendritic cells, which are hematopoietic cells, follicular dendritic cells are stromal-derived non-hematopoietic cells. Follicular dendritic cells have long been known to be reservoirs of membrane-bound Ag (Mitchell and Abbot, 1965; Nossal et al., 1968) and to retain Ag on their surface for extended periods (Heesters et al., 2013; Mandel et al., 1981). The prolonged retention of Ag by follicular dendritic cells supports the germinal center reaction and affinity maturation (Heesters et al., 2016), processes that are critical for the development of high-affinity antibody responses and immunological memory. Follicular dendritic cells express high levels of complement receptor 1 (CR1; CD35) and 2 (CR2; CD21), which capture and present complement-opsonized Ag. CR1/2-bound Ag is internalized into a   16 non-degradative endosomal recycling compartment where it is retained for months and periodically re-displayed on the cell surface (Heesters et al., 2013, 2014). Follicular dendritic cells also express low levels of FcγRIIB, which can capture and retain immune complexes (Batista and Harwood, 2009). The capture, concentration, and recycling of these Ags to the cell surface by these receptors regulate the availability of Ag to B cells.  Figure 1.2 Ag capture by follicular dendritic cells, subcapsular macrophages, and dendritic cells (A) Follicular dendritic cells can directly interact with the conduit network in B cell follicles, where they can sample small (<70 kDa) Ags. Top panel is an electron micrograph depicting this interaction, and the bottom panel is a schematic for clarification. (B) Ags that are opsonized by the C3d component of complement are captured by complement receptors expressed on the apical surface of subcapsular sinus macrophages. The Ag is then transported into the B cell follicle where it can activate cognate B cells. In addition, the Ag can be transferred to the CR1 or CR2 complement receptors on non-cognate B cells and then delivered to CR1 or CR2 on follicular dendritic cells. Follicular dendritic cells retain Ags for long periods of time by shuttling them through non-degradative vesicular recycling pathways. Follicular dendritic cells present surface-bound Ag to B cells. (C) Follicular dendritic cells can also acquire Ags from conventional dendritic cells in the lymph node. Additionally, non-cognate B cells can capture Ags in the extrafollicular medullary region of the lymph node and transfer them to follicular dendritic cells via complement receptors. Image reproduced from Heesters et al., 2014, with permission.    17 1.3 The B cell immune synapse  1.3.1 Immune synapses  The interaction of B cells with Ag-bearing APCs results in the formation of an immune synapse. Immune cells such as T cells, B cells, and natural killer (NK) cells form specialized, contact-dependent interaction sites with APCs that are broadly referred to as immune synapses. At these transient cell-cell communication junctions, signaling enzymes are activated, initiating the complex pathways that result in immune cell activation. Immune synapses were first observed in T cells (Monks et al., 1998). In response to lipid bilayers decorated with peptide-MHC complexes, TCR microclusters initially form at the periphery of the T cell-APC contact site and over time are reorganized to create a radially symmetric pattern of supramolecular activating clusters (SMAC). In mature immune synapses, TCRs are concentrated into a central SMAC (cSMAC) along with the co-receptor CD28 and PKC-θ. Integrins such as LFA-1, stabilize the interaction with the APC but are excluded from the cSMAC, instead forming a concentric ring around the cSMAC that is termed the peripheral SMAC (pSMAC). Actomyosin structures that form at the pSMAC are important for sustained TCR signaling and Ag affinity discrimination (Hong et al., 2017; Murugesan et al., 2016; Yi et al., 2012). The distal SMAC (dSMAC), which forms outside the pSMAC ring, contains proteins with large ectodomains such as CD43 and CD45 (Davis and van der Merwe, 2006). The formation of an immune synapse in T cells and NK cells is critical to their function. The formation of receptor-ligand microclusters supports the assembly of “signalosomes”, which amplify downstream signaling processes (Dustin and Choudhuri, 2016; Hartman et al., 2009; Werlen and Palmer, 2002). Importantly, cytoskeletal-dependent processes at the immune synapse contribute to signal amplification by controlling the spatial organization of activating receptor clusters relative to signal enhancing co-receptors and inhibitors (Hammer et al., 2019). Moreover, in T cells, adhesive contacts made at the immune synapse support the serial interactions between TCRs and Ags that occur over hours to support full T cell activation (Bromley et al., 2001). In the case of cytotoxic T cells and NK cells, immune synapse formation is important for the directed section of lytic granules towards target cells, and is facilitated by depletion of actin from the cSMAC (Mace and Orange, 2012).    18 Immune synapse formation in B cells involves a similar reorganization of membrane proteins (Harwood and Batista, 2010) with three spatially separated regions that have distinct actin structures and organization of surface proteins (Wang and Hammer, 2019) (see Figure 1.3). When B cells encounter Ag-bearing membranes, BCR-Ag microclusters initially form throughout the contact site. The microclusters then undergo centripetal movement and into a cSMAC. A pSMAC enriched in integrins and CD19 forms around the cSMAC. The cSMAC is thought to be a site at which BCR-bound Ags are internalized so that they can be delivered to MHC II loading compartments, processed and presented to T cells in order to elicit T cell help (Batista et al., 2001; Natkanski et al., 2013; Yuseff et al., 2013). At the pSMAC, actin is assembled into concentric rings of linear contractile actomyosin filaments that resemble the lamella in migrating cells. In T cells, contractility generated by these concentric rings of linear filaments connected by non-muscle myosin IIA at the inner face of the peripheral branched actin network promotes TCR centralization (Hong et al., 2017; Murugesan et al., 2016; Yi et al., 2012) and these structures may also contribute to the centralization of BCR-Ag microclusters (Tolar, 2017). The dSMAC is composed of a lamellipodial-like branched actin network that drives BCR-induced cell spreading (Wang and Hammer, 2019). Potential negative regulators of BCR signaling, such as the transmembrane tyrosine phosphatases CD45 and CD148 are segregated away from BCRs and accumulate in the dSMAC (Harwood and Batista, 2011). This spatial reorganization of the BCR, other membrane proteins, and intracellular structures that accompanies immune synapse formation enhances the two major functions of the BCR, signal transduction and Ag internalization. Because BCR microclusters recruit signaling enzymes to form microsignalosomes (described above in section 1.1.4), immune synapse formation amplifies BCR signaling. Immune synapse formation also promotes efficient Ag internalization. The internalization of Ags that are extracted from the APC membrane allows B cells to process Ags and present them to T cells in order to elicit T cell help (Yuseff et al., 2013). As described below, the formation of an immune synapse, and thus the enhancement of BCR signaling and activation, requires remodeling of the actin cytoskeleton (Harwood and Batista, 2011; Li et al., 2019).   19 1.3.2 Cytoskeletal reorganization drives immune synapse formation  The actin cytoskeleton is critical for the dynamic changes in cell morphology and BCR reorganization that occur during immune synapse formation. The binding of membrane-bound Ag by the BCR causes rapid reorganization of BCRs into BCR-Ag microclusters (Harwood and Batista, 2010; Li et al., 2019; Tolar, 2011). When B cells are added to Ags that are tethered to planar lipid bilayers, microclusters are formed within the first minutes of Ag encounter. Initial BCR signaling leads to the activation of the Rap GTPase, which through an unknown mechanism, causes local activation of the actin severing protein cofilin. Cofilin-mediated actin severing initiates local breakdown of the actin cytoskeleton, which increases BCR mobility and presumably allows the concatenation of BCR nanoclusters into BCR microclusters (Freeman et al., 2015; Gold and Reth, 2019). The formation of BCR-Ag microclusters promotes the formation of microsignalosomes that function as the basic unit of BCR signaling. At the same time, the transient inactivation of members of the ezrin-radixin-moesin (ERM) family of proteins uncouples the submembrane actin cytoskeleton from the plasma membrane (Treanor et al., 2011). The subsequent phosphorylation and reactivation of ERM proteins around BCR microclusters promotes microcluster integrity. The association of the co-receptor CD19 with BCR microclusters is also dependent on the remodeling of the actin cytoskeleton (Depoil et al., 2008; Mattila et al., 2013). The interaction of CD19 with BCR microsignalosomes recruits and activates PI3K and Vav, transmitting signals to the pro-survival kinase Akt, the metabolic regulator mTOR, and proteins that regulate the actin cytoskeleton (Keppler et al., 2015).  BCR signaling also stimulates robust actin polymerization. Actin polymerization at the plasma membrane generates force that drives membrane protrusions (Mogilner and Oster, 1996; Pollard and Borisy, 2003). Localized BCR-induced actin polymerization at the site of B cell-APC contact exerts outward force on the B cell plasma membrane, causing the B cell to spread its membrane over the surface of the APC (Fleire et al., 2006; Song et al., 2013, 2014). Depleting key components of the BCR signaling pathway such as Btk, CD19, Vav, Rap, or PLCγ2 inhibits the B cell spreading response (Arana et al., 2008a; Depoil et al., 2008; Lin et al., 2008; Liu et al., 2011; Weber et al., 2008). Cell spreading increases the number of BCR microclusters that are formed and thereby increases BCR signaling (Fleire et al., 2006; Weber et al., 2008). This promotes further actin polymerization at the B cell immune synapse, creating a positive feedback   20 loop. The amplification of BCR signaling caused by this spreading response could increase the ability of B cells to respond to lower affinity Ags, and to membrane-bound Ags that are present at lower density. While the B cell scans the surface of the APC for Ags, adhesive contacts at B cell-APC contact site stabilize the B cell-APC interaction (Carrasco and Batista, 2006a). VLA-4 and LFA-1 integrins on the B cell bind to VCAM-1 and ICAM-1, respectively, on the APC. These adhesive contacts enhance B cell spreading, thereby increasing BCR signaling in response to low density and low affinity Ags (Carrasco and Batista, 2006b; Carrasco et al., 2004). The net result is that integrin-mediated adhesion lowers the Ag density and affinity threshold that is required for B cell activation (Carrasco and Batista, 2006a). Similarly, LFA-1-mediated adhesion in T cells increases the duration of TCR-induced Ca2+ signaling and increases the Ag sensitivity of the T cell by 100-fold (Dustin, 2012). Extracellular stimuli such as chemokines and Ags induce conformation changes in VLA-4 and LFA-1 integrins that increases their avidity and affinity for their ligands (Arana et al., 2008b). The Rap GTPase, which is required for B cell spreading and actin reorganization (Lin et al., 2008), is also required for chemokine- and Ag-dependent activation of integrins (McLeod et al., 2004). Rap activation recruits talin to the immune synapse, which upon binding to integrins, induces conformational change that increases ligand affinity (Comrie and Burkhardt, 2016). Talin also binds to the actin cytoskeleton, and transmits mechanical forces from actin retrograde flow to integrins, which increases integrin affinity (Calderwood et al., 2000; Comrie and Burkhardt, 2016). Integrin signaling activates downstream pathways that are critical for the B cell response including cell spreading, actin reorganization and cell survival (Arana et al., 2008b; Carrasco and Batista, 2006a; Koopman et al., 1994; McLeod et al., 2004). Therefore, integrin activation by chemokines and Ags can enhance BCR signaling by amplifying the cytoskeletal response during immune synapse formation.  The B cell spreading response is followed by membrane retraction that drives the aggregation of BCR-Ag microclusters as they move towards the center of the immune synapse. Generation of large BCR-Ag clusters can increase the physical interactions between BCRs, allowing BCR-associated Syk to access and phosphorylate more ITAMs and amplify Ag-induced signaling. Moreover, larger microclusters may more efficiently shield activated BCRs from negative regulators of BCR signaling, such as CD22 (Gasparrini et al., 2016). Additionally, the   21 centripetal movement of BCR-Ag microclusters could increase their interactions with clusters of signaling molecules such as CD19.  The centripetal movement and coalescence of BCR-Ag microclusters that results in the formation of a cSMAC (Fleire et al., 2006) involves both actin- and microtubule-dependent processes (Liu et al., 2012; Schnyder et al., 2011; Tolar, 2017; Treanor et al., 2011; Wang et al., 2017). As new actin is polymerized at the cell periphery, the elastic resistance of the plasma membrane causes the peripheral actin network to flow towards the center of the cell, which is termed actin retrograde flow (Mogilner and Oster, 1996). Actin retrograde flow is important for the centripetal movement of TCRs (Babich et al., 2012; Kumari et al., 2015) and actin dynamics have been shown to be important for this process in B cells (Liu et al., 2012; Treanor et al., 2011). The dynamic reorganization of the actin cytoskeleton also controls the localization of the microtubule cytoskeleton. Upon BCR signaling, the MTOC is polarized towards the immune synapse (Reversat et al., 2015; Yuseff et al., 2011, 2013) in an actin-dependent manner (Ibañez-Vega et al., 2019; Obino et al., 2016; Wang et al., 2017). In T cells, actin clearance at the center of the immune synapse allows for MTOC docking (Ritter et al., 2015; Stinchcombe et al., 2006). Tethering of the microtubule plus ends to the peripheral actin cytoskeleton is essential for MTOC polarization and requires proteins that link actin and microtubules such as IQGAP1 and ADAP (Combs et al., 2006; Stinchcombe et al., 2006) as well as the dynein motor protein (Martín-Cófreces et al., 2008; Quann et al., 2009; Yi et al., 2013). In B cells, MTOC polarization requires actin dynamics mediated by the Rap/cofilin pathway as well as actin-microtubule interactions mediated by CLIP-170 and IQGAP1 (Wang et al., 2017). Polarization of the MTOC and associated microtubules towards the B cell immune synapse promotes important immune synapse functions such as microcluster centralization, Ag extraction, and delivery of Ag to MHC II loading compartments (Schnyder et al., 2011; Yuseff et al., 2011).    22      23 Figure 1.3 B cell immune synapse formation The upper panel depicts an en face x-z cross-section of a B cell encountering an APC, spreading across its surface, gathering Ag, and then contracting to form an immune synapse. The lower panel depicts an x-y slice through the B cell at the B cell-APC contact site. (A) In resting B cells, the mobility of the BCR is restricted by the actin cytoskeleton. Because BCR signaling depends on BCR-BCR interactions, only a small fraction of BCRs are able to initiate downstream signaling pathways, as indicated by the blue stars in the upper panel that represent activated tyrosine kinases (e.g. Syk). (B) Upon Ag contact, BCR signaling promotes the local disassembly of the submembrane actin cytoskeleton via cofilin activation, increasing the mobility of BCRs (or BCR nanoclusters). This promotes the aggregation of BCRs (or the concatenation/ coalescence of BCR nanoclusters) as well as transient co-clustering with CD19 (illustrated in the lower panel), which allows more BCRs to recruit tyrosine kinases and activate signaling pathways (collision-coupling model). (C) Actin depletion at the center of the B cell-APC contact site is accompanied by actin polymerization at the periphery of the contact site, which drives B cell spreading. This allows the BCR to encounter more Ag and increases microcluster formation. BCR-Ag microclusters recruit and activate tyrosine kinases, in particular, Syk, leading to the formation of microsignalosomes that activate multiple BCR signaling pathways. (D) Subsequent membrane contraction is accompanied by the centripetal movement of BCR microclusters toward the center of the B cell-APC contact site. (E) The coalescence of BCR microclusters results in the formation of a cSMAC that is surrounded by clusters of activated integrins in the pSMAC. Potential negative regulators of BCR signaling, such as the tyrosine phosphatases CD45 and CD148, are segregated away from BCRs and accumulate in the dSMAC. During immune synapse formation, the MTOC and microtubule network is polarized towards the B cell-APC contact site. Reorientation of the microtubule network supports the coalescence of BCR microclusters into a cSMAC as well as Ag extraction and the delivery of BCR-Ag complexes to Ag processing compartments. Adapted from Abraham at el., 2016.       24 1.3.3 Ag extraction, processing, and presentation to T cells In addition to enhancing BCR signaling, immune synapse formation is important for B cells to acquire Ags from the APC. The aggregation of Ags at the cSMAC optimizes Ag extraction and the subsequent delivery of BCR-Ag complexes to endosomal compartments that process and load Ags onto MHC II (Natkanski et al., 2013; Yuseff et al., 2013). After B cells have acquired Ags, they migrate towards the T cell zones of the lymphoid organ in order to receive T cell help (Victora et al., 2010). The amount of Ag acquired by the B cell determines the amount of Ag available to present to T cells and is, therefore, a critical determinant of the capacity of B cells to elicit T cell help and become activated (Batista et al., 2001; Carrasco and Batista, 2006a). T follicular helper cells provide B cells with essential co-stimulatory signals, such as CD40 ligand, and cytokines such as IL-4 and IL-21 (Crotty, 2011). T cell help is required for germinal center formation and for the differentiation of long-lived plasma cells and memory cells (Crotty, 2011). Thus, Ag extraction and presentation to T helper cells are crucial to the generation of a long-lived and high-affinity antibody response.  B cells can extract Ags from APCs using either mechanical or proteolytic mechanisms, depending on the physical properties of the Ag-presenting substrate (Spillane and Tolar, 2017). B cells predominantly extract Ags from APCs via the mechanical mechanism, which involves actomyosin contractility (Natkanski et al., 2013; Spillane and Tolar, 2017). Mechanical forces generated by actomyosin pull on BCR-Ag complexes in an attempt to overcome the Ag’s affinity for the capture receptor on the APCs. This mechanism only allows high-affinity Ags to be extracted from the APC as the pulling force generated by actomyosin breaks most BCR-Ag bonds (Natkanski et al., 2013). When Ags are presented on follicular dendritic cells or conventional dendritic cells, B cells likely use this mechanical pathway. However, if the Ag-presenting substrate is very stiff (e.g. Ags tethered to planar lipid bilayers or polystyrene beads), B cells will employ the proteolytic mechanism to extract Ags. Lysosomes associated with the MTOC are positioned adjacent to the immune synapse upon MTOC polarization (Wang et al., 2017; Yuseff et al., 2011). Lysosomal contents are then secreted at the immune synapse and the released hydrolases can cleave the Ag such that it is released from the Ag-presenting surface and can be internalized by the BCR via receptor-mediated endocytosis. The mechanism by which B cells initiate this proteolytic Ag extraction mechanism after failed attempts to mechanically   25 extract Ag is not understood. Internalization of BCR-Ag complexes and their subsequent trafficking to endosomal compartments is thought to result in degradation of the BCR and the termination of BCR signaling (Stoddart et al., 2005). However, there is some evidence for on-going BCR signaling from early endosomes (Chaturvedi et al., 2011).   1.4 The actin cytoskeleton Changes in cell morphology are essential to many biological processes, including the formation of immune synapses in B cells (see Figure 1.3). The morphological changes required for immune synapse formation are driven by dynamic changes in the submembrane actin cytoskeleton. In addition to providing mechanical support for the cell membrane, the actin cytoskeleton has emerged as a key regulator of receptor signaling (Mattila et al., 2016), membrane organization (Kusumi et al., 2012), vesicle transport (Schuh, 2011), and transcription (Olson and Nordheim, 2010). Actin monomers (also called globular actin or G-actin) polymerize into helical filaments (also referred to as F-actin) that can be assembled into higher-order structures that are optimized to carry out specific cellular functions. For example, flat branching networks of actin filaments allow the cell to crawl on and spread over surfaces whereas bundles of linear unbranched filaments make finger-like projections that probe the external cell environment. At the immune synapse, branched actin structures are typically located at the periphery, with linear actin filaments bundled into actomyosin arcs at the inner face of the peripheral branched actin network. The center of the immune synapse is often depleted of actin structures. These distinct actin network architectures are assembled and disassembled by a complex network of actin regulatory proteins in a highly dynamic manner and can be rapidly remodeled and interconverted.      26 1.4.1 Actin assembly The nucleation of actin filaments is energetically unfavorable due to the instability of actin dimers and trimers (Pollard and Borisy, 2003). Hence, in vivo, actin filaments are generally nucleated by nucleation factors such as the actin-related protein 2/3 (Arp2/3) complex and formin proteins. Once a nucleus of 3-4 actin subunits is formed, actin monomers are added to the growing filament in the same orientation, giving actin polymers distinct polarity, with a “barbed” and a “pointed” end (Pollard and Borisy, 2003). Monomers can be added to or removed from either end of the filament but with different kinetic constants. At the barbed end, ATP-bound actin associates with a diffusion-limited rate constant and dissociates slowly (Pollard, 2016). At the pointed end, both association and dissociation of ATP-actin is much slower (Pollard, 2016). The net result is filament elongation at the barbed end. Once a filament is nucleated, elongation occurs readily, at a rate that is directly proportional to the concentration of actin monomers (Pollard and Borisy, 2003). The mismatch in rate constants between barbed and pointed ends results in the slow turnover or “treadmilling” of subunits along the filament. In vitro, the treadmilling of actin filaments is very slow under steady-state conditions (Pollard and Borisy, 2003). Once nucleated, purified actin polymerizes rapidly in vitro, resulting in long, relatively stable actin filaments and depletion of available monomers (Pollard, 2016). However, most cells maintain a large pool of actin monomers, and actin assembly and turnover occurs in seconds, much faster than filaments can turn over in vitro. The size of the pool of actin monomers that are available for polymerization is controlled by their nucleation state and proximity to membrane surfaces. To achieve this, cells express numerous actin-regulatory proteins that control the kinetics and localization of nucleation, elongation, and depolymerization.  The nucleotide state of actin monomers that are incorporated into filaments is critical for the function of actin in cells. Conformational changes in the actin monomer induced by polymerization increase the rate of ATP hydrolysis (Blanchoin and Pollard, 2002; Chou and Pollard, 2019). ATP hydrolysis is essential to maintain filament treadmilling as ADP-actin dissociates faster than ATP-actin (Pollard and Borisy, 2003). Nucleotide state also acts as an internal timer, indicating the local age of an actin filament and limiting the lifetime of actin filaments (Merino et al., 2019; Pollard and Borisy, 2003). Moreover, the nucleotide state influences the ability of actin-binding proteins to bind to actin filaments. For example, cofilin has   27 a higher affinity for ADP-actin, while the Arp2/3 complex has a higher affinity for ATP-actin. Thus, older segments of actin filaments containing primarily ADP-actin are more readily severed and debranched than newly polymerized segments (Andrianantoandro and Pollard, 2006; Pollard, 2007).   1.4.2 Actin nucleators Actin nucleation in cells requires nucleation proteins. Nucleators not only overcome the energetic barrier that restricts nucleation but also regulate the assembly of distinct actin networks in space and time. The Arp2/3 complex nucleates branched actin networks, formin proteins, and WASp homology 2 (WH2) domain-containing proteins such as spire nucleate linear actin filaments. The Arp2/3 complex is an essential actin nucleator as the deletion of its subunits is lethal in yeast and mice (Welch et al., 1997; Yae et al., 2006). The Arp2/3 complex binds to the side of existing mother filaments and nucleates the polymerization of a daughter filament by acting as the first two subunits (Pollard, 2007). The pointed end of the daughter filament is thus anchored to the side of the mother filament, with the barbed end elongating away from the site of nucleation, creating a Y-shaped branch with an angle of 70o (Mullins et al., 1998). Nucleation of actin by the Arp2/3 complex occurs at the plasma membrane, where nucleation promoting factors (NPFs) that activate the Arp2/3 complex are recruited and activated by GTPases (Campellone and Welch, 2010; Takenawa and Suetsugu, 2007). Moreover, polymerization at the barbed ends of branched actin networks pushes against the plasma membrane, which generates force that pushes the plasma membrane outward (Mogilner and Oster, 1996). The Arp2/3 complex is composed of seven subunits, two actin-related proteins, Arp2 and Arp3, which serve as the first two subunits of the daughter filaments, and 5 other subunits. The 5 other subunits (named actin-related protein complex 1-5 (ARPC1-5)), hold the Arp2 and Arp3 subunits apart, making the complex inactive (Pollard, 2016). As a consequence of this conformation, the purified Arp2/3 complex has little activity (Mullins et al., 1998; Welch et al., 1998). Additionally, binding of the Arp2/3 complex to the mother filament requires a conformational change, with the Arp2 and Arp3 subunits coming together to form a dimer   28 (Rouiller et al., 2008). This conformational change is induced by the binding of an NPF, which will be discussed in more detail in the next section.  In contrast to the Arp2/3 complex, formin proteins nucleate linear, unbranched actin filaments that are important for cellular processes such as formation of the cytokinetic ring, stress fibers, and filopodia (Goode and Eck, 2007). Formins promote actin nucleation by binding to and stabilizing energetically unfavorable actin dimers (Courtemanche, 2018). Formins also processively bind to the barbed end of actin filaments, enhancing elongation, and preventing capping (Paul and Pollard, 2009). Mammals have 15 formin proteins that each contain the formin homology 2 domain, which is essential for actin assembly (Goode and Eck, 2007). Interestingly, some formin proteins interact with and remodel the microtubule cytoskeleton and thus might be critical for microtubule-actin crosstalk (Goode and Eck, 2007). The precise functions of each mammalian formin is still being elucidated, but their importance is underlined by their involvement in many human diseases (Courtemanche, 2018).  Similar to actin nucleation by formins, other nucleator proteins that contain tandem WH2 domain repeats can bind 3-4 actin monomers and facilitate filament nucleation (Dominguez and Holmes, 2011). These WH2 domain-containing nucleators include Spire and cordon bleu. Little is known about how these proteins nucleate actin filaments and how their activity is regulated. Spire can interact with formin proteins to modulate their nucleation activity and can also bind to and bundle microtubules (Campellone and Welch, 2010), positioning Spire to be a key regulator of cytoskeletal interactions.   1.4.3 NPFs activate the Arp2/3 complex Arp2/3 complex-dependent actin networks are critical for a wide variety of cellular processes including phagocytosis, cell migration, and endocytosis. In order to nucleate actin structures that are optimized for these distinct purposes, the cell has many NPFs, each responding to distinct upstream signaling inputs and executing distinct cellular processes (Figure 1.4). The hematopoietic-specific WASp and the ubiquitous neural WASp (N-WASp) are the most well-studied NPFs (Campellone and Welch, 2010; Oda and Eto, 2013; Rotty et al., 2013; Sun et al., 2019; Takenawa and Suetsugu, 2007). Mutations in WASp or its binding partner WASp-interacting protein (WIP) result in Wiskott-Aldrich Syndrome, which is characterized by   29 severe immune dysregulation. Mutations in N-WASp result in neurological and cardiac abnormalities. WASp and N-WASp are activated by the Cdc42 GTPase as well as by polyphosphoinositides (such as PIP2) and SH3 domain-containing proteins such as Nck. WASp plays a critical role in processes requiring actin in immune cells such as immune synapse formation and phagocytosis. N-WASp directs Arp2/3 complex-dependent nucleation in a variety of cellular processes, including invadopodia formation, membrane ruffling, and endocytosis. The WASp-family verprolin-homologous protein (WAVE) family of NPFs, of which there are 3 mammalian isoforms, are broadly expressed and are primarily required for the formation of plasma membrane protrusions that drive cell migration (Oda and Eto, 2013; Rotty et al., 2013; Takenawa and Suetsugu, 2007). WAVE proteins form a complex with four accessory proteins, which together comprise the WAVE regulatory complex. WAVE complex activation depends on interactions with Rac1, Nck, or PIP3. Three more recently discovered WCA-domain containing NPFs, WASP homolog associated with actin, membranes, and microtubules (WHAMM), WASp and Scar homolog (WASH), and junction mediating and regulatory protein (JMY) diversify the functionality of the Arp2/3 complex (Rottner et al., 2010). WASH and WHAMM nucleate actin networks that move and shape intracellular compartments and can also associate with microtubules (Rottner et al., 2010). JMY is capable of both Arp2/3 complex-dependent and independent nucleation of actin filaments (Rottner et al., 2010). The number and diversity of NPFs equip cells with the ability to fine-tune Arp2/3 complex-dependent actin nucleation in order to execute a diverse set of cellular functions.       30   Figure 1.4 Functions of NPFs Branched actin networks drive a number of cellular processes. This is facilitated by a diverse network of NPFs. Each NPF responds to different upstream signals and then builds structures that execute distinct cellular processes. For more information, see text. Adapted, with permission, from Campellone and Welch, 2010.      31 1.4.4 Assembly of branched actin networks by the Arp2/3 complex  The Arp2/3 complex is one part of a complex molecular machine that controls the nucleation of branched actin networks at membrane surfaces (Figure 1.5). Besides the Arp2/3 complex, this machine includes existing actin filaments (mother filaments), actin monomers, NPFs, and capping proteins (Mullins et al., 2018). Regulation of this actin assembly machine and thus, the assembly of branched actin within the cell, is controlled by the activation of NPFs by upstream signaling pathways. NPFs are activated and recruited to the plasma membrane by Rho family GTPases. The accumulation of NPFs on the membrane stimulates the nucleation and elongation of a branched actin network with the growing barbed ends oriented towards the plasma membrane. NPFs activate the Arp2/3 complex via their WCA domain (also called VCA domain, where V stands for verprolin homology), which consists of a WH2 (W) domain, a connecting (C) motif, and an acidic (A) motif. The WH2 domain binds to actin monomers and brings them to the Arp2/3 complex to form the nucleus for filament growth. The CA regions bind to the Arp2/3 complex and induce a conformational change that enhances binding to the mother filament and aligns the Arp2 and Arp3 subunits so that they can act as a template for the nucleation of the daughter filament (Espinoza-Sanchez et al., 2018; Goley et al., 2004). The Arp2/3 complex must bind to two WCA sequences that are loaded with monomeric actin and to the mother filament before daughter filament nucleation occurs. After the daughter filament is nucleated, NPFs can enhance daughter filament elongation by delivering actin to the growing barbed end via their proline-rich region and WH2 domain. The rate of nucleation and elongation was thought to be dependent on the concentration of actin monomers in solution. However, recent studies have shown that by concentrating actin monomers onto membrane surfaces, NPFs enable branched actin networks to grow faster than can be expected than if monomer incorporation were diffusion-limited.  Recently, new roles for NPFs have been brought to light by in vitro studies. Bieling et al. demonstrated that when NPFs are not bound to monomeric actin, they can bind to the barbed ends of filaments, inhibiting elongation and tethering the network to the membrane (Bieling et al., 2018). Additionally, at high concentrations, NPFs can enhance filament elongation in an Arp2/3 complex-independent manner. This is achieved by increasing the availability of polymerization-competent monomeric actin at membrane surfaces. These authors also found that   32 although profilin-actin complexes cannot bind to WH2 domains, the proline-rich region of NPFs binds profilin-actin complexes and can transfer actin monomers to the WH2 domain. These findings suggest that most actin monomers that enter into actin networks are delivered by NPFs. Thus, all growing filaments within the network compete for the same pool of monomers and active NPFs. By regulating the concentration of actin monomers on membrane surfaces, NPFs also contribute to the negative feedback loop that controls network density and nucleation rates. Because Arp2/3 complex nucleation is autocatalytic (each new daughter filament can serve as a substrate for nucleation), branched actin networks could grow exponentially until all monomers are incorporated, thereby freezing the network. Instead, nucleation by the Arp2/3 complex is “product inhibited”, meaning that the more filaments there are in competition for the surface-associated pool of monomers, the slower the network grows. Hence, low-density networks with fewer barbed ends elongate faster, whereas high-density networks (with high nucleation rates) exhibit slower elongation rates (Mullins et al., 2018).  Once nucleated, monomers can be added to the barbed ends of actin filaments until the actin monomer pool is exhausted or the filaments are capped by capping proteins. Proteins that compete with capping proteins for barbed end-binding facilitate actin elongation (Chesarone and Goode, 2009; Edwards et al., 2014). Actin elongation factors, such as formin proteins and Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins, protect barbed ends from capping proteins and control the rate of filament elongation (Chesarone and Goode, 2009; Edwards et al., 2014; Pollard, 2016). Recently, the proline-rich domain of NPFs have also been implicated in filament elongation (Bieling et al., 2018).     33     34 Figure 1.5 Regulation of branched actin networks The location, shape, and dynamics of branched actin networks are regulated by a complex network of actin-binding and actin-regulatory proteins. Upon activation by the Cdc42 and Rac GTPases, the WASp and WAVE NPFs activate the Arp2/3 complex and provide actin monomers to the Arp2/3 complex. Nucleation of actin filaments occurs at the sides of pre-existing filaments such that the daughter filaments are assembled at a 70o angle relative to the mother filament. Daughter filaments elongate until capped by a capping protein. Rates of elongation are controlled by elongation factors such as formin and Ena/VASP proteins. Branched actin networks are disassembled in a two-step process. First, actin severing proteins (e.g. cofilin) and debranching proteins (e.g. GMFγ) fragment the network, preferentially removing older filament segments containing ADP-actin subunits. Then, actin-depolymerizing factors such as twinfilin and cyclase-associated protein (CAP) depolymerize the released filaments from the ends. The disassembly of branched actin networks and the liberation of monomers fuel actin assembly at the plasma membrane. Adapted from Pollard, 2007.       35 An essential player in this regulatory network are the capping proteins. Capping proteins bind to the barbed ends of actin filaments and inhibit monomer incorporation. Paradoxically, increasing the expression of capping proteins also increases the rate of cell migration (Hug et al., 1995) because it enhances Arp2/3 complex-dependent nucleation of branched actin networks in vitro and in vivo (Akin and Mullins, 2008). By terminating the growth of some filaments in the network, capping proteins increase the number of NPFs and actin monomers that are available to the Arp2/3 complex, thereby promoting filament nucleation (Akin and Mullins, 2008). This complex regulatory machine that includes the Arp2/3 complex, mother filaments, actin monomers, NPFs, and capping protein allows for exquisite spatiotemporal control over the architecture and dynamics of branched actin networks.   1.4.5 Disassembly of branched actin networks Actin monomer availability is a key regulator of Arp2/3 complex-mediated nucleation. Rapid polymerization cannot be sustained without replenishing the pool of monomeric actin. Proteins that disassemble and depolymerize actin filaments are therefore required to fuel actin polymerization by the Arp2/3 complex. The cofilin/actin-depolymerizing factor (ADF) family of proteins are essential for this process. Cofilin severs actin by binding to filaments and disrupting the interactions between actin subunits. The binding creates a twist in the filament, which promotes disassembly (Elam et al., 2013). In branched actin networks, this structural change also causes debranching (Chan et al., 2009; Gressin et al., 2015). In bundled actin networks, cofilin requires other factors such as actin interacting protein 1/WD-repeat protein 1(AIP1/Wdr1) to induce severing and filament disassembly (Gressin et al., 2015). Cofilin-mediated severing depends on the local density of cofilin bound to the filament. A current model for cofilin-mediated severing posits that AIP1/Wdr1 competes with cofilin for filament binding sites, creating interspersed cofilin-decorated and bare regions on filaments. Strain accumulates at the boundary between the cofilin-bound and bare regions and causes the filament to fragment Elam et al., 2013). Cofilin-mediated disassembly of Arp2/3 complex-generated branched actin networks is critical for actin turnover, and cofilin activity enhances the rate of actin-based motility (Carlier et al., 1997). Furthermore, local activation of cofilin results in a rapid burst of actin polymerization because cofilin-mediated severing increases the number of barbed ends that   36 can then be elongated (Ghosh et al., 2004). A similar process occurs in B cells that encounter surfaces that are coated with anti-Ig antibodies that cluster the BCR. Cofilin activity is required to remodel cortical actin at the contact site, generating barbed ends as well as actin monomers in order to drive B cell spreading (Freeman et al., 2011).  Cofilin is mostly associated with actin severing functions but can also debranch Arp2/3 complex-nucleated actin networks (Blanchoin et al., 2000; Chan et al., 2009). The glia maturation factor (GMF) proteins, which are members of the ADF homology family, also play a key role in disassembling Arp2/3-generated actin networks by promoting debranching (Goode et al., 2018). Unlike cofilin, GMF does not bind to actin and instead directly regulates the activity of the Arp2/3 complex (Gandhi et al., 2010). At high concentrations in vitro, GMF can inhibit the Arp2/3 complex by blocking the binding of the WCA domain of NPFs to the Arp2/3 complex (Gandhi et al., 2010). At lower concentrations, GMF stimulates the debranching of Arp2/3 complex-nucleated networks by inducing a conformational change that promotes branch disassembly (Gandhi et al., 2010; Ydenberg et al., 2013). GMF, therefore, is important for remodeling branched actin networks into linear arrays (Goode et al., 2018). However, all of these mechanistic studies have been conducted in yeast, and it is unclear whether mammalian GMFs have the same functions in vivo. Vertebrates express two GMF genes, GMFβ and GMFγ. These isoforms have different expression patterns, with GMFβ enriched in brain tissue, and GMFγ predominantly found in endothelial cells as well as the spleen and thymus (Inagaki et al., 2004; Zuo et al., 2013). GMFγ expression is important for cell migration in neutrophils, T cells, and monocytes (Aerbajinai et al., 2011, 2016; Lippert and Wilkins, 2012).  Coronins also mediate the turnover of Arp2/3 complex-nucleated actin networks. Coronins can bind to actin filaments, protecting newly-formed filaments from cofilin-mediated severing (Gandhi et al., 2010). However, coronin is also an inhibitor of the Arp2/3 complex. Coronins can directly inhibit the Arp2/3 complex (Humphries et al., 2002) and displace the Arp2/3 complex from branch junctions (Cai et al., 2007). Additionally, coronins can enhance cofilin activity on older filaments by interacting with the cofilin-activating phosphatase Slingshot (SSH), targeting it to the lamellipodia (Cai et al., 2007). Like cofilin, the contributions of coronins to actin turnover is required for efficient lamellipodial protrusions and cell migration   37 (Cai et al., 2007), making coronins important regulators of Arp2/3 complex-dependent processes (Campellone and Welch, 2010).  1.5 Regulation of the Arp2/3 complex-nucleated cytoskeleton at the B cell immune synapse  When B cells encounter their cognate Ag on the surface of an APC, BCR signaling induces substantial reorganization of the actin and microtubule cytoskeletons to enhance BCR signaling and Ag acquisition. A major target of BCR signaling is the activation of the Arp2/3 complex. The Arp2/3 complex is activated by Vav-mediated activation of the Cdc42-WASp/N-WASP/WIP and Rac-WAVE pathways (refer to Figure 1.3). The importance of this pathway is underscored by the primary immunodeficiency disease WAS, which is caused by mutations in the gene encoding the NPF, WASp. This syndrome is characterized by increased susceptibility to autoimmune disease, recurring infections, and predisposition to lymphomas and leukemias. These patients have major defects in T cell-dependent immunity that contribute to immune dysregulation. WASp-deficient T cells exhibit defects in migration, proliferation, and survival (Rivers and Thrasher, 2017; Sun et al., 2019). WASp deficiency in both human and mouse B cells leads to B cell-mediated autoimmunity (Becker-Herman et al., 2011; Recher et al., 2012) and WAS patients have elevated levels of IgE, IgA, and IgM autoantibodies (Candotti, 2018; Recher et al., 2012). Despite the presence of autoantibodies, WASp-deficient B cells have impaired Ag-specific antibody responses, especially to T cell-independent Ags (Recher et al., 2012). Moreover, B cells depleted of WASp display reduced BCR signaling, Ag internalization, and antibody responses (Liu et al., 2013).  The activation of WASp and N-WASp requires Cdc42 or SH3 domain-containing proteins such as Nck (Campellone and Welch, 2010). Upon Ag encounter, BCR-CD19 interactions induce the phosphorylation of CD19, which recruits the guanine nucleotide exchange factor Vav via its SH2 domain (Bustelo, 2000; Li et al., 1997; O’Rourke et al., 1998; Weng et al., 1994). CD19-mediated recruitment of Vav leads to phosphorylation of Vav by microsignalosome-associated tyrosine kinases such as Syk (Bustelo, 2000; Wu et al., 1997). Vav then activates Rac and Cdc42 GTPases, which in turn activate NPFs that are critical for activating the Arp2/3 complex-dependent nucleation of branched actin networks. In B cells,   38 many of these upstream activators of the Arp2/3 complex are required for B cell immune synapse formation and B cell antibody responses. Depleting Vav proteins impairs B cell spreading in response to membrane-bound Ag (Weber et al., 2008). Loss of Cdc42 impairs B cell development, plasma cell differentiation, and antibody responses in mice (Burbage et al., 2015; Guo et al., 2009), whereas depletion of Nck results in impaired BCR signaling and antibody responses (Castello et al., 2013). At the B cell immune synapse, active phosphorylated WASp localizes to the peripheral actin ring, where actin polymerization occurs. WASp-deficient B cells also have defects in microcluster aggregation and Ag extraction (Becker-Herman et al., 2011; Liu et al., 2011). A similar defect in B cell spreading and microcluster formation occurs in memory B cells from WAS patients (Bai et al., 2016). WASp activity also depends on its binding partner WIP. Loss of WIP in B cells results in impaired B cell migration and homing, as well as defective BCR signaling through the CD19-PI3K pathway, impaired germinal center formation, and decreased antibody responses (Bai et al., 2016; Keppler et al., 2015, 2018; Sun et al., 2019). Moreover, in mice lacking DOCK8, another activator of the Rac GTPase, expression and activation of both WASp and CD19 is reduced, leading to defects in B cell spreading, BCR clustering and BCR signaling (Randall et al., 2009; Sun et al., 2018).  The role of N-WASp in B cells is not fully understood. Liu et al. reported that N-WASp-deficient mice have elevated levels of autoantibodies and increased Ag sensitivity, indicating that N-WASp is a negative regulator of BCR signaling (Liu et al., 2013). However, Volpi et al. demonstrated that autoantibody production in mice lacking WASp requires the expression of N-WASp and that depleting both N-WASp and WASp lead to a complete abrogation of antibody responses to T-cell dependent Ags (Volpi et al., 2016). These findings indicate that there is some redundancy in the roles of WASp and N-WASp in promoting B cell activation (Volpi et al., 2016; Westerberg et al., 2012). Although roles for WASp, N-WASp, and WIP have been identified in B cells, a direct role for WAVE has yet to be established.  The Arp2/3 complex-dependent actin network is also regulated by actin disassembly factors. Actin disassembly at the inner face of the peripheral ring of branched actin liberates monomers to fuel Arp2/3 complex-dependent nucleation. Rap GTPase-dependent activity of the actin severing protein cofilin is required for B cell spreading, microcluster formation, APC-induced BCR signaling, and cSMAC formation (Freeman et al., 2011; Lin et al., 2008; Wang et   39 al., 2017). Disruption of actin depolymerization also results in immune dysfunction. Loss-of-function mutations in Wdr1, a protein that enhances the activity of cofilin, results in severe immunodeficiency disease associated with significant defects in neutrophil migration (Kuhns et al., 2016). As in WAS, patients with Wdr1 mutations exhibit autoinflammation and thrombocytopenia (Standing et al., 2017). Neutrophils in mice lacking Wdr1 have increased levels of actin and cofilin, resulting in inefficient migration (Kile et al., 2007). Recently, patients with Wdr1 deficiency were found to have aberrant T cell immune synapse formation and T cell activation (Pfajfer et al., 2018). These patients also had defects in B cell development and function (Pfajfer et al., 2018). Despite the clear requirement for actin disassembly factors at the B cell immune synapse, little is known about how these factors regulate B cell immune responses. In addition to cofilin, novel inhibitors of the Arp2/3 complex such as GMFγ, the coronin family, and arpin could be important regulators of actin assembly at the B cell immune synapse. In chapter 5, I investigate the role of cofilin and its regulators, Wdr1 and LIM kinase (LIMK) in BCR-induced actin dynamics, B cell immune synapse formation, and BCR signaling in response to APC-bound Ags. Although individual roles for regulators of the Arp2/3 complex have been extensively investigated, a direct role for the Arp2/3 complex in B cell immune synapse formation and function has not been established. In chapter 3, I investigate the role of the Arp2/3 complex in the spatial organization of BCRs and BCR microclusters and the B cell immune synapse. In chapter 4, I investigate the role of the Arp2/3 complex in regulating BCR signaling in response to APC-bound Ags. Recently, a WAS-like disease has been identified in patients with loss-of-function mutations in Arpc1B, the hematopoietic-specific isoform of the Arpc1 subunit of the Arp2/3 complex. These patients have defects in T cell responses that result in combined immunodeficiency and severe inflammation (Brigida et al., 2018; Kahr et al., 2017; Kuijpers et al., 2017; Somech et al., 2017; Volpi et al., 2019). However, the role of B cells in this disease is less well defined. Because the actin cytoskeleton is a critical regulator of both Ag-independent and Ag-induced BCR signaling, the Arp2/3 complex is likely to have a significant role in B cell function. For example, the submembrane actin cytoskeleton controls the interactions between BCRs and key signal amplifiers such as CD19. CD19 phosphorylation is especially important in B cell responses to membrane-bound Ag but not soluble Ag (Depoil et al., 2008). CD19 is   40 relatively immobile on the surface of the B cell, and its mobility is restricted by the CD81 tetraspanin network (Mattila et al., 2013). Actin reorganization upon Ag encounter increases BCR mobility, allowing BCRs to interact with immobilized CD19 (Mattila et al., 2013). However, defects in actin polymerization, such as WIP deficiency, result in diminished CD19 phosphorylation and PI3K signaling in response to Ag stimulation (Keppler et al., 2015). This suggests that although the breakdown of the actin cytoskeleton permits BCR-CD19 interactions, repolymerization of branched actin could be important for supporting these interactions to achieve downstream signaling. Whether the Arp2/3 complex-dependent actin networks enhance functional interactions between the BCR and CD19 is not known, and I investigate this question in chapter 4. Moreover, the molecular mechanisms that orchestrate the actin-dependent formation, movement, and spatial organization of BCR microclusters, which are critical for APC-induced B cell activation, and require actin polymerization are not well understood.     41 1.6 Hypothesis and specific aims  Consistent with the idea that the actin cytoskeleton regulates immune synapse formation and BCR signaling, mutations in Arp2/3 complex components, the NPFs that activate the Arp2/3 complex, and in actin-binding proteins that work in concert with the Arp2/3 complex (e.g. Wdr1) result in immune dysfunction.  Therefore, my overall hypothesis is that actin, the Arp2/3 complex, and other key actin regulatory proteins (e.g. Wdr1, cofilin) control the spatial patterning of receptors at the B cell immune synapse and thereby control APC-induced BCR signaling and APC-induced B cell activation.  The specific aims for my thesis project are to test the following hypotheses: 1. Branched actin networks nucleated by the Arp2/3 complex are required for the centripetal movement and coalescence BCR-Ag microclusters at the B cell immune synapse (Chapter 3). 2. The Arp2/3 complex-dependent centralization and coalescence of BCR-Ag microclusters amplifies BCR signaling, increases BCR-CD19 interactions, and promotes B cell activation (Chapter 4). 3. Actin disassembly factors (cofilin and its regulators Wdr1 and LIMK) work in concert with the Arp2/3 complex to control actin dynamics in B cells, thereby regulating the centralization and coalescence of BCR-Ag microclusters as well as BCR signaling (Chapter 5).   42 Chapter 2: Methods  2.1 Cell isolation and culture  2.1.1 Primary B cell isolation and culture Primary murine B cells were obtained from spleens from 8- to 12-week old C57BL/6J (Jackson Laboratories, #000664), MD4 (Goodnow et al., 1988) (Jackson Laboratories, #002595) or Nur77GFP (Moran et al., 2011) mice of either sex following protocols approved by the University of British Columbia Animal Care Committee. B cells were isolated using a negative selection B cell isolation kit (Stemcell Technologies, #19854A). B cells were cultured in RPMI-1640 medium (Sigma, #RNBG5535) supplemented with 5% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 1 mM pyruvate, 50 µM 2-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin (complete RPMI-1640 medium). Cells were cultured in a 5% CO2 atmosphere at 37°C.   2.1.2 B cell lines and cell culture A20 murine IgG+ B lymphoma cells (confirmed to be IgG+ by flow cytometry) were obtained from ATCC (#TIB-208, #CRL-1596, respectively). A20 D1.3 mouse B cells, which express a transgenic hen egg lysozyme (HEL)-specific BCR, were a gift from F. Batista (Ragon Institute, Cambridge, MA) (Batista and Neuberger, 1998). A20 and A20 D1.3 B cells were cultured in RPMI-1640 medium supplemented with 5% heat-inactivated FCS, 2 mM glutamine, 1 mM pyruvate, 50 µM 2-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin. Ramos and Raji D1.3 B cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 1 mM pyruvate, 50 µM 2-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were cultured in a 5% CO2 atmosphere at 37°C.       43 2.1.3 APC cell lines and cell culture  COS-7 cells (ATCC, #CRL-1651) were cultured in DMEM supplemented with 5% FCS, 2 mM glutamine, and 1 mM pyruvate. B16F1 cells (ATCC, #CRL-6323) were cultured in DMEM supplemented with 10% FCS, 2 mM glutamine, and 1 mM pyruvate (complete DMEM medium). Cells were cultured in a 5% CO2 atmosphere at 37°C.  2.2 Transfections  2.2.1 Transient transfection of B cell lines  A20, A20 D1.3, Ramos, and Raji D1.3 B cells (3 × 106 cells) were transiently transfected using AMAXA Cell Line Nucleofector Kit V (Lonza, #VCA-1003) or the Ingenio Electroporation Kit (Mirus, #MIR 50118). The cells were transfected with 1.5 µg of either control siRNA (ON-TARGETplus Non-Targeting Pool, Dharmacon, #D-00810-01-05), Actr3 siRNA (SMARTpool ON-TARGETplus, Dharmacon, #L-046642-01-0005), Wdr1 siRNA (SMARTpool ON-TARGETplus, Dharmacon, #L-047667-01-005), or cofilin siRNA (ON-TARGETplus, Dharmacon, #L-058638-01-0005) or with 2 µg of plasmid DNA. See Table 1 for a list of plasmid constructs that were used to express proteins in B cells.       44 Table 1 Plasmid constructs used to express proteins in B cells  Name Origin  β-actin-GFP Gift from R. Nabi (UBC) Photo-convertable-β-actin-GFP Gift from S. Bamji (UBC) mTagRFP-T-Lifeact7  Addgene, #54586 F-Tractin-GFP Gift from J. Hammer (NIH) Myosin IIA-GFP Addgene, #38297 (Jacobelli et al., 2009) GMFγ-GFP  Origene, #RG210289 pEGFP-C1 Clontech; discontinued mCherry2-C1 Addgene, #54563 pMSCV-Syk-mCherry  Addgene, #50045 pMSCV-mCherry-Vav1  Addgene, #50044       45 2.2.2 Transient transfection of APCs  COS-7 and B16F1 cells were transiently transfected using Lipofectamine 3000 (Thermo Fisher, #L3000008) with plasmid DNA encoding either mHEL-GFP, mHEL-HaloTag, or a single-chain anti-Igκ antibody. mHEL-GFP is a transmembrane protein consisting of the complete HEL and GFP proteins in the extracellular domain, fused to the transmembrane region and cytosolic domain of the H-2Kb protein (Batista et al., 2001). The mHEL-HaloTag fusion protein consists of the complete HEL protein, the transmembrane region and 23-amino acid cytoplasmic domain of H-2Kb, and the complete HaloTag protein (Figure 2.1) (Wang et al., 2018). The DNA fragment encoding the HaloTag protein was excised from the pHTC HaloTag CMV-neo vector (Promega, #G7711). To avoid excessively high levels of expression, the HEL-HaloTag construct was subcloned into the pβN1 expression vector, a derivative of pEGFP-C1 in which the EGFP gene was removed and the CMV promoter was replaced with the weaker β-actin promoter. The single-chain anti-Igκ antibody is a transmembrane protein consisting of the single-chain Fv with the variable regions from the 187.1 rat anti-Igκ monoclonal antibody, fused to the hinge and membrane-proximal domains of rat IgG1 and the transmembrane and cytoplasmic domains of the H-2Kb protein (Ait-Azzouzene et al., 2005). Where indicated, B16F1 cells were co-transfected with 1.5 µg of either control siRNA (ON-TARGETplus Non-Targeting Pool, Dharmacon, #D-001810-01-05) or Actr3 siRNA (SMARTpool ON-TARGETplus, Dharmacon, #L-046642-01-0005).      46    Figure 2.1 Plasmid map for mHEL-HaloTag The mHEL-HaloTag fusion protein consists of the complete wild-type HEL protein, the transmembrane region and 23-amino acid cytoplasmic domain of the H-2Kb protein, and the complete HaloTag protein. This HEL-HaloTag cDNA construct was subcloned into the pβN1 mammalian expression vector, a derivative of pEGFP-C1 in which the EGFP gene was removed and the CMV promoter was replaced with the β-actin promoter.      47 2.3 Cytoskeletal inhibitors To inhibit the Arp2/3 complex, cells were resuspended in modified HEPES-buffered saline (mHBS; 25 mM HEPES, pH 7.2, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 mg/ml glucose, 2 mM glutamine, 1 mM pyruvate, 50 µM 2-mercaptoethanol) supplemented with 2% FCS (mHBS-FCS) along with 100 µM of the Arp2/3 complex inhibitor CK-666 (Hetrick et al., 2013) (Calbiochem, #CAS 442633-00-3) or the inactive analog CK-689 (Hetrick et al., 2013) (Calbiochem, #CAS 170930-46-8) for 1 hr at 37°C. The cells were then stimulated with soluble or plate-bound anti-Ig or APCs. To inhibit myosin IIA, B cells were treated with 50 µM of (S)-nitro-blebbistatin (pnBB) (Cayman Chemicals, #CAS 856925-71-8) or DMSO for 30 min at 37°C.   2.4 Immunoblotting Cells were lysed in RIPA buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Igepal (Sigma-Aldrich), 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, 1 mM PMSF, 10 µg/ml leupeptin, 1 µg/ml aprotinin, 25 mM β-glycerophosphate, 1 µg/ml pepstatin A, 10 µg/ml soybean trypsin inhibitor, 1 mM Na3MoO4, 1 mM Na3VO4) and analyzed by immunoblotting. Filters were incubated overnight at 4°C with the primary antibodies described in Table 2. Immunoreactive bands were visualized using horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad #170–6515; 1:3000) or goat anti-mouse IgG (Bio-Rad #170–6516; 1:3000), followed by ECL detection (GE Life Sciences). Blots were quantified and imaged using a Li-Cor C-DiGit imaging system.      48 Table 2 Primary antibodies used for immunoblotting  Target Species Manufacturer, Catalogue number Dilution Arp3 Rabbit Santa Cruz, #sc-15390 1:1000 Arp2 Rabbit abcam, #ab128934 1:1000 p34/ARPC2 Rabbit Millipore, #07–227 1:1000 β-actin Mouse  Santa Cruz, #sc-47778 1:5000 CD79a Rabbit Gold et al., 1991 1:5000 pCD79a Rabbit Cell Signaling Technologies, #5173 1:1000 pCD19 Rabbit Cell Signaling Technologies, #3571 1:1000 CD19 Rabbit Cell Signaling Technologies, #3574 1:1000 pERK Rabbit Cell Signaling Technologies, #9101 1:1000 ERK Rabbit Cell Signaling Technologies, #9102 1:1000 pAkt Rabbit Cell Signaling Technologies, #9271 1:1000 Akt Rabbit Cell Signaling Technologies, #9272  1:1000  2.5 B cell spreading on immobilized anti-Ig  Glass coverslips were coated with 2.5 µg/cm2 goat anti-mouse IgG (for A20 B cells; Jackson ImmunoResearch, #115-005-008) or donkey anti-human IgM (for Ramos and Raji D1.3 B cells; Jackson ImmunoResearch, #109-005-043) and then blocked with 2% BSA in PBS, as described previously (Lin et al., 2008). B cells (7.5 × 104) were transfected or pre-treated as described above and added to the prepared coverslips in 0.1 ml of mHBS-FCS. At the indicated times, cells were fixed and stained as described in section 2.7.1 for confocal microscopy or in section 2.7.2 for STED microscopy. Alternatively, cells were transfected with fluorescent proteins to visualize actin filaments and imaged in real-time at 37°C using total internal reflection (TIRF) microscopy or TIRF-structured illumination microscopy (TIRF-SIM), as described in section 2.8.2 and 2.8.3, respectively.    49  2.6 B cell interactions with APCs  2.6.1 Preparation of APCs  APCs were transfected as described in section 2.2.1. Cells were cultured for 18-24 hr before being detached using enzyme-free dissociation buffer (0.5 mM EDTA, 100 mM NaCl, 1 mM glucose, pH 7.4). The cells (1.5 × 104) were then seeded onto coverslips that had been coated with 5 µg/ml fibronectin for 30 min. The cells were then cultured for 18-24 hr in complete DMEM medium. For APCs expressing mHEL-HaloTag, coverslips were washed once with mHBS-FCS and then incubated with Janelia Fluor 549 HaloLigand (Grimm et al., 2015) (1:20,000 in mHBS-FCS) for 15 min at 37°C. The coverslips were then washed once with mHBS-FCS and kept in mHBS-FCS until the B cells were added.   2.6.2 Interaction of primary murine B cells with APCs Primary murine B cells (1 × 106) from C57BL/6J or MD4 mice were added to APCs expressing the single-chain anti-Igκ surrogate Ag or to APCs expressing mHEL-HaloTag that was labeled with either the HaloTag-tetramethylrhodamine ligand (Promega, #G8251) or Janelia Fluor 549 (Grimm et al., 2015). After the indicated times at 37°C, the cells were fixed and stained as described in section 2.7.1. Alternatively, B cells and APCs were lysed in RIPA buffer and analyzed by immunoblotting as described in section 2.4.   2.6.3 Interaction of B cell lines with APCs  A20 B cells (5 × 105 in 0.1 ml mHBS-FCS) were added to anti-Igκ-expressing APCs. A20 D1.3 or Raji D1.3 B cells (5 × 105 in 0.1 ml mHBS-FCS) were added to APCs expressing mHEL-GFP or to APCs expressing mHEL-HaloTag that was labeled with either the HaloTag-tetramethylrhodamine ligand (Promega, #G8251) or with Janelia Fluor 549 (Grimm et al., 2015). After the indicated times at 37°C, the cells were fixed and stained as described in section 2.7.1. For live-cell imaging in which actin filaments were visualized, A20 D1.3 or Raji D1.3 B cells expressing either F-Tractin-GFP or LifeAct-GFP (5 × 105 in 0.1 ml mHBS-FCS) were added to APCs expressing mHEL-GFP or to APCs expressing mHEL-HaloTag that was labeled with   50 either the HaloTag- tetramethylrhodamine ligand (Promega, #G8251) or Janelia Fluor 549 (Grimm et al., 2015). The cells were then imaged by spinning disk microscopy or instant structured illumination microscopy (ISIM) as described in sections 2.8.1 and 2.8.4, respectively.  2.7 Cell staining for fluorescence microscopy  2.7.1 Immunostaining for fluorescence microscopy Cells were fixed with 4% PFA in PBS for 10 min at room temperature, then permeabilized for 3 min at room temperature with 0.1% Triton X-100 in PBS and blocked with 2% BSA in PBS for 30 min at room temperature. Primary antibodies were diluted in PBS with 2% BSA. Coverslips were incubated with primary antibodies (Table 3) for 1 hr at room temperature or overnight in the cold, washed with PBS, and then incubated for 30 min at room temperature with secondary antibodies (Table 4) diluted in PBS. Where indicated, actin filaments were stained using fluorophore-conjugated phalloidin (Table 5). Coverslips were washed and then mounted onto glass slides using ProLong Diamond anti-fade mounting reagent (Thermo Fisher, #P36965).  Table 3 Primary antibodies used for immunostaining  Target Species  Manufacturer, Catalogue number Dilution Incubation pZap70(Y319)/ pSyk(Y352) Rabbit  Cell Signaling Technologies, #2701 1:200 1 hr, room temp pCD79a(Y182) Rabbit Cell Signaling Technologies, #5173 1:400 Overnight, 4°C pCD19(Y531)  Rabbit Cell Signaling Technologies, #3571 1:200 1 hr, room temp p34-Arc/ARPC2 Rabbit EMD Millipore, #07-227 1:200 1 hr, room temp      51 Table 4 Secondary antibodies used for immunostaining  Target Species  Manufacturer, Catalogue number Dilution Incubation Alexa Fluor 488 anti-rabbit IgG Goat  Invitrogen, #A-11008    1:400    30 min, room temp  Alexa Fluor 568 anti-rabbit IgG Goat Invitrogen, #A11036 Alexa Fluor 647 anti-rabbit IgG Goat Invitrogen, #A21244 Alexa Fluor 647 anti-rat IgG Goat Invitrogen, #A-21247   Table 5 Fluorophore-conjugated phalloidin probes for visualization of actin filaments  Conjugated fluorophore Manufacturer, Catalogue number Dilution Alexa Fluor 488 Thermo Fisher, #A12379 1:400 Rhodamine Thermo Fisher, #R415 1:400 Alexa Fluor 532 Thermo Fisher, #A22282 1:200 Alexa Fluor 568 Thermo Fisher, #A12380 1:400 Alexa Fluor 647 Thermo Fisher, #A22287 1:400      52 2.7.2 Immunostaining for STED microscopy Cells that were allowed to spread on anti-IgG-coated coverslips were fixed with 4% PFA for 10 min at room temperature and permeabilized using 0.1% Triton X-100 in PBS. To visualize actin filaments, cells were stained with Alexa Fluor 532-conjugated phalloidin (Thermo Fisher, #A22282, 1:200) for 30 min at room temperature. Coverslips were mounted onto slides using ProLong Diamond anti-fade reagent (Thermo Fisher, #P36965).   2.7.3 Single-particle tracking (SPT) SPT using directly-conjugated Fab fragments of antibodies was performed as described previously (Abraham et al., 2017). Primary B cells from C57BL/6J mice were resuspended to 107 cells/ml in RPMI-1640 without phenol red (Life Technologies #32404014) plus 5 mM HEPES. The cells were then placed on ice and labeled for 5 min with 1 ng/ml Cy3-conjugated anti-mouse IgM Fab fragments (Jackson ImmunoResearch, #115-167-020), 100 ng/ml Cy3-conjugated Fab fragments of the 11–26c rat anti-mouse IgD monoclonal antibody (ATCC, #HB-250), or 10 ng/ml Cy3-conjugated Fab fragments of the 1D3 rat anti-mouse CD19 monoclonal antibody (ATCC, #HB-305). Fab fragments of the 11–26c antibody and 1D3 antibody were prepared by AbLab (Vancouver, Canada) using a Fab preparation kit (Pierce, #44985). If inhibitory drugs where used, they were present throughout the staining process, including washes. The labeled B cells were then attached to coverslips that had been coated with 0.25 µg/cm2 of the non-stimulatory M5/114 anti-MHC II monoclonal antibody (Millipore, #MABF33). The cells were then imaged using TIRF microscopy (TIRFM) as described in section 2.8.2.       53 2.8 Microscopy   2.8.1 Confocal microscopy Spinning disk confocal microscopy was performed using a system based on a Zeiss Axiovert 200M microscope with a 100X 1.45 NA oil Pan-Fluor objective and a QuantEM 512SC Photometrics camera (Intelligent Imaging Innovations). Z-stacks were acquired at 0.2-µm intervals. For live-cell imaging, z-stacks through the B cell-APC contact site were acquired every 12 s.    2.8.2 TIRF microscopy (TIRFM)  TIRFM was performed using an Olympus TIRFM system consisting of an inverted microscope (Olympus IX81) equipped with a 150X 1.45 NA TIRFM objective (Olympus), motorized filter wheel (Olympus), high-performance electron multiplier (EM)-charge-coupled device (CCD) camera (Photometrics Evolve), and real-time data acquisition software (Metamorph). For live-cell imaging of B cells spreading on anti-Ig coated coverslips, the TIRF plane was adjusted to obtain a penetration depth of 90-100 nm from the coverslip and the cells were imaged for 10 min at 1 frame per second. For single-particle tracking, the 561 nm solid-state diode laser was used to excite Cy3-labeled samples. The TIRF plane was adjusted to yield a penetration depth of 85–90 nm from the coverslip and the cells were imaged for 10 s at 33 frames per second.   2.8.3 High-resolution live-cell imaging of B cells spreading on anti-Ig-coated coverslips   A20 B cells expressing F-Tractin-GFP were imaged at 37°C using either a Zeiss LSM 880 Airyscan microscope in super-resolution mode with a 63X 1.4 NA objective or a TIRF-SIM microscope (DeltaVision OMX-SR, GE Healthcare Life Sciences) equipped with a 60X 1.42 NA objective (Olympus). Images were acquired at 1-2 s intervals. For Airyscan images, raw data were processed using automatically determined parameters. For TIRF-SIM images, raw data were reconstructed using Softworx (Applied Precision Ltd.; GE Healthcare Life Sciences) with a value of 0.01 for the Wiener filter constant.    54 2.8.4 High-resolution live-cell imaging of B cell-APC interactions F-Tractin-GFP-expressing A20 D1.3 B cells were added to mHEL-HaloTag-expressing COS-7 cell APCs that had been labeled with the Janelia Fluor 549 dye (Grimm et al., 2015) and then imaged using 3D-ISIM (York et al., 2013). Z-stacks through the B cell-APC contact site were acquired every 6.6 s and the images were deconvolved as described York et al., 2013). Briefly, the images were subjected to 10 iterations of Richardson-Lucy deconvolution with a Gaussian point spread function having a full width half maximum (0.22 µm lateral and 0.5 µm axial) that was derived from measurements of 100 nm yellow/green beads.    2.8.5 STED microscopy STED images were acquired using a Leica TCS SP8 laser scanning STED system equipped with a 592 nm depletion laser, a CX PL APO 100X 1.40 NA oil objective, and a Leica HyD high sensitivity detector. Image deconvolution was performed using Huygens software (Scientific Volume Imaging, Hilversum, Netherlands).   2.8.6 Fluorescence recovery after photobleaching (FRAP) COS-7 cells that had been transfected with actin-GFP (1.5 × 105 cells) were plated on fibronectin-coated coverslips and cultured overnight. Fluorescence recovery after photobleaching was performed as described previously (Freeman et al., 2011) using an Olympus FV1000 confocal microscope with a 100X 1.40 NA oil objective. Briefly, within a selected region of interest (ROI), the pre-bleached fluorescence intensity was measured, followed by photobleaching using the 405 nm laser (100% intensity for 3 s). Fluorescence recovery within the same ROI was then recorded for 40 s. FluoView v4.0 software (Olympus) was used to quantify the fluorescence within the ROI, which was then normalized to the pre-bleach fluorescence intensity.       55 Table 6 Microscopy systems used  Microscope Resolution  Technique  Application Spinning disk confocal Diffraction limited Multiple point scanning - Live-cell imaging of B cell-APC interactions - Optical sectioning of B cell-APC contact site after fixation   Laser scanning confocal  Diffraction limited Point scanning - FRAP - Optical sectioning of B cell-APC contact site after fixation  Stimulated emission depletion (STED) ~50 nm  (x-y) Point scanning - High spatial resolution imaging of fixed cells; actin structures Total internal reflection microscopy (TIRFM) Diffraction limited Widefield  - High temporal resolution live-cell imaging of B cells spreading on anti-Ig-coated coverslips - SPT TIRF-Structured illumination microscopy (TIRF-SIM) ~80 nm  (x-y) Widefield with modulated light - High spatial and temporal resolution live-cell imaging of B cells spreading on anti-Ig coated coverslips Instant structured illumination microscopy (ISIM) ~140 nm  (x-y) Multiple point scanning - High spatial resolution live-cell imaging of B cell-APC interactions  Airyscan  ~120 nm  (x-y) Point scanning - High spatial resolution live-cell imaging of B cells spreading on anti-Ig coated coverslips      56 2.9 Image analysis  2.9.1 Quantification of cell spreading area and actin organization   Cell contact area was quantified using FIJI (Schindelin et al., 2012) from images taken of the contact site between the B cell and the coverslip or the B cell and the APC. Actin staining was used to define the cell periphery. The percent of the cell area that was depleted of actin was also determined using FIJI. The outer face of the peripheral actin ring was used to define the cell edge and compute the total cell area and the inner face of the peripheral actin ring was used to delimit the central actin-depleted region of the cell. Kymographs were generated using FIJI software (see schematic in Figure 2.2).     Figure 2.2 Schematic of kymograph analysis Kymographs are 2-D space-time plots. (A) The vertical axis represents time and the horizontal axis represents the distance along a line that is fixed in space. The graph shows the time-dependent evolution of the fluorescence intensities for each pixel along the dotted distance line. Static objects, such as the blue dot in (B), yield vertical lines on the kymograph as their locations on the line (orange dashed lines in (A) and (B)) do not change over time. Mobile objects, such as the red dot in (B), yield lines that deviate from the horizontal, with dx/dt being proportional to the object’s velocity. Faster motion is indicated by lines with larger deviation from the vertical. (C) Example of kymograph analysis of a BCR-Ag microcluster (circled in red) moving from the cell periphery to the cell center along the orange line. The kymograph generated along the orange line is shown on the right. The track made by the microcluster circled in red is highlighted by the red arrowhead.    57 2.9.2 Identification of Ag clusters, quantification of fluorescence intensity, and cSMAC formation using ImageJ  In chapter 3 and chapter 5, the total fluorescence intensity of clustered Ag or signaling molecules at the B cell-APC interface and the microcluster area were quantified using “analyze particles” in ImageJ, which detects objects from a binarized image. Background subtraction was carried out using the “rolling ball” method (https://imagej.net/Rolling_Ball_Background_Subtraction), which calculates the average pixel intensity surrounding the pixels of interest and subtracts that from the original image. The area and total fluorescence intensity was then determined for each cluster, as was the number of clusters per cell and the total fluorescence within the clusters on each cell. This process was automated using a custom macro. Cells were determined to have formed a cSMAC if >90% of the total Ag fluorescence was contained within 2 clusters or less. The custom FIJI macros used in this thesis can be found at https://github.com/madscience12/FIJImacros/blob/master/APC_analyzer_MBM.ijm.  2.9.3 Identification of Ag clusters and quantification of fluorescence intensity using a custom MATLAB program In chapter 4, the total fluorescence intensity of clustered Ag or clustered signaling molecules at the B cell-APC interface was quantified using a custom MATLAB code written by co-author Joshua Scurll (Bolger-Munro et al., 2019). This approach generally yielded similar results as the ImageJ methodology described in section 2.9.2. Each image was filtered (i.e. convolved) with a 3 × 3 averaging filter to smooth noise. Then, the standard deviation of the background pixel intensities in the filtered image was estimated to be σ = 1.4826×MAD (F3×3(I)), where MAD (F3×3(I)) is the median absolute deviation of the pixel intensities in the filtered image, F3×3(I). A binary mask (Mask 1) was then defined by thresholding the pixel intensities in the filtered image at Nσ above the median intensity, where N=2 for pCD79, 2 for pSyk, and 3 for pCD19 experiments. Next, the original, unfiltered image I was filtered with Laplacian of Gaussian (LoG) filters of different widths h. Each LoG filter highlights fluorescent spots and edges of fluorescent features at a specific scale determined by h. Regions of approximately uniform pixel intensities become approximately zero after filtering with a LoG filter of width h   58 that is substantially smaller than the size of the uniform-intensity region. For each LoG-filtered image, LoGh(I), the standard deviation σh of the background pixel values was again estimated as described above, that is σh =1.4826 × LoGh (I). A second binary mask (Mask 2) was then defined by identifying pixels that exceeded a threshold Nσh in LoGh (I) at any scale, where N=3 for pCD79, 5 for pSyk, or 4 for pCD19 experiments. Additionally, any pixel that passed this threshold was removed from the mask if its greatest negative response to the different LoG filters was greater than its maximum positive response. This last step helps to sharpen the edges of fluorescent features that could have been excessively blurred by filters of large width. A final binary mask was defined by taking the intersection of Mask 1 and Mask 2. Hence, pixels were retained in the final mask only if they were present in both Mask 1 and Mask 2.  To quantify fluorescence in an image, background fluorescence was estimated by calculating the median intensity of the pixels outside of the mask, and this was subsequently subtracted from the whole image. Next, the intensities of pixels outside of the mask were set to 0 to leave only those pixels that were present in the mask. Finally, these pixel intensities were summed to obtain the total fluorescence intensity in the image. For each B cell, the total fluorescence intensity derived in this manner for a given signaling molecule was normalized to the total fluorescence intensity of clustered Ag on the same B cell. This custom MATLAB code can be found at https://bitbucket.org/jscurll/bolger-munro_image_analysis_scripts/src/master/quantify_fluorescence_overlap.m.  2.9.4 Quantification of fluorescence overlap To quantify overlap, images were first processed as described in section 2.9.2 and then a custom MATLAB code was used to calculate the Mander’s coefficients. This MATLAB code can be found at https://bitbucket.org/jscurll/bolger-munro_image_analysis_scripts/src/master/quantify_fluorescence_overlap.m.  2.9.5 Quantification of the distance between each microcluster and the center of the immune synapse  Custom FIJI macros were used to calculate the distance between each microcluster and the center of the immune synapse, as described (Bolger-Munro et al., 2019). Briefly, background   59 fluorescence was subtracted and a binary mask of the microclusters was generated. Then, the center of mass of the total Ag mask (all clusters, TCM) as well as the center of mass for each microcluster (MCM) was calculated for each frame in the movie. Because this was computed from binarized images, the fluorescent intensity did not influence the calculation. Next, the distance (DCM) between the TCM and MCM for each microcluster in each frame was calculated. Finally, the average DCM was calculated for each frame in the movie and plotted. See Figure 2.3 for schematic. This custom FIJI macro can be found at https://github.com/madscience12/FIJImacros/blob/master/distance_macro_MBM.ijm    Figure 2.3 Quantification of the distance between each microcluster and the center of the immune synapse Using a binary mask of the total Ag gathered at the B cell-APC contact site (visualized using fluorescently tagged mHEL), the center of mass of the total Ag gathered into clusters (Tcm, yellow dot) was computed. This represents the center of the IS. For each frame in the movie, the distance (Dcm, green lines) from the Tcm to the center of mass of each microcluster (Mcm) was calculated. The average Dcm was then calculated for every frame in the movie and used to generate plots of Dcm versus time. The figure shows schematic representations of microcluster distributions, relative to the center of the immune synapse, for 3 different time points after initiating B cell-APC contact. Scale bar: 5 µm.    60  2.9.6 SPT Single particles were detected and tracked using Icy bioimaging analysis software (De Chaumont et al., 2012) with the UnDecimated Wavelet Transform Detector plug-in and the Multiple Hypothesis Spot-Tracking plug-in (Chenouard et al., 2013, 2014). Diffusion coefficients and confinement diameters were determined as described previously (Abraham et al., 2017). The diffusion model assumes that displacements arise from a two-dimensional Brownian diffusion process. Diffusion coefficients were calculated for individual tracks using a maximum likelihood estimation approach that accounts for noise due to positional errors in acquisition as well as blurring during acquisition (Berglund, 2010).  2.10 Flow cytometry  2.10.1 Staining for surface BCR levels for flow cytometry A20, Ramos, or Raji D1.3 B cells were resuspended in ice-cold FACS buffer (PBS with 2% FCS). Fc receptors were blocked by adding 25 µg/ml of the 2.4G2 rat anti-mouse Fc receptor monoclonal antibody (ATCC, #HB-197) for 5 min on ice. Fluorophore-conjugated antibodies to detect cell surface BCRs were then added and the cells were incubated for 1 hr on ice. The cells were then washed 3 times with FACS buffer and then analyzed by flow cytometry as in section 2.10.7.    2.10.2 Staining for intracellular actin filaments by flow cytometry  A20, Ramos, or Raji D1.3 B cells were fixed with 4% PFA for 10 min at room temperature then washed once with FACS buffer. The cells were then permeabilized with 0.1% Triton X-100 in PBS for 5 min at room temperature, washed once with FACS buffer, and resuspended in FACS buffer with Alexa Fluor 647 phalloidin (Thermo Fisher, #A22287, 1:100) for 1 hr at room temperature. The cells were then washed three times with FACS buffer before being resuspended in FACS buffer and analyzed by flow cytometry.      61 2.10.3 Quantifying BCR-induced transcriptional responses using flow cytometry  COS-7 APCs expressing the anti-Igκ surrogate Ag (2 × 105 cells per well) were cultured overnight in 12-well plates. Primary B cells from Nur77GFP mice (106) were added to each well containing APCs. After 3 hr of co-culture, the B cells and APCs were removed by pipetting and GFP levels were quantified by flow cytometry.   2.10.4 Staining for upregulation of surface activation markers using flow cytometry  Untransfected COS-7 cells or COS-7 cells expressing the anti-Igκ surrogate Ag (2 × 105 cells per well) were cultured overnight in 12-well plates. Primary B cells from C57BL/6J or MD4 mice (106) were added to each well containing APCs. After co-culturing the cells for 18 hr, the B cells and APCs were removed from the wells by pipetting. To quantify cell surface expression of CD69 and CD86, the cells were resuspended in ice-cold FACS buffer (PBS with 2% FCS), Fc receptors were blocked by adding 25 µg/ml of the 2.4G2 monoclonal antibody (ATCC, #HB-197) for 5 min, and then the cells were stained for 30 min at 4°C with anti-CD69-FITC (eBioscience, #11-0691-82 1:200) or anti-CD86-APC (eBioscience, #17-0862-82; 1:200) plus DAPI (1:10,000). After staining, the cells were resuspended in FACS buffer and analyzed by flow cytometry.  2.10.5 Quantifying BCR-induced proliferation of B cells using flow cytometry APCs expressing the anti-Igκ surrogate Ag (2 × 105 cells per well) were cultured overnight in 12-well plates. Primary B cells from C57BL/6J mice were resuspended in complete RPMI-1640 medium and labeled with 2 µM CFSE (Invitrogen, #C1157) for 8 min. The B cells were then added to the APCs and cultured in complete RPMI-1640 medium with 5 ng/ml BAFF (R&D Systems, #8876-BF-010) and 5 ng/ml IL-4 (R&D Systems, #404 ML-010). After 24–72 hr the cells were resuspended by pipetting, stained with 7-AAD (Thermo Fisher, #A1310; 1:1000) to identify dead cells, and analyzed by flow cytometry.       62 2.10.6 Flow cytometry  Fluorescence was quantified using an LSR II cytometer (Becton Dickinson) and the data were analyzed using FlowJo software (Treestar). Forward and side scatter were used to gate on single B cells. DAPI or 7-AAD staining was used to exclude dead cells. The Ag-induced increase in fluorescence of surface activation markers was calculated as the geometric mean for B cells cultured with anti-Igκ-expressing COS-7 APCs, minus the geometric mean for B cells cultured with untransfected COS-7 cells that do not express the surrogate Ag. The gating strategies and sample calculations are shown in Figure 2.4 and Figure 2.5.   2.11 Statistical analysis Two-tailed paired t-tests were used to compare mean values for matched sets of samples. The Mann-Whitney U test was used to compare ranks in samples with many cells and high variability (e.g. dot plots for immunofluorescence signaling data). For signaling experiments, outliers were identified using Robust Regression and Outlier Removal (ROUT) in GraphPad Prism with Q set to 1% (Motulsky and Brown, 2006). Kolmogorov-Smirnov tests were used to compare SPT cumulative frequency distributions and FRAP curves.    63     Figure 2.4 Example of gating strategy for analysis of Nur77GFP expression (A) Schematic of gating strategy. Isolated primary murine B cells from Nur77GFP reporter mice were cultured by themselves for 3 hr (No APC) or cultured with either parental COS-7 cells (No-Ag-APC) or COS-7 cells expressing membrane-bound anti-Igκ (Ag-APC) for 3 hr. The cells were then analyzed for GFP expression by flow cytometry. (B) Representative flow cytometry analysis. Lymphocytes were gated on using SSC-A and FSC-A. Single cells were gated on using FSC-W and FSC-A. The geometric mean (GM) of the GFP fluorescence was calculated for GFP+-gated cells. To quantify Ag-induced increases in GFP fluorescence, the GM for GFP+ B cells that had been added to parental COS-7 APCs was subtracted from the GM of GFP+ B cells that had been added to anti-Igκ-expressing COS-7 APCs.      64  Figure 2.5 Example of gating strategy for analysis of CD69 and CD86 upregulation and blast cell formation (A) Schematic of gating strategy. Primary murine B cells from C57BL/6J or MD4 mice were cultured by themselves for 18 hr, or cultured with either parental COS-7 or B16F1 cells (No-Ag-APC) or COS-7 or B16F1 cells expressing membrane-bound anti-Igκ (Ag-APC) for 18 hr. Lymphocytes were gated on using SSC-A and FSC-A. Single cells were gated on using FSC-W and FSC-A. Dead cells were excluded by DAPI staining. The cells were then analyzed for cell surface expression of CD69 or CD86 by flow cytometry. (B) Representative flow cytometry analysis. To quantify Ag-induced increases in the expression of CD69 or CD86, the GM of the CD69+ or CD86+ B cells that had been added to parental COS-7 APCs was subtracted from the GM of CD69+ or CD86+ B cells that had been added to anti-Igκ-expressing COS-7 APCs. Blast cells were identified as SSC-Ahi and FSC-Ahi cells.       65 Chapter 3: Actin networks nucleated by the Arp2/3 complex control BCR organization at the immune synapse  3.1 Introduction  3.1.1 Reorganization of the actin cytoskeleton drives immune synapse formation The immune synapse is a transient and highly dynamic structure that is characterized by a rapidly evolving reorganization of plasma membrane components and signaling proteins. When B cells encounter Ag on the surface of an APC, BCRs undergo rapid actin-dependent spatial reorganization that amplifies BCR signaling (Harwood and Batista, 2010) and reduces the amount of Ag required for B cell activation (Batista et al., 2001; Depoil et al., 2008; Weber et al., 2008). Initial BCR signaling induces localized actin severing and transiently uncouples the cortical cytoskeleton from the plasma membrane (Freeman et al., 2011; Gupta et al., 2006; Treanor et al., 2011). These changes allow for increased mobility of BCRs within the plasma membrane and enable the formation of BCR microclusters that are initially distributed throughout the B cell-APC contact site (Batista et al., 2001; Depoil et al., 2008). Because BCR signaling output depends on BCR-BCR interactions, microcluster formation amplifies BCR signaling. This initial BCR signaling stimulates actin polymerization at the periphery of the B cell-APC contact site (Fleire et al., 2006). Actin polymerization exerts outward force on the plasma membrane to generate membrane protrusions (Mogilner and Oster, 1996). This allows the B cell to scan the APC surface for additional Ag, resulting in further BCR microcluster formation and enhancement of BCR signaling (Fleire et al., 2006; Weber et al., 2008). Subsequent retraction of the B cell membrane is accompanied by the centripetal movement of BCR-Ag microclusters, which coalesce to form a cSMAC (Fleire et al., 2006), characteristic of an immune synapse (Dustin, 2008). BCR-mediated extraction of Ag from the APC membrane, which allows B cells to present Ags and elicit T cell help, is thought to occur at the cSMAC in naive B cells (Nowosad et al., 2016; Yuseff et al., 2013). Although the actin-dependent formation, movement, and spatial organization of BCR microclusters are critical for APC-induced B cell activation, the molecular mechanisms that orchestrate this process are not fully understood.    66 3.1.2 Actin nucleation at the B cell immune synapse Actin polymerization at the periphery of the B cell-APC contact site drives the formation of membrane protrusions that scan the surface of the APC for Ags. Ag encounter and microcluster formation are key determinants of the magnitude of BCR signaling and whether or not B cell activation occurs (Fleire et al., 2006; Harwood and Batista, 2010). Although the actin-dependent formation, movement, and spatial organization of BCR microclusters are critical for APC-induced B cell activation, the molecular mechanisms that orchestrate this process are not fully understood.  Actin organization and dynamics are controlled by a large network of actin-regulatory proteins (see Figure 1.5) (Bezanilla et al., 2015; Chhabra and Higgs, 2007; Michelot and Drubin, 2011; Tolar, 2017). There are two main modes of actin polymerization (Pollard, 2007). Formin-mediated linear actin polymerization, which is controlled by the RhoA GTPase, generates thin membrane protrusions such as filopodia (Mattila and Lappalainen, 2008). Alternatively, branched actin polymerization is mediated by the 7-subunit Arp 2/3 complex, which binds to existing actin filaments and nucleates new filaments that grow at a 70° angle from the mother filament (Goley and Welch, 2006). Receptor-induced activation of the Cdc42 and Rac GTPases causes conformational changes in NPFs belonging to the WASp family, which allows them to activate the Arp2/3 complex (Rotty et al., 2013). The Arp2/3 complex nucleates the branched actin networks that underlie the formation of sheet-like membrane protrusions such as lamellipodia (Pollard and Borisy, 2003). As new actin is polymerized at the cell periphery, the elastic resistance of the plasma membrane causes the peripheral actin network to flow toward the center of the synapse (Ponti et al., 2004).  The peripheral actin network at the T cell immune synapse resembles that of the lamellipodia of migrating cells (Dustin, 2007; Dustin and Cooper, 2000). Depletion of the Arp2/3 complex decreases the capacity of T cells to form lamellipodia-like structures at the T cell immune synapse in response to membrane-bound Ags (Gomez et al., 2007). Instead, Arp2/3 complex-deficient T cells form filopodia-like structures that are dependent on the expression of formin proteins (Gomez et al., 2007). Moreover, actin polymerization that drives the centripetal (i.e. retrograde) actin flow is essential for TCR microcluster centralization and cSMAC formation (Babich et al., 2012; Hammer and Burkhardt, 2013; Yi et al., 2012). Although actin   67 dynamics are required for cSMAC formation in B cells (Liu et al., 2012; Treanor et al., 2011), a role for the Arp2/3 complex and actin retrograde flow has not been established.  3.1.3 The activation of the Arp2/3 complex  The importance of Arp2/3 complex-nucleated branched actin networks for B cell function is highlighted by the immune dysregulation caused by mutations that impair the activation of the Arp2/3 complex. Mutations in subunits of the Arp2/3 complex or in the NPFs that activate it result in increased susceptibility to infections, autoimmune complications and B-cell malignancies (Candotti, 2018; Kahr et al., 2017). B cells lacking WASp are hyperresponsive to BCR and TLR ligands (Becker-Herman et al., 2011; Recher et al., 2012), exhibit decreased migration in response to chemokines (Westerberg et al., 2012), and exhibit reduced B cell spreading and microcluster formation in response to membrane-bound Ags (Liu et al., 2011). WIP regulates the activity and distribution of WASp and stabilizes existing actin filaments (Candotti, 2018) and B cells lacking WIP have defective antibody responses, impaired migration, and altered actin organization (Keppler et al., 2015, 2018). Further upstream, the Rho GTPase Cdc42 activates WASp. B cell-specific deletion of Cdc42 in mice also results in severe impairment in antibody production, aberrant actin organization, and diminished BCR signaling (Burbage et al., 2015). Although Cdc42 and the WASp/WIP complex act upstream of the Arp2/3 complex, a role for Arp2/3 complex-dependent actin dynamics in B cell immune synapse formation and function has not been established.  3.1.4 Systems to study B cell immune synapse formation and function To elucidate the mechanisms that underlie immune synapse formation, it is critical to visualize the spatiotemporal reorganization of the BCR and key membrane proteins such as CD19 and CD22 in concert with reorganization of cytoskeletal structures and the recruitment of signaling proteins. BCR microcluster formation and actin reorganization has been studied primarily using Ags embedded in an artificial planar lipid bilayer (Carrasco et al., 2004; Depoil et al., 2008; Fleire et al., 2006; Lin et al., 2008) or Ags tethered to plasma membrane sheets (Hoogeboom et al., 2018; Natkanski et al., 2013; Nowosad and Tolar, 2017; Spillane and Tolar, 2017). Using these systems, the interface between the B cell and the Ag-presenting substrate can   68 be imaged in one x-y focal plane. This allows for high spatial and temporal resolution imaging, which is critical for these studies. Although these systems have contributed greatly to our current understanding of molecular events that occur during B cell immune synapse formation, they fail to accurately recapitulate the complex and biologically relevant mechanical properties of APCs such as Ag mobility.  Ag mobility is a key determinant of BCR organization and signaling, as mobile Ags (e.g. those embedded in a planar lipid bilayer) induce greater BCR signaling than immobile ligands (Ketchum et al., 2014). This is likely due to differences in the Ag-induced spatial reorganization of BCRs. Ags that are immobilized on a glass coverslip are useful for studying cytoskeletal rearrangements that occur early in immune synapse formation (i.e. cell spreading), but the immobilization of Ag limits the formation, growth and lateral movement of BCR microclusters (Ketchum et al., 2014) that amplify BCR signaling.  In contrast, Ags that are linked to an artificial planar lipid bilayer have unrestricted lateral mobility and support the coalescence of BCR microclusters into a cSMAC. This system is particularly powerful because it allows one to control the densities of Ag, integrin ligands and other molecules that are attached to the bilayer. This has enabled quantitative analysis of the relationship between Ag density, BCR signaling, and B cell activation by membrane-bound Ags. However, this system does not recapitulate the complexity of the APC membrane. Cell membranes, including those of APCs, are compartmentalized by the submembrane actin cytoskeleton. This compartmentalization affects the mobility of Ag and other molecules that are attached to the bilayer. The mobility of proteins within the APC membrane has been shown to impact the ability of APCs to activate lymphocytes. For example, during Ag presentation to T cells, the mobility of ICAM-1 on the surface of the APC must be restricted in order to support maximal T cell activation (Comrie et al., 2015). Moreover, planar lipid bilayers are considerably more rigid than cell membranes (Spillane and Tolar, 2017). Recently, it has become clear that the BCR is sensitive to mechanical force, such that increasing the stiffness of Ag-presenting surfaces causes greater recruitment of Syk and other signaling components (Wan et al., 2013, 2015). Thus, planar lipid bilayers will not elicit the same types of forces at the B cell immune synapse as intact APCs and this could impact BCR signaling. Additionally, because planar lipid bilayers are so rigid, B cells cannot readily extract Ags from their surface (Spillane and Tolar, 2017).   69 Hence, planar lipid bilayers do not accurately recapitulate the ligand mobility or substrate rigidity of APCs, which both have important functions in BCR signaling responses.  To address these limitations, Tolar and colleagues developed the use of plasma membrane sheets that are derived from mammalian cells (Hoogeboom et al., 2018; Natkanski et al., 2013; Nowosad and Tolar, 2017; Spillane and Tolar, 2017). Sonication of adherent cells leaves behind pieces of membrane that are adhered to the coverslip and to which Ags can be tethered. The major advantage of this system is that some of the cell’s cytoskeleton and membrane organization remain intact. However, B cells are then settled onto the cytosolic side of the membrane sheet and are therefore exposed to the intracellular domains of transmembrane proteins, which could interact with B cell surface molecules in unexpected ways. Additionally, Ags are tethered to plasma membrane sheets via annexin-V, which forms clusters. Therefore, Ags tethered to membrane sheets in this way are pre-clustered, which might not accurately represent the physiological organization of Ags on the surface of APCs (Nowosad and Tolar, 2017).  In vivo, the most effective APC for B cell activation is the follicular dendritic cell. Thus, using purified follicular dendritic cells would be the ideal system for studying B cell immune synapse formation. However, these cells are very rare and fragile (Sukumar et al., 2006). Instead, our lab and others have employed adherent cells such as COS-7 cells and B16 melanoma cells that can be easily transfected to express transmembrane forms of Ag (Wang et al., 2018). These cells spread when plated on coverslips coated with extracellular matrix proteins and become very flat and thin, which allows for the B cell-APC contact site to be imaged in one focal plane. Unlike follicular dendritic cells and subcapsular sinus macrophages, these cells lack ligands for murine integrins, allowing us to investigate the role of BCR signaling in immune synapse formation in the absence of additional signals.       70 3.1.5 Rationale and hypothesis  Upon recognition of molecules displayed on the surface of APCs, B cells reorganize the spatial distribution of activating receptors (i.e. BCRs), inhibitory proteins, scaffolding proteins that recruit signaling enzymes, and cytoskeletal elements, creating distinct molecular patterns that are optimized for cellular responses. The formation of BCR microclusters is a key determinant of B cell activation. However, the molecular mechanisms that control microcluster spatial organization at the B cell immune synapse are not fully understood. Because upstream activators of the Arp2/3 complex such as Rac, Cdc42 GTPases, and the WASp/WIP complex are important for immune synapse formation and B cell activation, I tested the hypothesis that branched actin networks nucleated by the Arp2/3 complex are required for the centripetal movement and coalescence BCR-Ag microclusters at the B cell immune synapse immune synapse. Using live, intact APCs and high-resolution imaging I demonstrated that B cells interacting with APCs generate dynamic actin-rich protrusions that scan the surface of APCs and promote the formation of BCR-Ag microclusters. Moreover, I employed live-cell imaging and quantitative image analysis to show that the Arp2/3 complex is critical for the formation of these dynamic protrusions and is required for the centralization of BCR-Ag microclusters and the subsequent formation of a cSMAC.      71 3.2 Results  3.2.1 B cells generate dynamic actin-rich membrane protrusions that scan Ag presenting surfaces for Ag When B cells encounter Ags or anti-Ig Abs (an Ag surrogate) that are attached to planar lipid bilayers or to rigid substrates such as coverslips, they extend radial membrane protrusions to search for Ags (Fleire et al., 2006). These protrusions are driven by actin polymerization at the inner face of the plasma membrane and resemble the leading edge of a migrating cell. To visualize how B cells probe the surface of APCs, I expressed F-Tractin-GFP in A20 D1.3 B cells, which express a transgenic hen egg lysosome (HEL)-specific BCR, in order to visualize filamentous actin. I then imaged their interaction with mHEL-HaloTag-expressing APCs using ISIM (York et al., 2013) to obtain high spatiotemporal resolution. I found that B cells continually extended and retracted short-lived and asymmetrical actin-rich protrusions across the surface of the APC (Figure 3.1A [coloured arrowheads], Video 1). This dynamic searching mechanism allowed A20 D1.3 B cells to scan a total area of ~100 µm2 on the surface of the APC over 4 min of contact (Figure 3.1B). BCR-Ag microclusters formed within actin-rich protrusions (Figure 3.1A, Video 1). These microclusters then moved toward the center of the synapse along with the actin structures in which they were embedded, especially as membrane protrusions were retracted (Figure 3.1A,C, Video 1). At the same time, actin filaments were cleared from the center of the contact site and BCR microclusters within this region coalesced with each other until a cSMAC was formed. As determined from the kymographs in Figure 3.1C, the velocities at which the peripheral actin structures and BCR-Ag microclusters moved toward the center of the synapse were similar, suggesting that actin retrograde flows within these protrusions drive the initial centripetal movement of these microclusters.      72     73 Figure 3.1 B cells generate actin-based protrusions in response to APC-bound Ags (A) A20 D1.3 B cells expressing F-Tractin-GFP (green) were added to COS-7 APCs expressing mHEL-HaloTag (magenta) and the B cell-APC contact site was imaged every 6.6 s for 4 min using ISIM. Images from Video 1 are shown. The different colored arrowheads in the upper panels (actin channel) represent membrane protrusions that were extended or retracted. The yellow circles in the lower panels indicate BCR-Ag microclusters (magenta) that were formed on new actin protrusions. (B) For each frame of Video 1, the cell edge, as defined by actin, was overlaid as a temporally-coded time series (left). The total area searched by the B cell over the 4-min period of observation is shown on the right. (C) Kymographs showing the time evolution of fluorescence signals along the yellow lines in the lower right panel of (A) depict the centripetal movement of Ag clusters and the surrounding actin structures. The velocity of a particle is proportional to the angle at which its track deviates from a vertical line along the time axis. See Figure 2.2 for additional explanation of kymograph analysis. (D) A20 B cells expressing F-Tractin-GFP were added to anti-IgG-coated coverslips and the contact site was imaged every 2 s for 10 min using a Zeiss Airyscan microscope. Images from Video 2 are shown. Blue arrowheads represent a long-lived membrane protrusion. (E) A temporally coded time series representing the edge of the cell (left) was generated from Video 2 and the total area searched by the B cell over the 10 min period of observation is shown (right). Scale bars: 5 µm.   Video 1 Dynamic actin-based protrusions and BCR-Ag microclusters at the B cell-APC contact site A20 D1.3 B cells expressing F-Tractin-GFP (green) were added to COS-7 APCs expressing mHEL-HaloTag (red) and the B cell-APC contact site was imaged using ISIM. Images taken every 6.6 s. Video playback is 10 frames per second (66X real speed). See also Figure 3.1A-C.   Video 2 Actin dynamics in B cells spreading on immobilized anti-IgG antibodies A20 B cells expressing F-Tractin-GFP were added to anti-IgG-coated coverslips and the contact site was imaged using a Zeiss Airyscan microscope. Images taken every 2 s. Video playback is 15 frames per second (30X real speed). See also Figure 3.1D,E.         74 In contrast to their behavior on APCs, B cells exhibited largely radial spreading when they contacted anti-IgG antibodies that were immobilized on a rigid glass coverslip (Figure 3.1D, Video 2; see also Figure 3.10). The cells underwent continual outward spreading, reaching a contact area of ~150 µm2 after 10 min (Figure 3.1E). B cells spreading on immobilized anti-Ig formed multiple large lamellipodia, some of which persisted for up to 10 min (for example, Figure 3.1D [blue arrowheads]). They also formed short dynamic filopodia, many of which were retracted or engulfed by newly formed lamellipodia. However, B cells encountering Ags that are mobile on the surface of an APC exhibited more complex and dynamic membrane probing and retraction behavior than when they encountered immobile Ags.  3.2.2 The Arp2/3 complex is important for centralization of BCR-Ag microclusters Branched actin networks that are nucleated by the Arp2/3 complex exert outward force on the plasma membrane, driving cell spreading. At the same time, the assembly and elongation of new actin filaments at the plasma membrane pushes the existing actin networks in the opposite direction, away from the plasma membrane and towards the center of the cell. This is called actin retrograde flow, and in lamellipodia it is dependent on the Arp2/3 complex. Because the results in Figure 3.1 indicated that BCR-Ag microclusters move centripetally at the same velocity as the surrounding actin network, I tested the hypothesis that Arp2/3 complex activity is required for the centralization of BCR-Ag microclusters. To address this question, I used two complementary and well-characterized approaches to ablate the function of the Arp2/3 complex in B cells. First, I used siRNA to deplete Arp3 expression. Immunoblotting showed that Arp3 siRNA reduced Arp3 expression by 73% compared to control siRNA-expressing B cells (Figure 2.3). Depletion of Arp3 was accompanied by a ~55% reduced expression of both Arp2 and p34, suggesting destabilization of the Arp2/3 complex (Figure 3.2), as observed previously (Nicholson-Dykstra and Higgs, 2008; Zhang et al., 2017). Second, I inhibited Arp2/3 complex activity using a pharmacological inhibitor, CK-666, which locks the Arp2/3 complex in an open conformation, preventing it from binding to existing actin filaments and then nucleating the formation of new actin filaments (Hetrick et al., 2013). B cells treated with CK-666 did not exhibit increased cell death compared to untreated cells or cells treated with CK-689, an inactive     75   Figure 3.2 siRNA-mediated knockdown of Arp3 in A20 B cells The cells were transfected with control siRNA or Arp3 siRNA. Cell extracts were analyzed by immunoblotting for the Arp3, Arp2, and p34 components of the Arp 2/3 complex as well as actin. Results from two independent experiments are shown.     76     Figure 3.3 CK-666 treatment does not decrease B cell viability (A) Primary murine B cells were treated with 100 µM CK-689 or CK-666, or left untreated for 1 hr. The cells were then stained with 7-AAD to identify dead cells and analyzed by flow cytometry. The percent of live cells (7-AAD negative) was quantified. Each dot represents an independent experiment. Bars represent the mean and error bars represent the SEM. A two-tailed paired t-test was used to calculate p values. (B) Primary murine B cells were treated with 100 µM CK-689 or CK-666 for the indicated lengths of time and the percent of live (7-AAD-negative) cells was determined. Representative graph from one of two independent experiments      77  Figure 3.4 CK-666 does not alter actin organization or dynamics in the COS-7 APCs (A) COS-7 cells expressing mHEL-HaloTag (magenta) were allowed to adhere to fibronectin overnight and then treated with 100 µM of the control drug (CK-689) or the Arp2/3 complex inhibitor (CK-666) for 30 min. The cells were then stained with phalloidin to visualize actin filaments. Scale bars: 5 µm. (B) FRAP analysis of membrane-proximal actin-GFP in COS-7 cells. COS-7 cells were transfected with actin-GFP and allowed to spread on fibronectin. The cells were then treated with 100 µM CK-689 or CK-666 for 30 min. Actin-GFP within the indicated ROI was photobleached and fluorescence recovery was measured at 2 s intervals for 1 min. Representative images are shown (left). Scale bar: 5 µm. Kymograph of actin recovery is shown in the middle panel. Red arrow indicates bleach event. Yellow arrow indicates repolymerization of actin at the cell edge. The recovery of actin-GFP fluorescence after photobleaching is graphed in the right panel (mean ± SEM for >17 cells from two experiments). The curves were not significantly different as assessed using the Kolmogorov-Smirnov test.   78 structural analog of CK-666 (Figure 3.3). Although the COS-7 APCs would also be exposed to CK-666 in these experiments, the CK-666 concentration that we used did not alter their actin organization or dynamics over a 30 min time course (Figure 3.4). To assess the role of the Arp2/3 complex in the movement and organization of BCR-Ag microclusters at the immune synapse, I performed real-time imaging of the contact site between A20 D1.3 B cells and mHEL-HaloTag-expressing APCs. To do this, I used an image analysis pipeline (described in section 2.9.2) to quantify, for each time frame in the video, the number of BCR-Ag microclusters on each cell that was imaged, the area and amount of Ag fluorescence associated with each cluster, and the total Ag fluorescence contained in all of the clusters on each B cell. From these measurements, I was able to quantify the centripetal movement of each BCR-Ag microcluster and determine the percent of cells that formed a cSMAC. I found that inhibiting or depleting the Arp2/3 complex greatly reduced the coalescence of BCR-Ag microclusters into a cSMAC (Figure 3.5A,B). In A20 D1.3 B cells that were transfected with control siRNA (Figure 3.5A, Video 3) or treated with CK-689 (Figure 3.5B, Video 5), an inactive analog of CK-666, BCR-Ag microclusters exhibited obvious centripetal movement. By 10 min after contacting the APCs, ~60% of the B cells had gathered >90% of the Ag fluorescence into one or two large clusters at the center of the synapse, which we defined as having formed a cSMAC (Figure 3.5A,B). In B cells in which the Arp2/3 complex was depleted or inhibited, BCR-Ag microclusters still formed (Figure 3.5A,B) and there was no change in the total amount of Ag that was gathered into microclusters, compared to control cells (Figure 3.5C,D, see also Figure 3.6B,D). However, many of the BCR-Ag microclusters in the Arp3 siRNA-expressing cells (Figure 3.5A, Video 4) and the CK-666-treated cells (Figure 3.5B, Video 6) did not exhibit persistent centripetal motion and only ~15% of the cells in which the Arp2/3 complex was depleted or inhibited formed a cSMAC by 10 min (Figure 3.5A,B). Consistent with these findings, cSMAC formation was also impaired when Arp2/3 complex activity was inhibited when primary splenic B cells from C57BL/6J mice, were added to APCs expressing the single chain anti-Igκ surrogate Ag, and when primary B cells from MD4 mice (which express a HEL-specific BCR) were added to APCs expressing the HEL-HaloTag Ag (Figure 3.6).  To further assess BCR-Ag microcluster coalescence and cSMAC formation, the distribution of Ag fluorescence into clusters over time was quantified. Cumulative frequency   79 distribution curves showed that the 50th percentile (cumulative frequency = 0.5) for the number of clusters per cell required to contain 90% of the Ag fluorescence after 10 min of B cell-APC contact was ~1 for the control siRNA-transfected A20 D1.3 B cells and CK-689-treated cells versus 4 for the Arp3 siRNA-transfected cells and CK-666-treated cells (Figure 3.7A,B). For example, after 10 min of contact with an APC, the control siRNA-transfected B cell shown in Figure 3.5A had coalesced all of the Ag into 4 clusters, one of which contained 97.7% of the Ag fluorescence (Figure 3.7C). By contrast, a similar amount of Ag was distributed among 16 distinct clusters in the Arp3 siRNA-transfected cell depicted in Figure 3.5A, and no individual cluster contained more than 43% of the total Ag fluorescence (Figure 3.7C). Similar results were obtained when comparing the CK-689-treated (control) and CK-666-treated cells (Figure 3.7D). Again, the control cell formed a cSMAC into which ~ 95% of the Ag was gathered whereas no cluster in the CK-666-treated cells contained more than 22% of the total Ag fluorescence. For all B cells analyzed in this manner, the number of BCR-Ag microclusters formed after 10 min in contact with an APC, as well as the distribution of the total Ag fluorescence among individual clusters, is shown in Figure 3.7E,F.     80      81 Figure 3.5 Arp2/3 complex function is important for centralization of BCR-Ag microclusters in A20 D1.3 B cells (A,B) A20 D1.3 B cells were transfected with control siRNA or Arp3 siRNA (A) or pre-treated for 1 hr with 100 µM of either the control compound CK-689 or the Arp2/3 complex inhibitor CK-666 (B). The cells were then added to mHEL-GFP-expressing COS-7 APCs and the B cell-APC contact site was imaged every 12 s for 10 min by spinning disk microscopy. Images are from Video 3 (Control siRNA) and Video 4 (Arp3 siRNA) or Video 5 (CK-689) and Video 6 (CK-666). The percent of cells for which >90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed. n > 35 cells from four independent experiments combined (A); n > 18 cells from three independent experiments combined (B). (C,D) For each B cell in (A) and (B), the total fluorescence intensity of Ag that was gathered into clusters at the B cell-APC contact site was determined for every frame in the video. The data are plotted as mean (line) ± SEM (shaded area) for 9 cells (control siRNA), 12 cells (Arp3 siRNA), 10 cells (CK-689) or 6 cells (CK-666) from representative experiments. The Mann-Whitney U test was used to calculate p values. ns, not significant. Scale bars: 5 µm.  Video 3 BCR-Ag microclusters coalesce into a cSMAC in B cells expressing control siRNA A20 D1.3 B cells expressing control siRNA were added to COS-7 APCs expressing mHEL-GFP (white) and the B cell-APC contact site was imaged using spinning disk microscopy. Images taken every 12 s. Video playback is 10 frames per second (120X real speed). Four representative cells are shown. The first cell in the video is also shown in Figure 3.5A.  Video 4 Impaired cSMAC formation in B cells expressing Arp3 siRNA A20 D1.3 B cells expressing Arp3 siRNA were added to COS-7 APCs expressing mHEL-GFP (white) and the B cell-APC contact site was imaged using spinning disk microscopy. Images taken every 12 s. The video is played back at 10 frames per second (120X real speed). Four representative cells are shown. The first cell in the video is also shown in Figure 3.5A.  Video 5 BCR-Ag microclusters coalesce into a cSMAC in CK-689-treated B cells A20 D1.3 B cells were pre-treated with 100 µM CK-689 for 1 hr and then added to COS-7 APCs expressing mHEL-GFP (white). The B cell-APC contact site was imaged using spinning disk microscopy. Images taken every 12 s. Video playback is 10 frames per second (120X real speed). Four representative cells are shown. The first cell in the video is also shown in Figure 3.5B.  Video 6 Impaired cSMAC formation in CK-666-treated B cells A20 D1.3 B cells were pre-treated with 100 µM CK-666 for 1 hr and then added to COS-7 APCs expressing mHEL-GFP (white). The B cell-APC contact site was imaged using spinning disk microscopy. Images taken every 12 s. Video playback is 10 frames per second (120X real speed). Four representative cells are shown. The first cell in the video is also shown in Figure 3.5B.    82  Figure 3.6 Arp2/3 complex function is important for centralization of BCR-Ag microclusters in ex vivo murine B cells (A,B) Ex vivo primary murine B cells from C57BL/6J mice were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Igκ surrogate Ag. The cells were fixed at the indicated times and stained with an antibody that recognizes the surrogate Ag. In (A), the percent of cells for which >90% of the total Ag fluorescence intensity was contained in one or two clusters was quantified and graphed. The mean (bars) ± SEM (red lines) from 11 independent experiments are graphed. n >15 cells per experiment. In (B), the total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified and the means ± SEM for the median values from 11 independent experiments are graphed. ns, not significant (p=0.3702, p=0.3573, p=0.3044, p=0.1644, p=0.4388, p=0.6089 for the 1, 3, 5, 10, 15, and 30 min time points, respectively); two-tailed paired t-test. (C,D) Ex vivo primary murine B cells from MD4 mice were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing mHEL-HaloTag before being fixed at the indicated times. In (C), the percent of cells for which >90% of the total Ag fluorescence intensity was contained in one or two clusters was quantified and graphed. The mean (bars) ± SEM (red lines) from 3 independent experiments are graphed. n >40 cells per experiment. In (D), the total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified and the means ± SEM for the median values from 3 independent experiments are graphed. Two-tailed paired t-test was used to calculate p values. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. **0.0011 **0.0030**0.0059% cells with a cSMAC5 10 15Time (min)020406080100*0.0229 *0.0385ns0.4001% cells with a cSMAC5 10 15Time (min)A B02461 3 5 10 15Time (min) 30Total Ag at B cell:APC contact site (AU x 106)ns ns ns ns ns nsCK-689 CK-666C D10864203 5 10 15Total Ag at B cell:APC contact site (AU x 106)Time (min) CK-689 CK-666nsp=0.4194*p=0.0277nsp=0.5251nsp=0.6056Primary splenic B cells from B6 micePrimary splenic B cells from MD4 mice020406080100 CK-689 CK-666CK-689 CK-666  83     84 Figure 3.7 The Arp2/3 complex controls the distribution of BCR-Ag microclusters at the B cell immune synapse (A,B) For each B cell analyzed in Figure 3.5 the number of Ag clusters per cell required to contain >90% of the total Ag fluorescence intensity after 10 min of contact with the APCs was quantified and is plotted as a cumulative frequency curve. Dots show the 50th percentile (cumulative frequency = 0.5) for the number of clusters per cell required to contain 90% of the Ag fluorescence after 10 min. In (A), n = 35 control siRNA-transfected cells and 48 Arp3 siRNA-transfected cells from four experiments. In (B), n = 18 CK-689-treated cells and 20 CK-666-treated cells from three experiments. (C,D) For the cells shown in Figure 3.5A (C) and Figure 3.5B (D), binary representations of the Ag clusters present after 10 min of APC contact are shown. The graphs depict the percent of the total Ag fluorescence intensity, in arbitrary units (AU), present in individual clusters. The numbers to the right are the total number of Ag clusters at 10 min. (E,F) For each B cell analyzed, the stacked bar plots show the fraction of the total Ag fluorescence intensity in individual clusters at 10 min after adding A20 D1.3 B cells to HEL-GFP-expressing COS-7 APCs. Each bar represents one cell. Each colored segment within a bar represents a single Ag cluster, the size of which is proportional to the fraction of the cell’s total Ag fluorescence intensity contained within that cluster. The number at the top of each bar is the total number of distinct Ag clusters present at the 10 min time point. Kolmogorov-Smirnov tests were used to calculate p values. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. Scale bars: 5 µm.     85 The failure to form a cSMAC when the Arp2/3 complex was depleted or inhibited was due to decreased merger of individual BCR microclusters into larger clusters. Consistent with this idea, the average size of BCR-Ag microclusters increased steadily over time in control cells as the microclusters merged but remained relatively unchanged over the 14 min of imaging in the Arp3 siRNA-treated cells and CK-666-treated cells (Figure 3.8A,B). Consequently, because microclusters merged, the number of individual microclusters per cell decreased over time in the control cells but remained similar, or increased, when Arp2/3 complex activity was inhibited (Figure 3.8C,D). Thus, BCR-mediated gathering of APC-bound Ags into a cSMAC at the center of the synapse is strongly dependent on the functions of the Arp2/3 complex. The extent of BCR-Ag microcluster coalescence and cSMAC formation was further analyzed by quantifying the distance of each microcluster on an individual B cell from the center of mass of the Ag fluorescence (i.e. the center of the immune synapse, see Figure 2.3). For each B cell, the average microcluster distance was calculated for each frame in the video and this value was normalized to the maximum average distance for that cell (Figure 3.8E,F). Utilizing this method of tracking microcluster movement relative to the center of the cell, as opposed to particle tracking algorithms, was required because the B cells often moved across the surface of the APC and the center of the B cell was not a static point (see Video 3). In A20 D1.3 B cells that were transfected with control siRNA or treated with CK-689, the normalized average distance from the center decreased over time, indicating that microclusters moved towards the center of the immune synapse. This can be seen in kymographs depicting microclusters moving centripetally from the cell periphery and merging with other microclusters at the center of the immune synapse (Figure 3.8G,H). However, in Arp3 siRNA-transfected and CK-666-treated A20 D1.3 B cells, the average distance of BCR microclusters from the center of the synapse remained relatively unchanged over the entire 14 min imaging period (Figure 3.8G,H). Thus, the centripetal movement of BCR-Ag microclusters is strongly dependent on the functions of the Arp2/3 complex.       86      87 Figure 3.8 The Arp2/3 complex is important for the centripetal movement of BCR-Ag microclusters towards the cSMAC (A-F) For each control siRNA- and Arp3 siRNA-transfected A20 D1.3 B cell (A,C,E) or CK-689- and CK-666-treated A20 D1.3 B cell (B,D,F) analyzed in Figures 3.5 and 3.7 the average area of the BCR-Ag microclusters (A,B) and the number of Ag clusters at the B cell-APC contact site (C,D) were determined for every frame in the video. The data are plotted as the mean (line) ± SEM (shaded area). In (E,F), the distance between each microcluster and the center of mass of Ag fluorescence was calculated for each cell for every frame in the video. The average distance from the center of Ag fluorescence for all microclusters on a single cell was then calculated for each time point and normalized to the maximum average distance for that cell. This normalized average distance from the center is plotted as mean (line) ± SEM (shaded area). See Figure 2.3 for additional explanation of this analysis. (G,H) Kymographs depicting the locations of microclusters along a line extending across the immune synapse. In the control cells, the microclusters depicted move centripetally and then coalesce with other microclusters for form a cSMAC. When the Arp2/3 complex is depleted (G) or inhibited (H), the microclusters are largely static. Cells represented here are the same as those shown in Figure 3.5 and are the first cells depicted in Video 3 (control siRNA), Video 4 (Arp3 siRNA), Video 5 (CK-689), and Video 6 (CK-666). The Mann-Whitney U test was used to calculate p values. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant. Scale bars: 5 µm.      88 3.2.3 The Arp2/3 complex is important for BCR-induced actin reorganization and dynamics at the immune synapse To understand how the Arp2/3 complex promotes BCR microcluster centralization and cSMAC formation, I sought to visualize the actin networks nucleated by the Arp2/3 complex at the B cell immune synapse in more detail. Ags or anti-Ig antibodies that are immobilized on coverslips are widely used model systems for imaging BCR-induced spreading and actin reorganization with high resolution. To assess the role of the Arp 2/3 complex in this response, I used stimulated emission depletion (STED) microscopy. A20 B cells that bound to anti-IgG coated coverslips extended broad lamellipodia and exhibited radial spreading (Figure 3.9A). This cell morphology was characterized by a dense peripheral ring of highly branched actin filaments surrounding an actin-depleted central area that contained thin actin filaments (Figure 3.9A). Within the peripheral branched actin network of the lamellipodia, linear actin filaments, which may be nucleated by formins, extended perpendicularly from the edge of the cell to the inner face of the peripheral actin ring (Figure 3.9A [yellow arrowheads]). In addition, actin arcs (Figure 3.9A [green arrowheads]) runnning parallel to the inner face of the peripheral actin ring and which were associated with myosin IIA (Figure 3.9B). This may be similar to the formin-dependent actomyosin arcs that have been described in T cells (Murugesan et al., 2016).  This actin organization at the Ag contact site was strongly dependent on Arp2/3 complex activity. When I depleted Arp3 with siRNA (Figure 3.10A), or inhibited the Arp2/3 complex in A20 B cells using CK-666 (Figure 3.10B), BCR-induced actin reorganization and cell spreading were dramatically altered. Unlike the control A20 B cells, which formed a highly dense and flat actin network at the periphery, Arp3-depleted and CK-666-treated B cells extended long and loosely packed filopodia-like fibers (Figure 3.10A,B). This aberrant actin reorganization resulted in substantially reduced cell spreading on anti-IgG-coated coverslips (Figure 3.10C,D). Although depleting or inhibiting the Arp2/3 complex dramatically altered actin organization at the periphery of the B cells, the linear actin arcs were still observed in Arp3-depleted and CK-666 treated B cells (Figure 3.10A,B [green arrowheads]. This is consistent with the idea that actomyosin arcs are formed via formin-mediated linear actin polymerization, and not via Arp2/3 complex-mediated branched actin polymerization (Murugesan et al., 2016).   89  Figure 3.9 Actin and myosin structures in A20 B cells spreading on immobilized anti-IgG (A) A20 B cells were allowed to spread on anti-IgG-coated coverslips for 15 min before being imaged by STED microscopy. Yellow arrowheads indicate linear actin structures embedded within the peripheral branched actin network. Green arrowheads indicate actin arcs. Four representative images are shown. Scale bars: 10 µm. (B) A20 B cells that had been transfected with myosin IIA-GFP were allowed to spread on anti-IgG-coated coverslips for 10 min. After phalloidin staining, the cells were imaged by spinning disk microscopy. Scale bars: 5 µm.   90 Because the assembly of actin structures that are nucleated by the Arp2/3 complex exerts forces on the plasma membrane, we hypothesized that retrograde flow of actin would be disrupted in B cells treated with the Arp2/3 inhibitor. To test this, A20 B cells expressing F-Tractin-GFP were treated with CK-689 or CK-666 and then added to anti-IgG-coated coverslips. TIRF-SIM was used to image actin dynamics with high spatial and temporal resolution. In control CK-689-treated cells, the actin networks were highly dynamic at the cell edge, with actin retrograde flow rates of ~2.5 µm/min (Figure 3.11, Video 7). In contrast, in B cells treated with the Arp2/3 complex inhibitor, the peripheral actin structures were nearly static (Figure 3.11, Video 8). Thus, Arp2/3 complex function is essential for BCR-induced actin reorganization and for peripheral actin dynamics.     91     92 Figure 3.10 The Arp2/3 complex is important for BCR-induced actin reorganization (A,B) A20 B cells were transfected with either control siRNA or Arp3 siRNA (A) or pre-treated for 1 hr with 100 µM of the control compound CK-689 or the Arp2/3 complex inhibitor CK-666 (B). Cells were then allowed to spread on anti-IgG-coated coverslips for the indicated times before being fixed, stained for actin, and imaged by STED microscopy. Images representative of >20 cells per condition are shown. Yellow arrowheads indicate linear actin structures embedded within the peripheral branched actin network. Green arrowheads indicate actin arcs. Scale bars: 5 µm. For the regions within the yellow boxes, the images were enlarged (scale bars: 2 µm) and the relative densities of the actin structures are shown as heat maps. (C,D) A20 B cells were transfected with either control siRNA or Arp3 siRNA (C) or pre-treated for 1 hr with µM CK-689 or CK-666 (D). The cells were then allowed to spread on anti-IgG-coated coverslips for the indicated times before being stained with phalloidin to visualize actin filaments. The B cell-coverslip contact site was imaged using spinning disk microscopy. The cell area was quantified, using actin staining to define the cell edge. For each data point the mean ± SEM is shown for >27 cells from a representative experiment. ****p<0.0001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test.      93   Figure 3.11 The Arp2/3 complex is important for BCR-induced actin reorganization and dynamics A20 B cells expressing F-Tractin-GFP were pre-treated for 1 hr with CK-689 or CK-666 and then added to anti-IgG-coated chamber wells. After 5 min, the cells were imaged by TIRF-SIM at 1 s intervals for 1 min. The left panels are the first images from Video 7 (CK-689-treated cells) and Video 8 (CK-689-treated cells), respectively (Scale bars: 5 µm). Yellow arrowheads indicate linear actin structures embedded within the peripheral branched actin network. Green arrowheads indicate actin arcs. The middle panels are enlargements of the areas within the yellow boxes in the left panels (Scale bars: 2 µm). The right panels are kymographs along the yellow dotted lines in the middle panels.  Video 7 Peripheral actin dynamics in CK-689-treated B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were pre-treated for 1 hr with 100 µM CK-689 and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRF-SIM. Images taken every 1 s. Video playback is 10 frames per second (10X real speed). See also Figure 3.11.  Video 8 Impaired peripheral actin dynamics in CK-666-treated B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were pre-treated for 1 hr with 100 µM CK-666 and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRF-SIM. Images taken every 1 s. Video playback is 10 frames per second (10X real speed). See also Figure 3.11.      94 3.2.4 The Arp2/3 complex-dependent actin structures surround BCR microclusters and drive their centralization How specific actin architectures allow B cells to scan APC membranes and then centralize the resulting BCR microclusters into a cSMAC is not known. To investigate the role of Arp2/3 complex-nucleated actin networks in these processes, I used ISIM to simultaneously image actin structures and BCR-Ag microclusters in F-Tractin-GFP-transfected A20 D1.3 B cells interacting with mHEL-HaloTag-expressing COS-7 cells. This allowed me to visualize the local actin structures that formed around BCR-Ag microclusters in real time. As shown in Video 1 and Video 9, control B cells extended and retracted dynamic, transient actin-rich protrusions over the surface of the APC, with both lamellipodial and filopodia-like structures being observed (Figure 3.12A [coloured arrowheads], Figure 3.13A). The high spatiotemporal resolution of ISIM revealed that BCR-Ag microclusters were embedded within actin-rich protrusions and moved toward the cell body as these protrusions were retracted (Figure 3.12A, Figure 3.13A, Video 9, see also Figure 3.1A). For example, the microcluster highlighted in Figure 3.12A is encaged within an actin-rich protrusion. In contrast, B cells that were treated with the Arp2/3 complex inhibitor CK-666 primarily extended long, linear, filopodia-like actin structures over the surface of the APC (Figure 3.12A, Figure 3.13A, Video 10). Most of these actin structures did not retract toward the cell body over the 6.5 min observation period. Moreover, BCR-Ag microclusters that formed on these protrusions did not translocate toward the center of the synapse or merge with other microclusters (Figure 3.12A, Figure 3.13A, Video 10). Although the architectures of the actin protrusions generated by control and CK-666-treated cells were dramatically different, the cells explored roughly the same area of the APC surface over the period of observation (Figure 3.12B,C, Figure 3.14A,B). This is in contrast to the significantly reduced cell spreading on rigid anti-Ig-coated surfaces that was observed when the Arp2/3 complex was depleted or inhibited (Figure 3.10C,D). Thus, Arp2/3 complex activity is required for B cells to spread on rigid surfaces but may be dispensable for B cells to effectively probe the APC surface and form BCR-Ag microclusters under the conditions tested. However, Arp2/3 complex-mediated actin polymerization, and the resulting actin retrograde flow, appears to be essential for the centralization of BCR-Ag microclusters. Consistent with this idea, kymograph analysis showed that peripheral BCR-Ag clusters in control cells moved toward   95 the center of the synapse together with, and at a similar velocity as the surrounding actin structures (Figure 3.13B). In contrast, when the Arp2/3 complex was inhibited, both the BCR-Ag clusters and the associated actin structures were relatively immobile (Figure 3.13B). Inhibition of the Arp2/3 complex results in dramatic alterations of the actin cytoskeleton at the immune synapse (Figure 3). It is possible that this disrupted actin architecture blocks contractile forces generated by myosin IIA (Ennomani et al., 2016). In T cells, contractile forces generated by myosin IIA are important for centralizing TCR microclusters into a cSMAC (Ilani et al., 2009; Jacobelli et al., 2004; Kumari et al., 2012; Murugesan et al., 2016; Yi et al., 2012). To assess the contribution of myosin contractility in our system, we treated A20 D1.3 B cells with (S)-nitro-blebbistatin (pnBB) to inhibit myosin IIA. When these cells were added to COS-7 APCs expressing mHEL-HaloTag, they were able to form cSMACs to the same extent as DMSO-treated cells (Figure 3.15A-C). These results suggest that APC-induced cSMAC formation is dependent on Arp2/3 complex activity, with little to no involvement of myosin contractility, at least under these experimental conditions. This result is consistent with a study in T cells (Babich et al., 2012), which seems to contradict the above mentioned studies that implicate myosin-based contractility in cSMAC formation in T cells. Nevertheless, my data suggest that Arp2/3 complex-dependent branched actin structures encage BCR-Ag microclusters, and that actin retrograde flow, but not myosin II-mediated contractility, drives their initial centripetal movement.      96       97 Figure 3.12 The Arp2/3 complex is important for actin and BCR microcluster dynamics at the B cell-APC immune synapse (A–C) A20 D1.3 B cells expressing F-Tractin-GFP (green) were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing mHEL-HaloTag (magenta). The cells were imaged every 6.6 s for 6 min using ISIM. Images from Video 9 and Video 10 are shown in (A). Arrowheads indicate new protrusion events. The yellow boxes indicate the regions that are enlarged in Figure 3.13. In (B) the cell edge from each frame in Video 9 (CK-689-treated cells) or Video 10 (CK-666-treated cells), as defined by peripheral actin, was overlaid as a temporally coded time series (left). The total area searched by the B cell over the 6-min period of observation is shown the right. In (C) the B cell-APC contact area is shown as a function of time for the cells in (A) (solid lines), and for another set of representative cells (dashed lines).   Video 9 Actin and BCR-Ag microcluster dynamics in CK-689-treated B cells interacting with APCs A20 D1.3 B cells expressing F-Tractin-GFP (green) were pre-treated for 1 hr with 100 µM CK-689 and then added to COS-7 APCs expressing mHEL-HaloTag (magenta). The B cell-APC contact site was imaged using ISIM. Images taken every 6.6 s. Video playback is 10 frames per second (66X real speed). See also Figure 3.12 and Figure 3.13.  Video 10 Impaired actin and BCR-Ag microcluster dynamics in CK-666-treated B cells interacting with APCs A20 D1.3 B cells expressing F-Tractin-GFP (green) were pre-treated for 1 hr with 100 µM CK-666 and then added to COS-7 APCs expressing mHEL-HaloTag (magenta). The B cell-APC contact site was imaged using ISIM. Images taken every 6.6 s. Video playback is 10 frames per second (66X real speed). See also Figure 3.12 and Figure 3.13.       98  Figure 3.13 Arp2/3 complex-dependent actin structures encage BCR-Ag microclusters on membrane protrusions (A) Images from Video 9 and Video 10 showing enlargements of the regions indicated by the yellow boxes in Figure 3.12. A20 D1.3 B cells expressing F-Tractin-GFP (green) were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing mHEL-HaloTag (magenta). The cells were imaged every 6.6 s for 6 min using ISIM. Scale bars: 2 µm. (B) The kymographs each represent a time series of images taken along the white dashed lines in (A).   99   Figure 3.14 Arp2/3 complex activity does not regulate B cell-APC contact area (A,B) A20 D1.3 B cells expressing F-Tractin-GFP (green) were pre-treated with 100 µM CK-689 or CK-666 and then added to COS-7 cells expressing mHEL-HaloTag (magenta). The cells were fixed at the indicated times and imaged by spinning disk microscopy. Representative images are shown (A) and the B cell-APC contact area (mean ± SEM), as defined by actin, is graphed (B). For each data point, n > 20 cells from three independent experiments except for the 1 min time point (n = 4 cells). ns, not significant (p=0.7219, p=0.1310, p=0.0722, p=0.1443, p=0.2930, p=0.6263 for the 1, 3, 5, 10, 15, and 30 min time points, respectively); two-tailed paired t-test. Scale bars: 5 µm.     100   Figure 3.15 Inhibition of myosin II does not affect cSMAC formation (A-C) A20 D1.3 B cells were pre-treated with DMSO (control) or 50 µM pnBB for 30 min and then added to COS-7 APCs expressing mHEL-HaloTag. The cells were then fixed at the indicated times and imaged by spinning disk microscopy. Representative images from 2 independent experiments are shown in (A). For each cell, the number of clusters required to contain >90% of the Ag fluorescence was calculated and graphed in (B). Each dot represents one cell and the median (red line) and interquartile ranges (black box) are shown for n>57 cells per condition. The percent of cells for which > 90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed in (C). Scale bars: 5 µm.     101 3.3 Discussion 3.3.1 Summary of findings Ag-bearing APCs are potent activators of B cells and play a key role in B cell-mediated immune responses. In particular, follicular dendritic cells displaying Ag on their surface promote the positive selection of germinal center B cells (Batista and Harwood, 2009). This leads to antibody affinity maturation and the differentiation of germinal center B cells into plasma cells or memory B cells. Hence it is important to understand how B cells are activated by APCs. The Ag-induced cytoskeletal reorganization that occurs at the B cell-APC contact site enhances the ability of the B cell to probe the surface of the APC for its cognate Ag. Cytoskeletal reorganization also drives dynamic changes in the spatial patterning of the BCR, which amplifies BCR signaling and promotes BCR-mediated Ag internalization. However, the molecular mechanisms that orchestrate this process are not well understood. In this chapter, I define a key role for the Arp2/3 complex in controlling the Ag-induced changes in the spatial patterning of BCR-Ag microclusters.  Using high spatiotemporal resolution imaging of actin structures within B cells that are interacting with APCs, I observed that B cells scan the surface of the APC using complex and dynamic actin-rich protrusions that constantly extend and retract across the APC surface. These transient lamellipodia- and filopodia-like structures were shaped by the activity of the Arp2/3 complex. Microclusters that formed on these protrusions were embedded in an Arp2/3 complex-dependent actin meshwork and moved centripetally towards the center of the B cell-APC contact site with actin retrograde flow. When Arp2/3 complex activity was blocked, B cells extended long linear filopodia-like protrusions across the surface of the APC. Using quantitative image analysis, I demonstrated that these protrusions supported the gathering of Ag into microclusters but that these microclusters failed to coalesce into a cSMAC. Therefore, Arp2/3 complex-mediated actin polymerization, and the resulting actin retrograde flow, appear to be essential for the centralization of BCR-Ag microclusters. Moreover, I showed that inhibiting the activity of the Arp2/3 complex blocked actin retrograde flow in B cells interacting with either anti-Ig-coated glass coverslips or Ag-bearing APCs. Interestingly, inhibiting myosin IIA did not result in decreased cSMAC formation in this experimental system. Thus, in response to Ag binding,   102 branched actin networks nucleated by the Arp2/3 complex control both probing events that are important for Ag scanning as well as the organization of BCR-Ag microclusters.   3.3.2 Ag probing behaviour at the B cell immune synapse When B cells interacted with Ag-bearing APCs, BCR-Ag microclusters formed rapidly throughout the contact site and at sites distant from the cell body. These latter microclusters formed on filopodia-like structures in both control cells and in cells in which Arp2/3 complex activity was blocked. In control cells, branched actin networks that were nucleated within these linear protrusions encased the BCR-Ag microclusters and exhibited centripetal flow that was required for BCR-Ag microcluster movement towards the center of the B cell-APC contact site. When the Arp2/3 complex was inhibited, lamellipodial branched actin protrusions were not generated in response to either APC-bound Ag or anti-Ig-coated coverslips, and instead, the formation of filopodia was greatly enhanced. This is consistent with the idea that the Arp2/3 complex and formins compete for a limited pool of actin monomers (Suarez and Kovar, 2016). In B cells in which the Arp2/3 complex was inhibited, the filopodia that formed were able to extend over the surface of the APC and gather Ag into microclusters. However, these filopodia and their associated BCR-Ag microclusters were not efficiently retracted towards the cell body. The importance of using live intact APCs to study the immune synapse is highlighted by these findings. In contrast to the highly dynamic lamellipodia and filopodia that B cells extend asymmetrically over the APC surface, the actin-rich protrusions that B and T cells form when they spread on Ag- bearing lipid bilayers or Ag-coated coverslips are predominantly symmetrical, sheet-like lamellipodia. This difference in spreading and probing behavior may reflect the physical properties of the Ag-bearing substrate. In fibroblasts and epithelial cells, stiffer substrates favor Rac1- and Arp2/3 complex-dependent formation of lamellipodia, whereas softer substrates favor Cdc42- and formin-dependent formation of filopodia (Collins et al., 2017; Wong et al., 2014). The greater prevalence of filopodia when B cells spread on APCs, compared to artificial lipid bilayers, is consistent with the finding that the APC membrane is less stiff than the artificial lipid bilayers that have been used to mimic APCs (Natkanski et al., 2013). Whether local variations in the stiffness of the APC membrane shape B cell probing behavior is not   103 known. However, the membranes of follicular dendritic cells and dendritic cells differ in their stiffness (Spillane and Tolar, 2017) and this could impact the type of probing behavior that B cells use to scan the surface of these APCs. 3.3.3 Actin retrograde flow is required for cSMAC formation Actin retrograde flow is important for the movement and spatial organization of membrane protein clusters at cellular interfaces. Upon stimulation of epidermal cells with epidermal growth factor (EGF), EGF receptors are transported along filopodia to the cell body by actin retrograde flow (Lidke et al., 2005). In T cells, actin polymerization and the resulting retrograde flow is required for both the formation of TCR microclusters and the transport of peripheral TCR microclusters to the cSMAC (Babich et al., 2012; Campi et al., 2005; Hammer and Burkhardt, 2013; Varma et al., 2006; Yi et al., 2012). However, a role for actin retrograde flow in B cell immune synapse formation had not been investigated. By using high-resolution real-time microscopy, I was able to observe Arp2/3 complex-dependent actin structures that formed around BCR-Ag microclusters such that the microclusters were surrounded by actin cages and moved centripetally with the actin retrograde flow at the same velocity. This is consistent with the observation that actin is required for the centripetal transport of BCR-Ag microclusters, as depolymerizing actin with latrunculin A arrests microclusters at the periphery (Liu et al., 2012; Treanor et al., 2011). Moreover, actin dynamics are essential for the aggregation of BCR-Ag microclusters at the immune synapse, as stabilizing actin filaments by treating cells with jasplakinolide also abrogates microcluster centralization at the B cell immune synapse (Liu et al., 2012). Additionally, when signaling molecules that are upstream of Arp2/3 complex activation are inhibited or depleted, the organization of BCR-Ag microclusters is disrupted (Arana et al., 2008a; Depoil et al., 2008; Keppler et al., 2018; Liu et al., 2011, 2012; Wan et al., 2013; Weber et al., 2008; Westerberg et al., 2010).  The mechanisms that couple microclusters to the retrograde flow of actin are not understood. Microclusters could be directly bound to or frictionally coupled to the actin cytoskeleton. In T cells, TCRs are thought to be transiently linked to actin and these linkages may create frictional coupling and viscous drag that moves TCR clusters centripetally with the actin retrograde flow (DeMond et al., 2008; Murugesan et al., 2016). Demond et al. used nanopatterned supported lipid bilayers that physically constrain the mobility of pMHCs to   104 demonstrate this frictional coupling (DeMond et al., 2008). They found that obstacles perpendicular to the direction of microcluster flow stop the actin-dependent transport of microclusters whereas barriers at intermediate angles slow microcluster speed, where the decrease in speed is dependent on the angle of the barrier relative to the preferred direction of flow. Thus, although actin is required for transport, the mechanism that links TCR microclusters to actin allows ‘slip’, suggesting an indirect linkage. The finding that actin filaments do not accumulate at these barriers, even though actin retrograde flow slows down, supports the frictional coupling model (Yu et al., 2010). If TCRs were directly coupled to the actin cytoskeleton, then movement around barriers would require actin reorganization at these sites (DeMond et al., 2008). This model predicts that the spatial sorting of molecules at the T cell immune synapse is dependent only on the strength of their coupling to the force that is generated by actin retrograde flow. The concept of a ‘molecular clutch’ in which actin filaments are transiently coupled to membrane proteins was first described by Mitchison and Kirschner (Mitchison and Kirschner, 1988). Recently, a ‘clutch’ that links LAT clusters to actomyosin flow has been identified (Ditlev et al., 2019). At the periphery of the T cell immune synapse, Nck, WASp and N-WASp form a connection between LAT clusters and the dendritic actin network. Without these scaffolding molecules, LAT clusters can still be moved by actin structures but less efficiently. As LAT clusters approach the more centrally located actomyosin arcs, a formin-dependent signal releases this clutch and LAT clusters can then be transported towards the center of the immune synapse without a direct connection to the cytoskeleton, consistent with the observation that actomyosin arcs sweep TCRs towards the center of the immune synapse (Ditlev et al., 2019; Murugesan et al., 2016). This supports the hypothesis that Ag receptor cluster translocation is a multistep process (see section 3.3.4 below for a more detailed discussion). My finding that peripheral BCR-Ag microclusters are surrounded by actin cages, and move centripetally at the same rate as the surrounding actin structures, would be consistent with this frictional coupling model for microcluster centralization. Previous work showed that actin structures and their interaction with the plasma membrane are important for BCR-Ag microcluster integrity and centralization. If B cells are allowed to form microclusters in response to an Ag lipid bilayer, and are then treated with latrunculin A, BCR-Ag microcluster   105 centralization is rapidly arrested (Treanor et al., 2011), similar to what is observed in T cells (Varma et al., 2006). Additionally, actin depolymerization results in the dispersion of BCR-Ag microclusters (Treanor et al., 2011). This suggests that protein-protein interactions within the microcluster are not sufficient to maintain microclusters over time and that the actin cytoskeleton promotes microcluster integrity by corraling BCRs (Treanor et al., 2011). Activation of ERM family proteins around BCR-Ag microclusters is also required to maintain microcluster integrity (Treanor et al., 2011). By linking the actin cytoskeleton to the plasma membrane, active ERM proteins help create physical barriers that limit BCR diffusion, thereby trapping BCRs and promoting microcluster stability. The physical constraints imposed by an actin cytoskeleton that is closely coupled to the plasma membrane not only maintains the integrity of BCR-Ag microclusters but may also drive their centralization. It is likely that BCR-Ag microclusters are swept towards the cell body as the actin meshwork undergoes retrograde flow, a mechanism similar to that which transports TCR microclusters. My finding that Arp2/3 complex activity is required for the centripetal transport of BCR-Ag microclusters suggests that Arp2/3 complex-dependent actin structures form the actin barriers that stabilize BCR-Ag microclusters and provide the force for their transport.  When B cells probe the APC surface, their ability to generate filopodia allows them to form BCR-Ag microclusters that are distant from the cell body. This ability to scan more distant parts of the APC surface for Ag may increase microcluster formation. Although filopodia are generated via formin-dependent actin polymerization, I found that inhibiting the Arp2/3 complex prevented the retraction of these filopodia such that microclusters that formed on them were not transported towards the center of the immune synapse. In neurons, the Arp2/3 complex is important for both lamellipodia and filopodia initiation and dynamics (Korobova and Svitkina, 2008). Depletion of the Arp2/3 complex impairs the frequency of filopodial extension and retraction events (Korobova and Svitkina, 2008). Although actin retrograde flow rates within filopodial extensions are not altered when the Arp2/3 complex is blocked (San Ruiz-Miguel and Letourneau, 2014), presumably because the flow is generated by formin-polymerized actin filaments pushing against the membrane, Arp2/3 complex-mediated actin retrograde flow at the base of the filopodium may be important for filopodial retraction (via a frictional coupling mechanism) (Bornschlogl et al., 2013). Consistent with this, I found that inhibiting the Arp2/3   106 complex in B cells resulted in many stable filopodia-like protrusions that did not retract towards the cell body over the observation period. There appeared to be some actin retrograde flow within these filopodia but BCR-Ag microclusters were not efficiently transported towards the cell body. This suggests that the Arp2/3 complex may also be important for generating a centripetally moving actin meshwork that surrounds BCR-Ag microclusters within filopodia and promotes their movement towards the cell body.  The role of formin-generated actin structures at the B cell immune synapse has not been investigated. When T cells spread on pMHC-decorated artificial lipid bilayers, linear actin filaments nucleated by formins extend perpendicularly from the cell edge, traversing the lamellipod (Murugesan et al., 2016). At the inner face of the peripheral actin ring, myosin organizes these filaments into concentric arcs (Murugesan et al., 2016; Yi et al., 2012). Although I saw similar actomyosin structures in B cells spreading on anti-Ig-coated glass coverslips, these features were hard to identify in B cells interacting with APCs. Future experiments could address the role of formins in establishing the organization of the B cell immune synapse.   3.3.4 The three-step model of microcluster transport BCR and TCR microcluster centralization, as well as cSMAC formation, has been proposed to involve three cytoskeleton-dependent mechanisms that act sequentially: actin retrograde flow, contractile forces generated by actomyosin arcs, and movement along microtubules that span the actin-poor central region of the immune synapse (Kumari et al., 2014; Schnyder et al., 2011) (Figure 3.16). I showed that Arp2/3 complex-dependent branched actin nucleation is essential for actin retrograde flow at the B cell periphery and is required for BCR-Ag microcluster centralization and cSMAC formation. Because Arp2/3 complex-generated branched actin networks, and the resulting actin retrograde flow, occurs primarily at the periphery of the B cell immune synapse, other transport mechanisms must drive the continued centripetal motion of BCR-Ag microclusters that results in cSMAC formation. In T cells spreading on pMHC-coated lipid bilayers, myosin-based contractility of actomyosin arcs at the inner face of the peripheral ring of branched actin has been shown to sweep TCR microclusters towards the center of the immune synapse (Murugesan et al., 2016). In B cells spreading on anti-Ig-coated coverslips I observed similar actomyosin arcs at the inner face of the peripheral actin   107 ring. Consistent with the idea that these linear actin arcs are generated by formins, these structures were also observed in B cells in which the Arp2/3 complex was inhibited, as has been observed in T cells (Murugesan et al., 2016). Although myosin IIA is important for B cells to extract Ags from APC membranes (Hoogeboom et al., 2018; Natkanski et al., 2013), its role in microcluster centralization and cSMAC formation is not clear. By analogy to T cells, Arp2/3 complex-dependent actin retrograde flow may be required for the initial movement of BCR microclusters from the periphery of the immune synapse to the more centrally-located actomyosin arcs, which further propel microcluster centralization. However, inhibiting myosin IIA in B cells did not impair cSMAC formation, suggesting that actomyosin contractility is not an absolute requirement for this process. Alternatively, B cells interacting with the COS-7 APCs used in my experiments may not effectively form actomyosin arcs. The formation of such formin-dependent actin structures may be enhanced when lymphocytes are on substrates with greater rigidity, such as supported lipid bilayers or Ag-coated coverslips. Indeed, actomyosin arcs were readily detectable in B cells plated on anti-Ig-coated coverslips. Another possibility is that actomyosin arc formation during B cell-APC interactions requires other receptor-ligand interactions that are not reproduced in this system. At the T cell immune synapse, the actomyosin arcs are found at the pSMAC and co-localize with the LFA-1 integrin. Signaling by integrins that have bound to their ligand on the APC may enhance the formation of these actomyosin arcs. Because the APCs I used in my experiments do not express ligands for murine LFA-1 or VLA-4, it would be important to test whether co-expressing the ligands for these integrins along with the surrogate Ag would enhance the formation of actomyosin arcs and reveal a role for myosin contractility in supporting the centralization of BCR-Ag microclusters.     108   Figure 3.16 The three-step model of Ag receptor centralization The centralization of both TCR and BCR receptor clusters has been proposed to depend on the coordinated activity of three spatially distinct cytoskeletal processes. For simplicity, this figure illustrates these processes in B cells. First, BCR-Ag microclusters which form at the periphery, are transported inward with actin retrograde flow. Next, BCR-Ag microclusters interact with actomyosin contractile arcs, which sweep them further towards the center of the B cell-APC contact site. Finally, BCR-Ag microclusters are transported along microtubules via the motor protein dynein to form the cSMAC. Adapted from Kumari et al., 2014.      109 In both T and B cells, the final step in cSMAC formation is thought to be the movement of microclusters along microtubules that span the actin-poor central region of the immune synapse (Kumari et al., 2014; Schnyder et al., 2011). Dynein-dependent movement of BCR and TCR microclusters along juxtamembrane microtubules is required for cSMAC formation in both B and T cells (Hashimoto-Tane et al., 2011; Schnyder et al., 2011). However, the mechanism by which microclusters switch from one cytoskeletal network to the other is not understood. The formation of branched actin at the periphery of the immune synapse is required for capturing the plus ends of microtubules and for moving the microtubule network towards the Ag-presenting surface (Wang et al., 2017). In B cells spreading on anti-Ig-coated coverslips, microtubule plus-end binding proteins, such as CLIP-170, contact the inner face of the dense peripheral actin ring, similar to what occurs at the leading edge of a migrating cell (Wang et al., 2017). The actin-microtubule crosslinking protein IQGAP1 plays an important role in capturing CLIP-170 and the plus ends of microtubules at the boundary between the actin-rich and actin-poor regions (Wang et al., 2017). This connection could facilitate the transfer of microcluster cargo between the actin and microtubule networks. Interestingly, in nerve growth cones, ‘pioneer’ leading edge microtubules associate with and run parallel to actomyosin arcs that are at the inner face of the membrane-proximal branched actin network (Schaefer et al., 2002). If microtubules associate with actin arcs in lymphocytes, this could be a mechanism that allows microclusters that have been swept inwards by the actomyosin arcs to associate with microtubules.  Although each of the cytoskeleton-dependent processes proposed in this three-step model may be necessary for cSMAC formation, it is likely that none are individually sufficient for cSMAC formation. My data is consistent with a sequential three-step model of microcluster centralization in which the first step, Arp2/3 complex-dependent actin retrograde flow, is a prerequisite for the subsequent myosin- and microtubule-dependent steps. In this model, actin retrograde flow at the periphery is required to deliver BCR-Ag microclusters to sites where they can become associated with actomyosin arcs and microtubules. Thus, inhibiting Arp2/3 complex activity may block a critical initial step in BCR-Ag microcluster centralization and prevent the subsequent actomyosin- and microtubule-based centralization that leads to cSMAC formation.     110 3.3.5 NPFs direct Arp2/3 complex-dependent actin assembly The activation of NPFs is critical for the activation and function of the Arp2/3 complex. As such, it will be interesting to determine the contributions of different NPFs to the formation, movement, and coalescence of BCR-Ag microclusters. The Arp2/3 complex can be activated by the WASp, N-WASp, and WAVE NPFs. Depletion of WASp in B cells results in reduced B cell spreading and microcluster formation in response to membrane-bound Ags (Liu et al., 2011). Conversely, depletion of N-WASP leads to increased actin accumulation at the immune synapse and decreased BCR microcluster aggregation (Liu et al., 2013).   In contrast to WASp and N-WASp, the role of the WAVE regulatory complex (WRC) in B cell immune synapse formation has not been investigated. In mice lacking the Hem1 subunit of the WRC, neutrophil function is impaired, as is T cell development and actin polymerization in response to anti-CD3 stimulation (Park et al., 2008). B cell development is also severely impaired in these mice (Park et al., 2008) but immune synapse formation was not assessed. In T cells, WAVE2 localizes to the immune synapse and is required for actin reorganization, conjugate formation with APCs, and TCR-induced calcium release (Nolz et al., 2006). When T cells encounter anti-CD3-coated substrates, WAVE localizes to the periphery of the spreading cell (Hartzell et al., 2016). Interestingly, in cytotoxic T lymphocytes (CTLs), WAVE-dependent protrusions occur at the cell periphery whereas WASp-dependent protrusions are seen more centrally (Tamzalit et al., 2019). These different protrusions perform distinct functions in the CTL killing response, with WAVE-dependent protrusions promoting target cell adhesion and WASp-dependent protrusions potentiating the release of lytic granules (Tamzalit et al., 2019). Whether the WRC has important roles at the B cell immune synapse that are distinct from those of WASp and N-WASp, and functions at different sites, remains to be determined.  As discussed in section 1.4.3 (Figure 1.4), accumulating evidence indicates that the different NPFs that activate the Arp2/3 complex function at different sites in the cell and perform distinct functions. For example, N-WASp is recruited to phagocytic and endocytic sites and is important for the formation of invadopodia whereas WAVE drives Arp2/3 complex activation at the lamellipodia (Campellone and Welch, 2010; Rottner et al., 2017). How this impacts immune cell function is not fully understood. By analogy to T cells, WASp and N-WASp could be important for B cells to internalize membrane-bound Ags whereas WAVE may be most   111 important for driving B cell spreading. In addition, the binding of different NPFs to clusters of receptors or transmembrane signaling proteins could specify different patterns or regulation of movement. In cell-free membrane reconstitution studies with purified proteins, the movement of LAT clusters by the actin cytoskeleton is promoted by the presence of Nck, WASp, and N-WASp (Ditlev et al., 2019). Interestingly, the movement of LAT-N-WASp and LAT-WASp clusters were influenced differently by actin dynamics, suggesting that the nature of the associated NPF dictates how the clusters are transported at the immune synapse. Thus, it would be interesting to investigate the contributions of different NPFs to the spatial organization of BCRs and the initiation of BCR signaling and B cell activation in response to APC-bound Ags.   3.3.6 Perspectives I identified a key role for Arp2/3 complex-dependent actin polymerization in generating protrusions that scan the surface of the APC for Ag. Because actin monomer availability is limited, actin polymerization at the leading edge must be coupled to depolymerization. Actin depolymerization must work cooperatively with actin polymerization to drive dynamic membrane protrusions and actin retrograde flow. Indeed, the centralization of BCR-Ag microclusters at the immune synapse requires the dynamic reorganization of the actin cytoskeleton. Stabilizing actin filaments with jasplakinolide abrogates both BCR-Ag microcluster centralization and BCR-induced tyrosine phosphorylation (Liu et al., 2012). Thus investigating how actin-depolymerizing proteins contribute to the organization of BCR-Ag microclusters will provide a deeper understanding of how B cells use the actin cytoskeleton to tune their response to APC-bound Ags. The actin severing protein, cofilin, is required for the initial remodeling of the actin cytoskeleton that occurs upon Ag binding (Freeman et al., 2011). Moreover, this initial actin remodeling is required for cell spreading and immune synapse formation, pointing to an important role for actin depolymerization in immune synapse formation (Freeman et al., 2011). In chapter 5, I identify an important role for actin-depolymerizing factors in B cell immune synapse formation and BCR signaling.  For membrane-bound Ags a cytoskeleton-dependent positive feedback loop amplifies BCR signaling. First, BCR signaling induces the remodeling of the actin cytoskeleton. The subsequent actin-dependent spatial reorganization of BCRs into microclusters amplifies BCR   112 signaling, which again induces actin reorganization (Harwood and Batista, 2010). As such, remodeling of the actin cytoskeleton reduces the amount of Ag required for B cell activation (Batista et al., 2001; Depoil et al., 2008; Freeman et al., 2015; Weber et al., 2008). Therefore, an important extension of the work discussed in this chapter is to investigate how Arp2/3 complex-dependent actin polymerization and the resulting spatial reorganization of BCR microcluster impacts BCR signaling output. Understanding how the cytoskeleton controls the threshold for B cell activation could contribute to the development of novel strategies for modulating B cell activation. The relationship between Arp2/3 complex-dependent BCR organization, BCR signaling and subsequent B cell activation will be investigated in the following chapter.      113 Chapter 4: Arp2/3 complex activity amplifies APC-induced BCR signaling and enhances B cell activation  4.1 Introduction  4.1.1 BCR signaling induces the formation of microsignalosomes B cell encounter of cognate Ag on the surface of APCs leads to the adhesion of the B cell to the APC, rapid cell spreading, and immune synapse formation. Triggering of the BCR results in the phosphorylation of the CD79a/b (Ig-α/β) signaling subunit of the BCR (Abraham et al., 2016; Dal Porto et al., 2004), which recruits the Syk tyrosine kinase to the BCR. Syk activity promotes the assembly of BCR-associated ‘microsignalosomes’, a signaling complex that includes the Btk tyrosine kinase, PLCγ2, and PI3K (Abraham et al., 2016). Formation of the microsignalosome increases BCR signaling efficiency and decreases the amount of Ag needed for B cell activation (Treanor et al., 2009; Weber et al., 2008). The combined effects of activating this signaling network are changes in the B cell cytoskeleton and gene expression patterns, as well as cell cycle entry. Full activation of the B cell, including proliferation and differentiation into antibody-forming cells, requires a second signal such as T cell help.  As discussed in chapter 3, the actin cytoskeleton plays a vital role in the early events of B cell activation. Initial BCR signaling triggers local actin breakdown, which increases the lateral mobility of BCRs and other membrane proteins within the plasma membrane, thereby promoting the formation of BCR-Ag microclusters. The formation of BCR microclusters is dependent on the reorganization of the submembrane actin cytoskeleton (Depoil et al., 2008; Treanor et al., 2011). Because microcluster formation amplifies BCR signaling and propagates the B cell spreading response, the extent of BCR clustering may be a critical determinant of whether the magnitude of BCR signaling exceeds the threshold for B cell activation (Harwood and Batista, 2010; Weber et al., 2008).  The formation of Ag receptor microclusters occurs in both B and T cells and, therefore, the microsignalosome is proposed to be the basic signaling unit for lymphocytes (Harwood and Batista, 2008). Observations in both B and T cells have implicated the spatial clustering of Ag   114 receptors as an essential mechanism for regulating Ag-induced signaling responses (Depoil et al., 2008; Doh and Irvine, 2006; Mossman et al., 2005; Weber et al., 2008). Formation of BCR microclusters is crucial for amplifying BCR signaling responses, and is dependent on the reorganization of the actin cytoskeleton (Fleire et al., 2006). BCR clustering in response to Ag allows the small amount of basally active Syk in the cytoplasm to phosphorylate nearby ITAMs, which then recruit and activate more Syk molecules and phosphorylate more ITAMs (including in BCRs not bound to Ags) (Mukherjee et al., 2013). This creates a positive feedback loop where cluster formation amplifies BCR signaling. This is further enhanced by Syk-dependent remodeling of the actin cytoskeleton and cell spreading, which promotes the formation of additional microclusters. Subsequent polymerization of actin around BCR-Ag microclusters is also critical for the stabilization and maintenance of microclusters, as disrupting the actin cytoskeleton after microclusters form leads to the dispersal of microclusters and the loss of proximal BCR signaling (Treanor et al., 2011).  Although the actin cytoskeleton regulates both BCR organization and BCR signaling, the relationship between actin-dependent spatial organization of BCR microclusters and BCR signaling output is not fully understood. Moreover, whether the spatial patterning of BCRs encodes information that impacts B cell activation responses is not known.   4.1.2 BCR-CD19 interactions amplify BCR signaling in response to membrane-bound Ags In addition to regulating the organization of BCRs in the plasma membrane, the actin cytoskeleton plays an important role in regulating the interactions between BCRs and important signal amplifiers such as CD19 (Mattila et al., 2013). CD19 is essential for the BCR signaling response to membrane-bound Ags, but is dispensable for soluble Ags (Depoil et al., 2008; Mattila et al., 2013). CD19 is phosphorylated by Syk, presumably when it is brought into close proximity to the BCR (Tuveson et al., 1993). Phosphorylated CD19 promotes microsignalosome formation and recruits critical downstream signaling molecules such as Vav and PI3K (Buhl et al., 1997; O’Rourke et al., 1998). In CD19-deficient B cells, B cell spreading, microcluster formation, and BCR signaling is severely impaired (Depoil et al., 2008). Thus, the extent and duration of BCR-CD19 interactions could be a critical determinant in the B cell response to   115 membrane-bound Ags.The spatial organization of receptor clusters relative to clusters of essential signal amplifiers may be a conserved principle for regulating receptor signaling. In mast cells, FcεRI clusters are separated from clusters of the adaptor protein LAT (Wilson et al., 2001) in resting cells but come together upon receptor ligation. Similarly, in T cells, TCRs are segregated from LAT in the resting state (Lillemeier et al., 2010) and concatenate into microclusters with spatially segregated domains upon stimulation (Yi et al., 2019). Thus, the spatial organization of BCRs relative to key signal amplifiers could be critical for optimizing BCR signaling responses to membrane-bound Ag.  The actin cytoskeleton could promote the interactions between BCRs and signal amplifiers by encaging BCRs and amplifiers within the same membrane compartment. This trapping can stabilize the interactions between BCRs and CD19 and promote processive kinase reactions that drive B cell activation. In chapter 3, I demonstrated that the Arp2/3 complex is required for the organization of BCR microclusters but not their formation. Whether the Arp2/3 complex-dependent spatial organization of BCR microclusters impacts BCR signaling outputs is unclear. Furthermore, whether the Arp2/3 complex is important for mediating BCR-CD19 interactions has not been studied.  4.1.3 Rationale and hypothesis The individual components of the BCR signaling network have been studied in detail, but their coordination in time and space remains poorly understood. Specifically, how the organization of BCRs into specific synaptic patterns modulates the quantity or quality of the signaling output, and ultimately the cellular response, is unclear. Therefore, I tested the hypothesis that the Arp2/3 complex-dependent spatial organization of BCR-Ag microclusters is crucial for amplifying BCR signaling and promoting B cell activation. Using several readouts to measure BCR signaling and B cell activation (see Figure 4.1), I demonstrate that Arp2/3 complex-dependent processes amplify BCR signaling and enhance the ability of APC-bound Ags to induce transcriptional responses and proliferation.      116  Figure 4.1 Readouts of BCR signaling and B cell activation Readouts used to assess B cell activation in response to anti-Igκ-expressing APCs. Primary murine B cells were pre-treated with CK-689 or CK-666 for 1 hr and then added to the APCs. The CK-689 or CK-666 was present in the co-culture for the entire length of the experiment unless otherwise indicated.     117 4.2 Results  4.2.1 Arp2/3 complex activity amplifies BCR signaling at the immune synapse How the spatial organization of the BCR at the immune synapse impacts BCR signaling and B cell activation is not fully understood. Because inhibition of the Arp2/3 complex prevented the centripetal movement of BCR microclusters into a cSMAC, but did not significantly alter the amount of Ag gathered into BCR microclusters (Figure 3.5C,D, Figure 6B,E), I was able to ask whether the Arp2/3 complex-dependent spatial patterning of BCR-Ag microclusters impacts BCR signaling output. I quantified proximal BCR signaling events at the contact site between ex vivo primary murine splenic B cells and COS-7 APCs expressing a single-chain anti-Igκ surrogate Ag on their surface (Freeman et al., 2011). A critical initial step in BCR signaling is phosphorylation of the tyrosine residues within the ITAMs present in the CD79a/b signaling subunit of the BCR (Dal Porto et al., 2004). This is required for the recruitment and activation of Syk, a tyrosine kinase that phosphorylates multiple proteins that are critical for BCR signaling and B cell activation. APC-induced phosphorylation of CD79a/b at the immune synapse was assessed by staining with an antibody that recognizes the phosphorylated ITAMs of both CD79a and CD79b. I found that CD79 phosphorylation occurred rapidly at the B cell-APC contact site and co-localized with BCR-Ag clusters, which were detected using an antibody that detects the surrogate Ag (Figure 4.2A). As shown in the previous chapter, the BCR-Ag microcluster coalesced into a tight cSMAC within 5–10 min in control cells but not in B cells treated with the Arp2/3 complex inhibitor CK-666 (Figure 4.2A). Using quantitative image analysis described in section 2.9.3, I quantified both the Ag and phospho-CD79 (pCD79) associated with BCR-Ag microclusters at the B cell-APC interface. I then determined the relationship between the amount of Ag gathered into clusters and the signaling output at those BCR-Ag microclusters. For each B cell, the total pCD79 fluorescence intensity present in clusters at the B cell-APC interface was divided by the total fluorescence intensity of clustered Ag. This ratio represents the amount of BCR signaling output per unit of bound Ag. In control B cells treated with CK-689, pCD79 levels were maximal at 5 and 10 min after the B cells were added to the APCs and declined thereafter (Figure 4.2B), perhaps due to internalization of BCR-Ag microclusters. Importantly, inhibiting Arp2/3 complex activity   118 significantly reduced the amount of BCR signaling generated per unit of Ag that was gathered into clusters (Figure 4.2C,D). Similar results were obtained when HEL-specific B cells from MD4 mice were added to COS-7 APCs expressing the mHEL-HaloTag Ag (Figure 4.3A,B). I also analyzed CD79 phosphorylation by immunoblotting, which would detect BCRs on the cell surface as well as ones that have been internalized. Indeed, immunoblotting showed that APC-induced CD79 phosphorylation increased continually over the first 30 min of B cell-APC contact (Figure 4.4A), in contrast to what was observed by immunostaining the B cell-APC contact site. Importantly, similar to what was observed by imaging the B cell-APC interface, treating the B cells with CK-666 reduced APC-induced CD79 phosphorylation, especially at the 15 and 30 min time points (Figure 4.4A). In contrast, CK-666 treatment did not impair the ability of soluble anti-Ig antibodies to stimulate phosphorylation of CD79 or CD19 (Figure 4.4B,C). This suggests that the Arp2/3 complex-dependent spatial patterning of BCR-Ag microclusters amplifies proximal BCR signaling in response to polarized, membrane-bound Ags but not soluble Ags.           119   Figure 4.2 Arp2/3 complex activity amplifies proximal BCR signaling Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Igκ surrogate Ag. The cells were fixed at the indicated times and stained with an antibody that recognizes the surrogate Ag and with an antibody that recognizes the phosphorylated ITAMs in CD79a and CD79b (pCD79). Images of representative cells are shown (A). For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Beeswarm plots in which each dot is one cell. The median (red line) and interquartile ranges (red box) for >39 cells for each time point from a representative experiment are shown (B). The median values for this ratio were determined in each experiment and graphed (C). The graph shows the median values of clustered pCD79/clustered Ag in CK-666-treated B cells as a percent of the median values in CK-689-treated control cells (=100%) for the 5 and 10 min time points from five independent experiments. The number of cells analyzed for each condition was >75 (expt 1), >7 (expt 2), >39 (expt 3), >116 (expt 4), and >75 (expt 5), respectively. The CK-666/CK-689 ratios from the 5 experiments were significantly different from 100% at 5 and 10 min but not at 15 or 30 min. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test. Scale bars: 2 µm.   120   Figure 4.3 The Arp2/3 complex amplifies proximal BCR signaling in B cells from MD4 mice Primary murine B cells from MD4 mice were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing mHEL-HaloTag. The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated ITAMs in CD79a and CD79b (pCD79). Images of representative cells are shown (A). For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Beeswarm plots in which each dot is one cell. The median (red line) and interquartile ranges (red box) for >71 cells for each time point from a representative experiment are shown (B). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test. Scale bars: 2 µm.     121      122 Figure 4.4 Arp2/3 complex activity increases proximal BCR signaling in response to APC-bound Ag but is dispensable for signaling in response to soluble Ags (A) Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Igκ surrogate Ag. (B,C) Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then stimulated with 10 µg/ml soluble anti-Igκ for the indicated times. pCD79 and total CD79a immunoblots are shown (left panels) and the pCD79/total CD79a ratios are graphed (right panels) (A,B). pCD19 and total CD79a (loading control) immunoblots are shown (left panels) and the pCD19/total CD79a ratios are graphed (right panels) (C). Dotted red line corresponds to the pCD79/total CD79a or pCD19/CD79a ratio value for unstimulated CK-689-treated B cells. Representative data from one of seven (A,B) or three (C) experiments are shown.         123 The Ag-induced binding of Syk to phosphorylated CD79a/b leads to the phosphorylation of Syk (Rowley et al., 1995). Phosphorylation on Y342 and Y346 increases Syk activity and generates binding sites for PLCγ2 and Vav (Geahlen, 2009). When ex vivo primary B cells interacted with APCs expressing the single-chain anti-Igκ surrogate Ag, co-localization of pSyk (Y346) with BCR-Ag clusters was observed within 3 min (Figure 4.5A,B). This rapid association of activated Syk with the BCR was not dependent on Arp2/3 complex activity. B cells treated with CK-666 had significantly lower levels of clustered pSyk at 3 min and 5 min after contacting the APCs, compared to control cells (Figure 4.5C,D). Similar results were obtained when HEL-specific primary B cells were added to APCs expressing the HEL-HaloTag Ag (Figure 4.6A,B), where CK-666 treatment significantly reduced the clustered pSyk/clustered Ag ratio at the 3, 5, and 10 min time points. Together, these data suggest that the Arp2/3 complex is required for the amplification of the earliest BCR signaling events, phosphorylation of the CD79a/b ITAMs and phosphorylation of tyrosine residues in Syk that are required for its activation.  4.2.2 Inhibiting the Arp2/3 complex increases tonic BCR signaling as well as BCR and CD19 diffusion Although treating B cells with the Arp2/3 complex inhibitor reduced the ability of APC-bound Ags to stimulate the phosphorylation of CD79 and Syk, CK-666-treated B cells exhibited higher levels of pCD79 prior to Ag encounter (Figure 4.4A,B). This was accompanied by increased phosphorylation of ERK and Akt, downstream targets of BCR signaling (Figure 4.7). Actin-based diffusion barriers restrict the lateral mobility of the BCR within the plasma membrane and increased BCR mobility is associated with increased tonic Ag-independent BCR signaling (Freeman et al., 2015; Treanor et al., 2010). Because Arp2/3 complex activity contributes to the formation of the submembrane cortical actin network, which creates diffusion barriers for transmembrane proteins, I tested the hypothesis that Arp2/3 complex activity limits Ag-independent BCR signaling by restricting BCR mobility prior to Ag encounter. We used single-particle tracking (SPT) to compare the diffusion and confinement properties of both IgM- and IgD-containing BCRs, in control versus CK-666-treated primary B cells. Indeed, the median     124      125 Figure 4.5 The Arp2/3 complex amplifies Syk phosphorylation Primary murine B cells that had been pre-treated with 100 µM CK-689 or CK-666 for 1 hr were added to COS-7 cells expressing the single-chain anti-Igκ Ag. The cells were fixed at the indicated times and stained for the surrogate Ag and phosphorylated Syk (pSyk). Representative images are shown (A). The co-localization of pSyk with BCR-Ag clusters is not dependent on Arp2/3 complex activity (B). The co-localization of pSyk and Ag clusters in the cells was quantified using Mander’s coefficient. In (C), the total fluorescence intensity of clustered pSyk was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site for each B cell. Beeswarm plots with the median and interquartile ranges for >112 cells for each time point from a representative experiment are shown. The median values for this ratio were determined and graphed (D). The graph shows the median values of clustered pSyk/clustered Ag for CK-666-treated B cells as a percent of the median values for CK-689-treated control cells (=100%) from three independent experiments at the 3 min time point. CK-666/CK-689 ratios were not significantly different from 100% at 5, 10, 15 and 30 min. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test. Scale bars: 2 µm.      126   Figure 4.6 The Arp2/3 complex amplifies Syk phosphorylation in MD4 mice Primary murine B cells from MD4 mice were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing mHEL-HaloTag Ag. The cells were fixed at the indicated times and stained for pSyk. Images of representative cells are shown (A). For each B cell, the total fluorescence intensity of clustered pSyk was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Beeswarm plots in which each dot is one cell. The median (red line) and interquartile ranges (red box) for >40 cells for each time point from a representative experiment are shown (B). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test. Scale bars: 2 µm.     127 diffusion coefficients for IgM and IgD were approximately 2.8-fold higher in CK-666-treated cells than in control CK-689-treated cells (Figure 4.8A,B). Consistent with their increased lateral mobility, both IgM and IgD had larger effective confinement diameters in CK-666-treated cells than in control CK-689-treated cells (Figure 4.8A,B). Thus, CK-666 treatment increases both BCR mobility and tonic BCR signaling. This suggests that the Arp2/3 complex contributes to the formation of actin-based barriers that normally limit BCR diffusion and tonic BCR signaling. CD19 is essential for B cell activation by membrane-bound Ags but not soluble Ags (Depoil et al., 2008; Xu et al., 2014). Initial BCR signaling leads to phosphorylation of CD19 on key tyrosine residues, allowing CD19 to recruit PI3K and Vav (Buhl et al., 1997; O’Rourke et al., 1998; Tuveson et al., 1993). CD19 is relatively immobile within the plasma membrane and this facilitates BCR-CD19 interactions and CD19-dependent amplification of BCR signaling (Mattila et al., 2013). Indeed, depolymerizing the actin cytoskeleton results in diminished CD19 phosphorylation and PI3K signaling in response to BCR stimulation (Keppler et al., 2015; Mattila et al., 2013). Using SPT, I found that treating B cells with CK-666 caused a 2.3-fold increase in the median diffusion coefficient for CD19 and increased its effective confinement diameter (Figure 4.8C). This suggests that Arp2/3 complex-dependent actin structures limit the lateral mobility of CD19 in the plasma membrane, which may be important for membrane-bound Ags to stimulate CD19 phosphorylation and augment BCR-CD19 interactions.       128     Figure 4.7 The Arp2/3 complex regulates tonic signaling in resting B cells Ex vivo primary murine B cells were treated with DMSO, 100 µM CK-689, or 100 µM CK-666 for 1 hr, or stimulated with anti-Igκ antibodies for 5 min. Cell extracts were analyzed by immunoblotting with antibodies against phospho-ERK (pERK) and total ERK, or phospho-Akt (pAkt) and total Akt. Representative blot are shown (left). Band intensities were quantified and the ratio of pERK/total ERK and pAkt/total Akt (right) relative to those in anti-Igκ-treated cells (=1.0) are graphed as the mean ± SEM for four (pERK) or five (pAkt) independent experiments. Two-tailed paired t-test.             129  Figure 4.8 The Arp2/3 complex regulates the lateral mobility of BCRs and CD19 in resting B cells Ex vivo primary murine splenic B cells were treated with 100 µM CK-689 or CK-666 for 1 hr. SPT was then carried out by labeling the cells at low stoichiometry with Cy3-labeled Fab fragments of antibodies to IgM (A), IgD (B) or CD19 (C). The cells were then settled onto non-stimulatory anti-MHC II-coated coverslips and imaged for 10 s at 33 Hz by TIRFM. Single-particle trajectories from representative cells are plotted using a color-coded temporal scale (left panels). Scale bars: 5 µm. Diffusion coefficients were calculated for the indicated number of tracks and cumulative frequency curves are shown (center panels). The diameter of maximum displacement over the 10 s period of observation (confinement diameter) was calculated for each track and cumulative frequency curves are shown (right panels). The dots on the curves indicate the median values. Representative data from one of three independent experiments ****p<0.0001; Kolmogorov-Smirnov test.     130 4.2.3 Arp2/3 complex activity is important for BCR-CD19 interactions To test the hypothesis that the Arp2/3 complex is required for increasing BCR-CD19 interactions, I first asked whether inhibiting Arp2/3 complex activity altered the ability of membrane-bound Ags to stimulate CD19 phosphorylation. I used phospho-specific antibodies to detect CD19 that is phosphorylated on Y531 (pCD19) and quantified the level of pCD19 at the contact site between primary splenic B cells and APCs expressing the single-chain anti-Igκ surrogate Ag (Figure 4.9A). In control cells, pCD19 levels were maximal at 3 min after the B cells were added to the APCs and declined thereafter (Figure 4.9B). Inhibiting the Arp2/3 complex significantly reduced pCD19 levels at the 3 min time point compared to control cells. Instead, when the Arp2/3 complex was inhibited, CD19 phosphorylation peaked at 5 min after addition to APCs (Figure 4.9B,C). However, this peak was significantly lower that the peak pCD19 value for control cells (at 3 min), indicating that the initial peak of APC-induced CD19 phosphorylation was diminished and delayed in CK-666-treated B cells, compared to the control CK-689-treated cells (Figure 4.9B,C). As observed for phosphorylation of CD79, inhibiting the Arp2/3 complex did not impair the ability of soluble anti-Ig antibodies to stimulate rapid and robust CD19 phosphorylation (see Figure 4.4C). This suggests that Arp2/3 complex-dependent actin structures provide spatial organization that is important for BCR-induced CD19 phosphorylation in response to spatially restricted membrane-bound Ags. Because the co-localization of pCD19 with the BCR could determine the extent to which CD19 amplifies BCR signaling, I next quantified the fraction of total pCD19 fluorescence that overlaps BCR-Ag microclusters, using the Manders’ coefficient. In control cells, much of the pCD19 fluorescence occurred within BCR-Ag clusters at all time points after initiating B cell-APC contact (Figure 4.9D). The pCD19-Ag overlap was significantly less in the CK-666-treated B cells than in the control cells at all time points. By contrast, the overlap between pSyk clusters and BCR-Ag clusters in CK-666-treated cells was either not significantly different from, or was slightly higher than in control cells over the first 15 min of B-APC interactions (see Figure 4.5B). Thus, unlike pSyk, which is strongly associated with signaling BCRs, the co-localization of pCD19 with the BCR is impacted by Arp2/3 complex activity. Importantly, I found that the total amount of pCD19 fluorescence that overlapped with BCR-Ag clusters was much lower in CK-666-treated cells than in control cells at the 3-min time point (Figure 4.9E). This reflects   131 both decreased CD19 phosphorylation and decreased BCR-pCD19 overlap in the CK-666-treated cells. Taken together, these findings indicate that actin networks nucleated by the Arp2/3 complex promote APC-induced CD19 phosphorylation and enhance the interaction of pCD19 with the BCR.     132    133 Figure 4.9 Arp2/3 complex activity increases CD19 phosphorylation in response to APC-bound Ags Primary murine B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 cells expressing the single-chain anti-Igκ surrogate Ag. Cells were fixed at the indicated time points and stained with an antibody that recognizes the surrogate Ag and with an antibody that recognizes phosphorylated CD19. Representative cells are shown (A). For each B cell, the total fluorescence intensity of clustered pCD19 was calculated. Beeswarm plots in which each dot represents one cell are plotted with the median (red line) and interquartile ranges (red box) for >125 cells per time point from a representative experiment (B). The median pCD19 fluorescence intensity was determined for >15 cells per experiment and graphed (C). For each experiment, the median pCD19 fluorescence intensity for CK-666-treated cells was expressed as a percent of the median value for CK-689-treated cells (=100%). This ratio is plotted for four independent experiments. For each cell in (B), the fraction of total pCD19 fluorescence that overlaps with BCR-Ag microclusters was quantified by calculating the Manders’ coefficient (D). For each cell in (B), the total fluorescence intensity of pCD19 that was within BCR-Ag microclusters in cells was quantified (E). ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; ns, not significant; Mann-Whitney U test. Scale bars: 2 µm.     134 4.2.4 Arp2/3 complex activity is important for BCR-induced B cell activation responses I next sought to determine if the early BCR signaling defects observed in B cells treated with the Arp2/3 complex inhibitor translated into reduced B cell activation responses at later times. As described below, I found that COS-7 APCs expressing the single chain anti-Igκ surrogate Ag induce primary B cells to undergo robust transcriptional responses, activation marker upregulation, cell size increase, and proliferation. Therefore, I assessed whether inhibiting Arp2/3 complex activity by treating the cells with CK-666 impacted these APC-induced B cell activation responses that occur after 3–72 hr of Ag stimulation. To assess BCR-induced transcriptional responses, I used B cells from Nur77GFP reporter mice in which GFP expression is under the control of the Nur77 promoter (Moran et al., 2011). Nur77 is an immediate early gene whose transcription is induced in lymphocytes by TCR or BCR engagement. Primary Nur77GFP B cells were incubated with COS-7 cells expressing the single chain anti-Igκ surrogate Ag, or with parental COS-7 cells (no surrogate Ag), in the presence of CK-689 or CK-666. GFP expression was measured by flow cytometry after 3 hr (see Figure 2.4 for gating strategy). In the absence of surrogate Ag expression by the COS-7 cells, Nur77GFP B cells did not adhere to the COS-7 cells and GFP expression was not increased relative to B cells that were cultured in the absence of COS-7 cells (see Figure 2.4B). By contrast, culturing Nur77GFP B cells with anti-Igκ-expressing COS-7 cells resulted in increased GFP expression compared to B cells that were cultured with parental COS-7 cells. Importantly, in the presence of the Arp2/3 inhibitor CK-666, this APC-induced increase in GFP expression was reduced to ~30% of the levels observed in Nur77GFP B cells treated with the control CK-689 drug (Figure 4.10A).  In the presence of APC-bound Ags, inhibition of Arp2/3 complex activity also impaired the ability of primary B cells to increase their cell surface levels of CD69, the canonical marker of lymphocyte activation, as well as CD86, a co-stimulatory ligand critical for T cell activation. Surface expression of CD69 and CD86 depends on BCR-induced, NF-κB-dependent transcription (Arvå and Andersson, 1999; Kohm et al., 2002; Sánchez-Mateos et al., 1989; Testi et al., 1994). Culturing primary B cells with anti-Igκ-expressing COS-7 APCs for 18 hr resulted in increased expression of CD69 and CD86 on B cells (Figure 4.10B,C), responses that were completely dependent on expression of the surrogate Ag on the APCs (see Figure 2.5B and Fig.   135 4.10D). Interestingly, I found that upregulation of CD69 could be stimulated by Ag-bearing APCs but not by immobilized anti-IgM (Fig. 4.10D). This highlights the importance of Ag mobility, which allows the growth and coalescence of BCR microclusters, in B cell activation. Similarly, soluble anti-IgM was also unable to induce CD69 upregulation (Fig. 4.10D). This is consistent with the idea that APCs improve the efficiency of B cell activation by concentrating Ags and forming an immune synapse with a high local density of Ag. When CK-666 treated B cells were added to COS-7 APCs, the APC-induced upregulation of CD69 and CD86 was inhibited by 40–50% (Figure 4.10B,C). This was not due to increased B cell death, as DAPI-positive dead cells were excluded from analysis (see Figure 2.5B). Importantly, CD86 upregulation in response to PMA plus ionomycin was not affected by CK-666 (Figure 4.10E). This suggests that inhibiting the Arp2/3 complex impairs proximal BCR signaling that is important for upregulation of CD86, as opposed to downstream events that are induced by both PMA plus ionomycin and BCR engagement. As part of their activation program, B cells increase in size as they enter S phase, reflecting an increase in the secretory machinery required for antibody synthesis (DeFranco et al., 1985; Kirk et al., 2010). Compared to resting lymphocytes, activated lymphoblasts exhibit an increase in both forward scatter (cell size) and side scatter (granularity) when analyzed by flow cytometry. Co-culturing primary B cells with anti-Igκ-expressing COS-7 APCs for 18 hr resulted in 30–55% of the B cells becoming larger, more granular blast cells (Figure 4.10F). However, in the presence of the Arp2/3 complex inhibitor CK-666, the percent of B cells that became blast cells was reduced to 5–16% (Figure 4.10F).       136       137 Figure 4.10 Arp2/3 complex activity is required for inducing transcriptional responses and cell cycle entry. (A) Primary ex vivo B cells from Nur77GFP mice were exposed to anti-Igκ-expressing COS-7 APCs (filled curves) or parental COS-7 cells (unfilled curves) for 3 hr. Histograms show GFP expression. See Figure 2.4 for gating strategy. The Ag-induced increase in GFP fluorescence was calculated as the geometric mean for B cells cultured with anti-Igκ-expressing APCs (dotted line) minus the geometric mean for B cells cultured with parental COS-7 cells. The graph shows the Ag-induced increase in GFP fluorescence in CK-666-treated B cells as a percent of the response in CK-689-treated control cells (=100%). Each dot is an independent experiment and the red line is the median. (B,C) Histograms showing CD69 (B) or CD86 (C) upregulation in primary ex vivo C57BL/6J B cells exposed to anti-Igκ-expressing APCs (filled curves) or parental COS-7 cells (unfilled curves) for 18 hr. The Ag-induced increases in CD69 or CD86 expression were calculated as in (A). See Figure 2.5 for gating strategy and representative calculations. Graphs show the increase in expression in CK-666-treated B cells as a percent of the response in CK-689-treated controls (=100%). Each dot is an independent experiment. The median (red line) and interquartile ranges (box) are shown. (D) Primary ex vivo B cells were added to anti-Igκ-expressing COS-7 APCs (filled blue bar) or parental COS-7 cells (unfilled blue bar), added to wells coated with plate-bound anti-IgM (light green bar), treated in suspension with 10 µg/ml soluble anti-IgM (dark green bar) or left unstimulated (unfilled green bar) for 18 hr. The graph shows the Ag-induced increase in CD69 fluorescence as calculated in (B). The data are plotted as the mean + SEM for replicate samples in a representative experiment. (E) Induction of CD86 expression in response to APCs (calculated as above) or to PMA + ionomycin (geometric mean for stimulated B cells minus geometric mean for unstimulated B cells) in the same experiment. Responses by CK-666-treated B cells are expressed as a percent of those in the CK-689-treated control cells. Results from two experiments are shown along with the average (bar). (F) B cells were treated as above and after 18 hr of co-culture, the percent of blast cells with increased forward and side scatter was determined by flow cytometry (see Figure 2.5 for gating strategy). Each dot is an independent experiment. The median (red line) and interquartile ranges are shown.      138 In this co-culture system, both the B cells and APCs are exposed to CK-666 for the entire duration of the experiment. To ensure that the observed reduction in CD69 and CD86 expression was not due to effects of CK-666 on the APCs, I used Arp3 siRNA to deplete the Arp2/3 complex in B16F1 murine melanoma cells expressing the single chain anti-Igκ Ag (Figure 4.11A,B). When primary B cells were added to these APCs, I observed that control and Arp3-depleted B16F1 APCs induced the upregulation of CD69 and CD86 to the same extent (Figure 4.11C,D). Moreover, B cells treated with CK-666 exhibited similarly impaired CD69 and CD86 responses, regardless of whether Arp3 was depleted or not in the APCs (Figure 4.11E,F). This indicates that the impaired upregulation of CD69 and CD86 observed in CK-666 treated cells is B cell-intrinsic. Because CK-666 was present during the entire B cell-APC co-culture in these experiments, it was also important to determine whether the impaired responses to APC-bound Ags were due to alterations in the initial BCR spatial reorganization and signaling, or to later events. To test this, I delayed the addition of CK-666. Pre-treating B cells with CK-666 for 1 hr prior to adding them to the APCs resulted in substantial inhibition of CD69 upregulation (Figure 4.12A). However, if the CK-666 was added 5 min after initiating the B cell-APC co-culture, or at later time points, it had very little effect on the BCR-induced upregulation of CD69 (Figure 4.12A). Adding CK-666 to the B cells at the same time that they were mixed with the APCs caused only partial inhibition of the CD69 response compared to pre-treating the B cells for 1 hr with the drug. This likely reflects the fact that some time is required for the CK-666 concentration in the B cells to reach a level that substantially inhibits Arp2/3 complex activity. Hence APC-induced B cell activation is only impaired if Arp2/3 complex activity is inhibited during the initial stages of B cell-APC interaction. Moreover, if CK-666 was present during the initial B cell-APC interaction, and then washed out after 5–120 min, CD69 upregulation was able to recover to nearly 100% of control values, regardless of when the washout was performed (Figure 4.12B). Once the drug was removed, the cells recovered their ability to aggregate BCR microclusters and form a cSMAC (Figure 4.12C-E) and B cell activation could proceed normally, consistent with the effects of CK-666 on actin dynamics being rapidly reversible (Yang et al., 2012). Together, these results argue that Arp2/3 complex-dependent processes that occur during the initial stages of B cell-APC interactions are required for B cell activation responses.    139      140 Figure 4.11 Depleting the Arp2/3 complex in APCs does not affect B cell activation responses The APCs used were untransfected B16F1 murine melanoma cells (white in panels C-F) or B16F1 cells transfected with either the single chain anti-Igκ alone (grey), anti-Igκ plus control siRNA (blue), or anti-Igκ plus Arp3 siRNA (green). (A) Schematic of experimental workflow. (B) B16F1 cells that had been transfected with control siRNA or Arp3 siRNA were analyzed by immunoblotting for Arp3 and actin. Results from one representative experiment are shown. (C,D) Primary murine B cells were added to the different APC populations for 18 hr. Cell surface levels of CD69 (C) and CD86 (D) were quantified by flow cytometry. The Ag-induced increases in CD69 and CD86 fluorescence were calculated as in Figure 2.5. The data are expressed relative to the increase in CD69 or CD86 expression stimulated by APCs that had been transfected with only the single chain anti-Igκ (the MFI for these cells is indicated by the dotted line on the histogram). Each dot represents an independent experiment and bars represent the median. (E) Primary B cells were pre-treated CK-689 (grey) or CK-666 (orange) for 1 hr and added to untransfected (No-Ag-APC) B16F1 cells (unfilled curves) or B16F1 cells that had been transfected with single chain anti-Igκ plus Arp3 siRNA (filled curves). The MFI for CK-689-treated cells that were added to Ag-bearing Arp3 siRNA-transfected APCs is indicated by the dotted line. (F) CK-666-treated B cells were added to B16F1 APCs expressing either membrane-bound anti-Igκ alone (grey filled bar), anti-Igκ plus control siRNA (blue filled bar), or anti-Igκ plus Arp3 siRNA (green filled bar). Ag-induced increases in CD69 and CD86 expression were calculated as in Figure 2.5. Each dot represents one independent experiment and bars represent the median. Two-tailed paired t-test.      141    142 Figure 4.12 Arp2/3 complex activity is required in the first 5 min of APC contact (A) Primary murine B cells were pre-treated with CK-689 or CK-666 for 1 hr before being added to anti-Igκ-expressing APCs or parental COS-7 cells (-60 min time point). Alternatively, the drugs were added at the same time that the B cells were added to the APCs or parental COS-7 cells (0 min), or at 5–60 min after initiating the co-culture. After 18 hr, the cells were stained and analyzed for cell surface expression of CD69 as in Figure 2.5. The increase in CD69 expression in CK-666-treated B cells is expressed as a percent of that for cells that were treated with CK-689 in the identical manner. For each point, the average ± range is shown for two experiments. (B) B cells were treated with CK-689 or CK-666 for 1 hr and then added to COS-7 APCs expressing membrane-bound anti-Igκ (Ag-APC) or to parental COS-7 cells (No-Ag-APC). The culture medium was removed at the indicated times after adding the B cells to the APCs and replaced with medium lacking CK-689 or CK-666. For t = 0, the drugs were washed out immediately before adding the B cells to the APCs. The Ag-induced increase in CD69 expression after 18 hr of co-culture was calculated as above and responses by CK-666-treated B cells are expressed as a percent of those for the CK-689-treated control cells. For each point, the average ± range is shown for two experiments. Where no error bars are shown, they were smaller than the symbol. (C–E) A20 D1.3 B cells were pre-treated with 100 µM CK-689 or CK-666 for 1 hr and then added to COS-7 APCs expressing mHEL-HaloTag. These ‘no washout’ cells were fixed after 10 min of B cell-APC contact. Alternatively, B cells were added to the APCs and after 5 min of contact the drug-containing medium was washed out and replaced with fresh medium (Drug washout). After another 5 min, the cells were fixed. Cells were imaged by spinning disk microscopy (D). Representative images are shown. For each cell, the number of clusters required to contain 90% of the Ag fluorescence was calculated and graphed in (E). Each dot represents one cell and the median (red line) and interquartile ranges (black box) are shown for n>25 cells per condition. Representative data from one of two independent experiments. The Mann-Whitney U test was used to calculate p values. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05. Scale bars: 5 µm.       143 In T cells, myosin-based contractility promotes TCR microcluster centralization and cSMAC formation, and the amplification of TCR-dependent signaling (Babich et al., 2012; Ilani et al., 2009; Jacobelli et al., 2004; Kumari et al., 2012; Murugesan et al., 2016; Yi et al., 2012). In Chapter 3, I found that inhibiting myosin IIA in B cells with pNBB did not inhibit BCR microcluster centralization and cSMAC formation, in contrast to the effect of CK-666 treatment. Hence, I hypothesised that pNBB would not significantly affect APC-induced B cell activation responses. When pnBB-treated cells were added to COS-7 APCs expressing membrane-bound anti-Igκ for 18 hr, they exhibited a slight, but not statistically significant reduction in CD69 and CD86 upregulation compared to DMSO-treated cells (Figure 4.13A,B). As discussed in chapter 3, when pnBB-treated A20 D1.3 B cells were added to COS-7 APCs expressing mHEL-HaloTag, they were able to form cSMACs to the same extent as DMSO-treated cells (Figure 3.15). These results suggest that APC-induced activation marker expression and cSMAC formation are dependent on Arp2/3 complex activity, whereas myosin contractility has little or no involvement, at least under these experimental conditions. In the presence of co-stimulatory cytokines such as BAFF and IL-4, Ag-activated B cells proliferate extensively. CFSE dilution assays showed that >90% of primary B cells that were co-cultured with anti-Igκ-expressing COS-7 APCs plus these cytokines for 72 hr underwent cell division (Figure 4.14A). Addition of CK-666 to the culture reduced the percent of B cells that had divided at least once to ~55% and the mean number of cell divisions was reduced from 2 to 0.7 (Figure 4.14A). This reduction in B cell proliferation was accompanied by increased cell death (Figure 4.14B), which is the alternative fate for B cells that are cultured in vitro in the absence of mitogenic stimuli. Note that when the Arp2/3 inhibitor CK-666 was added 24 hr after initiating the B cell-APC co-culture, B cell proliferation was not impaired (Figure 4.14A, right panel). This indicates that CK-666 does not block cell division and suggests that the critical functions of the Arp2/3 complex in APC-dependent B cell activation occur during the first 24 hr. Collectively, these findings indicate that the Arp2/3 complex-dependent processes are important for B cells to enter S phase and proliferate.     144  Figure 4.13 Inhibition of myosin does not affect upregulation of B cell activation markers Primary murine B cells were pre-treated with DMSO (control), 50 µM pnBB for 30 min, or 100 µM CK-666 for 1 hr and then added to COS-7 APCs expressing anti-Igκ for 18 hr. Cell surface levels of CD69 (A) and CD86 (B) were quantified by flow cytometry. The Ag-induced increases in CD69 and CD86 fluorescence were calculated as in Figure 2.5. Graphs show the increase in expression in CK-666-treated and pnBB-treated B cells as a percent of the response in DMSO-treated controls (=100%). Each dot represents an independent experiment.     145    Figure 4.14 Arp2/3 complex activity is required for B cell proliferation in response to APC-bound Ags CFSE-labeled primary murine B cells were pre-treated with CK-689 or CK-666 for 1 hr prior to being cultured with APCs in the presence of IL-4 and BAFF for 3 days (filled curves). The unfilled curves depict CFSE dilution at day 1. Representative data from one experiment is shown on the left and the average number of divisions per cell is indicated (A). The graph shows the percent of live cells that had proliferated by Day 3. Where indicated, CK-666 was added 24 hr after initiating the B cell-APC co-culture instead of 1 hr prior. The percent of dead B cells that stained with 7-AAD is shown in (B). In the graphs, each dot is an independent experiment and the bars indicate medians.       146 4.3 Discussion   4.3.1 Summary of findings How microcluster movement and coalescence and the organization of BCRs into specific synaptic patterns modulates the quantity or quality of the signaling output, and ultimately the cellular response, is not well understood. In chapter 3, I showed that the Arp2/3 complex is important for organizing BCR-Ag microclusters at the B cell immune synapse. In this chapter, I investigated how the Arp2/3 complex-dependent spatial patterning of BCR-Ag microclusters impacts BCR signaling, transcriptional responses, and B cell proliferation in response to APC-bound Ags. Using quantitative analysis of microscopy data, I showed that inhibiting Arp2/3 complex activity significantly reduced proximal BCR signaling (i.e. phosphorylation of CD79a/b and the association of pSyk with BCR-Ag microclusters) during the first 5 min of B cell-APC encounter. Importantly, the amount of pCD79 or pSyk per unit of gathered Ag was significantly decreased upon Arp2/3 complex inhibition, suggesting that the Arp2/3 complex is required for signal amplification in response to membrane-bound Ags. Next, I asked whether this translated into decreased B cell activation. Indeed, CK-666 treatment reduced the ability of Ag-bearing APCs to stimulate a transcriptional response at 3 hr, activation marker upregulation and cell cycle entry at 18 hr, and B cell proliferation at 72 hr. Importantly, I found that adding CK-666 to B cells shortly after they had engaged APCs did not impair the upregulation of B cell activation markers. As well, I found that Arp2/3 complex activity was not required for the upregulation of activation markers in response to PMA plus ionomycin, stimuli that bypass the BCR but initiate many of the same downstream signaling reactions. Taken together, these findings support the idea that Arp2/3 complex-dependent effects on BCR-Ag microclusters that occur within the first few minutes of B cell-APC interactions amplify initial BCR signaling reactions and that this impacts B cell responses that occur up to 3 days later. This actin-dependent signal amplification could be important for lowering the amount of membrane-bound Ag required to exceed the threshold for initiating downstream B cell responses. Importantly, these findings link the rapid reorganization of cytoskeletal elements, receptors (BCRs), proximal signaling enzymes (Syk), and signal amplifiers (CD19), supporting the idea that dynamically evolving spatial distributions of these   147 molecules encode information that impacts cellular responses. In the next sections, I discuss several potential mechanisms by which Arp2/3 complex-dependent actin polymerization might regulate the signaling output of BCR microclusters: stabilizing BCR microsignalosomes, increasing and maintaining BCR clustering, controlling the interactions of BCR microclusters with transmembrane regulators such as CD19, and exerting mechanical forces on Ag-bound BCRs (Figure 4.15).  4.3.2 Arp2/3 complex-dependent actin structures may stabilize BCR microsignalosomes  Arp2/3 complex-dependent actin polymerization could enhance BCR signaling by promoting the integrity of the BCR-Ag microclusters and by acting as a platform for the assembly of signaling complexes. When the actin-depolymerizing drug latrunculin A is added to B cells after they form microclusters, the microclusters become more diffuse and Syk phosphorylation decreases (Treanor et al., 2011). Similarly, reactivation of ERM proteins following microcluster formation is required to support proximal BCR signaling (Treanor et al., 2011). This suggests that actin polymerization at BCR microclusters is important for maintaining microcluster integrity and supporting downstream signaling. Using high resolution ISIM imaging, I observed actin structures surrounding BCR-Ag microclusters. By confining BCRs and associated signaling molecules, these actin structures could enhance the frequency and duration of the molecular interactions required for BCR signal transduction. Cooperation between microcluster components is important for BCR signaling. For example, PLCγ2 and Vav recruit and retain each other at the BCR-Ag microsignalosome (Weber et al., 2008). Prolonging the lifetime of BCR microclusters could stabilize these cooperative interactions that enhance BCR signaling. This idea is similar to the oligomerization-induced trapping model put forth by Kusumi and colleagues, in which large ligand-induced signaling complexes are more likely to be confined or trapped by the membrane-associated cytoskeleton (Ritchie et al., 2003).      148   Figure 4.15 The actin cytoskeleton regulates BCR signaling (A) In resting B cells, the actin cytoskeleton restricts BCR diffusion, thus limiting BCR-BCR and BCR-CD19 collisions. (B) Breakdown of the actin cytoskeleton increases BCR mobility, thus promoting BCR-BCR and BCR-CD19 interactions. This is required for BCR signaling and microcluster formation. (C) Repolymerization of the actin cytoskeleton around BCR microclusters promotes repeated collisions of BCRs and CD19 and stabilizes BCR microclusters, enhancing BCR signaling. (D) Actin retrograde flow promotes the aggregation of BCR microclusters and interactions with CD19. (E) Large, stable microclusters exhibit enhanced BCR signaling. Adapted from Tolar, 2017.      149 In T cells, signaling proteins such as PLCγ1 accumulate at actin foci that surround TCR microclusters (Kumari et al., 2015). The formation of these actin foci is dependent on WASp-mediated activation of the Arp2/3 complex (Kumari et al., 2015). Although proximal TCR signaling and microcluster formation are not affected by deletion of WASp or inhibition of the Arp2/3 complex, the recruitment and activation of PLCγ1 is impaired when these actin foci are not formed (Kumari et al., 2015). Recently, Arp2/3 complex-dependent actin foci have been observed in B cells (Roper et al., 2019). These foci co-localize with BCR signaling components such as BLNK and CD19 and are important for Ag extraction (Roper et al., 2019). Whether Arp2/3 complex-dependent actin structures surrounding BCR microclusters act as scaffolds that amplify and diversify BCR signaling remains to be determined. As I describe below in section 4.3.4, Arp2/3 complex-dependent structures are likely to be necessary for controlling or stabilizing the interactions between BCR microclusters and clusters of essential adaptor proteins such as CD19.  Although Ag-induced actin reorganization enhances BCR signaling in response to APC-bound Ags, the submembrane actin cytoskeleton limits the lateral mobility of the BCR in resting B cells and thereby prevents spontaneous BCR signaling. Indeed, disrupting the actin cytoskeleton with latrunculin A is sufficient to induce robust Ag-independent BCR signaling (Treanor et al., 2010). The cytoplasmic domain of CD79b is required for this actin-dependent control of BCR lateral diffusion (Treanor et al., 2010). I found that inhibiting Arp2/3 complex activity in resting primary B cells resulted in increased basal levels of pERK and pAkt and that this correlated with increased lateral mobility of both IgM-BCRs, IgD-BCRs, and CD19 within the plasma membrane. Hence, Arp2/3 complex-mediated actin polymerization is also important for limiting Ag-independent BCR signaling, which if dysregulated could lead to autoimmunity or B-cell malignancy.     150 4.3.3 Arp2/3 complex-dependent BCR clustering and spatial organization may amplify BCR signaling I found that the Arp2/3 complex is important for amplifying BCR signaling in response to membrane-bound Ags. Because both control and Arp2/3 complex-inhibitor treated B cells gathered approximately the same amount of Ag into microclusters, I was able to show that Arp2/3 complex inhibition decreased the levels of phosphorylated CD79, Syk, and CD19 per unit of gathered Ag compared to control cells. Furthermore, adding the Arp2/3 complex inhibitor to the cells after microclusters had grown in size and initiated peak proximal signaling responses did not reduce transcriptional responses associated with B cell activation. Therefore, the Arp2/3 complex-dependent signal amplification that occurs in the first few minutes of APC contact, when BCR-Ag microclusters are growing and coalescing, is important for initiating B cell activation responses. However, the relationship between microcluster organization and BCR signaling is not well understood.  The extent to which B cells spread on Ag-coated surfaces, gather Ag, and form BCR-Ag microclusters determines the magnitude of BCR signaling and whether B cell activation occurs. As such, the number of microclusters and the amount of Ag gathered within them is a critical determinant for BCR signaling and B cell activation (Harwood and Batista, 2010). My finding that Arp2/3 complex inhibition did not affect the amount of Ag gathered into clusters, or the number of microclusters formed at early time points, but reduced the amount of BCR signaling per unit of gathered Ag revealed an additional mechanism by which actin dynamics amplifies BCR signaling. This indicates that the spatiotemporal dynamics of BCR-Ag microcluster organization, as well as their interactions with clusters of co-receptors, enhances BCR signaling output and could reduce the amount of Ag required for B cell activation. Hence, Arp2/3 complex-dependent actin dynamics may ‘tune the threshold’ for B cell activation.  Arp2/3 complex-dependent actin dynamics could increase and prolong BCR-BCR interactions within microclusters that enhance BCR signaling. The formation of large and stable microclusters, which is dependent on Ag mobility and the actin cytoskeleton, increases BCR signaling output (Ketchum et al., 2014). When BCRs are in microclusters, recruited Syk molecules can phosphorylate nearby ITAMs on BCRs that are not bound to Ag (Mukherjee et al., 2013). This leads to further recruitment of Syk and ITAM phosphorylation via a positive   151 feedback loop that results in signal amplification. When BCR clusters are smaller, there are fewer ITAMs available, and signal amplification is not as efficient. Hence, the clustering of BCRs is critical for the amplification of BCR signaling. In chapter 3, I demonstrated that in control B cells, microcluster area increased most rapidly between 2 and 6 min after the B cells were added to the APCs, which corresponds with the peak of proximal BCR signaling. In the presence of the Arp2/3 complex inhibitor, small BCR-Ag clusters still formed but they did not merge. Accordingly, the amount of clustered pCD79 or pSyk relative to the amount of clustered Ag was decreased in CK-666 treated B cells, suggesting that microcluster coalescence amplifies BCR signaling in response to membrane-bound Ags. Additionally, when B cells interacted with immobilized plate-bound anti-Ig antibodies, they failed to upregulate activation markers efficiently. This is consistent with the idea that actin-dependent microcluster aggregation enhances the amount of BCR signaling per unit of bound Ag.  In T cells, the presence of large TCR oligomers increases the capacity of the cells to respond to low densities of peptide-MHC complexes (Ags) on the surface of APCs (Schamel et al., 2005). Moreover, previously stimulated T cells and memory T cells have larger pre-existing TCR oligomers on their surface compared to naive T cells (Kumar et al., 2011). Thus, large oligomeric complexes are important for increasing the sensitivity of TCRs for low-density Ags. Using loss-of-function approaches, I showed that Arp2/3 complex activity is important for the coalescence of BCR microclusters and for microcluster-based BCR signaling. These findings suggest that inhibiting the Arp2/3 complex disrupts the positive feedback loop in which actin-dependent microcluster coalescence amplifies BCR signaling. I predict that this would increase the Ag density and affinity thresholds for B cell activation. Consistent with this idea, deletion WASp in B cells leads to impaired antibody responses, especially in response to low dose Ag (Westerberg et al., 2005). Thus, by controlling BCR microcluster organization, the WASp-Arp2/3 complex pathway could tune the threshold for APC-induced B cell activation.   Microcluster size and BCR signaling are also controlled by the extracellular galectin lattice. Galectin-9-deficient B cells exhibit enhanced BCR microcluster formation and increased BCR signaling in response to membrane-bound Ags (Cao et al., 2018), likely reflecting the fact that galectin-9 enhances the association of IgM with the inhibitory molecules CD45 and CD22 (Cao et al., 2018). Interestingly, it has been proposed that in large microclusters, interior BCRs   152 are shielded from negative regulators such as CD22 (Gasparrini et al., 2016). This may be another mechanism by which Arp2/3 complex-dependent microcluster coalescence amplifies microcluster-based BCR signaling.  Because B cell activation must be tightly regulated, B cells likely employ multiple mechanisms to control the level of tonic Ag-independent BCR signaling as well as the relationship between Ag binding and BCR signaling output. The ability of APC-bound Ags to rapidly induce the formation of BCR signalosomes is likely critical for B cells to respond rapidly to small amounts of pathogen-derived molecules and provide protection that limits the spread of infection. By regulating BCR clustering and the interactions of the BCR with its positive and negative regulators, scaffolding networks such as the submembrane actin cytoskeleton and galectin lattice control the Ag density threshold required for activation and prevent inappropriate or deleterious B cell activation.   4.3.4 The Arp2/3 complex stabilizes BCR-CD19 interactions B cell activation in response to membrane-bound Ags requires the co-receptor CD19 (Depoil et al., 2008). By recruiting PI3K, PLCγ2, and Vav, CD19 acts as a signaling hub that promotes BCR signaling in response to membrane-bound Ags (Depoil et al., 2008; Weber et al., 2008). Initial BCR-CD19 interactions are dependent on the immobilization of CD19 by the tetraspanin network, but not actin (Mattila et al., 2013). However, I found that inhibiting Arp2/3 complex-dependent actin polymerization increased the lateral mobility of CD19 within the plasma membrane, suggesting that branched actin structures also restrain its mobility. This increased mobility of CD19 may decrease the initial rate at which productive BCR-CD19 collisions occur in resting B cells. In B cells that have bound to an APC-associated Ag, Arp2/3 complex-dependent actin structures could bring BCR-Ag and CD19 clusters together or establish physical barriers that maintain the interaction between these two species. Confinement of BCR and CD19 clusters within the same compartment could amplify BCR signaling by increasing the frequency and duration of BCR-CD19 interactions. In line with this idea, I found that inhibiting the Arp2/3 complex resulted in decreased APC-induced CD19 phosphorylation, as well as reduced overlap between BCR-Ag clusters and pCD19 clusters at 3 min after the initiating B cell-APC contact. By 5 min, the differences between CK-666-treated B cells and control cells   153 were smaller. Hence, inhibiting the Arp2/3 complex reduces and delays initial Ag-induced BCR-CD19 interactions. By activating PI3K, CD19 supports BCR-induced activation of Akt and mTOR, promoting B cell survival, growth, nutrient uptake and metabolic changes required for B cell activation, differentiation, and Ig class switching (Schweighoffer and Tybulewicz, 2018). I found that inhibition of the Arp2/3 complex leads to deceased cell survival and proliferation in response to APC-bound Ag. Thus, the Arp2/3 complex-dependent interactions between BCR and CD19 clusters could be crucial for CD19 to couple the BCR to PI3K signaling. This is consistent with the role of WIP in regulating both CD19 phosphorylation and PI3K signaling (Keppler et al., 2015). Taken together, these findings suggest that the WASp/WIP-mediated activation of the Arp2/3 complex drives the assembly of actin structures that can transiently co-localize BCR and CD19 clusters in an actin-based corral. This could enhance the frequency and duration of BCR-CD19 interactions, allowing for signal amplification and the CD19-mediated recruitment of PI3K and subsequent activation of PI3K-dependent signaling pathways.  In vivo, B cells are exposed to multiple signals, including chemokines, survival factors and danger signals. Signaling from these distinct receptors converge on the actin cytoskeleton. For example, signaling through the BCR, the chemokine receptor CXCL5, the survival factor receptor BAFFR, and the costimulatory receptor CD40 all activate PI3K via CD19 phosphorylation. Activation of this signaling cascade requires actin dynamics mediated by WIP (Keppler et al., 2015). Modulating the activity of the Arp2/3 complex via the CD19/PI3K pathway could by a mechanism by which a variety of receptors can prime B cells (i.e. increase BCR mobility, Ag-independent BCR-BCR collisions, and tonic BCR signaling (Freeman et al., 2015)) and lower their threshold for Ag-induced B cell activation. Thus, by controlling receptor organization and dynamics, the actin cytoskeleton could provide an important mechanism for integrating environmental stimuli to tune Ag sensitivity and the threshold of signaling required for B cell activation (Mattila et al., 2016).  4.3.5 Mechanosensitivity of BCR signaling Many receptors are regulated by mechanical forces and this is another possible mechanism by which Arp2/3 complex-mediated actin polymerization could amplify BCR signaling. In T cells, actin retrograde flow is essential for sustaining TCR signaling in response   154 to Ag-bearing artificial lipid bilayers (Babich et al., 2012; Kumari et al., 2015). Because the TCR is a mechanosensitive receptor, actin retrograde flow could exert tension on the TCR that enhances its signaling (Basu and Huse, 2017). The BCR is also highly sensitive to mechanical force, such that increasing the stiffness of Ag-presenting surfaces causes greater recruitment of Syk and other signaling components (Shaheen et al., 2017; Wan et al., 2013, 2015, 2018; Zeng et al., 2015). Hence, Arp2/3 complex-dependent actin retrograde flow could increase the mechanical tension on Ag-bound BCRs and amplify their signaling. Consistent with this idea, I observed that peak BCR signaling occurred during the first 5 min of APC encounter when BCR-Ag microclusters are undergoing the most significant centripetal movement and presumably the most tension through the BCR. Because many studies investigating the role of substrate stiffness on B cell responses were conducted using immobilized Ags, it will be important to investigate how substrate rigidity and ligand mobility act together to regulate B cell responses to membrane-bound Ags. If the BCR is mechanosensitive, then conditions that increase drag forces through the BCR should enhance receptor signaling. Understanding how the mechanical properties of BCR interactions with Ags that are mobile within cell membranes could suggest new ways to modulate BCR signaling output. This could lead to the development of novel therapeutics that modulate these properties in order to control aberrant B cell activation or enhance the B cell response to vaccination or infection.  4.3.6 Arp2/3 complex-dependent actin dynamics control Ag affinity and density thresholds For membrane-bound Ags, the magnitude of BCR signaling is dependent on Ag density and affinity. Therefore, a cytoskeletal-dependent positive feedback loop could reduce the threshold of Ag affinity and density required for inducing a B cell response. An important extension of the work in this chapter is to investigate how the actin cytoskeleton modulates (or ‘tunes’) these affinity and density thresholds. I hypothesize that inhibiting actin nucleation by the Arp2/3 complex disrupts this positive feedback loop and would therefore increase the Ag density and affinity required to trigger B cell activation. I observed that B cells lacking Arp2/3 complex activity can gather the same amount of Ag as control-treated cells. However, BCR signaling is reduced in these cells and B cell activation is impaired. This suggests that Arp2/3 complex-  155 dependent BCR organization is required for amplifying BCR signaling in response to membrane-bound Ag, effectively lowering the Ag density and affinity required for activation. It will be interesting to test whether impaired Arp2/3 complex activity would have a more dramatic effect at suboptimal Ag density or affinity.  4.3.7 Functions of the B cell cSMAC  I have shown that the Arp2/3 complex reduces BCR signaling especially during the first 5 minutes of Ag encounter. Moreover, I demonstrate that these early signaling Arp2/3 complex-regulated events are sufficient to drive the transcriptional responses that lead to B cell activation. This suggests that events preceding cSMAC formation are critical for inducing B cell activation responses. Although the cSMAC was originally thought to be a site of active signaling, more recent studies have challenged this idea. Depoil et al. showed that BCR signaling occurs only at the peripheral microclusters and not at the cSMAC (Depoil et al., 2008) and the loss of N-WASp in B cells, which blocks cSMAC formation, results in increased BCR signaling (Liu et al., 2013). In contrast to the findings of Depoil et al., I did observe BCR signaling at the cSMAC. However, the amount of pCD79 and pSyk per unit of gathered Ag was much lower at time points after cSMAC formation (e.g. 10-15 min after adding the B cells to the APC) than during the initial few minutes of B cell-APC contact. Hence, my findings may also support the idea that the B cell cSMAC is a site of signal attenuation. However, when I analyzed APC-induced phosphorylation of CD79 by immunoblotting total cell extracts that would include intracellular BCRs (Figure 4.4A), instead of by imaging only the B cell-APC contact site, I found that CD79 phosphorylation was greater at 15-30 min after initiating B cell-APC contact than at the 5 min time point when the cSMAC is just starting to form. Although both assays showed that inhibiting the Arp2/3 complex resulted in decreased CD79 phosphorylation, further analysis of the inter-relationship between cSMAC formation, BCR internalization, and BCR signaling would be informative.   In T cells, initial studies also suggested that the cSMAC was a site of signal attenuation (Mossman et al., 2005; Varma et al., 2006). However, it has been postulated that the termination of TCR signaling at the immune synapse only occurs in response to strong Ags (Cemerski et al., 2008). Lee et al. connected these seemingly contradictory findings using experimental results   156 and computational simulations to dissect the role of immune synapse formation in T cell signaling and activation (Lee et al., 2003). They found that the formation of the cSMAC facilitates full phosphorylation of TCRs by Lck and that only fully phosphorylated receptors were internalized at the cSMAC. Thus, the immune synapse could be an “adaptive controller” that either enhances or attenuates TCR signaling depending on Ag strength. My data suggest that the actin-driven coalescence of BCR-Ag microclusters is critical for the amplification of BCR signaling at the B cell-APC contact site but that this occurs mainly at early time points before BCR-Ag microclusters have coalesced into a cSMAC.  A major function of cSMAC formation may be to optimize BCR-mediated Ag acquisition, as the localized concentration of Ags facilitates Ag extraction, which is required to elicit T cell help. In B cells, Ag internalization is required for T cell help and maximal B cell activation. Ag internalization occurs primarily at large Ag clusters (Natkanski et al., 2013), and the cSMAC is thought to be a centralized site for efficient Ag internalization (Harwood and Batista, 2011). I did not test the contribution of the Arp2/3 complex to Ag extraction by B cells. However, it has recently been shown that the Arp2/3 complex assembles actin foci that interact with BCR microclusters to facilitate Ag internalization (Roper et al., 2019). In any case, the increased ability of large BCR-Ag microclusters to internalize Ag suggests that the Arp2/3 complex-dependent aggregation of BCR-Ag microclusters enhance both initial signaling by the BCR and its Ag acquisition function.  4.3.8 Is the Arp2/3 complex-nucleated actin network required for the humoral immune response? Consistent with my findings that Arp2/3 complex activity regulates both tonic BCR signaling and APC-induced B cell activation, upstream activators of the Arp2/3 complex are important regulators of B cell activation. Murine B cells lacking WASp, an NPF that activates the Arp2/3 complex, exhibit reduced cell spreading, BCR-Ag microcluster formation, and BCR-induced tyrosine phosphorylation in response to Ag-bearing lipid bilayers (Liu et al., 2011). The WASp-interacting protein WIP regulates the activity and subcellular distribution of WASp and also stabilizes existing actin filaments. Murine B cells lacking WIP have defective CD19-mediated PI3K signaling and antibody responses (Keppler et al., 2015). The Cdc42 GTPase   157 regulates WASp activation, and B-cell-specific loss of Cdc42 results in aberrant actin organization, diminished BCR signaling, and severe impairment in antibody production (Burbage et al., 2015). Thus, the Cdc42-WASp/WIP-Arp2/3 complex pathway of actin assembly is likely to be critical for actin-mediated regulation of BCR signaling and the B cell antibody response in vivo. To directly test this idea, B cell-specific deletion of a component of the Arp2/3 complex is required, as discussed in the next section.   4.3.9 Perspectives In line with its central role in many aspects of B cell function, human mutations that impair the activation of the Arp2/3 complex result in disease. Mutations in WASp or WIP result in WAS, an X-linked immunodeficiency disorder (Candotti, 2018). Similarly, missense mutations in the ARPC1B gene have recently been described as causing a WAS-like disease (Kahr et al., 2017). Although WAS is associated with increased susceptibility to infections, many patients also develop autoimmunity and B-cell malignancies (Candotti, 2018). My finding that the loss of Arp2/3 complex activity in B cells results in increased tonic BCR signaling but impaired APC-induced B cell activation may help explain this paradox. In resting B cells, Arp2/3 complex-dependent actin structures limit the clustering of BCRs to restrain signaling. Loss of Arp2/3 complex-dependent structures in resting cells could increase Ag-independent signaling and allow the spontaneous assembly of large BCR clusters that support Ag-independent signaling, as the case for the activated B cell-like subtype of DLBCL where such clusters deliver essential survival signals (Davis et al., 2010). In the context of Ag binding, Arp2/3 complex-dependent structures are required for organizing BCR clusters into spatial patterns that amplify BCR signaling. Indeed, I found that inhibiting Arp2/3 complex activity resulted in decreased BCR signaling and B cell activation in response to APC-bound Ags. Hence, the actin cytoskeleton both restrains spontaneous B cell activation and supports Ag-induced B cell activation  WASp, Cdc42, and WIP, all of which act upstream of the Arp2/3 complex, are critical for humoral immune responses in vivo (Burbage et al., 2015; Keppler et al., 2015). This suggests that the Arp2/3 complex is important for humoral immunity. Mutations that inactivate the Arp2/3 complex may have a more severe phenotype than mutations in Cdc42 or WASp because the   158 Arp2/3 complex can also be activated via Rac-dependent activation of the WAVE NPF, and perhaps by other NPFs. Investigating the role of the Arp2/3 complex in vivo is an important extension of the work discussed in this chapter. Although Arpc3 mutant mice are unable to develop past the blastocyte stage (Yae et al., 2006), Cre-lox technology has enabled tissue-specific deletion of the subunits of the Arp2/3 complex. Arp3 knockout in adult excitatory neurons alters dendritic spine development and maintenance (Kim et al., 2013). Similarly, cell type-specific knockout of Arp2 impairs integrin-dependent processes in macrophages (Rotty et al., 2017). Cre-lox-mediated deletion of a critical subunit of the Arp2/3 complex in mature B cells would allow us to directly test the hypothesis that Arp2/3 complex is important for the in vivo trafficking and activation of mature B cells. Mb1-Cre and CD19-Cre mice have been widely used for Cre-lox-mediated deletion of genes in B lineage cells and could be crossed to mice harboring a floxed allele of one of the Arp2/3 complex subunits. However, if the Arp2/3 complex is important for actin-dependent processes such as adhesion and migration, which are required for B cell development, loss of the Arp2/3 complex may prevent the development of mature B cells. To specifically study the role of the Arp2/3 complex in mature B cells would require use of inducible-Cre systems or Cre that is expressed under the control of a promoter that is only active in mature B cells. To do this, one could cross mice expressing floxed alleles of Arpc4 to Mb-1 mer-Cre-mer mice (Hobeika et al., 2018), which express a Cre-estrogen receptor fusion protein under the control of the Mb-1 promoter. Tamoxifen injection would initiate Cre-mediated deletion of floxed genes only in B cells. Alternatively, Arpc4-floxed mice could be crossed to CD21-Cre mice in which Cre is only expressed in transitional and mature B cells (Kraus et al., 2004). In the next paragraphs I discuss how one would analyze B cell development, activation, and in vivo function in mice in which the Arp2/3 complex is depleted only in mature B cells. First, it will be important to characterize the transitional and mature B cell subsets in wild type mice and in mice lacking the Arp2/3 complex. Immature transitional T1 B cells (B220+ CD21lo CD24hi) home to the spleen and differentiate into T2 B cells (B220+ CD21hi CD24hi CD23+). These cells then give rise to circulating follicular (B220+ CD23hi CD21int) B cells and non-circulating splenic marginal zone (B220+ CD23lo CD21hi) B cells (Melchers, 2015; Pillai et al., 2005). Marginal zone B cells are important first responders to blood-borne Ags and produce   159 natural Abs against conserved molecular patterns present on pathogens (Cerutti et al., 2013; Pillai et al., 2005). The development of marginal zone B cells requires these cells to migrate to the marginal sinus of the spleen, where they are retained (Pillai et al., 2005). Deletion of genes that are important for migration and adhesion such as Pyk2, myosin IIA, WASp, and WIP results in decreased numbers of marginal zone B cells (Curcio et al., 2007; Guinamard et al., 2000; Hoogeboom et al., 2018; Recher et al., 2012; Westerberg et al., 2012). Thus, I predict that Arpc4 knockout mice will lack marginal zone B cells. B-1 B cells might also be affected by loss of Arpc4. B-1 B cells play a key role in immune defense against microbial Ags, and their development is influenced by BCR signal strength in response to self Ags (Baumgarth, 2017). Thus, it will be important to quantify the numbers of the B-1a (B220+ CD11b+ CD5+) and B-1b (B220+ CD11b+ CD5-) subsets in the spleen and peritoneal cavity. B cells lacking both WASp and N-WASp have reduced numbers of B-1b cells in the peritoneum (Westerberg et al., 2012), as do B cells lacking myosin IIA (Hoogeboom et al., 2018). Finally, because serum Ig concentrations depend on the development of mature B cells subsets, we will also use commercial ELISAs to measure circulating titers of serum IgM, IgG, and IgA. T-independent and T-dependent humoral responses can be tested by challenging mice with the hapten 4-hydroxy-3-nitrophenyl (NP) conjugated to either Ficoll (NP-Ficoll; T-independent Ag) or keyhole limpet hemocyanin (NP-KLH; T-dependent Ag). Because interfering with the Arp2/3 complex disrupts Ag internalization in B cells (Roper et al., 2019) and cell migration in many cell types (Schaks et al., 2019), I predict that mice lacking Arpc4 in the B cell compartment will likely have severely impaired responses to T-dependent Ags. They may also have impaired responses to T-independent Ags. WAS patients have recurrent bacterial infections associated with impaired antibody responses to T-independent polysaccharide Ags (Ochs et al., 1980). Moreover, in WASp-deficient mice, both T-dependent and T-independent antibody responses are severely impaired, with the T-independent response being almost lost completely (Westerberg et al., 2005).  During the germinal center response, activated B cells compete for Ags displayed on follicular dendritic cells, which leads to affinity maturation, Ig class switching, and the development of long-lived plasma cells and memory cells. The germinal center response involves multiple rounds of B cell-APC contacts, B cell migration, and in the case of T-  160 dependent Ags, interactions with T follicular helper cells. All of these processes depend on remodeling of the actin cytoskeleton. Thus, loss of Arpc4 could impair the germinal center response. Indeed, in mice lacking both WASp and N-WASp, as well as mice lacking WIP, germinal center formation was impaired (Dahlberg et al., 2015; Keppler et al., 2015; Westerberg et al., 2012). To test the effect of Arpc4 deletion on the germinal center response, the percentage of B220+ B cells expressing the germinal center markers GL7 and CD95 can be assessed by flow cytometry on day 13 post immunization with NP-KLH. The resulting NP-specific antibody titers and Ig class switching can be assessed using ELISA. WASp and N-WASp double knockout B cells also exhibit impaired affinity maturation of the antibody response. To assess affinity maturation in B cells lacking Arpc4, one can measure the affinity of NP-specific B cells to BSA that is covalently coupled to 4 NP (NP4-BSA) or 26 NP (NP26-BSA) molecules. NP26-BSA binds to both high and low-affinity antibodies, whereas only high affinity antibodies will bind to NP4-BSA. The NP4/NP26 ratio of NP-specific antibodies is therefore a measure of affinity maturation. Based on my findings and those in mice lacking WASp and N-WASp, I predict that Arpc4 deletion in B cells would lead to a significantly impaired germinal center response that is associated with decreased production of high affinity antibodies.  If the Arp2/3 complex is found to be important for the humoral immune response, drugs that target B cell specific regulators of Arp2/3 complex activity could be potential therapeutics for the modulation of B cell responses. Because the Arp2/3 complex is ubiquitously expressed and important for a wide range of biological functions, directly targeting the Arp2/3 complex is not practical for therapeutic purposes. However, drugs that selectively target regulators of this pathway that are highly expressed in B cells could be used to limit B cell activation in B cell-mediated autoimmune diseases, inflammatory diseases, and allergy. The recent developments of novel drug delivery systems, such as liposomes, nanoparticles, and SynNotch T cells, could allow B cell-specific delivery of drugs that modulate cytoskeletal dynamics. Liposomes and nanoparticles, which can be loaded with therapeutics and decorated with targeting ligands are now approved for clinical use, mostly for the delivery of anti-cancer drugs (Pattni et al., 2015; Wang et al., 2012). Recently, CD22 and CXCR4-targeted liposomes have been developed for the treatment of CLL (Boons, 2010; McCallion et al., 2019). SynNotch T cells have recently been developed to modulate immune cell responses and specifically deliver therapeutic payloads   161 (Roybal et al., 2016). In this system, T cells are engineered to express the SynNotch receptor, which consists of a customizable extracellular ligand-binding domain, the core regulatory domain of the Notch receptor (which is cleaved upon receptor ligation), and a customizable cytoplasmic domain that controls transcriptional responses (which is released upon cleavage of the Notch regulatory domain). SynNotch T cells specific for CD19-expressing cells have been developed (Roybal et al., 2016). Upon CD19 binding, these T cells can deliver custom payloads such as cytokines, cytotoxic proteins, immunoregulatory molecules and even antibodies. Using these approaches, it may be possible to selectively deliver therapeutics targeting the Arp2/3 complex pathway in order to modulate the B cell response in disease.       162 Chapter 5: The role of actin disassembly factors in the B cell spreading response, BCR microcluster organization and BCR signaling  5.1 Introduction B cell activation depends on the magnitude of BCR signaling in response to Ag. Remodeling of the actin cytoskeleton facilitates B cell spreading and the aggregation of BCR-Ag microclusters, processes that initiate BCR signaling in response to APC-bound Ag. In chapters 3 and 4, I demonstrated that Arp2/3 complex-dependent actin dynamics are essential for BCR-Ag microcluster aggregation, amplification of BCR signaling at the immune synapse, and B cell activation in response to APC-bound Ags. However, the rapid actin polymerization that drives cell spreading also requires the activity of actin disassembly factors that generate new actin monomers to fuel polymerization. Thus, actin polymerization by the Arp2/3 complex occurs concurrently with the actions of actin disassembly factors such as ADF/cofilin and together these proteins drive actin remodeling at the B cell immune synapse. Actin disassembly factors are likely also important for B cells to extend and retract lamellipodia as well as actin retrograde flow, BCR microcluster centralization, and BCR signaling in response to APC-bound Ags. The ADF/cofilin family of proteins consists of small actin-binding proteins that were first discovered for their ability to bind to (cofilin is short for co-filamentous protein) and sever actin filaments (Nishida et al., 1984). Unicellular eukaryotes such as yeast have one ADF/cofilin protein whereas most mammals express three isoforms: ADF (also known as destrin), cofilin-1, and cofilin-2 (Lappalainen et al., 1998; Vartiainen et al., 2002). Cofilin-1, herein referred to as cofilin, is ubiquitously and is most abundantly expressed in non-muscle cells. ADF is less abundant but enriched in epithelial and brain tissues whereas cofilin-2 is a muscle-specific isoform (Vartiainen et al., 2002). Although some studies have identified specific functions for these isoforms, their individual roles in actin dynamics are not well defined (Rochelle et al., 2013; Vartiainen et al., 2002; Wang et al., 2016). Cofilin promotes actin disassembly by severing filaments as well as actin polymerization by freeing actin monomers for incorporation into filaments. Thus, cofilin is critical for actin dynamics.  The activity of cofilin depends on its ability to bind to the side of actin filaments. Phosphorylation of serine 3 of cofilin prevents this interaction by inhibiting the ability of cofilin   163 to bind actin filaments (Bravo-Cordero et al., 2013). Phosphorylation of cofilin at this site is mediated by LIM kinase (LIMK) 1/2 (Arber et al., 1998; Yang et al., 1998), testicular protein kinases (TESKs) (Toshima et al., 2001a, 2001b), and Nck-interacting kinase (NRK) (Nakano et al., 2003). TESKs are predominantly expressed in the testis (Toshima et al., 1995) and NRK is predominantly expressed in skeletal muscle (Kanai-Azuma et al., 1999). Thus LIMK 1 and 2 are likely the most important upstream negative regulators of cofilin in B cells. LIMKs are activated via phosphorylation by the Rho-associated protein kinase (ROCK), which in turn is activated by the Rho GTPase, or by p21-activated kinase (PAK), which is activated by Rac or Cdc42 (Scott and Olson, 2007). Dephosphorylation of cofilin causes a conformational change that allows cofilin to bind actin filaments and carry out its severing activity. The primary phosphatases that regulate this process belong to the slingshot (SSH) family (Kanellos and Frame, 2016; Niwa et al., 2002). SSH binds to actin filaments, which enhances its capacity to activate cofilin (Nagata-Ohashi et al., 2004). Moreover, the phosphatase activity of SSH and therefore its ability to dephosphorylate cofilin is increased by PI3K in response to extracellular stimuli (Nishita et al., 2004, 2005). Cofilin activity is also modulated downstream of Ca2+ signaling in cells, which activates both SSH (via calcineurin) (Pandey et al., 2007; Wang et al., 2005; Zhang et al., 2012) and LIMK (via Ca2+/calmodulin‐dependent protein kinase (CaMK) II and IV) (Saito et al., 2013; Takemura et al., 2009). The complexity of the cofilin activation networks is highlighted by the observation that SSH can also interact with LIMKs, inactivating them, and further enhancing cofilin activation (Soosairajah et al., 2005). Cofilin activity is also regulated by the membrane lipid PIP2 in a pH-dependent manner. PIP2 binds to cofilin in the actin-binding site, inhibiting the capacity of cofilin to bind to actin filaments. Cofilin clusters PIP2 at the plasma membrane and changes in PIP2 membrane concentration can regulate membrane-proximal cofilin activity (Zhao et al., 2010). Extracellular signals that cause the hydrolysis of PIP2 promote the release of cofilin from its inhibitory interaction with PIP2, which stimulates localized membrane protrusions and controls the direction of migration (Mouneimne et al., 2004, 2006; van Rheenen et al., 2007). Cofilin activity can also be regulated by pH. The inhibitory interaction between PIP2 and cofilin is pH sensitive, with decreased PIP2-cofilin binding at higher pH. Thus, higher pH increases the local release and activation of cofilin (Frantz et al., 2008; Zhao et al., 2010). Cofilin-mediated severing of actin   164 filaments is also sensitive to pH, with greater severing at higher pH (Yeoh et al., 2002). Moreover, pH can regulate the interaction between cofilin and cortactin. Cortactin inhibits cofilin activity, and at high pH cofilin is released from cortactin inhibition (Magalhaes et al., 2011). Recently, other proteins that enhance cofilin-mediated actin disassembly have been identified, including AIP1/Wdr1 (Chen et al., 2015; Gressin et al., 2015; Jansen et al., 2015; Kueh et al., 2008; Nadkarni and Brieher, 2014; Nomura et al., 2016; Rodal et al., 1999; Shi et al., 2013), MICAL (Grintsevich et al., 2016, 2017), coronins (Jansen et al., 2015; Kueh et al., 2008; Mikati et al., 2015), twinfilin (Johnston et al., 2015) and CAP (Johnston et al., 2015; Kotila et al., 2019). AIP1/Wdr1 (herein referred to as Wdr1) is particularly important for immune function as highlighted by the severe immunodeficiency disease caused by loss-of-function mutations in Wdr1, which is characterized by defective adhesion and motility of neutrophils and monocytes as well as aberrant T and B cell activation (Pfajfer et al., 2018). Wdr1 binds to cofilin-decorated actin filaments and increases the rate of cofilin-mediated actin severing (Chen et al., 2015; Gressin et al., 2015; Jansen et al., 2015; Kueh et al., 2008; Nadkarni and Brieher, 2014; Nomura et al., 2016; Rodal et al., 1999; Shi et al., 2013). Currently, there are two models for how Wdr1 enhances cofilin-mediated severing. In the first, Wdr1 competes with cofilin for filament binding sites, creating boundaries between cofilin-decorated and bare regions. This causes the accumulation of strain at these boundaries, leading to severing (Elam et al., 2013; Gressin et al., 2015). Alternatively, Wdr1 could disrupt the interaction between cofilin and actin filaments in a way that enhances severing (Aggeli et al., 2014).  In B cells, cofilin activity is required for B cell spreading, microcluster formation, and BCR signaling (Freeman et al., 2011). BCR-mediated dephosphorylation and activation of cofilin requires signaling through the Rap GTPase (Freeman et al., 2011). Because cofilin activity drives the cytoskeletal reorganization events that are critical for immune synapse formation, it is likely to be a key node in the BCR signaling pathway that controls APC-induced B cell activation. As such, cofilin-regulatory proteins such as Wdr1 and LIMK may be prime targets for modulating B cell activation. Indeed, both B and T cells from patients with Wdr1 mutations exhibit aberrant spreading (Pfajfer et al., 2018). Moreover, the Rho-ROCK-LIMK pathway controls immune synapse formation and Ca2+ signaling in T cells (Thauland et al., 2017). In this system, inhibiting cofilin activity by expressing constitutively active ROCK or by   165 depleting cofilin with siRNA results in smaller immune synapses and defective Ca2+ release. Conversely, inhibiting ROCK activity, which increases the amount of active cofilin, results in larger immune synapses with increased Ca2+ release. Given the role of cofilin and its regulators in controlling actin dynamics in lymphocytes, in this chapter I tested the hypothesis that actin severing mediated by the Wdr1-LIMK-cofilin axis is required for the regulation of immune synapse formation, APC-induced BCR signaling, and cSMAC formation.       166 5.2 Results   5.2.1 Wdr1 and LIMK regulate cofilin activity in B cells To investigate the role of actin disassembly at the B cell immune synapse, we used three approaches to modulate cofilin activity in B cells (Figure 5.1A). To reduce cofilin activity directly, we used siRNA to deplete cofilin expression in A20 B cells. We also depleted the expression of Wdr1 in A20 B cells using siRNA. Because Wdr1 enhances cofilin activity, we predicted that Wdr1 depletion would decrease cofilin activity in B cells. Immunoblotting showed that both cofilin and Wdr1 expression were dramatically reduced compared to cells transfected with a control siRNA (Figure 5.1B,C). To enhance cofilin activity, we inhibited LIMK using the pharmacological inhibitor, LIMKi3.  To test whether depleting Wdr1 expression or inhibiting LIMK altered cofilin activity, we assessed the BCR-induced dephosphorylation (activation) of cofilin in B cells expressing control or Wdr1 siRNA and in B cells treated with LIMKi3. In A20 B cells that were transfected with control siRNA, stimulation with soluble anti-IgG resulted in a transient decrease in the level of the phosphorylated inactive form of cofilin (p-cofilin) (Figure 5.1D,E). This is consistent with our previous findings that BCR stimulation induces dephosphorylation and activation of cofilin, which increases the mobility of BCRs and enhances BCR signaling (Freeman et al., 2011, 2015). We found that A20 B cells transfected with Wdr1 siRNA (Figure 5.1D,E), as well as A20 B cells and primary murine B cells treated with LIMKi3 (Figure 5.1F,G), consistently exhibited very low levels p-cofilin, even though total cofilin levels remained unchanged.  We then sought to understand how the decreased p-cofilin levels observed in Wdr1-depleted B cells affected actin networks in resting B cells. To address this, we compared the concentration of intracellular actin filaments in A20 B cells expressing control and Wdr1 siRNA. Although there was an increase the amount of dephosphorylated (active) cofilin, Wdr1-depleted A20 B cells had higher levels of actin filaments than control cells, as judged by phalloidin staining (Figure 5.2). This supports the idea that Wdr1 promotes actin severing. The increased levels of active cofilin in Wdr1-depleted cells could reflect the fact that SSH is activated by filamentous actin (Nagata-Ohashi et al., 2004), which makes cofilin activation sensitive to the cellular concentrations of actin filaments.    167      168 Figure 5.1 Wdr1 and LIMK regulate cofilin activity in B cells  Cofilin activity in murine B cells was modulated depleting either cofilin or Wdr1 or by inhibiting LIMK (A). A20 B cells were transfected with control siRNA or with either cofilin siRNA (B) or Wdr1 siRNA (C). Cell extracts were analyzed by immunoblotting for cofilin and total actin (loading control) (B) or for Wdr1 and the L chain of IgG (loading control) (C). Representative results from 3 independent experiments are shown. (D,E) A20 B cells were transfected with control siRNA or Wdr1 siRNA and stimulated with 20 µg/ml soluble anti-IgG for the indicated times. p-cofilin and total cofilin immunoblots are shown (D). The p-cofilin/total cofilin ratios were normalized to the ratio at time = 0 and the mean ± SEM from three independent experiments are graphed (E). (F-I) A20 B cells (F,G) or primary murine B cells (H,I) were pre-treated for 1 hr with DMSO, 50 µM LIMKi3 (A20 B cells) or 1 µM (primary B cells) LIMKi3 before being stimulated with 20 µg/ml soluble anti-IgG for the indicated times. p-cofilin and total cofilin immunoblots are shown (F,H). The p-cofilin/total cofilin ratios were normalized to levels at time = 0 and the mean ± SEM from three independent experiments are graphed (G,I).      169    Figure 5.2 Wdr1 siRNA knockdown increases cellular filamentous actin levels Untransfected A20 cells or A20 B cells that were transfected with control or Wdr1 siRNA were stained with rhodamine-phalloidin to detect filamentous actin. Intracellular filamentous actin levels were then quantified by flow cytometry. Dashed curves represent unstained B cells. Filled curves represent phalloidin-stained B cells. Representative data from one of two independent experiments are shown.      170 5.2.2 The Wdr1-LIMK-cofilin pathway is important for B cell spreading on immobilized anti-Ig Because BCR-Ag microcluster aggregation and BCR signaling is regulated by the Arp2/3 complex-dependent nucleation of branched actin, we asked whether cofilin-mediated actin severing was important for regulating actin remodeling at Ag contact sites. When A20 B cells were added to anti-IgG coated coverslips, they extended broad lamellipodia-like protrusions that are dependent on the Arp2/3 complex (see chapter 3, Figure 3.10). This system mimics the initial stages of B cell Ag encounter and provides a simple readout for branched actin dynamics. Previously, our lab has shown that inhibiting cofilin with cell-permeable cofilin-blocking peptides impairs B cell spreading on anti-Ig coated coverslips (Freeman et al., 2011). Consistent with this, A20 B cells transfected with cofilin siRNA exhibited a significantly reduced cell spreading area than control siRNA transfected B cells (Figure 5.3A,B). After 15 and 30 min of contact with anti-IgG-coated coverslips, control A20 B cells had a dense ring of branched actin at the periphery of the substrate contact site that surrounded a region of hypodense actin (Figure 5.3A). In A20 B cells expressing cofilin siRNA, the peripheral actin ring did not encircle a central actin-depleted region. Instead, we observed an accumulation of actin arc-like structures at the center of the contact site (Figure 5.3A). To quantify the effect of cofilin depletion on actin organization at the contact site, we calculated the percent of the total cell area that was depleted of actin filaments after 30 min of cell spreading. Control siRNA-expressing A20 B cells cleared actin from ~30% of the total cell-substrate contact area. In contrast, cofilin siRNA-expressing A20 B cells cleared actin from only ~10% of the total cell area. Similarly, A20 B cells transfected with Wdr1 siRNA exhibited a significantly impaired spreading response at 10, 15, and 30 min after addition to anti-IgG coated coverslips (Figure 5.3D,E) with reduced actin clearance at the center of the contact site after 30 min of spreading. Thus, depletion of either cofilin or Wdr1 results in decreased cell spreading, reduced actin clearance, and an impaired ability to form a distinct ring of peripheral actin. This indicates that both cofilin and Wdr1 are important for the spreading-associated actin remodeling that is initiated by BCR signaling. Moreover, the similar phenotypes resulting from depletion of cofilin and Wdr1 are consistent with the idea that Wdr1 is required for the cofilin-mediated severing of actin filaments.   171 We next asked whether enhancing cofilin activity by inhibiting its negative regulator, LIMK, would alter B cell spreading on immobilized anti-IgG. In T cells, inhibiting the signaling pathways that activate LIMK, and thereby increasing cofilin activity, results in an increased immune synapse area (Thauland et al., 2017). This presumably increases actin turnover and the liberation of actin monomers, which supports actin polymerization at the cell periphery. Thus, we hypothesized that inhibiting LIMK activity in B cells would increase cell spreading area. Surprisingly, pre-treating A20 B cells with LIMKi3 significantly reduced B cell spreading compared to DMSO-treated A20 B cells (Figure 5.3G,H). However, in contrast to cofilin depletion or Wdr1 depletion, LIMKi3-treated B cells exhibited a thinner ring of peripheral actin (Figure 5.3H,I). This could be a consequence of increased actin severing at the inner face of the peripheral actin ring. Taken together, these results indicate that LIMK activity regulates BCR-induced actin remodeling and that cofilin activity must be finely tuned to maximize the branched actin polymerization that drives B cell spreading.  To visualize how modulating cofilin, Wdr1, or LIMK modulates the spreading response in real time, A20 B cells expressing F-Tractin-GFP were added to anti-IgG coated coverslips and imaged using TIRFM. As in Figure 5.3, depletion of either cofilin or Wdr1 resulted in decreased cell spreading compared to control siRNA-transfected cells (Figure 5.4A). Similarly, inhibition of LIMK resulted in decreased cell spreading (Figure 5.4B). Kymograph analysis revealed that cofilin and Wdr1 depletion, as well as LIMK inhibition, resulted in impaired actin dynamics such that the peripheral actin network was static and the retrograde actin flow was largely ablated, as compared to control cells (Figure 5.4A,B). Taken together, these data show that interfering with the LIMK-cofilin-Wdr1 regulatory network results in abnormal peripheral actin structures and actin dynamics in B cells spreading on anti-Ig-coated coverslips.        172      173 Figure 5.3 The Wdr1-LIMK-cofilin network regulates B cell spreading on immobilized anti-Ig A20 B cells were transfected with control siRNA or cofilin siRNA (A-C), transfected with control siRNA or Wdr1 siRNA (D-F), or pre-treated with DMSO or 50 µM LIMKi3 for 1 hr (G-I). Cells were then allowed to spread on anti-IgG-coated coverslips for the indicated times before being fixed, stained with rhodamine-phalloidin, and imaged by confocal microscopy (A,D,G). Representative images are shown. Scale bars: 10 µm. In (B, E, H) the cell area was quantified using the actin staining to define the cell edge. In the beeswarm plots each dot represents one cell and the median (blue line) and interquartile ranges (black box) for >30 cells are shown for each time point. Representative data from one of four (B), three (E), or six (H) independent experiments. p-values were determined using the Mann-Whitney U test. In (C, F, I) the percent of the total cell area at the substrate contact site that was cleared of filamentous actin was quantified using actin staining to define both the outer cell edge and the central region of the cell in which actin was depleted. Bar graphs represent the median of three (C), four (F), and six (I) independent experiments. Two-tailed paired t-test.      174   Figure 5.4 The Wdr1-LIMK-cofilin network is important for BCR-induced actin dynamics A20 B cells expressing F-Tractin-GFP were transfected with control siRNA, cofilin siRNA, or Wdr1 siRNA (A), or pre-treated with DMSO or 50 µM LIMKi3 for 1 hr (B). Cells were then added to anti-IgG-coated coverslips and imaged by TIRFM at 1 s intervals for 10 min. The top panels are the final frames from Video 11 (control siRNA), Video 12 (cofilin siRNA), Video 13 (Wdr1 siRNA), Video 14 (DMSO-treated), and Video 15 (LIMKi3-treated). The middle panels are kymographs along the yellow lines in the top panels. In the bottom panels, the cell edge in each frame, as defined by actin, was overlaid as a temporally-coded time series. Scale bars: 5 µm.       175 Video 11 Peripheral actin dynamics in control siRNA-expressing B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were transfected with control siRNA and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRFM. Images taken every 1 s for 10 min. Video playback is 60 frames per second (60X real speed). See also Figure 5.4.  Video 12 Peripheral actin dynamics in cofilin siRNA-expressing B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were transfected with cofilin siRNA and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRFM Images taken every 1 s for 10 min. Video playback is 60 frames per second (60X real speed). See also Figure 5.4.  Video 13 Peripheral actin dynamics in Wdr1 siRNA-expressing B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were transfected with Wdr1 siRNA and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRFM. Images taken every 1 s for 10 min. Video playback is 60 frames per second (60X real speed). See also Figure 5.4.  Video 14 Peripheral actin dynamics in DMSO-treated B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were pre-treated for 1 hr with DMSO and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRFM. Images taken every 1 s for 10 min. Video playback is 60 frames per second (60X real speed). See also Figure 5.4.  Video 15 Peripheral actin dynamics in LIMKi3-treated B cells plated on immobilized anti-IgG A20 B cells expressing F-Tractin-GFP were pre-treated for 1 hr with 50 µM LIMKi3 and then added to anti-IgG-coated coverslips. The contact site was imaged using TIRFM. Images taken every 1 s for 10 min. Video playback is 60 frames per second (60X real speed). See also Figure 5.4.      176 5.2.3 Cofilin-mediated actin disassembly is important for actin organization at the immune synapse To further investigate the altered spreading morphology that we observed when cofilin activity was modulated, we used STED microscopy. When control cells were adding to anti-IgG coated coverslips, protrusions containing branched actin networks, as well as a central actin-depleted region, was first observed at 5 min. By 10 min, the B cells had assembled a thick peripheral ring of branched actin that gave way to linear filaments running parallel to the inner face of this ring. These linear filaments formed actin arcs that surrounded the actin-depleted region at the center of the contact site (control cells in Figure 5.5A,B,C). As observed in Figure 5.3, A20 B cells expressing cofilin siRNA were unable to clear actin from the center of the contact site. Instead, many of these cells accumulated concentric actin rings at the center of the contact site (Figure 5.5A). Wdr1-depleted A20 B cells also exhibited defective actin clearance at the center of the contact site (Figure 5.5B). However, these cells did not organize actin into concentric actin arcs at the center of the contact site as in cofilin-depleted B cells. Conversely, A20 B cells treated with the LIMK inhibitor exhibited large actin-depleted regions and thin peripheral actin rings. Interestingly, depleting cofilin or its co-activated Wdr1, as well as increasing cofilin activity by inhibiting LIMK, delayed the ability of the cells to form the peripheral ring of branched actin. Compared to control cells, the peripheral actin structures were disorganized at the earlier time points in cells in which the Wdr1-LIMK-cofilin network was perturbed and this was associated with decreased cell spreading. Thus, properly regulated cofilin activity is important for establishing the peripheral branched actin structures that promote the spreading of B cells on anti-Ig coated coverslips.      177    178 Figure 5.5 The Wdr1-LIMK-cofilin network shapes the actin architecture at the Ag contact site A20 B cells were transfected with control siRNA or cofilin siRNA (A-C), control siRNA or Wdr1 siRNA (D-F), or pre-treated with DMSO or 50 µM LIMKi3 for 1 hr (G-I). The cells were then allowed to spread on anti-IgG-coated coverslips for the indicated times before being fixed, stained for actin, and imaged by STED microscopy. Representative images are shown. Scale bars: 5 µm.     5.2.4 Cofilin is important for APC-induced BCR signaling and cSMAC formation  In chapters 3 and 4, I demonstrated that Arp2/3 complex-dependent actin dynamics at the B cell immune synapse is required for the aggregation of BCR-Ag microclusters and amplification of BCR signaling. Because actin turnover is required to fuel Arp2/3 complex-dependent actin polymerization, we next asked whether actin disassembly mediated by cofilin is important for BCR signaling and cSMAC formation in response to APC-bound Ag. When control siRNA-expressing A20 D1.3 B cells were added to COS-7 APCs expressing mHEL-HaloTag, BCR-Ag microclusters formed rapidly at the B cell-APC contact site and co-localized with pCD79 clusters (Figure 5.6A). The BCR-Ag microclusters then coalesced into a cSMAC within 10 min (Figure 5.6B). To investigate the relationship between the amount of Ag gathered into clusters and the signaling output at those BCR-Ag microclusters, the total pCD79 fluorescence intensity present in clusters at the B cell-APC interface was quantified and divided by the total fluorescence intensity of clustered Ag for each B cell. Control cells exhibited maximal pCD79 per unit of clustered Ag at 3 min after the B cells were added to the APCs, with BCR signaling output declining thereafter (Figure 5.6C). This is consistent with the findings presented in chapter 4. In an initial experiment, when cofilin was depleted using siRNA the B cells were still able to form BCR-Ag microclusters at the B cell-APC contact site but cSMAC formation was impaired (Figure 5.6A,B). Cofilin depletion did not significantly reduce the amount of Ag gathered into microclusters (Figure 5.6C). However, in this initial experiment I found that depleting cofilin significantly reduced the amount of BCR signaling generated per unit of Ag that was gathered into clusters (Figure 5.6D). These findings suggest that cofilin-mediated actin network disassembly impacts the coalescence of BCR-Ag microclusters, which is important for amplifying proximal BCR signaling in response to membrane-bound Ags.    179 These findings are consistent with previous findings from our laboratory that showed that APC-induced BCR-Ag microcluster formation and microcluster-based phosphotyrosine signaling were significantly reduced in A20 B cells expressing cofilin S3D, a phosphomimetic mutant that may act as a dominant negative form of cofilin (Freeman et al., 2011). Expressing SSH-CS, a catalytically inactive form of SSH, which may act as a dominant negative and prevent cofilin activation, also inhibited BCR-Ag microcluster formation and BCR signaling (Freeman et al., 2011). However, because the mechanisms by which cofilin S3D and SSH-CS impact the function of endogenous cofilin is not clear, siRNA-mediated depletion of cofilin provides more direct evidence for a role in BCR organization and signaling at the immune synapse.     180    181 Figure 5.6 Cofilin is important for BCR-Ag microcluster organization and amplification of proximal BCR signaling A20 D1.3 B cells were transfected with control siRNA or cofilin siRNA and then added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) The percent of cells for which > 90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed. (C) The total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. (D) For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. All data are from a single experiment. n >30 cells for each time point. Mann-Whitney U test was used to calculate p values.      182 5.2.5 The cofilin regulatory proteins Wdr1 and LIMK are important for BCR-Ag microcluster organization and proximal BCR signaling   Because cofilin is important for BCR-Ag microcluster organization and BCR signaling in response to membrane-bound Ags, I investigated whether the cofilin regulators Wdr1 and LIMK also regulate these responses. Again, control A20 D1.3 B cells that were added to COS-7 APCs expressing mHEL-HaloTag rapidly formed BCR-Ag microclusters that coalesced into a cSMAC after 10 to 15 min (Figure 5.7A,B, Figure 5.8A,B, Figure 5.9A). Wdr1-depleted B cells were able to form BCR-Ag microclusters and cSMACs and gathered a similar amount of Ag into microclusters as control cells (Figure 5.7A,B,C). However, the amount of pCD79 generated per unit of Ag that was gathered into clusters was significantly reduced in Wdr1-depleted B cells compared to the cells transfected with control siRNA (Figure 5.7D,E). In A20 D.13 B cells that were treated with the LIMK inhibitor, LIMKi3, cSMAC formation at 15 and 30 min after APC encounter was reduced (Figure 5.8A,B). Moreover, these cells exhibited significantly reduced amounts of clustered pCD79 per unit of clustered Ag (Figure 5.8,C,D). Similar results were obtained when the HEL-specific primary B cells from MD4 mice were added to COS-7 APCs expressing the mHEL-HaloTag Ag (Figure 5.9).     183    184 Figure 5.7 Wdr1 is important for BCR-Ag microcluster organization and amplification of proximal BCR signaling in A20 D1.3 B cells A20 D1.3 B cells were transfected with control siRNA or Wdr1 siRNA and then added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) The percent of cells for which > 90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed. (C) The total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. (D) For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. All data are from a single experiment. n >25 cells for each time point. Mann-Whitney U test was used to calculate p values.     185    186 Figure 5.8 LIMK activity is important for BCR-Ag microcluster organization and for amplification of proximal BCR signaling in A20 D1.3 B cells A20 D1.3 B cells were pre-treated with DMSO or 50 µM LIMKi3 for 1 hr and then added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) The percent of cells for which > 90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed. (C) The total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. (D) For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. All data are from a single experiment. n >24 cells for each time point, except for the control siRNA 3 min time point where n=8. Mann-Whitney U test was used to calculate p values.      187  Figure 5.9 LIMK activity is important for BCR-Ag microcluster organization and amplifying proximal BCR signaling in B cells from MD4 mice Primary murine B cells from MD4 mice were pre-treated with DMSO or 50 µM LIMKi3 for 1 hr and then added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) The percent of cells for which > 90% of the total Ag fluorescence intensity was contained in one or two clusters is graphed. (C) The total fluorescence intensity of Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. (D) For each B cell, the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag at the B cell-APC contact site. Each dot is one cell. The median (blue line) and interquartile ranges (black box) are shown. All data are from a single experiment. n >30 cells for each time point. Mann-Whitney U test was used to calculate p values.    188 5.3 Discussion  5.3.1 Summary of findings In vivo, B cells are most effectively activated by Ags presented on the surface of APCs (Batista and Harwood, 2009). The formation of an immune synapse is critical for amplifying BCR signaling in response to APC-bound Ags and facilitates the internalization of BCR-bound Ags, which is critical for B cell activation. Immune synapse formation requires extensive remodeling of the actin cytoskeleton, including Arp2/3 complex actin polymerization that drives B cell spreading. Actin polymerization must be coupled to depolymerization to maintain an actin monomer concentration that supports polymerization (Carlier and Shekhar, 2017; Krause and Gautreau, 2014). Moreover, the severing of the cortical actin cytoskeleton is a rate-limiting step in the morphological changes that occur during immune synapse formation. Previously, our lab showed that cofilin is critical for the severing of the cortical actin cytoskeleton in response to BCR stimulation (Freeman et al., 2011). Importantly, inhibition of cofilin impairs BCR-induced spreading, immune synapse formation, and MTOC polarization (Freeman et al., 2011; Wang et al., 2017). Hence, we hypothesized that cofilin and cofilin-regulating proteins such as Wdr1 and LIMK could modulate BCR microcluster formation and IS formation during APC-induced B cell activation. We found that cofilin, Wdr1, and LIMK are all important for the assembly of peripheral actin structures that drive B cell spreading and that they contribute to the amplification of APC-induced BCR signaling and support the centralization of BCR-Ag microclusters into a cSMAC.  5.3.2 Regulation of cofilin by phosphorylation   Cofilin activity is controlled by phosphorylation on serine 3, a key residue in the actin-binding face. Phosphorylation of this serine residue inhibits actin binding, thus reducing the actin severing activity of cofilin (Bravo-Cordero et al., 2013). The activity of cofilin is therefore controlled by kinases (LIMK) and phosphatases (SHH) that modulate the phosphorylation state of this site. The binding of Wdr1 to actin filaments enhances cofilin-mediated severing, and accordingly we found that depleting Wdr1 led to increased filamentous actin in B cells. However, surprisingly, depleting Wdr1 decreased the levels of phosphorylated (inactive) cofilin in B cells. Because the amount of cofilin was unchanged, this indicates that Wdr1 depletion   189 increased the amount of active, unphosphorylated cofilin. Similar observations have been reported in developing zebrafish neutrophils where Wdr1 depletion results in constitutive activation of cofilin but the accumulation of actin filaments (Bowes et al., 2019). Although these findings seem paradoxical, they can be explained by the complex regulatory networks that control the activity of actin-binding proteins. Actin filaments are stabilized by saturating concentrations of cofilin (Andrianantoandro and Pollard, 2006; Gressin et al., 2015; Nadkarni and Brieher, 2014). Cofilin-mediated severing is caused by the destabilization of monomer interaction at the boundaries between cofilin-decorated and cofilin-bare regions (Elam et al., 2013; Nadkarni and Brieher, 2014). By creating cofilin-bare regions on actin filaments, Wdr1 enhances cofilin activity (Nadkarni and Brieher, 2014). Thus, reducing Wdr1 expression results in cofilin saturation and stabilization of actin filaments. The activity of the cofilin phosphatase, SSH, is increased by its binding to actin filaments. Therefore, increasing the cytoplasmic concentration of actin filaments increases actin-dependent SSH activity, resulting in enhanced cofilin dephosphorylation (activation). In line with this idea, our lab has shown that treating B cells with the actin stabilizing drug jasplakinolide results in dramatic cofilin dephosphorylation (Lei, 2010). Conversely, reducing the amount of filamentous actin in B cells via treatment with latrunculin A leads to increased cofilin phosphorylation (Lei, 2010). These findings illustrate the complexity of the regulatory networks that define the size and architecture of actin structures in B cells, highlighting feedback loops in which cells can respond to changes in filamentous actin concentration by regulating the activity of cofilin.   5.3.3 Actin disassembly factors control actin organization and dynamics in B cells Cofilin activity is required for coupled actin filament disassembly and polymerization in vitro (Carlier et al., 1997) and controls the size and shape of actin protrusions in cells (Delorme et al., 2007; Ghosh et al., 2004; Hotulainen et al., 2005). When cofilin is locally activated at the leading edge of a lamellipod, an actin protrusion is generated at that site (Ghosh et al., 2004). Globally increasing cofilin activity leads to a broadening of the leading edge in migrating cells (Delorme et al., 2007) and cofilin expression is critical for cell motility (Hotulainen et al., 2005). Cofilin is thought to contribute to the generation of actin protrusions that are required for cell motility in two ways. First, cofilin-mediated severing generates new barbed ends, at which actin   190 elongation can occur (Ghosh et al., 2004). Second, cofilin-mediated severing replenishes the pool of actin monomers within cells (Hotulainen et al., 2005). This requires Wdr1 to optimize the spacing of cofilin molecules on actin filaments such that filament severing is favored (Okreglak and Drubin, 2010). Thus, both cofilin activity and Wdr1 are likely to be important for driving B cell spreading. Indeed, we found that the LIMK-cofilin-Wdr1 network is important for B cells to spread on anti-Ig coated coverslips. Depleting or inhibiting any of these proteins resulted in reduced cell spreading, supporting the idea that actin turnover is required for the B cell spreading response. This finding is consistent with our previous findings that interfering with cofilin activity or function by expressing the phosphomimetic mutant (inactive) form of cofilin or by expressing an inactive form of SSH (which cannot dephosphorylate and activate cofilin) abrogates B cell spreading (Freeman et al., 2011).  Modulating cofilin activity by targeting Wdr1 or LIMK also led to changes in the actin architecture at the B cell-coverslip contact site. Cofilin-depleted B cells were unable to clear actin from the center of the contact site. A similar disruption of actin organization was observed in A20 B cells expressing the dominant-negative form of SSH (Freeman et al., 2015). This is consistent with findings in developing neurons, which adopt a similar shape as a spreading B cell with a dense actin network at the periphery and an actin-depleted region in the center (Flynn et al., 2012). Cofilin deficiency leads to “congestion of the intracellular space” and impaired neurite outgrowth. Similarly, localized inactivation of cofilin in the lamellipodia of neuronal cells results in the expansion of the peripheral actin ring (Vitriol et al., 2013) that results from decreased filament disassembly, as opposed to an increase in actin polymerization.  Wdr1-depleted B cells exhibited a similarly impaired spreading phenotype as cofilin-depleted B cells, with reduced actin clearance at the center of the contact site. In neutrophils of developing zebrafish, Wdr1 depletion results in an accumulation of actin filaments (Bowes et al., 2019). In both of these systems, Wdr1 depletion results in an increase in active cofilin, which likely stabilizes actin filaments. Thus, Wdr1 depletion inhibits cofilin-mediated severing and phenocopies cofilin depletion. Similarly, in Drosophila, depletion of flare, the Drosophila homolog of Wdr1, produces a similar phenotype to the depletion of twinstar, the Drosophila homolog of cofilin (Chu et al., 2012; Ren et al., 2007). Both of these mutants exhibit an accumulation of actin filaments and an increase in actin network stability. In contrast to these   191 findings, B cells from patients with loss-of-function mutations in Wdr1 have an enhanced spreading response to immobilized anti-Ig (Pfajfer et al., 2018). However, this experiment was done in conjunction with CpG DNA stimulation of the B cells. Our laboratory showed that TLR stimulation leads to the “dynamization” of the actin cytoskeleton and impacts the activity of many actin-regulatory proteins (Freeman et al., 2015). A potential link between TLR signaling and cofilin activation is protein kinase D (PKD). PKD is a negative regulator of SHH and a positive regulator of LIMK (Ohashi, 2015). TLR signaling has been shown to activate PKD in macrophages (Park et al., 2009). Upon TLR stimulation of B cells, PKD signaling could act via SSH and LIMK to dephosphorylate and activate cofilin, perhaps allowing B cell spreading even in the absence of Wdr1. An important future experiment will be to replace endogenous Wdr1 in A20 cells with the mutant forms found in patients in order to directly test how those mutations affect B cell actin organization and dynamics.  In contrast to cofilin depletion and Wdr1 depletion, inhibiting LIMK resulted in a thinner peripheral actin ring. The increased activation of cofilin in these cells may promote the severing of actin filaments that comprise the peripheral actin ring. Both B cells depleted of Wdr1 and those treated with the LIMK inhibitor had low levels of phosphorylated cofilin. Why then does the increase in the amount of active cofilin lead to drastically different phenotypes in Wdr1-depleted and LIMKi3-treated B cells? In LIMKi3-treated B cells, Wdr1 is still available to direct the severing function of cofilin, even at high levels of active cofilin. In contrast, in Wdr1-depleted B cells, active cofilin saturates actin filaments and stabilizes them. These findings highlight the interplay between LIMK/SSH-mediated regulation of cofilin activation and Wdr1-mediated regulation of cofilin spacing on actin filaments.  Spatiotemporal regulation of cofilin activity by LIMK may be essential for receptor-induced actin remodeling. A study showing that depletion of either LIMK or cofilin in Jurkat T cells reduces SDF-1-induced chemotaxis indicates the importance of rapidly turning cofilin on and off (Nishita et al., 2005). This study found that cofilin-deficient Jurkat cells assembled large protrusions at the cell periphery whereas LIMK depletion resulted in “immature” protrusion events. These authors suggested that cofilin is important for the turnover of lamellipodial protrusions but that LIMK-mediated suppression of cofilin activity is required in the early stages of lamellipodia formation to allow time for the assembly of actin protrusions. In B cells, BCR-  192 induced activation of cofilin could be important for the initial remodeling of the submembrane actin cytoskeleton in order to liberate actin monomers and generate barbed ends as substrates for new actin polymerization. Subsequently, LIMK activity could transiently suppress cofilin activity to allow for large polymerization events. New BCR-Ag microclusters that form within these actin protrusions could locally reactivate cofilin to increase the velocity of actin retrograde flow and facilitate retraction of these protrusions. Experimental approaches that enable acute disruption of cofilin activity at the site of actin protrusions could provide important insights into the mechanisms controlling actin turnover at the B cell immune synapse.  5.3.4 Actin disassembly factors are important for immune synapse formation in B cells In chapter 3 and 4 I showed that actin dynamics mediated by the activity of Arp2/3 complex are critical for B cell responses to APC-bound Ags. By driving actin retrograde flow, the Arp2/3 complex plays a critical role in regulating BCR signaling in response to membrane-bound Ags. Actin retrograde flow also requires the disassembly of aged actin filaments by actin disassembly factors (Delorme et al., 2007; Flynn et al., 2012; Hotulainen et al., 2005). Inactivation of cofilin decreases the speed of actin retrograde flow (Flynn et al., 2012; Ohashi et al., 2011; Vitriol et al., 2013). Therefore, we examined the role of the LIMK-cofilin-Wdr1 network in BCR microcluster formation and BCR signaling in response to membrane-bound Ags. We showed that interfering with any of these proteins reduces BCR signaling at the B cell-APC contact site. Moreover, we observed a decrease or delay in the aggregation of BCR microclusters at the B cell immune synapse. This is consistent with the idea that BCR signaling is enhanced by the actin dynamics that drive BCR-Ag microcluster aggregation. Depleting B cells of either cofilin or Wdr1 likely stabilizes the actin network and creates barriers that prevent BCR aggregation. When LIMK is inhibited, cofilin hyperactivity could prevent the formation of actin protrusions. Recently, a role for LIMK in immune synapse formation in T cells has been described (Thauland et al., 2017). These authors found that inhibiting LIMK reduces the amount of phosphorylated (inactive) cofilin and increases Ca2+ signaling in response to APCs. Similar results were observed upon inhibition ROCK, a kinase that activates LIMK. Additionally, they found that ROCK inhibition results in increased immune synapse area whereas expression of a constitutively active form of ROCK reduces the synapse area. This is in contrast to our data in   193 which LIMK inhibition blocked B cell spreading. However, ROCK also controls the activity of other actin-regulatory proteins that are critical for B cell immune synapse formation including myosin and ERM proteins (Amano et al., 2010).    Disruption of the LIMK-cofilin-Wdr1 network could also affect BCR aggregation and immune synapse formation by impeding the polarization of the MTOC. Rap GTPase-dependent cofilin activity and cell spreading is required for the polarization of the MTOC to the immune synapse (Wang et al., 2017). MTOC polarization establishes a network of microtubules at the immune synapse that contributes to the aggregation of BCR-Ag microclusters at the cSMAC. Therefore, the observed defects in cSMAC formation caused by targeting the LIMK-cofilin-Wdr1 network could be a result of impaired microtubule organization. Interestingly, LIMK has recently been shown to control microtubule stability and mitotic spindle formation (Prunier et al., 2017). Modulating LIMK activity could therefore change the structure and integrity of immune synapse-associated microtubules. Thus, experimental approaches that allow precise spatiotemporal control of LIMK activity at the site of actin protrusions could yield new insights into cytoskeletal dynamics at the B cell immune synapse.  5.3.5 Perspectives  The precise control of B cell activation is essential for ensuring appropriate B cell responses. Aberrant B cell activation can lead to autoimmunity and B cell malignancies. Here, we have shown that the regulation of cofilin activity in B cells is important for cytoskeletal remodeling events that support BCR signaling. An important next step will be to use real-time imaging to determine how depleting cofilin or Wdr1, or inhibiting LIMK, impacts BCR and actin organization at the immune synapse. By simultaneously imaging BCR-Ag microcluster organization and actin dynamics as in chapter 3, we can gain a more thorough understanding of how the components of the LIMK-cofilin-Wdr1 network organize the immune synapse and amplify BCR signaling. The experimental paradigm that I established in chapter 4 could then be used to investigate how the LIMK-cofilin-Wdr1 network regulates B cell activation.  Previously, our lab has shown that BCR-induced activation of cofilin requires activation of the Rap GTPase (Freeman et al., 2011). Whether the activity of LIMK is also regulated by BCR signaling is not known. LIMK is activated downstream of Cdc42 and Rac via PAK   194 (Edwards et al., 1999; Scott and Olson, 2007). Thus, an interesting future experiment will be to assess LIMK phosphorylation after BCR stimulation with soluble or membrane-bound Ags. Additionally, it will be important to corroborate the findings that we obtained using LIMKi3 by using siRNA to deplete LIMK, as has been done in the Jurkat T cell line (Nishita et al., 2005).  Local inhibition or activation of cofilin within a B cell protrusion would likely affect both actin and BCR-Ag microcluster dynamics. Spatiotemporal control of cofilin activity can be achieved using optogenetic approaches. For example, using chromophore-assisted laser inactivation, Vitriol et al. were able to locally inactivate cofilin within a lamellipod (Vitriol et al., 2013). These authors fused the phototoxic GFP-like protein KillerRed (Bulina et al., 2006) to cofilin. When KillerRed is activated by green light, it generates reactive oxygen species that damage proteins (Sano et al., 2014). By inactivating cofilin at the base of an actin protrusion at the B cell immune synapse, we could examine the role of cofilin in actin retrograde flow, protrusion extension and retraction, and BCR microcluster dynamics. I predict that local cofilin inactivation at these sites would increase the concentration of actin filaments and therefore decrease the velocity of retrograde actin flow and microcluster centralization. Recently, a reversible photoactivatable cofilin analog has been developed (Stone et al., 2019). In this technique, called Z-lock, cofilin is fused to a light oxygen voltage (LOV) sensing domain and to Zdk, a small protein that binds to the LOV domain. This interaction forms a bridge that sterically blocks the actin-binding site of cofilin. Upon excitation with green light, the intramolecular LOV-Zdk interaction is disrupted, exposing cofilin’s actin-binding site. This would allow us to locally increase cofilin activity at specific sites at the immune synapse, which may locally increase actin retrograde flow (Delorme et al., 2007).  In B cells, cofilin activity is likely required for two temporally distinct processes. First, initial BCR signaling induces local activation of cofilin, which remodels the actin cortex, allowing for actin polymerization and the formation of actin protrusions at the immune synapse. Cofilin-mediated actin severing supports this process by increasing the availability of actin monomers that can be rapidly incorporated into filaments by actin nucleation and elongation pathways. At later times during the spreading response, cofilin activity at the base of actin protrusions contributes to the turnover of actin filaments that is required for actin retrograde flow and the eventual retraction of the protrusions. Without this function of cofilin, the spreading actin   195 network freezes as actin monomers are depleted. The contributions of these functions are difficult to tease apart without the ability to control cofilin activity in space and time.              196 Chapter 6: Overall discussion  6.1 Summary of main findings Understanding the molecular mechanisms that control BCR signaling and B cell activation has significant implications for health and disease. Using high spatiotemporal resolution imaging of the interactions between B cells and APCs, I observed that B cells scan the surface of APCs by constantly extending and retracting dynamic actin-rich protrusions. This is in contrast to the radially symmetric spreading exhibited by B cells interacting with anti-Ig-coated coverslips and planar lipid bilayers. This dynamic scanning behaviour is driven by Arp2/3 complex activity, which together with regulators of actin disassembly, generates retrograde actin flow. New BCR-Ag microclusters that form on these protrusions become embedded within the actin network and move toward the cell body with actin retrograde flow as these protrusions are retracted. By using quantitative microscopy, I demonstrated that Arp2/3 complex activity is important for driving the fusion of BCR-Ag microclusters and the eventual formation of the cSMAC. Importantly, I showed that Arp2/3 complex activity is required for amplifying proximal BCR-signaling events in response to membrane-bound Ags but is dispensable for responses to soluble Ags. Additionally, I showed that the Arp2/3 complex activity increases interactions between BCR-Ag microclusters and clusters of phosphorylated CD19, which amplifies and diversifies BCR signaling reactions. These findings suggest that the Arp2/3 complex-dependent movement of BCRs enhances BCR-BCR and BCR-CD19 interactions that are critical for amplifying BCR signaling. Finally, I showed that the activity of the Arp2/3 complex during the initial interactions of B cells with APCs is important for activating transcriptional responses and B cell proliferation in response to APC-bound Ag. In chapter 5, I demonstrated that the actin disassembly factor cofilin, and its regulators Wdr1 and LIMK, are also important for some of these processes. These findings support the idea that actin dynamics are required for the centripetal transport of BCR-Ag microclusters, which promotes the formation of large stable BCR-Ag microclusters and increases BCR-CD19 interactions, both of which are important for amplifying BCR signaling responses to membrane-bound Ags.     197 6.2 Contributions of the Arp2/3 complex-dependent actin dynamics to the three-step model of BCR-Ag microcluster centralization  6.2.1 The Arp2/3 complex is important for actin retrograde flow at the cell periphery  In chapter 3 I showed that the Arp2/3 complex is important for generating retrograde actin flow that transports BCR-Ag microclusters towards the center of the immune synapse. In chapter 5, I showed that interfering with actin disassembly factors also impacts actin organization and dynamics at the B cell immune synapse. These findings support a major role for the dynamics of branched actin networks in controlling the organization of BCR-Ag microclusters at the B cell immune synapse. This is consistent with observations in T cells that retrograde actin flow is critical for TCR microcluster centralization and T cell immune synapse formation (Babich et al., 2012; Murugesan et al., 2016). The translocation of BCR-Ag microclusters towards the center of the cSMAC has been proposed to involve three cytoskeleton-dependent mechanisms that act sequentially: actin retrograde flow, actomyosin contraction, and movement along microtubules that span the actin-depleted central region of the immune synapse. Inhibiting actin retrograde flow by blocking Arp2/3 complex activity or interfering with cofilin activity may block a critical first step in BCR-Ag microcluster centralization and prevent the subsequent myosin- and microtubule-based centralization that leads to cSMAC formation.   6.2.2 Actomyosin structures at the immune synapse   The dense branched actin network at the periphery of the immune synapse is transitioned into linear actin filaments that are organized into concentric arcs by myosin IIA at the pSMAC. These linear filaments are dependent on the activity of formins, at least in T cells (Murugesan et al., 2016), and resemble the transverse arcs that form just behind the lamellipodium of a migrating cell. In T cells, these actomyosin arcs are important for sweeping TCR microclusters towards the cSMAC (Murugesan et al., 2016). A direct role for myosin in BCR-Ag microcluster centralization has not been established, but has been proposed by Tolar and by Wang and Hammer (Tolar, 2017; Wang and Hammer, 2019). In the three-step model of BCR centralization, myosin contractility would occur after BCR-Ag microclusters are delivered to the actomyosin arcs at the pSMAC by actin retrograde flow. However, inhibition of the Arp2/3 complex could   198 disrupt this process. Drastic changes in actin architecture that occur upon inhibition of the Arp2/3 complex could inhibit myosin contractility (Ennomani et al., 2016), thereby blocking the centralization of BCR-Ag microclusters. However, inhibiting the Arp2/3 complex results in enhanced formation of actomyosin arcs in T cells (Murugesan et al., 2016), which suggests that Arp2/3 complex inhibition could enhance actomyosin contractility. Although actomyosin arcs still formed in CK-666-treated B cells, at least when they spread on immobilized anti-Ig, I found that inhibiting myosin contractility did not affect BCR centralization, BCR signaling, or B cell activation. This suggests that myosin activity might not be important for the processes that amplify BCR signaling during the first few minutes of B cell-APC interactions, the magnitude of which impacts whether or not B cell activation responses occur. An important limitation of this experiment is that it lacked a positive control to ensure that pnBB treatment blocked other myosin-dependent processes in the B cells. A complementary loss-of-function approach, such as the use of myosin IIA siRNA, along with a positive control to show that myosin activity is inhibited (e.g. Ag extraction), would help resolve the role of myosin and actomyosin arcs in BCR reorganization.  Although I observed actomyosin arcs in B cells that spread on anti-Ig-coated coverslips, it was difficult to identify actomyosin arcs at the B cell-APC contact site. This could be because the formation of actomyosin arcs is enhanced by integrin activation. In T cells, activated integrins accumulate at the pSMAC, suggesting that this region is important for integrin-mediated adhesions (Yi et al., 2012). Myosin contractility is required for the clustering and accumulation of LFA-1 clusters at the pSMAC (Yi et al., 2012). Thus, actomyosin structures might become more apparent when B cells interact with APCs expressing integrin ligands.  In the APC system that I used, I deliberately excluded integrin ligands in order to dissect how cytoskeletal regulation impacts APC-induced BCR signaling without the additional complexity of integrin signaling. However, most physiological APCs for B cells, e.g. follicular dendritic cells, express integrin ligands. Hence, an important extension of my work is to use APCs expressing integrin ligands to investigate the relative contributions of actin retrograde flow and myosin contractility to microcluster centralization, BCR signaling, and B cell activation responses. Under these conditions, myosin inhibition might have a greater effect on BCR-Ag microcluster centralization and B cell activation. If integrin-mediated adhesion increases the   199 formation of actomyosin arcs and enhances BCR-Ag microcluster centralization and BCR signaling, I predict that this enhancement would be negated when the Arp2/3 complex is inhibited. Integrin activation, clustering, and localization at the immune synapse requires Ag receptor signaling and actin dynamics (Comrie and Burkhardt, 2016), both of which are significantly decreased when the Arp2/3 complex is inhibited. Similarly, integrin activation is impaired and integrins are mislocalized in macrophages in which the Arp2/3 complex has been depleted (Rotty et al., 2017). Based on these findings, I predict that inhibiting Arp2/3 complex activity, and therefore actin retrograde flow, in B cells would result in a greater relative reduction in BCR signaling and B cell activation when the APCs express integrin ligands, compared to the reductions that I observed using APCs that lacked the relevant ligands for B cell integrins. However, more complex scenarios are possible. Because Arp2/3 complex inhibition increases the formation of actomyosin arcs, which promote the clustering of integrins at the pSMAC (Yi et al., 2012), then Arp2/3 complex inhibition could enhance the contributions of integrins to BCR organization and signaling at the immune synapse while impairing the direct effects of BCR signaling on these processes. It is possible that myosin contractility is not required for the initial microcluster dynamics that amplify BCR signaling and transcriptional responses that I measured, but for events that occur later at the immune synapse. Myosin activity is required for Ag extraction (Hoogeboom et al., 2018; Natkanski et al., 2013) and mice lacking myosin IIA are unable to initiate robust antibody responses (Hoogeboom et al., 2018). B cell activation depends on the ability to acquire Ag and present it to T cells to elicit T cell help (Batista et al., 2001; Carrasco and Batista, 2006a). Thus, myosin could act downstream of the initial Arp2/3 complex-dependent gathering of Ag into a cSMAC to extract Ags from the membrane of APCs. Indeed, Ag extraction occurs most frequently at large clusters (Natkanski et al., 2013). Consistent with this idea, Nowosad et al. found that BCR-Ag microcluster size increased over time and that a cSMAC was formed before the onset of pulling forces that are associated with Ag extraction (Nowosad et al., 2016). Thus, I would predict that Arp2/3 complex-dependent actin retrograde flow must first organize BCR-Ag microclusters into large clusters that can then be efficiently internalized by the B cell in a myosin-dependent manner. Inhibiting the Arp2/3 complex or actin disassembly factors that are important for actin retrograde flow and cSMAC formation would therefore result in decreased   200 Ag extraction. In support of this idea, it has recently been shown that the Arp2/3 complex is important for extracting Ags from plasma membrane sheets (Roper et al., 2019). Thus, even though myosin-dependent processes might not impact initial BCR signaling, the upregulation of CD69 and CD86, or B cell proliferation, as I observed, their role in Ag extraction may explain why myosin in critical for the antibody response to T-dependent Ags in vivo.   6.2.3 Arp2/3 complex-dependent regulation of microtubules The third step in the centralization of Ag receptor microclusters is the dynein-dependent transport of microclusters along microtubules (Hashimoto-Tane et al., 2011; Schnyder et al., 2011). When actin retrograde flow is blocked, BCR-Ag microclusters may never reach the sites at which microtubules interact with the inner face of the peripheral branched actin network. Alternatively, inhibiting the Arp2/3 complex could disrupt either the cortical capture of microtubule plus ends or the organization of juxtamembrane microtubules at the immune synapse, leading to impaired microtubule-dependent transport of BCR-Ag microclusters. In B cells, the actin-microtubule crosslinking protein IQGAP1 is highly enriched at the peripheral ring of branched actin and is essential for MTOC reorientation (Wang et al., 2017). Moreover, several NPFs such as WASH and WHAMM associate with microtubules (Rottner et al., 2010) and could integrate actin remodeling with reorientation of the microtubule network. Moreover, branched actin networks can control the organization and distribution of microtubules by regulating tubulin acetylation (Shi et al., 2019). Further work is required to fully understand how reorganization of the actin and microtubule cytoskeletons is coordinated at the B cell immune synapse.  In addition to a potential role for the Arp2/3 complex in organizing the microtubule cytoskeleton, blocking the Arp2/3 complex could interfere with the polarization of the MTOC to the immune synapse. In resting B cells the Arp2/3 complex is partitioned into two distinct pools, a “cortical pool”, that localizes to the submembrane actin cytoskeleton and a “centrosomal pool”, that is located at the MTOC and links this organelle to the nucleus (Obino et al., 2016). Upon BCR stimulation, both pools are recruited to the immune synapse to stimulate actin polymerization, freeing the MTOC from its nuclear tether so that it can polarize to the immune synapse (Obino et al., 2016). Wang et al. found that cofilin-mediated actin dynamics and the   201 formation of a ring of branched actin at the periphery of B cell immune synapse is required for polarization of the MTOC (Wang et al., 2017). Surprisingly, Obino et al. found no defect in MTOC polarization towards Ag-coated beads when B cells were treated with CK-666 (Obino et al., 2016). Whether this is true for B cell-APC interactions remains to be determined.   6.2.4 An updated view of the three-step model for microcluster centralization and cSMAC formation   BCR-Ag microcluster centralization depends on the coordinated activity of three distinct cytoskeletal processes (Figure 3.16). My findings indicate that the Arp2/3 complex-mediated actin retrograde flow is essential for initiating BCR-Ag microcluster formation. Although myosin- and microtubule-based microcluster movement may be required for later steps in BCR centralization, they are unable to support cSMAC formation without the required initial step of retrograde flow-mediated microcluster centralization, which is dependent on the Arp2/3 complex. Inhibiting the Arp2/3 complex may block the delivery of BCR-Ag microclusters to sites where they can engage myosin- and microtubule-dependent mechanisms of transport. The different actin networks assembled at the immune synapse, as well as the microtubule network are regulated in a coordinated manner in order to establish this three-step mechanism. This likely involves many different regulatory proteins and proteins that connect these networks.   6.3 Actin dynamics may control Ag density and affinity thresholds for membrane-bound Ags Proper B cell function depends on the precise control of BCR signaling. In the presence of pathogens, BCR signaling must exceed a threshold to trigger B cell activation. Processes that increase tonic BCR signaling or decrease the amount of BCR signaling required for B cell activation could make B cells more sensitive to small amounts of dangerous Ags but could also contribute to inappropriate or chronic BCR signaling that leads to autoimmune diseases or cancer. The magnitude of BCR signaling is directly related to the amount and duration of Ag binding to BCRs. Fleire et al. demonstrated that increasing the density of Ag or the BCR-Ag binding affinity increases the extent of B cell spreading, the amount of Ag gathered into BCR-Ag microclusters, and the magnitude of BCR signaling (Fleire et al., 2006). These authors also   202 demonstrated that there is a minimum Ag density and affinity that is required for triggering B cell activation, as well as a density and affinity ceiling at which BCR signaling and B cell activation are maximal. The Ag density and affinity thresholds are interrelated as increasing the Ag density, and thus the number of Ag-bound BCRs, reduces the Ag affinity threshold.  The threshold for BCR signaling that is required to induce B cell activation depends on the interplay between Ag density, affinity, the actin cytoskeleton, and BCR mobility (Figure 6.1). Stimulation of B cells with TLR ligands increases actin turnover dynamics and consequently increases the lateral mobility of BCRs on the surface of the B cells and the level of Ag-independent BCR signaling. The net result is that the amount of Ag required to induce maximal BCR signaling is reduced (Freeman et al., 2015). In this way, danger signals such as TLR ligands, can “prime” B cells by rendering them more sensitive to small amounts of pathogen-associated Ags. Moreover, I showed that Arp2/3 complex-dependent actin dynamics amplifies BCR signaling in response to membrane-bound Ags, and this may lower the Ag density and affinity thresholds for APC-induced B cell activation. Arp2/3 complex activity amplifies BCR signaling in multiple ways. Initial BCR signaling stimulates actin polymerization that drives B cell spreading, leading to the formation of additional microsignalosomes. This stimulates further actin polymerization, and as I showed, generates actin retrograde flow that increases the amount of microcluster-based BCR signaling per unit of Ag bound. I found that inhibiting or depleting the Arp2/3 complex in B cells decreased BCR signaling output, as assessed by the levels of pCD79 and pSyk per unit of Ag that was gathered into clusters. The same was true for B cells in which the LIMK-cofilin-Wdr1 network had been targeted. Thus, by controlling the spatial organization of BCR-Ag microclusters, both actin assembly and disassembly factors modulate the relationship between Ag binding and BCR signaling. Based on my findings, I predict that inhibiting the Arp2/3 complex or actin disassembly factors would increase the Ag density and affinity thresholds so that B cells might be unable to respond to low-affinity Ags or Ags that are present at low density on the surface of APCs. Consistent with this idea, B cell-specific deletion of WASp in mice impairs antibody responses to low amounts of Ag (Westerberg et al., 2005).  Because B cell activation also requires B cells to extract Ags and present them to T cells, the affinity threshold for B cell activation is also impacted by actin-dependent forces that are exerted on the BCR. Insufficient force exerted on the BCR by Arp2/3 complex-dependent actin   203 retrograde flow, or by actomyosin contractility, could result in an inability to extract Ags from the APC and present them to T cells. Alternatively, if the forces exerted on the BCR exceed the strength of the BCR-Ag bond, especially in the case of low-affinity Ags, it could rupture the BCR-Ag bond and prevent Ag extraction. This allows B cells to discriminate between high- and low-affinity Ags and preferentially mount antibody responses to high-affinity Ags (Natkanski et al., 2013). In this way, myosin contractility has been shown to mediate “affinity discrimination” by B cells by favoring the extraction of high-affinity Ags (Natkanski et al., 2013). Thus, actin-dependent forces may tune the Ag affinity thresholds for B cell activation. This may be a key element of the germinal center response in which B cells with the highest Ag affinity are positively selected based on their ability to present Ags to T follicular helper cells. In this way, the germinal center response leads to affinity maturation of the antibody response.       204  Figure 6.1 Actin dynamics may control Ag density and affinity thresholds for membrane-bound Ags  In resting B cells, the actin cytoskeleton (green lines) limits BCR-BCR and BCR-CD19 interactions in order to restrict Ag-independent BCR signaling to a level that supports cell survival but which is below the threshold of signaling required for B cell activation. When Ag-induced BCR signaling reaches a certain threshold, the B cell becomes activated. Stimulation of B cells with TLR ligands induces actin remodeling that increases the lateral mobility of BCRs (purple tracks within the actin corrals) on the surface of the B cell and increases tonic Ag-independent BCR signaling. This may lower the amount of Ag required to initiate B cell activation and “prime” B cells to respond to small amounts of microbial Ags. This may help B cells discriminate between inert Ags and pathogen-associated Ags. Innate-like marginal zone (MZ) B cells are naturally primed, having increased actin dynamics, IgM mobility and tonic BCR signaling, and can rapidly respond to Ags. Similarly, loss-of-function mutations in key actin regulatory proteins may decrease the density of actin-based diffusion barriers, resulting in increased BCR mobility and BCR-BCR collisions, which increases BCR clustering and signaling. This may lower the Ag density or affinity threshold for autoantigens that do not cause B cell activation in normal individuals. In activated B cells, increased actin dynamics initially increases BCR mobility but subsequent actin remodeling maintains the integrity of BCR microclusters that assemble microsignalosomes.    205 Human mutations that impair the activation of the Arp2/3 complex, such as loss-of-function mutations in WASp, WIP, and ARPC1B, cause immunodeficiency (Candotti, 2018; Kahr et al., 2017). This can be explained by the decrease in BCR signaling and B cell activation responses we observed when Arp2/3 activity is blocked. These patients also suffer from autoimmune diseases and have a higher incidence of B-cell malignancies (Candotti, 2018). My finding that the loss of Arp2/3 complex activity in B cells results in increased tonic BCR signaling but impaired APC-induced B cell activation may help explain this paradox. By increasing BCR mobility, Arp2/3 complex inhibition could lower the Ag density threshold for autoantigens. In this way, Arp2/3 complex-deficient B cells could be “primed” for autoimmunity. Thus, in resting B cells, the Arp2/3 complex may be important to maintain tonic BCR signaling below a threshold for activation, but upon Ag encounter, the Arp2/3 complex-mediated actin remodeling directs the organization of BCRs that amplifies Ag-induced BCR signaling. An important question is whether loss-of-function mutations in key cytoskeletal regulators always result in both immunodeficiency and autoimmunity. Patients with mutations in DOCK8, which has a role in WASp activation also display a mix of immunodeficiency and inflammation. They develop atopic dermatitis but suffer from recurring infections and have low levels of serum IgM, high levels of IgE, and impaired IgG responses (Zhang et al., 2009). In DOCK8-deficient murine B cells, immune synapse formation is impaired (Randall et al., 2009; Sun et al., 2018). Although Randall et al. demonstrated a key role for DOCK8 in immune synapse formation, they found it to be dispensable for Ca2+ signaling, upregulation of the activation markers CD86 and CD69, and the initiation of proliferation in response to BCR stimulation by soluble anti-IgM antibodies (Randall et al., 2009). However, consistent with my finding that the Arp2/3 complex is only important for B cell activation in response to membrane-bound Ags, a more recent study has shown that DOCK8 is indeed important for BCR signaling in response to membrane-bound Ags (Sun et al., 2018). These findings support the idea that the actin-dependent organization of BCRs is critical for amplifying BCR signaling and B cell activation. Because large Ags such as bacteria and viruses are presented to B cells by subcapsular macrophages and other APCs, actin-dependent amplification of BCR signaling is likely to be essential for the generation of protective antibodies that prevent recurring infections.    206 Patients with mutations in Wdr1 suffer from both immunodeficiency and autoimmunity (Kile et al., 2007; Pfajfer et al., 2018; Standing et al., 2017). However, the B cell contribution to this disease phenotype is not well understood. Pfajfer et al. recently demonstrated that patient-derived Wdr1-depleted B cells exhibit altered spreading in response to immobilized anti-Ig antibodies. Whether this translates into altered B cell activation was not investigated. The autoimmune phenotype that develops in patients with Wdr1 mutations appears to be distinct from that observed in WAS patients and, at least in part, dependent on neutrophils (Kile et al., 2007). It is not clear whether B cells also contribute to the development of autoimmunity in these patients. However, Pfajfer et al. demonstrated that Wdr1 loss-of-function mutations result in an increase in immature transitional B cells in the periphery, which could indicate that these B cells are able to escape negative selection in the bone marrow (Pfajfer et al., 2018). Some of these B cells could contribute to autoimmunity.   6.4 The Arp2/3 complex in other B cell subsets  6.4.1 Immune synapse formation and central tolerance  As described previously, patients with mutations that prevent Arp2/3 complex activation develop autoimmune diseases, perhaps because the loss of Arp2/3 complex-regulated actin dynamics alters BCR signaling and interferes with central tolerance mechanisms. During B cell development, B cells are exposed to autoantigens in the bone marrow. If the binding of self-Ag to the BCR elicits a strong signaling response, these cells are deleted or rendered anergic. B cells with autoantigen-specific BCRs but impaired BCR signaling responses may escape this negative selection by not robustly responding to the autoantigen. Thus, these cells could persist and could react strongly to autoantigen in peripheral tissues, resulting in autoimmunity. Indeed, patients with mutations in WASp have increased levels of autoantibodies (Candotti, 2018). In the bone marrow, developing B cells could encounter Ags presented by stromal cells. Thus, disrupting the Arp2/3 complex activation pathway could result in diminished BCR signaling at the immune synapse, such that B cells with high-affinity autoreactive BCRs escape negative selection. Interestingly, autoimmunity complications are also present in patients with common variable immunodeficiency (Agarwal and Cunningham-Rundles, 2019), which can be caused by loss-of-  207 function mutations in CD19 (Kanegane et al., 2007; van Zelm et al., 2006). Therefore, BCR-CD19 interactions at the immune developmental synapse could also be important for amplifying BCR signaling in response to self-Ags and initiating the deletion of high-affinity autoreactive B cells. These findings provide further support for the idea that Arp2/3 complex-dependent processes that regulate BCR-CD19 interactions have functional consequences for B cell development, selection, and activation.  6.4.2 Germinal center B cells  After B cells have formed immune synapses and acquired Ags from APCs, they migrate towards the T cell zones of secondary lymphoid organs in order to receive T cell help (Victora et al., 2010). B cells that have received T cell help can then enter the germinal center (Victora and Nussenzweig, 2012). The germinal center reaction is critical for the development of high-affinity and long-lived antibody responses as well as the differentiation of memory B cells. The germinal center reaction occurs in two spatially and functionally distinct zones (Mesin et al., 2016; Victora and Nussenzweig, 2012). In the dark zone, B cells proliferate and randomly generate somatic mutations in the genes encoding Ig variable regions (somatic hypermutation). In the light zone, B cells compete for Ag presented by follicular dendritic cells and for T cell help. Repeated cycling between these zones results in antibody affinity maturation and the differentiation of germinal center B cells into plasma cells or memory B cells.  Follicular dendritic cells displaying Ag on their surface promote the positive selection of germinal center B cells (Batista and Harwood, 2009). Initial studies suggested that germinal center B cells were unresponsive to stimulation by soluble Ags (Victora et al., 2010) but it has recently been shown that membrane-bound Ags can elicit robust BCR signaling in germinal center B cells (Nowosad et al., 2016). Although this signaling is not sufficient to fully activate germinal center B cells as T cell help is required (Nowosad et al., 2016). Importantly, B cells with higher affinity for Ag acquire more Ag from follicular dendritic cells, which allows them to receive more T cell help, leading to increased clonal expansion and affinity maturation of the antibody response (Mesin et al., 2016; Victora and Nussenzweig, 2012; Victora et al., 2010). Thus, BCR signaling that promotes actomyosin-dependent Ag extraction plays an important indirect role in the activation of germinal center B cells.    208 The ability of germinal center B cells to extract high-affinity Ags from follicular dendritic cells is facilitated by the unique immune synapse organization that forms between these cells. At the germinal center immune synapse, Ag microclusters are not centralized to form a cSMAC (Nowosad et al., 2016). Instead, microclusters do not fuse and are kept at the periphery. This synaptic architecture may be optimized for the exertion of mechanical force across the BCR-Ag bond as germinal center B cells exert more force on the BCR-Ag bond than naive B cells. This allows germinal center B cells to extract only high-affinity Ags. This is in contrast to naive B cells, which are programmed to enhance BCR signaling and Ag extraction in response to low-affinity Ags. Thus, germinal centers could leverage differential regulation of their actin cytoskeleton to favor the extraction of high-affinity Ags in order to drive affinity maturation and the production of high-affinity antibodies.  How might the Arp2/3 complex contribute to germinal center immune synapse formation? Instead of the centripetally flowing branched actin network observed in naive B cell immune synapses, in germinal center B cells actin accumulates into peripheral punctate structures that are associated with BCR-Ag microclusters and high myosin contractility (Nowosad et al., 2016). These structures resemble the actin foci that have been observed in T cells and in naive B cells. These structures are dependent on WASp activity in T cells (Kumari et al., 2015) and Arp2/3 complex activity in B cells (Roper et al., 2019). Inhibiting the Arp2/3 complex could disrupt the formation of these actin foci-like structures and impair Ag affinity discrimination and Ag extraction. Moreover, Arp2/3 complex-dependent formation of these structures around BCR-Ag microclusters in germinal center B cells could enhance proximal BCR signaling by stabilizing the microcluster. It would be interesting to investigate whether inhibiting the Arp2/3 complex would result in decreased proximal BCR signaling in germinal center B cells, as it does in naive B cells, and whether this impacts the affinity maturation process.  BCR signaling at these peripheral BCR-Ag microclusters could also be potentiated by mechanical stimuli. In naive B cells, the BCR is highly sensitive to mechanical force, such that increasing the stiffness of Ag-presenting surfaces causes greater recruitment of Syk and other signaling components (Shaheen et al., 2017; Wan et al., 2013, 2015, 2018; Zeng et al., 2015). Microclusters in germinal center B cells have increased myosin activity that could enhance BCR signaling. The Arp2/3 complex could contribute to the force generated by myosin at these   209 structures. As mentioned previously, inhibition of the Arp2/3 complex impairs actin foci assembly in naive B cells (Roper et al., 2019). Myosin activity is not required for the assembly of these structures (Kumari et al., 2015; Roper et al., 2019). However, the Arp2/3 complex may be required to first assemble actin structures around BCR-Ag microclusters, which provides a platform for the myosin contractility that drives Ag extraction. Thus, I would predict that inhibiting the Arp2/3 complex in germinal center B cells would result in a defect in Ag extraction and a reduced ability to obtain T cell help. In naive B cells, B cell spreading is essential for the formation of BCR microclusters and the amplification of BCR signaling (Fleire et al., 2006; Weber et al., 2008; see chapters 3 and 4). Recent evidence suggests that germinal center B cells may not spread over the surface of follicular dendritic cells during the germinal center reaction (Kwak et al., 2018). 3D imaging of germinal center B interacting with planar lipid bilayers revealed that these cells instead generate dynamic actin-rich protrusions that contact the APC, which the authors called “pods” (Kwak et al., 2018). These pods gather Ags at the tip and exert pulling forces on BCR-Ag microclusters. Thus, instead of a single coherent point of contact covering the entire region of the immune synapse as in naive B cells, germinal center B cells seem to form multiple distinct points of contact. The authors suggest that pods limit the size of Ag clusters, reducing Ag avidity (the combined effect of multiple BCRs binding the same Ag molecule) and therefore allowing for testing of Ag affinity (the strength of a single BCR-Ag interaction). Because these pod-like protrusions are actin-rich, they could be assembled by the Arp2/3 complex. Thus, it will be interesting to investigate the contribution of the Arp2/3 complex to pod formation and affinity discrimination in germinal center B cells. Moreover, investigations into the structure and function of immune synapses formed by other B cell subtypes, such as marginal zone B cells and transitional B cells, will be important future directions for the field.   6.5 Conclusions  In this thesis, I showed that the spatial organization of BCR-Ag microclusters controlled by the Arp2/3 complex and actin disassembly factors is critical for amplifying B cell responses to APC-bound Ags. I demonstrated that when B cells form immune synapses with live intact APCs, they use complex and dynamic actin-rich protrusions to scan the surface of the APC for   210 Ags. This dynamic probing behavior is mediated by Arp2/3 complex activity, which also generates retrograde actin flow. Arp2/3 complex-dependent retrograde actin flow drives the initial centripetal movement of BCR microclusters and is required for cSMAC formation. I demonstrated that Arp2/3 complex-dependent processes amplify proximal BCR signaling by controlling the BCR-BCR and BCR-CD19 interactions that are required for signal amplification. I also showed that disassembly of actin networks at the immune synapse mediated by cofilin and its regulators are important for this process. These results suggest that actin dynamics mediated by the combined activity of the Arp2/3 complex and actin disassembly factors is critical for organizing BCR-Ag microclusters in order to amplify the response to membrane-bound Ags. Importantly, I showed that by controlling early events in B cell immune synapse formation, the Arp2/3 complex is important for enhancing transcriptional responses and B cell proliferation in response to membrane-bound Ag. Taken together, these findings advance our understanding of how the actin cytoskeleton tunes BCR signaling in response to membrane-bound Ags. 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