Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

The role of the MEK/ERK pathway in regulating cytoskeleton-dependent B cell responses to immobilized… Peters, Victoria 2020

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Notice for Google Chrome users:
If you are having trouble viewing or searching the PDF with Google Chrome, please download it here instead.

Item Metadata

Download

Media
24-ubc_2020_november_peters_victoria.pdf [ 11.16MB ]
Metadata
JSON: 24-1.0394585.json
JSON-LD: 24-1.0394585-ld.json
RDF/XML (Pretty): 24-1.0394585-rdf.xml
RDF/JSON: 24-1.0394585-rdf.json
Turtle: 24-1.0394585-turtle.txt
N-Triples: 24-1.0394585-rdf-ntriples.txt
Original Record: 24-1.0394585-source.json
Full Text
24-1.0394585-fulltext.txt
Citation
24-1.0394585.ris

Full Text

  The role of the MEK/ERK pathway in regulating cytoskeleton-dependent B cell responses to immobilized and cell-bound antigens  by  Victoria Peters  B.Sc., The University of Ottawa, 2018  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)   September 2020  © Victoria Peters, 2020    ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis entitled:  The role of the MEK/ERK pathway in regulating cytoskeleton-dependent B cell responses to immobilized and cell-bound antigens  submitted by Victoria Peters in partial fulfillment of the requirements for the degree of Master of Science in Microbiology and Immunology  Examining Committee: Michael R Gold, Microbiology and Immunology Supervisor  Ninan Abraham, Microbiology and Immunology Supervisory Committee Member  Cal Roskelley, Cell and Developmental Biology Supervisory Committee Member Chinten James Lim, Pediatrics Additional Examiner    iii Abstract The immune synapse (IS) is a special polarized structure that optimizes the functions of the B cell receptor (BCR), signal transduction and antigen internalization. The B cell IS is formed when B cells bind antigens (Ags) that are displayed on the surface of Ag-presenting cells (APCs), creating an area of contact between the two cells. The binding of the BCR to the Ag on the APC triggers rapid and dynamic reorganization of the BCRs and the cytoskeleton. This allows the cell to spread across the surface of the APC and induces the formation of BCR microclusters throughout the contact site. The microclusters then move centripetally and coalesce into the central supramolecular activation cluster (cSMAC) of an IS. A major signaling pathway initiated by the binding of the BCR to Ags is the Ras/MEK/ERK pathway. Although ERK activity has been implicated in B cell survival and proliferation, the role of ERK in regulating BCR-induced cytoskeletal reorganization, IS formation, and APC-induced BCR signaling has not been investigated. I showed that ERK activity is important for B cells to spread on immobilized Ag. Inhibiting ERK resulted in decreased spreading and altered actin organization. By imaging B cell-APC interactions, I also showed that inhibiting ERK activity impaired IS formation, resulting in decreased APC-induced BCR signaling and delayed cSMAC formation. Thus, ERK may regulate actin-dependent processes that are important for B cell responses to immobilized and APC-bound Ags.        iv Lay Summary   B cells are a type of immune cell that produces antibodies when activated by foreign molecules (antigens). Antibodies provide essential protection against pathogens. B cell activation is tightly controlled because inappropriate B cell activation and the production of antibodies against parts of one’s own body can lead to autoimmune disease. When a B cell recognizes an antigen via its B cell receptor (BCR), signals cause changes in the B cell’s actin-based cytoskeleton. These changes allow the B cell to extend protrusions that help it encounter more antigen molecules and that promote its activation. Using loss-of-function approaches and microscopy, I showed that a signaling molecule called ERK controls the BCR-induced shape remodeling that enhances B cell activation. This study identifies ERK as a key component that links the BCR to cell changes that promote B cell activation.    v Preface  Dr. Michael Gold and I conceptualized the project. I performed and analyzed all the experiments in chapter 3. Madison Bolger-Munro wrote image analysis scripts that I used. Microscopy training and support was provided by the UBC Life Sciences Institute Imaging Facility.   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).                vi Table of contents Abstract .......................................................................................................................................... iii Lay Summary ................................................................................................................................ iv Preface ............................................................................................................................................. v Table of contents ........................................................................................................................... vi List of Tables .................................................................................................................................. x List of Figures ............................................................................................................................... xi List of Abbreviations ................................................................................................................... xiv Acknowledgements ..................................................................................................................... xvii Chapter 1: Introduction ................................................................................................................. 1 1.1 B cell development and humoral immunity ............................................................................ 1 1.1.1 B cells and disease .................................................................................................................................. 2 1.1.2 B cell deficiency ..................................................................................................................................... 4 1.1.3 B cells in autoimmunity ......................................................................................................................... 4 1.2 BCR structure and signaling .................................................................................................... 5 1.2.1 BCR organization ................................................................................................................................... 9 1.2.2 Antigen presentation by APCs ............................................................................................................. 11 1.2.3 B cell immune synapse ......................................................................................................................... 11 1.3 The actin cytoskeleton ............................................................................................................. 14 1.3.1 Dynamic actin structures ...................................................................................................................... 15 1.3.2 Actin regulators – Cofilin ..................................................................................................................... 16 1.3.3 Actin regulators – formins and the Arp2/3 complex ............................................................................ 18   vii 1.3.4 The role of the Arp2/3 complex in B cell spreading, IS formation and BCR microcluster centralization ....................................................................................................................................................... 21 1.3.5 NPFs in B cells ..................................................................................................................................... 22 1.3.6 Regulation of NPFs by phosphorylation .............................................................................................. 24 1.4 The MEK/ERK pathway ......................................................................................................... 25 1.4.1 Kinases ................................................................................................................................................. 26 1.4.2 Structure of ERK .................................................................................................................................. 27 1.4.3 Activation of the Ras/Raf/MEK/ERK pathway ................................................................................... 27 1.4.4 Diversity of ERK substrates ................................................................................................................. 30 1.4.5 The role of ERK in B cells ................................................................................................................... 31 1.4.6 The MEK/ERK pathways regulates cytoskeletal organization ............................................................ 33 1.4.7 Stathmin regulates microtubule organization and dynamics ................................................................ 35 1.5 Systems to study BCR-induced actin remodeling and APC-induced IS formation .......... 35 1.6 Hypothesis and specific aims .................................................................................................. 38 Chapter 2: Methods ...................................................................................................................... 39 2.1 Cell isolation and culture ........................................................................................................ 39 2.1.1 Primary B cell isolation and culture ..................................................................................................... 39 2.1.2 B cell lines ............................................................................................................................................ 39 2.1.3 Antigen presenting cells (APCs) .......................................................................................................... 39 2.2 siRNA-mediated gene silencing .............................................................................................. 40 2.2.1 siRNA transduction .............................................................................................................................. 40 2.3 Chemical inhibitors .................................................................................................................. 40 2.4 Analysis of BCR signaling in response to anti-Ig Abs .......................................................... 41   viii 2.4.1 BCR-induced phosphorylation of ERK, stathmin, Akt, cofilin, and HS1 in response to soluble anti-IgG 41 2.4.2 BCR-induced phosphorylation of ERK and stathmin in response to immobilized anti-IgG ............... 42 2.4.3 Protein quantification of immunoblotting ............................................................................................ 42 2.5 B cell spreading on anti-Ig or HEL coated coverslips .......................................................... 44 2.5.1 Cell spreading and fixation .................................................................................................................. 44 2.5.2 Immunostaining .................................................................................................................................... 45 2.5.3 Confocal microscopy and image analysis ............................................................................................ 46 2.5.4 STED microscopy ................................................................................................................................ 47 2.6 B cell interactions with APCs ................................................................................................. 47 2.7 Statistical analysis .................................................................................................................... 48 Chapter 3: Results ........................................................................................................................ 49 3.1 Introduction .............................................................................................................................. 49 3.2 Results ....................................................................................................................................... 52 3.2.1 Optimizing the use of chemical inhibitors against the MEK/ERK pathway ........................................ 52 3.2.2 Inhibiting either MEK or ERK reduces B cell spreading on immobilized anti-IgG ............................ 58 3.2.3 MEK inhibition and ERK inhibition have distinct effects on actin organization ................................ 63 3.2.4 Inhibiting either MEK or ERK inhibits MTOC polarization but the ERK substrate stathmin is not required for B cell spreading .............................................................................................................................. 66 3.2.5 Inhibition of the MEK/ERK pathway inhibits BCR-induced spreading to a greater extent at sub-optimal densities of immobilized anti-IgG ......................................................................................................... 70 3.2.6 MEK/ERK inhibition reduces B cell spreading on Ag-coated coverslips ........................................... 76 3.2.7 Effect of ERK2 depletion on B cell spreading ..................................................................................... 79 3.2.8 Inhibition of the MEK/ERK pathway reduces cSMAC formation at the IS ........................................ 84   ix 3.2.9 Inhibition of the MEK/ERK pathway reduces the ability of A20D1.3 cells to spread on APCs and decreases microcluster-based BCR signaling ..................................................................................................... 88 3.2.10 ERK2 depletion does not significantly alter B cell spreading on APCs or APC-induced BCR signaling 94 3.2.11 Inhibition of MEK or ERK in primary B cells impacts cell spreading on APC and reduces or delays APC-induced BCR signaling ................................................................................................................... 99 3.2.12 Stathmin is not important for BCR microcluster centralization or APC-induced BCR signaling 106 Chapter 4: Discussion ................................................................................................................ 108 4.1 Summary of the main findings ............................................................................................. 108 4.1.1 Regulation of B cell spreading and actin dynamics by ERK ............................................................. 111 4.1.2 Differences between the effects of the MEK inhibitor and the ERK inhibitor may reflect MEK-dependent regulation of cofilin that is independent of ERK ............................................................................ 114 4.1.3 Stathmin cannot be ruled out for its role in B cell spreading and APC-induced BCR signaling ....... 115 4.1.4 The role of the MEK/ERK pathway at the B cell IS .......................................................................... 116 4.1.5 ERK induced activation of WAVE2 may stabilize BCR microclusters by creating actin foci ......... 117 4.1.6 MEK/ERK inhibition has different effects on primary B cells and A20 B cells ............................... 119 4.1.7 A role for ERK1 in APC-induced BCR signaling? ............................................................................ 120 4.1.8 ERK may stabilize BCR-CD19 interactions ...................................................................................... 121 4.1.9 ERK may regulate microtubules through the Arp2/3 complex .......................................................... 121 4.2 Perspectives ............................................................................................................................ 122 4.3 Conclusions ............................................................................................................................. 125 Bibliography ............................................................................................................................... 126     x List of Tables  Table 2.1 siRNAs …………………………………………………………………………..……40 Table 2.2 Chemical inhibitors …………………………………………………………..….……41 Table 2.3 Primary antibodies used for immunoblotting…………………………………..….….43 Table 2.4 Secondary antibodies used for immunoblotting …………………………..……….…44 Table 2.5 Primary antibodies used for immunostaining…………………………………...…….45 Table 2.6 Secondary antibodies used for immunostaining………………………………………46 Table 2.7 Phalloidin reagents used for immunostaining ……………………..…………….……46 Table 4.1 Summary of effects of MEK/ERK inhibition and depletion ……………….…...…..109    xi List of Figures Figure 1.1 B cell receptor (BCR) signaling pathways   …………………………………………..6 Figure 1.2. Stages of B cell immune synapse formation …………………………………..……13 Figure 1.3 Actin dynamics ………………………………………………………………………20 Figure 1.4. The MEK/ERK signaling pathway is activated by the BCR..………….……………29 Figure 3.1. Multiple downstream targets of the MEK/ERK pathway regulate actin and microtubule dynamics……………………………………………………………………………50 Figure 3.2. Effects of MEK and ERK inhibitors on BCR-induced phosphorylation of ERK, stathmin, and cofilin……………………………………………………………………………...54 Figure 3.3. The ERK inhibitor FR180204 and MEK inhibitor U0126 does not inhibit BCR-induced phosphorylation of Akt or HS1..………………………………………………………..55 Figure 3.4. Immobilized anti-Ig activates the MEK/ERK pathway – inhibition by the ERK inhibitor FR180204 and the MEK inhibitor U0126……………………………………………...57 Figure 3.5. Inhibition of the MEK/ERK pathway impairs BCR-induced cell spreading and actin reorganization……………………………………………………………………………………60 Figure 3.5. Continued……………………………………………………………………………61 Figure 3.6. The MEK inhibitor U0126 reduces B cell spreading on anti-IgG-coated coverslips in a dose-dependent manner………………………………………………………………………...62 Figure 3.7. Inhibiting MEK or ERK activity alters peripheral actin structures………………….65 Figure 3.8. Inhibition of the MEK/ERK pathway impairs BCR-induced MTOC polarization….68 Figure 3.9. Stathmin depletion does not affect B cell spreading on immobilized anti-IgG……..69 Figure 3.10 The MEK inhibitor Selumenitib inhibits anti-IgG-induced activation of the ERK/stathmin pathway………………………………………………………………………….71   xii  Figure 3.11. The ERK inhibitor FR180204 reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG………………………………………………………...73 Figure 3.12. The MEK inhibitor U0126 reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG……………………………………………………………….74 Figure 3.13 The MEK inhibitor selumetinib reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG………………………………………………………...75 Figure 3.14. Inhibitors of the MEK/ERK pathway reduce A20D1.3 B cell spreading on Ag-coated coverslips…………………………………………………………………………………77 Figure 3.14. Continued…………………………………………………………………………..78 Figure 3.15. siRNA-mediated depletion of ERK2 reduces B cell spreading on anti-IgG-coated coverslips………………………………………………………………………………………...81 Figure 3.16 siRNA-mediated depletion of ERK2 reduces B cell spreading on coverslips coated with different densities of anti-IgG……………………………………………………………....82 Figure 3.17. siRNA-mediated depletion of ERK2 does not reduce A20D1.3 B cell spreading on Ag-coated coverslips……………………………………………………………………………..83 Figure 3.18. Inhibiting ERK delays cSMAC formation but MEK inhibition does not……….....87 Figure 3.19. B cell spreading on the surface of APCs is reduced following inhibition of MEK or ERK………………………………………………………………………………………………90 Figure 3.20. Inhibition of either MEK or ERK decreases BCR signaling in response to APC-bound Ags………………………………………………………………………………………..91 Figure 3.20. Continued…………………………………………………………………………..92 Figure 3.21. Depleting ERK2 does not reduce B cell spreading on APCs………………………96   xiii  Figure 3.22. Depleting ERK2 does not impair APC-induced BCR signaling or cSMAC formation…………………………………………………………………………………………97 Figure 3.23. Inhibiting MEK in primary B cells reduces cell spreading on APCs……………..101 Figure 3.24. ERK inhibition with 30 μM FR180204 delays early APC-induced BCR signaling………………………………………………………………………………………...102 Figure 3.25. MEK inhibition reduces early APC-induced BCR signaling……………………..104 Figure 3.26. Depleting stathmin does not affect APC-induced microcluster formation, microcluster-based BCR signaling, or cSMAC formation……………………………………..107 Figure 4.1. Proposed roles of MEK and ERK in regulating actin dynamics…………………...110 Figure 4.2. Proposed function of the MEK/ERK pathway in Arp2/3 complex-dependent B cell spreading………………………………………………………………………………………..123     xiv List of Abbreviations Ag: antigen APC: antigen-presenting cell BLNK: B cell linker protein BSA: bovine serum albumin CREB: cAMP response element binding protein cSMAC: central supramolecular activation cluster DAG: diacylglycerol DC: dendritic cell DMEM: Dulbecco’s modified Eagle medium dSMAC: distal supramolecular activation cluster ER: endoplasmic reticulum F-actin: filamentous actin FAK: focal adhesion kinase FDC: follicular dendritic cell FN: fibronectin G-actin: globular actin GAP: GTPase-activating protein GEF: guanine nucleotide exchange factor  Ig: immunoglobulin  IP3: inositol trisphosphate  ITAM: immunoreceptor tyrosine-based activation motif  JNK: c-Jun N-terminal kinase   xv KD: knockdown  LIMK: LIM kinase MFI: mean fluorescence intensity  mHBS: modified HEPES-buffered saline mHEL: transmembrane form of hen egg lysozyme  MTOC: microtubule organizing centre NK cell: natural killer cell N-WASP: neural Wiskott-Aldrich Syndrome protein PAK: p21-activated kinase PBS: phosphate-buffered saline PFA: paraformaldehyde  PI3K: phosphoinositide 3-kinase PIP2: phosphatidylinositol 4,5-bisphosphate PIP3: phosphatidylinositol (3,4,5)-trisphosphate PKD: protein kinase D PLCγ2: phospholipase C gamma 2 pSMAC: peripheral supramolecular activation cluster PTK: protein tyrosine kinase RSKs: ribosomal S6 kinases   ROCK: Rho-associated protein kinase   ROI: region of interest S: serine SH: Src homology   xvi SSH: Slingshot SHP-1: Src homology 2 domain containing phosphatase-1   SHIP1: Src homology 2 domain containing inositol polyphosphate 5-phosphate 1   STED: stimulated emission depletion   T: threonine   TBST: tris-buffered saline tween 20  TCR: T cell receptor 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  WT: wild type   XLA: X-linked agammaglobulinemia  Y: tyrosine      xvii Acknowledgements  I would first and foremost like to thank my supervisor, Dr. Michael Gold for his support, guidance, and mentorship. Your immense patience has been very appreciated! Thank you for sharing your passion for science and giving me so many opportunities I otherwise would not have been provided. I have grown so much more confident in the world of science through your support. I would also like to thank my committee members, Drs. Ninan Abraham and Cal Roskelley for their advice and support.  I would like to thank all the members of the Gold/Matsuuchi lab. Thank you for helping me with everything, especially cleaning up after me and answering my many questions. Kate Choi, May Dang-Lawson and Madison Bolger-Munro, thank you for teaching me everything I know! I would also like to acknowledge the Department of Microbiology and Immunology. The community atmosphere has been incredibly supportive, and I am thankful for the lifelong friends I have made along the way. Kaitlyn Beehler, thank you for telling me about Sci Hub, I literally could not have written this thesis without it.  Finally, I would like to thank my family. To my parents, Susan and Geoffrey Peters, thank you so much for the sacrifices you have made. I am especially thankful you instilled a desire for learning in Andrew, Graham, Spencer and me (readers are leaders!). I attribute my successful completion of graduate school entirely to our nightly meals around the table. Pusan Seters, it’s time to write your book(s). And thank you Sam Frawley for moving across the country with me, even though “Ontario is nicer”.     1 Chapter 1: Introduction  1.1 B cell development and humoral immunity B lymphocytes are an essential arm of the adaptive immune system that defend the body against infection by producing antibodies (Abs) and secreting cytokines [1]. B-lymphocytes mediate the humoral immune response by secreting highly specific Abs against foreign substances (antigens) that can neutralize the antigen (Ag) and promote its clearance by innate immune cells [2]. B cells constitute approximately 15% of peripheral blood leukocytes and arise from hematopoietic stem cells in the bone marrow (BM).  Each B cell expresses on its surface multiple copies of B cell receptor (BCR) complexes, which on a single B cell all have the identical unique Ag specificity. This allows the population of B cells in the body to recognize a wide variety of Ags and provide specific protection against many potential pathogens. Following interaction with the Ag for which it is specific, the B cell becomes activated and proliferates. Activated B cells can differentiate into plasma cells that produce antibodies or into memory B cells that provide long-lived protection against subsequent infection with the same pathogen [2]. In addition to producing protective Abs, B cells also contribute to immunity by producing both inflammatory and anti-inflammatory cytokines and initiating T cell activation by displaying MHC II proteins that have bound Ag-derived peptides [3].   Early B cell development and commitment to the B cell lineage occurs prenatally in the fetal liver, before continuing in the BM after birth [4]. Lymphocytes are derived from hematopoietic stem cells (HSCs) and undergo a series of ordered differentiation steps that produce mature, naïve B cells in the peripheral blood [5]. Early progenitor B cells are characterized by VDJ recombination at the Ig heavy chain locus. The resulting µ heavy chain   2 polypeptide forms disulfide bonds with the Vpre-B and l5 surrogate light chains, and then associates non-covalently with the Iga/Igb (CD79a/CD79b) signaling subunit to produce a pre-B-cell receptor (pre-BCR) [3]. Signals from the pre-BCR provide essential survival signals, suppress the expression of the surrogate light chain genes, and drive VJ recombination at the Ig light chain loci. The resulting k or l light chain can then associate with newly synthesized µ heavy chains and the CD79a/CD79b signaling subunit to produce IgM-BCRs that are expressed on the surface of the B cell [6]. During early development, B cells express only IgM-BCRs but mature B cells express both IgM- and IgD-BCRs, which on a single cell have the same Ag specificity [7–9]  1.1.1 B cells and disease Failures in B cell development or activation result in immunodeficiency diseases whereas inappropriate B cell activation and the failure to eliminate or control self-reactive B cells can result in autoimmune diseases [10]. As discussed in section 1.1.3, immunodeficiencies result in impaired immune response to foreign pathogens and increased susceptibility to infection. B cell-mediated autoimmune diseases are common and often debilitating (see section 1.1.4). B cells can initiate and amplify pathogenic processes in both Ab-dependent and Ab-independent manners [11]. B cells undergo tightly regulated developmental processes in which both central and peripheral tolerance allows for the removal of potentially harmful auto-reactive B cells from the repertoire [12]. Normally self-reactive B cells undergo apoptosis or become anergic when they encounter a self-Ag for which they are specific [12]. If a B cell escapes this process, an immune response against a self-Ag can be mounted [12]. Therefore, a failure to induce immune tolerance can result in autoimmunity [13].    3 Processes that are associated with B cell development and activation such as Ig gene rearrangement, receptor editing, Ig class switching, somatic hypermutation, and rapid proliferation can introduce mutations that result in malignant transformation. Leukemias and lymphomas can arise during B cell development or activation when mutations result in the loss of growth control and the activation of cell survival pathways [11]. Additionally, unregulated BCR signaling can lead to B cell malignancies such as B cell chronic lymphocytic leukemia (B-CLL) [14] and activated B cell-like diffuse large B cell lymphoma (ABC-DLBCL) [15].   Because autoreactive B cells produce autoantibodies and inflammatory cytokines, and also initiate T cell activation, B cells are now thought of as an important therapeutic target for a variety of autoimmune diseases [16–20]. These approaches for targeting B cells have also been used to treat B cell malignancies. Rituximab, which binds to CD20 on normal and pathogenic B cells, has been used to treat B cell malignancies such as DLBCL and CLL [16]. Rituximab is also used as a treatment for autoimmune diseases such as rheumatoid arthritis and multiple sclerosis [17,18], as it can directly kill B cells and also trigger their killing by innate immune cells. Because some B cell malignancies are driven by unregulated BCR signaling, components of the BCR signaling pathway are major targets for the development of therapeutics. Spleen tyrosine kinase (Syk), which is required for most signaling events downstream of the BCR, has become an important target for the treatment of B cell malignancies [19,20]. Similarly, Ibrutinib, an inhibitor of Bruton’s tyrosine kinase (Btk), another essential component of BCR signaling pathway, has been used to treat B cell malignancies such as CLL and mantle cell lymphoma [20,21]. Thus, understanding the mechanisms that control B cell activation could provide new insights into the pathogenesis of immune diseases and suggest potential treatments.    4 1.1.2 B cell deficiency B cell deficiencies that result in impaired immune responses to pathogens and can result from mutations in genes that are important for B cell development or B cell activation. Examples of primary or inherited B cell immunodeficiencies include severe combined immunodeficiencies (SCID), where both T and B cells do not develop, and X-linked agammaglobulinemia (XLA), where B cells do not develop properly. In both cases, patients exhibit severe deficits in Ab production [22]. Another B cell immunodeficiency is common variable immune deficiency (CVID), where CD19, a co-receptor for the BCR, is not expressed properly, resulting in reduced B cell activation and Ab production [23]. Hyper-IgM syndrome is due to a defect in CD40L, a co-stimulatory ligand that is essential for Ig class switching [24]. This syndrome is characterized by elevated serum IgM levels with an absence of IgG, IgA, and IgE, resulting in an extreme susceptibility to bacterial infections. Therefore, determining the signaling mechanisms involved in B cell activation can help identify the cause of B cell deficiencies and suggest potential approaches for restoring B cell function via gene therapy.  1.1.3 B cells in autoimmunity Failure to deplete or silence self-reactive B cells can result in immune responses against self-Ags, potentially leading to autoimmune disease. B cells can contribute to autoimmunity via Ag presentation, cytokine production, and Ab production. Abs directed against self-Ags, as well as the presentation of self-Ags to T cells, can promote autoimmune disorders such as systemic lupus erythematous, rheumatoid arthritis, diabetes and multiple sclerosis (MS) [25–27]. B cells can also have anti-inflammatory functions, which primarily involves their ability to produce the anti-inflammatory cytokine IL-10. B cell-specific deletion of the gene encoding IL-10 in mice   5 leads to the accumulation of pro-inflammatory T cells and exacerbates arthritis [28], indicating that the anti-inflammatory functions of B cells are important for limiting autoimmunity.  Thus, defects in B cell activation that reduce the production of IL-10 by B cells could also contribute to autoimmunity.    1.2 BCR structure and signaling B cell recognition of Ag is necessary for subsequent B cell activation and effector function [29]. Ag-specific humoral immune responses are initiated by the binding of Ag to the BCR. The BCR plays two critical functions in B cell biology, the first being the initiation of intracellular signaling in response to Ag binding and the second being the internalization of BCR-bound Ag so that it can be delivered to Ag processing/MHC II-loading compartments [30]. BCR signaling forms the foundation for essential cellular decisions during B cell development, selection and activation [31]. Upon Ag binding the BCR activates signaling pathways that control cytoskeletal remodeling, metabolic activity, gene expression, cell survival, proliferation, and the differentiation of B cells into Ab-producing cells [30]. BCR structure and BCR signaling pathways are summarized in Figure 1.1. The BCR consists of a membrane-bound immunoglobulin (mIg) molecule and the associated Igα (CD79a)/Igβ (CD79b) signaling subunit [30,32]. The mIg subunit is composed of two Ig heavy chains and two Ig light chains. Igα and Igβ each contain a single immunoreceptor tyrosine-based activation motif (ITAM) within their cytoplasmic tail. The ITAM is a conserved sequence motif that contains two precisely spaced tyrosine residues [33,34]. Phosphorylation of the ITAM tyrosine residues is the key event that initiates BCR signaling following Ag binding [35].   6  Ag-induced clustering of the BCR, together with its association with the co-receptor CD19, initiates BCR signaling by activating Src family kinases and the Syk tyrosine kinase,   Figure 1.1 B cell receptor (BCR) signaling pathways. Ag binding induces BCR clustering and the formation of BCR microsignalosomes, a complex that initiates downstream signaling pathways. The formation of the signalosome is mediated by protein tyrosine phosphorylation. Src-family kinases, such as Lyn, phosphorylate the ITAMs of the CD79a/b subunit, which creates a binding site for the Src homology (SH2) domains in the Syk tyrosine kinase. Syk initiates signaling pathways that lead to cytoskeletal rearrangement, metabolic reprogramming, and changes in gene expression. The interaction of BCRs with CD19 amplifies BCR signaling and is important for activating phosphoinositide 3-kinase (PI3K). Phosphatidylinositol 3,4,5-trisphosphate (PIP3), the second messenger generated by PI3K promotes the membrane recruitment and activation of proteins that contain pleckstrin homology (PH) domains. BCR-induced activation of PLCγ2 results in cleavage of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). DAG activates PKC enzymes as well as activators of the Ras and Rap GTPases. See text for more detail. Multiple arrows or dotted lines indicate multiple steps. Illustrations reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com), [30].   7 which then phosphorylate the CD79a and CD79b ITAMs [29,30,32,36,37]. ITAM phosphorylation promotes the recruitment and activation of additional Syk molecules. The phosphorylation of both ITAM tyrosine residues creates a binding site for the tandem SH2 domains of Syk, which then phosphorylates and activates downstream components of the BCR signaling pathways. Phosphotyrosine-SH2 domain interactions promote the assembly of BCR-associated microsignalosomes, which are complexes that contain signaling enzymes such as phosphoinositide 3-kinase (PI3K), the BTK tyrosine kinase, and phospholipase C gamma 2 (PLCγ2) [38]. These components of the signalosome are responsible for producing key second messengers that regulate cellular processes such as cytoskeletal reorganization, gene expression and BCR-mediated Ag internalization [38].  		 The activation of three non-receptor protein tyrosine kinases (PTKs) (Lyn, Syk and Btk) is required for proper BCR signal transduction. Deficiencies in any one of these PTKs can result in impaired B cell development or function [39,40].  Syk is essential for coupling the BCR to distal signal transduction pathways as it phosphorylates and interacts with the adaptor molecule B cell linker protein (BLNK), which is also known as SLP-65 [41,42]. BLNK then acts as a molecular scaffold for the activation of multiple signaling pathways by bringing together PLCγ2 and Btk via phosphotyrosine-SH2 domain interactions. PLCγ2 activation requires phosphorylation by Btk, and Btk loss-of-function mutations in XLA patients result in decreased activation of PLCγ2 and greatly impaired B cell activation in response to Ag [43].  PLCγ activation results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG), second messengers that activate downstream signaling pathways. IP3 binds to its receptor on the endoplasmic reticulum (ER), which causes the release of Ca2+ from the ER into the cytoplasm. The resulting increase in cytoplasmic free Ca2+ increases the binding   8 of Ca2+ to calmodulin, resulting in the activation of the phosphatase calcineurin. Calcineurin dephosphorylates the transcription factor NFAT, allowing it to translocate into the nucleus to drive transcription [44]. DAG, the other second messenger, activates the Ras GTPase and protein kinase C (PKC) enzymes. PKCbII is critical for BCR signaling as it plays a key role in activation of the NFκB transcription factor [31]. DAG also activates RasGRP family guanine nucleotide exchange factor (GEF) proteins. RasGRP1 and 3 activate the Ras GTPase, which initiates activation of the Raf/MEK/ERK pathway. Activated Ras controls a signaling cascade that activates extracellular-signal-regulated kinase (ERK), which phosphorylates multiple substrates including key transcription factors such as AP-1 [30]. RasGRP2 activates the Rap1 and Rap2 GTPases, which control BCR-induced actin reorganization [45,46].  BCR-induced cell survival is mediated by the PI3K/Akt pathway. Syk-mediated phosphorylation of CD19, BCAP, and other adaptor proteins creates a binding site for the SH2 domains in the p85 subunit of PI3K, which facilitates the translocation of PI3K to the plasma membrane. At the plasma membrane, PI3K can phosphorylate phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the membrane lipid PIP3. PIP3 recruits proteins that contain PH domains, such as the PDK1 and Akt kinases, to the plasma membrane. Akt is phosphorylated by PDK1 and other kinases. Activated Akt increases cell metabolism by promoting glucose uptake and by activating the mTOR complex [47]. Akt-mediated phosphorylation also inhibits the FOXO transcription factors that promote apoptosis [48].   Another important target of BCR signaling that is regulated by PI3K is Vav, a GEF that activates the Rac GTPases. Vav is recruited to the plasma membrane via the binding of its PH domain to PIP3 and the binding of its SH2 domain to phosphotyrosine-containing motifs on CD19. Subsequent phosphorylation of Vav by Syk increases its GEF activity. Vav can then   9 activate Rac, an upstream activator of the p38 and JNK MAP kinase pathways and a major regulator of BCR-induced actin remodeling [49].   BCR signaling can also be negatively regulated by the B cell specific Siglec family member CD22 and the FcRgIIB. CD22 has three tandem cytosolic immunoreceptor inhibitory motifs (ITIMs) that are phosphorylated by activated Lyn after BCR clustering [50]. The phosphorylated CD22 ITIMs recruit the SHP-1 and SHIP1 phosphatases via their SH2 domains. SHP-1 dephosphorylates the ITAM motifs of the BCR whereas SHIP1 dephosphorylates PIP3 and opposes PI3K signaling [51]. Mutations in the CD22/Lyn/SHP-1 pathway lead to autoimmunity in mice [52]. FcRgIIB binds to Ag-Ab immune complexes. When immune complexes bring the BCR and FcRgIIB close to each other, BCR-associated tyrosine kinases phosphorylate the ITIM in the cytoplasmic domain of the FcRgIIB, leading to the recruitment of SHIP1 and inhibition of PI3K signaling. This is important for terminating BCR signaling, and therefore controlling B cell activation, when a sufficient Ab response has been mounted.   1.2.1 BCR organization Determining the mechanism of B cell activation is contingent on understanding how BCRs are organized within the plasma membrane and how this organization impacts BCR signaling. BCRs on the surface of B cells are not randomly distributed, but rather are compartmentalized within the plasma membrane along with other proteins [53,54]. Initial models of B cell activation had proposed that BCRs exist as monomers in the plasma membrane and that BCR signaling is initiated by Ag-induced BCR clustering (also called crosslinking) [55]. However, super resolution imaging has shown that BCRs in naïve mature B cells exist as separate IgM and IgD nanoclusters with approximately 20-120 BCRs in each cluster [54].    10  Membrane compartments are created by two mechanisms. The first is via lipids that self-organize into lipid rafts or microdomains [56]. The other mechanism is via the membrane-associated actin cytoskeleton that forms pickets within the plasma membrane to create networks of membrane associated proteins [57,58]. BCRs and other membrane proteins on the surface of the B cell exist in dense “hot spots” or protein islands within these compartments. These protein-protein interactions are thought to promote the formation of BCR nanoclusters [59]. This pre-clustering of BCRs may promote tonic BCR signaling in resting cells, which is essential to maintain B cell survival [60]. Upon Ag engagement, BCR nanoclusters aggregate further into larger protein complexes known as BCR microclusters, which recruit signaling molecules to form the microsignalosomes described in section 1.2.   The mobility of BCR nanoclusters on resting B cells is limited by the membrane-associated actin cytoskeleton [61,62], which is anchored to the plasma membrane via the interaction of ezrin with the cytoplasmic domains of transmembrane proteins [63]. Ag-induced BCR signaling causes localized cofilin-mediated severing of the actin filaments as well as the inactivation of ezrin, resulting in the uncoupling of the actin network from the plasma membrane [62,64]. This increased lateral BCR mobility allows for increased BCR:BCR collisions as well as BCR collisions with the co-receptor CD19 [62]. This aggregation of nanoclusters results in the formation of BCR microclusters, which ultimately leads to the formation of BCR microsignalsomes that initiate BCR signaling events. This model suggests that BCR nanoclusters are relatively confined in resting B cells. Prior to Ag encounter, BCR signaling is limited by preventing the interaction of BCRs with each other and with the activating co-receptor CD19 [53]. Therefore, BCR organization promotes appropriate BCR signaling in both resting and Ag-stimulated B cells     11 1.2.2 Antigen presentation by APCs  BCR oligomerization and the resulting BCR signaling can be induced by the binding of multivalent Ags, Ag arrays displayed on the surface of other cells, or experimentally with anti-Ig Abs. Although B cells can respond well to soluble Ags, robust activation of B cells can be simulated by Ags that are bound to the surfaces of other cells such as follicular dendritic cells (FDCs) and subcapsular sinus macrophages [53,64]. Following an encounter with an antigen-presenting cell (APC), B cells undergo a two-phase spreading and contraction response [65]. The B cell extends lamellipodia across the APC in order to maximize the amount of Ag encountered. This response is dependent on BCR signaling pathways that initiate the reorganization of the actin cytoskeleton [66]. Furthermore, the extent of which the B cell spreads on the APC is dependent on the affinity and density of Ag on the surface of the APC. As the B cell spreads across the surface of the APC, multiple BCR-Ag microclusters form throughput the contact area [66]. Subsequently, the B cell contracts and BCR-Ag microclusters move centripetally and coalesce, forming a central supramolecular activation complex (cSMAC) where Ag internalization is thought to occur. The amount of Ag gathered into BCR microclusters is directly related to the magnitude of APC-induced BCR signaling as well as the amount of Ag that is internalized and ultimately presented to T cells. Thus, a complete description of B cell activation should incorporate both the molecular signaling cascades and the associated cytoskeleton rearrangements that drive BCR spatial reorganization at the immune synapse (IS).   1.2.3 B cell immune synapse  The activation of B cells by APCs leads to the formation of an immunological synapse (IS) at the B cell:APC contact site [67] (Figure 1.2). When B cells encounter membrane-bound   12 Ags, reorganization of the actin cytoskeleton drives B cell spreading and IS formation [30]. Initial BCR signaling promotes localized disassembly of the submembrane actin cytoskeleton, which increases BCR mobility and allows BCR nanoclusters to aggregate into BCR-microclusters. The formation of microclusters is thought to initiate signaling based on the collision-coupling model [30]. In this model, BCR-BCR collisions initiate BCR signal transduction by increasing the transphosphorylation of BCR ITAMs within the microcluster [53,68]. Actin polymerization at the periphery of the contact site drives B cell spreading, which is accompanied by depletion of F-actin from the center of the B cell:APC contact site. As the B cell spreads, the B cell encounters more Ag molecules on the surface of the APC, increasing BCR microcluster formation. The BCR microclusters then recruit and activate tyrosine kinases, including Syk, which results in the formation of microsignalosomes and robust BCR signaling [38]. Ensuing B cell membrane contraction occurs within 5-10 min after contacting an APC and is accompanied by the movement of BCR microclusters towards the center of the B cell:APC contact site [68], where they coalesce into a central supramolecular activation cluster (cSMAC).  In both T and B cells, microcluster centralization and cSMAC formation have been proposed to involve three cytoskeleton-dependent mechanisms that act sequentially: retrograde flow of actin from the periphery of the cell towards the center of the IS, actomyosin-based contractile forces, and movement along microtubules that span the actin-poor central region of the IS [67,69]. In the last phase of IS formation, the coalescence of BCR microclusters results in the formation of a cSMAC that is surrounded by activated integrins, which form the peripheral supramolecular activation cluster (pSMAC) [70]. The cSMAC is a region of intense BCR signaling and a site where BCR-bound Ags are internalized for delivery to MHC II loading     13        Figure 1.2. Stages of B cell immune synapse formation. The image depicts a B cell encountering an APC, spreading on its surface, gathering Ag, and then contracting to form an immune synapse (IS). (A) IS formation begins following the binding of the BCR to its cognate Ag on the APC. In resting B cells, the lateral mobility of BCRs is restricted by the actin cytoskeleton, which limits BCR-BCR collisions and BCR signaling prior to Ag encounter. Upon Ag binding, BCR signaling promotes the local disassembly of the submembrane actin cytoskeleton through the activation of cofilin, resulting in increased BCR mobility. This allows the aggregation of BCR nanoclusters into microclusters as well as the transient co-clustering of BCRs with CD19. (B) BCR-induced actin polymerization at the periphery of the cell exerts outward pressure on the membrane, which promotes spreading of the B cell on the surface of the APC. At the same time, actin is depleted at the center of the B cell:APC contact site. Spreading allows the B cell to encounter more Ag and increases BCR microcluster formation, which results in the formation of microsignalosomes that activate BCR signaling pathways.  (C) Subsequent membrane contraction is accompanied by centripetal movement of BCR microclusters toward the center of the B cell:APC contact site. (D) This coalescence of the BCR microclusters results in the formation of a cSMAC that is surrounded by clusters of activated integrins, which form a pSMAC. At the cSMAC, BCR-bound Ags are internalized for subsequent processing and presentation to T cells. Adapted, with permission, from [71].      14 compartments. During IS formation, the microtubule network is moved toward the B cell:APC contact site, bringing the microtubule-organizing center (MTOC) and the microtubule network close to the plasma membrane at the B cell:APC contact site [72–74]. Juxtamembrane microtubules support the coalescence of the BCR microclusters into the cSMAC. Concomitantly, microtubule-associated lysosomes and MHC II loading compartments are brought close to the cSMAC, where BCR-bound Ags are internalized [75,76].   1.3 The actin cytoskeleton  IS formation is a highly dynamic process that requires extensive reorganization of cellular components. As previously mentioned, the actin cytoskeleton provides forces that drive IS formation and thereby promote APC-induced B cell activation. In resting B cells, the submembrane actin cytoskeleton prevents the spontaneous aggregation of BCR nanoclusters, allowing only low-level tonic BCR signaling in the steady state [77]. Upon Ag encounter, BCR-induced actin remodeling promotes B cell spreading, which increases Ag binding and BCR signaling. The subsequent membrane contraction to form the cSMAC increases the efficiency of Ag internalization. Actin polymerization also controls the trafficking of internalized Ag to Ag-processing compartments [78]. Hence, the actin cytoskeleton is an important regulator of BCR signaling and B cell activation. Consistent with this idea, mutations in a variety of actin-regulatory proteins result in impaired B cell activation and immune responses. Identifying cytoskeletal-regulatory proteins that control B cell signaling and activation provide new insights into B cell function.      15 1.3.1 Dynamic actin structures  Actin, a 42-kDa protein, is one of the most abundant proteins in the cell, comprising approximately 1-5% of total cellular protein in most cell types [71]. The actin cytoskeleton is made of a network of filamentous actin fibers that support many cellular functions [79].  Some of these functions include determining cell shape, supporting cell movement, establishing polarity, controlling intracellular trafficking, providing structural scaffolds, and supporting cell division [80,81]. The actin cortex is a dense meshwork of F-actin filaments that underlies the plasma membrane to form the “skeleton of the cell” [79]. The actin cortex is responsible for defining cell shape and opposing mechanical stress [53].   The formation of F-actin is a result of the polymerization of globular actin monomers, or G-actin, with the two states existing in a dynamic equilibrium [53]. The actin monomer is composed of a single polypeptide chain with four subdomains (SD 1-4). The polypeptide is folded in its tertiary U-shaped structure [82]. In order to form new actin filaments, the self-assembly of actin monomers into a trimer is required. This rate-limiting nucleation step is required to form a seed for further actin polymerization [79]. The filament is then extended by the addition of actin monomers in the same orientation [83,84]. The two polarized ends of the actin filament are termed the barbed end and the pointed end, based on the orientation of the myosin heads that bind to the actin filament. Actin filament growth and shrinkage results from the addition and loss of G-actin monomers from the filament ends [79,82], which occur at different rates [85]. At steady state, ATP-bound actin monomers are more readily added to the barbed end of the filament, with slower addition at the pointed end [79]. Over time, the ATP that is bound to actin monomers within a filament is converted to ADP+Pi and then ADP. This results in the accumulation of ADP-actin near the pointed ends [86]. These older actin filament   16 segments are preferentially targeted for depolymerization by proteins such as cofilin. The overall rate of actin polymerization and depolymerization is termed actin dynamics. Actin dynamics is dependent on the concentration of G-actin in the cell and is controlled by multiple regulatory proteins.   Actin networks support many functions within the cell and can be rearranged to have different physical properties. One way in which actin filaments can be organized is into branched arrays, such as those within the lamellipodia of migrating cells. Branched arrays can exert an outward force on the membrane of the cell to generate forces required for locomotion or altering cell shape [87]. An alternate organization of actin filaments is into linear bundles where filaments are arranged either in parallel or antiparallel fashion [88,89]. Parallel actin bundles are often found in finger-like membrane protrusions such as filopodia, and have all their barbed ends facing in the same direction [53,82]. Conversely, anti-parallel actin filament bundles associate with motor proteins to establish contractile actin networks such as stress fibers [88]. The actin filaments must be crosslinked by proteins in order to build different bundled actin structures. Actin bundling proteins include L-plastin, fascin, fimbrin, filamin, formins, and a-actinin [88,90]. Additionally, actin structures can be quickly assembled and disassembled as the cell moves and multiple types of actin structures can exist at the same time in a single cell. The ability of the cell to remodel its actin cytoskeleton into different structures at specific locations within the cell is important for a rapid response to external stimuli.   1.3.2 Actin regulators – Cofilin   Actin monomer availability is a key regulator of cytoskeletal dynamics. Rapid polymerization cannot be accomplished without replenishing the pool of available actin   17 monomers. Therefore, proteins that disassemble actin filaments are required to fuel actin polymerization and thus cytoskeletal reorganization. Cofilin is an actin-severing protein and a major regulator of actin dynamics [91]. Cofilin is a 20-kDa protein that binds to older ADP-actin-containing filaments and changes their properties to promote filament disassembly [92,93]. This process of actin severing occurs in a concentration-dependent manner, much like actin polymerization. At high concentrations cofilin binds to actin filaments with a 1:1 ratio of cofilin to actin subunits [92]. The Wdr1 protein enhances cofilin-mediated severing by competing with cofilin for filament binding sites. This creates boundaries between cofilin-decorated and bare regions, increasing the strain at these boundaries, which results in actin severing [94,95]. In small patches of cofilin-decorated actin, twists are introduced such that the filament will buckle if other forces are applied [79,96]. Motor proteins often generate these forces. Hence, cofilin binding does not itself induce severing but instead destabilizes regions of actin filaments. Applied mechanical force is required for the subsequent filament severing [96].    Cofilin plays an essential role in actin turnover dynamics and it aids in replenishing the pool of G-actin monomers needed for actin assembly [97] (Figure 1.3). As such, cofilin is often enriched in areas where extensive actin turnover takes place, for example at the leading edge of motile cells or ruffling membranes [96,98]. Moreover, cofilin can promote the debranching of older ADP-bound filaments in actin networks such as those found at the leading edge of a migrating cell [93]. When cofilin is inactivated, actin filament turnover decreases and F-actin accumulates due to an increased rate of polymerization relative to depolymerization [99].   The activity of cofilin is spatially and temporally regulated by post-translational modification and by cofilin-binding proteins [100]. Phosphorylation and dephosphorylation are the primary post-translational modifications that regulate cofilin activity. LIMK-mediated   18 phosphorylation of cofilin on S3 inhibits its ability to bind to F-actin [92]. Cofilin S3 is dephosphorylated by the Slingshot (SSH) phosphatases [101]. In B cells, BCR signaling results in the transient dephosphorylation of cofilin, thus increasing cofilin-mediated actin severing [102].      1.3.3 Actin regulators – formins and the Arp2/3 complex All actin structures are formed based on two principles. The first is nucleation, where actin-nucleating proteins initiate filament formation, and the second is polymerization. Polymerization is where G-actin monomers are added to the end of a filament [80,88]. Although actin monomers are able to self-assemble, this form of assembly is slow and results in unstable polymerization intermediates of actin dimers and trimers that will rapidly dissociate, making this energetically costly [53,103]. Hence, actin-nucleating proteins and other actin-associated proteins promote actin polymerization, bundling, capping and branching, and these proteins are essential for rapidly remodeling the cytoskeleton in response to external stimuli.   Actin nucleators, such as formin proteins, are able to stimulate the formation of an actin filament by seeding polymerization from a pool of profilin-bound actin monomers [83,84,87]. Formin proteins are recruited to the plasma membrane where they are activated by Rho GTPases and initiate the formation of linear unbranched actin filaments [87].  The Arp2/3 complex is a multi-subunit complex of proteins that is responsible for the branching of actin filaments to form lamellipodial protrusions (Figure 1.3). The Arp2/3 complex binds to a pre-existing primer, or mother filament, and initiates the formation of a new daughter filament at a 70o angle, resulting in a Y-shaped branched structure [83,104]. The Arp2/3 complex must be activated by a nucleation-promoting factor (NPF) [81]. Several NPFs can stimulate   19 Arp2/3 mediated actin nucleation, with the major NPFs functioning at the plasma membrane being Wiskott-Aldrich syndrome protein (WASp) and WASp-family verprolin-homologous protein (WAVE) [84]. WASp is activated by Cdc42-GTP whereas WAVE acts downstream of Rac-GTP [105]. The binding of these activated NPFs to the Arp2/3 complex results in a conformational change that allows the Arp2/3 complex to bind to the side of an existing actin filament and then bind an actin monomer [81]. This results in the formation of a trimeric complex of Arp2, Arp3 and the actin monomer, where more actin monomers can be added onto the pre-existing monomer to generate a new actin filament [81]. Further extension of the daughter filament is mediated by actin elongation factors such as formins and enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) proteins [87,106,107].  The activation and location of the different Arp2/3 NPFs determine when and where these branched actin networks will be assembled within the cell. Additionally, capping proteins bind to the free barbed end to prevent further filament elongation but also to link the filaments to membrane-associated proteins [108,109]. The actions of the Arp2/3 complex and capping proteins need to be coordinated so that actin-based forces can be distributed along the leading edge of a migrating cell or the broad lamellipodia of spreading cells [110].      20    Figure 1.3 Actin dynamics. (1) Branched actin polymerization is initiated by the Arp2/3 complex. The Arp2/3 complex is activated by the Cdc42 à WASp pathway and the Racà WAVE2 pathway. BCR signaling activates both Cdc42 and Rac. Activated nucleation-promoting factors (NPFs) induce a conformational change in the Arp2/3 complex that allows it to bind to the side of an actin filament and nucleate a daughter filament to create a branched network. (2) Elongation proteins, such as VASP, transfer actin monomers to the ends of uncapped growing filaments. (3) The pool of available actin monomers is limited. As a result, the cell requires sustained actin depolymerization to replenish the pool. The actin-severing protein cofilin, and its co-factor Wdr1, bind to actin filaments and exert tension that promotes severing of the filament. (4) CAP1 and cofilin work together to depolymerize the severed filaments into actin monomers. CAP1 also works together with profilin to load the liberated actin monomers with ATP. Image provided by M. Gold.      21 1.3.4 The role of the Arp2/3 complex in B cell spreading, IS formation and BCR microcluster centralization BCR signaling stimulates branched actin polymerization, which is likely due to the activity of the Arp2/3 complex. Recent work from our lab identified a role for the Arp2/3 complex in controlling B cell spreading, actin retrograde flow, and the centralization of BCR microclusters into a cSMAC at the IS [69]. This Arp2/3 complex-dependent movement and coalescence of BCR microclusters amplifies APC-induced BCR signaling and thus B cell activation.  BCR-induced actin remodeling can be readily visualized when B cells spread on immobilized anti-Ig Abs to determine the role of various cytoskeletal proteins in controlling these processes. Inhibiting Arp2/3 activity results in altered organization of the peripheral actin ring when B cells spread on immobilized anti-Ig [69]. Instead of a dense peripheral ring of highly branched F-actin that underlies broad lamellipodia in a radially spreading cell, cells in which Arp2/3 complex activity is inhibited exhibit greatly reduced spreading and disorganized peripheral actin structures that are dominated by loosely packed filopodial-like protrusions [69]. This is consistent with the idea that formins and the Arp2/3 complex compete for the same limited pool of actin monomers [111]. As B cells spread, the assembly of actin networks at the periphery of the Ag contact site is accompanied by the depletion of F-actin from the center of the contact site [112]. The ring of actin at the periphery of the cell generates forces that initiate the formation of symmetrical radial membrane protrusions that resemble lamellipodia [2,66,112]. As new actin is polymerized at the periphery of the cell, the resistance of the plasma membrane causes the peripheral actin network to flow inward toward the cell, termed actin retrograde flow [113]. Because preventing Arp2/3   22 nucleating activity alters the actin structures that are responsible for exerting force on the plasma membrane, reducing nucleation by the Arp2/3 complex also results in reduced actin retrograde flow [69]. Additionally, because the Arp2/3 complex is important for actin retrograde flow, preventing Arp2/3 complex activity in B cells reduces the cells’ ability to retract actin-rich protrusions when they scan the surface of APCs [69]. Retraction of protrusions allows BCR microclusters that have formed on these protrusions to move towards the cell body and subsequently coalesce into a cSMAC. This impaired coalescence of BCR microclusters that occurs when the Arp2/3 complex is inhibited results in impaired BCR signaling, with less microcluster-based BCR signaling generated per unit of bound Ag [69]. In T cells, actin retrograde flow is also necessary for optimal and sustained T cell receptor (TCR) signaling in response to peptide-MHC complexes that are tethered to artificial lipid bilayers [114]. Because the TCR is a mechanosensitive receptor, forces exerted on the TCR by actin retrograde flow may enhance its signaling [115]. The BCR is also sensitive to mechanical forces [116,117]. Hence, Arp2/3 complex-dependent forces may amplify BCR signaling by increasing the mechanical tension on BCRs that are bound to APC-associated Ags.   1.3.5 NPFs in B cells  The activity of the Arp2/3 complex is regulated by NPFs that are activated via upstream signaling pathways. Receptor-induced activation of NPFs at the cell membrane stimulates the nucleation and elongation of a branched actin network that grows towards the plasma membrane. NPFs bind to actin monomers and then to the Arp2/3 complex to form the nucleus for filament growth. NPFs also induce a conformational change in the Arp2/3 complex that enhances its binding to the mother filament [118]. Interestingly, at high concentrations, NPFs can also   23 enhance filament elongation in an Arp2/3 complex-independent manner by increasing the availability of polymerization-competent monomeric actin at the membrane surface [119].  WASp is an essential NPF in lymphocytes, as illustrated by the immunodeficiency syndrome WAS, which is caused by mutations in the gene encoding WASp or the WASp-interacting protein WIP. Patients with this syndrome present with increased susceptibility to autoimmune disease, predisposition to lymphomas and leukemias, and reoccurring infections [120,121]. T cells isolated from WASp-deficient patients display defects in migration, proliferation, and survival [122,123]. Likewise, murine B cells that lack WASp exhibit reduced BCR signaling, Ag internalization, and Ab responses [124]. At the B cell IS, activated WASp localizes to the peripheral actin ring at the site of actin polymerization. Additionally, WASp-deficient B cells have defects in BCR microcluster aggregation and Ag extraction from APCs [125,126]. Similar defects are seen in primary B cells from WAS patients, where B cell spreading and BCR microcluster formation are impaired [127].  The other major NPF responsible for Arp2/3 complex activation is WAVE. There are three isoforms of WAVE, WAVE1-3, which promote Rac1-dependent actin polymerization [128]. WAVE 1 and WAVE3 are primarily expressed in neuronal cells whereas WAVE2 is present at high levels in hematopoietic cells [129]. WAVE is part of the multi-subunit WAVE regulatory complex (WRC), which includes Sra-1, Abi-1/2, Hem-1, and HSPC300 [130,131]. Mice deficient in WAVE die during gestation and display defects in development and cell migration [132]. In T cells, WAVE2 localizes to the IS after TCR stimulation and plays a critical role in the formation of lamellipodia and T cell spreading [133]. Our lab also has preliminary data showing that depletion of WAVE2 reduces cell spreading in B cells. And when a protein   24 component of the WRC called HEM1 is depleted from B cells, BCR-induced spreading is also reduced [134].  The activation of WASp and the WRC are controlled by the Cdc42 and Rac GTPases, respectively [135]. Hence, the signaling pathways by which the BCR activates Cdc42 and Rac are critical for the BCR to induce Arp2/3 complex-dependent actin polymerization. Following Ag encounter, BCR-CD19 interactions induce the phosphorylation of CD19, allowing CD19 to recruit Vav, a GEF for Rac and Cdc42 [136–139]. The recruitment of Vav to CD19 leads to the phosphorylation of Vav by tyrosine kinases such as Syk [139,140]. Vav then activates the Cdc42 and Rac GTPases, which in turn activate WASp and WAVE2, respectively. Depleting Vav impairs B cell spreading in response to membrane-bound Ag, similar to preventing Arp2/3 complex activity [141]. Moreover, loss of Cdc42 impairs B cell development, plasma cell differentiation, and Ab responses in mice [142,143]. DOCK8 is another Rac GEF and mice lacking this protein display reduced WASp and CD19 activation, leading to defects in B cell spreading, BCR clustering and BCR signaling [144,145].  There are also three recently discovered NPFs that activate the Arp2/3 complex at different cellular locations, WASP homology associated with actin, membranes, and microtubules (WHAMM), WASp and Scar homolog (WASH), and junction mediating and regulatory protein (JMY) [105]. These NPFs diversify the functionality of the Arp2/3 complex   1.3.6 Regulation of NPFs by phosphorylation NPFs are recruited to the plasma membrane by Rho family GTPases, where subsequent phosphorylation results in their full activation. Phosphorylation is a rapid and reversible mechanism for controlling protein function [146] and is one of the most common and important   25 post-translational modifications [146]. There are many actin-regulatory proteins that are controlled by receptor-induced phosphorylation, including cofilin, HS1, CAP1 and Vav [147–150]. WASp, one of the NPFs that activate the Arp2/3 complex, requires phosphorylation of Y291 for its activation [151]. WRC activity requires phosphorylation of both WAVE2 and Abi1 [152], modifications that are mediated, at least in part, by ERK [153]. In primary mammary epithelial cells, ERK co-localizes with the WRC at lamellipodial leading edges, consistent with the idea that it phosphorylates WAVE2 and Abi1 [154].    1.4 The MEK/ERK pathway  Signal transduction pathways are essential regulators of cellular responses to extracellular cues. The Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway is an evolutionarily conserved signaling pathway that controls cell cycle progression, apoptosis, protein synthesis, and cell growth in a wide variety of organisms. Germ line mutations in components of the Ras/Raf/MEK/ERK pathway cause developmental disorders including Noonan syndrome, Costello syndrome and cardio-facio-cutaneous syndrome [155]. This pathway couples receptors such as the BCR to downstream kinases that control cell growth and to transcription factors that modulate gene expression [156]. The core of this pathway consists of three tiers: Raf, a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) that phosphorylates and activates MEK, a MAPK kinase (MAPKK), which then phosphorylates and activates the ERK MAPK [156,157]. This signaling pathway is activated by the Ras GTPase. Receptor signaling activates GEFs that convert Ras to its active GTP-bound state that can recruit and activate Raf. Traditionally, the Raf/MEK/ERK pathway has been synonymous with cell   26 cycle progression and transcription factor activation. However, as described below there is emerging evidence that ERK also regulates the cytoskeleton [152,154,158,159].    1.4.1 Kinases  Non-receptor kinases are essential elements of intracellular signaling that are activated by extracellular stimuli such as cytokines, growth factors, or Ags. Protein phosphorylation on serine, threonine, and tyrosine residues was first described in eukaryotes and shown to be a key mechanism for regulating enzyme activity and cellular functions [160]. Kinases are molecular switches that exist in either an inactive or active state [161].  Serine/threonine kinases are composed of an N-terminal lobe, which binds the phosphate donor ATP, and a C-terminal lobe, which binds the substrate and initiates the transfer of the phosphate group [160]. Many kinases are activated by phosphorylation on at least one serine or threonine residue in their activation loop [162]. All protein kinases catalyze the same reaction, the transfer of the γ-phosphate of ATP to the hydroxyl group of a serine, threonine or tyrosine residue [162]. The activation loop changes conformation when the kinase switches between the active and inactive states [163]. In ERK, for example, phosphorylation at T183 in the activation loop induces a conformational change that exposes a hydrophobic surface, which allows the formation of homodimers [164]. This dimerization is required for nuclear translocation of ERK [165]. Studies of the function of kinases has been complicated by the fact that many cells express multiple isoforms of a single kinase. This makes it difficult to assess the function of these molecules in a single-gene deletion study [166]. However, the magnitude and complexity of the lymphocyte serine/threonine phospho-proteome is now appreciated [167,168]. For example, there are approximately 10,000 distinct phosphorylation modifications in the Jurkat T-cell   27 leukemia line, of which more than 600 are regulated in response to TCR signaling [169–171]. In this thesis I will be focusing on the serine/threonine kinase ERK and its effect on the cytoskeleton in B cells.    1.4.2 Structure of ERK  There are two isoforms of ERK found in all cells, ERK1 (44 kDa) and ERK2 (42 kDa), with ERK2 being the most abundant isoform in B cells [172]. However, human ERK1 and ERK 2 are 82% identical in sequence and share many if not all functions [173]. Thus, ERK 1 and ERK 2 tend to be referred to collectively as ERK1/2 [174]. ERK is the main substrate of MEK and requires dual phosphorylation on the conserved TEY motif within the activation loop for full activity [175]. This corresponds to phosphorylation at T202/Y204 for human ERK1 and T185/Y187 for human ERK2.  1.4.3 Activation of the Ras/Raf/MEK/ERK pathway   A switch-like biochemical response allows cells to convert external stimuli into binary cellular decisions [176,177]. BCR signaling activates Ras, which initiates the Raf/MEK/ERK cascade (Figure 1.4) [178,179]. There are three isoforms of the 21-kDa Ras protein that are encoded by separate genes: H-Ras, N-Ras and K-Ras [180]. The Ras proteins are anchored to the cytoplasmic face of the plasma membrane by C-terminal acyl groups. Ras proteins cycle between an active GTP-bound state and an inactive GDP-bound state, acting as a binary switch that controls the Raf/MEK/ERK pathway. BCR-mediated activation of Ras, and hence ERK, occurs in two phases [181]. First, PLCγ2-dependent production of DAG recruits the RasGRP1 and RasGRP3 GEFs to the plasma membrane, where they are activated by PKC-mediated   28 phosphorylation [182,183]. Subsequently, Ras-GTP initiates a positive feedback loop in which the Ras-GEF mSOS amplifies BCR-induced Ras activation [181,184].   The Raf serine/threonine kinase is activated by Ras-GTP. There are three isoforms of Raf, each encoded by a separate gene: A-Raf, B-Raf and Raf-1. The Raf proteins all have three functional domains: CR1, CR2 and CR3. The CR1 domain is responsible for Ras binding. The CR2 domain is the regulatory domain and the CR3 domain is the kinase domain [161]. Raf was originally identified as a retroviral oncogene (v-Raf) and all Raf kinases are activated by the Ras GTPases [185]. Recruitment of Raf to the plasma membrane via its binding to Ras-GTP is the initial event in Raf activation [186–188]. This is followed by the dimerization of Raf kinase to act on downstream substrates [189]. MEK proteins are the main targets of Raf. This family of dual-specificity kinases has both serine/threonine and tyrosine kinase activity. MEK is activated by Raf via phosphorylation at S218 and S222 [190]. The MEK proteins contain a regulatory domain and a MAP kinase-binding domain that binds to and activates ERK.      29  Figure 1.4. The MEK/ERK signaling pathway is activated by the BCR. The mIg subunit of the BCR binds Ag and the Iga/Igb (CD79a/CD79b) subunit transduces signals to the cell interior. The Src family kinases (Lyn, Blk and Fyn), as well as the Syk and Btk tyrosine kinases are activated and then promote the formation of a ‘signalosome’ that contains Btk, the BLNK adaptor protein, and PLCg2. DAG produced by PLCγ2 recruits Ras to the plasma membrane, where it is activated by PKC-mediated phosphorylation. Further activation of Ras requires the recruitment of the mSOS Ras-GEF to the membrane, resulting in a positive feedback loop that amplifies Ras activation. GTP-bound Ras recruits the Raf kinase to the membrane. Activated Raf binds to and activates MEK1/2, which then activates the ERK1 and ERK2 MAP kinases. Illustrations reproduced courtesy of Cell Signaling Technology, Inc. (www.cellsignal.com).     30 1.4.4 Diversity of ERK substrates  ERK regulates multiple biological processes by phosphorylating substrate proteins [173]. Over 160 substrates downstream of ERK have been identified [153]. The ERK substrates can be categorized into several groups: transcription factors, protein kinases and phosphatases, cytoskeletal and scaffold proteins, and receptors and signaling molecules [173]. The transcription factor Elk1 is one of the most well-known targets of the ERK signaling cascade [191,192]. Although many ERK substrates are localized in the nucleus, there are many cytosolic substrates as well. As discussed below in section 1.4.6, ERK phosphorylates multiple proteins that control the cytoskeleton and cell morphology [173].  The ribosomal S6 kinases (RSKs) are key targets of ERK signaling. RSK is a multifunctional ERK effector that regulates multiple cellular processes [193]. The identification of substrates of RSK has been difficult due to the lack of specific inhibitors. However, it is well known to promote proliferation by stimulating growth-related protein synthesis in the G1 phase of the cell cycle via its phosphorylation of the ribosomal S6 subunit [193]. In B cells RSK also phosphorylates the cAMP-response element-binding protein (CREB), which contributes to the BCR-induced survival response in mature B cells [194].  Paxillin is another substrate of ERK signaling. Paxillin is a focal adhesion protein that regulates cell morphology and motility in response to integrin signaling and receptor engagement [159]. Paxillin is an adaptor/scaffolding protein whose function is tightly regulated by its phosphorylation pattern. Phosphorylation of paxillin by ERK is thought to regulate integrin-mediated adhesion and enhances cell spreading and adhesion in thymoma cells [195]. ERK also interacts with and phosphorylates other cytoskeletal elements including vinexin (also known as SORBS), which promotes cell spreading[196,197].    31 ERK also plays an essential role in development [172]. Prolonged MEK1/2 inhibition impairs the development of embryonic stem cells [198] whereas prolonged ERK1/2 inhibition results in senescence-like growth suppression [199]. ERK is found in almost all cell types, including lymphocytes, where it controls many functions.   1.4.5 The role of ERK in B cells In B cells ERK plays both a positive (survival/proliferation) and negative (apoptosis/deletion) role, depending on the maturational and activation state of the cell [200–202]. The ERK pathway promotes the survival of B cell progenitors by linking pre-B cell receptor signaling to transcriptional events that are necessary for early B cell development. [203,204]. Disrupting the genes encoding both ERK1 and ERK2 results in defective pre-BCR-mediated cell expansion, which is dependent on ERK-mediated phosphorylation of the Elk1 and CREB transcription factors [203].  Productive rearrangement of the Ig heavy and light chain V gene segments in B lymphocyte progenitors results in the expression of a mature BCR. Newly generated B cells that enter the spleen undergo a process termed positive selection, where cells that express a BCR are allowed to continue development. The positive selection process is dependent on Ag-independent tonic signals transduced by the BCR. The Ras/ERK pathway appears to be essential for generating these tonic BCR survival signals. Activation of Ras rescues the differentiation of immature/transitional B cells with reduced levels of cell surface Ig. Conversely, ERK inhibition impairs the development of mature B cells [205]. This suggests that tonic BCR signaling regulates the differentiation of immature into mature B cells via ERK.    32 Negative selection is the removal of autoreactive B cells that are specific for self-Ags. However, autoreactive B cells can escape negative selection in the bone marrow (central deletion) or in circulation (peripheral tolerance). Patients affected by systemic lupus erythematosus and other autoimmune diseases have an increased number of autoreactive B cells that have escaped negative selection [13,206]. Expressing constitutively active Ras in B-lineage cells allows autoreactive B cells to overcome this negative selection and leads to the production of autoantibodies [207,208]. ERK activation appears to be necessary but sufficient for activated Ras to overcome tolerogenic signals [205]. The ERK pathway is also required for multiple aspects of germinal center (GC) response where Ag-activated B cells undergo somatic hypermutation, affinity maturation, and then differentiation into either plasma cells or memory B cells [209]. Dysregulation of this process can lead to the survival of autoreactive plasma cells and memory B cells that can contribute to autoimmune disease. ERK has also been implicated in the maintenance of memory B cells [210]. Upon Ag stimulation, memory B cells are susceptible to apoptosis, but can be rescued via an anti-apoptotic effect mediated through the Ras/ERK cascade [210]. The MEK/ERK pathway opposes apoptosis by phosphorylating Bim, Bcl-2 and caspase-9, proteins that regulate apoptosis [211]. Inhibition of MEK activity impairs Ab production by IgG memory B cells [210] and is required for a subset of B cell responses to Ag [201]. ERK2 has a unique role in the efficient generation of Ag-specific IgG-bearing memory B cells, providing a survival signal that allows these cells to remain in the spleen and produce a more robust response [212]. ERK2 may also phosphorylate caspase-9 and inhibit its activity, thereby promoting the survival of IgG-expressing memory B cells [213]. ERK activity also supports the cytokine-dependent differentiation of B cells into Ab-producing plasma cells by inducing the expression of Prdm1,   33 the gene that encodes the Blimp1 protein, a master regulator of plasma cell differentiation [214]. ERK signaling also supports TLR4-induced plasma cell differentiation by integrating signaling inputs from the BCR and cytokines [215].   1.4.6 The MEK/ERK pathways regulates cytoskeletal organization   Although the role of ERK in cytoskeletal organization has not been investigated in B cells, a number of studies have implicated ERK in regulating actin and microtubule dynamics in other cell types.  In human epithelial cells, ERK is required for membrane ruffling and extension [216,217]. In epithelial cells, ERK co-localizes with the WRC and phosphorylates WAVE2 and Abi1, which promotes the binding of the WRC to the Arp2/3 complex. [218]. ERK can phosphorylate WAVE2 on S308, S343, and T346, which increases its NPF activity [109,219]. As a result, ERK is important for actin-dependent leading-edge advancement during cell migration [154]. In macrophages, ERK activity stabilizes protruding lamellipodia and ERK inhibition results in the production of transient, unstable protrusions [218] similar to what is observed when WAVE2 is depleted. In T cells, WAVE2 localizes to the periphery of the T cell IS, where actin polymerization promotes TCR microcluster centralization [133]. When plated on anti-CD3/anti-CD28-coated coverslips, T cells undergo radial spreading that is driven by the formation of lamellipodia [220]. Depleting WAVE2 in T cells inhibits this T cell spreading response [133]. And patients with HEM1-deficient B cells, which is a component of the WRC, show defective BCR-induced spreading [134]. Similarly, preliminary data from our lab indicates that depleting WAVE2 in the A20 B cell line reduces the ability of these cells to spread radially on immobilized anti-IgG Abs (unpublished data). Because WRC activity is dependent on ERK-  34 mediated phosphorylation, inhibiting ERK could phenocopy the effects of depleting WAVE2 in B cells.  Although ERK has classically been thought of as a regulator of genes that promote proliferation, differentiation and survival [158], it also regulates cell movement. ERK promotes cytoskeletal contractility by phosphorylating and activating myosin light chain kinase (MLCK), which then phosphorylates myosin light chain (MLC) [221]. Phosphorylation of MLC by MLCK increases myosin ATPase activity, which is essential for actomyosin contractility [222]. In T cells, MEK may regulate cofilin activity, which supports T cell movement by enabling actin treadmilling and actin retrograde flow [99,223]. As well, MEK activity can uncouple Rho from its downstream effector, ROCK, resulting in increased cofilin activity and reorganization of the actin cytoskeleton [224].   ERK may also play a role in regulating microtubule dynamics. At the B cell: APC IS, actin-dependent forces are essential for the initial movement of peripheral BCR microclusters towards the center of the IS. However, the central region of the IS is depleted of dense actin structures. In B cells the microtubule-based motor protein dynein is responsible for translocating Ag-bound BCRs along the microtubule network towards the actin-depleted center of the contact site [72]. This requires the polarization of the microtubule-organizing center (MTOC) towards the APC contact site [73]. The microtubule-regulatory protein, stathmin, is a direct substrate of ERK [225]. In T cells, both ERK activity and stathmin-dependent microtubule growth are required for TCR-induced MTOC polarization towards the IS [226,227] and for effective targeted cell killing by cytotoxic T cells [226]. Preliminary work from our lab showed that BCR signaling induces robust ERK-dependent phosphorylation of stathmin and that ERK inhibition prevents BCR-induced MTOC polarization (T. Jou, unpublished data). Because microtubules are   35 essential for cSMAC formation in B cells, ERK-dependent stathmin phosphorylation may contribute to BCR microcluster centralization and BCR signaling.   1.4.7 Stathmin regulates microtubule organization and dynamics Stathmin, or oncoprotein 18/Op18, is a cytosolic phosphoprotein that is a direct substrate of ERK [228]. Stathmin destabilizes microtubules either by sequestering free tubulin dimers or by directly inducing microtubule catastrophe [229]. Stathmin has been implicated in the regulation of cell motility [229], a process with many similarities to cell spreading. Stathmin activity is controlled by serine phosphorylation [229]. The N-terminus of stathmin contains four serine residues (S16, S25, S38 and S63) that are phosphorylated by different kinases. Rac-dependent S16 phosphorylation plays a role in T helper cell differentiation [229]. S25 and S38 can be phosphorylated by multiple kinases belonging to the MAPK family, including ERK1/2 [230,231]. Phosphorylation of stathmin on these residues abrogates its ability to sequester tubulin dimers and enables increased microtubule growth. In T cells, stathmin is rapidly phosphorylated in response to TCR engagement and localizes to the IS [226]. Because stathmin is strongly phosphorylated in response to BCR engagement and may be important for BCR-induced MTOC polarization, I hypothesized that ERK-regulated stathmin phosphorylation contributes to IS formation in B cells.   1.5 Systems to study BCR-induced actin remodeling and APC-induced IS formation  APC-induced IS formation involves initial BCR spreading on the surface of the APC, followed by membrane contraction and the coalescence of BCR microclusters into a cSMAC. The initial spreading step can be studied with high spatial and temporal resolution by plating B   36 cells on immobilized anti-Ig antibodies or Ags. Unlike the B cell:APC interaction, the contact site between the B cell and a coverslip is completely contained in a single focal plane. This allows one to perform high spatial resolution imaging, including super-resolution microscopy, to visualize the complex and dynamic cytoskeletal network and identify the proteins that contribute to BCR-induced actin remodeling [69,73,232]. Another advantage to this system is that the density of immobilized anti-Ig antibodies and Ags can be easily controlled. This enables quantitative analysis of the relationship between Ag density and actin remodeling. Although this system has greatly contributed to our current knowledge of cytoskeletal events that occur during cell spreading and IS formation, it fails to properly model the complex mechanical properties of APCs such as Ag mobility. Ags that are immobilized on glass coverslips are useful for studying cytoskeletal rearrangement that occur during cell spreading, but at the same time the immobilization of Ags has many limitations. In particular, when Ags are not mobile the formation, growth, and mobility of BCR microclusters are reduced [233]. This means BCRs do not move centripetally or coalesce into a cSMAC, processes that amplify BCR signaling [69]. To avoid these limitations, BCR-induced actin reorganization, BCR microcluster formation, and B cell IS formation have been studied extensively using Ags that are tethered to an artificial planar lipid bilayer [66,234–236]. This system allows the interface between the B cell and Ag-presenting substrate to be imaged in one x-y focal plane. However, a drawback to this system is that it again fails to accurately replicate certain properties of biological membranes. In particular, cell membranes are compartmentalized by the submembrane actin cytoskeleton, which affects the mobility of the Ag within the membrane. The mobility of proteins within the plasma membrane of APCs has been shown to influence their ability to activate T cells [237]. As well, BCRs are sensitive to mechanical force and increasing the   37 stiffness of Ag-presenting surfaces, such as with the lipid bilayer, induces greater recruitment of Syk and other BCR signaling components [116,238]. Thus, planar lipids will elicit increased forces at the B cell IS than an intact APC, potentially impacting BCR signaling [239]. To overcome this issue, our lab and others have employed flat, adherent cells such as COS-7 cells that can be readily transfected to express transmembrane forms of Ag [69,240].  Coverslips are coated with extracellular matrix proteins, which induces the COS-7 cells to spread flat and thin, allowing for the B cell-APC contact site to be imaged in one focal plane. COS-7 cells lack the ligands for murine integrins, which allows us to study the effect of BCR signaling on IS formation in the absence of additional signaling.                 38 1.6 Hypothesis and specific aims    Our lab has shown that the Arp2/3 complex is necessary for BCR-induced cell spreading as well as BCR microcluster centralization and BCR signaling in response to APC-bound Ags [69]. Because the WRC activates the Arp2/3 complex, and its activity depends on ERK-mediated phosphorylation, I proposed that ERK activity is required for B cell spreading as well as APC-induced BCR microcluster centralization and BCR signaling.  My overall hypothesis is that the MEK/ERK/stathmin pathway controls the actin and microtubule dynamics that regulate B cell spreading, IS formation, and APC-induced BCR signaling.   To test this hypothesis, I had the following specific objectives: 1. Test the effect of inhibiting the MEK/ERK/stathmin pathway on BCR-induced B cell spreading and actin remodeling 2. Test the effect of inhibiting the MEK/ERK/stathmin pathway on APC-induced microcluster formation and centralization as well as BCR signaling in response to APC-bound Ags.      39 Chapter 2: Methods 2.1 Cell isolation and culture  2.1.1 Primary B cell isolation and culture  B cells were isolated from spleens of 6-12 week old MD4 mice [191] (Jackson Laboratories, #002595) using a negative selection B cell isolation kit (Stemcell Technologies, #19854). The University Animal Care Committee approved all protocols. B cells were cultured in RPMI-1640 supplemented with 5% heat-inactivated FBS, 2 mM glutamine, 1 mM pyruvate, 50 μM 2-mercaptoethanol, 50 U/mL penicillin and 50 μg/mL streptomycin (complete RPMI medium). Primary B cells were used for experiments immediately after isolation.  2.1.2 B cell lines   The A20 murine IgG+ B cell line was obtained from the ATCC (#TIB-208) and cultured in complete RPMI medium. A20D1.3 cells, which express the D1.3 transgenic BCR that binds hen egg lysozyme (HEL) [192], were a gift from F. Batisita (Cancer Research UK, London, UK).   2.1.3 Antigen presenting cells (APCs)   COS-7 green African monkey kidney epithelial cells (ATCC, #CRL-1651) were cultured in DMEM supplemented with 5% heat-inactivated FBS, 2 mM glutamine, 1 mM pyruvate, 50 U/mL penicillin and 50 μg /mL streptomycin (complete DMEM medium).     40 2.2 siRNA-mediated gene silencing  2.2.1 siRNA transduction  ON-TARGETplus siRNA SMARTpools were used to decrease the expression of target proteins. A20 and A20D1.3 B cells were transduced with 2 μg of stathmin (op18) siRNA, 4 μg of Mapk1 (ERK2) siRNA, or 2-4 μg control siRNA using AMAXA Nucleofector Kit V (Lonza). The cells were then cultured in complete RPMI medium for 24 hr before being used in experiments.  Table 2.1 siRNAs siRNA Manufacturer Catalogue Number Mouse stathmin (Op18) Dharmacon, GE Life Sciences L-041608-00-0005 Mouse ERK2 (MAPK1) L-040613-00-0005 Non-targeting Pool D-001810-01-05  2.3 Chemical inhibitors  B cells (1 x 106/mL) were suspended in modified HEPES-buffered saline (mHBS; 25 mM sodium HEPES pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 g/L glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol) and pre-treated with selective inhibitors of MEK, ERK, GSK-3, or the Arp2/3 complex before being stimulated with soluble anti-IgG antibodies or added to anti-IgG coated coverslips, HEL-coated coverslips, or APCs.      41 Table 2.2 Chemical inhibitors  Inhibitor Manufacturer Catalogue Number Target Final Concentration References U0126 Sigma-Aldrich 662005 MEK 0.1-60 μM [193] Selumetinib Selleckchem S1008 MEK 3 μM [194] FR180204 Tocris Bioscience 3706 ERK 0.1-60 μM [195] CK666 Millipore 182515 Arp2/3 complex 100 μM [196] CHIR99021 Stem Cell Technologies 72052 GSK-3 0.1-60 μM [197]  2.4 Analysis of BCR signaling in response to anti-Ig Abs 2.4.1 BCR-induced phosphorylation of ERK, stathmin, Akt, cofilin, and HS1 in response to soluble anti-IgG  A20 Cells (5 x106 in 0.25 mL mHBS) were pre-treated with chemical inhibitors or an equivalent volume of DMSO for 1 hr at 37°C before being stimulated with 20 µg/mL goat anti-mouse IgG Abs (Jackson ImmunoResearch, #115-005-008). Reactions were terminated by adding 0.25 mL cold phosphate-buffered saline (PBS) (ThermoFisher Scientific Gibco, #10010-023) containing 1 mM Na3VO4 and placing cells on ice. The cells were then centrifuged at 2800 rpm in a microfuge for 3 min and the cell pellet was solubilized by adding 100 µL cold radioimmunoprecipitation assay (RIPA) buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% IGEPAL [Sigma-Aldrich, #CA-630], 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate   42 (SDS), 2 mM ethylenediaminetetraacetic (EDTA), protease inhibitors [1mM phenylmethylsulphonyl fluoride (PMSF), 10 μg/mL leupeptin, 10 μg/mL pepstatin, 1 μg/mL aprotinin] and phosphatase inhibitors [25 mM β-glycerophosphate, 1mM Na3VO4, Na3MoO4]). Samples were kept on ice and vortexed periodically for 20 min and then centrifuged at 14,000 rpm at 4°C in a microfuge for 15 min to pellet cellular debris.  2.4.2 BCR-induced phosphorylation of ERK and stathmin in response to immobilized anti-IgG  A20 B cells (105 in 0.1 mL mHBS) were pre-treated with chemical inhibitors or an equivalent volume of DMSO for 1 hr at 37°C before being added to the wells of a 12-well tissue culture plate that had been coated with 2.5 μg/cm2 goat anti-mouse IgG for 5-30 min. Reactions were terminated by placing the plate on ice. The mHBS was aspirated before lysing the cells with 100 µL RIPA buffer (30 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% IGEPAL [Sigma-Aldrich, #CA-630], 0.5% sodium deoxycholate, 0.1% SDS, 2 mM EDTA, protease inhibitors [1mM PMSF, 10 μg/mL leupeptin, 10 μg/mL pepstatin, 1 μg/mL aprotinin] and phosphatase inhibitors [25 mM β-glycerophosphate, 1mM Na3VO4, Na3MoO4]). The plates were kept on ice and RIPA buffer was pipetted up and down vigorously before centrifuging the cell lysates at 14,000 rpm at 4°C in a microfuge for 15 min to pellet cellular debris. The samples were then analyzed by immunoblotting.   2.4.3 Protein quantification of immunoblotting  Protein concentrations were determined using a bicinchoninic acid (BCA) assay (ThermoFisher Scientific-Pierce, #23225). Absorbance readings were carried out with an   43 Ultrospec 2011 Pro spectrophotometer (Sigma-Aldrich GE Healthcare, #80-2112-21). The cell lysates were subsequently diluted 1:5 with 5X reducing SDS-PAGE sample buffer (62.5 mM Tris-HCl, pH6.8, 2% glycerol, 2% SDS, 100 mM DTT, 0.02% bromophenol blue), boiled for 5 min and loaded on a gel immediately or stored at -80°C.   SDS-PAGE and immunoblotting was performed as described [198]. The Abs used are described in Table 2.2. Bands were visualized using an enhanced chemiluminescence (ECL) reagent (Millipore-Sigma, #WBKLS0500). The blots were imaged and bands were quantified using a C-digit scanner and Image Studio Lite software (Li-cor). Total pixel intensities for bands detected using phospho-specific Abs were normalized to loading control bands for the same sample. To re-probe blots, the nitrocellulose membranes were incubated with Ab stripping buffer (25 mM HCl in TBST [20 mM Tris-HCl, pH 8, 150 mM NaCl, 0.1% Tween-20]) for 1 hr at room temperature and then washed with TBST before incubating with another primary Ab.   Table 2.3 Primary antibodies used for immunoblotting  Antibody  Manufacturer Catalogue Number Dilution Mouse anti-beta actin Santa Cruz Sc-47778 1:3000 Rabbit anti-phospho- stathmin (pS25) Abcam  Ab194752 1:1000  Rabbit anti-stathmin Cell Signaling Technologies 3352 Rabbit anti-ERK1/2 9102 Rabbit anti-phospho-ERK1/2 (pT202/pY204) 4377 Rabbit anti-cofilin 2218 Rabbit anti-phospho-cofilin 3313   44 (pS3) Rabbit anti-phospho-CD79a (pY182) 5173 Rabbit anti-Mb1 (CD79a) [241] Rabbit anti-Akt 9272 Rabbit anti-phospho-Akt (pS473) 9271 Rabbit anti-HS1 4557  Rabbit anti-phospho-HS1 (pY397) 4507    Table 2.4 Secondary antibodies used for immunoblotting  Antibody  Manufacturer Catalogue Number Dilution Horseradish peroxidase-conjugated goat anti-rabbit IgG  Bio-Rad 170-6515 1:3000 Horseradish peroxidase-conjugated goat anti-mouse IgG 170-6516  2.5 B cell spreading on anti-Ig or HEL coated coverslips 2.5.1 Cell spreading and fixation  Glass coverslips were coated with 0.63 – 2.5 μg/cm2 goat anti-mouse IgG or 0.22 – 0.44 μg/cm2 HEL (NANOCS, #LSN-BN-1) in PBS for 1 hr at room temperature and then blocked with 1 mg/mL BSA in PBS as described [199]. A20 cells (106 cells) in 1 mL mHBS were treated with a chemical inhibitor or vehicle control for 1 hr at 37°C, as described in section 2.4, before adding 100 μL (105 cells) of the cell suspension to the coverslips. After allowing the cells to   45 spreading for 5-30 min at 37°C, the cells were fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. The cells were then permeabilized for 3 min at room temperature with 0.1% (W/V) Triton X-100 in PBS and blocked with 2% bovine serum albumin (BSA) in PBS before being stained for F-actin and tubulin.   2.5.2 Immunostaining  All Abs were diluted in PBS with 2% BSA. Coverslips were incubated with primary Abs (Table 2.5) for 1 hr at room temperature or overnight at 4°C. The coverslips were then washed with PBS before being incubated with secondary Abs (Table 2.6) for 1 hr at room temperature. F-actin was stained using fluorophore-conjugated phalloidin (Table 2.7). For stimulated emission depletion (STED) microscopy, the cells were stained with Alex 532-conjugated-phalloidin (ThermoFisher, #A12380) (1:400) for 1 hr at room temperature. The coverslips were then washed with PBS and mounted onto glass slides using ProLong Diamond anti-fade mounting reagent (Molecular Probes-Invitrogen, #P36961).   Table 2.5 Primary antibodies used for immunostaining Antibody Species Manufacturer Catalogue Number Dilution Anti-α-tubulin Rabbit Abcam ab52866 1:400 Anti-α-tubulin Rat ab6161 Anti-pCD79a (pY182) Rabbit Cell Signaling Technologies 5173S     46 Table 2.6 Secondary antibodies used for immunostaining Antibody Species Manufacturer Catalogue Number Dilution Alexa Fluor 488 anti-rabbit IgG Goat Molecular Probes-Invitrogen A-11008 1:350 Alexa Fluor 647 anti-rabbit IgG A-21247  Table 2.7 Phalloidin reagents used for immunostaining  Phalloidin conjugate Manufacturer Catalogue Number Dilution Rhodamine-Phalloidin  Thermo Fisher R415 1:400 Alexa Fluor 647-Phalloidin A-22287 1:200  2.5.3 Confocal microscopy and image analysis   Laser scanning confocal microscopy was performed using an Olympus IX81/Fluoview FV1000 confocal microscope based on an IX81 inverted microscope with 60X or 100X NA 1.40 oil objective. Spinning disk confocal microscopy was performed using a system based on Zeiss Axiovert 200M microscope with a 100X NA 1.45 oil objective and a QuantEM 512SC Photometrics camera for image acquisition (Quorum Technologies). Images taken of the contact site between the B cell and the coverslip were analyzed using Slidebook v6.0 software (3i Inc., Denver, CO). The cell area in the lowest confocal plane, i.e. closest to the coverslip, was quantified using FIJI software [242] from images taken of the contact site between the B cell and the coverslip. Actin staining was used to define the cell periphery. The percent of the cell area that was depleted of actin was determined using FIJI. The outer face of the peripheral actin ring   47 was used to define the cell edge and calculate the total cell area while the inner face of the peripheral actin ring was used to delimit the central actin-depleted region of the cell.   2.5.4 STED microscopy   STED super-resolution microscopy was performed using a TCS SP8 laser scanning STED system (Leica) equipped with 592 and 600 nm depletion lasers, a CX PL APO 100X NA 1.40 oil objective, and HyD high sensitivity detectors (Leica). Acquisition was performed using LASX software (Leica). Time-gated detection was set from 0.3-6 ns. Image deconvolution was performed using Huygens software (Scientific Volume Imaging, Hilversum, Netherlands).   2.6 B cell interactions with APCs  COS-7 cells were transiently transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The cells were transfected with 0.25 μg of a plasmid encoding a transmembrane form of HEL (mHEL) fused to the HaloTag protein [200] and then cultured for 18-24 hr before being used in experiments. The mHEL-HaloTag-expressing COS-7 cells were then detached using enzyme-free cell dissociation buffer (0.5 mM EDTA, 100 mM NaCl, 1 mM glucose, pH 7.4), resuspended in complete DMEM medium and added to 18-mm coverslips that have been coated with 5 μg/mL fibronectin. After allowing the COS-7 cells to adhere and spread for 16-24 hr at 37°C, the DMEM medium was replaced with a 1:2000 dilution of HaloTag tetramethylrhodamine ligand (Promega, #G8251) in PBS for 15 min at 37°C. Following this incubation, the COS-7 cells were washed with room temperature PBS. Subsequently, B cells (5 x 105 cells in 0.1mL mHBS) were added to the APCs for 3-30 min at 37°C before adding 4% PFA to fix the cells and preserve B cell-APC conjugates. The cells were   48 permeabilized, blocked, and immunostained as in section 2.5.1 with rabbit anti-pCD79 and Alexa Fluor 647-phalloidin. The cells were then imaged by spinning disk microcopy as in section 2.5.3.  Images of the plane of B cell-APC contact were taken and within a single experiment all images were acquired using identical settings. Custom FIJI macros (https://github.com/madscience12/FIJImacros/blob/master/APC_analyzer_MBM.ijm) described in [69] were used to quantify the amount of mHEL-HaloTag Ag and pCD79 fluorescence intensity present in clusters and quantify the cluster area. Each image was filtered (i.e. convolved) with 3 x 3 averaging filter to smooth noise. A binary mask was then defined by thresholding the pixel intensities in the filtered image. To quantify fluorescence in an image, background fluorescence was estimated by calculating the median intensity of the pixels outside the mask, and this value was subsequently subtracted from the image. Next, the intensity of pixels outside of the mask was 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 pCD79 was also normalized to the total fluorescence intensity of clustered Ag on the same B cell as a measure of BCR signaling output per unit of Ag bound.   2.7 Statistical analysis  Mann Whitney U tests were used to compare the means of data pooled from multiple experiments. Student’s two-tailed paired t-tests were used to compare the mean values for matched sets of samples from multiple experiments.     49 Chapter 3: Results  3.1 Introduction   In response to polarized Ag arrays (immobilized Ags, Ags on beads, Ag-bearing APCs), BCR signaling induces dramatic actin remodeling, which is coordinated with polarization of the MTOC towards the Ag contact site. When the Ag is mobile within a membrane, these cytoskeletal changes drive the formation of a classical IS. The growth, centralization, and coalescence of BCR microclusters amplifies BCR signaling whereas cSMAC formation and the movement of the microtubule network to the IS is thought to promote the internalization of BCR-bound Ags and their delivery to Ag-processing/MHC II-loading compartments. BCR-induced actin remodeling at Ag contact sites is dependent on the Arp2/3 complex, which is regulated by the upstream NPFs WASp, N-WASp, and the WRC. Importantly, sustained actin polymerization requires cofilin-dependent actin severing and depolymerization in order to liberate actin monomers for new polymerization. Similarly, microtubule growth and reorganization is dependent on the supply of tubulin dimers, which are normally sequestered by stathmin. Protein kinases and phosphatases can rapidly and reversibly regulate the activity and interactions of many proteins, including cytoskeletal regulators. Indeed, the activity of the Arp2/3 complex, the NPFs that activate the Arp2/3 complex, cofilin and stathmin are all regulated by serine/threonine phosphorylation.  The MEK/ERK pathway regulates cytoskeleton-dependent cellular processes (Figure 3.1). In terms of actin dynamics, MEK can inhibit Rho-dependent activation of ROCK [243]. Because ROCK activates LIMK, a negative regulator of cofilin, MEK is a positive regulator of cofilin and inhibiting MEK (and increasing the activity of the ROCK/LIMK module) reduces cofilin’s actin-severing activity [224,243–245]. ERK can potentially act upstream of multiple    50   Figure 3.1. Multiple downstream targets of the MEK/ERK pathway regulate actin and microtubule dynamics. BCR engagement by Ag results in the activation of multiple signaling cascades, one of them being the MEK/ERK pathway. MEK can inhibit RhoA-dependent activation of ROCK and mDia, which has profound effects on cofilin-mediated actin severing and on filopodia formation, respectively. ERK-dependent phosphorylation of the WAVE2 and Abi1 components of the WRC regulates its ability to activate the Arp2/3 complex, which drives the formation of lamellipodia. ERK also phosphorylates paxillin and FAK, proteins that integrate receptor signaling, actin dynamics, and integrin-mediated adhesion. Another ERK substrate is p90RSK, which phosphorylates and inhibits myosin phosphatase target subunit 1 (MYPT1). This increases myosin II-mediated tension that drives membrane protrusion. ERK also phosphorylates stathmin, which inhibits the ability of stathmin to sequester tubulin dimers and allows increased microtubule polymerization.     51 actin regulators. ERK-dependent phosphorylation of the WAVE2 and Abi1 components of the WRC are is important for the WRC to activate the Arp2/3 complex [219]. The Gold lab has also shown that paxillin, a scaffolding protein that links multiple cytoskeletal regulators, is phosphorylated by ERK after BCR engagement [246]. Schaller and colleagues have shown that the phosphorylation of paxillin by ERK is essential for macrophage cell spreading [247]. ERK also phosphorylates and regulates p90 RSK [193,248,249]. This allows RSK to then inhibit myosin phosphatase target subunit 1 (MYPT1), which increases myosin II-mediated tension for lamella expansion and edge dynamics that drive membrane protrusions [250].  The MEK/ERK pathway may also link the BCR to regulation of the microtubule network. Stathmin is a direct substrate of ERK [225,251] and our lab has shown that BCR clustering results in ERK-dependent phosphorylation of stathmin (Tim Jou, unpublished data). Thus, an ERK/stathmin module could link BCR signaling to changes in microtubule dynamics or organization. Based on these connections between MEK, ERK, and key cytoskeletal regulators, I tested the hypothesis that MEK activity and ERK activity are required for BCR-induced B cell spreading and actin remodeling, and for optimal APC-induced BCR signaling at the IS. The development of potent and selective cell-permeable inhibitors of MEK and ERK has facilitated the identification of downstream targets and cellular processes that are regulated by the MEK/ERK pathway. The MEK1/2 inhibitor U0126 binds in a non-competitive manner to a pocket that is adjacent to, but not overlapping with, the ATP-binding site and the ERK-binding site [252]. U0126 effectively blocks MEK-dependent phosphorylation and activation of ERK and has been a powerful tool for investigating the role of the MEK/ERK pathway [201]. U0126 is highly specific for MEK and shows little effect on the kinase activities of other protein kinases such as PKC, Raf, or JNK [253]. A more selective and highly potent non-ATP-competitive   52 inhibitor of MEK, the benzimidazole AZD6244 (Selumetinib), has been described recently [254]. The ability to directly probe the actions of ERK was enabled by the development of the first ERK-selective inhibitor FR180204 [255]. FR180204 inhibits both ERK1 and ERK2 in an ATP-competitive manner, thereby blocking signaling transduction via the ERK pathway [255]. To investigate the role of the MEK/ERK pathway in BCR-induced actin remodeling and IS formation, I used all three of these drugs, as well as ERK2-specific siRNA, as loss-of-function approaches. siRNA-mediated knockdown was also used to probe the role of stathmin in these processes.    Using confocal microscopy and quantitative image analysis, I demonstrated that actin remodeling is dependent on the MEK/ERK pathway and is required for optimal BCR-induced spreading on immobilized Ag. I also found that inhibiting the MEK/ERK pathway impairs APC-induced BCR signaling. In contrast, depleting stathmin did not appear to affect these processes.  3.2 Results  3.2.1 Optimizing the use of chemical inhibitors against the MEK/ERK pathway To investigate the role of the MEK/ERK signaling pathway in B cells, I employed selective chemical inhibitors of these kinases. To establish optimal concentrations of these inhibitors, I pre-treated A20 B cells for 1 hr with different concentrations prior to stimulating them with soluble anti-IgG. I then used immunoblotting with phospho-specific Abs to assess the phosphorylation of cofilin, as well as MEK-dependent phosphorylation of ERK and ERK-dependent phosphorylation of stathmin.  Because U0126 inhibits MEK, and ERK is a substrate of MEK, I expected that U0126 would prevent both the phosphorylation of ERK and its downstream substrates, including   53 stathmin. Indeed, I found that BCR-induced stathmin phosphorylation was inhibited by >80% by 10 µM U0126, indicating that concentrations greater than 10 µM would effectively inhibit ERK activity (Figure 3.2). The ERK inhibitor FR180204 blocks the enzymatic activity of ERK but should not block the MEK-dependent phosphorylation of ERK on its activating TEY motif. BCR-induced phosphorylation of stathmin was inhibited by >80% from 20-30 µM FR180204 whereas ERK phosphorylation was only substantially reduced at 30-60 µM FR180204 (Figure 3.2). I also assessed cofilin phosphorylation since MEK has been implicated in activating cofilin in rat kidney cells by inhibiting the ROCK/LIMK pathway, which in turn inhibits cofilin [224]. The inactive form of cofilin is phosphorylated by LIMK on S3 and activated cofilin is dephosphorylated on S3. I found that a high concentration (60 µM) of the MEK inhibitor U0126 resulted in increased cofilin phosphorylation, consistent with the idea that MEK activity promotes the dephosphorylation of cofilin. However, cofilin phosphorylation did not appear to be altered by the ERK inhibitor FR180204 (Figure 3.2). Moreover, treating A20 cells with 30 µM of either the MEK inhibitor or the ERK inhibitor did not reduce BCR-induced phosphorylation of Akt or HS1, compared to the DMSO vehicle control (Figure 3.3). Thus, these compounds appear to selectively inhibit the MEK/ERK pathway as opposed to inhibiting all BCR signaling pathways.      54   Figure 3.2. Effects of MEK and ERK inhibitors on BCR-induced phosphorylation of ERK, stathmin, and cofilin. (A) A20 B cells were incubated with or without 5 μg/mL anti-IgG for 15 min at 37ºC. Immunoblotting with phospho-specific Abs shows the levels of phospho-ERK (pERK), phospho-stathmin (pStathmin), and phospho-cofilin (pCofilin) in resting versus stimulated cells. (B) A20 B cells were pre-treated with increasing concentrations of the ERK inhibitor FR180204 or the MEK inhibitor U0126 for 1 hr. The cells were then incubated with 5 μg/mL anti-IgG for 15 min. Immunoblots show pERK and total ERK, pStathmin and total stathmin, and pCofilin and total cofilin. Actin was used as a loading control. A representative set of blots from one of 3 independent experiments is shown. (C) The band intensities for pERK, pStathmin, and pCofilin were normalized to the values for the total proteins for the same sample. This ratio is graphed as relative band densities for the blots shown in (B).     55      Figure 3.3. The ERK inhibitor FR180204 and MEK inhibitor U0126 does not inhibit BCR-induced phosphorylation of Akt or HS1. (A) A20 B cells were treated with 30 μM of either the ERK inhibitor FR180204 or the MEK inhibitor U0126 for 1 hr at 37ºC. Cells were then incubated with or without 5 μg/mL anti-IgG for 15 min at 37ºC. Immunoblots show pHS1 and total HS1 as well as pAkt and total Akt. A representative set of blots from one of 3 independent experiments is shown. The band intensities for (B) pAkt and (C) pHS1 were normalized to the values for the total proteins for the same sample. Mean +/- SD for three independent experiments.     56 When B cells respond to immobilized Ags on a rigid substrate, BCR signaling may be influenced by forces that are exerted on the BCR when it binds to the immobile Ag. As well, the cytoskeletal reorganization that drives B cell spreading could also impact BCR signaling. Therefore, before determining the effects of the MEK and ERK inhibitors on B cell spreading, I assessed whether immobilized anti-Ig activated the MEK/ERK pathway and whether the activation of MEK and ERK could be blocked by the same concentrations of inhibitors used in Figure 3.3. To address this question, A20 B cells were pre-treated with the inhibitors and then allowed to spread for 5-30 min in wells that had been coated with 2.5 μg/cm2 anti-IgG. Again,  phospho-ERK (pERK) levels were used as a readout for MEK activity and phospho-stathmin (pStathmin) levels were used as a readout for ERK activity. In control cells, robust phosphorylation of ERK and stathmin was observed when the cells were plated on immobilized anti-IgG. As for BCR stimulation by soluble anti-IgG, pre-treating A20 B cells with the MEK inhibitor completely blocked ERK phosphorylation and treatment with 30 μM of either the MEK or ERK inhibitor completely blocked the phosphorylation of stathmin at all time points (Figure 3.4). This indicates that stimulation of the BCR by immobilized anti-Ig activates the MEK/ERK/stathmin pathway and this can be effectively blocked by both the MEK inhibitor and ERK inhibitor.          57        Figure 3.4. Immobilized anti-Ig activates the MEK/ERK pathway – inhibition by the ERK inhibitor FR180204 and the MEK inhibitor U0126. A20 B cells were pretreated with DMSO or with 30 μM of the MEK inhibitor U0126 or the ERK inhibitor FR180204 for 1 hr. The cells were then allowed to spread on anti-IgG-coated coverslips for the indicated times. For the 0 min time point, cells were lysed directly with RIPA buffer instead of being added to anti-IgG-coated coverslips. Blots show phospho-ERK (pERK), phospho-stathmin (pStathmin), and total stathmin (loading control). Results from the one experiment that was performed are shown.          58 3.2.2 Inhibiting either MEK or ERK reduces B cell spreading on immobilized anti-IgG To explore the role of MEK and ERK in BCR-induced actin remodeling and B cell spreading, A20 B cells were treated with selective inhibitors of these kinases and then allowed to spread on a coverslip coated with anti-IgG antibodies. Rhodamine-phalloidin staining was used to image F-actin. This allowed us to identify the perimeter of the cell to calculate the cell area at the contact site with the coverslip. It also allowed us to visualize the characteristics of actin reorganization exhibited by B cells spreading on BCR ligands and the formation of a thick ring of peripheral actin surrounding an actin-depleted central region of the cell.  I found that inhibiting either MEK or ERK significantly reduced B cell spreading and altered the BCR-induced actin remodeling that drives cell spreading. A20 B cells that were treated with DMSO, the solvent control, exhibited progressive radial outward spreading from 5 min to 30 min, forming broad lamellipodia with a thick peripheral actin ring and clearance of actin from the center of the contact site. However, A20 cells that were treated with 30 µM of either the MEK inhibitor (U0126) or the ERK inhibitor (FR180204) exhibited significantly reduced spreading compared to the control cells at all time points, especially at the later times, i.e. 15 min and 30 min (Figure 3.5A-C). These concentrations of U0126 and FR180204 completely inhibited BCR-induced phosphorylation of ERK and stathmin (Figure 3.3) and Figure 3.6 shows that 30 µM U0126 caused maximal reduction in cell spreading. A lower concentration of U0126, 10 µM, partially inhibited the phosphorylation of ERK and stathmin (Figure 3.3) and also reduced the spreading of A20 B cells on immobilized anti-IgG, although to a lesser extent than 30 µM (Figure 3.6).   The reduced spreading of cells treated with the MEK inhibitor or the ERK inhibitor was associated with reduced actin clearance at the center of the cell as well as thinner peripheral F-  59 actin ring (Figure 3.5D-F). As B cells spread on rigid surfaces, the formation of lamellipodial protrusions is accompanied by depletion of F-actin structures for the center of the contact site. To quantify the extent of actin clearance, the F-actin staining was converted to a binary image. The periphery of the cell was used to calculate the total area of the cell at the contact site and after thresholding, the area of the cell without F-actin staining was quantified. This allowed us to calculate the percent of the total cell area that was cleared of F-actin. Compared to control cells, inhibiting the activity of either MEK or ERK reduced the extent to which actin was cleared from the center of the contact site after 30 min (Figure 3.5D,E). Thus, inhibiting the MEK/ERK pathway impaired BCR-induced actin reorganization at the Ag contact site. The peripheral ring of F-actin was that forms when B cells spread was also altered when the MEK/ERK pathway was inhibited. The width of the peripheral actin ring was measured at 5 random sites for each cell. For control cells, the mean peripheral actin ring width in 3 independent experiments was 2.67 μm whereas the peripheral actin rings in cells in which ERK or MEK was inhibited had mean widths of 1.9 µm and 1.5 μm, respectively (Figure 3.5F). Because cell spreading is driven by outward forces generated by the peripheral ring of branched actin, the thinner peripheral actin rings in cells in which MEK or ERK was inhibited could account for the decreased spreading exhibited by these cells.        60  Figure 3.5. Inhibition of the MEK/ERK pathway impairs BCR-induced cell spreading and actin reorganization. A20 B cells were pre-treated for 1 h with DMSO (control) or with 30 μM of either the ERK inhibitor FR180204 or the MEK inhibitor U0126. The cells were then allowed to spread on anti-IgG-coated glass coverslips for the indicated times and stained for actin (red) and tubulin (green). (A) Representative images. Scale bars: 20 μm.        61  Figure 3.5. Continued. A20 B cells were pre-treated for 1 hr with DMSO (control) or with 30 μM of either the ERK inhibitor FR180204 or the MEK inhibitor U0126. The cells were then allowed to spread on anti-IgG-coated glass coverslips for the indicated times and stained for actin (red). (B,C) A representative scatter plot of the cell areas for N > 100 cells per time point from one experiment is shown (panel B). Each dot is one cell and the bars represent the median and SD.  Panel C shows the mean +/- SEM for the median values from 3 experiments. (D,E) A representative scatter plot showing the percent of the central region of the cell that was depleted of F-actin for N > 100 cells per time from one experiment (panel D). Panel E shows the mean +/- SEM for the median values from 3 experiments. (F) The width of the peripheral actin ring was calculated for N > 75 cells that had been allowed to spread for 30 min. The graph shows the mean +/- SEM of the median values from 3 experiments. The Mann-Whitney U test (panels B, D) and Student’s paired T-test (panels C, E, F) was used to determine p values.       62                      Figure 3.6. The MEK inhibitor U0126 reduces B cell spreading on anti-IgG-coated coverslips in a dose-dependent manner. A20 B cells were pre-treated for 1 hr with the indicated concentrations of the MEK inhibitor U0126. The cells were then allowed to spread on anti-IgG-coated glass coverslips for 30 min before quantifying cell areas for N >50 cells per point. The graph shows mean +/- SEM for the median values from three independent experiments. Students paired T-test was used to determine p values. *p<0.05; **p<0.01.       0 10 30 60050100150200Concentration (µM)Cell Area (µm2 )30 min** **  63 3.2.3 MEK inhibition and ERK inhibition have distinct effects on actin organization When A20 B cells are plated on immobilized anti-IgG they spread in a radial manner, forming broad lamellipodial protrusions that are dependent on Arp2/3 complex-mediated branched actin polymerization [69]. The Arp2/3 complex normally competes with formins for ATP-loaded actin monomer. When Arp2/3 complex activity is inhibited, A20 B cells plated on immobilized anti-Ig exhibit disorganized peripheral actin organization with long filopodial protrusions that are characteristic of formin-dependent linear actin polymerization [69]. Because the ERK-dependent phosphorylation of the WRC is important for its ability to activate the Arp2/3 complex, I hypothesized that ERK inhibition might also result in increased formation of filopodia.  To test this, I pre-treated A20 B cells with either the MEK inhibitor or the ERK inhibitor before allowing the cells to spread on immobilized anti-IgG for 30 min. In addition to imaging the cells, I used an ImageJ plug-in to quantify the circularity of individual cells. Circularity is a measurement of how close to a perfect circle an object is and is calculated using the formula 4π(area)/(perimeter)2. A perfect circle has a circularity of 1.0 since the area is πr2 and the perimeter is 2πr. Circularity values <1 indicate how far an object deviates from being circular. I found that control A20 B cells that had spread on immobilized anti-Ig for 30 min were largely round and has a relatively smooth periphery. In contrast, the A20 B cells that had been pre-treated with the ERK inhibitor FR180204 exhibited a number of small filopodia-like protrusions and had a significantly lower circularity value than the control cells (Figure 3.7A,B). Because MEK is the upstream activator or ERK, I predicted that U0126-treated cells would have the same phenotype as FR180204-treated cells. Surprisingly, A20 B cells in which MEK was inhibited did not form filopodia and exhibited higher circularity values than the control cells (Figure 3.7A,B).   64 This could reflect ERK-independent effects of MEK, for example on the ROCK/LIMK/cofilin module, as shown in Figure 3.1. To further assess the altered spreading morphology that was observed when ERK activity was inhibited, I used stimulated emission depletion (STED) super-resolution microscopy to image the cells at higher resolution. Our lab has previously used this approach to observe the alteration of the peripheral actin structure following inhibition of the Arp2/3 complex [61]. A20 B cells were treated with DMSO as a control, or with the MEK inhibitor or the ERK inhibitor before being allowed to spread for 30 min on coverslips coated with anti-IgG (Figure 3.7C). As our lab has observed previously [61], control DMSO-treated cells exhibited radial spreading and formed a dense peripheral ring of highly branched F-actin that surrounded an actin-depleted central area. This actin-depleted region contained some thin actin filaments as well as a number of actin foci. Within the peripheral branched actin network of the lamellipodia, linear actin filaments, which can be nucleated by formins, extended perpendicularly from the edge of the cell to the inner face of the peripheral actin ring.  In contrast, inhibiting ERK or MEK altered this BCR-induced actin reorganization in distinct ways. When ERK activity was inhibited with FR180204, the cells did not form broad lamellipodia with branched actin organization. Instead the cells formed a number of long, loosely packed filopodia-like projection, as was seen by confocal microscopy. This altered actin organization is suggestive of reduced Arp2/3 complex, perhaps reflecting the role of ERK in activating the WRC. When MEK was inhibited by treating the cells with U0126, the cells displayed broad lamellipodia but exhibited reduced actin clearance from the center of the contact site as well as actin arcs running parallel to the inner face of the peripheral actin ring. In T cells, actomyosin arcs are assembled via formin-mediated linear actin polymerization [256]. Because a   65 similar expansion of actin arcs was not caused by inhibiting ERK in A20 B cells, my data suggests that inhibiting MEK may promote assembly of these arcs via pathways that are independent of ERK.    Figure 3.7. Inhibiting MEK or ERK activity alters peripheral actin structures.  A20 B cells were pre-treated with DMSO or with 30 μM of either the ERK inhibitor FR180204 or the MEK inhibitor U0126 for 1 hr and then allowed to spread for 30 min on coverslips coated with 2.5 μg/cm2 anti-IgG. The cells were then stained with Alexa Fluor 488-conjugated phalloidin. (A) Representative confocal microscopy images. Yellow triangles indicate filopodial protrusions. (B) Scatter plot showing the circularity index from one experiment. The mean +/- SD is shown for N >100 cells. The Mann-Whitney U test was used to determine p values. (C) Representative STED microscopy. Scale bars: 5 μm.     66 3.2.4 Inhibiting either MEK or ERK inhibits MTOC polarization but the ERK substrate stathmin is not required for B cell spreading The ERK substrate stathmin regulates microtubule growth and is required for TCR-induced MTOC polarization towards the IS [226]. I showed in Figures 3.2 and 3.4 that inhibiting either MEK or ERK completely blocked BCR-induced phosphorylation of stathmin. Phosphorylation of stathmin induces the release of tubulin dimers from stathmin and supports microtubule growth. I found that inhibiting either MEK or ERK activity reduced the percent of cells that exhibited MTOC polarization when they spread on immobilized anti-IgG. Tubulin staining was used to visualize the microtubule network and the MTOC was defined as a bright fluorescent spot at which microtubules converged. MTOC polarization was defined as the MTOC being present in the closest plane to the anti-IgG-coated coverslip. In control cells ~60% of the cells exhibited MTOC polarization. Cells treated with either the MEK inhibitor or the ERK inhibitor reduced the percent of cells with polarized MTOCs to ~40% (Figure 3.8). Thus, the MEK/ERK pathway, potentially, acting via stathmin, contributes to BCR-induced MTOC polarization. In T cells, phosphorylated stathmin localizes to the IS [226]. Due to stathmin’s proximity to the Ag contact site, and its role in MTOC polarization, I hypothesized that ERK-dependent phosphorylation of stathmin contributes to B cell spreading. If so, this could be one reason why BCR-induced spreading is reduced when the MEK/ERK pathway is inhibited. To assess the contribution of stathmin in cell spreading, I used siRNA to selectively deplete stathmin. Although immunoblotting showed that this reduced that the levels of stathmin protein by more that 80% (Figure 3.9A), no reduction in anti-Ig-induced spreading was observed, as compared to control cells (Figure 3.9B,C). Additionally, the peripheral actin organization did not seem to be   67 altered, compared to control cells, and the width of the peripheral actin ring was not reduced when stathmin was depleted (Figure 3.9E). This is in contrast to the impaired spreading and altered actin organization observed when chemical inhibitors were used to inhibit the activity of either MEK or ERK. This indicates that the MEK/ERK pathways regulates BCR-induced spreading in a manner that is independent of stathmin. Interestingly, depleting stathmin also did not impair BCR-induced polarization of the MTOC towards the Ag contact site (Figure 3.9E), in contrast to what has been reported in T cells [226].       68  Figure 3.8. Inhibition of the MEK/ERK pathway impairs BCR-induced MTOC polarization. A20 B cells were pre-treated for 1 h with DMSO (control) or with 30 μM of either the ERK inhibitor FR180204 or the MEK inhibitor U0126. The cells were then allowed to spread on anti-IgG-coated glass coverslips for 30 min before being immunostained for tubulin (green). (A) Representative image of tubulin staining in the confocal plane closest to the coverslip. MTOC polarization towards the immobilized anti-Ig is indicated by a bright point of microtubule convergence in this confocal slice. (B) The percent of cells exhibiting MTOC polarization was determined for N > 30 cells in each experiment. The mean +/- SEM from three independent experiments is shown. Students paired T-test was used to determine p values. *p<0.05; **p<0.01       69    Figure 3.9. Stathmin depletion does not affect B cell spreading on immobilized anti-IgG. A20 B cells were transfected with 2 μg of control (CTL) or stathmin (STM) siRNA. After 24 hr, the cells were allowed to spread on anti-IgG-coated coverslips for the indicated times and then stained for actin and tubulin. (A) Immunoblot shows stathmin depletion. (B) Representative images of the confocal plane closest to the coverslip showing tubulin (green) and actin (red) staining. Scale bars: 5 μm. (C) Representative scatter plot from one experiment showing the cell area at the Ag contact site. N >100 cells per point. The Mann-Whitney U test was used to calculate p values. ****p<0.0001, ns, not significant. (D) In each experiment the median value was determined for N >75 cells per condition. The graph shows the mean +/- SEM for these values from three independent experiments. Students paired T-test was used to determine p values. (E) Median width of the peripheral actin ring in cells that had spread for 30 min. For each cell, the width of the peripheral actin was measured at 5 random sites and the median width for at least 30 cells from 3 independent experiments was determined. The mean +/- SEM for these values from three independent experiments is shown.  (F) Percent of cells with the MTOC in the confocal plane closest to the glass coverslip. Mean +/- SEM for three experiments.    70 3.2.5 Inhibition of the MEK/ERK pathway inhibits BCR-induced spreading to a greater extent at sub-optimal densities of immobilized anti-IgG  Before assessing the effects of inhibiting the MEK/ERK pathway on B cell spreading in response to sub-optimal densities of immobilized anti-Ig. a newer and more selective MEK inhibitor was added to the repertoire of inhibitors for a more robust analysis of the role of MEK in regulating cell spreading. The optimal concentration required for Selumetinib to inhibit ERK and stathmin phosphorylation was determined by stimulating B cells with soluble anti-IgG and immunoblotting for phospho-ERK and phospho-stathmin. The phosphorylation of Akt was used to assess the Selumetinib non-selectively inhibits ERK-independent BCR signaling pathways. Selumetinib concentrations greater than 1 µM caused a significant reduction in BCR-induced phosphorylation of ERK and stathmin but had no effect on Akt phosphorylation (Figure 3.10).  Because coating the coverslips with 2.5 μg/cm2 anti-IgG might induce very strong BCR signals, I tested whether inhibiting MEK or ERK would have a greater effect on BCR spreading at lower, sub-optimal densities of anti-IgG. To test this, I coated the coverslips with sequential 2-fold dilutions of anti-Ig such that the density ranged from 0.625 μg/cm2 to 2.5 μg/cm2 anti-IgG. For control DMSO-treated A20 B cells, the mean contact area after 30 min was ~30% greater at the highest density of anti-IgG tested (2.5 μg/cm2) compared to the lower density of anti-IgG (0.625 μg/cm2), with the majority of the increase occurring between 0.625 μg/cm2 and 1.86 μg/cm2). This showed that these lower densities of anti-IgG were sub-optimal for inducing B cell spreading and represent a “sensitized” system for detecting the effects of inhibiting specific components of BCR signaling pathways. Using this approach, I found that the ERK inhibitor FR180204 caused a greater percent reduction in spreading area when the coverslips were coated with 0.625 μg/cm2 or 1.25 μg/cm2 anti-IgG than with high densities (Figure 3.11). At the highest    71       Figure 3.10 The MEK inhibitor Selumenitib inhibits anti-IgG-induced activation of the ERK/stathmin pathway. A20 B cells were pre-treated with increasing concentrations of the MEK inhibitor Selumenitib for 1 hr at 37ºC. The cells were then either left unstimulated (0 min) or stimulated with 5 μg/mL anti-IgG for 15 min at 37ºC. Immunoblots show pAkt and total Akt, pERK and total ERK, and pStathmin and total Stathmin from a single experiment.                 72    density of anti-IgG, inhibiting ERK reduced the mean cell spreading area by 12% whereas at 0.625 μg/cm2 anti-IgG inhibiting ERK reduced the mean cell spreading area by 12% whereas at 0.625 μg/cm2 anti-IgG inhibiting ERK resulted in a 40% decrease in the spreading area (Figure 3.11C). Similarly, the MEK inhibitor U0126 caused the greatest reduction in A20 B cell spreading at the lowest density of anti-IgG tested, 0.625 μg/cm2, where the mean spreading area was reduced by 40% (Figure 3.12). U0126 treatment also reduced A20 cell spreading when the coverslips were coated with 1.86 μg/cm2 and 2.5 μg/cm2, although to a lesser extent than when the anti-IgG coating density was 0.625 μg/cm2. However, in the same experiments inhibiting MEK had very little effect when the coverslips were coated with 1.25 μg/cm2 anti-IgG. The more specific MEK inhibitor, Selumetinib, which inhibited anti-IgG-induced phosphorylation of ERK and stathmin at concentrations of 1-30 µM (Figure 3.10) also inhibited A20 cell spreading but this was only evident when the coverslips were coated with 0.625 μg/cm2 anti-IgG, where spreading was reduced by 20%. At the highest density of anti-IgG, spreading was not significantly reduced (Figure 3.13). Nevertheless, these data show that using a sensitized system in which BCR stimulation is sub-optimal can help reveal pathways that are important for the spreading response. Together, these results indicate that the dose-dependent spreading of A20 B cells on immobilized anti-IgG involves the MEK/ERK pathway, particularly at sub-optimal densities of anti-IgG.       73    Figure 3.11. The ERK inhibitor FR180204 reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG. Coverslips were coated with the indicated amounts of anti-IgG. A20 B cells were pre-treated with DMSO or 30 μM of the ERK inhibitor FR180204 before being allowed to spread on the coverslips for 30 min. The cells were then fixed and stained with phalloidin-rhodamine. (A) Representative images are shown. Scale bar: 10 μm. (B) Representative scatter plot from one experiment showing the cell spreading area for N >75 cells per condition. The bars represent the median and SD. The Mann-Whitney U test was used to calculate p values. (C) Mean +/- SEM for the median cell area values from 3 independent experiments. Students paired T-test was used to determine p values.   74   Figure 3.12. The MEK inhibitor U0126 reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG. Coverslips were coated with the indicated amounts of anti-IgG. A20 B cells were pre-treated with DMSO or 30 μM of the MEK inhibitor U0126 before being allowed to spread on the coverslips for 30 min. The cells were then fixed and stained with phalloidin-rhodamine. (A) Representative images are shown. Scale bar: 10 μm. (B) Representative scatter plot from one experiment showing the cell spreading area for N >75 cells per condition. The bars represent the median and SD. The Mann-Whitney U test was used to calculate p values. (C) Mean +/- SEM for the median cell area values from 3 independent experiments. Students paired T-test was used to determine p values.    75  Figure 3.13 The MEK inhibitor Selumetinib reduces A20 B cell spreading to a greater extent at lower densities of immobilized anti-IgG. Coverslips were coated with the indicated amounts of anti-IgG. A20 B cells were pre-treated with DMSO or 3 μM of the MEK inhibitor Selumetinib before being allowed to spread on the coverslips for 30 min. The cells were then fixed and stained with phalloidin-rhodamine. (A) Representative images are shown. Scale bar: 10 μm. (B) Representative scatter plot from one experiment showing the cell spreading area for N >75 cells per condition. The bars represent the median and SD. The Mann-Whitney U test was used to calculate p values. (C) Mean +/- SEM for the median cell area values from 3 independent experiments. Students paired T-test was used to determine p values.    76 3.2.6 MEK/ERK inhibition reduces B cell spreading on Ag-coated coverslips Anti-Ig Abs that cluster the BCR and initiate BCR signaling and B cell activation responses have long been used to study BCR function. However, most anti-Ig Abs do not bind to the Ag-binding site of the BCR and therefore interact with this receptor in a fundamentally different manner than cognate Ag. Work by Reth and colleagues has suggested that the binding of either monovalent or polyvalent Ags to the Ag-binding site of the BCR induces a conformational change in the BCR that leads to receptor activation, which precedes and promotes BCR clustering [68,257–259].  Therefore, I tested whether the MEK/ERK pathway is important for Ag-induced B cell spreading. To do this, I coated coverslips with biotinylated HEL and assessed the ability of A20 D1.3 B cells, which express a transgenic BCR specific for HEL, to spread on this surface. Similar to when the coverslips were coated with anti-Ig, the B cells tended to spread more, i.e. greater contact area after 30 min, when the slides were coated with higher densities of HEL (Figure 3.14). Importantly, inhibiting either MEK or ERK reduced A20 D1.3 B cell spreading at all of the densities of HEL that were used to coat the coverslips. Thus, MEK activity and ERK activity are important for Ag-induced B cell spreading.     77  Figure 3.14. Inhibitors of the MEK/ERK pathway reduce A20D1.3 B cell spreading on Ag-coated coverslips. A20D1.3 B cells were pre-treated with DMSO, 30 μM of the ERK inhibitor FR180204, 30 μM of the MEK inhibitor U0126, or 3 μM of the selective MEK inhibitor Selumetinib. The cells were then allowed to spread for 30 min on coverslips that had been coated with the indicated amounts of Ag (biotinylated HEL). The cells were then fixed and stained with phalloidin-rhodamine. (A) Representative images are shown. Scale bar: 10 μm.   78   Figure 3.14. Continued. A20D1.3 B cells were pre-treated with DMSO, 30 μM Fr180204 (A,B,C), 30 μM U0126 (A,D,E) or 3 μM selumetinib (A,F,G) before being added to HEL-coated coverslips for 30 min. The coverslips were coated with the indicated amounts of biotinylated HEL. (B,D,F) Representative scatter plots from one experiment showing the cell spreading area for N >75 cells per condition. The bars represent the median and SD. The Mann-Whitney U test was used to calculate p values. (C,E,G) Mean +/- SEM for the median cell area values from 3 independent experiments. Students paired T-test was used to determine p values.   79 3.2.7 Effect of ERK2 depletion on B cell spreading  Inhibiting MEK and ERK activity with the small chemical inhibitors U0126 and FR180204, respectively, reduced B cell spreading on immobilized anti-Ig and Ag. However, because chemical inhibitors can have off-target effects, in particular inhibiting other kinases, I used siRNA-mediated depletion as a complementary approach to assess the role of the MEK/ERK pathway in BCR-induced cell spreading. B cells express both ERK1 and ERK2 [260]. Although ERK1 and ERK2 are thought to have many overlapping functions, the two isoforms also appear to have unique functions from one another. This is highlighted by studies showing that mouse embryos lacking ERK1 are viable whereas loss of ERK2 results in embryonic lethality [261,262]. Although B cells express both ERK1 and ERK2, the ERK2 isoform is more abundant [260] (Figure 3.15 A,B). Hence, I tested whether depleting ERK2 in A20 B cells would phenocopy the effects of treating these cells with chemical inhibitors of MEK or ERK and reduce the ability of the cells to spread on either immobilized anti-IgG or immobilized HEL.   To assess the contribution of ERK2 in B cell spreading, siRNA was used to selectively deplete ERK2 (Figure 3.15A,B). ERK2 expression was reduced by 70% with little effect on ERK1 expression. When control siRNA- and ERK siRNA-transfected A20 cells were allowed to spread on coverslips coated with 2.5 µg/cm2 anti-IgG, a modest reduction in cell contact area was observed at most time points (Figure 3.15C-E). The reduced spreading caused by depletion of ERK2 was most evident at 30 min, the latest time point that was assessed. This was similar to what was observed when A20 cells were treated with the MEK inhibitor U0126 or the ERK inhibitor FR180204 (Figure 3.5A-C). As in Figures 3.11-3.13, I also asked whether the effect of depleting ERK2 would be greater at sub-optimal densities of immobilized anti-IgG. Control   80 siRNA- and ERK2 siRNA-transfected cells were allowed to spread for 30 min on coverslips coated with 0.625 μg/cm2 to 2.5 μg/cm2 anti-IgG. Again, modest reductions in the spreading area were observed when ERK2 was depleted (Figure 3.16). This was most evident when the coverslips were coated with 1.25 μg/cm2 or 1.86 μg/cm2 anti-IgG. However, in these experiments depleting ERK2 did not reduce cell spreading when the coverslips were coated with 0.625 μg/cm2 anti-IgG. Overall, depleting ERK2 in A20 cells reduced the ability of the cells to spread on immobilized anti-IgG, but to a lesser extent than the small molecule inhibitors of MEK or ERK. Nevertheless, these findings are consistent with the idea that ERK2 contributes to the BCR-induced actin remodeling that promotes B cell spreading, at least in response to immobilized anti-IgG.  In contrast, partial depletion of ERK2 did not reduce the ability of A20 D1.3 B cells to spread on immobilized HEL (Figure 3.17) even though the chemical inhibitors of MEK and ERK did reduce the extent to which these cells spread on immobilized HEL (Figure 3.14). The greater inhibition of anti-IgG- and HEL-induced B cell spreading by the chemical inhibitors of MEK and ERK, compared to ERK2 siRNA, may reflect the fact that the chemical inhibitors completely suppressed the activity of both ERK1 and ERK2, as indicated by the complete inhibition of BCR-induced stathmin phosphorylation (Figures 3.2 and 3.4). In contrast, in ERK2 siRNA-transfected cells ERK1 was still expressed and ERK2 expression was only reduced by 70-80%.   This residual ERK activity may be sufficient to support BCR-induced cell spreading.       81  Figure 3.15. siRNA-mediated depletion of ERK2 reduces B cell spreading on anti-IgG-coated coverslips. A20 B cells were transfected with 4 μg of control (CTL) siRNA or ERK2 siRNA. After 24 h, the cells were allowed to spread for the indicated times on coverslips coated with 2.5 μg/cm2 anti-IgG. The cells were then stained for actin. (A) Immunoblot analysis of ERK2 and actin (loading control) levels. (B) Quantification of ERK1 and ERK2 protein levels in control siRNA- and ERK2 siRNA-transfected cells. The relative density of the band is expressed in arbitrary units (AU). (C) Representative images of the confocal plane closest to the coverslip showing actin staining. Scale bars: 10 μm. (D) Representative scatter plot from one experiment showing the cell area at the Ag contact site. N >100 cells per point. The Mann-Whitney U test was used to calculate p values. The data in panels A-D are from the same experiment. (E) For each experiment the median cell area was determined for N >75 cells per condition. The graph shows the mean +/- SEM for the median cell areas from three independent experiments.      82    Figure 3.16. siRNA-mediated depletion of ERK2 reduces B cell spreading on coverslips coated with different densities of anti-IgG. A20 B cells were transfected with 4 μg of control or ERK2 siRNA. After 24 hr, the cells were allowed to spread for 30 min on coverslips that had -been coated with the indicated concentrations of anti-IgG. The cells were then stained with rhodamine-phalloidin. (A) Representative images of the confocal plane closest to the coverslip showing and actin staining. Scale bar: 10 μm. (B) Representative scatter plot from one experiment showing the cell area at the Ag contact site. N >100 cells per point. The Mann-Whitney U test was used to calculate p values. ****p<0.0001, ns, not significant. (C) For each experiment the median value of the cell area was determined for N >75 cells per condition. The graph shows the mean +/- SEM for the median cell areas from three independent experiments.   83  Figure 3.17. siRNA-mediated depletion of ERK2 does not reduce A20D1.3 B cell spreading on Ag-coated coverslips. A20D1.3 B cells were transfected with 4 μg of control (CTL) siRNA or ERK2 siRNA. After 24 hr, the cells were allowed to spread for 30 min on coverslips that had been coated with the indicated concentrations of biotinylated HEL. The cells were then stained with rhodamine-phalloidin. (A) Immunoblot analysis of ERK2 and actin (loading control) levels. (B) Quantification of ERK1 and ERK2 protein levels in control siRNA- and ERK2 siRNA-transfected cells. The relative density of the band is expressed in arbitrary units (AU). (C) Representative images of the confocal plane closest to the coverslip showing actin staining. Scale bars: 10 μm. (D) Representative scatter plot from one experiment showing the cell area at the Ag contact site. N >100 cells per point. The Mann-Whitney U test was used to calculate p values. The data in panels A-D are from the same experiment. (E) In each experiment the median cell area was determined for N >75 cells per condition. The graph shows the mean +/- SEM for the median cell areas from three independent experiments.   84 3.2.8 Inhibition of the MEK/ERK pathway reduces cSMAC formation at the IS How the spatial organization of the BCR at the IS is controlled, and how this impacts BCR signaling and B cell activation, is not fully understood. The formation of BCR microclusters is a key determinant of B cell activation and the cytoskeleton regulates the formation and centralization of microclusters. Bolger-Munro et al. showed that while the BCR microclusters can still form when the Arp2/3 complex is depleted or inhibited, the centralization of microclusters is greatly impaired and reduces BCR signaling and B cell activation [69]. This work showed that the Arp2/3 complex activity is important for generating actin retrograde flow, which drives BCR microcluster centralization at the IS to create a cSMAC [69]. Because the WRC is one of the two major NPFs that activate the Arp2/3 complex, and ERK-dependent phosphorylation of WAVE2 and Abi1 is important for WRC activity, I asked if ERK activity is there for required for BCR microcluster centralization.  To address this question, A20D1.3 B cells, which express a HEL-specific BCR, were treated with either the MEK inhibitor U0126 or the ERK inhibitor FR180204. These cells were then added to COS-7 cells that express a fluorescently tagged, transmembrane form of HEL (mHEL-HaloTag) on their surface. After allowing the B cells to interact with these APCs for 3 to 30 min, the contact site between the A20D1.3 B cells and the COS-7 APCs was then imaged by confocal microscopy. In control DMSO-treated A20D1.3 B cells, distinct BCR-Ag microclusters were observed at 3 and 5 min after the B cells were added to the APCs (Figure 3.18A). These microclusters then underwent progressive coalescence into larger clusters. In many of the B cells, the BCR-Ag microclusters eventually merged into one large cSMAC. To quantify the percent of cells that formed a cSMAC, I used the approach developed previously in our lab [69]. At each time point, the number of BCR-Ag microclusters on each cell was calculated, as well as   85 the amount of Ag fluorescence associated with each cluster and the total Ag fluorescence contained in all of the clusters on each B cell. When at least 90% of the total fluorescence was contained in two or less clusters, the cell was deemed to have formed a cSMAC.    In control DMSO-treated A20D1.3 B cells, ~45-50% of the cells had formed a cSMAC by 10 min after contacting the APCs (Figure 3.18A,B). As a control for inhibition of cSMAC formation, the A20D1.3 B cells were pre-treated with the Arp2/3 complex inhibitor, CK-666. CK-666 locks the Arp2/3 complex in an open conformation, preventing it from binding existing actin filaments and nucleating the formation of new actin filaments [263]. Previous work in our lab has shown that CK-666 treatment, as well as siRNA-mediated depletion of the Arp3 subunit of the Arp2/3 complex, reduces cSMAC formation [69]. Consistent with these results, I found that CK-666 treatment impaired BCR microcluster centralization and coalescence and reduced cSMAC formation by more than 50%, such that less than 20% of cells formed a cSMAC at 10 min (Figure 3.18B). Importantly, A20D1.3 B cells that had been treated with the ERK inhibitor FR180204 also showed reduced BCR microcluster centralization and cSMAC formation, with only 30% of cells forming a cSMAC after 10 min (Figure 3.18B). However, the MEK inhibitor U0126 reduced cSMAC formation in only one of three independent experiments but had no effect in the other two experiments (Figure 3.18B). To determine whether inhibiting ERK activity prevented or just delayed cSMAC formation, I quantified the percent of cells that formed a cSMAC at later times. Regardless of how the A20D1.3 B cells were treated, cSMAC formation increased steadily over time (Figure 3.18C). The cells treated with the Arp2/3 inhibitor continued to exhibit reduced cSMAC formation at 15 and 30 min. In contrast, when ERK was inhibited the cells exhibited impaired microcluster centralization and cSMAC formation at early times but by 30 min displayed the same extent of cSMAC formation as the solvent control by the later time   86 point. Thus, preventing ERK activity appears to reduce and delay the initial centripetal movement of BCR-Ag microclusters and their coalescence into a cSMAC, although not to the same extent as when the Arp2/3 complex is inhibited.        87  Figure 3.18. Inhibiting ERK delays cSMAC formation but MEK inhibition does not. A20D1.3 B cells were pre-treated for 1 hr with DMSO (control) or with either 30 μM of the ERK inhibitor FR180204 or the MEK inhibitor U0126. The cells were then added to a monolayer of mHEL-HaoTag-expressing COS-7 APCs. The cells were fixed and permeabilized at the indicated times. Spinning disk confocal microscopy was used to image the contact site between the B cell and the APC. (A) Representative images are shown. Scale bar: 2 μm.  (B) Percent of cells that formed a cSMAC at 10 min. Cells were considered to have formed a cSMAC if >90% of the Ag fluorescence was contained in 1 or 2 clusters. Each dot represents the percent of cells that formed a cSMAC in a given experiment. The mean +/- SEM is shown for three independent experiments. (C) Percent of cells that formed a cSMAC at each time point. Mean +/- SEM from 3-4 independent experiments per time point. Students paired T-test was used to determine p values. ***p<0.001; ****p<0.0001; ns, no significance.    88 3.2.9 Inhibition of the MEK/ERK pathway reduces the ability of A20D1.3 cells to spread on APCs and decreases microcluster-based BCR signaling  Because B cell spreading on immobilized Ag was impaired following inhibition of MEK or ERK, I asked whether this was also the case when B cells interacted with APCs displaying Ags that were mobile within the plasma membrane. Staining for F-actin and imaging the B cell:APC contact site allowed me to visualize the perimeter of B cells that were in contact with APCs and calculate their area. I found that pre-treated A20D1.3 B cells with either the MEK inhibitor or the ERK inhibitor tended to reduce the ability of B cells to spread on APCs at all time points, especially at 5 and 10 min after the B cells were added to APCs (Figure 3.19).  Bolger-Munro et al. showed that the Arp2/3 complex-dependent centripetal movement of BCR-Ag microclusters amplifies BCR signaling output [69]. Inhibiting the activity of the Arp2/3 complex prevents actin retrograde flow, which is required for BCR microcluster centralization at the IS. When the centripetal movement of microclusters is blocked in this manner, BCR signaling output is reduced. Because ERK-mediated phosphorylation of the WRC may contribute to activation of the Arp2/3 complex, I asked whether inhibiting the MEK/ERK pathway would reduce microcluster-based BCR signaling at the IS. To do this, I quantified proximal BCR signaling at the contact site between A20D1.3 B cells and COS-7 APCs expressing mHEL-HaloTag. 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 [29]. This is required for the recruitment and activation of Syk, a PTK that phosphorylates multiple proteins that are critical for BCR signaling and B cell activation. APC-induced phosphorylation of CD79a/b was assessed by staining with an Ab that recognizes the phosphorylated ITAMs of both CD79a and CD79b. In addition to quantifying the total amount of phospho-CD79 (pCD79) associated with   89 BCR-Ag microclusters at the B cell:APC contact site for each cell, the total amount of Ag fluorescence in these clusters was quantified for the same cells. For each B cell, the total pCD79 fluorescence intensity in BCR-Ag clusters was then divided by the total fluorescence intensity of clustered Ag. This ratio represents the amount of BCR signaling output per unit of Ag within BCR-Ag microclusters.    A20D1.3 B cells were pre-treated with either DMSO (control), the ERK inhibitor FR180204 or the MEK inhibitor U0126 and then added to mHEL-HaloTag-expressing COS-7 APCSs. Inhibiting either MEK or ERK resulted a significant decrease in the amount of clustered pCD79 per cell at almost all time points (Figure 3.20B). This reduction in proximal BCR signaling could be due either to decreased clustering of Ag into BCR microclusters or decreased BCR signaling output per unit of Ag gathered. Inhibiting ERK did not alter Ag gathering, except for a minor reduction at the 3 min time point (Figure 3.20C). Because ERK inhibition reduced the amount of clustered pCD79 fluorescence, but not the amount of Ag fluorescence, cells treated with the ERK inhibitor exhibited less BCR signaling per unit of clustered Ag than the control cells, with this being statistically significant at the 5, 10, and 15 min time points (Figure 3.20D). In contrast, inhibiting MEK by treating the B cells with U0126 reduced both pCD79 fluorescence (Figure 3.20E) and the amount of Ag that was gathered into clusters (Figure 3.20F). As a result, except at the 30 min time point, BCR signaling output per unit of Ag gathered in cells was similar, and sometimes higher, in the cells in which MEK was inhibited than in the control cells (Figure 3.20G). Hence, both inhibiting MEK and inhibiting ERK reduces the amount of APC-induced pCD79 phosphorylation for different reasons. Inhibiting ERK reduced BCR signaling output per amount of Ag gathered whereas inhibiting MEK reduced Ag gathering, leading to less microcluster-based BCR signaling.   90  Figure 3.19. B cell spreading on the surface of APCs is reduced following inhibition of MEK or ERK. A20D1.3 B cells were pre-treated with DMSO or with 30 μM of the ERK inhibitor FR180204 or the MEK inhibitor U0126 for 1 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 and phalloidin. (A) Representative images of B cells that had interacted with APCs for 5 min. Anti-phospho-CD79 fluorescence is shown. The perimeter of each cell is identified with the yellow line. Scale bar: 2 µm. (B) Representative dot plot from a single experiment where each dot represents the area of a single B cell interacting with a COS-7 APC. For each treatment condition, N> 30 cells were analyzed for each time point. (C) Mean +/- SEM for the median cell area for APC-associated A20D1.3 B cells from 3 independent experiments. In all experiments, N> 30 cells were analyzed for each point.      91                        Figure 3.20. Inhibition of either MEK or ERK decreases BCR signaling in response to APC-bound Ags. A20D1.3 B cells were pre-treated with DMSO (grey), 30 μM of the ERK inhibitor FR180204 (orange) or 30 μM of the MEK inhibitor U0126 (red) for 1 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (pCD79). (A) Representative images of A20B1.3 cells that had interacted with the APCs for 5 min. Scale bar: 2 µm.    92     93 Figure 3.20. Continued. A20D1.3 B cells were pre-treated with DMSO (grey), 30 μM of the ERK inhibitor FR180204 (orange) or 30 μM of the MEK inhibitor U0126 (red) for 1 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (pCD79). (B-G) For each cell, the total amount of clustered pCD79 fluorescence (panels B and E) and clustered Ag fluorescence (panels C and F) at the B cell-APC contact site was quantified. These values were used to calculate BCR signaling output, which is the ratio of clustered pCD79 fluorescence divided by the clustered Ag fluorescence (panels D and G) for each cell. The scatter plots show the background-corrected fluorescence intensity or normalized BCR signaling output for N> 50 cells per point. The median and interquartile ranges are shown. The Mann-Whitney U test was used to calculate p values. *p<0.05; **p<0.01; ***p<0.001; ****, p<0.0001; ns, not significant.                 94 3.2.10 ERK2 depletion does not significantly alter B cell spreading on APCs or APC-induced BCR signaling    I found that inhibiting ERK activity using a cell-permeable chemical inhibitor reduced APC-induced B cell spreading, BCR signaling, and microcluster centralization. To try to confirm these findings, I used siRNA to selectively deplete ERK2 (Figure 3.21A). The levels of ERK2 protein were reduced by 88%, with minimal effects on ERK1 levels. When control siRNA- and ERK2 siRNA-transfected A20D1.3 B cells were added to mHEL-HaloTag-expressing COS-7 cells, no consistent change in cell contact area was observed (Figure 3.21C-E). In one of three experiments ERK2 depletion reduced B cell spreading on the APCs at the 3 min time point and the same was true in two experiments at the 10 min time point. However, unlike the MEK and ERK inhibitors, depleting ERK2 in A20D1.3 B cells did not consistently reduce the ability of the cells to spread on COS-7 cells. As for spreading on immobilized anti-Ig, this may reflect the fact that the chemical inhibitors completely suppressed the activity of both ERK1 and ERK2, as indicated by the complete inhibition of BCR-induced stathmin phosphorylation (Figures. 3.2 and 3.4). In contrast, ERK2 siRNA-transfected cells still expressed ERK1 and ERK2 expression was only reduced by 80-90%. This residual ERK activity may be sufficient to support BCR-induced cell spreading.   To determine if depleting ERK2 impaired cSMAC formation or APC-induced BCR signaling, control siRNA- and ERK siRNA-transfected A20D1.3 B cells were added to mHEL-HaloTag-expressing COS-7 cells and stained for pCD79. The amount of clustered pCD79 clustered Ag at the B cell:APC contact site, as well as the amount of BCR signaling per unit of gathered, was quantified as in Figure 3.20. In both control and ERK2-depleted cells, BCR-Ag microclusters formed rapidly at the B cell:APC contact site and co-localized with pCD79 clusters   95 (Figure 3.22A). The ERK2-depleted cells exhibited only small differences in the amount of clustered pCD79 and Ag at the 3 and 5 min time points (Figure 3.22B,C). The BCR signaling output ratio was significantly reduced in the ERK2-depleted A20D1.3 B cells at the 3 min time point but slightly higher at the 5 min time point (Figure 3.22D), perhaps reflecting a delay in the centripetal movement of BCR-Ag microclusters, which is thought to amplify microcluster-based BCR signaling. However, the control and ERK2-depleted cells formed cSMACs to the same extent (Figure 3.22E). Hence substantial depletion of ERK was not sufficient to reduce APC-induced cSMAC formation and BCR signaling to the same extent as treating the cells with the highly potent ERK inhibitor FR180204.     96   Figure 3.21. Depleting ERK2 does not reduce B cell spreading on APCs. A20D1.3 B cells were transfected with 4 μg of control (CTL) siRNA or ERK2 siRNA and then cultured for 24 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (pCD79) and phalloidin. (A) Representative ERK1/2 and actin (loading control) blots. (B) Quantification of ERK1 and ERK2 protein levels in control siRNA- and ERK2 siRNA-transfected cells. The relative densities of the bands are expressed in arbitrary units (AU). (C) Representative images of A20B1.3 cells that had interacted with the APCs for 5 min. Actin staining (yellow) shows the cell perimeter. Scale bar: 2 μm. (D) Representative scatter plot from one experiment showing the cell areas at the APC contact site. N >30 cells per point. The Mann-Whitney U test was used to calculate p values. (E) In each experiment the median cell area was determined for N >30 cells per condition. The graph shows the mean +/- SEM for the median cell areas from three independent experiments. *p<0.05; ****p<0.0001; ns, not significant.   97    98 Figure 3.22. Depleting ERK2 does not impair APC-induced BCR signaling or cSMAC formation. A20D1.3 B cells were transfected with 4 μg of control (CTL) siRNA or ERK2 siRNA and then cultured for 24 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (pCD79). (A) Representative confocal images of the B cell:APC contact site. Scale bars: 2 μm. (B-D) Scatter plot graphs show the background-corrected fluorescence intensity of clustered pCD79 (panel B) and Ag (panel C) at the B cell-APC contact site. For each cell, the ratio of clustered pCD79 fluorescence divided clustered Ag fluorescence was calculated (panel D). For each point the median and SD are shown for N> 50 cells. The Mann-Whitney U test was used to calculate p values. (E) Percent of cells that formed a cSMAC, as defined by >90% of the Ag fluorescence intensity being contained in 1-2 clusters. N >50 cells per condition. The graph shows the mean +/- SEM for these values for three independent experiments. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant.                      99 3.2.11 Inhibition of MEK or ERK in primary B cells impacts cell spreading on APC and reduces or delays APC-induced BCR signaling   Inhibition of either MEK or ERK in A20D1.3 B cells reduced cell spreading on APCs, and ERK inhibition reduced both cSMAC formation and APC-induced BCR signaling (Figure 3.20). To extend this to a more physiological system, I next asked whether the same was true in primary B cells. B cells isolated from MD4 mice were added to COS-7 APCs expressing mHEL-HaloTag and cell spreading area, cSMAC formation, and proximal BCR signaling were quantified. Inhibiting MEK reduced the ability of the primary B cells to spread on the surface of the APC, especially at earlier time points, whereas inhibiting ERK had little effect (Figure 3.23B-C). This is in contrast to the A20D1.3 treated with chemical inhibitors, where both the MEK and ERK inhibited cells reduced cell spreading on COS-7 cells (Figure 3.19). However, in Figure 3.17 both the FR180204 and U0126 treated cells reduced spreading, but the MEK inhibitor U0126 had a greater effect, consistent to what is observed in Figure 3.23. In regard to BCR-induced B cell signaling in primary B cells, DMSO treated control cells displayed maximal pCD79 levels at 3 min after being added to APCs and a rapid decline thereafter (Figure 3.24B and Figure 3.25B). ERK inhibition appeared to delay signaling, as these cells exhibited reduced pCD79 levels at 3 min (Figure 3.24B). In the FR180204-treated cells, pCD79 levels were lower than in the control cells at 3 min after the B cells were added to the APCs but peaked at 5 min, at which time pCD79 levels were higher in the FR180204-treated cells than in the control cells. This suggests that inhibiting ERK activity delays APC-induced BCR signaling. Because ERK inhibition did not affect Ag gathering (Figure 3.24C), the cells with reduced ERK kinase activity had reduced BCR signaling output per unit of gathered Ag gathered at 3 min but higher ratios at 5 min, compared to the control cells (Figure 3.24D). Thus,   100 as shown in A20D1.3 B cells, ERK activity amplifies BCR signaling during the initial stages of B cell:APC interactions. Furthermore, the average number of clusters of Ag gathered at the B cell: APC contact was determined (Figure 3.23E). This is an alternative method of looking at cSMAC formation, ie where the mean number of clusters becomes less than or equal to 2. Control cells had a maximum average number of clusters of 4 at 3 min, while ERK inhibited cells averaged 5 at the same time point. This indicates the primary B cells treated with the ERK inhibitor have delayed coalescence of BCR-Ag microclusters at the IS. This is consistent with the BCR-signaling results, as the coalescence of BCR microclusters into a cSMAC induces increased signaling, and a reduction in BCR-microcluster centralization impairs proximal signaling events. This data suggests that ERK activity is important for the amplification of early BCR events of phosphorylation of the CD79a/b ITAMs required for B cell activation.  Inhibition of MEK activity in primary B cells also resulted in reduced pCD79 levels and BCR signaling generated per unit of gathered Ag at the 3, 5, and 10 min time points, compared to control cells (Figure. 3.25B,D). However, at 15 min after adding the B cells to the APCs, the amount of Ag gathered was lower in the U0126-treated cells (Figure 3.25C) but the total pCD79 and BCR signaling output ratio were higher than in control cells (Figure. 3.25B,D). MEK inhibition also reduced pCD79 levels at early time points in the A20D1.3 B cells and impaired Ag gathering at later times points (Figure 3.20E,F). Although microcluster coalescence and cSMAC formation was not reduced or delayed in the primary B cells (Figure 3.25, E), MEK inhibition appears to alter the kinetics of BCR signal amplification at the IS.       101  Figure 3.23. Inhibiting MEK in primary B cells reduces cell spreading on APCs. Primary murine splenic B cells were pretreated for 1 hr with DMSO (control) or with 30 μM of the ERK inhibitor FR180204 or the MEK inhibitor U0126 for 1 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained for pCD79 and for actin to define the perimeter of the B cell. (A) Representative images from (A) Representative images of B cells that had interacted with APCs for 5 min. pCD79 staining is shown. The perimeter of each cell is identified by the yellow line. Scale bars: 2 μm. (B) Representative dot plot from a single experiment where each dot represents the area of a single B cell interacting with a COS-7 APC. For each treatment condition, N> 30 cells were analyzed for each time point. (C) Mean +/- SEM for the median cell area for APC-associated splenic B cells from 3 independent experiments. In all experiments, N> 30 cells were analyzed for each point.          102      103 Figure 3.24. ERK inhibition with 30 μM FR180204 delays early APC-induced BCR signaling. Primary B cells were pre-treated with DMSO (grey) or with 30 μM of the ERK inhibitor FR180204 (orange) for 1 hr. The cells were then added to COS-7 cells expressing the mHEL-HaloTag Ag (red). The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (green). (A) Representative images from a single experiment are shown. (B and C) Dot plot graphs show the background-corrected fluorescence intensity of clustered pCD79 (panel B) and Ag (panel C) at the B cell-APC contact site. (D) For each cell, the ratio of pCD79 fluorescence intensity was divided by the Ag fluorescence intensity and the ratio was graphed. For panels B-D, N> 75 cells per point. The median and interquartile ranges are shown. The Mann-Whitney U test was used to calculate p values. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant. (E) In each experiment the median number of distinct Ag clusters per cell was determined for N >75 cells per condition. Each dot is the median value from one experiment. The graph shows the mean +/- SEM for the median values from three independent experiments.                       104          105 Figure 3.25. MEK inhibition reduces early APC-induced BCR signaling. Primary B cells were pre-treated with DMSO (grey) or with 30 μM of the MEK inhibitor U0126 (red) for 1 hr. The cells were then added to COS-7 cells expressing the mHEL-HaloTag Ag (red). The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (green).  (A) Representative images from a single experiment are shown.  (B and C) Dot plot graphs show the background-corrected fluorescence intensity of clustered pCD79 (panel B) and Ag (panel C) at the B cell-APC contact site. (D) For each cell, the ratio of pCD79 fluorescence intensity was divided by the Ag fluorescence intensity and and the ratio was graphed. For panels B-D, N> 75 cells per point. The median and interquartile ranges are shown. The Mann-Whitney U test was used to calculate p values. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant (E) In each experiment the median number of distinct Ag clusters per cell was determined for N >75 cells per condition. Each dot is the median value from one experiment. The graph shows the mean +/- SEM for the median values from three independent experiments.                       106 3.2.12 Stathmin is not important for BCR microcluster centralization or APC-induced BCR signaling  Our lab has shown that microtubules are required for the centralization of BCR microclusters into cSMACs at the IS [264]. Dynein motor proteins mediate the movement of BCR-Ag microclusters along microtubules from the periphery of the cell to the center of the IS [72]. Stathmin is a microtubule regulating protein that is directly activated by ERK and I showed that inhibiting ERK activity reduces MTOC polarization towards anti-IgG-coated coverslips (Figure 3.8). Because inhibiting ERK with FR180204 reduces cSMAC and APC-induced BCR signaling, I asked whether stathmin acts downstream of ERK to support these responses. To test this, I used siRNA to deplete stathmin in A20D1.3 B cells and then analyzed their responses to COS-7 APCs expressing mHEL-HaloTag. In both control siRNA-transfected A20D1.3 B cells and stathmin-depleted cells, BCR-Ag microclusters formed rapidly at the B cell:APC contact site and co-localized with pCD79 clusters (Figure 3.26A). Both control and stathmin-depleted cells exhibited maximum total pCD79 and BCR signaling per unit of clustered Ag at 5 min after the B cells were added to the APCs. Overall, stathmin depletion did not cause obvious differences in pCD79, Ag gathering, or BCR signaling output compared to control cells at the 3, 5, 10, or 15 min time points (Figure 3.26B-D). As well, stathmin depletion did not impair cSMAC formation (Figure 3.26E). In both the control and stathmin-depleted >40% of the cells formed a cSMAC by 10 min and >60% by 30 min (Figure 3.26E). These findings suggest that stathmin does not contribute to BCR microcluster organization or signaling at the IS and is not responsible for the reduced microcluster centralization and APC-induced BCR signaling that occurs when ERK activity is inhibited. Thus, ERK must regulate these processes via substrates other than stathmin.     107                              Figure 3.26. Depleting stathmin does not affect APC-induced microcluster formation, microcluster-based BCR signaling, or cSMAC formation. A20D1.3 B cells were transfected with 2 μg of control siRNA or stathmin siRNA and then cultured for 24 hr. The cells were then added to COS-7 APCs expressing the mHEL-HaloTag Ag. The cells were fixed and permeabilized at the indicated times and then stained with anti-phospho-CD79 (pCD79). (A) Representative confocal images of the B cell:APC contact site. Scale bars: 5 μm. (B-D) Scatter plot graphs show the background-corrected fluorescence intensity of clustered pCD79 (panel B) and Ag (panel C) at the B cell-APC contact site. For each cell, the ratio of clustered pCD79 fluorescence divided clustered Ag fluorescence was calculated (panel D). For each point the median and SD are shown for N >53 cells. (E) Percent of cells that formed a cSMAC, as defined by >90% of the Ag fluorescence intensity being contained in 1-2 clusters. N >53 cells per condition. The graph shows the mean +/- SEM for these values for three independent experiments.    108 Chapter 4: Discussion 4.1 Summary of the main findings  How ERK signaling controls cytoskeletal dynamics to regulate the B cell response to both immobilized and mobile APC-bound Ags is not known. In chapter 3, I showed that the MEK/ERK pathway is important for B cell spreading, peripheral actin organization, and APC-induced BCR microcluster centralization and signaling. Using quantitative image analysis of microscopy data, I showed that inhibiting the MEK/ERK pathway significantly reduces cell spreading, in response to both APC-bound and immobilized Ags and that this is not dependent on the downstream substrate stathmin. Moreover, the morphology of spreading cells is altered following chemical inhibition of both MEK and ERK and the organization of the actin cytoskeleton was changed. Additionally, I found that ERK activity is important for BCR-Ag microcluster centralization at the IS, but that this is not dependent on MEK activity. Furthermore, both MEK and ERK inhibition reduced or delayed proximal BCR signaling events following interacting with an APC in A20D1.3 B cells and in primary B cells. Taken together, these findings support the idea that ERK signaling regulates cytoskeletal dynamics in B cells (Fig 4.1).  In the next sections, I discuss several potential mechanisms by which ERK may be regulating Arp2/3 complex activity or other regulatory proteins to control actin dynamics and thus signaling output of BCR microclusters. This includes stabilizing BCR microsignalosomes, increasing and maintaining BCR clustering, controlling the interactions of BCR microclusters with the co-receptor CD19, and exerting mechanical forces on Ag-bound BCRs. Additionally, ERK may be important for regulating microtubule dynamics, which is also important for BCR microcluster centralization.    109 Table 4.1 Summary of effects caused by MEK/ERK inhibition or depletion  Process LOF approach    ERK inhibitor ERK2 siRNA MEK inhibitor A20 cell spreading on immobilized anti-IgG Reduced spreading  Reduced spreading Reduced spreading   A20D1.3 cell spreading on immobilized HEL Reduced spreading No Change Reduced spreading A20D1.3 cell spreading on APCs Reduced spreading No Change Reduced spreading     A20D1.3  clustered pCD79 on APCs Reduced signaling No change Reduced signaling Primary B cell clustered pCD79 on APCs Delayed signaling N/A Reduced signaling     A20D1.3  clustered Ag on APCs No change  No change Reduced signaling Primary B cell clustered Ag on APCs No change  Not assessed Reduced signaling     A20D1.3  pCD79/Ag on APCs (signal amplification) Reduced signaling  No change  No change  Primary B cell clustered Ag on APCs (signal amplification) Delayed signaling  Not assessed Reduced signaling     A20D1.3  cSMAC formation on APCs Delayed No change No change Primary B cells  cSMAC formation on APCs Delayed  Not assessed No change     110        Figure 4.1. Proposed roles of MEK and ERK in regulating actin dynamics. ERK may promote branched actin polymerization by phosphorylating the WAVE2 and Abi1 components of the WRC. This is required for the WRC to activate the Arp2/3 complex. Additionally, MEK may inhibit LIMK, which would result in increased cofilin activity, thus altering actin dynamics independently from ERK. Image adapted from M. Gold.               111 4.1.1 Regulation of B cell spreading and actin dynamics by ERK Cell spreading is a complex process that requires the dynamic spatiotemporal integration of signals from receptors to coordinate cell polarity and cytoskeletal remodeling. In this thesis, I showed that ERK activity is important for B cell spreading on immobilized and APC-bound Ag. In cells in which either MEK or ERK was inhibited, the thickness of the peripheral actin ring was greatly reduced. To generate a pushing force, cells use the energy of actin polymerization [85]. The assembly of branched actin filament networks provides the pushing forces required for cell spreading, and these are the structures that form thick peripheral actin rings. Because the peripheral actin ring exerts force on the plasma membrane to allow B cells to spread, if the ring is not as thick there may be decreased outward force exerted on the plasma membrane, and this may explain why cells in which either MEK or ERK are inhibited spread less on immobilize and APC-bound Ag. Further studies are required to elucidate the mechanisms by which ERK controls cell spreading in B cells. ERK-mediated phosphorylation is required for the WRC to activate the Arp2/3 complex. This may be one of the main mechanisms by which ERK contributes to BCR-induced spreading. To support this idea, we could directly test whether treating B cells with the MEK or ERK inhibitors results in reduced phosphorylation of the WAVE2 and Abi1 components of the WRC. Our lab has preliminary data that WAVE2 depletion reduces spreading in A20 B cells (unpublished data), and recent work has shown that HEM1-deficicent B cells exhibit defective spreading [134]). If ERK acts primarily through WRC phosphorylation to promote B cell spreading, then experiments using ERK inhibitors in conjunction with depleting components of the WRC should not further reduce spreading compared to cells treated with just the chemical inhibitor or depletion alone. However, if the combination of depleting components of the WRC,   112 such as WAVE2, and chemically inhibiting ERK further reduces cell spreading, this would indicate that ERK acts on other substrates in addition to the WRC to control processes such as cell spreading.  ERK may also promote B cell spreading by targeting other cytoskeletal regulatory protein such as FAK, paxillin, and p90 Ribosomal S6 Kinase (RSK). FAK has been shown to control cell spreading and the formation of protrusive lamellipodia via a direct association with the Arp2/3 complex [265,266]. FAK binds directly to Arp3 and this association can enhance Arp2/3-dependent actin polymerization. FAK can also regulate actin cytoskeleton dynamics by coordinating the activation of the Rac, Cdc42, and RhoA GTPases [267]. FAK also plays a role in discriminating substrate stiffness [268], that governs mechanosensitive-dependent lymphocyte activation [238,269–271]. As a result, FAK-deficient B cells have been shown to have reduced spreading [246,268].  Paxillin is best known as a component of focal adhesions and is phosphorylated in response to BCR signaling [246], as well as provideing docking sites that facilitate the assembly of multiprotein complexes that include FAK [272,273]. Paxillin recruits multiple cytoskeletal regulators, such as Rac and Cdc42, that promote cell adhesion and spreading [274]. The proteins that paxillin interacts with are involved in the organization of the cytoskeleton and range from vinculin and talin, that bind  directly to the cytoskeleton, and to regulators of cytoskeletal dynamics such as ADP-ribosylation factor, GAP and PAK [272]. Serine phosphorylation of paxillin has been reported to regulate protrusion dynamics in migrating cells [275]. ERK has been shown to phosphorylate paxillin on S106, S231, and S290 [276] and this promotes cell movement, presumably by promoting the assembly of adhesive complexes and associated actin structures [158]. Additionally, our lab has shown that phosphorylated paxillin co-localizes with   113 BCR-Ag microclusters (Kate Choi, unpublished data). Hence, ERK-dependent phosphorylation of paxillin could contribute to BCR-induced cell spreading. To test this hypothesis, paxillin could be depleted from B cells using siRNA or CRISPR technology or replaced with a form that cannot be phosphorylated at the ERK phosphorylation sites such as S106A, S231A and S290A. B cells with either the paxillin depletion or non ERK phosphorylatable mutation could then spread on anti-Ig coated coverslips and compared for spreading and morphology against  WT cells.   While branched actin polymerization at the cell periphery that exerts forces on the plasma membrane drive cell spreading, contractile forces generated by myosin II motors pull on the actin network to increase membrane tension, limit the extension of membrane protrusions, and promote their retraction [277–279]. By enhancing branched actin polymerization at the cell periphery, ERK activity can stabilize membrane protrusions by overcoming myosin-based membrane tension and contraction [154,280]. Inhibiting the MEK/ERK pathway in B cells would reduce WRC-dependent actin polymerization by the Arp2/3 complex, which decreases the generation of outward forces that promote cell spreading and enhances the ability of myosin-based contractility to limit membrane protrusion.   When lymphocytes interact with APCs, they also undergo an initial spreading response that is followed by membrane retraction that is driven by myosin-based contractility [59,76,281]. This membrane contraction may contribute to the centralization of BCR microclusters and the formation of a cSMAC. ERK can increase myosin-based contractility by activating p90 RSK, which then phosphorylates MYPT1 on S507 [250]. This modification of MYPT1 prevents it from binding to myosin and inhibiting its activity. Hence, when B cells interact with APCs, ERK may contribute to both the initial cell spreading, as described in the previous paragraph, and to   114 the subsequent membrane contraction that drives cSMAC formation.  To test whether the ERK/p90 RSK/MYPT1 pathway contributes to B cell contraction, one could express a mutant form of MYPT1 with an S507A mutation in A20 cells and determine if that enhances B cell spreading on immobilized anti-Ig or prevents B cell contractility and cSMAC formation when B cells interact with APCs.      4.1.2 Differences between the effects of the MEK inhibitor and the ERK inhibitor may reflect MEK-dependent regulation of cofilin that is independent of ERK Cofilin activity is necessary for coupled actin filament disassembly and polymerization [282] and regulates the size and shape of actin protrusions in cells [283–285]. Actin retrograde flow requires the disassembly of aged actin filaments by actin disassembly factors [283,284,286]. Inactivation of cofilin decreases the speed of actin retrograde flow [286–288], a process necessary for BCR coalescence at the IS. MEK can inhibit LIMK activity and recently, a role for LIMK in IS formation in T cells has been described [289]. These authors demonstrated that inhibiting LIMK reduces the amount of phosphorylated, and thus inactive cofilin and increases Ca2+signaling in response to APCs. Because I observed differences between the effects of the MEK inhibitor and the ERK inhibitor on B cell morphology, as well as in APC-induced microcluster centralization and BCR signaling, it is possible that blocking MEK may decrease cofilin activity whereas blocking ERK does not. In fact, a MEK-cofilin signaling module operates within the lamellipodium of migrating T cells [101]. This group also showed that MEK controls the speed and directionality of T cell migration by regulating cofilin. Our lab has previously shown that cofilin activity is required for B cell spreading on immobilized Ag [290]. A MEK-LIMK module may therefore contribute to this response.   115 4.1.3 Stathmin cannot be ruled out for its role in B cell spreading and APC-induced BCR signaling  Stathmin is an oncoprotein that directly regulates microtubule dynamics through phosphorylated inactivation and dephosphorylated activation [291]. As a microtubule destabilizing protein that contributes to tubulin dimer/polymer sequestration, stathmin controls microtubule dynamics through several mechanisms. One mechanism is that it stimulates microtubule dynamics by increasing catastrophes at the plus end, where the polymer interacts with actin filaments [292,293]. Once phosphorylated by ERK, stathmin becomes inactivated and releases itself from tubulin polymers, which can then contribute to microtubule assembly.  I used MEK and ERK inhibitors to block the phosphorylation of stathmin on S25, which maintains stathmin in an active state. Although inhibiting the MEK/ERK pathway reduced B cell spreading and APC-induced BCR signaling, siRNA-mediated depletion of stathmin did not. This suggests that stathmin, and perhaps microtubules, are not involved in these processes. Our lab has previously shown that treating A20 B cells with nocodazole to depolymerize microtubules has no effect on spreading on immobilized anti-IgG (data unpublished). However, our lab has found that treating A20 cells with nocodazole prevents cSMAC formation [73]. This it likely because microtubule stability influences motor protein activity [294], which is required to move BCR-Ag microclusters from the periphery of the IS towards the center of the IS to form a cSMAC. Because I did not see a change in BCR-Ag microcluster clustering following stathmin depletion, this could indicate that although microtubules play a role in cSMAC formation, and stathmin regulates microtubule dynamics, stathmin does not limit microtubule reorganization in A20 cells. Perhaps other microtubule regulating proteins, such as kinesin-related protein XKCM1 are able to compensate when stathmin is depleted [295].    116 Additionally, to further understand the role of ERK-mediated phosphorylation of stathmin, a S25A, S16A and S38A amino acid substitution could be introduced into the endogenous stathmin in A20 cells using CRISPR/Cas9 technology. This would prevent the phosphorylation of stathmin and thus keep it in an activated state, similar to what occurs when the MEK or ERK inhibitor are used. Cell spreading assays following an amino acid switch should be performed to ultimately determine the role of stathmin regulation during B cell spreading  4.1.4 The role of the MEK/ERK pathway at the B cell IS The control of BCR signaling is critical for regulating the activation of B cells during an infection. Proper regulating of BCR signaling prevents inappropriate B cell activation that can lead to autoimmunity and malignancies. IS formation is an important B cell function that facilitates B cell activation by optimizing BCR signaling as well as Ag uptake. The aberrant control of IS formation can result in the dysregulation of BCR signaling and immune functions such as Ag uptake, Ab production, and activation of T cells. The abnormal activation of B cells can lead to autoimmunity due to the production of autoantibodies and proinflammatory cytokines. Autoreactive B cells can present self-Ags and activate T cells to drive autoimmune diseases.  The IS is critical for B cell activation and it was previously believed that the cSMAC was a site of active signaling. However, it is now believed that BCR signaling occurs mainly at peripheral BCR microclusters, as opposed to the cSMAC [235].The principal role of the cSMAC may be to optimize BCR-mediated Ag acquisition. This is because the localized concentration of Ags allows for efficient Ag extraction, which is necessary for eliciting T cell help. The   117 internalization of Ag normally occurs at large Ag clusters [296], and the cSMAC is known to be a site for efficient Ag internalization [112]. Because I found that inhibiting ERK inhibition with FR180204 delayed APC-induced BCR signaling, there may be an ERK-dependent aggregation of BCR-Ag microclusters that both enhances initiation signaling by the BCR and its Ag acquisition. However, whether this would result in altered BCR-mediated Ag extraction was not tested.  4.1.5 ERK induced activation of WAVE2 may stabilize BCR microclusters by creating actin foci BCR signaling is enhanced when the integrity of BCR-Ag microclusters is increased. This requires a platform for which the assembly of signaling complexes can occur. When the actin-depolymerizing drug latrunculin A is added to B cells after they’ve formed microclusters, the microclusters become more diffused and proximal BCR signaling events are decreased [62]. The actin polymerization at BCR microclusters is important for maintaining microcluster integrity that is needed to support downstream signaling events. In T cells, signaling proteins accumulate at actin foci that surround TCR microclusters [297]. WASp-mediated activation of the Arp2/3 complex is necessary for the formation of these actin foci [297]. Additionally, Arp2/3 complex-dependent actin foci have been observed in B cells, where the foci co-localize with BCR signaling components [298]. As such, if ERK activity is required for WRC activation, and thus activation of the Arp2/3 complex, then this could account for why BCR signaling, as judged by pCD79 levels, was reduced and delayed in cells that were treated with the ERK inhibitor. When BCRs are organized into microclusters, Syk is recruited to phosphorylate nearby ITAMs on BCRs that are not bound to Ag [299]. This causes a positive feedback loop as more   118 Syk is recruited and ITAMs are further phosphorylated, resulting in signal amplification. When BCR clusters are smaller, there are fewer ITAMs available and signal amplification of BCR signaling is not as efficient. Therefore, the clustering of BCRs is critical for the amplification of BCR signaling. Consistent with this idea, T cells with larger TCR oligomers have an increased capacity to respond to low densities of peptide-MHC complexes on the surface of APCs [300]. Memory T cells and T cells have larger pre-existing TCR oligomers on their surface, in comparison to naïve T cells, making them more sensitive to low densities of peptide-MHC complexes [301]. It is not known whether the same is true for memory B cells. I showed that with the use of the ERK inhibitor FR180204, small microclusters still form but do not merge as rapidly as in control cells. This may delay the coalescence of BCR microclusters and microcluster-based BCR signaling. This is consistent with the idea that microcluster coalescence amplifies BCR signaling in response to membrane-bound Ag. As a result, ERK may be important for maintaining the positive feedback loop in which actin-dependent microcluster coalescence amplifies BCR signaling. Mutations that cause reduced ERK signaling could result in B cells having a higher Ag density threshold for B cell activation. Thus, controlling BCR microcluster organization through the MEK/ERK pathway may tune the threshold for APC-induced B cell activation.  In addition to controlling Ag-induced microcluster-based BCR signaling, tonic Ag-independent BCR signaling is limited by the actin cytoskeleton. Indeed, inhibiting the Arp2/3 complex results in increased tonic BCR signaling. Because ERK-dependent activation of the WRC activates the Arp2/3 complex, it would be interesting to test whether treating B cells with the ERK inhibitor also increases BCR mobility and tonic BCR signaling. Increased tonic BCR signaling could lead to inappropriate or deleterious B cell activation. Hence, by regulating the   119 BCR mobility in resting B cells, and by potentially stabilizing Ag-induced BCR clustering, ERK-dependent regulation of the actin cytoskeleton may limit tonic BCR signaling but support Ag-induced BCR signaling.   4.1.6 MEK/ERK inhibition has different effects on primary B cells and A20 B cells It is important to note the differences between primary B cells and A20 B cells in order to compare the results obtained with the two cell types. First, primary cells are smaller than A20 B cells, with a smaller cytosol: nucleus ratio. This may reduce the concentration of inhibitor in the medium that is required for achieve the same intracellular concentration in primary cells, compared to A20 B cells. Therefore, performing experiments with primary B cells using the same concentration of chemical inhibitor used with A20 B cells may result in increased cell toxicity and off-target affects. Alternatively, tumor cells such as A20 B cells may have mechanisms for pumping drugs out of the cytoplasm. In addition, the passage of A20 B cells over time may result in the accumulation of mutations that can affect signaling pathways and cellular responses.  Another important difference between these two cell types is the class of Ig expressed on the cell surface. Naïve primary B cells co-express the IgM and IgD isotypes as part of their BCR whereas A20 B cells express IgG [302]. Even though all BCRs activate the same signaling pathways, B cells expressing IgG show an enhanced response to Ag stimulation compared to those expressing IgM/IgD [303]. This is because IgG has an extra signaling motif in its cytoplasmic domain called the immunoglobulin tail tyrosine (ITT) motif [304]. Nck, an upstream regulator of actin dynamics, binds to the ITT, which may enhanced BCR-induced changes in actin dynamics in response to Ag [305]. During normal B cell activation, Ig class switching to   120 IgG or IgE incorporates this ITT motif into the BCRs, which may increase the resulting memory B cell’s sensitivity for Ag and improve reactivation in secondary immune reactions. Additionally, IgG-expressing B cells, such as A20 and A20 D1.3 B cells, exhibit a stronger ERK2 activation response than IgM-expressing B cells, such as primary B cells, due to the ITT motif in the cytoplasmic tail of the IgG heavy chain [212].   4.1.7 A role for ERK1 in APC-induced BCR signaling? I found that siRNA-mediated depletion of ERK2 from A20D1.3 B cells did not reproduce the effects of the ERK inhibitor, FR1809204 in reducing and delaying APC-induced BCR signaling. There are several reasons why this may be the case. Because the ERK siRNA did not completely deplete all of the ERK2, the residual amount of ERK activity may be sufficient to support B cell responses to APCs. Alternatively, the chemical inhibitors inhibit both ERK 1 and ERK2, whereas the siRNA depleted only the ERK2 isoform. Because ERK1 and ERK2 share many substrates, ERK1, together with the residual amount of ERK2, could compensate for the reduction of ERK2. It is also important to note that the COS-7 cells used as APCs are exposed to the chemical inhibitors while the B cells interacting with them. This raises the possibility that the ERK inhibitor alters B cells responses to APCs by acting on the APCs themselves. Selectively depleting ERK from the B cells avoids this issue. An important next step would be to repeat these experiments by depleting both ERK1 and ERK2. If complete depletion of both ERK1 and ERK2 can be achieved, but this does not reduce or delay BCR microcluster centralization and signaling, then this would suggest that the results obtained the with ERK inhibitor FR182004 are due to off-target affects.     121 4.1.8 ERK may stabilize BCR-CD19 interactions   B cell activation in response to membrane bound Ags requires the co-receptor CD19 [235]. This co-receptor acts as a signaling hub by recruiting PI3K, PLC𝝲2 and Vav, which enhances BCR signaling [141,235]. Bolger-Munro et al. showed that inhibiting Arp2/3 complex-dependent actin polymerization increased the lateral mobility of CD19 within the plasma membrane. This indicates that branched actin structures limit the mobility of CD19 within the plasma membrane. The confinement of BCR and CD19 clusters within the same compartment, or actin cage, amplifies BCR signaling as it increases the frequency and duration of BCR-CD19 interactions. An interesting future experiment would be to investigate if inhibiting the MEK/ERK pathway reduces the co-localization of CD19 and BCR clusters within the plasma membrane. If reduced co-localization is observed following inhibition, this would suggest that the MEK/ERK pathway is important for regulating the polymerization of branched actin networks that maintain CD19 and BCR clusters within the same area.   4.1.9 ERK may regulate microtubules through the Arp2/3 complex The centralization of Ag-bound BCR microclusters to form cSMACs requires dynein-dependent transportation along microtubules [72,75]. Actin retrograde flow is essential for BCR-Ag microclusters to reach the sites at which microtubules interact with the inner face of the peripheral branched actin network. This interaction is dependent on the cortical capture of microtubule plus ends and the polarization of the microtubules to form a juxtamembrane at the immune synapse. There are numerous NPFs, such as WASH and WHAMM, that associate with microtubules and could play a role in integrating actin remodeling with reorientation of the   122 microtubule network [105]. Even more, the Arp2/3 complex may play a role in organizing the microtubule cytoskeleton. In unstimulated B cells, the Arp2/3 complex localizes to both cortical actin and to the MTOC, where it links the MTOC to the nucleus [104]. Following BCR stimulation the Arp2/3 complex is recruited to the immune synapse to induce actin polymerization. This frees the MTOC from the nucleus, allowing it to polarize towards the immune synapse [104]. Because ERK may play a role in activating the Arp2/3 complex through phosphorylation of the WRC, ERK may also contribute to microtubule reorganization through the ERK/WRC/Arp2/3 complex module. As a result, B cells with impaired ERK activity may not be able to exceed the threshold for BCR activation as MTOC reorientation is required for the formation of the IS, which supports B cell signaling. This may also explain why cells treated with the ERK inhibitor FR180204 display delayed cSMAC formation.   4.2 Perspectives            I identified a role for the MEK/ERK pathway in cytoskeletal-dependent responses to immobilized and membrane-bound Ags. In B cells, ERK activation results in the regulation of transcription factors responsible for the expression of genes required for proliferation and differentiation. Therefore, it is likely that defects in ERK signaling would impair multiple steps in B cell activation. Here I have shown for the first time that ERK activity is important for cytoskeletal remodeling events that support cell spreading and BCR signaling.  We hypothesize that ERK is responsible for phosphorylating WAVE2, which is required for maximal Arp2/3 complex-dependent actin nucleation, which then drives B cell spreading and BCR microcluster centralization (Figure 4.2). A key follow-up experiment would be to immunoprecipitate WAVE2 and use phospho-specific Abs to directly confirm that ERK   123 phosphorylates WAVE2 and the associated Abi1 protein in B cells. Selective depletion of ERK1 versus ERK2 would reveal whether one or both ERK isoforms phosphorylate the WRC in B cells.   Figure 4.2. Proposed function of the MEK/ERK pathway in Arp2/3 complex-dependent B cell spreading. The spreading of B cells on immobilized Ag is driven by the formation of broad lamellipodia that results from Arp2/3 complex-dependent nucleation of branched actin networks. The Arp2/3 complex can be activated by multiple NPFs, of which WASp and the WRC have been implicated in peripheral actin dynamics in lymphocytes. WASp and the WRC are activated by Cdc42-GTP and Rac-GTP, respectively. ERK-dependent phosphorylation of the WAVE2 and Abi1 components of the WRC has been demonstrated in epithelial cells and is required for WRC-dependent lamellipodia formation and cell motility. We propose that the MEK/ERK pathways acts in a similar manner in B cells to support WRC-dependent activation of the Arp2/3 complex, which is composed of Wave2 and Abi1 among other proteins.      124 In this thesis I used loss-of-function approaches, chemically inhibiting the MEK/ERK pathway and depleting ERK2 using siRNA. An interesting next step would be to use gain-of-function approaches to assess what the effects of hyperactivation of the MEK/ERK pathway would be on B cell spreading and APC-induced BCR signaling. This could be done by transfecting A20 B cells or A20 D1.3 B cells with plasmids encoding activated Ras or constitutively active mutant forms of MEK or ERK [306–309]. Alternatively, we could overexpress MEK or ERK in B cell lines, as Ordan et al. have done [310]. These approaches would determine whether hyperactivation of the Ras/ERK pathway alters B cell function and provide further insights into the role of ERK in regulating cytoskeleton-dependent B cell responses to APC-bound and immobilized Ags.  Moreover, because experiments performed in this thesis used murine B cell lines and primary B cells, further studies on the role of the MEK/ERK pathway in regulating B cell responses to Ags should utilize human B cells. This could reveal whether using chemical inhibitors of the MEK/ERK pathway, or B cell-specific depletion of ERK can modulate Ag-induced activation of normal human B cells or inhibit the growth and survival of B cells from patients with aberrant ERK activity, such as in Hodgkin disease, chronic lymphocytic leukemia, and mantle cell lymphoma [311,312].   Because MEK and ERK are ubiquitously expressed and are important for a wide range of biological functions, directly targeting ERK would not be practical for therapeutic purposes. However, identifying downstream substrates of the MEK/ERK pathway that are responsible for controlling cytoskeletal dynamics, and which are more highly expressed in B cells than in other cell types, could be used to limit B cell activation in various B cell-mediated autoimmune disease, allergy and inflammatory diseases. Great strides have been made in the development of   125 highly selective potent inhibitors of MEK, including binimetinib (Array Biopharma), trametinib (Novartis Pharmaceuticals), and cobimetinib (Genetech) [313]. However, a major setback with this current selection of therapeutics is the tendency of tumours to become resistant to MEK inhibitors [313]. However, ERK inhibitors could overcome this acquired drug resistance to MEK inhibitors.   4.3 Conclusions  In this thesis, I have shown that the serine/threonine kinase ERK is important for regulating actin dynamics in B cells and for promoting actin-dependent processes such as B cell spreading and APC-induced BCR signaling. I demonstrated that inhibiting MEK and ERK activity via chemical inhibitors reduces the thickness of the ring of peripheral actin, actin clearance from the center of the cell, and microtubule polarization when B cells spread on immobilized Ag, especially when the surface is coated with lower densities of the Ag. I also demonstrated that inhibiting ERK activity, but not MEK, reduces the centripetal movement of BCR microclusters, which is required for cSMAC formation and optimal BCR signaling. Taken together, these findings advance our knowledge of how the ERK signaling pathway regulates B cell responses to APC-bound and surface-bound Ags. Understanding the complex signaling pathways that control B cell activation will further drive the development of novel therapeutics that can be used to modulate B cell responses in health and disease.   126 Bibliography [1]	 P.	Shen,	S.	Fillatreau,	Antibody-independent	functions	of	B	cells:	A	focus	on	cytokines,	Nat.	Rev.	Immunol.	15	(2015)	441–451.	https://doi.org/10.1038/nri3857.	[2]	 N.E.	Harwood,	F.D.	Batista,	Early	Events	in	B	Cell	Activation,	Annu.	Rev.	Immunol.	28	(2010)	185–210.	https://doi.org/10.1146/annurev-immunol-030409-101216.	[3]	 T.W.	Lebien,	T.F.	Tedder,	B	lymphocytes:	How	they	develop	and	function,	Blood.	112	(2008)	1570–1580.	https://doi.org/10.1182/blood-2008-02-078071.	[4]	 E.	Montecino-Rodriguez,	K.	Dorshkind,	B-1	B	Cell	Development	in	the	Fetus	and	Adult,	Immunity.	36	(2012)	13–21.	https://doi.org/10.1016/j.immuni.2011.11.017.	[5]	 W.E.	Gathings,	A.R.	Lawton,	M.D.	Cooper,	Immunofluorescent	studies	of	the	development	of	pre-B	cells,	B	lymphocytes	and	immunoglobulin	isotype	diversity	in	humans,	Eur.	J.	Immunol.	7	(1977)	804–810.	https://doi.org/10.1002/eji.1830071112.	[6]	 L.J.	McHeyzer-Williams,	M.G.	McHeyzer-Williams,	Antigen-Specific	Memory	B	Cell	Development,	Annu.	Rev.	Immunol.	23	(2005)	487–513.	https://doi.org/10.1146/annurev.immunol.23.021704.115732.	[7]	 C.	Gutzeit,	K.	Chen,	A.	Cerutti,	The	enigmatic	function	of	IgD:	some	answers	at	last,	Eur.	J.	Immunol.	48	(2018)	1101–1113.	https://doi.org/10.1002/eji.201646547.	[8]	 K.	Chen,	A.	Cerutti,	The	function	and	regulation	of	immunoglobulin	D,	Curr.	Opin.	Immunol.	23	(2011)	345–352.	https://doi.org/10.1016/j.coi.2011.01.006.	[9]	 R.	Geisberger,	M.	Lamers,	G.	Achatz,	The	riddle	of	the	dual	expression	of	IgM	and	IgD,	Immunology.	118	(2006)	429–437.	https://doi.org/10.1111/j.1365-  127 2567.2006.02386.x.	[10]	 D.J.	Rawlings,	G.	Metzler,	M.	Wray-Dutra,	S.W.	Jackson,	Altered	B	cell	signalling	in	autoimmunity,	Nat.	Rev.	Immunol.	17	(2017)	421–436.	https://doi.org/10.1038/nri.2017.24.	[11]	 F.	Martin,	A.C.	Chan,	B	CELL	IMMUNOBIOLOGY	IN	DISEASE:	Evolving	Concepts	from	the	Clinic,	Annu.	Rev.	Immunol.	24	(2006)	467–496.	https://doi.org/10.1146/annurev.immunol.24.021605.090517.	[12]	 N.	Manjarrez-Orduño,	T.D.	Quách,	I.	Sanz,	B	cells	and	immunological	tolerance,	J.	Invest.	Dermatol.	129	(2009)	278–288.	https://doi.org/10.1038/jid.2008.240.	[13]	 S.	Yurasov,	H.	Wardemann,	J.	Hammersen,	M.	Tsuiji,	E.	Meffre,	V.	Pascual,	M.C.	Nussenzweig,	Defective	B	cell	tolerance	checkpoints	in	systemic	lupus	erythematosus,	J.	Exp.	Med.	201	(2005)	703–711.	https://doi.org/10.1084/jem.20042251.	[14]	 F.	Bosch,	R.	Dalla-Favera,	Chronic	lymphocytic	leukaemia:	from	genetics	to	treatment,	Nat.	Rev.	Clin.	Oncol.	16	(2019)	684–701.	https://doi.org/10.1038/s41571-019-0239-8.	[15]	 R.E.	Davis,	V.N.	Ngo,	G.	Lenz,	P.	Tolar,	R.M.	Young,	P.B.	Romesser,	H.	Kohlhammer,	L.	Lamy,	H.	Zhao,	Y.	Yang,	W.	Xu,	A.L.	Shaffer,	G.	Wright,	W.	Xiao,	J.	Powell,	J.K.	Jiang,	C.J.	Thomas,	A.	Rosenwald,	G.	Ott,	H.K.	Muller-Hermelink,	R.D.	Gascoyne,	J.M.	Connors,	N.A.	Johnson,	L.M.	Rimsza,	E.	Campo,	E.S.	Jaffe,	W.H.	Wilson,	J.	Delabie,	E.B.	Smeland,	R.I.	Fisher,	R.M.	Braziel,	R.R.	Tubbs,	J.R.	Cook,	D.D.	Weisenburger,	W.C.	Chan,	S.K.	Pierce,	L.M.	Staudt,	Chronic	active	B-cell-receptor	signalling	in	diffuse	large	B-cell	  128 lymphoma,	Nature.	463	(2010)	88–92.	https://doi.org/10.1038/nature08638.	[16]	 G.	Salles,	M.	Barrett,	R.	Foà,	J.	Maurer,	S.	O’Brien,	N.	Valente,	M.	Wenger,	D.G.	Maloney,	Rituximab	in	B-Cell	Hematologic	Malignancies:	A	Review	of	20	Years	of	Clinical	Experience,	Adv.	Ther.	34	(2017)	2232–2273.	https://doi.org/10.1007/s12325-017-0612-x.	[17]	 R.	Li,	K.R.	Patterson,	A.	Bar-Or,	Reassessing	B	cell	contributions	in	multiple	sclerosis,	Nat.	Immunol.	19	(2018)	696–707.	https://doi.org/10.1038/s41590-018-0135-x.	[18]	 J.L.	Barnas,	R.J.	Looney,	J.H.	Anolik,	B	cell	targeted	therapies	in	autoimmune	disease,	Curr.	Opin.	Immunol.	61	(2019)	92–99.	https://doi.org/10.1016/j.coi.2019.09.004.	[19]	 G.M.	Deng,	V.C.	Kyttaris,	G.C.	Tsokos,	Targeting	syk	in	autoimmune	rheumatic	diseases,	Front.	Immunol.	7	(2016)	1–5.	https://doi.org/10.3389/fimmu.2016.00078.	[20]	 K.D.	Puri,	J.A.	Di	Paolo,	M.R.	Gold,	B-Cell	Receptor	Signaling	Inhibitors	for	Treatment	of	Autoimmune	Inflammatory	Diseases	and	B-Cell	Malignancies,	Int.	Rev.	Immunol.	32	(2013)	397–427.	https://doi.org/10.3109/08830185.2013.818140.	[21]	 J.A.	Burger,	A.	Wiestner,	Targeting	B	cell	receptor	signalling	in	cancer:	Preclinical	and	clinical	advances,	Nat.	Rev.	Cancer.	18	(2018)	148–167.	https://doi.org/10.1038/nrc.2017.121.	[22]	 D.	Vetrie,	I.	Vořechovský,	P.	Sideras,	J.	Holland,	A.	Davies,	F.	Flinter,	L.	Hammarström,	C.	Kinnon,	R.	Levinsky,	M.	Bobrow,	C.I.E.	Smith,	D.R.	Bentley,	The	gene	involved	in	X-linked	agammaglobulinaemia	is	a	member	of	the	src	family	of	protein-tyrosine	kinases,	Nature.	361	(1993)	226–233.	https://doi.org/10.1038/361226a0.	  129 [23]	 C.	Bacchelli,	S.	Buckridge,	A.J.	Thrasher,	H.B.	Gaspar,	Translational	Mini-Review	Series	on	Immunodeficiency:Molecular	defects	in	common	variable	immunodeficiency,	Clin.	Exp.	Immunol.	149	(2007)	401–409.	https://doi.org/10.1111/j.1365-2249.2007.03461.x.	[24]	 P.	Revy,	T.	Muto,	Y.	Levy,	F.	Geissmann,	A.	Plebani,	O.	Sanal,	N.	Catalan,	M.	Forveille,	R.	Dufourcq-Lagelouse,	A.	Gennery,	I.	Tezcan,	F.	Ersoy,	H.	Kayserili,	A.G.	Ugazio,	N.	Brousse,	M.	Muramatsu,	L.D.	Notarangelo,	K.	Kinoshita,	T.	Honjo,	A.	Fischer,	A.	Durandy,	Activation-induced	cytidine	deaminase	(AID)	deficiency	causes	the	autosomal	recessive	form	of	the	hyper-IgM	syndrome	(HIGM2),	Cell.	102	(2000)	565–575.	https://doi.org/10.1016/S0092-8674(00)00079-9.	[25]	 J.	Antel,	A.	Bar-Or,	Roles	of	immunoglobulins	and	B	cells	in	multiple	sclerosis:	From	pathogenesis	to	treatment,	J.	Neuroimmunol.	180	(2006)	3–8.	https://doi.org/10.1016/j.jneuroim.2006.06.032.	[26]	 J.C.W.	Edwards,	L.	Szczepański,	J.	Szechiński,	A.	Filipowicz-Sosnowska,	P.	Emery,	D.R.	Close,	R.M.	Stevens,	T.	Shaw,	Efficacy	of	B-cell-targeted	therapy	with	rituximab	in	patients	with	rheumatoid	arthritis,	N.	Engl.	J.	Med.	350	(2004)	2572–2581.	https://doi.org/10.1056/NEJMoa032534.	[27]	 O.T.M.	Chan,	M.P.	Madaio,	M.J.	Shlomchik,	The	central	and	multiple	roles	of	B	cells	in	lupus	pathogenesis,	Immunol.	Rev.	169	(1999)	107–121.	https://doi.org/10.1111/j.1600-065X.1999.tb01310.x.	[28]	 N.A.	Carter,	R.	Vasconcellos,	E.C.	Rosser,	C.	Tulone,	A.	Muñoz-Suano,	M.	Kamanaka,	M.R.	Ehrenstein,	R.A.	Flavell,	C.	Mauri,	Mice	Lacking	Endogenous	IL-10–Producing	  130 Regulatory	B	Cells	Develop	Exacerbated	Disease	and	Present	with	an	Increased	Frequency	of	Th1/Th17	but	a	Decrease	in	Regulatory	T	Cells,	J.	Immunol.	186	(2011)	5569–5579.	https://doi.org/10.4049/jimmunol.1100284.	[29]	 J.M.	Dal	Porto,	S.B.	Gauld,	K.T.	Merrell,	D.	Mills,	A.E.	Pugh-Bernard,	J.	Cambier,	B	cell	antigen	receptor	signaling	101,	Mol.	Immunol.	41	(2004)	599–613.	https://doi.org/10.1016/j.molimm.2004.04.008.	[30]	 L.	Abraham,	J.C.	Wang,	M.	Bolger-Munro,	M.R.	Gold,	Encyclopedia	of	Immunobiology	2016	review-final.pdf,	(2016)	40–52.	[31]	 M.R.	Gold,	To	make	antibodies	or	not:	Signaling	by	the	B-cell	antigen	receptor,	Trends	Pharmacol.	Sci.	23	(2002)	316–324.	https://doi.org/10.1016/S0165-6147(02)02045-X.	[32]	 P.	Cruz-Tapias,	J.	Castiblanco,	N.E.	Correa,	G.	Montoya-Ortíz,	Autoimmunity	From	Bench	to	Bedside,	2013.	[33]	 J.C.	Cambier,	New	nomenclature	for	the	Reth	motif	(or	ARH1/TAM/ARAM/YXXL),	Immunol.	Today.	16	(1995)	110.	https://doi.org/10.1016/0167-5699(95)80105-7.	[34]	 M.	Reth,	Antigen	receptor	tail	clue	[5],	Nature.	338	(1989)	383–384.	https://doi.org/10.1038/338383b0.	[35]	 H.	Flaswinkel,	M.	Reth,	Dual	role	of	the	tyrosine	activation	motif	of	the	Ig-alpha	protein	during	signal	transduction	via	the	B	cell	antigen	receptor.,	EMBO	J.	13	(1994)	83–89.	https://doi.org/10.1002/j.1460-2075.1994.tb06237.x.	[36]	 N.	Engels,	J.	Wienands,	The	signaling	tool	box	for	tyrosine-based	costimulation	of	lymphocytes,	Curr.	Opin.	Immunol.	23	(2011)	324–329.	  131 https://doi.org/10.1016/j.coi.2011.01.005.	[37]	 T.A.	Packard,	J.C.	Cambier,	B	lymphocyte	antigen	receptor	signaling:	initiation,	amplification,	and	regulation,	F1000Prime	Rep.	5	(2013)	40.	https://doi.org/10.12703/P5-40.	[38]	 B.	Treanor,	N.E.	Harwood,	F.D.	Batista,	Microsignalosomes:	Spatially	resolved	receptor	signalling,	Biochem.	Soc.	Trans.	37	(2009)	1014–1018.	https://doi.org/10.1042/BST0371014.	[39]	 A.M.	Cheng,	B.	Rowleyt,	W.	Paot,	A.	Haydayt,	J.B.	Bolent,	T.	Pawson,	Syk	tyrosine	kinase	required	for	mouse	viability	and	B-cell	development,	378	(1995)	303–306.	[40]	 M.	Turner,	P.J.	Mee,	P.S.	Costello,	O.	Williams,	A.A.	Price,	L.P.	Duddy,	M.T.	Furlong,	R.L.	Geahlen,	V.L.J.	Tybulewiczt,	Perinatal	lethality	and	blocked	B-cell	development	in	mice	lacking	the	tyrosine	kinase	Syk,	378	(1995)	298–302.	[41]	 J.	Wienands,	J.	Schweikert,	B.	Wollscheid,	H.	Jumaa,	P.J.	Nielsen,	M.	Reth,	SLP-65:	A	new	signaling	component	in	B	lymphocytes	which	requires	expression	of	the	antigen	receptor	for	phosphorylation,	J.	Exp.	Med.	188	(1998)	791–795.	https://doi.org/10.1084/jem.188.4.791.	[42]	 C.	Fu,	C.W.	Turck,	T.	Kurosaki,	A.C.	Chan,	BLNK:	A	central	linker	protein	in	B	cell	activation,	Immunity.	9	(1998)	93–103.	https://doi.org/10.1016/S1074-7613(00)80591-9.	[43]	 Y.J.	Kim,	F.	Sekiya,	B.	Poulin,	Y.S.	Bae,	S.G.	Rhee,	Mechanism	of	B-Cell	Receptor-Induced	Phosphorylation	and	Activation	of	Phospholipase	C-γ2,	Mol.	Cell.	Biol.	24	(2004)	9986–9999.	https://doi.org/10.1128/mcb.24.22.9986-9999.2004.	  132 [44]	 G.R.	Crabtree,	E.N.	Olson,	NFAT	signaling:	Choreographing	the	social	lives	of	cells,	Cell.	109	(2002)	67–79.	https://doi.org/10.1016/S0092-8674(02)00699-2.	[45]	 S.J.	McLeod,	M.R.	Gold,	Activation	and	function	of	the	Rap1	GTPase	in	B	lymphocytes,	Int.	Rev.	Immunol.	20	(2001)	763–789.	https://doi.org/10.3109/08830180109045589.	[46]	 K.	Katagiri,	A.	Maeda,	M.	Shimonaka,	T.	Kinashi,	RAPL,	a	Rap1-binding	molecule	that	mediates	Rap1-induced	adhesion	through	spatial	regulation	of	LFA-1,	Nat.	Immunol.	4	(2003)	741–748.	https://doi.org/10.1038/ni950.	[47]	 J.	Jellusova,	R.C.	Rickert,	The	PI3K	pathway	in	B	cell	metabolism,	Crit.	Rev.	Biochem.	Mol.	Biol.	51	(2016)	359–378.	https://doi.org/10.1080/10409238.2016.1215288.	[48]	 X.	Zhang,	N.	Tang,	T.J.	Hadden,	A.K.	Rishi,	Akt,	FoxO	and	regulation	of	apoptosis,	Biochim.	Biophys.	Acta	-	Mol.	Cell	Res.	1813	(2011)	1978–1986.	https://doi.org/10.1016/j.bbamcr.2011.03.010.	[49]	 T.	Kurosaki,	H.	Shinohara,	Y.	Baba,	B	Cell	Signaling	and	Fate	Decision,	Annu.	Rev.	Immunol.	28	(2010)	21–55.	https://doi.org/10.1146/annurev.immunol.021908.132541.	[50]	 S.	Coughlin,	M.	Noviski,	J.L.	Mueller,	A.	Chuwonpad,	W.C.	Raschke,	A.	Weiss,	J.	Zikherman,	An	extracatalytic	function	of	CD45	in	B	cells	is	mediated	by	CD22,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	112	(2015)	E6515–E6524.	https://doi.org/10.1073/pnas.1519925112.	[51]	 P.M.	Waterman,	J.C.	Cambier,	The	conundrum	of	inhibitory	signaling	by	ITAM-containing	immunoreceptors:	Potential	molecular	mechanisms,	FEBS	Lett.	584	  133 (2010)	4878–4882.	https://doi.org/10.1016/j.febslet.2010.09.029.	[52]	 A.J.	Gross,	J.R.	Lyandres,	A.K.	Panigrahi,	E.T.L.	Prak,	A.L.	DeFranco,	Developmental	Acquisition	of	the	Lyn-CD22-SHP-1	Inhibitory	Pathway	Promotes	B	Cell	Tolerance,	J.	Immunol.	182	(2009)	5382–5392.	https://doi.org/10.4049/jimmunol.0803941.	[53]	 P.K.	Mattila,	F.D.	Batista,	B.	Treanor,	Dynamics	of	the	actin	cytoskeleton	mediates	receptor	cross	talk:	An	emerging	concept	in	tuning	receptor	signaling,	J.	Cell	Biol.	212	(2016)	267–280.	https://doi.org/10.1083/jcb.201504137.	[54]	 P.K.	Mattila,	C.	Feest,	D.	Depoil,	B.	Treanor,	B.	Montaner,	K.L.	Otipoby,	R.	Carter,	L.B.	Justement,	A.	Bruckbauer,	F.D.	Batista,	The	Actin	and	Tetraspanin	Networks	Organize	Receptor	Nanoclusters	to	Regulate	B	Cell	Receptor-Mediated	Signaling,	Immunity.	38	(2013)	461–474.	https://doi.org/10.1016/j.immuni.2012.11.019.	[55]	 J.	Yang,	M.	Reth,	Receptor	Dissociation	and	B-cell	Activation,	B	Cell	Recept.	Signal.	(2015).	https://doi.org/10.1007/82.	[56]	 D.	Lingwood,	K.	Simons,	Lipid	rafts	as	a	membrane-organizing	principle,	Science.	327	(2010)	46–50.	https://doi.org/10.1126/science.1174621.	[57]	 K.	Ritchie,	A.	Kusumi,	Single-particle	tracking	image	microscopy,	Methods	Enzymol.	360	(2003)	618–634.	https://doi.org/10.1016/S0076-6879(03)60131-X.	[58]	 C.	Dietrich,	B.	Yang,	T.	Fujiwara,	A.	Kusumi,	K.	Jacobson,	Relationship	of	lipid	rafts	to	transient	confinement	zones	detected	by	single	particle	tracking,	Biophys.	J.	82	(2002)	274–284.	https://doi.org/10.1016/S0006-3495(02)75393-9.	[59]	 J.	Li,	W.	Yin,	Y.	Jing,	D.	Kang,	L.	Yang,	J.	Cheng,	Z.	Yu,	Z.	Peng,	X.	Li,	Y.	Wen,	X.	Sun,	B.	Ren,	C.	Liu,	The	coordination	between	B	cell	receptor	signaling	and	the	actin	  134 cytoskeleton	during	B	cell	activation,	Front.	Immunol.	10	(2019)	1–13.	https://doi.org/10.3389/fimmu.2018.03096.	[60]	 K.P.	Lam,	R.	Kühn,	K.	Rajewsky,	In	vivo	ablation	of	surface	immunoglobulin	on	mature	B	cells	by	inducible	gene	targeting	results	in	rapid	cell	death,	Cell.	90	(1997)	1073–1083.	https://doi.org/10.1016/S0092-8674(00)80373-6.	[61]	 B.	Treanor,	D.	Depoil,	A.	Gonzalez-Granja,	P.	Barral,	M.	Weber,	O.	Dushek,	A.	Bruckbauer,	F.D.	Batista,	The	Membrane	Skeleton	Controls	Diffusion	Dynamics	and	Signaling	through	the	B	Cell	Receptor,	Immunity.	32	(2010)	187–199.	https://doi.org/10.1016/j.immuni.2009.12.005.	[62]	 B.	Treanor,	D.	Depoil,	A.	Bruckbauer,	F.D.	Batista,	Dynamic	cortical	actin	remodeling	by	ERM	proteins	controls	BCR	microcluster	organization	and	integrity,	J.	Exp.	Med.	208	(2011)	1055–1068.	https://doi.org/10.1084/jem.20101125.	[63]	 D.	Pore,	N.	Gupta,	Ezrin-Radixin-Moesin	family	proteins	in	the	regulation	of	B	cell	immune	response,	Crit.	Rev.	Immunol.	35	(2015)	15–31.	https://doi.org/10.1016/j.physbeh.2017.03.040.	[64]	 Y.	Sasaki,	T.	Kurosaki,	Immobile	BCRs:	The	Safety	on	the	Signal	Trigger,	Immunity.	32	(2010)	143–144.	https://doi.org/10.1016/j.immuni.2010.02.007.	[65]	 Y.R.	Carrasco,	F.D.	Batista,	B	cell	recognition	of	membrane-bound	antigen:	an	exquisite	way	of	sensing	ligands,	Curr.	Opin.	Immunol.	18	(2006)	286–291.	https://doi.org/10.1016/j.coi.2006.03.013.	[66]	 S.J.	Fleire,	J.P.	Goldman,	Y.R.	Carrasco,	M.	Weber,	D.	Bray,	F.D.	Batista,	B	cell	ligand	discrimination	through	a	spreading	and	contraction	response,	Science	(80-.	).	312	  135 (2006)	738–741.	https://doi.org/10.1126/science.1123940.	[67]	 B.H.	Hosseini,	I.	Louban,	D.	Djandji,	G.H.	Wabnitz,	J.	Deeg,	N.	Bulbuc,	Y.	Samstag,	M.	Gunzer,	J.P.	Spatz,	G.J.	Hämmerling,	Immune	synapse	formation	determines	interaction	forces	between	T	cells	and	antigen-presenting	cells	measured	by	atomic	force	microscopy,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	106	(2009)	17852–17857.	https://doi.org/10.1073/pnas.0905384106.	[68]	 B.	Treanor,	B-cell	receptor:	From	resting	state	to	activate,	Immunology.	136	(2012)	21–27.	https://doi.org/10.1111/j.1365-2567.2012.03564.x.	[69]	 M.	Bolger-Munro,	K.	Choi,	J.M.	Scurll,	L.	Abraham,	R.S.	Chappell,	D.	Sheen,	M.	Dang-Lawson,	X.	Wu,	J.J.	Priatel,	D.	Coombs,	J.A.	Hammer,	M.R.	Gold,	Arp2/3	complex-driven	spatial	patterning	of	the	BCR	enhances	immune	synapse	formation,	BCR	signaling	and	b	cell	activation,	Elife.	8	(2019)	1–40.	https://doi.org/10.7554/eLife.44574.	[70]	 B.	Alarcón,	D.	Mestre,	N.	Martínez-Martín,	The	immunological	synapse:	A	cause	or	consequence	of	T-cell	receptor	triggering?,	Immunology.	133	(2011)	420–425.	https://doi.org/10.1111/j.1365-2567.2011.03458.x.	[71]	 E.	Kuokkanen,	V.	Šuštar,	P.K.	Mattila,	Molecular	control	of	B	cell	activation	and	immunological	synapse	formation,	Traffic.	16	(2015)	311–326.	https://doi.org/10.1111/tra.12257.	[72]	 T.	Schnyder,	A.	Castello,	C.	Feest,	N.E.	Harwood,	T.	Oellerich,	H.	Urlaub,	M.	Engelke,	J.	Wienands,	A.	Bruckbauer,	F.D.	Batista,	B	Cell	Receptor-Mediated	Antigen	Gathering	Requires	Ubiquitin	Ligase	Cbl	and	Adaptors	Grb2	and	Dok-3	to	Recruit	Dynein	to	the	  136 Signaling	Microcluster,	Immunity.	34	(2011)	905–918.	https://doi.org/10.1016/j.immuni.2011.06.001.	[73]	 J.C.	Wang,	J.Y.J.	Lee,	S.	Christian,	M.	Dang-Lawson,	C.	Pritchard,	S.A.	Freeman,	M.R.	Gold,	The	Rap1-cofilin-1	pathway	coordinates	actin	reorganization	and	MTOC	polarization	at	the	B	cell	immune	synapse,	J.	Cell	Sci.	130	(2017)	1094–1109.	https://doi.org/10.1242/jcs.191858.	[74]	 D.	Obino,	A.M.	Lennon-Duménil,	A	critical	role	for	cell	polarity	in	antigen	extraction,	processing,	and	presentation	by	B	lymphocytes,	Adv.	Immunol.	123	(2014)	51–67.	https://doi.org/10.1016/B978-0-12-800266-7.00001-7.	[75]	 A.	Hashimoto-Tane,	T.	Yokosuka,	K.	Sakata-Sogawa,	M.	Sakuma,	C.	Ishihara,	M.	Tokunaga,	T.	Saito,	Dynein-Driven	Transport	of	T	Cell	Receptor	Microclusters	Regulates	Immune	Synapse	Formation	and	T	Cell	Activation,	Immunity.	34	(2011)	919–931.	https://doi.org/10.1016/j.immuni.2011.05.012.	[76]	 T.	Ilani,	G.	Vasiliver-Shamis,	S.	Vardhana,	A.	Bretscher,	M.L.	Dustin,	T	cell	antigen	receptor	signaling	and	immunological	synapse	stability	require	myosin	IIA,	Nat.	Immunol.	10	(2009)	531–539.	https://doi.org/10.1038/ni.1723.	[77]	 S.A.	Freeman,	V.	Jaumouillé,	K.	Choi,	B.E.	Hsu,	H.S.	Wong,	L.	Abraham,	M.L.	Graves,	D.	Coombs,	C.D.	Roskelley,	R.	Das,	S.	Grinstein,	M.R.	Gold,	Toll-like	receptor	ligands	sensitize	B-cell	receptor	signalling	by	reducing	actin-dependent	spatial	confinement	of	the	receptor,	Nat.	Commun.	6	(2015).	https://doi.org/10.1038/ncomms7168.	[78]	 N.	Filigheddu,	V.F.	Gnocchi,	M.	Coscia,	M.	Cappelli,	P.E.	Porporato,	R.	Taulli,	Santiago	M.	Di	Pietro,*	Juan	M.	Falco	´n-Pe	´rez,*	Danie	`le	Tenza,†	Subba	R.G.	Setty,‡	Michael	S.	  137 Marks,‡	Grac	¸a	Raposo,†	and	Esteban	C.	Dell’Angelica*,	Mol.	Biol.	Cell.	18	(2007)	986–994.	https://doi.org/10.1091/mbc.E06.	[79]	 E.M.	De	La	Cruz,	M.L.	Gardel,	Actin	mechanics	and	fragmentation,	J.	Biol.	Chem.	290	(2015)	17137–17144.	https://doi.org/10.1074/jbc.R115.636472.	[80]	 D.	C.	Wickramarachchi,	A.N.	Theofilopoulos,	D.H.	Kono,	Immune	pathology	associated	with	altered	actin	cytoskeleton	regulation,	Autoimmunity.	43	(2010)	64–75.	https://doi.org/10.3109/08916930903374634.	[81]	 E.D.	Goley,	M.D.	Welch,	The	ARP2/3	complex:	An	actin	nucleator	comes	of	age,	Nat.	Rev.	Mol.	Cell	Biol.	7	(2006)	713–726.	https://doi.org/10.1038/nrm2026.	[82]	 M.F.	Carlier,	J.	Pernier,	P.	Montaville,	S.	Shekhar,	S.	Kühn,	Control	of	polarized	assembly	of	actin	filaments	in	cell	motility,	Cell.	Mol.	Life	Sci.	72	(2015)	3051–3067.	https://doi.org/10.1007/s00018-015-1914-2.	[83]	 M.A.	Chesarone,	A.G.	Dupage,	B.L.	Goode,	Unleashing	formins	to	remodel	the	actin	and	microtubule	cytoskeletons,	Nat.	Rev.	Mol.	Cell	Biol.	11	(2010)	62–74.	https://doi.org/10.1038/nrm2816.	[84]	 C.T.	Skau,	C.M.	Waterman,	Specification	of	Architecture	and	Function	of	Actin	Structures	by	Actin	Nucleation	Factors,	Annu.	Rev.	Biophys.	44	(2015)	285–310.	https://doi.org/10.1146/annurev-biophys-060414-034308.	[85]	 T.D.	Pollard,	What	We	Know	and	Do	Not	Know	About	Actin,	Handb.	Exp.	Pharmacol.	(2016).	https://doi.org/10.1007/164.	[86]	 J.	Lehtimaki,	M.	Hakala,	P.	Lappalainen,	Actin	Filament	Structures	in	Migrating	Cells,	Handb.	Exp.	Pharmacol.	(2016)	251–263.	https://doi.org/10.1007/164.	  138 [87]	 M.A.	Chesarone,	B.L.	Goode,	Actin	nucleation	and	elongation	factors:	mechanisms	and	interplay,	Curr.	Opin.	Cell	Biol.	21	(2009)	28–37.	https://doi.org/10.1016/j.ceb.2008.12.001.	[88]	 L.	Blanchoin,	R.	Boujemaa-Paterski,	C.	Sykes,	J.	Plastino,	Actin	dynamics,	architecture,	and	mechanics	in	cell	motility,	Physiol.	Rev.	94	(2014)	235–263.	https://doi.org/10.1152/physrev.00018.2013.	[89]	 M.	Bezanilla,	A.S.	Gladfelter,	D.R.	Kovar,	W.L.	Lee,	Cytoskeletal	dynamics:	A	view	from	the	membrane,	J.	Cell	Biol.	209	(2015)	329–337.	https://doi.org/10.1083/jcb.201502062.	[90]	 R.	Li,	G.G.	Gundersen,	Beyond	polymer	polarity:	How	the	cytoskeleton	builds	a	polarized	cell,	Nat.	Rev.	Mol.	Cell	Biol.	9	(2008)	860–873.	https://doi.org/10.1038/nrm2522.	[91]	 V.L.J.	Tybulewicz,	R.B.	Henderson,	Rho	family	GTPases	and	their	regulators	in	lymphocytes,	Nat.	Rev.	Immunol.	9	(2009)	630–644.	https://doi.org/10.1038/nri2606.	[92]	 K.	Ohashi,	Roles	of	cofilin	in	development	and	its	mechanisms	of	regulation,	Dev.	Growth	Differ.	57	(2015)	275–290.	https://doi.org/10.1111/dgd.12213.	[93]	 J.R.	Bamburg,	B.W.	Bernstein,	Roles	of	ADF/cofilin	in	actin	polymerization	and	beyond,	F1000	Biol.	Rep.	2	(2010)	1–7.	https://doi.org/10.3410/B2-62.	[94]	 W.A.	Elam,	H.	Kang,	E.M.	De	La	Cruz,	Biophysics	of	actin	filament	severing	by	cofilin,	FEBS	Lett.	587	(2013)	1215–1219.	https://doi.org/10.1016/j.febslet.2013.01.062.	[95]	 L.	Gressin,	A.	Guillotin,	C.	Guérin,	L.	Blanchoin,	A.	Michelot,	Architecture	Dependence	  139 of	Actin	Filament	Network	Disassembly,	Curr.	Biol.	25	(2015)	1437–1447.	https://doi.org/10.1016/j.cub.2015.04.011.	[96]	 E.M.	De	La	Cruz,	J.L.	Martiel,	L.	Blanchoin,	Mechanical	heterogeneity	favors	fragmentation	of	strained	actin	filaments,	Biophys.	J.	108	(2015)	2270–2281.	https://doi.org/10.1016/j.bpj.2015.03.058.	[97]	 T.D.	Pollard,	G.G.	Borisy,	Cellular	Motility	Driven	by	Assembly	Review	and	Disassembly	of	Actin	Filaments,	Cell.	112	(2003)	453–465.	https://doi.org/10.1007/BF02073506.	[98]	 R.B.	Dickinson,	Forcing	filament	fragmentation	with	cofilin,	Biophys.	J.	108	(2015)	2094–2095.	https://doi.org/10.1016/j.bpj.2015.04.002.	[99]	 G.	Kanellos,	M.C.	Frame,	Cellular	functions	of	the	ADF/cofilin	family	at	a	glance,	J.	Cell	Sci.	129	(2016)	3211–3218.	https://doi.org/10.1242/jcs.187849.	[100]	C.	Chan,	C.C.	Beltzner,	T.D.	Pollard,	Cofilin	Dissociates	Arp2/3	Complex	and	Branches	from	Actin	Filaments,	Curr.	Biol.	19	(2009)	537–545.	https://doi.org/10.1016/j.cub.2009.02.060.	[101]	M.	Klemke,	E.	Kramer,	M.H.	Konstandin,	G.H.	Wabnitz,	Y.	Samstag,	An	MEK-cofilin	signalling	module	controls	migration	of	human	T	cells	in	3D	but	not	2D	environments,	EMBO	J.	29	(2010)	2915–2929.	https://doi.org/10.1038/emboj.2010.153.	[102]	J.J.	Bravo-Cordero,	M.A.O.	Magalhaes,	R.J.	Eddy,	L.	Hodgson,	J.	Condeelis,	Functions	of	cofilin	in	cell	locomotion	and	invasion,	Nat.	Rev.	Mol.	Cell	Biol.	14	(2013)	405–417.	https://doi.org/10.1038/nrm3609.	  140 [103]	T.D.	Pollard,	Rate	constants	for	the	reactions	of	ATP-	and	ADP-actin	with	the	ends	of	actin	filaments,	J.	Cell	Biol.	103	(1986)	2747–2754.	https://doi.org/10.1083/jcb.103.6.2747.	[104]	D.	Obino,	F.	Farina,	O.	Malbec,	P.J.	Sáez,	M.	Maurin,	J.	Gaillard,	F.	Dingli,	D.	Loew,	A.	Gautreau,	M.I.	Yuseff,	L.	Blanchoin,	M.	Théry,	A.M.	Lennon-Duménil,	Actin	nucleation	at	the	centrosome	controls	lymphocyte	polarity,	Nat.	Commun.	7	(2016).	https://doi.org/10.1038/ncomms10969.	[105]	K.	Rottner,	J.	Hänisch,	K.G.	Campellone,	WASH,	WHAMM	and	JMY:	Regulation	of	Arp2/3	complex	and	beyond,	Trends	Cell	Biol.	20	(2010)	650–661.	https://doi.org/10.1016/j.tcb.2010.08.014.	[106]	M.	Edwards,	A.	Zwolak,	D.A.	Schafer,	D.	Sept,	R.	Dominguez,	J.A.	Cooper,	Capping	protein	regulators	fine-tune	actin	assembly	dynamics,	Nat.	Rev.	Mol.	Cell	Biol.	15	(2014)	677–689.	https://doi.org/10.1038/nrm3869.	[107]	T.D.	Pollard,	Actin	and	Actin-Binding	Proteins,	Cold	Spring	Harb.	Perspect.	Biol.	8	(2016)	a018226.	https://doi.org/10.1101/cshperspect.a018226.	[108]	T.	Oikawa,	H.	Yamaguchi,	T.	Itoh,	M.	Kato,	T.	Ijuin,	D.	Yamazaki,	S.	Suetsugu,	T.	Takenawa,	Ptdlns(3,4,5)P3	binding	is	necessary	for	WAVE2-induced	formation	of	lamellipodia,	Nat.	Cell	Biol.	6	(2004)	420–426.	https://doi.org/10.1038/ncb1125.	[109]	O.	Nakanishi,	S.	Suetsugu,	D.	Yamazaki,	T.	Takenawa,	Effect	of	WAVE2	phosphorylation	on	activation	of	the	Arp2/3	complex,	J.	Biochem.	141	(2007)	319–325.	https://doi.org/10.1093/jb/mvm034.	[110]	M.R.	Mejillano,	S.	Kojima,	D.A.	Applewhite,	F.B.	Gertler,	T.M.	Svitkina,	G.G.	Borisy,	  141 Lamellipodial	Versus	Filopodial	Mode	of	the	Actin	Nanomachinery,	Cell.	118	(2004)	363–373.	https://doi.org/10.1016/j.cell.2004.07.019.	[111]	C.	Suarez,	D.R.	Kovar,	Internetwork	competition	for	monomers	governs	actin	cytoskeleton	organization,	Nat.	Rev.	Mol.	Cell	Biol.	17	(2016)	799–810.	https://doi.org/10.1038/nrm.2016.106.	[112]	N.E.	Harwood,	F.D.	Batista,	The	cytoskeleton	coordinates	the	early	events	of	B-cell	activation,	Cold	Spring	Harb.	Perspect.	Biol.	3	(2011)	1–15.	https://doi.org/10.1101/cshperspect.a002360.	[113]	A.	Mogilner,	G.	Oster,	Cell	motility	driven	by	actin	polymerization,	Biophys.	J.	71	(1996)	3030–3045.	https://doi.org/10.1016/S0006-3495(96)79496-1.	[114]	A.	Babich,	S.	Li,	R.S.	O’Connor,	M.C.	Milone,	B.D.	Freedman,	J.K.	Burkhardt,	F-actin	polymerization	and	retrograde	flow	drive	sustained	PLCγ1	signaling	during	T	cell	activation,	J.	Cell	Biol.	197	(2012)	775–787.	https://doi.org/10.1083/jcb.201201018.	[115]	R.	Basu,	M.	Huse,	Mechanical	Communication	at	the	Immunological	Synapse,	Trends	Cell	Biol.	27	(2017)	241–254.	https://doi.org/10.1016/j.tcb.2016.10.005.	[116]	Z.	Wan,	X.	Chen,	H.	Chen,	Q.	Ji,	Y.	Chen,	J.	Wang,	Y.	Cao,	F.	Wang,	J.	Lou,	Z.	Tang,	W.	Liu,	The	activation	of	IgM-	or	isotype-switched	IgG-	and	IgE-BCR	exhibits	distinct	mechanical	force	sensitivity	and	threshold,	Elife.	4	(2015)	1–24.	https://doi.org/10.7554/eLife.06925.	[117]	Z.	Wan,	S.	Zhang,	Y.	Fan,	K.	Liu,	F.	Du,	A.M.	Davey,	H.	Zhang,	W.	Han,	C.	Xiong,	W.	Liu,	B	Cell	Activation	Is	Regulated	by	the	Stiffness	Properties	of	the	Substrate	Presenting	the	Antigens,	J.	Immunol.	190	(2013)	4661–4675.	  142 https://doi.org/10.4049/jimmunol.1202976.	[118]	S.	Espinoza-Sanchez,	L.A.	Metskas,	S.Z.	Chou,	E.	Rhoades,	T.D.	Pollard,	Conformational	changes	in	Arp2/3	complex	induced	by	ATP,	WASp-VCA,	and	actin	filaments,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	115	(2018)	E8642–E8651.	https://doi.org/10.1073/pnas.1717594115.	[119]	P.	Bieling,	S.D.	Hansen,	O.	Akin,	T.	Li,	C.C.	Hayden,	D.A.	Fletcher,	R.D.	Mullins,	WH2	and	proline-rich	domains	of	WASP-family	proteins	collaborate	to	accelerate	actin	filament	elongation,	EMBO	J.	37	(2018)	102–121.	https://doi.org/10.15252/embj.201797039.	[120]	W.	Song,	C.	Liu,	A.	Upadhyaya,	The	pivotal	position	of	the	actin	cytoskeleton	in	the	initiation	and	regulation	of	B	cell	receptor	activation,	Biochim.	Biophys.	Acta	-	Biomembr.	1838	(2014)	569–578.	https://doi.org/10.1016/j.bbamem.2013.07.016.	[121]	S.	Murugesan,	J.	Hong,	J.	Yi,	D.	Li,	J.R.	Beach,	L.	Shao,	J.	Meinhardt,	G.	Madison,	X.	Wu,	E.	Betzig,	J.A.	Hammer,	Formin-generated	actomyosin	arcs	propel	t	cell	receptor	microcluster	movement	at	the	immune	synapse,	J.	Cell	Biol.	215	(2016)	383–399.	https://doi.org/10.1083/jcb.201603080.	[122]	E.	Rivers,	A.	Thrasher,	Wiskott-Aldrich	syndrome	protein:	emerging	mechanisms	in	immunity,	Eur.	J.	Immunol.	(2017).	https://doi.org/10.1002/eji.201646715.	[123]	X.	Sun,	Y.	Wei,	P.P.	Lee,	B.	Ren,	C.	Liu,	The	role	of	WASp	in	T	cells	and	B	cells,	Cell.	Immunol.	341	(2019)	103919.	https://doi.org/10.1016/j.cellimm.2019.04.007.	[124]	C.	Liu,	X.	Bai,	J.	Wu,	S.	Sharma,	A.	Upadhyaya,	C.I.M.	Dahlberg,	L.S.	Westerberg,	S.B.	Snapper,	X.	Zhao,	W.	Song,	N-WASP	Is	Essential	for	the	Negative	Regulation	of	B	Cell	  143 Receptor	Signaling,	PLoS	Biol.	11	(2013).	https://doi.org/10.1371/journal.pbio.1001704.	[125]	S.	Becker-Herman,	A.	Meyer-Bahlburg,	M.A.	Schwartz,	S.W.	Jackson,	K.L.	Hudkins,	C.	Liu,	B.D.	Sather,	S.	Khim,	D.	Liggitt,	W.	Song,	G.J.	Silverman,	C.E.	Alpers,	D.J.	Rawlings,	WASp-deficient	B	cells	play	a	critical,	cell-intrinsic	role	in	triggering	autoimmunity,	J.	Exp.	Med.	208	(2011)	2033–2042.	https://doi.org/10.1084/jem.20110200.	[126]	C.	Liu,	H.	Miller,	K.L.	Hui,	B.	Grooman,	S.	Bolland,	A.	Upadhyaya,	W.	Song,	A	Balance	of	Bruton’s	Tyrosine	Kinase	and	SHIP	Activation	Regulates	B	Cell	Receptor	Cluster	Formation	by	Controlling	Actin	Remodeling,	J.	Immunol.	187	(2011)	230–239.	https://doi.org/10.4049/jimmunol.1100157.	[127]	X.	Bai,	Y.	Zhang,	L.	Huang,	J.	Wang,	W.	Li,	L.	Niu,	H.	Jiang,	R.	Dai,	L.	Zhou,	Z.	Zhang,	H.	Miller,	W.	Song,	X.	Zhao,	C.	Liu,	The	early	activation	of	memory	B	cells	from	Wiskott-Aldrich	syndrome	patients	is	suppressed	by	CD19	downregulation,	Blood.	128	(2016)	1723–1734.	https://doi.org/10.1182/blood-2016-03-703579.	[128]	T.	Takenawa,	H.	Miki,	WASP	and	WAVE	family	proteins:	Key	molecules	for	rapid	rearrangement	of	cortical	actin	filaments	and	cell	movement,	J.	Cell	Sci.	114	(2001)	1801–1809.	[129]	S.	Suetsugu,	H.	Miki,	T.	Takenawa,	Identification	of	two	human	WAVE/SCAR	homologues	as	general	actin	regulatory	molecules	which	associate	with	the	Arp2/3	complex,	Biochem.	Biophys.	Res.	Commun.	260	(1999)	296–302.	https://doi.org/10.1006/bbrc.1999.0894.	[130]	A.	Steffen,	K.	Rottner,	J.	Ehinger,	M.	Innocenti,	G.	Scita,	J.	Wehland,	T.E.B.	Stradal,	Sra-  144 1	and	Nap1	link	Rac	to	actin	assembly	driving	lamellipodia	formation,	EMBO	J.	23	(2004)	749–759.	https://doi.org/10.1038/sj.emboj.7600084.	[131]	S.	Eden,	R.	Rohatgi,	A.	V.	Podtelejnikov,	M.	Mann,	M.W.	Kirschner,	Mechanism	of	regulation	of	WAVE1-induced	actin	nucleation	by	Rac1	and	Nck,	Nature.	418	(2002)	790–793.	https://doi.org/10.1038/nature00859.	[132]	D.	Yamazaki,	S.	Suetsugu,	H.	Miki,	Y.	Kataoka,	S.-I.	Nishikawa,	T.	Fujiwara,	N.	Yoshida,	T.	Takenawa,	WAVE2	is	required	for	directed	cell	migration	adn	cadriovascular	development,	Nature.	424	(2003)	448–452.	https://doi.org/10.1038/nature01822.	[133]	J.C.	Nolz,	T.S.	Gomez,	P.	Zhu,	S.	Li,	R.B.	Medeiros,	Y.	Shimizu,	J.K.	Burkhardt,	B.D.	Freedman,	D.D.	Billadeau,	The	WAVE2	complex	regulates	actin	cytoskeletal	reorganization	and	CRAC-mediated	calcium	entry	during	T	cell	activation,	Curr.	Biol.	16	(2006)	24–34.	https://doi.org/10.1016/j.cub.2005.11.036.	[134]	S.A.	Cook,	W.A.	Comrie,	M.C.	Poli,	M.	Similuk,	A.J.	Oler,	A.J.	Faruqi,	D.B.	Kuhns,	S.	Yang,	A.	Vargas-Hernández,	A.F.	Carisey,	B.	Fournier,	D.E.	Anderson,	S.	Price,	M.	Smelkinson,	W.	Abou	Chahla,	L.R.	Forbes,	E.M.	Mace,	T.N.	Cao,	Z.H.	Coban-Akdemir,	S.N.	Jhangiani,	D.M.	Muzny,	R.A.	Gibbs,	J.R.	Lupski,	J.S.	Orange,	G.D.E.	Cuvelier,	M.	Al	Hassani,	N.	Al	Kaabi,	Z.	Al	Yafei,	S.	Jyonouchi,	N.	Raje,	J.W.	Caldwell,	Y.	Huang,	J.K.	Burkhardt,	S.	Latour,	B.	Chen,	G.	ElGhazali,	V.K.	Rao,	I.K.	Chinn,	M.J.	Lenardo,	HEM1	deficiency	disrupts	mTORC2	and	F-actin	control	in	inherited	immunodysregulatory	disease,	Science.	369	(2020)	202–207.	https://doi.org/10.1126/science.aay5663.	[135]	K.G.	Campellone,	M.D.	Welch,	A	nucleator	arms	race:	cellular	control	of	actin	assembly,	Nat.	Rev.	Mol.	Cell	Biol.	11	(2010)	237–251.	  145 https://doi.org/10.1038/nrm2867.	[136]	X.	Li,	D.	Sandoval,	L.	Freeberg,	R.H.	Carter,	Role	of	CD19	tyrosine	391	in	synergistic	activation	of	B	lymphocytes	by	coligation	of	CD19	and	membrane	Ig.,	J.	Immunol.	158	(1997)	5649–5657.	[137]	L.M.	O’Rourke,	R.	Tooze,	M.	Turner,	D.M.	Sandoval,	R.H.	Carter,	V.L.J.	Tybulewicz,	D.T.	Fearon,	CD19	as	a	Membrane-Anchored	Adaptor	Protein	of	B	Lymphocytes:	Costimulation	of	Lipid	and	Protein	Kinases	by	Recruitment	of	Vav,	Immunity.	8	(1998)	635–645.	https://doi.org/10.1016/S1074-7613(00)80568-3.	[138]	W.K.	Weng,	L.	Jarvis,	T.W.	LeBien,	Signaling	through	CD19	activates	Vav/mitogen-activated	protein	kinase	pathway	and	induces	formation	of	a	CD19/Vav/phosphatidylinositol	3-kinase	complex	in	human	B	cell	precursors,	J.	Biol.	Chem.	269	(1994)	32514–32521.	[139]	X.R.	Bustelo,	Regulatory	and	signaling	properties	of	the	Vav	family.,	Mol.	Cell.	Biol.	20	(2000)	1461–77.	https://doi.org/10.1128/mcb.20.5.1461-1477.2000.	[140]	J.	Wu,	Q.	Zhao,	T.	Kurosaki,	A.	Weiss,	The	Vav	binding	site	(Y315)	in	ZAP-70	is	critical	for	antigen	receptor-mediated	signal	transduction,	J.	Exp.	Med.	185	(1997)	1877–1882.	https://doi.org/10.1084/jem.185.10.1877.	[141]	M.	Weber,	B.	Treanor,	D.	Depoil,	H.	Shinohara,	N.E.	Harwood,	M.	Hikida,	T.	Kurosaki,	F.D.	Batista,	Phospholipase	C-γ2	and	Vav	cooperate	within	signaling	microclusters	to	propagate	B	cell	spreading	in	response	to	membrane-bound	antigen,	J.	Exp.	Med.	205	(2008)	853–868.	https://doi.org/10.1084/jem.20072619.	[142]	F.	Guo,	C.S.	Velu,	H.L.	Grimes,	Y.	Zheng,	Rho	GTPase	Cdc42	is	essential	for	B-  146 lymphocyte	development	and	activation,	Blood.	114	(2009)	2909–2916.	https://doi.org/10.1182/blood-2009-04-214676.	[143]	M.	Burbage,	S.J.	Keppler,	F.	Gasparrini,	N.	Martínez-Martín,	M.	Gaya,	C.	Feest,	M.-C.	Domart,	C.	Brakebusch,	L.	Collinson,	A.	Bruckbauer,	F.D.	Batista,	Cdc42	is	a	key	regulator	of	B	cell	differentiation	and	is	required	for	antiviral	humoral	immunity,	J.	Exp.	Med.	212	(2015)	53–72.	https://doi.org/10.1084/jem.20141143.	[144]	K.L.	Randall,	T.	Lambe,	A.	Johnson,	B.	Treanor,	E.	Kucharska,	H.	Domaschenz,	B.	Whittle,	L.E.	Tze,	A.	Enders,	T.L.	Crockford,	T.	Bouriez-Jones,	D.	Alston,	J.G.	Cyster,	M.J.	Lenardo,	F.	Mackay,	E.K.	Deenick,	S.G.	Tangye,	T.D.	Chan,	T.	Camidge,	R.	Brink,	C.G.	Vinuesa,	F.D.	Batista,	R.J.	Cornall,	C.C.	Goodnow,	Dock8	mutations	cripple	B	cell	immunological	synapses,	germinal	centers	and	long-lived	antibody	production,	Nat.	Immunol.	10	(2009)	1283–1291.	https://doi.org/10.1038/ni.1820.	[145]	X.	Sun,	J.	Wang,	T.	Qin,	Y.	Zhang,	L.	Huang,	L.	Niu,	X.	Bai,	Y.	Jing,	X.	Xuan,	H.	Miller,	Y.	Zhao,	W.	Song,	X.	Tang,	Z.	Zhang,	X.	Zhao,	C.	Liu,	Dock8	regulates	BCR	signaling	and	activation	of	memory	B	cells	via	WASP	and	CD19,	Blood	Adv.	2	(2018)	401–413.	https://doi.org/10.1182/bloodadvances.2017007880.	[146]	F.	Ardito,	M.	Giuliani,	D.	Perrone,	G.	Troiano,	L.	Lo	Muzio,	The	crucial	role	of	protein	phosphorylation	in	cell	signalingand	its	use	as	targeted	therapy	(Review),	Int.	J.	Mol.	Med.	40	(2017)	271–280.	https://doi.org/10.3892/ijmm.2017.3036.	[147]	K.	Mizuno,	Signaling	mechanisms	and	functional	roles	of	cofilin	phosphorylation	and	dephosphorylation,	Cell.	Signal.	25	(2013)	457–469.	https://doi.org/10.1016/j.cellsig.2012.11.001.	  147 [148]	Y.	Yamanashi,	T.	Fukuda,	H.	Nishizumi,	T.	Inazu,	K.I.	Higashi,	D.	Kitamura,	T.	Ishida,	H.	Yamamura,	T.	Watanabe,	T.	Yamamoto,	Role	of	tyrosine	phosphorylation	of	HS1	in	B	cell	antigen	receptor-	mediated	apoptosis,	J.	Exp.	Med.	185	(1997)	1387–1392.	https://doi.org/10.1084/jem.185.7.1387.	[149]	G.L.	Zhou,	H.	Zhang,	H.	Wu,	P.	Ghai,	J.	Field,	Phosphorylation	of	the	cytoskeletal	protein	CAP1	controls	its	association	with	cofilin	and	actin,	J.	Cell	Sci.	127	(2014)	5052–5065.	https://doi.org/10.1242/jcs.156059.	[150]	M.	López-Lago,	H.	Lee,	C.	Cruz,	N.	Movilla,	X.R.	Bustelo,	Tyrosine	Phosphorylation	Mediates	Both	Activation	and	Downmodulation	of	the	Biological	Activity	of	Vav,	Mol.	Cell.	Biol.	20	(2000)	1678–1691.	https://doi.org/10.1128/mcb.20.5.1678-1691.2000.	[151]	A.	Dovas,	D.	Cox,	Regulation	of	WASp	by	phosphorylation	activation	or	other	functions?,	Commun.	Integr.	Biol.	3	(2010)	101–105.	https://doi.org/10.4161/cib.3.2.10759.	[152]	N.	Lu,	C.J.	Malemud,	Extracellular	signal-regulated	kinase:	A	regulator	of	cell	growth,	inflammation,	chondrocyte	and	bone	cell	receptor-mediated	gene	expression,	Int.	J.	Mol.	Sci.	20	(2019)	1–18.	https://doi.org/10.3390/ijms20153792.	[153]	A.	Jacob,	D.	Cooney,	M.	Pradhan,	K.	Mark	Coggeshall,	Convergence	of	signaling	pathways	on	the	activation	of	ERK	in	B	cells,	J.	Biol.	Chem.	277	(2002)	23420–23426.	https://doi.org/10.1074/jbc.M202485200.	[154]	M.C.	Mendoza,	E.E.	Er,	W.	Zhang,	B.A.	Ballif,	H.L.	Elliott,	G.	Danuser,	J.	Blenis,	ERK-MAPK	Drives	Lamellipodia	Protrusion	by	Activating	the	WAVE2	Regulatory	Complex,	  148 Mol.	Cell.	41	(2011)	661–671.	https://doi.org/10.1016/j.molcel.2011.02.031.	[155]	Y.	Gao,	K.	Kawano,	S.	Yoshiyama,	H.	Kawamichi,	X.	Wang,	A.	Nakamura,	K.	Kohama,	Myosin	light	chain	kinase	stimulates	smooth	muscle	myosin	ATPase	activity	by	binding	to	the	myosin	heads	without	phosphorylating	the	myosin	light	chain,	Biochem.	Biophys.	Res.	Commun.	305	(2003)	16–21.	https://doi.org/10.1016/S0006-291X(03)00690-9.	[156]	S.	Schubbert,	K.	Shannon,	G.	Bollag,	Hyperactive	Ras	in	developmental	disorders	and	cancer,	Nat.	Rev.	Cancer.	7	(2007)	295–308.	https://doi.org/10.1038/nrc2109.	[157]	L.S.	Steelman,	W.H.	Chappell,	S.L.	Abrams,	C.R.	Kempf,	J.	Long,	P.	Laidler,	S.	Mijatovic,	D.	Maksimovic-Ivanic,	F.	Stivala,	M.C.	Mazzarino,	M.	Donia,	P.	Fagone,	G.	Malaponte,	F.	Nicoletti,	M.	Libra,	M.	Milella,	A.	Tafuri,	A.	Bonati,	J.	Bäsecke,	L.	Cocco,	C.	Evangelisti,	A.M.	Martelli,	G.	Montalto,	M.	Cervello,	J.A.	McCubrey,	Roles	of	the	Raf/MEK/ERK	and	PI3K/PTEN/Akt/mtor	pathways	in	controlling	growth	and	sensitivity	to	therapy-implications	for	cancer	and	aging,	Aging	(Albany.	NY).	3	(2011)	192–222.	https://doi.org/10.18632/aging.100296.	[158]	S.	Tanimura,	K.	Takeda,	ERK	signalling	as	a	regulator	of	cell	motility,	J.	Biochem.	162	(2017)	145–154.	https://doi.org/10.1093/jb/mvx048.	[159]	M.C.	Brown,	C.E.	Turner,	Paxillin :	Adapting	to	Change,	(2004)	1315–1339.	[160]	S.F.F.	Pereira,	L.	Goss,	J.	Dworkin,	Eukaryote-Like	Serine/Threonine	Kinases	and	Phosphatases	in	Bacteria,	Microbiol.	Mol.	Biol.	Rev.	75	(2011)	192–212.	https://doi.org/10.1128/mmbr.00042-10.	[161]	M.	Cakir,	A.B.	Grossman,	Targeting	MAPK	(Ras/ERK)	and	PI3K/Akt	pathways	in	  149 pituitary	tumorigenesis,	Expert	Opin.	Ther.	Targets.	13	(2009)	1121–1134.	https://doi.org/10.1517/14728220903170675.	[162]	M.	Huse,	J.	Kuriyan,	The	conformational	plasticity	of	protein	kinases,	Cell.	109	(2002)	275–282.	https://doi.org/10.1016/S0092-8674(02)00741-9.	[163]	L.N.	Johnson,	M.E.M.	Noble,	D.J.	Owen,	Active	and	inactive	protein	kinases:	Structural	basis	for	regulation,	Cell.	85	(1996)	149–158.	https://doi.org/10.1016/S0092-8674(00)81092-2.	[164]	B.J.	Canagarajah,	A.	Khokhlatchev,	M.H.	Cobb,	E.J.	Goldsmith,	Activation	mechanism	of	the	MAP	kinase	ERK2	by	dual	phosphorylation,	Cell.	90	(1997)	859–869.	https://doi.org/10.1016/S0092-8674(00)80351-7.	[165]	A.	V.	Khokhlatchev,	B.	Canagarajah,	J.	Wilsbacher,	M.	Robinson,	M.	Atkinson,	E.	Goldsmith,	M.H.	Cobb,	Phosphorylation	of	the	MAP	kinase	ERK2	promotes	its	homodimerization	and	nuclear	translocation,	Cell.	93	(1998)	605–615.	https://doi.org/10.1016/S0092-8674(00)81189-7.	[166]	T.	Kinases,	D.	Finlay,	D.	Cantrell,	The	Coordination	of	T-cell	Function,	(2011).	[167]	E.	Kotelnikova,	N.A.	Kiani,	D.	Messinis,	I.	Pertsovskaya,	V.	Pliaka,	M.	Bernardo-Faura,	M.	Rinas,	G.	Vila,	I.	Zubizarreta,	I.	Pulido-Valdeolivas,	T.	Sakellaropoulos,	W.	Faigle,	G.	Silberberg,	M.	Masso,	P.	Stridh,	J.	Behrens,	T.	Olsson,	R.	Martin,	F.	Paul,	L.G.	Alexopoulos,	J.	Saez-Rodriguez,	J.	Tegner,	P.	Villoslada,	MAPK	pathway	and	B	cells	overactivation	in	multiple	sclerosis	revealed	by	phosphoproteomics	and	genomic	analysis,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	116	(2019)	9671–9676.	https://doi.org/10.1073/pnas.1818347116.	  150 [168]	C.	Huang,	S.R.	Foster,	A.D.	Shah,	O.	Kleifeld,	M.	Canals,	R.B.	Schittenhelm,	M.J.	Stone,	Phosphoproteomic	characterization	of	the	signaling	network	resulting	from	activation	of	chemokine	receptor	CCR2,	J.	Biol.	Chem.	(2020)	jbc.RA119.012026.	https://doi.org/10.1074/jbc.ra119.012026.	[169]	M.	Carrascal,	D.	Ovelleiro,	V.	Casas,	M.	Gay,	J.	Abian,	Phosphorylation	analysis	of	primary	human	T	lymphocytes	using	sequential	IMAC	and	titanium	oxide	enrichment,	J.	Proteome	Res.	7	(2008)	5167–5176.	https://doi.org/10.1021/pr800500r.	[170]	V.	Mayya,	D.H.	Lundgren,	S.L.L.	Hwang,	K.	Rezaul,	L.	Wu,	J.K.	Eng,	V.	Rodionov,	D.K.	Han,	Quantitative	phosphoproteomic	analysis	of	T	Cell	receptor	signaling	reveals	system-wide	modulation	of	protein-protein	interactions,	Sci.	Signal.	2	(2009).	https://doi.org/10.1126/scisignal.2000007.	[171]	V.	Nguyen,	L.	Cao,	J.T.	Lin,	N.	Hung,	A.	Ritz,	K.	Yu,	R.	Jianu,	S.P.	Ulin,	B.J.	Raphael,	D.H.	Laidlaw,	L.	Brossay,	A.R.	Salomon,	A	new	approach	for	quantitative	phosphoproteomic	dissection	of	signaling	pathways	applied	to	T	cell	receptor	activation,	Mol.	Cell.	Proteomics.	8	(2009)	2418–2431.	https://doi.org/10.1074/mcp.M800307-MCP200.	[172]	R.	Buscà,	J.	Pouysségur,	P.	Lenormand,	ERK1	and	ERK2	map	kinases:	Specific	roles	or	functional	redundancy?,	Front.	Cell	Dev.	Biol.	4	(2016)	1–23.	https://doi.org/10.3389/fcell.2016.00053.	[173]	S.	Yoon,	R.	Seger,	The	extracellular	signal-regulated	kinase:	Multiple	substrates	regulate	diverse	cellular	functions,	Growth	Factors.	24	(2006)	21–44.	  151 https://doi.org/10.1080/02699050500284218.	[174]	A.C.	Lloyd,	of	Biology	Minireview	BioMed	Central	Distinct	functions	for	ERKs ?,	Lloydia	(Cincinnati).	(2006).	[175]	R.	Roskoski,	ERK1/2	MAP	kinases:	Structure,	function,	and	regulation,	Pharmacol.	Res.	66	(2012)	105–143.	https://doi.org/10.1016/j.phrs.2012.04.005.	[176]	S.L.	Spencer,	P.K.	Sorger,	Measuring	and	modeling	apoptosis	in	single	cells,	Cell.	144	(2011)	926–939.	https://doi.org/10.1016/j.cell.2011.03.002.	[177]	W.Y.C.	Huang,	Q.	Yan,	W.C.	Lin,	J.K.	Chung,	S.D.	Hansen,	S.M.	Christensen,	H.L.	Tu,	J.	Kuriyan,	J.T.	Groves,	Phosphotyrosine-mediated	LAT	assembly	on	membranes	drives	kinetic	bifurcation	in	recruitment	dynamics	of	the	Ras	activator	SOS,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	113	(2016)	8218–8223.	https://doi.org/10.1073/pnas.1602602113.	[178]	K.	Nagai,	M.	Takata,	H.	Yamamura,	T.	Kurosaki,	Tyrosine	Phosphorylation	of	Shc	Is	Mediated	through	Lyn	and	Syk	in	B	Cell	Receptor	Signaling,	J.	Biol.	Chem.	270	(1995)	6824–6829.	[179]	D.	D’Ambrosio,	K.L.	Hippen,	J.C.	Gambier,	Distinct	mechanisms	mediate	SHC	association	with	the	activated	and	resting	B	cell	antigen	receptor,	Eur.	J.	Immunol.	26	(1996)	1960–1965.	https://doi.org/10.1002/eji.1830260842.	[180]	A.J.	Laude,	I.A.	Prior,	Palmitoylation	and	localisation	of	RAS	isoforms	are	modulated	by	the	hypervariable	linker	domain,	J.	Cell	Sci.	121	(2008)	421–427.	https://doi.org/10.1242/jcs.020107.	[181]	J.D.	McLaurin,	O.D.	Weiner,	Multiple	sources	of	signal	amplification	within	the	B-cell	Ras/MAPK	pathway,	Mol.	Biol.	Cell.	30	(2019)	1610–1620.	  152 https://doi.org/10.1091/mbc.E18-09-0560.	[182]	S.L.	Stang,	A.	Lopez-Campistrous,	X.	Song,	N.A.	Dower,	P.M.	Blumberg,	P.A.	Wender,	J.C.	Stone,	A	proapoptotic	signaling	pathway	involving	RasGRP,	Erk,	and	Bim	in	B	cells,	Exp.	Hematol.	37	(2009)	122–134.	https://doi.org/10.1016/j.exphem.2008.09.008.	[183]	J.C.	Stone,	Regulation	of	Ras	in	lymphocytes:	Get	a	GRP,	Biochem.	Soc.	Trans.	34	(2006)	858–861.	https://doi.org/10.1042/BST0340858.	[184]	J.	Das,	M.	Ho,	J.	Zikherman,	C.	Govern,	M.	Yang,	A.	Weiss,	A.K.	Chakraborty,	J.P.	Roose,	Digital	Signaling	and	Hysteresis	Characterize	Ras	Activation	in	Lymphoid	Cells,	Cell.	136	(2009)	337–351.	https://doi.org/10.1016/j.cell.2008.11.051.	[185]	L.S.	Steelman,	S.C.	Pohnert,	J.G.	Shelton,	R.A.	Franklin,	F.E.	Bertrand,	J.A.	McCubrey,	JAK/STAT,	Raf/MEK/ERK,	PI3K/Akt	and	BCR-ABL	in	cell	cycle	progression	and	leukemogenesis,	Leukemia.	18	(2004)	189–218.	https://doi.org/10.1038/sj.leu.2403241.	[186]	E.	Choy,	V.K.	Chiu,	J.	Silletti,	M.	Feoktistov,	T.	Morimoto,	D.	Michaelson,	I.E.	Ivanov,	M.R.	Philips,	Endomembrane	trafficking	of	ras:	The	CAAX	motif	targets	proteins	to	the	ER	and	Golgi,	Cell.	98	(1999)	69–80.	https://doi.org/10.1016/S0092-8674(00)80607-8.	[187]	V.K.	Chiu,	T.	Bivona,	A.	Hach,	J.B.	Sajous,	J.	Silletti,	H.	Wiener,	R.L.	Johnson,	A.D.	Cox,	M.R.	Philips,	Ras	signalling	on	the	endoplasmic	reticulum	and	the	Golgi,	Nat.	Cell	Biol.	4	(2002)	343–350.	https://doi.org/10.1038/ncb783.	[188]	A.	Apolloni,	I.A.	Prior,	M.	Lindsay,	R.G.	Parton,	J.F.	Hancock,	H-ras	but	Not	K-ras	  153 Traffics	to	the	Plasma	Membrane	through	the	Exocytic	Pathway,	Mol.	Cell.	Biol.	20	(2000)	2475–2487.	https://doi.org/10.1128/mcb.20.7.2475-2487.2000.	[189]	X.	Nan,	T.M.	Tamgüney,	E.A.	Collisson,	L.J.	Lin,	C.	Pitt,	J.	Galeas,	S.	Lewis,	J.W.	Gray,	F.	McCormick,	S.	Chu,	Ras-GTP	dimers	activate	the	Mitogen-Activated	Protein	Kinase	(MAPK)	pathway,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	112	(2015)	7996–8001.	https://doi.org/10.1073/pnas.1509123112.	[190]	W.	Li,	H.	Chong,	K.L.	Guan,	Function	of	the	Rho	Family	GTPases	in	Ras-stimulated	Raf	Activation,	J.	Biol.	Chem.	276	(2001)	34728–34737.	https://doi.org/10.1074/jbc.M103496200.	[191]	H.	Gille,	A.	Sharrocks,	P.	Shaw,	Phosphorylation	of	transcription	factor	p62TCF	by	MAP	kinase	stimulates	ternary	complex	formation	at	c-fos	promoter,	Nature.	359	(1992)	710–713.	[192]	R.	Marais,	J.	Wynne,	R.	Treisman,	The	SRF	accessory	protein	Elk-1	contains	a	growth	factor-regulated	transcriptional	activation	domain,	Cell.	73	(1993)	381–393.	https://doi.org/10.1016/0092-8674(93)90237-K.	[193]	C.	Hauge,	M.	Frödin,	RSK	aand	MSK	in	MAP	kinase	signalling,	J.	Cell	Sci.	119	(2006)	3021–3023.	https://doi.org/10.1242/jcs.02950.	[194]	J.T.	Blois,	J.M.	Mataraza,	I.	Mecklenbräuker,	A.	Tarakhovsky,	T.C.	Chiles,	B	cell	receptor-induced	cAMP-response	element-binding	protein	activation	in	B	lymphocytes	requires	novel	protein	kinase	Cδ,	J.	Biol.	Chem.	279	(2004)	30123–30132.	https://doi.org/10.1074/jbc.M402793200.	[195]	Z.X.	Liu,	C.F.	Yu,	C.	Nickel,	S.	Thomas,	L.G.	Cantley,	Hepatocyte	growth	factor	induces	  154 ERK-dependent	paxillin	phosphorylation	and	regulates	paxillin-focal	adhesion	kinase	association,	J.	Biol.	Chem.	277	(2002)	10452–10458.	https://doi.org/10.1074/jbc.M107551200.	[196]	T.S.	Lewis,	J.B.	Hunt,	L.D.	Aveline,	K.R.	Jonscher,	D.F.	Louie,	J.M.	Yeh,	T.S.	Nahreini,	K.A.	Resing,	N.G.	Ahn,	Identification	of	novel	MAP	kinase	pathway	signaling	targets	by	functional	proteomics	and	mass	spectrometry,	Mol.	Cell.	6	(2000)	1343–1354.	https://doi.org/10.1016/S1097-2765(00)00132-5.	[197]	M.	Mitsushima,	A.	Suwa,	T.	Amachi,	K.	Ueda,	N.	Kioka,	Extracellular	signal-regulated	kinase	activated	by	epidermal	growth	factor	and	cell	adhesion	interacts	with	and	phosphorylates	vinexin,	J.	Biol.	Chem.	279	(2004)	34570–34577.	https://doi.org/10.1074/jbc.M402304200.	[198]	J.	Choi,	A.J.	Huebner,	K.	Clement,	R.M.	Walsh,	A.	Savol,	K.	Lin,	H.	Gu,	B.	Di	Stefano,	J.	Brumbaugh,	S.Y.	Kim,	J.	Sharif,	C.M.	Rose,	A.	Mohammad,	J.	Odajima,	J.	Charron,	T.	Shioda,	A.	Gnirke,	S.	Gygi,	H.	Koseki,	R.I.	Sadreyev,	A.	Xiao,	A.	Meissner,	K.	Hochedlinger,	Prolonged	Mek1/2	suppression	impairs	the	developmental	potential	of	embryonic	stem	cells,	Nature.	548	(2017)	219–223.	https://doi.org/10.1038/nature23274.	[199]	T.K.	Hayes,	N.F.	Neel,	C.	Hu,	P.	Gautam,	M.	Chenard,	B.	Long,	M.	Aziz,	M.	Kassner,	K.L.	Bryant,	M.	Pierobon,	R.	Marayati,	S.	Kher,	S.D.	George,	M.	Xu,	A.	Wang-Gillam,	A.A.	Samatar,	A.	Maitra,	K.	Wennerberg,	E.F.	Petricoin,	H.H.	Yin,	B.	Nelkin,	A.D.	Cox,	J.J.	Yeh,	C.J.	Der,	Long-Term	ERK	Inhibition	in	KRAS-Mutant	Pancreatic	Cancer	Is	Associated	with	MYC	Degradation	and	Senescence-like	Growth	Suppression,	Cancer	Cell.	29	  155 (2016)	75–89.	https://doi.org/10.1016/j.ccell.2015.11.011.	[200]	S.B.	Gauld,	D.	Blair,	C.A.	Moss,	S.D.	Reid,	M.M.	Harnett,	Differential	Roles	for	Extracellularly	Regulated	Kinase-Mitogen-Activated	Protein	Kinase	in	B	Cell	Antigen	Receptor-Induced	Apoptosis	and	CD40-Mediated	Rescue	of	WEHI-231	Immature	B	Cells,	J.	Immunol.	168	(2002)	3855–3864.	https://doi.org/10.4049/jimmunol.168.8.3855.	[201]	J.D.	Richards,	S.H.	Davé,	C.-H.G.	Chou,	A.A.	Mamchak,	A.L.	DeFranco,	Inhibition	of	the	MEK/ERK	Signaling	Pathway	Blocks	a	Subset	of	B	Cell	Responses	to	Antigen,	J.	Immunol.	166	(2001)	3855–3864.	https://doi.org/10.4049/jimmunol.166.6.3855.	[202]	L.	Rui,	C.G.	Vinuesa,	J.	Blasioli,	C.C.	Goodnow,	Resistance	to	CpG	DNA-induced	autoimmunity	through	tolerogenic	B	cell	antigen	receptor	ERK	signaling,	Nat.	Immunol.	4	(2003)	594–600.	https://doi.org/10.1038/ni924.	[203]	T.	Yasuda,	H.	Sanjo,	G.	Pagès,	Y.	Kawano,	H.	Karasuyama,	J.	Pouysségur,	M.	Ogata,	T.	Kurosaki,	Erk	Kinases	Link	Pre-B	Cell	Receptor	Signaling	to	Transcriptional	Events	Required	for	Early	B	Cell	Expansion,	Immunity.	28	(2008)	499–508.	https://doi.org/10.1016/j.immuni.2008.02.015.	[204]	H.	Nagaoka,	Y.	Takahashi,	R.	Hayashi,	T.	Nakamura,	K.	Ishii,	J.	Matsuda,	A.	Ogura,	Y.	Shirakata,	H.	Karasuyama,	T.	Sudo,	S.I.	Nishikawa,	T.	Tsubata,	T.	Mizuochi,	T.	Asano,	H.	Sakano,	T.	Takemori,	Ras	mediates	effector	pathways	responsible	for	pre-B	cell	survival,	which	is	essential	for	the	developmental	progression	to	the	late	pre-B,	cell	stage,	J.	Exp.	Med.	192	(2000)	171–181.	https://doi.org/10.1084/jem.192.2.171.	[205]	S.L.	Rowland,	C.L.	DePersis,	R.M.	Torres,	R.	Pelanda,	Ras	activation	of	Erk	restores	  156 impaired	tonic	BCR	signaling	and	rescues	immature	B	cell	differentiation,	J.	Exp.	Med.	207	(2010)	607–621.	https://doi.org/10.1084/jem.20091673.	[206]	J.	Samuels,	Y.S.	Ng,	C.	Coupillaud,	D.	Paget,	E.	Meffre,	Impaired	early	B	cell	tolerance	in	patients	with	rheumatoid	arthritis,	J.	Exp.	Med.	201	(2005)	1659–1667.	https://doi.org/10.1084/jem.20042321.	[207]	S.A.	Greaves,	J.N.	Peterson,	R.M.	Torres,	R.	Pelanda,	Activation	of	the	MEK-ERK	pathway	is	necessary	but	not	sufficient	for	breaking	central	B	cell	tolerance,	Front.	Immunol.	9	(2018).	https://doi.org/10.3389/fimmu.2018.00707.	[208]	L.S.	Teodorovic,	C.	Babolin,	S.L.	Rowland,	S.A.	Greaves,	D.P.	Baldwin,	R.M.	Torres,	R.	Pelanda,	Activation	of	Ras	overcomes	B-cell	tolerance	to	promote	differentiation	of	autoreactive	B	cells	and	production	of	autoantibodies,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	111	(2014).	https://doi.org/10.1073/pnas.1402159111.	[209]	J.	Adem,	A.	Hämäläinen,	A.	Ropponen,	J.	Eeva,	M.	Eray,	U.	Nuutinen,	J.	Pelkonen,	ERK1/2	has	an	essential	role	in	B	cell	receptor-	and	CD40-induced	signaling	in	an	in	vitro	model	of	germinal	center	B	cell	selection,	Mol.	Immunol.	67	(2015)	240–247.	https://doi.org/10.1016/j.molimm.2015.05.017.	[210]	Y.	Takahashi,	A.	Inamine,	S.I.	Hashimoto,	S.	Haraguchi,	E.	Yoshioka,	N.	Kojima,	R.	Abe,	T.	Takemori,	Novel	role	of	the	Ras	cascade	in	memory	B	cell	response,	Immunity.	23	(2005)	127–138.	https://doi.org/10.1016/j.immuni.2005.06.010.	[211]	L.A.	O’Reilly,	E.A.	Kruse,	H.	Puthalakath,	P.N.	Kelly,	T.	Kaufmann,	D.C.S.	Huang,	A.	Strasser,	MEK/ERK-Mediated	Phosphorylation	of	Bim	Is	Required	to	Ensure	Survival	of	T	and	B	Lymphocytes	during	Mitogenic	Stimulation,	J.	Immunol.	183	(2009)	261–  157 269.	https://doi.org/10.4049/jimmunol.0803853.	[212]	H.	Sanjo,	M.	Hikida,	Y.	Aiba,	Y.	Mori,	N.	Hatano,	M.	Ogata,	T.	Kurosaki,	Extracellular	Signal-Regulated	Protein	Kinase	2	Is	Required	for	Efficient	Generation	of	B	Cells	Bearing	Antigen-Specific	Immunoglobulin	G,	Mol.	Cell.	Biol.	27	(2007)	1236–1246.	https://doi.org/10.1128/mcb.01530-06.	[213]	L.A.	Allan,	N.	Morrice,	S.	Brady,	G.	Magee,	S.	Pathak,	P.R.	Clarke,	Inhibition	of	caspase-9	through	phosphorylation	at	Thr	125	by	ERK	MAPK,	Nat.	Cell	Biol.	5	(2003)	647–654.	https://doi.org/10.1038/ncb1005.	[214]	T.	Yasuda,	K.	Kometani,	N.	Takahashi,	Y.	Imai,	Y.	Aiba,	T.	Kurosaki,	ERKs	induce	expression	of	the	transcriptional	repressor	Blimp-1	and	subsequent	plasma	cell	differentiation,	Sci.	Signal.	4	(2011)	1–11.	https://doi.org/10.1126/scisignal.2001592.	[215]	L.	Rui,	J.I.	Healy,	J.	Blasioli,	C.C.	Goodnow,	ERK	Signaling	Is	a	Molecular	Switch	Integrating	Opposing	Inputs	from	B	Cell	Receptor	and	T	Cell	Cytokines	to	Control	TLR4-Driven	Plasma	Cell	Differentiation,	J.	Immunol.	177	(2006)	5337–5346.	https://doi.org/10.4049/jimmunol.177.8.5337.	[216]	A.A.	Brahmbhatt,	R.L.	Klemke,	ERK	and	RhoA	differentially	regulate	pseudopodia	growth	and	retraction	during	chemotaxis,	J.	Biol.	Chem.	278	(2003)	13016–13025.	https://doi.org/10.1074/jbc.M211873200.	[217]	M.G.H.	Scott,	V.	Pierotti,	H.	Storez,	E.	Lindberg,	A.	Thuret,	O.	Muntaner,	C.	Labbe-Jullie,	J.A.	Pitcher,	S.	Marullo,	Cooperative	Regulation	of	Extracellular	Signal-Regulated	Kinase	Activation	and	Cell	Shape	Change	by	Filamin	A	and		-Arrestins,	Mol.	Cell.	Biol.	  158 26	(2006)	3432–3445.	https://doi.org/10.1128/mcb.26.9.3432-3445.2006.	[218]	S.D.	Smith,	Z.M.	Jaffer,	J.	Chernoff,	A.J.	Ridley,	PAK1-mediated	activation	of	ERK1/2	regulates	lamellipodial	dynamics,	J.	Cell	Sci.	121	(2008)	3729–3736.	https://doi.org/10.1242/jcs.027680.	[219]	C.M.	Danson,	S.M.	Pocha,	G.B.	Bloomberg,	G.O.	Cory,	Phosphorylation	of	WAVE2	by	MAP	kinases	regulates	persistent	cell	migration	and	polarity,	J.	Cell	Sci.	120	(2007)	4144–4154.	https://doi.org/10.1242/jcs.013714.	[220]	S.C.	Bunnell,	V.	Kapoor,	R.P.	Trible,	W.	Zhang,	L.E.	Samelson,	Dynamic	actin	polymerization	drives	T	cell	receptor-induced	spreading:	A	role	for	the	signal	transduction	adaptor	LAT,	Immunity.	14	(2001)	315–329.	https://doi.org/10.1016/S1074-7613(01)00112-1.	[221]	R.L.	Klemke,	S.	Cai,	A.L.	Giannini,	P.J.	Gallagher,	P.	De	Lanerolle,	D.A.	Cheresh,	Regulation	of	cell	motility	by	mitogen-activated	protein	kinase,	J.	Cell	Biol.	137	(1997)	481–492.	https://doi.org/10.1083/jcb.137.2.481.	[222]	R.S.	Adelstein,	Regulation	of	contractile	proteins	by	phosphorylation,	J.	Clin.	Invest.	72	(1983)	1863–1866.	https://doi.org/10.1172/JCI111148.	[223]	S.J.	Spratley,	L.I.	Bastea,	H.	Döppler,	K.	Mizuno,	P.	Storz,	Protein	kinase	D	regulates	cofilin	activity	through	p21-activated	kinase	4,	J.	Biol.	Chem.	286	(2011)	34254–34261.	https://doi.org/10.1074/jbc.M111.259424.	[224]	G.	Pawlak,	D.M.	Helfman,	MEK	mediates	v-Src-induced	disruption	of	the	actin	cytoskeleton	via	inactivation	of	the	Rho-ROCK-LIM	kinase	pathway,	J.	Biol.	Chem.	277	(2002)	26927–26933.	https://doi.org/10.1074/jbc.M202261200.	  159 [225]	K.A.	Burkhard,	F.	Chen,	P.	Shapiro,	Quantitative	analysis	of	ERK2	interactions	with	substrate	proteins:	Roles	for	kinase	docking	domains	and	activity	in	determining	binding	affinity,	J.	Biol.	Chem.	286	(2011)	2477–2485.	https://doi.org/10.1074/jbc.M110.177899.	[226]	E.L.	Filbert,	M.	Le	Borgne,	J.	Lin,	J.E.	Heuser,	A.S.	Shaw,	Stathmin	Regulates	Microtubule	Dynamics	and	Microtubule	Organizing	Center	Polarization	in	Activated	T	Cells,	J.	Immunol.	188	(2012)	5421–5427.	https://doi.org/10.4049/jimmunol.1200242.	[227]	X.	Chen,	D.S.J.	Allan,	K.	Krzewski,	B.	Ge,	H.	Kopcow,	J.L.	Strominger,	CD28-stimulated	ERK2	phosphorylation	is	required	for	polarization	of	the	microtubule	organizing	center	and	granules	in	YTS	NK	cells,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	103	(2006)	10346–10351.	https://doi.org/10.1073/pnas.0604236103.	[228]	A.K.	Pullikuth,	A.D.	Catling,	Scaffold	mediated	regulation	of	MAPK	signaling	and	cytoskeletal	dynamics:	A	perspective,	Cell.	Signal.	19	(2007)	1621–1632.	https://doi.org/10.1016/j.cellsig.2007.04.012.	[229]	J.	Nemunaitis,	Stathmin	1:	A	protein	with	many	tasks.	New	biomarker	and	potential	target	in	cancer,	Expert	Opin.	Ther.	Targets.	16	(2012)	631–634.	https://doi.org/10.1517/14728222.2012.696101.	[230]	I.A.	Leighton,	P.	Curmi,	D.G.	Campbell,	P.	Cohen,	A.	Sobel,	The	phosphorylation	of	stathmin	by	MAP	kinase,	Mol.	Cell.	Biochem.	127–128	(1993)	151–156.	https://doi.org/10.1007/BF01076766.	[231]	T.A.C.	and	M.G.	Ulrica	MARKLUND’,	NiMas	LARSSON’,	Goran	BRATTSAND’,	Orjan	  160 OSTERMAN’,	Serine	16	of	oncoprotein	18	is	a	major	cytosolic	target	for	the	Ca2+/calmodulin-dependent	kinase-G,	Eur.	J.	Biochem.	225	(1994)	53–60.	[232]	F.	Pournia,	M.	Dang-Lawson,	K.	Choi,	V.	Mo,	P.D.	Lampe,	L.	Matsuuchi,	Identification	of	serine	residues	in	the	connexin43	carboxyl	tail	important	for	BCR-mediated	spreading	of	B-lymphocytes,	J.	Cell	Sci.	133	(2020).	https://doi.org/10.1242/jcs.237925.	[233]	C.	Ketchum,	H.	Miller,	W.	Song,	A.	Upadhyaya,	Ligand	mobility	regulates	B	cell	receptor	clustering	and	signaling	activation,	Biophys.	J.	106	(2014)	26–36.	https://doi.org/10.1016/j.bpj.2013.10.043.	[234]	Y.R.	Carrasco,	S.J.	Fleire,	T.	Cameron,	M.L.	Dustin,	F.D.	Batista,	LFA-1/ICAM-1	interaction	lowers	the	threshold	of	B	cell	activation	by	facilitating	B	cell	adhesion	and	synapse	formation,	Immunity.	20	(2004)	589–599.	https://doi.org/10.1016/S1074-7613(04)00105-0.	[235]	D.	Depoil,	S.	Fleire,	B.L.	Treanor,	M.	Weber,	N.E.	Harwood,	K.L.	Marchbank,	V.L.J.	Tybulewicz,	F.D.	Batista,	CD19	is	essential	for	B	cell	activation	by	promoting	B	cell	receptor-antigen	microcluster	formation	in	response	to	membrane-bound	ligand,	Nat.	Immunol.	9	(2008)	63–72.	https://doi.org/10.1038/ni1547.	[236]	K.B.L.	Lin,	S.A.	Freeman,	S.	Zabetian,	H.	Brugger,	M.	Weber,	V.	Lei,	M.	Dang-Lawson,	K.W.K.	Tse,	R.	Santamaria,	F.D.	Batista,	M.R.	Gold,	The	Rap	GTPases	Regulate	B	Cell	Morphology,	Immune-Synapse	Formation,	and	Signaling	by	Particulate	B	Cell	Receptor	Ligands,	Immunity.	28	(2008)	75–87.	https://doi.org/10.1016/j.immuni.2007.11.019.	  161 [237]	C.J.	Hsu,	W.T.	Hsieh,	A.	Waldman,	F.	Clarke,	E.S.	Huseby,	J.K.	Burkhardt,	T.	Baumgart,	Ligand	mobility	modulates	immunological	synapse	formation	and	T	cell	activation,	PLoS	One.	7	(2012).	https://doi.org/10.1371/journal.pone.0032398.	[238]	Z.	Wan,	S.	Zhang,	Y.	Fan,	K.	Liu,	F.	Du,	A.M.	Davey,	H.	Zhang,	W.	Han,	C.	Xiong,	W.	Liu,	B	Cell	Activation	Is	Regulated	by	the	Stiffness	Properties	of	the	Substrate	Presenting	the	Antigens,	J.	Immunol.	190	(2013)	4661–4675.	https://doi.org/10.4049/jimmunol.1202976.	[239]	K.M.	Spillane,	P.	Tolar,	B	cell	antigen	extraction	is	regulated	by	physical	properties	of	antigen-presenting	cells,	J.	Cell	Biol.	216	(2017)	217–230.	https://doi.org/10.1083/jcb.201607064.	[240]	J.C.	Wang,	M.	Bolger-Munro,	M.R.	Gold,	Imaging	the	Interactions	Between	B	Cells	and	Antigen-Presenting	Cells,	in:	Methods	Mol.	Biol.,	Humana	Press,	New	York,	NY,	2018:	pp.	131–161.	https://doi.org/10.1007/978-1-4939-7474-0_10.	[241]	M.R.	Gold,	L.	Matsuuchi,	R.B.	Kelly,	A.L.	DeFranco,	Tyrosine	phosphorylation	of	components	of	the	B-cell	antigen	receptors	following	receptor	crosslinking,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	88	(1991)	3436–3440.	https://doi.org/10.1073/pnas.88.8.3436.	[242]	J.	Schindelin,	I.	Arganda-Carreras,	E.	Frise,	V.	Kaynig,	M.	Longair,	T.	Pietzsch,	S.	Preibisch,	C.	Rueden,	S.	Saalfeld,	B.	Schmid,	J.Y.	Tinevez,	D.J.	White,	V.	Hartenstein,	K.	Eliceiri,	P.	Tomancak,	A.	Cardona,	Fiji:	An	open-source	platform	for	biological-image	analysis,	Nat.	Methods.	9	(2012)	676–682.	https://doi.org/10.1038/nmeth.2019.	[243]	R.M.	Klein,	A.	Spofford,	L.	S.,	A.	Ortiz,	A.E.	Aplin,	B-RAF	regulation	of	Rnd3	  162 participates	in	actin	cytoskeletal	and	focal	adhesion	organization.,	Mol.	Biol.	Cell.	18	(2008)	3250–3263.	https://doi.org/10.1091/mbc.E07.	[244]	J.	Niu,	Q.	Mo,	H.	Wang,	M.	Li,	J.	Cui,	Z.	Li,	Z.	Li,	Invasion	inhibition	by	a	MEK	inhibitor	correlates	with	the	actin-based	cytoskeleton	in	lung	cancer	A549	cells,	Biochem.	Biophys.	Res.	Commun.	422	(2012)	80–84.	https://doi.org/10.1016/j.bbrc.2012.04.109.	[245]	C.A.	Pritchard,	L.	Hayes,	L.	Wojnowski,	A.	Zimmer,	R.M.	Marais,	J.C.	Norman,	B-Raf	Acts	via	the	ROCKII/LIMK/Cofilin	Pathway	To	Maintain	Actin	Stress	Fibers	in	Fibroblasts,	Mol.	Cell.	Biol.	24	(2004)	5937–5952.	https://doi.org/10.1128/mcb.24.13.5937-5952.2004.	[246]	K.W.K.	Tse,	M.	Dang-Lawson,	R.L.	Lee,	D.	Vong,	A.	Bulic,	L.	Buckbinder,	M.R.	Gold,	B	cell	receptor-induced	phosphorylation	of	Pyk2	and	focal	adhesion	kinase	involves	integrins	and	the	rap	GTPases	andis	required	for	B	cell	spreading,	J.	Biol.	Chem.	284	(2009)	22865–22877.	https://doi.org/10.1074/jbc.M109.013169.	[247]	X.	Cai,	M.	Li,	J.	Vrana,	M.D.	Schaller,	Glycogen	Synthase	Kinase	3-	and	Extracellular	Signal-Regulated	Kinase-Dependent	Phosphorylation	of	Paxillin	Regulates	Cytoskeletal	Rearrangement,	Mol.	Cell.	Biol.	26	(2006)	2857–2868.	https://doi.org/10.1128/mcb.26.7.2857-2868.2006.	[248]	P.P.	Roux,	S.A.	Richards,	J.	Blenis,	Phosphorylation	of	p90	Ribosomal	S6	Kinase	(RSK)	Regulates	Extracellular	Signal-Regulated	Kinase	Docking	and	RSK	Activity,	Mol.	Cell.	Biol.	23	(2003)	4796–4804.	https://doi.org/10.1128/mcb.23.14.4796-4804.2003.	[249]	K.	Sawicka,	A.	Pyronneau,	M.	Chao,	M.V.L.	Bennett,	R.S.	Zukin,	Elevated	erk/p90	  163 ribosomal	S6	kinase	activity	underlies	audiogenic	seizure	susceptibility	in	Fragile	X	mice,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	113	(2016)	E6290–E6297.	https://doi.org/10.1073/pnas.1610812113.	[250]	S.C.	Samson,	A.	Elliott,	B.D.	Mueller,	Y.	Kim,	K.R.	Carney,	J.P.	Bergman,	J.	Blenis,	M.C.	Mendoza,	P90	ribosomal	S6	kinase	(RSK)	phosphorylates	myosin	phosphatase	and	thereby	controls	edge	dynamics	during	cell	migration,	J.	Biol.	Chem.	294	(2019)	10846–10862.	https://doi.org/10.1074/jbc.RA119.007431.	[251]	J.	Lovrić,	S.	Dammeier,	A.	Kieser,	H.	Mischak,	W.	Kolch,	Activated	Raf	induces	the	hyperphosphorylation	of	stathmin	and	the	reorganization	of	the	microtubule	network,	J.	Biol.	Chem.	273	(1998)	22848–22855.	[252]	M.F.	Favata,	K.Y.	Horiuchi,	E.J.	Manos,	A.J.	Daulerio,	D.A.	Stradley,	W.S.	Feeser,	D.E.	Van	Dyk,	W.J.	Pitts,	R.A.	Earl,	F.	Hobbs,	R.A.	Copeland,	R.L.	Magolda,	P.A.	Scherle,	J.M.	Trzaskos,	Identification	of	a	novel	inhibitor	of	mitogen-activated	protein	kinase	kinase,	J.	Biol.	Chem.	273	(1998)	18623–18632.	https://doi.org/10.1074/jbc.273.29.18623.	[253]	D.T.	Dudley,	L.	Pang,	S.J.	Decker,	A.J.	Bridges,	A.R.	Saltiel,	A	synthetic	inhibitor	of	the	mitogen-activated	protein	kinase	cascade,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	92	(1995)	7686–7689.	https://doi.org/10.1073/pnas.92.17.7686.	[254]	H.	Huynh,	K.C.	Soo,	P.K.H.	Chow,	E.	Tran,	Targeted	inhibition	of	the	extracellular	signal-regulated	kinase	kinase	pathway	with	AZD6244	(ARRY-142886)	in	the	treatment	of	hepatocellular	carcinoma,	Mol.	Cancer	Ther.	6	(2007)	138–146.	https://doi.org/10.1158/1535-7163.MCT-06-0436.	  164 [255]	M.	Ohori,	T.	Kinoshita,	M.	Okubo,	K.	Sato,	A.	Yamazaki,	H.	Arakawa,	S.	Nishimura,	N.	Inamura,	H.	Nakajima,	M.	Neya,	H.	Miyake,	T.	Fujii,	Identification	of	a	selective	ERK	inhibitor	and	structural	determination	of	the	inhibitor-ERK2	complex,	Biochem.	Biophys.	Res.	Commun.	336	(2005)	357–363.	https://doi.org/10.1016/j.bbrc.2005.08.082.	[256]	S.	Murugesan,	J.	Hong,	J.	Yi,	D.	Li,	J.R.	Beach,	L.	Shao,	J.	Meinhardt,	G.	Madison,	X.	Wu,	E.	Betzig,	J.A.	Hammer,	Formin-generated	actomyosin	arcs	propel	T	cell	receptor	microcluster	movement	at	the	immune	synapse,	J.	Cell	Biol.	215	(2016)	383–399.	https://doi.org/10.1083/jcb.201603080.	[257]	W.W.A.	Schamel,	M.	Reth,	Monomeric	and	oligomeric	complexes	of	the	B	cell	antigen	receptor,	Immunity.	13	(2000)	5–14.	https://doi.org/10.1016/S1074-7613(00)00003-0.	[258]	M.	Reth,	J.	Wienands,	W.W.A.	Schamel,	An	unsolved	problem	of	the	clonal	selection	theory	and	the	model	of	an	oligomeric	B-cell	antigen	receptor,	Immunol.	Rev.	176	(2000)	10–18.	https://doi.org/10.1034/j.1600-065X.2000.00610.x.	[259]	M.	Reth,	Antigen	receptors	on	B	lymphocytes,	Annu.	Rev.	Immunol.	10	(1992)	97–121.	https://doi.org/10.1146/annurev.immunol.10.1.97.	[260]	M.R.	Gold,	J.S.	Sanghera,	J.	Stewart,	S.L.	Pelech,	Selective	activation	of	p42	mitogen-activated	protein	(MAP)	kinase	in	murine	B	lymphoma	cell	lines	by	membrane	immunoglobulin	cross-linking.	Evidence	for	protein	kinase	C-independent	and	-dependent	mechanisms	of	activation,	Biochem.	J.	287	(1992)	269–276.	https://doi.org/10.1042/bj2870269.	  165 [261]	C.	Frémin,	M.K.	Saba-El-Leil,	K.	Lévesque,	S.L.	Ang,	S.	Meloche,	Functional	redundancy	of	ERK1	and	ERK2	MAP	kinases	during	development,	Cell	Rep.	12	(2015)	913–921.	https://doi.org/10.1016/j.celrep.2015.07.011.	[262]	R.	Lefloch,	J.	Pouysségur,	P.	Lenormand,	Single	and	Combined	Silencing	of	ERK1	and	ERK2	Reveals	Their	Positive	Contribution	to	Growth	Signaling	Depending	on	Their	Expression	Levels,	Mol.	Cell.	Biol.	28	(2008)	511–527.	https://doi.org/10.1128/mcb.00800-07.	[263]	B.	Hetrick,	M.S.	Han,	L.A.	Helgeson,	B.J.	Nolen,	Small	Molecules	CK-666	and	CK-869	Inhibit	Actin-Related	Protein	2/3	Complex	by	Blocking	an	Activating	Conformational	Change,	Chem.	Biol.	20	(2013)	701–712.	https://doi.org/10.1016/j.chembiol.2013.03.019.	[264]	J.C.	Wang,	J.Y.J.	Lee,	M.	Dang-Lawson,	C.	Pritchard,	M.R.	Gold,	The	Rap2c	GTPase	facilitates	B	cell	receptor-induced	reorientation	of	the	microtubule-organizing	center,	Small	GTPases.	1248	(2018)	1–11.	https://doi.org/10.1080/21541248.2018.1441626.	[265]	R.D.	Mullins,	J.A.	Heuser,	T.D.	Pollard,	The	interaction	of	Arp2/3	complex	with	actin:	Nucleation,	high	affinity	pointed	end	capping,	and	formation	of	branching	networks	of	filaments,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	95	(1998)	6181–6186.	https://doi.org/10.1073/pnas.95.11.6181.	[266]	B.	Serrels,	A.	Serrels,	V.G.	Brunton,	M.	Holt,	G.W.	McLean,	C.H.	Gray,	G.E.	Jones,	M.C.	Frame,	Focal	adhesion	kinase	controls	actin	assembly	via	a	FERM-mediated	interaction	with	the	Arp2/3	complex,	Nat.	Cell	Biol.	9	(2007)	1046–1056.	  166 https://doi.org/10.1038/ncb1626.	[267]	J.	Hoon,	M.	Tan,	C.-G.	Koh,	The	Regulation	of	Cellular	Responses	to	Mechanical	Cues	by	Rho	GTPases,	Cells.	5	(2016)	17.	https://doi.org/10.3390/cells5020017.	[268]	S.	Shaheen,	Z.	Wan,	Z.	Li,	A.	Chau,	X.	Li,	S.	Zhang,	Y.	Liu,	J.	Yi,	Y.	Zeng,	J.	Wang,	X.	Chen,	L.	Xu,	W.	Chen,	F.	Wang,	Y.	Lu,	W.	Zheng,	Y.	Shi,	X.	Sun,	Z.	Li,	C.	Xiong,	W.	Liu,	Substrate	stiffness	governs	the	initiation	of	b	cell	activation	by	the	concerted	signaling	of	PKCβ	and	focal	adhesion	kinase,	Elife.	6	(2017).	https://doi.org/10.7554/eLife.23060.	[269]	K.T.	Bashoura,	A.	Gondarenko,	H.	Chen,	K.	Shen,	X.	Liu,	M.	Huse,	J.C.	Hone,	L.C.	Kam,	CD28	and	CD3	have	complementary	roles	in	T-cell	traction	forces,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	111	(2014)	2241–2246.	https://doi.org/10.1073/pnas.1315606111.	[270]	R.S.	O’Connor,	X.	Hao,	K.	Shen,	K.	Bashour,	T.	Akimova,	W.W.	Hancock,	L.C.	Kam,	M.C.	Milone,	Substrate	Rigidity	Regulates	Human	T	Cell	Activation	and	Proliferation,	J.	Immunol.	189	(2012)	1330–1339.	https://doi.org/10.4049/jimmunol.1102757.	[271]	M.	Saitakis,	S.	Dogniaux,	C.	Goudot,	N.	Bufi,	S.	Asnacios,	M.	Maurin,	C.	Randriamampita,	A.	Asnacios,	C.	Hivroz,	Different	TCR-induced	T	lymphocyte	responses	are	potentiated	by	stiffness	with	variable	sensitivityDifferent	TCR-induced	T	lymphocyte	responses	are	potentiated	by	stiffness	with	variable	sensitivity,	Elife.	6	(2017)	1–29.	https://doi.org/10.7554/eLife.23190.	[272]	C.E.	Turner,	Paxillin	interactions,	J.	Cell	Sci.	113	(2000)	4139–4140.	[273]	A.M.	López-Colomé,	I.	Lee-Rivera,	R.	Benavides-Hidalgo,	E.	López,	Paxillin:	A	crossroad	in	pathological	cell	migration,	J.	Hematol.	Oncol.	10	(2017)	1–15.	https://doi.org/10.1186/s13045-017-0418-y.	  167 [274]	J.T.	Parsons,	A.R.	Horwitz,	M.A.	Schwartz,	Cell	adhesion:	Integrating	cytoskeletal	dynamics	and	cellular	tension,	Nat.	Rev.	Mol.	Cell	Biol.	11	(2010)	633–643.	https://doi.org/10.1038/nrm2957.	[275]	A.	Nayal,	D.J.	Webb,	C.M.	Brown,	E.M.	Schaefer,	M.	Vicente-Manzanares,	A.R.	Horwitz,	Paxillin	phosphorylation	at	Ser273	localizes	a	GIT1-PIX-PAK	complex	and	regulates	adhesion	and	protrusion	dynamics,	J.	Cell	Biol.	173	(2006)	587–599.	https://doi.org/10.1083/jcb.200509075.	[276]	D.J.	Webb,	M.J.	Schroeder,	C.J.	Brame,	L.	Whitmore,	J.	Shabanowitz,	D.F.	Hunt,	A.R.	Horwitz,	Paxilin	phosphorylation	sites	mapped	by	mass	spectrometry,	J.	Cell	Sci.	118	(2005)	4925–4929.	https://doi.org/10.1242/jcs.02563.	[277]	G.	Giannone,	B.J.	Dubin-Thaler,	H.G.	Döbereiner,	N.	Kieffer,	A.R.	Bresnick,	M.P.	Sheetz,	Periodic	lamellipodial	contractions	correlate	with	rearward	actin	waves,	Cell.	116	(2004)	431–443.	https://doi.org/10.1016/S0092-8674(04)00058-3.	[278]	A.	Ponti,	M.	Machacek,	S.L.	Gupton,	C.M.	Waterman-Storer,	G.	Danuser,	Two	distinct	actin	networks	drive	the	protrusion	of	migrating	cells,	Science	(80-.	).	305	(2004)	1782–1786.	https://doi.org/10.1126/science.1100533.	[279]	W.A.	Sayyad,	L.	Amin,	P.	Fabris,	E.	Ercolini,	V.	Torre,	The	role	of	myosin-II	in	force	generation	of	DRG	filopodia	and	lamellipodia,	Sci.	Rep.	5	(2015)	1–12.	https://doi.org/10.1038/srep07842.	[280]	M.C.	Mendoza,	M.	Vilela,	J.E.	Juarez,	J.	Blenis,	G.	Danuser,	ERK	reinforces	actin	polymerization	to	power	persistent	edge	protrusion	during	motility,	Sci.	Signal.	8	(2015)	ra47.	https://doi.org/10.1126/scisignal.aaa8859.	  168 [281]	A.	Chaudhuri,	B.	Bhattacharya,	K.	Gowrishankar,	S.	Mayor,	M.	Rao,	Spatiotemporal	regulation	of	chemical	reactions	by	active	cytoskeletal	remodeling,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	108	(2011)	14825–14830.	https://doi.org/10.1073/pnas.1100007108.	[282]	M.F.	Carlier,	V.	Laurent,	J.	Santolini,	R.	Melki,	D.	Didry,	G.X.	Xia,	Y.	Hong,	N.H.	Chua,	D.	Pantaloni,	Actin	depolymerizing	factor	(ADF/cofilin)	enhances	the	rate	of	filament	turnover:	Implication	in	actin-based	motility,	J.	Cell	Biol.	136	(1997)	1307–1322.	https://doi.org/10.1083/jcb.136.6.1307.	[283]	V.	Delorme,	M.	Machacek,	C.	DerMardirossian,	K.L.	Anderson,	T.	Wittmann,	D.	Hanein,	C.	Waterman-Storer,	G.	Danuser,	G.M.	Bokoch,	Cofilin	Activity	Downstream	of	Pak1	Regulates	Cell	Protrusion	Efficiency	by	Organizing	Lamellipodium	and	Lamella	Actin	Networks,	Dev.	Cell.	13	(2007)	646–662.	https://doi.org/10.1016/j.devcel.2007.08.011.	[284]	P.	Hotulainen,	E.	Paunola,	M.K.	Vartiainen,	P.	Lappalainen,	Actin-depolymerizing	factor	and	cofilin-1	play	overlapping	roles	in	promoting	rapid	F-actin	depolymerization	in	mammalian	nonmuscle	cells,	Mol.	Biol.	Cell.	16	(2005)	649–664.	https://doi.org/10.1091/mbc.E04-07-0555.	[285]	M.	Ghosh,	X.	Song,	G.	Mouneimne,	M.	Sidani,	D.S.	Lawrence,	J.S.	Condeelis,	Cofilin	Promotes	Actin	Polymerization	and	Defines	the	Direction	of	Cell	Motility,	Science	(80-.	).	304	(2004)	743–746.	https://doi.org/10.1126/science.1094561.	[286]	K.C.	Flynn,	F.	Hellal,	D.	Neukirchen,	S.	Jacob,	S.	Tahirovic,	S.	Dupraz,	S.	Stern,	B.K.	Garvalov,	C.	Gurniak,	A.E.	Shaw,	L.	Meyn,	R.	Wedlich-Söldner,	J.R.	Bamburg,	J.V.	Small,	W.	Witke,	F.	Bradke,	ADF/Cofilin-Mediated	Actin	Retrograde	Flow	Directs	Neurite	  169 Formation	in	the	Developing	Brain,	Neuron.	76	(2012)	1091–1107.	https://doi.org/10.1016/j.neuron.2012.09.038.	[287]	K.	Ohashi,	S.	Fujiwara,	T.	Watanabe,	H.	Kondo,	T.	Kiuchi,	M.	Sato,	K.	Mizuno,	LIM	kinase	has	a	dual	role	in	regulating	lamellipodium	extension	by	decelerating	the	rate	of	actin	retrograde	flow	and	the	rate	of	actin	polymerization,	J.	Biol.	Chem.	286	(2011)	36340–36351.	https://doi.org/10.1074/jbc.M111.259135.	[288]	E.A.	Vitriol,	A.L.	Wise,	M.E.	Berginski,	J.R.	Bamburg,	J.Q.	Zheng,	Instantaneous	inactivation	of	cofilin	reveals	its	function	of	F-actin	disassembly	in	lamellipodia,	Mol.	Biol.	Cell.	24	(2013)	2238–2247.	https://doi.org/10.1091/mbc.E13-03-0156.	[289]	T.J.	Thauland,	K.H.	Hu,	M.A.	Bruce,	M.J.	Butte,	Cytoskeletal	adaptivity	regulates	T	cell	receptor	signaling,	Sci.	Signal.	10	(2017)	1–11.	https://doi.org/10.1126/scisignal.aah3737.	[290]	S.A.	Freeman,	V.	Lei,	M.	Dang-Lawson,	K.	Mizuno,	C.D.	Roskelley,	M.R.	Gold,	Cofilin-Mediated	F-Actin	Severing	Is	Regulated	by	the	Rap	GTPase	and	Controls	the	Cytoskeletal	Dynamics	That	Drive	Lymphocyte	Spreading	and	BCR	Microcluster	Formation,	J.	Immunol.	187	(2011)	5887–5900.	https://doi.org/10.4049/jimmunol.1102233.	[291]	M.J.	Lin,	S.J.	Lee,	Stathmin-like	4	is	critical	for	the	maintenance	of	neural	progenitor	cells	in	dorsal	midbrain	of	zebrafish	larvae,	Sci.	Rep.	6	(2016)	1–15.	https://doi.org/10.1038/srep36188.	[292]	L.D.	Belmont,	T.J.	Mitchison,	Identification	of	a	protein	that	interacts	with	tubulin	dimers	and	increases	the	catastrophe	rate	of	microtubules,	Cell.	84	(1996)	623–631.	  170 https://doi.org/10.1016/S0092-8674(00)81037-5.	[293]	B.	Howell,	N.	Larsson,	M.	Gullberg,	L.	Cassimeris,	Dissociation	of	the	tubulin-sequestering	and	microtubule	catastrophe-	promoting	activities	of	oncoprotein	18/stathmin,	Mol.	Biol.	Cell.	10	(1999)	105–118.	https://doi.org/10.1091/mbc.10.1.105.	[294]	S.	Uchida,	G.P.	Shumyatsky,	Deceivingly	dynamic:	Learning-dependent	changes	in	stathmin	and	microtubules,	Neurobiol.	Learn.	Mem.	124	(2015)	52–61.	https://doi.org/10.1016/j.nlm.2015.07.011.	[295]	S.	Kline-Smith,	C.	Walczak,	The	Microtubule-destabilizing	Kinesine	XKCM1	Regulates	Microtubule	Dynamics	and	Instability	in	Cells,	Mol.	Biol.	Cell.	13	(2002)	3901–3914.	https://doi.org/10.1091/mbc.E01.	[296]	E.	Natkanski,	W.-Y.	Lee,	B.	Mistry,	A.	Casal,	J.E.	Molloy,	P.	Tolar,	B	Cells	Use	Mechanical	Energy	to	Discriminate	Antigen	Affinities,	Science.	340	(2013)	1587–1590.	https://doi.org/10.1126/science.1237572.	[297]	S.	Kumari,	D.	Depoil,	R.	Martinelli,	E.	Judokusumo,	G.	Carmona,	F.B.	Gertler,	L.C.	Kam,	C.	V.	Carman,	J.K.	Burkhardt,	D.J.	Irvine,	M.L.	Dustin,	Actin	foci	facilitate	activation	of	the	phospholipase	C-γ	in	primary	T	lymphocytes	via	the	WASP	pathway,	Elife.	2015	(2015)	1–31.	https://doi.org/10.7554/eLife.04953.	[298]	S.I.	Roper,	L.	Wasim,	D.	Malinova,	M.	Way,	S.	Cox,	P.	Tolar,	B	cells	extract	antigens	at	arp2/3-generated	actin	foci	interspersed	with	linear	filaments,	Elife.	8	(2019)	1–24.	https://doi.org/10.7554/eLife.48093.	[299]	S.	Mukherjee,	J.	Zhu,	J.	Zikherman,	R.	Parameswaran,	T.	Kadlecek,	Q.	Wang,	B.	Au-  171 Yeung,	H.	Ploegh,	J.	Kuriyan,	J.	Das,	A.	Weiss,	Monovalent	and	Multiplevalent	Ligation	of	the	B	Cell	Receptor	Exhibit	Differential	Dependence	Upon	Syk	and	Src	Family	Kinases,	Sci.	Signal.	6	(2013)	1–27.	https://doi.org/10.1126/scisignal.2003220.Monovalent.	[300]	W.W.A.	Schamel,	I.	Arechaga,	R.M.	Risueño,	H.M.	van	Santen,	P.	Cabezas,	C.	Risco,	J.M.	Valpuesta,	B.	Alarcón,	Coexistence	of	multivalent	and	monovalent	TCRs	explains	high	sensitivity	and	wide	range	of	response,	J.	Exp.	Med.	202	(2005)	493–503.	https://doi.org/10.1084/jem.20042155.	[301]	R.	Kumar,	M.	Ferez,	M.	Swamy,	I.	Arechaga,	M.T.	Rejas,	J.M.	Valpuesta,	W.W.A.	Schamel,	B.	Alarcon,	H.M.	van	Santen,	Increased	Sensitivity	of	Antigen-Experienced	T	Cells	through	the	Enrichment	of	Oligomeric	T	Cell	Receptor	Complexes,	Immunity.	35	(2011)	375–387.	https://doi.org/10.1016/j.immuni.2011.08.010.	[302]	M.	Poggianella,	M.	Bestagno,	O.R.	Burrone,	The	Extracellular	Membrane-Proximal	Domain	of	Human	Membrane	IgE	Controls	Apoptotic	Signaling	of	the	B	Cell	Receptor	in	the	Mature	B	Cell	Line	A20,	J.	Immunol.	177	(2006)	3597–3605.	https://doi.org/10.4049/jimmunol.177.6.3597.	[303]	C.	Wakabayashi,	T.	Adachi,	J.	Wienands,	T.	Tsubata,	A	distinct	signaling	pathway	used	by	the	IgG-containing	B	cell	antigen	receptor,	Science	(80-.	).	298	(2002)	2392–2395.	https://doi.org/10.1126/science.1076963.	[304]	N.	Engels,	L.M.	König,	W.	Schulze,	D.	Radtke,	K.	Vanshylla,	J.	Lutz,	T.H.	Winkler,	L.	Nitschke,	J.	Wienands,	The	immunoglobulin	tail	tyrosine	motif	upgrades	memory-type	BCRs	by	incorporating	a	Grb2-Btk	signalling	module,	Nat.	Commun.	5	(2014).	  172 https://doi.org/10.1038/ncomms6456.	[305]	G.M.	Rivera,	S.	Antoku,	S.	Gelkop,	N.Y.	Shin,	S.K.	Hanks,	T.	Pawson,	B.J.	Mayer,	Requirement	of	Nck	adaptors	for	actin	dynamics	and	cell	migration	stimulated	by	platelet-derived	growth	factor	B,	Proc.	Natl.	Acad.	Sci.	U.	S.	A.	103	(2006)	9536–9541.	https://doi.org/10.1073/pnas.0603786103.	[306]	R.	Buscà,	P.	Abbe,	F.	Mantoux,	E.	Aberdam,	C.	Peyssonnaux,	A.	Eychène,	J.P.	Ortonne,	R.	Ballotti,	Ras	mediates	the	cAMP-dependent	activation	of	extracellular	signal-regulated	kinases	(ERKs)	in	melanocytes,	EMBO	J.	19	(2000)	2900–2910.	https://doi.org/10.1093/emboj/19.12.2900.	[307]	Q.	Zhang,	R.	Gong,	J.	Qu,	Y.	Zhou,	W.	Liu,	M.	Chen,	Y.	Liu,	Y.	Zhu,	J.	Wu,	Activation	of	the	Ras/Raf/MEK	Pathway	Facilitates	Hepatitis	C	Virus	Replication	via	Attenuation	of	the	Interferon-JAK-STAT	Pathway,	J.	Virol.	86	(2012)	1544–1554.	https://doi.org/10.1128/jvi.00688-11.	[308]	K.	Smorodinsky-Atias,	T.	Goshen-Lago,	A.	Goldberg-Carp,	D.	Melamed,	A.	Shir,	N.	Mooshayef,	J.	Beenstock,	Y.	Karamansha,	I.	Darlyuk-Saadon,	O.	Livnah,	N.G.	Ahn,	A.	Admon,	D.	Engelberg,	Intrinsically	active	variants	of	Erk	oncogenically	transform	cells	and	disclose	unexpected	autophosphorylation	capability	that	is	independent	of	TEY	phosphorylation,	Mol.	Biol.	Cell.	27	(2016)	1026–1039.	https://doi.org/10.1091/mbc.E15-07-0521.	[309]	A.	Khokhlatchev,	S.	Xu,	J.	English,	P.	Wu,	E.	Schaefer,	M.H.	Cobb,	Reconstitution	of	mitogen-activated	protein	kinase	phosphorylation	cascades	in	bacteria.	Efficient	synthesis	of	active	protein	kinases,	J.	Biol.	Chem.	272	(1997)	11057–11062.	  173 https://doi.org/10.1074/jbc.272.17.11057.	[310]	M.	Ordan,	C.	Pallara,	G.	Maik-Rachline,	T.	Hanoch,	F.L.	Gervasio,	F.	Glaser,	J.	Fernandez-Recio,	R.	Seger,	Intrinsically	active	MEK	variants	are	differentially	regulated	by	proteinases	and	phosphatases,	Sci.	Rep.	8	(2018)	1–16.	https://doi.org/10.1038/s41598-018-30202-5.	[311]	B.	Zheng,	P.	Flumara,	Y.	V.	Li,	G.	Georgakis,	V.	Snell,	M.	Younes,	J.N.	Vauthey,	A.	Carbone,	A.	Younes,	MEK/ERK	pathway	is	aberrantly	active	in	Hodgkin	disease:	A	signaling	pathway	shared	by	CD30,	CD40,	and	RANK	that	regulates	cell	proliferation	and	survival,	Blood.	102	(2003)	1019–1027.	https://doi.org/10.1182/blood-2002-11-3507.	[312]	W.	Liao,	S.	Sharma,	Modulation	of	B-cell	receptor	and	microenvironment	signaling	by	a	guanine	exchange	factor	in	B-cell	malignancies,	Cancer	Biol.	Med.	13	(2016)	277–285.	https://doi.org/10.20892/j.issn.2095-3941.2016.0026.	[313]	H.	Chin,	D.	Lai,	G.	Falchook,	Extracellular	signal-regulated	kinase	(ERK)	inhibitors	in	oncology	clinical	trials,	J.	Immunother.	Precis.	Oncol.	2	(2019)	10.	https://doi.org/10.4103/jipo.jipo_17_18.	      

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            data-media="{[{embed.selectedMedia}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.24.1-0394585/manifest

Comment

Related Items