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The role of BCR signalling and the gap junction protein Cx43 in B lymphocyte cytoskeletal rearrangements Machtaler, Steven Brian Alfred 2011

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The role of BCR signalling and the gap junction protein Cx43 in B lymphocyte cytoskeletal rearrangements by Steven Brian Alfred Machtaler B.Sc., Okanagan University College, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF   DOCTOR OF PHILOSOPHY  in  The Faculty of Graduate Studies (Zoology) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2011 ? Steven Brian Alfred Machtaler, 2011  ii  Abstract Regulation of the cytoskeleton is an essential process for normal B lymphocyte development and immune system regulation.  Though this biological process is important to normal function, its regulation is not completely understood.  An important receptor required to initiate antigen-mediated cytoskeletal rearrangements in B lympocytes is the B cell antigen receptor (BCR).  This is a multimeric protein complex that contains two signalling proteins, Ig? and Ig?, which become phosphorylated after antigen engagement leading to signalling cascades which result in cytoskeletal rearrangements and differentiation.  The gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells, forming intercellular channels for the transfer of small molecules between adjacent cells as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 has recently been shown to regulate cell adhesion and migration in neurons and glioma cells, biological processes dependent on the rearrangement of the cytoskeleton, however its role in B lymphocytes remains unknown.  The aim of this thesis was to determine the importance of the B cell antigen receptor (BCR) member Ig? and the gap junction protein connexin43 (Cx43) to the regulation of the B cell cytoskeleton.  I show here that the cytoplasmic domain of Ig? was necessary for maximal BCR-mediated cytoskeletal rearrangements, and that the cytoplasmic domain of Ig? was not sufficent to conferthis phenotype.  In order to determine if Cx43 was required for B cell cytoskeletal rearrangements, both loss-of and gain-of-function approaches were used.  I show that Cx43 was necessary for sustained BCR, integrin and chemokine-mediated Rap1 activation, as well as B cell spreading, adhesion, motility and migration.  I also identified that the C-terminal domain of Cx43 was necessary for these processes, suggesting that this may be a site where proteins which regulate the cytoskeleton are recruited to.  This thesis provides the first evidence that Cx43 is essential for regulation of the B cell cytoskeleton.     iii  Preface  The following information describes the relative contributions of all collaborators and co-authors for the work presented in this thesis.  A version of Figure 2-4 and Figure 2-5 from chapter 2 were published and reprinted with permission:   Jang, C., Machtaler, S., and Matsuuchi, L. (2010). The role of Ig-[alpha]/[beta] in B cell antigen receptor internalization. Immunology Letters 134, 75-82.  A version of Figure 2-5, Figure 2-6 and 2-7 were published and reproduced/adapted with permission:   Steven Machtaler*?, May Dang-Lawson*?, Kate Choi*, Caren Jang*?, Christian C. Naus? and Linda Matsuuchi*??.  The gap junction protein Cx43 regulates B lymphocyte spreading and adhesion.  The Journal of Cell Science. (2011) vol 124: 2611-2521 (DOI: 10.1242/jcs.089532). I was responsible for designing the study, collecting and analyzing the data in chapter 2 with the exception of Figure 2-4B and D as well as Figure 2-5B which were created by Caren Jang.   A version of chapter 3 has been published and reproduced/adapted with permission:   Steven Machtaler*?, May Dang-Lawson*?, Kate Choi*, Caren Jang*?, Christian C. Naus? and Linda Matsuuchi*??.  The gap junction protein Cx43 regulates B lymphocyte spreading and adhesion.  The Journal of Cell Science. (2011) vol 124: 2611-2521 (DOI: 10.1242/jcs.089532).    I was responsible for designing the experiments, collecting and analyzing the data, making the figures, carrying out the statistical analysis and writing the manuscript.  May Dang-Lawson, senior technician and lab manager, was responsible for carrying out the Rap1 activation assays and for the Cx43 shRNA retroviral transduction of WEHI231 B cells.   Kate Choi was a directed studies student under my supervision and was responsible for the iv  Cx43 phosphorylation experiment in Figure 3-3C.  Caren Jang a MSc student in the lab was responsible for determining the localization of Cx43-GFP in the endosomes and lysosomes of J558 B cells Figure 3-9, middle and bottom panels.  The Cx43+\- mice were provided by our collaborator, Dr. Christian Naus, Dept of Cellular and Physiological Sciences, UBC, LSI Director and a co-author on the J of Cell Science paper.  The data in chapter 4 was collected by myself and May Dang-Lawson.  I was responsible for designing the experiments, collecting and analyzing the data, making the figures and carrying out the statistical analysis.  May Dang-Lawson was responsible for carrying out the Rap1 activation assays.     v  Table of contents Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of contents ..................................................................................................................... v List of tables............................................................................................................................ ix List of figures ........................................................................................................................... x List of abbreviations ............................................................................................................ xiv Acknowledgements ............................................................................................................. xvii 1 Introduction ..................................................................................................................... 1 1.1 The role of B cells in the immune response ............................................................... 1 1.2 B cell development ..................................................................................................... 4 1.2.1 The role of adhesion and migration in hematopoietic stem cells ........................ 4 1.2.2 Development of B cell subsets within the bone marrow .................................... 7 1.2.3 Development of B cells outside of the bone marrow ........................................ 11 1.2.4 Mature B cell subsets ........................................................................................ 12 1.3 B cell migration ........................................................................................................ 18 1.3.1 Chemoattractants and their receptors ................................................................ 18 1.3.2 Chemokine induced polarity ............................................................................. 21 1.3.3 Chemokine induced integrin activation ............................................................ 23 1.3.4 Integrin inside-out signalling ............................................................................ 26 1.3.5 Integrin outside-in signalling ............................................................................ 29 1.3.6 Transendothelial migration ............................................................................... 31 1.3.6.1 Selectin-mediated capture and rolling ....................................................... 32 1.3.6.2 Integrin-mediated firm adhesion ............................................................... 33 1.3.6.3 Intravascular motility, cytoskeletal rearrangement, force generation and leukocyte motility ........................................................................................................ 34 1.3.6.4 Paracelllular and transcellular transmigration ........................................... 40 1.4 The B cell antigen receptor (BCR) ........................................................................... 42 1.4.1.1 BCR components ....................................................................................... 43 1.4.1.2 BCR assembly and surface trafficking ...................................................... 46 1.4.1.3 BCR distribution on the membrane ........................................................... 48 1.4.1.4 Antigen-mediated BCR activation............................................................. 50 1.4.1.5 BCR-mediated signalling .......................................................................... 54 1.4.1.6 BCR-mediated spreading and immune synapse formation ....................... 58 vi  1.5 The gap junction protein families............................................................................. 63 1.6 The gap junction protein connexin43 (Cx43) .......................................................... 66 1.6.1 Cx43 structure and tissue distribution............................................................... 67 1.6.2 Regulation of Cx43 channel functions .............................................................. 68 1.6.3 Emerging roles for Cx43 as a regulator of migration ....................................... 70 1.6.4 Cx43 in the immune system .............................................................................. 71 1.7 Rationale, objectives and aims ................................................................................. 74 2 The role of the cytoplasmic domain of Ig? in BCR-mediated cytoskeletal rearrangements ..................................................................................................................... 76 2.1 Synopsis .................................................................................................................... 76 2.2 Introduction .............................................................................................................. 77 2.3 Methods and materials ............................................................................................. 79 2.3.1 Antibodies and reagents .................................................................................... 79 2.3.2 Cells and culture ............................................................................................... 80 2.3.3 Transfections ..................................................................................................... 80 2.3.4 Western blotting ................................................................................................ 81 2.3.5 Scanning electron microscopy .......................................................................... 82 2.3.6 B cell spreading assay and actin-rich membrane projection quantification ..... 83 2.3.7 Particulate antigen bead assay .......................................................................... 84 2.3.8 BCR surface expression .................................................................................... 86 2.4 Results ...................................................................................................................... 87 2.4.1 Expression of the mutant C? and ?trunc BCRs in the J558 B cells.................. 87 2.4.2 The Ig? mutants C? and ?trunc are signalling competent ................................ 90 2.4.3 J558 plasmacytoma cells with a reconstituted BCR were able to make signalling dependent membrane protuberances, but were not able to spread radially. ... 93 2.4.4 The cytoplasmic domain of Ig? is required for maximal formation of BCR-mediated membrane protrusions ...................................................................................... 96 2.4.5 The cytoplasmic domain of Ig? is required for an optimal response to a particulate antigen ............................................................................................................ 99 2.5 Discussion .............................................................................................................. 102 3 The characterization of the GJ protein Cx43 expressed in B lymphocytes and its role in BCR-mediated membrane spreading and B cell adhesion .................................. 107 3.1 Synopsis .................................................................................................................. 107 3.2 Introduction ............................................................................................................ 108 3.3 Materials and methods ........................................................................................... 111 3.3.1 Plasmids .......................................................................................................... 111 vii  3.3.2 Antibodies ....................................................................................................... 111 3.3.3 Cells and cell growth ...................................................................................... 112 3.3.4 Transfection and retroviral transduction ......................................................... 112 3.3.5 B cell stimulation and preparation of cell extracts.......................................... 113 3.3.6 Rap1 activation assay ...................................................................................... 114 3.3.7 Cell spreading assay ........................................................................................ 115 3.3.8 Scanning electron microscopy (SEM) ............................................................ 116 3.3.9 B cell adhesion to bEND.3 cells ..................................................................... 116 3.3.10 Statistics .......................................................................................................... 118 3.4 Results .................................................................................................................... 119 3.4.1 Cx43 is expressed in immature and mature B cells and is a target of BCR signalling ....................................................................................................................... 119 3.4.2 Cx43 is required for sustained activation of Rap1 GTPase ............................ 122 3.4.3 Cx43 expression is necessary for BCR-mediated B cell spreading ................ 125 3.4.4 Wild type Cx43 is sufficient to confer BCR-mediated radial cell spreading and enhance Rap1 activation in J558 plasmacytoma cells: The C-terminal tail of Cx43 is required for this response. ............................................................................................. 132 3.4.5 Cx43-GFP enhances LFA-1 mediated Rap1 activation and spreading .......... 143 3.4.6 Cx43-GFP expression increases the adhesion of B cells to bEND.3 endothelial cells?? ........................................................................................................................ 147 3.5 Discussion .............................................................................................................. 151 4 The role of the GJ protein Cx43 in B cell motility, migration and transendothelial migration .............................................................................................................................. 159 4.1 Synopsis ................................................................................................................. 159 4.2 Introduction ............................................................................................................ 159 4.3 Methods and materials ........................................................................................... 161 4.3.1 Plasmids .......................................................................................................... 161 4.3.2 Antibodies and inhibitors ................................................................................ 162 4.3.3 Cells and growth conditions............................................................................ 162 4.3.4 B cell stimulation and preparation of cell extracts.......................................... 163 4.3.5 Rap1 activation assay ...................................................................................... 164 4.3.6 Mobility of Cx43-GFP and actin-GFP in transfected WEHI 231 B cells using fluorescence recovery after photobleaching (FRAP). ................................................... 164 4.3.7 TIRF microscopy ............................................................................................ 165 4.3.8 Cell motility assays ......................................................................................... 166 4.3.9 B cell migration............................................................................................... 167 viii  4.3.10 Transendothelial migration of WEHI 231 B cells .......................................... 168 4.3.11 Immunofluorescence ....................................................................................... 169 4.3.12 Statistics .......................................................................................................... 169 4.4 Results .................................................................................................................... 170 4.4.1 Cx43 expression is necessary for maximal VLA-4-mediated Rap1 activation and migration on fibronectin ......................................................................................... 170 4.4.2 Cx43 is necessary for CXCL12 activation of Rap1 and migration ................ 176 4.4.3 Cx43 is important for transendothelial migration ........................................... 180 4.4.4 Cx43 membrane localization is determined by the F-actin cytoskeleton ....... 184 4.5 Discussion .............................................................................................................. 187 5 Concluding chapter ..................................................................................................... 193 5.1 Summary of main findings ..................................................................................... 193 5.2 General discussion.................................................................................................. 196 5.2.1 How might Cx43 regulate morphology?......................................................... 196 5.2.2 Could there be a role for Cx43-mediated GJIC in B-lymphocytes? ............... 202 5.2.3 Is coupling necessary for gap junction protein functions?.............................. 205 5.2.4 Cx43 as a regulator of Rap1 GTPase in non-lymphoid cells? ........................ 205 5.2.5 Could Cx43 be a novel regulator of B cell cancers? ....................................... 207 5.2.6 Contributions................................................................................................... 208 5.2.7 Strengths and limitations................................................................................. 210 5.2.8 Future directions ............................................................................................. 212 5.2.8.1 What is the molecular mechanism by which Cx43 regulated Rap1 activation in B cells? .................................................................................................. 212 5.2.8.2 B cell development .................................................................................. 213 5.2.8.3 Regulation of the immune response ........................................................ 214 5.2.8.4 The role of Cx43 in B cell cancers .......................................................... 216 5.3 Conclusion .............................................................................................................. 216 References ............................................................................................................................ 217    ix  List of tables Table 1-1 Summary of mature B cell subsets. ........................................................................ 17 Table 1-2 Common integrins expressed on B cells. ............................................................... 26 Table 3-1 Potential connexin-cytoskeleton interacting proteins. .......................................... 154    x  List of figures Figure 1-1 The multiple roles of B lymphocytes. ..................................................................... 3 Figure 1-2 Proposed model for B cell precursor migration within the adult bone marrow. ..... 6 Figure 1-3 The developmental stages of B cell maturation. ..................................................... 7 Figure 1-4 The cell surface markers present at the different developmental stages after B cell lineage commitment. ................................................................................................................. 8 Figure 1-5. The recruitment of the Pre-BCR into a membrane microdomain (lipid raft) and subsequent proliferation/anti-apoptotic signals. ..................................................................... 10 Figure 1-6 Anatomy and localization of B cell subsets within the spleen. ............................. 13 Figure 1-7 G-protein coupled receptor signalling in lymphocytes. ........................................ 20 Figure 1-8 The regulation of GTPase activity. ....................................................................... 23 Figure 1-9 Integrin heterodimer conformation and domains. ................................................. 25 Figure 1-10 Inside-out activation of integrins. ....................................................................... 28 Figure 1-11 Integrin outside-in signalling pathways. ............................................................. 31 Figure 1-12 The lymphocyte adhesion cascade. ..................................................................... 32 Figure 1-13 The actin treadmilling model for protrusion of the leading edge ........................ 36 Figure 1-14 Structure of the B cell antigen receptor ............................................................... 43 Figure 1-15 Sites within a lymph node where a B cell can encounter antigen ....................... 51 Figure 1-16 Model of B cell microcluster formation, spreading and immune synapse formation ................................................................................................................................. 59 Figure 1-17 Immune synapse patterns in T cell model systems ............................................. 62 Figure 1-18 Schematic representation of the gap junction families Innexins, Pannexins and Connexins ............................................................................................................................... 64 xi  Figure 1-19 Schematic representation of mammalian Cx43................................................... 68 Figure 2-1 Overview of B cell spreading assay from section 2.3.6 ........................................ 84 Figure 2-2  Overview of particulate antigen bead assay described in Section 2.3.7 .............. 86 Figure 2-3 Schematic representation of the WT BCR, and the mutant BCRs C? and ?trunc 87 Figure 2-4 C? and ?trunc are expressed in J558 15-25 B cells. ............................................. 89 Figure 2-5 BCR signalling competency of J558 ?m3, C? and ?trunc cell lines .................... 93 Figure 2-6 BCR-mediated cell spreading of J558 and WEHI 231 B cells on anti-BCR (anti-IgM) coated coverslips. ........................................................................................................... 96 Figure 2-7 The cytoplasmic domain of Ig? is required for optimal BCR cytoskeletal rearrangements in J558 cells ................................................................................................... 99 Figure 2-8 Assays for binding to particulate Ab reveal that the formation of actin-rich cup is reduced when the cytoplasmic domain of Ig? is swapped with Ig? or deleted. ................... 101 Figure 3-1 Overview of B cell spreading assays described in Section 3.3.7 ........................ 116 Figure 3-2 Overview of B cell adhesion to bEND.3 cells described in Section 3.3.9 .......... 118 Figure 3-3 Cx43 is expressed in B cells and is a target of BCR signalling. ......................... 121 Figure 3-4 Reduction of Cx43 expression alters the ability of B cells to sustain activation of the Rap1 GTPase................................................................................................................... 124 Figure 3-5 Decreased Cx43 expression impairs BCR-induced spreading in WEHI 231 B cells. ...................................................................................................................................... 128 Figure 3-6 Decreased Cx43 expression impairs BCR-induced spreading in splenic B cells from Cx43 heterozygous mice. ............................................................................................. 130 Figure 3-7 Expression of Cx43-GFP enhances BCR-mediated B cell spreading in WEHI 231 B cells.................................................................................................................................... 132 xii  Figure 3-8 Endogenous Cx43, Cx43-GFP and Cx43?T-GFP expression and localization in B cells. ...................................................................................................................................... 134 Figure 3-9 Cx43-GFP localization in J558?m3 B cells ........................................................ 135 Figure 3-10 Wild type Cx43-GFP but not the C-terminal tail truncated Cx43?T-GFP supports sustained activation of the Rap1 GTPase and B cell spreading. ............................ 138 Figure 3-11 WT Cx43-GFP expression is sufficient to restore/enhance BCR-mediated spreading. .............................................................................................................................. 140 Figure 3-12 Wild type Cx43-GFP expression is not sufficient to initiate BCR-mediated cell spreading of J558?m3 cells expressing mutated BCRs. ....................................................... 142 Figure 3-13 Cx43-GFP expression enhances LFA-1 induced Rap1 activation and cell spreading. .............................................................................................................................. 146 Figure 3-14 Cx43-GFP expression enhances chemokine mediated Rap1 activation and B cell adhesion to bEND.3 endothelial cells. .................................................................................. 150 Figure 4-1 Overview of B cell motility assay described in Section 4.3.8 ............................ 166 Figure 4-2 Overview of transwell migration assay described in Section 4.3.9 .................... 168 Figure 4-3 Overview of transendothelial migration assay described in Section 4.3.10 ....... 169 Figure 4-4 Motility of WEHI 231 B cells decreases after Cx43 expression is knocked down................................................................................................................................................ 173 Figure 4-5 Knockdown of Cx43 does not alter actin-GFP dynamics ................................... 175 Figure 4-6 Cx43 is a target of chemokine signalling and important for expression in CXCL12 mediated Rap1 activation and cell migration of WEHI 231 B cells ..................................... 179 Figure 4-7 Knockdown of Cx43 expression in WEHI 231 B cells results in a decrease in transendothelial migration (TEM) across two different endothelial cell layers. .................. 184 xiii  Figure 4-8 Mobility of Cx43-GFP decreases after cytochalasin D disruption of the cytoskeleton. ......................................................................................................................... 186 Figure 5-1 Overview model of the role Cx43 plays in regulation of the B cell cytoskeleton............................................................................................................................................... 198 Figure 5-2 Proposed molecular mechanism of how Cx43 regulates B cell morphology ..... 202    xiv  List of abbreviations aa Amino acid Ab Antibody ABP Actin Binding Protein ADF Actin depolymerizing factor APC Antigen presenting cell Arp Actin-Related protein ATP Adenosine triphosphate BCA Bicinchoninic acid  BCR B cell antigen receptor BLNK B cell linker protein BSA bovine serum albumin  Btk Bruton's tyrosine kinase Ca2+ Calcium cAMP Cyclic Adenosine monophosphate CD Cluster of differentiation CIP Calf Intestinal phosphotase CLP Common lymphoid progenitor cell Cx Connexin DAG Diacylglycerol DC Dendritic cell Dictyostelium Dictyostelium discoideum  DNA Deoxyribonucleic acid ECM Extracellular Matrix EDTA Ethylenediaminetetraacetic acid ER Endoplasmic reticulum ERK Extracellular-signal-regulated kinases FACS Fluorescence-activated cell sorting F-actin Filamentous actin  FDC Follicular dendritic cell FITC Fluoroscein isothiocyanate  FRAP Fluorescence recovery after photobleaching FRET Fluorescence resonance energy transfer G Guanine G-actin Globular actin  GAP GTPase activating protein GDP Guanosine diphosphate GEF Guanine nucleotide exchange factors GFP Green Fluorescent protein GJ Gap Junction   xv  GJIC Gap junction intercellular communication GPCR G-protein coupled receptor GTP Guanosine triphosphate HEV High endothelial venule HIP55 Hematopoietic progenitor kinase 1 -interacting protein of 55 kDa HRP Horseradish peroxidase HS1 Haematopoietic lineage cell-specific gene protein 1 HSC Hematopoietic stem cell ICAM Inter-Cellular Adhesion Molecule  IFN Interferron Ig Immunoglobulin IL Interleukin IP3 Inositol trisphosphate ITAM Immunoreceptor Tyrosine based Activation Motif  kDa Kilodalton LFA Lymphocyte function-associated antigen LMPP Lymphoid-primed multipotent progenitor cell LPS Lipopollysaccharide MAP Mitogen-activated protein MAPK Mitogen-activated protein kinase MHC Major histocompatibility complex min Minute MLC Myosin light chain MPP Multipotent progenitor cell MZ Marginal zone NF-AT Nuclear factor of activated T-cells NF-kB Nuclear factor kappa-light-chain-enhancer of activated B cells NK Natural Killer PAGE Polyacrylamide gel electrophoresis  PALS Periarteriolar lymphoid sheath PBS Phosphate buffered saline PI3K Phosphoinositde-3-OH kinase  *PIP2 Phosphatidylinositol-(3-4)-biphosphate  PIP2 Phosphatidylinositol-(4-5)-biphosphate  PIP3 Phosphatidylinositol-(3,4,5)-trisphosphate  PKC Protein Kinase C PLCg Phospholipase C Gamma PMSF Phenylmethylsulfonyl fluoride  PSGL1 P-selectin glycoprotein 1  pTyr Phosphotyrosine xvi  RAG Recombinase Activating Gene RT Room temperature S1P Sphingosine-1-phosphate SDS Sodium dodecylsulfate  SEM Scanning electron microscopy SH2 Src homology 2 domain SHIP-1 SH2-containing inositol phosphatase-1 shRNA Short hairpin RNA SLP SH2 domain-containing leukocyte protein SOS Son of Sevenless Syk Spleen tyrosine kinase TCR T cell receptor TEM Transepithelial migration TGF Transforming growth factor TLR Toll-like receptor TM Transmembrane TNF Tumour necrosis factor VDJ Variable Diversity Joining VE Vascular endothelial VLA Very late antigen VVO Vesiculo-vacuolar organelles  WASp Wiskott-Aldrich Syndrome Protein WT Wild-type YFP Yellow flourescent protein ZO Zonula occludens      xvii  Acknowledgements I would first like to thank my supervisor Dr. Linda Matsuuchi for her support, encouragement and belief in me over the years.  I appreciate all that she has taught me about research and the importance of communication with other scientists in order to gain new perspectives on your work.  I would also like to thank my committee members, Dr. Ninan Abraham, Dr. Vanessa Auld, Dr. Christian Naus and past members Dr. John Gosline and Dr. Nelly Pante for their insight and encouragement.  I would like to thank Dr. Michael Gold for his many helpful suggestions on my project and critical readings of my manuscripts.   I have been fortunate to work with many gifted researchers over the course of my degree.  I would like to thank Dr. Kevin Lin, Dr. Kathy Tse and Spencer Freeman for their many helpful discussions.  I would like to thank my lab mates (present and past), specifically Caren Grande, Dr. Teresa Jackson and Kate Choi.  I would like to thank May Dang-Lawson and John Bechberger for their technical expertise and troubleshooting.  I would like to thank Derrick Horne from UBC Bioimaging for his help with the scanning electron microscopy.  I would like to thank the LSI Imaging for their training and advice on microscopy.  As well, I would like to thank my family and friends for their support throughout my education.  Finally I would like to thank my wife Emily, for her help, support and encouragement.   You have been an inspiration to me and make me a better person.    1  1 Introduction 1.1 The role of B cells in the immune response Throughout evolution a series of barriers have been established which an invading pathogen has to overcome in order colonize and establish themselves in a mammalian host.  These barriers are separated into three main parts: physical barriers, the innate immune system and the acquired adaptive immune system.   The first obstacles an invading pathogen will encounter are the physical barriers.  These include the epithelium, the resident flora, mucosa of the gastro-intestinal, respiratory and reproductive tracts, the production of anti-microbial peptides by the surface epithelial cells (known as defensins) (Gudmundsson and Agerberth, 1999) and the production of the anti-bacterial enzyme lysozyme (Medzhitov and Janeway, 2000).  These barriers have two common features: they are present at the site of ongoing interaction with invading microbes and they are focused at the invading microbe and not on the host cells (Medzhitov and Janeway, 2000).  The physical barriers that are present in the host are broad and relatively non-specific, but if the invading pathogen manages to circumvent them, it then has to negotiate a more specific defense, the innate immune system. Activation of the innate immune system requires the specific recognition of repeating or common patterns on the surface of the infectious microorganism (Medzhitov and Janeway, 2000).  The role of the innate immune system is: 1) to initially recognize the invading pathogen and provide signals via cytokines/chemokines to recruit members of the acquired/adaptive immune system, 2) to induce inflammation at the site of infection and 3) to begin the destruction and removal of the invading pathogen (Kobayashi et al., 2002; Takeda  2  and Akira, 2001). Cellular components of the innate immune system include: dendritic cells, neutrophils, basophils, eosinophils, natural killer cells, macrophages and monocytes. These cells are involved in the destruction of pathogens and the production of cytokines.  The innate immune system also consists of mast cells, which are involved in the inflammatory response, as well as intraepithelial (??) T cells and B-1 cells which act as sentinels at common sites of invasion (Kobayashi et al., 2002; Takeda and Akira, 2001). The strategy that the innate immune system employs is the recognition of markers on the invading pathogen that have repeating patterns which are not subject to variation and are essential to the survival of the pathogen.  This is accomplished through pattern recognition receptors like the Toll-like Receptors (TLRs) located on dendritic cells, neutrophils, macrophages and monocytes and low diversity rearrangements of the antigen receptors of the (??) T cells and B-1 Cells (Kobayashi et al., 2002; Takeda and Akira, 2001).  These receptors recognize molecules containing repeating patterns, such as liposaccharide (LPS) found in Gram-negative bacteria and viral dsRNA (Kobayashi et al., 2002; Medzhitov and Janeway, 2000). The advantage of this system is that it is very fast and effective when encountering pathogens; however, some pathogens can either evade or overwhelm this system and invade the host.  Therefore, the more specific acquired immune system must be activated to effectively eradicate the infection (Mittrucker and Kaufmann, 2000).  The two main cellular components of the acquired immune system are T and B lymphocytes.  The strategy that the acquired immune system employs is to have a large number of antigen-specific B and T cells that can each recognize and respond to a specific antigen (Gold and Matsuuchi, 1995).  In the case of B cells, when the B cell antigen receptor (BCR) of a single, unique B cell binds to an antigen the result is clonal expansion and  3  maturation of the antigen-specific  B cell into an antibody producing cell (plasma cell) which secretes antibodies specific to the antigen.  These antibodies, if they bind to the pathogen, will aid in the eradication of the infection (Gold and Matsuuchi, 1995; Harwood and Batista, 2008).  Along with this role in antibody production, B cells are also required for activation of the T cell immune response, where they can act as an antigen presenting cell (APC).  In addition, they release immunomodulatory cytokines which influence T cells and DC?s, regulate lymphoid tissue organization, wound healing and transplanted tissue rejection and influence tumour development and immunity Figure 1-1 (LeBien and Tedder, 2008).   Figure 1-1 The multiple roles of B lymphocytes.   Highlighted examples of B cell functions in mammals. Reprinted with permission from  (LeBien and Tedder, 2008).  4  1.2 B cell development 1.2.1 The role of adhesion and migration in hematopoietic stem cells The development of mammalian B lymphocytes are the result of fate decisions made by hematopoietic stems cells (HSCs) within bone marrow niches.  The differentiation of HSCs into B cells depends on their ability to interact with adhesion molecules, chemokines and activate transcription factors which induce binary cell fate decisions and lineage commitment (Hardy et al., 2007; Pillai and Cariappa, 2009).    HSCs are a unique population of precursor cells which possess the capacity for self renewal and differentiation into all the blood lineages (Massberg et al., 2007).  This function is possible due to their ability to move to niches which support the development of each hematopoietic cell type (Laird et al., 2008).  HSCs form in developing mouse embryos by embryonic day 7.5 (E7.5),  in the yolk sac, placenta and aorta-gonad-mesonephros, although the source and timing remain debated (Gekas et al., 2005; Laird et al., 2008; Medvinsky and Dzierzak, 1996; Samokhvalov et al., 2007).  The HSCs express high levels of the adhesion molecule VE-Cadherin (CD144) and integrin CD41/?2b which allows them to adhere and remain in these specific niches (Fraser et al., 2002; Laird et al., 2008; Taoudi et al., 2005).   At E9.0 the HSCs begin to express the cytokine receptor c-kit which binds to the chemokine KitL (Laird et al., 2008; Yoder et al., 1997).  By E11.5-12.5, the expression levels of VE-Cadherin and integrin CD41/?2b drop in the HSCs, resulting in their release from their current niche.  They then follow KitL gradients to the fetal liver where they receive signals promoting survival, rapid expansion and differentiation.   HSCs remain in the fetal liver until 1-2 days prior to birth when they become much more responsive to the chemokine CXCL12, following CXCL12 gradients to the spleen and bone marrow where they remain rapidly  5  proliferating until 3-4 weeks after birth when they become quiescent (Bowie et al., 2006; Christensen et al., 2004; Laird et al., 2008).  From here the HSCs occupy niches through interaction with osteoblasts that are dependent on the homotypic interaction of N-Cadherin, integrins and high density calcium salts (Adams and Scadden, 2006; Wilson et al., 2004).   The interaction of HSCs with osteoblasts is important for maintaining their quiescent, self-renewing phenotype, but in order to differentiate into B lymphocytes they have to leave that niche and interact with cells and signals that drive B cell lineage commitment (Adams and Scadden, 2006; Nagasawa, 2006).  HSCs destined for the B cell lineage migrate away from osteoblasts and interact with bone marrow reticular cells which express high levels of CXCL12 (CXCL12hi )(Figure 1-2).  Though both HSCs and the earliest B cell precursors have been found in contact with these CXCL12hi cells, HSCs make several lineage commitments before being restricted to B cells (Nagasawa, 2006).   HSCs generate a non-self renewing progenitor population termed MPPs (multipotent progenitor) which gives rise to all blood cell lineages.  This becomes restricted to the lymphoid lineage at the LMPP (lymphoid-primed multipotent progenitor) stage defined as Lin-KIT+SCA-1+FLT+ which is thought to give rise to the CLP (common lymphoid progenitor) (Lin-SCA-1lowKITlowIL7R+) leading to both T and B cells (Figure 1-3) (Nagasawa, 2006).   6   Figure 1-2 Proposed model for B cell precursor migration within the adult bone marrow.  HSCs are located in niches near the endothelium or in contact with bone osteoblasts.  They migrate towards CXCL12 expressing reticular cells where they differentiate into Pre-Pro B cells.  These B cell precursors leave the CXCL12hi reticular cells and move towards IL7 expressing cells within the bone marrow.  The transition to the Pre-B cell is marked by movement away from these IL7 expressing cells and clonal expansion.  After successful BCR rearrangement, the majority of immature B cells then migrate out of the bone marrow towards the spleen where they develop into mature B cells.  After successful T-cell dependent activation in the spleen, the mature B cells develop into long-lived plasma cells, which migrate back to the bone marrow.  Reprinted with permission from  (Nagasawa, 2006).   7   Figure 1-3 The developmental stages of B cell maturation.  A schematic representation of the stages that a HSC passes through on route to becoming a B cell.  Below each developmental stage is the cell surface protein characterization of that lineage.  HSC: hematopoietic stem cell, MPP: multipotent progenitor cell, LMPP: lymphoid-primed multipotent progenitor cell, CLP: Common lymphoid progenitor cell, - : no cell surface expression, + : cell surface expression, low : Low cell surface expression.   Reprinted with permission from  (Nagasawa, 2006).  1.2.2 Development of B cell subsets within the bone marrow   The differentiation of CLPs into the earliest B cell progenitors are defined based on the status of their Ig heavy and light chain rearrangements and the expression of specific proteins on their surface and within their cytoplasm (Bartholdy and Matthias, 2004).  The earliest B cell progenitor is the pre-pro B cell, which expresses the B cell lineage marker B220 (CD45) and CD43 (leukostatin).  These cells migrate towards CXCL12hi reticular cells within the bone marrow, interacting with them until they transit into Pro-B cells (Figure 1-2) (Nagasawa, 2006).  Pro-B cells migrate away from the CXCL12hi reticular cells and towards IL7 expressing cells within the bone marrow where they receive signals mediating both  8  survival and the initiation of Ig heavy chain rearrangement (Figure 1-2) (Nagasawa, 2006).  The Pro-B cell stage is marked by the  expression of components of the Pro-BCR and CD19 (Figure 1-4) (M?rtensson and Ceredig, 2000).  The pro-BCR consists of Ig?, Ig?, the surrogate light chain (Vpre-B and ?5) and Calnexin (M?rtensson and Ceredig, 2000; Nagata et al., 1997).  The cell surface expression of these proteins as a complex marks the transition into the Pre-BI stage.  This stage is marked by the first of the germline Ig heavy chain rearrangements, the joining of the D (diversity) and J (joining) regions via the activity of the RAG1/2 genes (Figure 1-4) (M?rtensson and Ceredig, 2000).    Figure 1-4 The cell surface markers present at the different developmental stages after B cell lineage commitment.  Schematic representation of B cell stages after commitment to B cell lineage in the bone marrow.  Below the representation of each developmental stage are the characteristic expression markers, the status of their Ig heavy and light chain rearrangement and the B cell receptor composition.  RAG: Recombinase activating gene, VpreB1, ?5: Surrogate light chains (SL), VDJ: Varriable Diversity Joining, ?m: membrane Ig?, G: Germline.  Reprinted with permission from  (M?rtensson and Ceredig, 2000).  9   The transition from the Pre-BI cell to the large Pre-BII cell is marked by the successful joining of the Ig heavy chain V (variable) region to the DJ regions, the surface expression of the heavy chain with associated surrogate light chain (Pre-BCR) (Figure 1-4), the initiation of proliferation and the migration away from IL7 expressing cells within the bone marrow (Figure 1-2) (M?rtensson and Ceredig, 2000; Nagasawa, 2006).  When a complete Pre-BCR is assembled and trafficked to the cell surface, it is reported to be organized into specialized lipid membrane microdomains, known as lipid rafts, independent of receptor engagement (Figure 1-5) (Kurosaki, 2002).  The Ig?/? components of the Pre-BCR become phosphorylated within their immunoreceptor tyrosine based activation motif (ITAM) regions, most likely by the src-family kinase Lyn that is found localized within these specialized membrane microdomains.  The phosphorylation of the ITAMs of the Ig?/? heterodimer causes the recruitment of Syk and the activation of PI3K, resulting in the activation of the transcription factor NF-?B and proliferation/anti-apoptotic signals (Figure 1-5) (Kurosaki, 2002). After proliferation has taken place, the large Pre-BII cells differentiate into small Pre-BII cells.  These cells are identical to the large Pre-BII cells with respect to surface markers, however, they are smaller than their predecessors because they have stopped proliferating.  Once the proliferation of the Pre-BII cell has ceased, the light chain undergoes VJ rearrangements in order to provide another unique antigen binding site (M?rtensson and Ceredig, 2000; Senn et al., 2003).  This newly re-arranged light chain (Ig? or Ig?) then replaces the surrogate light chain and associates with the heavy chain (Kurosaki, 2002; M?rtensson and Ceredig, 2000).     10   Figure 1-5. The recruitment of the Pre-BCR into a membrane microdomain (lipid raft) and subsequent proliferation/anti-apoptotic signals.   Schematic of the potential mechanism for initiation of signals from the Pre-BCR (green: Ig heavy chain, surrogate light chain, Ig? and Ig?).  In this model, recruitment of the Pre-BCR into a lipid raft results in Lyn-mediated phosphorylation of Ig?/? and NF-?B translocalization into the nucleus where it can initiate proliferation/anti-apoptotic signals initiated through the kinases Syk, PI3K, Tec/BTK and the adaptor protein BLNK.  Reprinted with permission from  (Kurosaki, 2002).   The complete membrane-bound BCR on the cell surface then represents the immature B cell stage (M?rtensson and Ceredig, 2000).  Immature B cells are further divided into two sub-groups: transitional (T) 1 and T2 cells.  T1 B cells move from the bone marrow, into the blood and towards the spleen in response to the action of the chemokine CXCL13 (Gunn et al., 1998; Lo et al., 2003; Su and Rawlings, 2002).  Once in the blood, the B cell begins the process of negative selection (Su and Rawlings, 2002).  Negative selection occurs when the antigen binding domains on the immature BCR bind with high affinity to soluble self-antigen within the blood or trapped in the periarterial lymphatic sheeth (Healy et al., 1997; Su and  11  Rawlings, 2002).  If the BCR binds with high affinity to self antigen, there are low calcium oscillations and an increase in the activated transcription factor NF-AT and ERK/pp90rsk (Healy et al., 1997).  The result of this is the anergy or apoptosis of the immature B cell that is autoreactive (Su and Rawlings, 2002).   1.2.3 Development of B cells outside of the bone marrow  The migration of the B cell from the bone marrow to the spleen marks the Transition (T)1 B cells (Su and Rawlings, 2002).  These cells home to the spleen in response to the action of the chemokines CXCL13, CCL19 and CCL21 produced by the follicular dendritic cells located within the white pulp of the spleen (Gunn et al., 1998; Lo et al., 2003).  The B cells then enter the periarteriolar lymphoid sheath (PALS) where blood borne self antigens are caught by the spleen, further driving negative selection (Su and Rawlings, 2002).  The surviving T1 cells then migrate into the primary follicles, expressing IgD and CD21 on their surface, marking the transition to T2 B cells.  T2 B cells then interact with self-antigen, presumably presented by follicular dendritic cells, initiating BCR-signals which drives development into mature B cell subsets (Pillai and Cariappa, 2009; Su and Rawlings, 2002).  This process, termed positive selection, is thought to check for the generation of a functional receptor while simultaneously generating instructive signals for lineage commitment (Pillai et al., 2004).  If T2 B cells bind antigen with very high affinity or do not bind any antigen at all, they undergo either apoptosis or anergy.  If the T2 B cells bind antigen with high affinity, but below the threshold of initiating anergy, they develop into mature follicular B cells, whereas if they bind antigen with low affinity, and in conjunction with Notch/DL1  12  signalling, they develop into marginal zone B cells (Pillai and Cariappa, 2009; Su and Rawlings, 2002).  1.2.4 Mature B cell subsets Mature B cells subsets are defined based on their anatomical location, cell surface protein complement and their function in the immune response (Table 1-1). Though the different B cell subsets may have different specific functions, they can be broadly categorized into adaptive and innate-like responses. Follicular B cells are the most common and well studied of the mature B cell subsets. Follicular B cells develop, as previously mentioned, from T2 immature B cells after low self-antigen recognition during positive selection (Pillai and Cariappa, 2009). They reside in B cell follicles of the spleen (Figure 1-6), comprising ~70% of the total splenic B cells, but also can be found in lymph nodes and the bone marrow.  These B cells are highly migratory and exit their splenic niches in the B cell follicles, migrate through the circulatory and lymphatic system, to other lymphatic sites (Allman and Pillai, 2008).  Follicular B cells are short lived and continually renewed from newly generated immature B cells.  Their main function is in the T cell dependent immune response.  Here, BCR recognition and subsequent co-stimulatory activation through CD40 binding to CD154 on T cells drives rapid expansion, migration and differentiation into one of two fates; either migration into extrafollicular areas where they proliferate and differentiate into short-lived plasma cells or migration into a B cell follicle, generation of a germinal center and development into both short and long lived plasma cells (Allen et al., 2007a; Oracki et al., 2010).  Within the germinal centers, the activated follicular B cells undergo a process known as somatic hypermutation where the Ig  13  variable region undergoes DNA point mutations, resulting in affinity changes with the goal of producing a higher affinity binding site to the specific antigen.  Along with somatic hypermutation, the Ig heavy chain also undergoes a class switch, resulting in secretion of antibodies (ie. IgG, IgA, IgE) with different binding properties (Oracki et al., 2010).   Figure 1-6 Anatomy and localization of B cell subsets within the spleen. Schematic representation of the white pulp of the spleen and the localization of B cell subsets in it.  Follicular B cells (FO) are located in the B cell follicles (yellow), and after activation they can be located in germinal centers or in extrafollicular spaces in the red pulp.  Marginal zone (MZ) B cells are located in the marginal zone surrounding the marginal sinus.  When these cells come into contact with blood-borne cognate antigen, they can leave their niche in the marginal zone and move into the B cell follicle where they pass off the antigen to follicular dendritic cells.  DZ: germinal center dark zone, LZ: germinal center light zone, T: T cell.  14  Another subset of mature B cells located in the spleen are Marginal Zone (MZ) B cells.  MZ B cells develop from T2 immature B cells when, during positive selection, they recognize self-antigen with high affinity as well as receiving signals from the cell surface protein Notch binding to its ligand DL-1 on endothelial cells of the red pulp venules (Pillai and Cariappa, 2009).  This B cell subset is largely sessile (does not recirculate) and is located at the marginal zone between the red and white pulp of the spleen (Figure 1-6), where it is uniquely poised to come in contact with blood borne antigens (Cinamon et al., 2008; Pillai and Cariappa, 2009).  MZ B cell function is considered innate-like due to their limited antigen recognition repertoire, which primarily recognizes microbial polysaccharides and self antigen, and their high expression of the complement receptor CD21 (Allman and Pillai, 2008).  They primarily respond to T-independent antigens via the toll-like receptor TLR4 which recognizes the bacterial cell wall component LPS.  The activation of MZ B cells results in relocation into the B cell follicle, rapid proliferation and differentiation into short-lived plasmablasts (Cinamon et al., 2008; LeBien and Tedder, 2008).  MZ B cells have also been implicated in T cell dependent immune response (Allman and Pillai, 2008).  After activation, MZ B cells can migrate into the B cell follicle where they can transfer antigen to follicular dendritic cells, for subsequent presentation to follicular B cells (Cinamon et al., 2008; Ferguson et al., 2004).   Similar to MZ B cells, the mature B1 B cell subset are also defined as innate-like B cells (Baumgarth, 2011).  These B cells are generated before and for the first few weeks after birth, residing primarily in the pleural/peritoneal cavities and intestines where they maintain a stable, self-renewing population (Baumgarth, 2011).  They comprise ~5% of the total B cells and their major function is the production of natural antibodies (antibodies present  15  without any previous known exposure to the antigen recognized by the antibody) that are specific for evolutionarily persistent antigens (ie. influenza, Streptococcus pneumonia).  There are two distinct subsets of B1 B cells: CD5+ B1a cells and CD5- B1b cells.  B1a cells respond to BCR-independent signals, initiating migration from mucosal sites to lymph nodes, where they differentiate, without proliferation, into short lived plasmablasts, secreting large amounts of IgM or IgA  (Allman and Pillai, 2008; Baumgarth, 2011). In contrast to this, B1b cells response is BCR dependent, resulting homing to the spleen/lymph node, clonal expansion and differentiation into antibody secreting plasma cells.  This subset can develop into ?memory? B1b cells which reside in the peritoneum and confer long-term protection (Baumgarth, 2011). Recently, another mature B cell subset has been identified which is responsible for down regulating autoimmunity as well as infection and allergy-associated inflammation (Mizoguchi and Bhan, 2006; Vitale et al., 2010).  These cells, B regulatory cells (Breg), have been reported to down regulate the immune response through both contact dependent and independent mechanisms.  Bregs present self antigen and induce the down-modulation of self-reactive T cells by down-regulating their antigen receptors.  They can also secrete the immune-suppressive cytokines IL-10 and TGF? which work in concert to suppress the activation of dendritic cells, macrophages and T effector cells as well as blocking inflammation (Vitale et al., 2010).  Bregs also recruit T-regulatory cells, activating them via an IL-10 mechanism, aiding in their suppressive phenotype (Vitale et al., 2010).  The development of these regulatory cells still remains debated.  There are two prevailing theories, that Bregs can be generated from all activated B cells through the stepwise  16  activation of TLRs, BCR and CD40 (Lampropoulou et al., 2010) or that they originate from a Breg progenitor cell (DiLillo et al., 2010).        Table 1-1 Summary of mature B cell subsets.   B regulatory cells Memory B cellsB1a B1b Follicular B cells Marginal Zone B cells Short lived Long livedDevelopment ? Low self-angiten BCR signallingHigh self-antigen BCR signalling plus Notch/DL1 signallingGenerated after activation of mature B cellsGenerated after activation of follicular B cells generated after antigen activation and persists without an immunizing agent: develop in germinal centersAnatomical location(~1% of splenic population): MZHighly migratory and move through the blood and lymph; reside in follicles of spleen (~70% of splenic B cells), lymph nodes, bone marrowMarginal zone (interface between red and white pulp in spleen) (~ 15% of splenic B cells): MZ-like B cells have been reported to be found in the pancreatic lymph node of diabetic NOD miceExtra-follicular B cells: short lived and non-migratory.  Develop from MZ/B1 B cells after T-independent Ag activationIntra-follicular B cells: develop in germinal centers and migrate to the spleenMarginal Zone of spleen; (5% of peripheral B cells)phenotypeCD5+ CD19hi CD1d mid CD23- CD43+ IgMhi IgD lowCD5- CD19hi CD1d mid CD23- CD43+ IgMhi IgD lowCD5+ CD19hi CD1dhi CD21hi/mid CD23+/_ CD43- IgMhi IgDlow/midCD5- CD19mid CD1d hi CD23+ CD43- IgMlow IgD hiCD5- CD19mid CD1d hi CD21hi CD23- CD43- IgMhi IgD lowSurface Ig- B220- Synd-1+ Flt3- MHCII+ CXCR4++ Pax5- IRF4hi Blimp-1++Surface Ig- B220- Synd-1+ Flt3- MHCII- CXCR4+++ Pax5- IRF4hi Blimp-1+++Human: CD27+ Isotype switched Ig, IgD-, CD80hi CD86hi CD95hi,  CD23low.  Mouse: no conclusive marker in miceT cell dependence dependent and independentdependent and independent, but primarily T independent response to blood-borne antigensT cell independent and T cell dependent: terminal differentiation phenotype for MZ and B1 B cells, proposed that Follicular B cells pass through this stage on route to long lived plasma cellsT cell dependent T cell dependent Ig repertoire IgM, IgA, IgG, IgE IgM, IgG1 High avidity Ig secretion High affinity Ig secretion Potentially allActivationTLR activation, IL5, IL10, independent of BCR signallingBCR signallingTLR signalling for the initial Il-10 secretion phase and BCR/CD40 engagement for survival and expansionBCR signalling; synergistic CD40 and TLR signallingBCR and TLR activation -CXCL12 bone marry CXCL12hi cellsBCR signallingProliferation No, maintain stable population Yes, in response to BCR Yes, in response to BCR yes in response to TLR activation yes in response to BCR Somatic hypermutation no no - yes no - - noimmunosuppressive adaptive immune responseInnate-like: respond to blood-borne pathogens: may play a role in adaptive response by activation of follicular B cells or transport of antigen to follicular dendritic cellsSecrete high avidity antibody Secrete high affinity antibodyrapidly responds to subsequent infection with a previously seen antigenproduction of natural antibodies (both self antigen [i.e.. Oxidized lipids, apoptosed cells] and pathogen associated molecules [microbial polysaccharides]) long term production of T-independent antibodiesSecrete Il-10 which suppresses T cell activationcreate high affinity antibodies to specific pathogenic antigensproduction of natural antibodiesHas high affinity BCR on surfaceTolerance under non-inflammatory conditionsdifferentiate into either long or short lived plasma blasts as well as memory B cellsRapid differentiation into short lived plasmablasts at early stages of infection to T-independent antigenSuppress activated T cell proliferation and cytokine productioncapture complement (iC3b) bound antigen complexes via CD21 which signals migration into the follicle where they transfer the antigen to follicular dendritic cellsActivates Treg cells May act as an APC to CD4+ T cellsfunctionInnate-likePlasma cellsIgM and IgAB1 B cellsindependentgenerated before birth and the first few weeks after birthB2 cellsMajority in Pleural/peritoneal cavities and intestine, but also found in bone marrow, spleen, blood, lymph nodes lung17  18  1.3 B cell migration The ability to move from an anatomical location in the body to another, and within that site, is not only essential for the development of B cells, but also for their ability to initiate an antibody-mediated immune response.  Mature B cells follow chemoattractant gradients, either bound to a surface (haptotaxis) or soluble (chemotaxis), allowing them to move to and from lymphoid organs in search of cognate antigen (Cyster, 2005; Thelen and Stein, 2008).  The effect of binding to a chemoattractant is two-fold.  First, the binding initiates signals which polarize the cell giving it a direction to move; and second, it initiates signals which activate integrins on their surface, allowing them to adhere to cells/substrate and generate the force required to move (Stein and Nombela-Arrieta, 2005).    1.3.1 Chemoattractants and their receptors There are two main groups of chemoattractants that regulate the movements of B cells: chemokines and lipid chemoattractants.  Chemokines are small (8-12 kDa), structurally related polypeptides which are classified into four groups based on the number and spacing of cysteine (C) residues in their N-terminus as C, CC, CXC or CXXXC (Cyster, 1999; Stein and Nombela-Arrieta, 2005).  Of the over 40 identified chemokines, the majority are produced at sites of inflammation for the recruitment of innate immune cells and effector T cells.  A small subset, termed homeostatic or lymphoid chemokines, are required for the function and organization of B cells (Cyster, 2005).  The lymphoid chemokines relevant to B cells include CXCL12, CXCL13, CCL19 and CCL21.  The lipid chemoattractant important for B cell homeostatitc migration is sphingosine-1-phosphate (S1P) (Cyster, 2005).  S1P is produced from the metabolism of the membrane lipid sphingomyelin and subsequent  19  phosphorylation by S1Pkinases (Rivera et al., 2008).  S1P is abundant in the blood (100 nM to ?M concentrations) produced by erythrocytes, and in the lymphatic fluid (~100 nM) produced by lymphatic endothelial cells; however its concentration in tissues is very low.  Its major role in B cells is in the egress from secondary lymphoid organs into the blood/lymph and maintenance of MZ B cells in the marginal zone (Cyster, 1999; Rivera et al., 2008).   Both chemokines and S1P function through binding G-protein coupled receptors (GPCR) located on the surface of the B cells (summarized in Figure 1-7).  There have been more than 20 chemokine receptors identified and five known S1P receptors (Rivera et al., 2008).  In general, chemokines bind to their respective GPCR as a monomer, however chemokines in areas of high concentration or chemokine rich areas can form dimers or higher order aggregates through binding glycosaminoglycans. This can result  in both homo and heteromeric chemokines and an enhancement or synergy of signals leading to enchanced leukocyte activation, however the mechanism of action remains poorly understood (Allen et al., 2007c; Thelen and Stein, 2008).  The binding of a chemokine to a GPCR results in exchange of GTP with GDP on the G?i subunit, activating it and initiating the dissociation from the ?? subunits.  This then allows both the G?-GTP and G?? subunits to independently bind and activate downstream effectors (Kehrl, 2006) including activation of phosphoinositde-3-OH kinase (PI3K) and phospholipase C? (PLC?) pathways (Figure 1-7).  The downstream effect of this is changes in cell polarity, integrin activation and rearrangement of the cytoskeleton (Kinashi, 2005).  20   Figure 1-7 G-protein coupled receptor signalling in lymphocytes. Schematic representation of chemokine/chemoattractant-mediated signalling initiated through GPCR?s.  The activation of GPCR?s results in signals that generate the formation of a leading edge, trailing edge or uropod, cell polarity and the activation of integrins. Reprinted with permission from  (Kinashi, 2005)  21  1.3.2 Chemokine induced polarity Polarity in lymphocytes is defined by the formation of a leading edge at the anterior of the migrating cell and an uropod at the trailing edge (Sanchez-Madrid and Angel del Pozo, 1999).  Much of the information relating to how chemokines and their cognate receptors induce changes in lymphocyte cell polarity has come from studies in the amoeba Dictyostelium discoideum (Dictyostelium) and mammalian neutrophils. Historically, the question of how chemokines induce changes in polarity was a much debated topic.  It was originally proposed that chemokine-induced polarization was induced by an accumulation of chemokine receptor (Zigmond et al., 1981) the hypothesis being that the asymmetric distribution of these receptors enhance the detected signal, reinforcing the polarity of the cells.  Problems arose with this hypothesis when the distribution of chemokine receptors was determined to be influenced by the activation and fixation methods used (Servant et al., 1999).  Using a GFP-tagged chemokine receptor, it was later reported that GPCR?s were uniformly distributed around the membrane of a chemotaxing neutrophil (Servant et al., 1999), though there is conflicting evidence for this in T cells where the chemokine receptor CXCR4 polarizes to the leading edge (Shimonaka et al., 2003).  The dissociation of the G protein ?? subunits also did not show a strong polarization, instead the second messenger phosphatidylinositol-(3,4,5)-trisphosphate (PIP3), the product of the phosphorylation of membrane lipids by PI3K, was shown to localize at the leading edge of migrating Dictyostelium and neutrophils (Jin et al., 2000; Meili et al., 1999; Servant et al., 2000; Wang et al., 2002).  The importance of PIP3 production in the establishment of polarity and migration was shown in both Dictyostelium and neutrophils by a decrease in migration after genetic knockdown or pharmacological inhibition (Funamoto et al., 2001; Wang et al., 2002).   22  The localization of PIP3 at the leading edge was shown to be controlled by the phosphatase SHIP-1, which degrades PIP3 and restricts its localization to the leading edge where it can recruit proteins with PH domains, however its importance still remains debated (Franca-Koh et al., 2007; Funamoto et al., 2001; Nishio et al., 2007). The GPCR induced production of PIP3 by PI3K is not the only factor in determining lymphocyte polarization as the establishment of polarity also requires the action of members of the Rho and Ras GTPase family (Thelen and Stein, 2008).  These small GTPases are activated by guanine nucleotide exchange factors (GEFs) which exchange bound GDP with GTP, allowing these proteins to bind and activate effector proteins.  Their activation is ?turned off? by the action of GTPase activating proteins (GAP?s) which catalyzes the conversion of GTP to GDP (Figure 1-8) (Tybulewicz and Henderson, 2009).  The GTPase Rho-A is required for uropod formation and retraction where as Rac and Cdc42 are required for leading edge formation (lamellipodia) and stabilization respectively  (Kinashi, 2005; Thelen and Stein, 2008).  Though the activation of these GTPases is important for polarity, their action is regulated by the Ras-like GTPase Rap1 (Gerard et al., 2007; Thelen and Stein, 2008).  Rap1 is activated through the GPCR-mediated activity of Phospholipase C, which is activated by the G?? subunits, and hydrolysis of the membrane phospolipid PIP2 into IP3 and DAG (Figure 1-7).  This in turn activates calcium and DAG-regulated GEFs resulting in the exchange of GTP with GDP on Rap1 and its activation (Kinashi, 2005).    Rap1 has been shown to be important for polarization in T cells, where constitutively active Rap1 results in a polarized phenotype (Gerard et al., 2007).  In this study they provide evidence that activated Rap1 can recruit and activate the Par complex, which is important for polarization of migrating astrocytes, asymmetric cell division in yeast, apical-basal polarity in Drosophila  23  ectoderm and apical-basolateral polarity in mammalian epithelial cells, via the activation of Cdc42, Tiam1 and Rac1 (Etienne-Manneville and Hall, 2003; Gerard et al., 2007).  Activated Rap1 has also been shown to recruit and activate the effector RapL, which is required for localization and activation of the integrin LFA-1 at the leading edge of polarized T cells (Bos, 2005; Katagiri et al., 2003).   Figure 1-8 The regulation of GTPase activity. Schematic representation of GTPase activation and inactivation mediated by GEFs and GAP?s respectively.  GEFs catalyze the exchange of GDP with GTP, resulting in GTPase activation, where as GAP?s catalyze the conversion of GTP to GDP resulting in GTPase inactivation.   1.3.3 Chemokine induced integrin activation Chemokine signalling is also important for activating integrins in lymphocytes, modulating their ability to bind to their ligands and mediate adhesion and facilitating migration.  Integrins are heterodimeric surface proteins which interact with extracellular matrix (ECM) proteins and ligands on the surface of other cells generating an adhesive force  24  (Hynes, 2002; Luo et al., 2007).  This adhesive force is essential for normal B cell function where blocked or impaired integrin function results in defects in B cell development, migration to the spleen/secondary lymphatic organs and membrane-bound antigen activation (Carrasco et al., 2004; Lo et al., 2003; Ryan and Tang, 1995).  The integrin heterodimer is composed of members of two subunit families, ?-subunits and ?-subunits, which are non-covalently associated (Figure 1-9).  There have been 18 described ?- and  8 ?-subunits which have been shown to form 24 heterodimers in vertebrates, but only 10 ?- and 3 ?-subunits have been described in lymphocytes (Table 1-2) (Luo et al., 2007; Shattil et al., 2010).    25   Figure 1-9 Integrin heterodimer conformation and domains. Crystal structure of the inactivated (bent: left) and activated conformation (extended: right) of an ? (red) ? (blue) integrin heterodimer.  Reprinted with permission from  (Shattil et al., 2010).         26  Table 1-2 Common integrins expressed on B cells.   Listed are the integrin heterodimers (and common names) expressed on B cells and their major ligands.  Based on information from (Luo et al., 2007). Integrin heterodimer Ligand ?L?2 (LFA1), ICAM-1, 2, 3, 5 ?4?1 (VLA-4) VCAM-1, Fibronectin ?4?7 (LPAM-1) MAdCAM-1, Fibronectin ?1?1 (VLA-1) Collagen, Laminin ?2?1 (VLA-2) Collagen, Laminin  Integrins bind to their cognate ligand and cluster together only after a conformational change which exposes the ligand binding site (Shattil et al., 2010).  Solitary integrins exist on the cell surface in one of three conformations, a closed/bent conformation which has low ligand binding affinity, an intermediate extended conformation which is activated but has a closed ligand binding site and an extended/open conformation which is activated and ligand bound (Shattil et al., 2010).  When these integrins are activated, they cluster together to form hetero-oligomers, increasing the overall strength of adhesion to the ligand (avidity) (Abram and Lowell, 2009).  The regulation of integrin conformation and clustering is very complex and is initiated by both signalling through chemokine receptors and the BCR (inside-out) and/or by binding multivalent integrin-ligands like ECM proteins (outside-in) (Abram and Lowell, 2009; Shattil et al., 2010).    1.3.4 Integrin inside-out signalling Inside-out signalling is defined as events that lead to conformational changes in integrins resulting in increased ligand binding and clustering (Abram and Lowell, 2009).  Regulation of this affinity change through inside-out signalling is regulated by two major  27  proteins; Rap1 which is required for activation of cytosolic proteins which bind to and change the conformation of integrins, and talin, the major adaptor protein that connects ? subuint to the F-actin cytoskeleton (Figure 1-10) (Abram and Lowell, 2009).  The importance for Rap1 activation in lymphocyte integrin activation has been demonstrated by expression of a constitutively active Rap1 isoform, which in T cells and B cells leads to activated LFA-1 and VLA-4 and an increase in adhesion (Durand et al., 2006; McLeod et al., 2004; Shimonaka et al., 2003).  Conversely, Rap1 knockout and expression of a constitutively active Rap-GAP, which keeps Rap1 in its inactive form, reduced integrin adhesion and migration (Chu et al., 2008; Li et al., 2007; McLeod et al., 2002; McLeod et al., 2004).  GPCR signalling results in the activation of PKC and PLC?, both of which have been implicated in Rap1 activation (Abram and Lowell, 2009; Medeiros et al., 2005; Pasvolsky et al., 2007).  The activation of Rap1 results in the recruitment and activation of two Rap1 effector proteins, RAPL and RIAM.   As previously described, RAPL associates with the ? subunit after Rap1-mediated activation and is important for both integrin localization and activation (Figure 1-10) (Katagiri et al., 2006; Katagiri et al., 2003)  RIAM is also activated in a Rap1-dependent manner and associates with the cytosolic adaptor talin and is localized to the ? subunit (Han et al., 2006). The binding of both of these proteins to their respective integrin domains is essential for proper activation, as demonstrated by a loss in adhesion when their expression is reduced (Han et al., 2006; Katagiri et al., 2006; Katagiri et al., 2003).   28   Figure 1-10 Inside-out activation of integrins. Schematic representation of the molecular mechanisms which lead to GPCR/BCR-mediated integrin activation.  Reprinted with permission from  (Abram and Lowell, 2009)  The activation and binding of talin to the ? integrin tail is essential in mediating the initial step in integrin activation regulated by inside-out signalling;  separation of the cytoplasmic tails resulting in extension of the extracellular domain (Figure 1-10) (Luo et al., 2007).  Talin is a 270 kDa protein which has a 47 kDa N-terminal subunit which contains a FERM domain (that binds to the integrin ? tail) and 220 kDa flexible rod tail subunit which has binding sites for the F-actin binding protein vinculin.  Knockout of talin results in a decrease in high affinity integrin-ligand binding  (Moser et al., 2009). Talin is normally held in an auto-inhibited conformation in the cytoplasm, however binding to activated RIAM  29  recruits talin to the membrane were it can potentially interact with the modified membrane lipid PIP2 (Goksoy et al., 2008; Martel et al., 2001; Shattil et al., 2010).  The binding of talin to PIP2 releases its auto-inhibition and allows it to interact with ? integrin tails (Goksoy et al., 2008; Martel et al., 2001).   The activated talin binds to the ? chain, outcompeting the ? chain for its binding site, which allows for the separation of the tails and extension of the ecto-domain (Vinogradova et al., 2002).  Talin is also important in integrin clustering.  Because talin has two integrin binding domains, and has the ability to dimerize, it can potentially bind and cluster up to 4 integrins (Moser et al., 2009).  The clustering of integrins is not only important for regulating ligand avidity, but also marks the transition to outside-in signalling and functional activation of cell adherence, spreading and cytoskeletal rearrangements (Abram and Lowell, 2009).  1.3.5 Integrin outside-in signalling Outside-in signalling is defined as the events that occur after ligand-meditated clustering of integrins on the cell surface (Abram and Lowell, 2009).  Where inside-out signalling controls the activation of integrins and ability to bind ligand in lymphocytes, outside-in signalling is required for transmitting signals required for firm adhesion, spreading and resistance to sheer forces (Abram and Lowell, 2009; Giagulli et al., 2006).  These signals include activation of Src and Syk family kinases, GTPases including Vav, Cdc42 and Rap, the actin regulator WASp and closely resembles antigen-receptor signalling (Abram and Lowell, 2007).  Similar to inside-out activation of integrins, the initial step in outside-in signalling is a conformational change resulting in the separation of the transmembrane and cytoplasmic domains of the ? and ? domains (Zhu et al., 2007).  When these domains are  30  genetically bound to each other via disulphide bonds, the surface integrins are still able to cluster, but there is no subsequent activation of downstream signalling (Zhu et al., 2007).  After this conformational change, there is an activation of members of the Src family kinases, though the mechanisms of their activation remains debated (Abram and Lowell, 2009).  The importance for the activation of the Src family kinases in outside-in signalling was shown in neutrophils after genetic knockout of the Src-family kinases Hck and Fgr (Giagulli et al., 2006).  Though these neutrophils were able to adhere to integrin ligands, the duration of adhesion was shorter and they were unable to arrest on inflamed muscle venules under flow.  A similar effect was identified in Syk deficient neutrophils which showed defects in ? integrin-ligand induced adhesion and spreading (M?csai et al., 2002).  Along with the Src and Syk non-receptor tyrosine kinases, the adaptor protein SLP76 has also been shown to be required for transmission of outside-in signals in neutrophils (Koretzky et al., 2006; Newbrough et al., 2003).  SLP76 is a downstream target of Syk and its knockout in mouse neutrophils resulted in a decrease in integrin-mediated spreading and inability to activate Vav and PLC?2 (Newbrough et al., 2003).  Similary, a knockout of the GEF Vav in neutrophils leads to a defect in sheer-resistant adhesion and spreading as well as decreased activation of PLC?2 (Graham DB, 2007; Pearce et al., 2007).  PLC?2 catalyzes the conversion of PIP2 into DAG and IP3, resulting in the release of calcium from stores in the ER and activation of members of the Rho-family of GTPases (Abram and Lowell, 2009).  The primary effectors of integrin outside-in signalling are the GTPases Rap1, Rac, Rho and Cdc42 which mediate the changes in the cytoskeleton required for firm adhesion and spreading.  Of particular importance for the rearrangement of the cytoskeleton is the activation of the actin nucleator WASp by Cdc42 and the activation of Rap1 (Lin et al., 2008; Zhang et al., 2006).  Defects in  31  WASp result in deficiencies in lymphocyte integrin-mediated clustering, adhesion, migration and spreading (Zhang et al., 2006).  Similarly, inactivation of Rap1 in B lymphocytes results in a reduction in LFA-1- mediated cell spreading (Lin et al., 2008).   Figure 1-11 Integrin outside-in signalling pathways. Schematic representation of the signalling pathways initiated after an activated integrin heterodimer binds to its ligand.  Reprinted with permission from  (Abram and Lowell, 2009).  1.3.6 Transendothelial migration  Integrin-mediated firm adhesion to the endothelium is an essential step in transendothelial migration (TEM), the biological process that allows lymphocytes to leave circulation and enter target tissues (Ley et al., 2007).  Though the process of TEM was originally described by Henri Dutrochet in 1824, the mechanisms have only recently started  32  to emerge (Dutrochet, 1824) .  In 1991 a model was proposed of leukocyte extravasation through the endothelium which involved three steps: 1) reversible rolling mediated by L-selectin, 2) leukocyte activation by the endothelium and 3) integrin-mediated adhesion (Butcher, 1991).  In 2007 this model was updated and expanded to seven steps: 1) selectin and integrin-mediated capture, 2) rolling on endothelium, 3) activation and slow rolling, 4) arrest, 5) intraluminal crawling, 6) para- and transcellular migration and 7) migration through the basement membrane (Figure 1-12) (Ley et al., 2007).     Figure 1-12 The lymphocyte adhesion cascade. Schematic representation of the steps required for lymphocyte adhesion and migration through an endothelial cell layer.  The purple cell represents a generic lymphocyte, the blue rectangular cells represent endothelial cells, the boxes indicate the key molecules that participate in each step.  Reprinted with permission from  (Ley et al., 2007).  1.3.6.1 Selectin-mediated capture and rolling   Selectins are expressed on bone-marrow derived cells as well as endothelial cells and mediate the initial stage of leukocyte interaction with the endothelium (Kansas, 1996).  There  33  are three major selectins expressed, L-selectin, P-selectin and E-selectin.  L-selectin is expressed by leukocytes where as both P- and E-selectin are expressed by the endothelium (Kansas, 1996).  There is ~ 52% homology between the C-type lectin domain of all three selectins as well as sharing the same ligand, P-selectin glycoprotein 1 (PSGL1), which is expressed by both leukocytes and endothelial cells (Kansas, 1996).  When lymphocytes interact with endothelium that is expressing both E- and P-selectin and PSGL1, there is an interaction between the selectin/ligand pairs which results in the lymphocytes becoming tethered to the endothelium and roll along it.  This bond has a very high on/off rate and is dependent on sheer force, termed a ?catch bond?, and does not occur in its absence (Marshall et al., 2003).  Though selectins are important for this process of capture and rolling, the ?4 integrins VLA-4 and ?4?7 as well as the ?2 integrin LFA-1 have also been implicated as being involved (Kerfoot and Kubes, 2002; Salas et al., 2004; Singbartl et al., 2001).   Interestingly, neutrophils rolling on immobilized E-selectin were able to induce a partial activation of LFA-1 (Chesnutt et al., 2006).  It is possible that in this context, the selectin ligation induces this partial integrin activation in order to further slow down the lymphocyte on the endothelium, aiding it in scanning the endothelium for surface-bound chemokines which initiates the next step in TEM (Ley et al., 2007).    1.3.6.2 Integrin-mediated firm adhesion   As the lymphocyte is rolling on the endothelium, contact with surface bound chemokines will induce a rapid activation of integrins and trigger firm adhesion to the endothelium (Ley et al., 2007).  Inflammatory cytokines activate endothelial cells resulting in the expression of the integrin ligands ICAM-1 and VCAM-1, as well as chemokines  34  including CXCL12 and CXCL13 on their luminal surface (Ley et al., 2007).  This provides a surface that is conducive to lymphocyte recruitment.  This integrin-mediated firm adhesion is regulated by both the inside-out and outside-in signalling previously mentioned in sections 1.3.4 and 1.3.5.  The result of firm adhesion is that the lymphocyte can move along and scan the surface of the endothelial cells for sites permissive to pass through.  1.3.6.3 Intravascular motility, cytoskeletal rearrangement, force generation and leukocyte motility The movement of lymphocytes along the surface of endothelial cells is mediated by a change in forces between protrusion of the leading edge (lamellipodium) and contraction of the trailing edge (uropod) (Lammermann et al., 2008).  The protrusive force at the leading edge is generated by the polymerization of highly branched F-actin (Giannone et al., 2007; Pollard and Borisy, 2003).  The chemokine-induced polarization of lymphocytes (section 1.3.2) results in the accumulation of activated Rac at the leading edge of a migrating cell allowing for the formation of the lamellipodia and the generation of forward migration.  Force at the leading edge of the lamellipodia is generated from polymerization of F-actin which pushes against the plasma membrane, allowing for a thin layer of plasma membrane to move forward.  Actin exists in two forms, a monomeric globular actin (G-actin) which is bound by profilin and filamentous actin (F-actin) which is a double helix polymer of G-actin arranged head to tail in order to give it molecular polarity (Pollard, 2007; Pollard and Borisy, 2003).   F-actin has a head, or barbed end, and a tail, or pointed end.  The conversion of G-actin to F-actin does spontaneously occur and can drive migration at ~0.4 ?m/min, however the kinetics of this reaction are too slow to support the migration rates reported for most  35  cells, including lymphocytes which have been reported to move between 5-20 ?m/min (Pollard, 2007; Pollard and Borisy, 2003; Shulman et al., 2009).  This actin-mediated force at the leading edge is mediated by a family of actin nucleating proteins, which include WASp and the Arp2/3 complex.  As previously described, the activation of GPCRs and integrins results in the activation of members of the Rho-family of GTPases, including Rac, RhoA and Cdc42.  These GTPases, specifically RhoA which is required for lamellipodia formation, can induce the activation of the actin nucleator WASp.  WASp is an adaptor protein which contains a specialized domain in its c-terminus (VCA domain) which is normally sequestered in an auto-inhibited conformation (Pollard and Borisy, 2003).  Activation of WASp by Rho GTPases results in recruitment to the plasma membrane via PIP2 binding and a reversal of the auto-inhibited conformation which exposes the VCA domain.  The VCA domain can then interact with free G-actin monomers and the Arp2/3 complex (Pollard, 2007; Pollard and Borisy, 2003).  The interaction between WASp, G-actin and the Arp2/3 induces a conformational change in Arp2/3, allowing it to associate with an F-actin filament.  Binding of the Arp2/3 complex to a pre-existing F-actin filament releases it from WASp and then initiates the polymerization of a new F-actin filament at a 70? angle to the original filament in the direction of the barbed end.  The new F-actin filament grows outward, pushing against the plasma membrane driving the lamellapod forward.  These new filaments at the leading edge are quickly capped by capping proteins, stopping the polymerization reaction.    36   Figure 1-13 The actin treadmilling model for protrusion of the leading edge Model of a potential mechanism for the actin-mediated protrusion of the leading edge.  1) An extracellular stimuli leads to 2) the activation of GTPases and accumulation of PIP2 at the plasma membrane.  3) WASp/Scar proteins are activated which 4) bind, activate and localize Arp2/3 to a pre-existing actin filament.  This results in 5) the elongation of the barbed end which 6) grows and pushes against the plasma membrane, driving it forward. 7) Quickly after the initiation of a new barbed end, it is capped by a capping protein which terminates elongation.  8) As the filament ages 9) it becomes severed by ADF/cofilin releasing G-actin monomers.  10) These monomers are bound by profilin which can then 11) recycle the G-actin monomers to the leading edge.  Reprinted with permission from  (Pollard and Borisy, 2003).  Though this capping may seem counterproductive to locomotion, it serves an important role in the generation of a forward force.  Long F-actin filaments are quite flexible and would be ineffective at pushing the membrane forward; however, short actin polymers  37  are much more rigid, and can initiate this forward force.  How these short polymers actually exert a force on the membrane was described by the ?elastic Brownian ratchet model? (Mogilner and Oster, 1996).  Actin filaments at the very tip of the leading edge are arranged in an orthogonal network and are heavily crosslinked.  As the new filaments are made, they are constantly cycling between a bent and straight orientation.  When the bent filaments straighten near the membrane, it pushes on the membrane exerting a force that drives the membrane forward.  Because of this, the optimal length of actin filament was calculated to be between 30-150 nm, any longer and the pushing initiated by straightening is ineffective (Mogilner and Oster, 1996; Pollard and Borisy, 2003).  They also suggest that if the opposing force generated by the membrane is too great, the actin filaments can re-orient themselves to be perpendicular to the membrane, resulting in the micro spikes and filament bundles as seen in fibroblasts and keratinocytes respectively (Mogilner and Oster, 1996). The generation of a forward force by actin filaments has to be opposed by something within the cell, or actin polymerization would solely drive new filaments backwards (Pollard and Borisy, 2003).   In non-lymphoid cells, this retrograde force is opposed by both cross linkage of the cytoskeleton and the coupling of integrins to both the cytoskeleton and the extracellular matrix in specialized structures known as focal adhesions (Giannone et al., 2007; Pollard and Borisy, 2003).  However, leukocytes do not make focal adhesions; instead the integrins are more evenly distributed on the plasma membrane.  They still likely couple the cytoskeleton to the extracellular environment (integrin ligands on endothelial cells) but their contribution to opposing this force is under the limit of detection by conventional traction-force microscopy and is not well understood (L?mmermann and Sixt, 2009; Smith et al., 2007).    38  Along with actin polymerization playing an important role in migration, actin depolymerization plays an equally important role (Pollard and Borisy, 2003).  The cell is a bounded compartment, therefore actin polymerization cannot continue for long without being balanced by actin depolymerization, which is mainly accomplished through the action of members of the actin depolymerizing factor (ADF)/cofilin family (Ichetovkin et al., 2000; Pollard and Borisy, 2003).  Active cofilin accumulates behind the leading edge of migrating cells where it can bind to the pointed ends of F-actin filaments, inducing their depolymerization (Svitkina and Borisy, 1999).  This generates G-actin monomers which can be bound by profilin for recycling to the leading edge and continuation of leading edge polymerization (Pollard and Borisy, 2003).  Active cofilin can also play an important role in the initiation of leading edge formation, by severing pre-existing filaments providing barbed ends for elongation (Ichetovkin et al., 2002).  As a lymphocyte migrates along the surface, not only does it have to negotiate protrusive forces at the front of the cell, but it also has to de-adhere and retract the trailing edge (uropod) (L?mmermann and Sixt, 2009).  This de-adhesion and retraction have recently been shown to be mediated by non-muscle myosin 2A in neutrophils and T cells (Jacobelli et al., 2009; Jacobelli et al., 2010; Smith et al., 2007).  The force of myosin 2A pulling on the trailing edge has a twofold effect: first, the rear-mediated compression aids in eliminating the existing adhesions at the rear of the cell and second, it provides a propulsive force generated by the rear of the cell which aids in lymphocyte ?amoeboid-like? fast walking (Jacobelli et al., 2009; Jacobelli et al., 2010; Morin et al., 2008). The balance between these forces is important for mediating the different forms of motility observed in leukocytes, in particular T cells.  T cells fluctuate between two, and  39  possibly three different modes of motility (Jacobelli et al., 2009; Jacobelli et al., 2010; Shulman et al., 2009).  When T cells encounter a high adhesive substrate, similar to sites of transendothelial migration or HEVs, their movement has been described as ?sliding? and ?millipede-like? (Jacobelli et al., 2009; Shulman et al., 2009).  In this ?sliding? mode of motility, the T cells maintain contact with an in vitro integrin substrate over a large surface area, with reduced velocity and is independent of myosin 2A activity (Jacobelli et al., 2009; Jacobelli et al., 2010).  Here, the forward forces are thought to be generated solely by actin polymerization at the leading edge (Jacobelli et al., 2009).  Similar to this, T cells crawling in vitro on the surface of an endothelial monolayer under sheer flow conditions maintain contact with the endothelial cells through the action of high affinity LFA-1 clusters, described as focal dots which are evenly distributed along the T cell:endothelial cell interface (Shulman et al., 2009).  In this model, the T cells initiate finger-like projections, or fillopods, which nucleate from the focal dots and were hypothesized to scan the endothelial surface for sites permissive for TEM.  This mode of motility was described as ?millipede-like? and does share many similarities with the ?sliding? motility, i.e. large contact area, but it is not known if it is dependent on myosin 2A contraction.  When T cells transit into areas of low adhesion with high confinement, for instance after they have transited through the endothelial monolayer and are in a 3D extracellular matrix or within a secondary lymphoid organ, they move via myosin 2A dependent ?amoeboid-like walking? motility (Jacobelli et al., 2009; Jacobelli et al., 2010).  In this type of motility, the T cell makes few contacts with the substrate which have a small surface area.  In the rear of the cell, myosin 2A compressions aid in propelling the cell forward and likely facilitate new adhesions at the front (Jacobelli et al., 2009).  This method of motility enables rapid cell movement and is ideally suited to  40  movement within a 3D matrix with low adhesion (Jacobelli et al., 2009; Jacobelli et al., 2010).  1.3.6.4 Paracelllular and transcellular transmigration When a lymphocyte crawling on the surface of the endothelium has found a site permissive to TEM, it initiates signals resulting in passage through the endothelial monolayer (diapedesis) in one of two ways; either through the junction between the cells (paracellular migration) or directly through the endothelial cell (transcellular migration) (Ley et al., 2007).  The majority of diapedesis occurs via paracelluar migration which is dependent on the localized and temporal loss of junctional assemblies between the endothelial cells (Luster et al., 2005).  One possible mechanism for how leukocytes initiate this destabilization is through LFA-1:ICAM-1 signalling and induction of the Rho-family GTPases (Greenwood et al., 2003; Millan and Ridley, 2005).  Here, the cytoplasmic domain of ICAM-1 in rat brain endothelial cells was critical for mediating the TEM of T cells, as well as the activation of RhoA.  Lymphocyte -induced activation of RhoA in endothelial cells results in the formation of stress fibers localized at intercellular junctions (Hordijk et al., 1999).  Lymphocyte -mediated RhoA activation and calcium efflux in the endothlelial cells also results in the phosphorylation of myosin light chain (MLC) via the activation of MLC-kinase, which applies a tensile force on the junction, pulling them apart (Ley et al., 2007; Millan and Ridley, 2005).  The lymphocyte can then interact with junctional molecules including PECAM1, JAM-A, B, C, ICAM-2 and CD99, which mediate their transit through the endothelial junction (Ley et al., 2007) .   Interestingly, as T cells are migrating through an endothelial monolayer under sheer flow, they accumulate high affinity LFA-1 at the apical  41  contact site between the T cell and endothelial cell (Shulman et al., 2009).  This finding, coupled with the finding that T cells deficient in myosin 2A accumulate on the HEV and show defects in TEM (Jacobelli et al., 2010) suggests that lymphocytes may be both pulling themselves through the junctions and pushing themselves through via actin-mediated polymerization.  Transcellular migration was originally described by Feng et al in guinea pig neutrophil migration to the skin in response to fMLP (Feng et al., 1998).  This pathway is utilized by the minority (5-20%) of leukocytes transmigrating through the endothelium (Ley et al., 2007).  Here, at sites of endothelial thinning, it is proposed that leukocytes will migrate through specialized, caveolin-1 rich pores in the endothelium which resemble vesiculo-vacuolar organelles (VVOs) (Feng et al., 2002; Millan et al., 2006).  VVOs form from the fusion of caveolin-1 rich vesicles which can facilitate the passage of solutes and macromolecules (Millan and Ridley, 2005).  Clustering of ICAM-1 in areas rich in these caveolin-1 rich vesicles was proposed to initiate this VVO channel, providing passage for the leukocytes through the endothelial monolayer (Millan et al., 2006).   Once the lymphocytes have passed through the endothelial monolayer they need to transit through the extracellular matrix (ECM) consisting of laminins and collagen (Ley et al., 2007).  This site represents a region of low adhesion and high confinement, where lymphocytes likely transit through in a myosin-dependent mechanism of motility described in the previous section.  After transiting through the venule, B lymphocytes can then move to the B cell follicles within the spleen and lymph nodes where they can search for cognate antigen via the B cell antigen receptor (BCR).  42  1.4 The B cell antigen receptor (BCR) The processes that regulate migration allow B cells to enter niches where they can come into contact with a vast array of antigens, which they can interact with though the B cell antigen receptor (BCR).  The BCR, located on the surface of the B lymphocyte, is the key receptor involved in antigen binding and signal transduction.  It is a multimeric protein complex that consists of four unique polypeptides arranged into two functional subunits; the antigen binding unit (two immunoglogulin (Ig) heavy chains that are disulfide linked to two Ig light chains) and the signalling subunit (the Ig?/? heterodimer, which is also linked together via disulfide bonds) (Figure 1-14) (Gold and Matsuuchi, 1995; Schamel and Reth, 2000b).  These two subunits are non-covalently associated together (Schamel and Reth, 2000b).  The BCR has two specific roles in the activation and development of the B lymphocyte: the first is to deliver signals to the B lymphocyte via antigen induced BCR clustering and the second is to internalize the antigen, where it is broken down in an endosomal compartment and presented via MHCII molecules on the surface of the B cell for recognition by T cells (Siemasko and Clark, 2001).  Because of the vast heterogeneity of potential pathogenic antigens, each na?ve B cell has a unique antigen binding domain (Pike and Ratcliffe, 2002).  This is accomplished through unique germline rearrangements of the DNA that encodes for the antigen binding region creating a specific and unique antigen binding site. This site is subsequently modified via somatic hypermutation after encountering cognate antigen in order to increase the specificity of the BCR to the foreign antigen (Roth and Craig, 1998).  The formation of the antigen specific BCRs occurs throughout the development of the B cell (M?rtensson et al., 2002).  43   Figure 1-14 Structure of the B cell antigen receptor The BCR consists of a signalling subunit and an antigen (Ag) binding subunit.  The signalling subunit consists of one Ig? (green) that is disulphide linked to one Ig? (orange).  This heterodimer is non-covalently associated with the Ag binding subunit, mIgM.  The Ag binding subunit consists of two Ig heavy chains (here Ig?: red) and two Ig light chains (here Ig?: blue).   1.4.1.1 BCR components The BCR on immature and mature B cells is characterized by the presence of the Ig?/? heterodimer that is non-covalently associated with the Ig?2/(Light chain)2 heterotetramer or with an Ig?2/(Light chain)2 heterotetramer (Figure 1-14).  These four components comprise the structure of the BCR on na?ve B cells (Reth, 1992). Murine Ig? is a 34-kDa glycoprotein that was first described by Hombach et al.  in 1988 (Hombach et al., 1988).  It contains an extracellular immunoglobulin (Ig) domain of  44  109 amino acids (aa), a transmembrane (TM) domain of 22 aa and a cytoplasmic domain of 61 aa that contains the Immunoreceptor Tyrosine based Activation Motif (ITAM) (Hombach et al., 1990a; Reth, 1989).  This ITAM motif is defined by the consensus sequence ?DxxxxxxxDxxYxxLxxxxxxxYxxL? where the negatively charged aspartic acid (D) can be replaced with a glutamic acid (E) and the terminal leucine (L) can be replaced with isoleucine (I) and ?X? represents any aa (Reth, 1989).  The Ig? protien is the product of the mb-1 gene (Matsuo et al., 1991) that was first described by Sakaguchi et al. in 1988 (Sakaguchi et al., 1988).  Murine Ig? is the 39-kDa glycoprotein product of the b29 gene, which was first described by Hermanson et al. in 1988 (Hermanson et al., 1988).   It contains a 128 aa extracellular domain, 22 aa transmembrane domain and a 48 aa cytoplasmic tail (Hombach et al., 1990a).  It, like the Ig?, also contains an ITAM motif in its cytoplasmic tail (Reth, 1989, 1992).   The membrane bound murine Ig? protein is a 67-78 kDa glycoprotein that is expressed on the cell surface as a ?2L2 tetramer (Gold and Matsuuchi, 1995; Vitetta et al., 1971).  The Ig? protein is comprised of four joined regions.  The VDJ rearrangements (section 1.2.2) are contiguous with four constant region domains of 110 aa each (Kehry et al., 1979) where the V region forms part of the site of antigen binding pocket (Senn et al., 2003). The murine Ig? protein has a 25 aa transmembrane domain and a 3aa cytoplasmic domain that consists of lysine-valine-lysine (Reth, 1992).  The Ig? chain is glycosylated with five N-linked oligosacharides in the constant region of the heavy chain (Finley et al., 1990), contributing to its variable molecular weight on SDS-Page gels.  45  The murine light chain attached to the Ig heavy chain can exist as one of two isotypes, the Ig? light chain or the Ig? light chain (Langman and Cohn, 1995).  Both of these proteins are between 25 and 28 kDa (Gold and Matsuuchi, 1995).  The successful completion of the pre-BCR signals the recombination of the light chain genes.  Both the ? and ? consist of V and J segments that undergo rearrangement to produce a unique antigen binding domain when coupled with the heavy chain (Tonegawa, 1983).  The light chain V region, like the V region of the heavy chain, when folded and associated together into the antigen binding pocket, these chains have the ability to bind antigen.  There are, therefore, two antigen binding sites on the mature Ig?/light chain component of the BCR (Senn et al., 2003). The mature BCR is also characterized by the heavy chain isotype switching from Ig? to predominantly Ig? (Reth, 1992).  Ig?, as well as the membrane versions of the other four classes of immunoglobulins, can associate with the Ig?/? heterodimer (Venkitaraman et al., 1991).  One major difference between Ig? and Ig? is the presence of a hinge region amino terminal to the constant (C) domain in Ig? (Reth, 1992).  Another difference between the two isoforms of surface antigen receptor is the size of the associated Ig? protein.  The Ig? associated with Ig? is 32 kDa and with Ig? is 33 kDa due to differential glycosylation (Campbell et al., 1991; Gold et al., 1991). During the maturation of the B lymphocyte, the heavy chain component of the BCR changes from primarily Ig? to primarily Ig? (Scher et al., 1983).  Although the heavy chain of the BCR is changed, it is still found associated with the Ig?/? heterodimer.  In fact, the Ig?/? heterodimer is found associated with all five heavy chain isotypes (Venkitaraman et al., 1991; Wienands et al., 1990).  Ig? is also shown to be associated with a single Ig?/?  46  heterodimer via interaction with the transmembrane region where the heterodimer similarly acts as a signal transducer through phosphorylation of tyrosine residues within their ITAM motifs (Gold et al., 1991; Schamel and Reth, 2000a).  This interaction between Ig? and the Ig?/? heterodimer is proposed to be more stable than the interaction between Ig? and the Ig?/? heterodimer where Ig? may be able to out compete the Ig? for the limited pool of Ig?/? (Schamel and Reth, 2000a).  Though the role of the Ig?/? heterodimer in the signal transduction of antigen binding to the heavy chain is conserved between the two isotypes (Ig? and Ig?), the requirement of the heterodimer in the trafficking of the BCR to the surface is different, where it is required by Ig? but not by Ig? (Wu et al., 1997).  1.4.1.2 BCR assembly and surface trafficking The multimeric BCR is found on the surface of the B cell as a four-chain complex consisting of the Ig-?, Ig-?, Ig-? and Ig-light chains (Gold and Matsuuchi, 1995).  These chains are assembled within the ER under the control of chaperone proteins.  These chaperone proteins are involved in proper folding of the proteins and the retention of the BCR complex until fully assembled (Foy and Matsuuchi, 2001).  On the surface of the B lymphocyte the Ig?/light chain is found non-covalently associated with the Ig?/? heterodimer (Hombach et al., 1990b).  This interaction takes place within the ER where the components of the BCR (Ig?/? heterodimer and IgM) are assembled and packaged for transport through the Golgi apparatus to the cell surface (Matsuuchi et al., 1992; Wienands et al., 1990).  It was previously proposed that the BCR complex consisted of the Ig?2light chain2 tetramer which was associated with two Ig?/? heterodimers (Gold and  47  Matsuuchi, 1995).  However, in 2000 Schamel and Reth showed that the Ig?2?2 tetramer and the Ig?2?2 tetramer were associated with only one heterodimer each.  This interaction is facilitated through the interactions of the Ig?/? and Ig?/? heterodimer transmembrane region.  The transmembrane region of the Ig? protein contains 9 hydrophilic amino acids that line up on one side of its ?-helical structure, see model (Schamel and Reth, 2000a).  This side of the alpha helix is the proposed site of interaction between the two complexes (Hombach et al., 1990b; Schamel and Reth, 2000a).  Since the Ig?2?2 tetramer is a symmetrical protein complex, the side that is not associated with the heterodimer is proposed to be able to interact with other BCR complexes, proposedly forming a complex oligomeric structure (Matsuuchi and Gold, 2001; Schamel and Reth, 2000a).  The Ig?/? heterodimer has a crucial role in the trafficking of the Ig? BCR to the cell surface (Matsuuchi et al., 1992).  This was assessed by transfection of the BCR components Ig?, Ig?, Ig? and Ig? proteins into a non-lymphoid cell line.  When all four of the proteins were present a functional BCR was expressed on the cell surface (Matsuuchi et al., 1992).   However, when either of the chains of the Ig?/? heterodimer were absent from the cell, there was no expression of the BCR on the cell surface, nor was it able to move successfully though the Golgi (Matsuuchi et al., 1992).  It was later shown that the extracellular region of the Ig? chain is important in the non-covalent association between the Ig?/? heterodimer and Ig? (Condon et al., 2000).  The exchange of a proline with a leucine in the extracellular domain of Ig? was able to stop the association of Ig?/? heterodimer with the heavy chain, causing an inability of the heavy chain to traffic to the cell surface (Condon et al., 2000).    48  The Ig? and Ig? chains were shown to be associated with the chaperone protein calnexin (Grupp et al., 1995).  This appears to be important in regulating the trafficking of the BCR to the cell surface.  The Ig?/? heterodimer appears to facilitate the dissociation of the calnexin molecule from the heavy chain, allowing the trafficking of the BCR to the cell surface.  When the Ig? transmembrane region is mutated, there is only a transient interaction with calnexin, allowing Ig?/? heterodimer independent trafficking to the cell surface (Grupp et al., 1995).  Calnexin and the Ig?/? heterodimer also appear to be important in the proper folding and assembly of the BCR complex within the ER (Wu et al., 1997).    1.4.1.3 BCR distribution on the membrane Currently, there are two differing models as to the distribution of the BCR on the surface of B cells.  The first, originally proposed by Schamel and Reth in 2000, suggests that both IgD and IgM BCRs are present on the surface as an oligomer as opposed to a monomer (Schamel and Reth, 2000a).  They later expanded on this model using a FRET-based assay to suggest that these BCR-oligomers were the dominant, auto-inhibited form of the BCR at the surface, and that they transitioned between large, inactive oligomers and active monomers (Yang and Reth, 2010b).  The inactive BCR monomers are proposed to have a closed conformation signalling domain, making it less accessible to activating kinases.  Dissociation of BCR monomers from the oligomer are proposed to lead to accessibility of their signalling domains, allowing them to bind to and cluster together around multivalent antigen, initiating signalling (Yang and Reth, 2010b).  49  In contrast to this model, Tolar and colleagues, also using a FRET-based system, were unable to detect energy transfer between individual BCRs suggesting that the BCR is indeed on the surface as a monomer (Tolar et al., 2005).  In this model, binding of BCR to antigen induces a conformational change in the Ig?/? subunits resulting in their separation which is required for activation (Tolar et al., 2005).  They later went on to show that the binding of a BCR to a monovalent, membrane bound antigen induces BCR clustering, which is mediated by a conformational change in the Ig heavy chain (Tolar et al., 2009).  It is possible that, although there was no observed FRET between different BCRs on the surface in this system in resting cells, the BCRs may be held in a conformation which separates the fluorophores past what is acceptable for FRET to occur.  Though this is possible, the approach utilized by Tolar et al by having the flourophores on the end of flexible linkers would make this scenario unlikely. The organization of the BCR on the cell surface of resting B cells has also recently been shown to be dependent on the action of the F-actin cytoskeleton and the membrane:cytoskeleton linker ezrin (Treanor et al., 2010).  Using single particle tracking and TIRF microscopy, Treanor and collegues show that the BCR is corralled in areas that are low in F-actin and ezrin.  Within these areas, the BCR is mobile, but its mobility significantly decreases when it moves through these F-actin/ezrin rich regions, and this confinement is dependent on the cytoplasmic tail of Ig? (Treanor et al., 2010).   When these cytoskeletal corrals are pharmacologically disrupted, it results in BCR-mediated signalling comparable to antigen binding.  How this change in diffusion initiates BCR signalling is unknown, but it is possible that the liberation of the BCR from its confined corral allows it to come into contact with protein islands and/or membrane microdomains (lipid rafts) similar to what is seen in T  50  cells (Lillemeier et al., 2006).  In T cells, these protein islands are enriched in cholesterol, linked to the F-actin cytoskeleton and contain signalling components necessary for activation (Lillemeier et al., 2006). In this model, the BCR on resting B cells would be held in an inactive, mobile state in areas of low F-actin/ezrin and occasionally move through protein islands in areas of high F-actin/ezrin leading to the tonic BCR signalling observed in B cells (Treanor et al., 2010).    1.4.1.4 Antigen-mediated BCR activation B cells can rapidly detect and mount a response to na?ve, unprocessed antigen after exposure (Batista and Harwood, 2009).  The activation of B cells through the BCR was originally proposed to be mediated solely through crosslinking, i.e. binding a multivalent antigen (Yang and Reth, 2010a).  This idea stemmed from the observation that stimulation with a monovalent F(ab) fragment was unable to activate B cells where as using a divalent F(ab)2 fragment resulted in a robust activation response (Woodruff et al., 1967).  Due to this, it was assumed that all BCR-mediated B cell activation was due to soluble, multivalent particulate antigens (which include bacteria, virus particles, Ab and complement-bound antigen generally larger than 70 kDa); however it is now evident that the majority of BCR-mediated activation occurs via recognition of membrane-bound particulate antigen (Carrasco and Batista, 2006b; Depoil et al., 2008).  Because the probability of a B cell coming into contact with cognate antigen while transiting through the blood stream is quite low, they spend the majority of their time residing within secondary lymphoid organs including the splenic white pulp, lymph nodes and Peyers patches in the gastrointestinal tract which are designed to filter and collect antigen (Cesta, 2006; Cyster, 2010).  As a B cell transits from  51  the blood stream, through the HEV and into the lymph node it comes into contact with a specialized population of dendritic cells (DC?s) surrounding the HEV as they follow a CXCL13 gradient, through the T cell paracortex, to the B cell follicle (Figure 1-15A) (Cyster, 2010; Qi et al., 2006; Wykes et al., 1998).  This population of DC?s has the capacity to take in antigen and present it on its surface without degrading it, allowing for activation of B cells (Qi et al., 2006).  If the antigen presented by these cells is not complimentary to their BCR, then the migrating B cells continue through the paracortex/T cell zone and into the B cell follicle in search of antigen (Cyster, 2010).        Figure 1-15 Sites within a lymph node where a B cell can encounter antigen Schematic representation of locations where B cells come into contact with antigen within a lymph node. As the B cell (blue) leaves the HEV within the T cell zone, it comes into contact with A) dendritic cells (DC?s) outside of the follicle.  If this does not present cognate antigen, A)B)C)D) 52  the B cell migrates into the B cell follicle where it comes into contact with B) subcapsular macrophages (SCS M?), C) Follicular dendritic cells (FDC) or D) soluble antigen of less than 70 kDa.  Reprinted with permission from  (Cyster, 2010).  Lymphatic fluid, and material carried within it, drains from afferent lymphatic vessels into the subcapsular sinus, the space between the collagenous capsule and the lymphocyte-rich cortex (Cyster, 2010).  B cell follicles are situated in close proximity to the subcapsular sinus and regulate the entry of lymph-containing antigen based on its size (Batista and Harwood, 2009).  Small soluble antigen of less than 70 kDa (i.e. low-molecular-mass toxins) have been shown to enter the B cell follicle through two different mechanisms.  First, small soluble antigen was reported to enter the B cell follicle rapidly, likely through small pores of 0.1 ? 1 ?m in diameter on its surface (Batista and Harwood, 2009; Pape et al., 2007).  This antigen was sufficient to activate B cells and trigger their migration to the B cell follicle: T cell zone boundary for MHCII-mediated presentation to T cells (Okada et al., 2005).  Though the diffusion of soluble antigen through small pores in the follicle is an attractive hypothesis, the existence of these pores remains debated.  Because the studies that identified these small pores used static, electron micrographs it was possible that these pores represent sites where cells have recently migrated through the sinus wall (Batista and Harwood, 2009).  In support of this, a second mechanism has been proposed for the traffick of small antigens to the B cell follicle.  Small conduits connecting the subcapsular sinus to the lymph node B cell follicle were recently identified, and provide a mechanism for small antigen entry, as well as for entry of chemokines, into the B cell follicle (Roozendaal et al., 2009). Though small antigen can enter the follicle through pores/conduits, larger particulate antigen needs to be actively transported into the follicle for B cell recognition.  Two groups  53  of cells have been implicated in display of particulate antigen in the follicle: follicular dendritic cells (FDCs) located within the follicle and subcapsular macrophages which are localized to the outer edges of the follicle (Figure 1-15B and C) (Cyster, 2010).  FDCs were originally proposed to be the cells solely responsible for antigen presentation, however the mechanism of how these cells actually acquired particulate antigen while being confined to the follicle remained a mystery.  In the spleen, the white pulp is surrounded by a marginal zone which contains a specialized subset of B cells known as marginal zone B cells (described in section 1.2.4) (Cinamon et al., 2008).  When these cells come into contact with blood borne antigen, they have recently been shown to migrate from the marginal zone and into the B cell follicle, transporting antigen to the FDC?s (Cinamon et al., 2008).  Though similar in structure to the spleen, lymph nodes do not contain a marginal zone or marginal zone B cells, therefore a different mechanism for antigen transport to FDC?s must be employed.  Particulate antigen entering the lymph node accumulates rapidly on a group of cells surrounding the B cell follicle known as subcapsulary macrophages.  These cells rapidly acquire particulate antigen from the subcapsulary sinus and display it on their follicular-facing side in an intact form for recognition by B cells (Carrasco and Batista, 2007; Phan et al., 2007).  At this point two different mechanisms have been established for B cell activation.  Phan et al showed that follicular B cells can bind this non-cognate antigen through complement receptors located on its surface and transport antigen it to the center of the follicle where it is passed to FDC?s, which can retain and present the antigen for long periods of time (Phan et al., 2007).  If the B cells is able to recognize this presented antigen via the BCR, then it slows down, interacts and forms an activation structure known as an immunological synapse resulting in B cell activation and migration to the T cell/B cell  54  boundary (Carrasco and Batista, 2007; Phan et al., 2007).   The immediate effect of activation through antigen binding is the activation of downstream signalling pathways resulting in B cell proliferation and differentiation. Along with the initiation of B cell spreading, B cell adhesion is of critical importance to B cell development and activation.  The binding of leukocyte function-associated molecule-1 (LFA-1, CD11a/CD18, ?L?2) and very late antigen-4 (VLA-4, ?4?1) on B cells to intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) on an antigen presenting cell is proposed to reduce the threshold for B cell activation by increasing the duration of attachment and promoting cell spreading (Carrasco and Batista, 2006a; Carrasco et al., 2004).  This results in optimal BCR signalling and subsequent differentiation of B cells into plasma cells and memory B cells (Batista et al., 2001).   1.4.1.5 BCR-mediated signalling When the unique antigen binding sites of the BCR bind foreign antigen the result is the activation of signalling cascades and the upregulation of genes that are associated with B cell activation (Pierce, 2002).  The crosslinking of the mature BCR results in the activation of three distinct signalling pathways mediated by PI3 kinase (PI3K), Ras/MAP kinase and PLC-?/Rap1 (Gold, 2002; Gold et al., 2000). Upon BCR aggregation and stimulation, the BCR is thought to be recruited into membrane microdomains, or lipid rafts, which act as platforms for BCR signalling (Cheng et al., 1999; Pierce, 2002; Saeki et al., 2003).  Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids (Fridriksson et al., 1999).  Within  55  these lipid rafts are ?raft-associated? proteins such as the src-family kinase Lyn and raftlin.  Here, lipid rafts act as a ??sorting mechanism?? separating proteins involved in BCR activation (i.e. Lyn) from those that are involved in inactivation (i.e. the tyrosine phosphatase CD45) or in raft maintenance (i.e. raftlin) (Cheng et al., 1999; Niiro and Clark, 2003; Saeki et al., 2003).    The binding of antigen to the BCR causes an increase in the tyrosine phosphorylation within the B cell (Gold et al., 1990; Gold et al., 1991).  This tyrosine phosphorylation occurs on the ITAMs of the Ig? and ? chains as well as to tyrosines on other downstream signalling molecules (Gold et al., 1991). The antigen bound BCR moves into the lipid raft where the ITAMs of the Ig? and ? are phosphorylated by the src-family kinase Lyn (Cheng et al., 1999) which is highly enriched within raft microdomains.  Lyn contains myristoylated and palmitoylated amino acid residues which anchors the protein within the lipid raft (Casey, 1995; Pierce, 2002).   The phosphorylated ITAM motifs of Ig?/? can then act as recruiting sites for other signalling molecules containing SH2 domains, thus perpetuating signalling through the BCR.  One of the downstream signalling pathways initiated after Ig?/? phosphorylation is the PI3K pathway. PI3K is a lipid kinase who?s substrate is the phospholipid phosphatidylinositol biphosphate (PI-4,5-P2 or PIP2).  PI3K phosphorylates the PIP2, converting it into phosphatidylinositol triphosphate (PI-3,4,5-P3 or PIP3), which is the substrate for plekstrin homology (PH) domain containing proteins within the cytoplasm, allowing for their recruitment to the plasma membrane (Gold et al., 2000). PIP3 can then be converted into phosphatidylinositol biphosphate (PI-3,4-P2 or *PIP2) by the action of the lipid phosphatase SHIP, which is also a substrate for PH domain containing proteins (Gold et al.,  56  2000).  Different PH-domain containing proteins have different affinities for PIP3 and *PIP2.  For example, the PH domains of some signalling proteins (for example Btk) have higher affinity for PIP3 than for *PIP2, while the other proteins (e.g. Akt) have PH domains with a higher affinity for *PIP2 (Gold et al., 2000).  This allows for the finely tuned regulation of protein recruitment to the plasma membrane.   The crosslinking of the BCR also causes the recruitment of PI3K to the plasma membrane via through its interaction with CD19 and BCAP (Tuveson et al., 1993).  After the initiation of BCR signalling, Lyn can phosphorylate the transmembrane protein CD19, which acts as a recruitment site for PI3K as well as Vav and Lyn (Kurosaki et al., 2010).  BCAP, an adaptor protein, also becomes phosporylated after BCR signalling and interacts with the p85 regulatory subunit of PI3K where it can co-operatively recruit PI3K to CD19 (Inabe et al., 2002; Okada et al., 2000).    There are many components of different signalling pathways that require PH domain-mediated membrane recruitment for activation, therefore PI3K is likely to be important in many pathways (Gold et al., 2000). Accordingly, mice with a targeted gene disruption of the PI3K regulatory subunit p85 exhibit impaired B cell development, reduced numbers of mature B cells, and reduced B cell proliferative responses (Suzuki et al., 1999).  The tyrosine phosphorylated ITAMs of the BCR serves as docking sites for the tyrosine kinase Syk (Kurosaki et al., 1995).  Syk binds to the two phosphorylated tyrosines on the Ig?/? through its dual SH2 domains and is subsequently phosphorylated by Lyn (Kurosaki et al., 1994; Rowley et al., 1995b).  The ITAMs of Ig? are not the only phosphorylated tyrosines on the intracellular domain, but non-ITAM tyrosines at positions 176 and 204 also play a role in the activation of BCR signalling (Engels et al., 2001).  The adaptor protein  57  BLNK (also known as SLP-65) is hypothesized to bind to the non-ITAM phospho-tyrosine at position 204 and then become phosphorylated by Syk (Engels et al., 2001).  BLNK can then bind and recruit other intracellular signalling molecules allowing for their activation (Engels et al., 2001).  BLNK is thought to link activated Syk to PLC? activation and allow PLC? to break down phosphoinositol di-phosphate (PIP2) into the inositol tri-phosphate (IP3) and diacylglycerol (DAG), allowing for calcium flux within the cell (Ishiai et al., 1999).  The products of the PIP2 breakdown can cause the activation of calcineurin and the activation of the transcription factor NF-AT, which then translocates into the nucleus (Gold, 2000).  The increased intracellular calcium, along with DAG, can also cause the activation of the kinase PKC, which can activate the downstream transcription factor NF-?? (Krappmann et al., 2001). Translocation of NF-?B and NFAT into the nucleus results in activation of genes involved in cell proliferation, survival, and differentiation.  As well as being important in calcium flux, DAG can activate guanine nucleotide exchange factor (GEF) proteins such as RasGRP2 (CalDAG-GEF1), which is a key regulator of the Rap GTPases (Gold, 2002).  Activated Syk also regulates the mitogen activated protein (MAP) Kinase pathway (Gold et al., 2000; Hashimoto et al., 1998; Richards et al., 1996).  Syk and PLC-? can activate the GTPase Ras through regulation of the GEF Son of Sevenless (SOS) and through activation of PKC?. SOS and PKC? recruit RasGDP to the plasma membrane, where it is subsequently activated (Kurosaki et al., 2010).  Activated Ras then binds to the MAP3K Raf-1, which subsequently activates MEK1/2, in turn activating ERK1/2 (Gold et al., 2000; Kurosaki et al., 2010).  Activation of ERK results in its dimerization and translocation into the nucleus where it transcriptionally regulates proteins such as Fos, Jun and Ets and the regulation of growth and differentiation (Dong et al., 2002).  58  1.4.1.6 BCR-mediated spreading and immune synapse formation Classically, the signalling events that occur downstream of BCR crosslinking have been mapped out utilizing biochemical BCR crosslinking experiments using soluble anti-BCR antibodies, inducing signalling independent of rearrangement of the cytoskeleton.  Though classical signalling experiments have been invaluable for studying the components downstream of BCR signalling, it is now appreciated that the majority of B cell activation in vivo takes place after recognition of membrane-restricted antigen (Depoil et al., 2008; Fleire et al., 2006).  It is now apparent that the recognition of physically restrained antigen involves the formation of BCR microclusters, the re-organization of the cytoskeleton and alterations in B cell morphology resulting in an immune synapse (Harwood and Batista, 2009).   The first observable event that occurs after BCR recognition of membrane tethered antigen is clustering of the BCR into microclusters, which act as small signalling platforms propagating BCR signals (Figure 1-16) (Harwood and Batista, 2009).  BCR microclusters contain between 50-500 individual BCR complexes and can consist of IgM alone, IgD alone or both (Depoil et al., 2008).  BCR signalling defective cells are still capable of forming these microclusters, therefore the question remained what is driving the clustering of the BCR (Depoil et al., 2008).  Recently it has been shown that BCR-signalling leads to a localized decoupling of the F-actin cytoskeleton from the plasma membrane through phosphorylation of ezrin and a disassembly of F-actin filaments  (Treanor et al., 2010).  This is hypothesized to allow BCR?s that are seggregated within F-actin-poor regions of the plasma membrane to aggregate, forming a BCR microcluster (Batista et al., 2010).  A subsequent study identified that a domain within the heavy chain of IgM (C?4) drives  59  clustering of BCRs within this microcluster, which is required for the initiation of signalling (Tolar et al., 2009).    Figure 1-16 Model of B cell microcluster formation, spreading and immune synapse formation Schematic representation of a B cell interaction with a membrane bound antigen as seen from the B cell:antigen-containing membrane interface.  When a B cell comes into contact with a membrane-bound cognate antigen, it initiates BCR microcluster formation (red dots).  These microclusters interact transiently with CD19 (green dots) initiating signals (yellow stars) which drives B cell spreading and the formation of more microclusters.  Following B cell spreading, B cell contraction takes place where the BCR is concentrated into a central aggregate.  This central aggregate is the site of antigen internalization.  Reprinted with permission from  (Harwood and Batista, 2010).  After the initial BCR microcluster event, signalling is initiated from these clusters.  It was observed that these clusters were rich in phosphotyrosine (pTyr) and accumulate Syk- 60  GFP, both markers of BCR signalling, as well as lacking the phosphatase CD45 (Depoil et al., 2008).  The primary function of these initial microclusters is thought to initiate membrane spreading across the antigen-containing surface in order to gather more antigen and form more microclusters in order to pass the threshold for B cell activation (Depoil et al., 2008; Harwood and Batista, 2009).   This spreading is initiated only after encountering high-affinity antigen and is dependent on BCR-mediated signals which drive actin polymerization (Fleire et al., 2006).  Using the chicken DT40 B cell knockout cell system, it was established that both Lyn and Syk are required to initiate B cell spreading and that PLC?2, the GEF Vav, BLNK and Btk are required to propagate the spreading response (Weber et al., 2008).  The action of the Rap1 GTPase was also shown to be required for a BCR-mediated spreading response (Lin et al., 2008).  Here, inactivation of Rap1 by expression of a dominant negative GAP resulted in the inability to initiate spreading as well as accumulate antigen (Lin et al., 2008).  Along with these downstream signalling proteins, CD19, a member of the B cell co-receptor complex, has also been shown to be essential for propagating spreading (Depoil et al., 2008).  Here, Depoil et al report that CD19 transiently interacts with microclusters and enhances the BCR signalling required for a spreading response (Depoil et al., 2008).   Along with the initiation of B cell spreading, integrin-mediated B cell adhesion to the antigen presenting cell (APC) is of critical importance to B cell activation.  The binding of leukocyte function-associated molecule-1 (LFA-1, CD11a/CD18, ?L?2) and very late antigen-4 (VLA-4, ?4?1) on B cells to intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1), respectively, on APCs is hypothesized to reduce the threshold for B cell activation by antigen-containing lipid bilayers.  This reduction in  61  threshold is mediated by increasing the duration of attachment and promoting cell spreading (Carrasco and Batista, 2006a; Carrasco et al., 2004).   Following spreading on a lipid bilayer, B cells undergo membrane contraction and formation of an immune synapse (Fleire et al., 2006).  The mature B cell immune synapse is characterized by the segregation of the BCR in a central aggregate, the central supermolecular activation complex (cSMAC) and the adhesion molecules LFA-1 and VLA-4 in a peripheral SMAC (pSMAC) (Harwood and Batista, 2009).  Though the formation of an immune synapse is seen in CD4 and CD8 positive T cells, B cells and natural killer cells, the precise function of it is unknown (Harwood and Batista, 2009).  In the case of B cells, it is thought that the cSMAC represents an area of signal termination and internalization, similar to that of T cells (Harwood and Batista, 2009; Varma et al., 2006).  There are, however, differences between the two immune synapses.  In B cells, CD45 is not concentrated in the cSMAC as in T cells, but appears more evenly distributed, which may change the location of signal attenuation.  The organization of the immune synapse into a cSMAC and pSMAC in a ?bullseye? configuration may also be an artifact of the in vitro lipid bilayer conditions used.  In the classical lipid bilayer setup, both the antigen and integrin ligands are infinitely mobile, which may not be the case as seen with an APC (Figure 1-17A and B).  In fact, when T cells are incubated on a membrane-tethered antigen bearing lipid bilayer which physically restricts the movement of the proteins, a ?multi-focal immune synapse? is observed (Figure 1-17B) (Dustin et al., 2006).  This multifocal immune synapse reflects what is seen between a T cell and primary dendritic cell, where it is thought that the DC cytoskeleton is limiting the mobility of integrin ligands leading to sustained T cell activation (Dustin et al., 2006).     62   Figure 1-17 Immune synapse patterns in T cell model systems A) Image of a T cell:dendritic cell immune synapse formed where green is the T cell receptor (TCR) and green is PKC?, a signalling marker for CD28.  This immune synapse is known as a multifocal immune synapse.  B) A T cell immune synapse formed on a planar lipid bilayer with TCR in green and ICAM-1 is labeled red. A well formed, classical ?bullseye? cSMAC (green) and pSMAC (red) is formed.  Here, the integrin ligand and membrane bound antigen embedded in the lipid bilayer are infinitely mobile. This resembles what is seen in B cells.  C) A T cell immune synapse formed on a planar lipid bilayer where chrome lines (white dashed lines) restrict the mobility of the integrin and antigen on the lipid bilayer. This resembles the immune synapses seen between T cells and DC?s.  Reprinted with permission from  (Dustin et al., 2006).  The evidence for the formation of an immune synapse between an immune cell and an APC in vivo has come from fixed samples, however due to the dynamic nature of immune synapse formation and the apparent variety in their appearance, their actual formation is difficult to assess (Barcia et al., 2006; Friedman et al., 2010).  Recently, the first convincing evidence for their formation in vivo between live T cells and antigen primed DCs within a lymph node was observed (Friedman et al., 2010).  Here, using a GFP-tagged TCR and two-photon microscopy, T cells were observed interacting with the antigen-primed DCs, slowing down and internalizing their TCR after contact, all hallmarks of activation.  However, the accumulation of TCR between the T cell and DC was a rare occurrence and was not required for TCR internalization (Friedman et al., 2010).  What this study nicely highlights is that  63  there are differences between immune synapse formation in vivo and in vitro, and with advances in two-photon microscopy it may soon be possible to observe the actual organization of immune synapses within live animals.  The end result of B cell interaction with an APC presenting cognate antigen and subsequent activation is that  they acquire, internalize and process the antigen for presentation to T cells (Batista et al., 2001; Cyster, 2010).  The activation of B cells results in their upregulation of the chemokine receptor CCR7, which follows a CCL19/21 gradient to the T cell:B cell boundary.  Here the B cell acts as an APC, presenting the processed antigen for recognition by T cells.  Movement to this boundary maximizes the possibility for interaction with a cognate T cell, the end result being development of the B cell into an antibody-secreting plasma cell and an acquired immune response (Cyster, 2010).   1.5 The gap junction protein families The need to synchronize cellular processes and spread signals across large populations of cells is common to all multicellular organisms.  This can be accomplished by signals generated through ligand/receptor pairs as seen in BCR signalling, or by the efflux of extracellular and cytoslolic molecules through tetraspan integral membrane protein channels between cells (gap junctions, abbreviated GJ) or between cells and the environment (hemichannels) (Scemes et al., 2009).  There are three families of tetraspan, gap junction-forming proteins: Innexins which represent invertebrate gap junctions, Connexins which represent chordate GJs and the newly identified chordate Pannexins, (Figure 1-18) the vertebrate analog of the Innexins (Phelan, 2005; Scemes et al., 2009).  64   Figure 1-18 Schematic representation of the gap junction families Innexins, Pannexins and Connexins A) Schematic representation of the tetraspan structure of innexins, pannexins and connexins.  The dots represent cysteine residues, the grey lines represent the plasma membrane.  Pannexins contain a glycosylation site (branched red line) on the second extracellular loop.  B) Schematic representation of the potential functions of innexins, pannexins and connexins.  Invertebrate innexins (top) can act as both a gap junction (between two cells) and a hemichannel (between the cell and environment).  Vertebrate pannexins (bottom) function only as a hemichannel. Vertebrate connexins (bottom) function as both a hemichannel and gap junction.  Reprinted with permission from  (Scemes et al., 2009)   The innexin family of gap junction forming proteins were originally identified as the structural proteins of gap junctions in Drosophila and C. elegans. Currently there are 8 inx genes found in Drosophila and 25 in C. elegans which are mostly involved in electrical coupling in nervous system (eg, shank, unc-7); however others,  eg. ogre, are required for post-embryonic development of the larvae (Phelan, 2005).  Interestingly, innexin genes have also been identified in the genomes of polydnaviruses (where they are identified as viral innexins or vinexins), which are symbiotic proviruses of parasitic Hymenoptera (Kroemer and Webb, 2004).  There have been four vinnexin genes identified but their function remains purely speculative (Kroemer and Webb, 2004).  Though they have no sequence homology to the vertebrate connexins, they share similarity in their structure and topology.  Both their N A) B) 65  and C-terminal domains are cytoplasmic and span the membrane four times, with two extracellular loops containing cysteine residues required to dock to an adjacent innexin hemichannel (Figure 1-18A).  The major difference in this domain is the presence of only two cysteine residues per extracellular loop, where as connexins have three (Phelan, 2005; Scemes et al., 2009).   They, like connexins, can also function as both hemichannels and gap junction channels (Figure 1-18B) (Phelan, 2005).  The connexin family of gap junction proteins are a diverse group found in most tissues in chrodates.  There are approximately 20-21 different connexins expressed, depending on the species, with all sharing a similar membrane topology (Figure 1-18A) (Phelan, 2005).  The differences between the connexin members are due to different sizes of the cytosolic loop and C-terminal domain.  Connexins, like innexins, are required for electrical coupling of cells.  For example, gap junctions are required for the electrical co-ordination of contraction in cardiac cells and coordinating insulin release from pancreatic beta cells (Cao et al., 1997; S?ez et al., 1997).  As well as gap junctions, connexins can also act as hemichannels (Figure 1-18B).  Though this is a controversial topic, there is evidence that they may be functional in retina development through ATP release by retinal pigment epithelial cells acting in a paracrine function to propagate calcium signals (Pearson et al., 2005; Scemes et al., 2009).  Interestingly, a new role for connexins in neuronal development and migration has been indentified, which will be discussed in section 1.6.3 (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  A third family of gap junction forming proteins, pannexins, was recently identified in chrodates in 2003, which appear to be the homologs of invertebrate innexins (Baranova et al., 2004; Bruzzone et al., 2003).  There are three members of this family, pannexin 1-3, and  66  share 25-46% similarity innexins, but have no sequence homology to the vertebrate connexins (MacVicar and Thompson, 2009).  Like innexins, pannexins contain two extracellular cysteine residues in each extracellular loop, however there is also a glycosylation site which is thought to prevent gap junction formation if carbohydrate side chains are present (Figure 1-18A) (MacVicar and Thompson, 2009; Scemes et al., 2009).  Because of this, they are hypothesized to only play a role as a hemichannel (Figure 1-18B).  Pannexins are highly expressed in neurons where it is hypothesized that they may influence synaptic physiology by inhibiting glutamate release through release of ATP via the pannexin pore (MacVicar and Thompson, 2009). Along with a role in neurons, pannexin 1 has also been proposed to be involved in the immune response, by associating with the P2X7 receptor and formation of the inflammosome in macrophages (Kanneganti et al., 2007; Pelegrin and Surprenant, 2006).  Here it is hypothesized that the breakdown products of phagocytosed bacteria within endosomes are transported into the cytoplasm through pannexin-1 channels where they interact with cryopyrin resulting in the activation of caspase 1 and an anti-bacterial response independent of TLR signalling (Kanneganti et al., 2007).  1.6 The gap junction protein connexin43 (Cx43)  Cx43 is the most well studied of the connexins due to its widespread expression in tissues (34) and cell types (46) and is the most predominant connexin expressed in most cell lines including lymphocytes (Laird, 2006; Solan and Lampe, 2009).  Genetic knockout of Cx43 results in mice that die shortly after birth due to heart malformation, making study in mouse models dependent on conditional knockouts or in utero electroporation techniques (Cina et al., 2009; Elias et al., 2007; Reaume et al., 1995).    67  1.6.1 Cx43 structure and tissue distribution Cx43 is a 43 kDa membrane protein that spans the membrane four times.  Its topology includes intracellular amino-terminal and an intracellular carboxyl-terminal domains, two extracellular loops, and a single cytoplasmic loop (Solan and Lampe, 2009).  The two extracellular loops contain three cysteine residues each, which are required for docking of adjacent hemichannels to form a gap junction (Solan and Lampe, 2009).  The carboxyl-terminal tail of Cx43 contains a proline-rich domain, two tyrosines and ten serine residues, the phosphorylation of which regulates Cx43 channel function  (Lampe and Lau, 2004).  Cx43 monomers combine to form hexameric hemichannels (connexons ) within the trans golgi network.  These connexons, once on the cell surface, can associate with connexons on adjacent cells to form GJs that allow the passage of ions and small molecules (generally less than 1 kDa) such as Ca++, ATP and cAMP (Laird, 2006; Musil and Goodenough, 1993).   68   Figure 1-19 Schematic representation of mammalian Cx43 Cysteine residues involved in gap junction formation are represented as blue boxes and the c-terminal proline-rich region is indicated in green.  Phosphorylation sites implicated in gap junction function are indicated as follows: Tyrosines 247 and 268 are represented as yellow boxes, Serines 255, 262, 279, 282 and 368 are represented as red boxes.  1.6.2 Regulation of Cx43 channel functions One of the most studied and well known roles of Cx43 is as a gap junction channel that passes small molecules and ions between adjacent cells (Solan and Lampe, 2009).  The regulation of the Cx43 channel is accomplished by the phosphorylation and dephosphorylation of the C-terminal domain as well as several other factors which include calcium concentrations, intracellular pH and voltage (Herv? et al., 2007).  The phosphorylation of the C-terminal domain of Cx43 domain has been reported to be mediated through the actions of the kinases PKC, MAPK and src (Cottrell et al., 2003; Lampe et al.,  69  2000; Lin et al., 2001; S?ez et al., 1997; Solan and Lampe, 2008). PKC has been shown to phosphorylate Cx43 on serines 262 and 368 which can lead to a reduction in channel conductivity (Lampe et al., 2000; S?ez et al., 1997).  Similarily, MAPKs phosphorylate Cx43 on serines 255, 279 and 282 resulting in the downregulation of channel conductivity due to a reduced open probability (Cottrell et al., 2003; Solan and Lampe, 2008).  The phosphorylation of tyrosines 268 and 247 by src is also important for channel regulation.  Here, src is thought to be recruited to the proline rich domain of the C-terminal tail of Cx43 via its SH3 domain and phosphorylate tyrosine 268.  This provides a potential SH2 domain which recruits tyrosine kinases to phosphorylate tyrosine 247, resulting in channel closure (Lin et al., 2001).   Along with the recruitment of kinases which regulate channel conductivity, Cx43 also recruits adaptor proteins which link Cx43 gap junctions to the cytoskeleton (Herv? et al., 2007).  The junctional protein Zonula occludens-1 (ZO-1) is a 220 kDa cytosolic protein which tethers transmembrane junctional proteins to the actin cytoskeleton (Herv? et al., 2007).  It was reported as interacting with the very C-terminus of Cx43 (Giepmans and Moolenaar, 1998) where it was thought to stabilize and regulate GJ plaque size (Hunter et al., 2005).  Recently it has been shown that ZO-1 also regulates the transition of undocked connexons into gap junctions, thereby regulating the prevalence of hemichannels vs gap junctional intercellular communication (GJIC) (Rhett et al., 2011).  The C-terminal domain of Cx43 has also been shown to interact with the adaptor protein drebrin and co-localize with cortactin (Butkevich et al., 2004; Squecco et al., 2006).  Drebrin is a brain-restricted actin binding protein that belongs to the Drebrin/actin binding protein (abp) family (Asada et al., 1994).  It was shown, using a combination of co-immunoprecipitation and FRET-based  70  analysis to interact with the C-terminal domain of Cx43 where it is thought to stabilize the formation of gap junctions by linking them to the F-actin cytoskeleton (Butkevich et al., 2004).  Cortactin, another actin-binding protein, was also shown to interact with Cx43 using a combination of co-immunoprecipitation and immunofluorescent co-localization, though its function is not known (Squecco et al., 2006).  1.6.3 Emerging roles for Cx43 as a regulator of migration Along with its role in cell-cell communication, Cx43 also regulates glioma and neural cell motility and migration (Bates et al., 2007; Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  Knockdown of Cx43 protein levels using shRNA constructs or by using a neuronal progenitor specific conditional knockout mice results in reduced neuronal migration in the developing cortex of mouse embryos (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  In this context, it is hypothesized that Cx43 is providing adhesive contacts which interact with the cytoskeleton, allowing for proper migration to take place (Elias et al., 2007).  This is not solely restricted to neurons, in a screen to identify genes that are involved in epithelial cell migration, Simpson and colleagues used siRNA knockdown and a high throughput wound healing assay to identify genes involved in epithelial cell migration and adhesion.  When they knocked down Cx43 expression they observed that the cells showed minimal adhesion and erratic migration (Simpson et al., 2008).   Though Cx43 is widely accepted as important for neuronal migration, the role of the C-terminal domain of Cx43 in this process remains debated.   In 2007, Elias et al used in utero cotransfection of Cx43 shRNA  and a C-terminal truncated Cx43 in a developing rat  71  brain to show that the extracellular domain, and not the C-terminal domain of Cx43 was required for proper developing neuronal migration (Elias et al., 2007).  In contrast to this report Bates et al., 2007 showed, using the Cx43-low expressing C6 glioma cell line, that over-expression of a C-terminal tail truncated Cx43 in C6 glioma cells has a dominant negative effect on motility compared to cells where WT Cx43 is over-expressed (Bates et al., 2007).  In agreement with these results, Cima et al., 2009 used in utero electroporation of a C-terminal truncated Cx43 on a neuron-progenitor specific conditional knockout background to show that the C-terminal domain is required for neuronal migration to the cortical plate (Cina et al., 2009).  In 2010,  Elias et al. were able to show, using in utero cotransfection of Cx43 shRNA  and a C-terminal truncated Cx43 into the developing rat brain, that the C-terminal domain of Cx43 was, in fact, required for the transgenital to radial migration switch of developing interneurons (Elias et al., 2010).  Though there is evidence for the C-terminal domain of Cx43 being required for neuronal migration, the molecular mechanism remains unknown.  1.6.4 Cx43 in the immune system Cx43 is expressed in many cells of the immune system including T cells, B cells, NK cells, macrophages and dendritic cells (Eugenin et al., 2003; Krenacs and Rosendaal, 1995; Oviedo-orta et al., 2000).  Although Cx43 is expressed in B cells (Oviedo-orta et al., 2000), its function is not understood.  Cx43 knockout mice die perinatally from hypoxia due to malformation of the heart (Reaume et al., 1995), making assessment of the adult immune system difficult. However, heterozygote Cx43 mice do exhibit a reduction in T and B lymphocyte numbers during development (Montecino-Rodriguez and Dorshkind, 2001).  It is  72  not clear however if the defect is intrinsic to the B cell or due to defects in other cell types that interact with them. The majority of research regarding Cx43 in the immune system has focused on GJIC between different cell types.  Using a gap junction channel permeable dye, in vitro co-cultures of T cells and B cells have been shown to communicate via GJIC, where pharmaceutical blockage of the pore did result in a reduction in cytokine production  (Oviedo-orta et al., 2000).  GJIC has also been shown to occur in an immune synapse model between lymphocytes and APCs.  Within a germinal center, follicular B cells and follicular dendritic cells have been shown to form Cx43 gap junctions and transfer dye between them (Krenacs et al., 1997; Krenacs and Rosendaal, 1995).  T cells have also been shown to form functional gap junctions between themselves and macrophages (Bermudez-Fajardo et al., 2007) and dendritic cells (Elgueta et al., 2009).  Here, primary T cells and bone marrow derived dendritic cells form functional gap junctions as measured by dye transfer.  When the channel is blocked, there is a decrease in T cell activation indicated by a reduction in proliferation, CD69 expression and IL-2 secretion (Elgueta et al., 2009).  Though there is convincing evidence that there is GJIC between lymphocytes and APCs, what is being passed through the channel and how it affects activation is unknown. Recently, GJIC has been implicated in antigenic cross presentation.  The principal mechanism by which the immune system eliminates abnormal cells (tumours and virally infected cells) is by activation of cytotoxic T cells (CD8+ T cells).  However, in order for this activation to occur, APCs need to present the MHC type I bound antigen from the abnormal cells to T cells within secondary lymphoid tissues.  This occurs through the process of cross presentation, which involves the transfer of endogenous antigen from a cell to an APC, which  73  can then present it to T cells for initiation of a T cell response. Though this process is essential, the mechanisms by which it occurs are not clear (Rock, 2003).  One potential mechanism has been identified as through the passage of antigen from an infected/abnormal cell to an APC through Cx43 gap junctions (Mendoza-Naranjo et al., 2007; Neijssen et al., 2005).  Both macrophages and dendritic cells have been shown to receive antigen smaller than 1800 daltons from target cells (melanomas and influenza infected cells respectively) in a gap junction dependent manner (Mendoza-Naranjo et al., 2007; Neijssen et al., 2005).  In the influenza infection model, the gap junction dependent transfer of antigen was sufficient to activate CD8+ T cells as measured by IFN? production (Neijssen et al., 2005).   Along with GJIC between lymphocytes and APCs, the role of GJIC between B cells and macrophages migrating across an endothelial monolayer has been investigated.  Cx43 has been investigated in B cell transendothelial migration, where it has been shown that B cells and endothelial cells form functional gap junctions (Oviedo-Orta et al., 2002).  Using an endothelial monolayer loaded with a GJ permeable dye, B cells accumulated the dye as they passed through the monolayer; however, blocking the GJ pore, severely limiting GJ communication, had no effect on TEM (Oviedo-Orta et al., 2002).  In contrast to this, blockage of GJIC communication in macrophages was shown to reduce their transit across the blood brain barrier (Eugenin et al., 2003).  Though both cell types show GJIC with the endothelial cells, the role that it actually plays is not clear.     74  1.7 Rationale, objectives and aims The overall aim of this thesis is to gain insights into how B lymphocytes are able to regulate their cytoskeleton. Regulation of the cytoskeleton is essential for normal B cell development and function.  As described earlier in this section, B cells develop in the bone marrow, they need to move and interact with different cells in order to receive the growth and differentiation signals to mature.  In order to complete this developmental program, B cells need to exit the bone marrow, enter circulation and home to the spleen.  This process requires that they change their shape in order to migrate through the stromal cell layer and into the bloodstream. They then adhere to the endothelial blood vessels of the spleen, triggering their exit from circulation and entrance into the white pulp.  Within the white pulp, they can encounter antigen presented on the surface of macrophages and dendritic cells.  If they recognize the membrane-bound particulate antigen, they reorganize their cytoskeleton and spread over the cell surface in order to gather antigen and initiate activation signals through the formation of an immune synapse.  This then results in the B cell migration to areas within the spleen where they can interact with T cells to initate an immune response.  Though these changes are mediated by different signals, they all require the reorganization of the cytoskeleton.  The gap junction protein Cx43 has been traditionally studied as a channel that allows the transport of small, soluble material of less than 1 kDa between cells. Recently, a new role has been described for Cx43, as a regulator of neuronal and glial migration, a biological process governed by reorganization of the cytoskeleton.  Similarily, Cx43 has also been shown to interact and recruit proteins that link it to the actin cytoskeleton. These results suggest that Cx43 is required not only for GJIC, but may also play a prominent role in  75  regulation of the cytoskeleton.  Though Cx43 has been shown to be expressed in B cells, its role is currently unknown. There are two major hypotheses in this thesis that were tested: 1) There are domains of the BCR that are important for the reorganization of the cytoskeleton. 2) The gap junction protein Cx43 is an important regulator of B lymphocyte cytoskeletal rearrangements. Aims: 1) To determine which domain of the BCR is important for BCR-mediated cell spreading. 2) To characterize the expression of Cx43 in B lymphocytes and determine if it is important for B cell function (including B cell spreading, adhesion, migration and TEM). 3) To determine if the C-terminal domain of Cx43 is important for the effects determined in Aim 2 above.  * see below. 4) To uncover the potential mechanism by which Cx43 regulates B cell morphology. * - This thesis focuses on the role of the C-terminal domain of Cx43 in the regulation of B cell morphology.  The roles of other domains of Cx43 which may be important to B cell morphology, including the extracellular loops (which contain cysteine residues important for gap junction coupling) and role of the pore are being investigated by other members of the lab and are outside of the scope of this thesis.  76  2 The role of the cytoplasmic domain of Ig? in BCR-mediated cytoskeletal rearrangements 2.1 Synopsis The focus of this thesis has been on the cytoplasmic domains of Ig? and Ig? since both contain ITAM?s that are required to recruit signalling proteins that transmit signals initiated after BCR activation.  Though these cytoplasmic tails are similar in structure, there are sequence differences that result in the recruitment of different signalling molecules.  Analyses of B cells expressing mutated BCRs have shown that there is a differential requirement for Ig? and Ig? in B cell development, BCR internalization and mobility in the membrane; however it is not known if there is a differential requirement for Ig? and Ig? in transmitting signals that lead to BCR-mediated cytoskeletal rearrangement.  Therefore, the aim of this chapter was to focus on the tails of these proteins and determine their importance. We show here using the J558 plasmacytoma transfected cell line as a gain-of-function model system that the cytoplamsic domain of Ig? is required for maximal BCR-mediated signalling and initiation of cytoskeletal rearrangements. Though the model used here provides evidence that the cytoplasmic domains of Ig? and Ig? are both required for maximal BCR-mediated cytoskeletal rearrangements, we noted that a WT BCR was also unable to support the ability of J558 plasmacytoma cells to initate BCR-mediated cell spreading radially, as observed when B lymphoma cells were examined using similar assays.  The subsequent data chapters address this finding as we identify another protein that is not expressed in terminally differentiated B plasmacytoma cell lines including J558 but is present in immature and mature B cells, which is required for BCR-mediated radial cell spreading.  77  2.2 Introduction The B cell antigen receptor (BCR) consists of a signalling subunit (Ig? and Ig?) and an antigen (Ag) binding subunit (mIgM) which are non-covalently associated and expressed on the surface of B lymphocytes (Hombach et al., 1990b). The BCR has two key roles in the activation and development of B lymphocytes and in the immune response: the first is to deliver signals to the B lymphocyte via Ag induced BCR clustering and the second is to internalize Ag, where it is broken down in an endosomal compartment and presented by MHC molecules on the surface of the B cell for recognition by helper T cells (Siemasko and Clark, 2001).   The binding of Ag to the BCR causes an increase in the tyrosine phosphorylation (P-Tyr) within the immunoreceptor tyrosine based activation motifs (ITAMs) of Ig?/? and on the tyrosines of downstream signalling molecules (Campbell et al., 1991; Gold et al., 1990; Gold et al., 1991). The Ag bound BCR can be recruited into membrane domains and the ITAMs of Ig?/? are phosphorylated by the tyrosine kinase Lyn, which is enriched within these domains (Cheng et al., 1999).  The phosphorylated Ig?/? acts as recruitment sites for other signalling molecules containing SH2 domains (Pawson and Nash, 2000).  The recruitment of the signalling complex to the ITAMs of the BCR causes the activation of three main signalling pathways: the PI3 kinase (PI3K) pathway, Ras/MAP kinase pathway and PLC-? pathway (Gold et al., 2000). Crosslinking and activation of the BCR leads to dynamic changes to B cell morphology (Jugloff and Jongstra-Bilen, 1997; Melamed et al., 1991).  When a mature B lymphocyte comes into contact with a membrane-bound Ag it responds by rapidly spreading over the Ag- 78  bearing membrane, followed by a rapid membrane contraction (Fleire et al., 2006).  This cell spreading and subsequent contraction serves to gather as much Ag as possible and then concentrates it into a central aggregate. This spreading phenomenon is both BCR signalling and actin-dependent (Fleire et al., 2006; Weber et al., 2008).   The spreading and subsequent clustering of the BCR into one locus is also seen as the first step in the formation of an immunological synapse.  The immunological synapse is the interface between the B cell and the Ag presenting cell.  It consists of a central aggregate of BCR surrounded by the integrins VLA-4 and LFA and is thought to lower the threshold for BCR activation (Carrasco and Batista, 2006).  This structure is an important site of cell:cell communication in B cells and results in changes in B cell differentiation into an antibody-producing plasma cell and the activation of the acquired immune system.  The Ag is then internalized through a process known as receptor mediated endocytosis for later presentation to T cells (Batista et al., 2001). In this chapter the focus was on the role of the cytoplasmic domain of Ig? in BCR-mediated cytoskeletal rearrangements. Our hypothesis was that there is a differential requirement for the cytoplasmic domains of Ig? and Ig? in BCR mediated cytoskeletal rearrangements, which is in turn mediated by differences in B cell signalling.  When the cytoplasmic domain of Ig? is mutated or swapped with Ig?, there will be either a reduction or enhancement in normal signalling pathways that regulate changes in the B cell cytoskeleton.  This was addressed by the stable expression in the J558 plasmacytoma cell line of a WT BCR, and two mutated BCRs.  The mutated BCRs contained either an Ig? with a cytoplasmic tail of Ig? (C?), resulting in a BCR with two beta tails, or a truncated Ig? (?trunc), resulting in a BCR with only one beta tail.  Reconstitution of a WT or mutated BCR  79  was not sufficient to restore the ability to initate BCR-mediated radial cell spreading; however the cytoplasmic domain of Ig? was required for maximal BCR-mediated production of actin-rich protuberances.  We also showed using an immune synapse assay that a WT BCR is required for maximal polarized response to a surrogate particulate antigen.  These data suggest that a WT BCR is necessary for maximal BCR-mediated cytoskeletal rearrangements in plasmacytoma cell lines, and that the Ig? tail is not sufficient to elicit the same response.  2.3 Methods and materials  2.3.1 Antibodies and reagents Polyclonal goat and rabbit anti-mouse Ig? antibody (Ab) (Ig? chain specific) were from Jackson ImmunoResearch Labs, Inc.; West Grove Pennsylvania. The polyclonal rabbit anti-mouse Ig? light chain Ab was purchased from Bethyl Laboratories (Montgomery, Texas). The polyclonal rabbit anti-mouse Ig? cytoplasmic domain Ab was produced against a 34 amino acid peptide from the Ig? cytoplasmic tail (187-220) was developed by the Matsuuchi Lab (University of British Columbia, Vancouver BC) and was previously described (Gold et al., 1991).  The polyclonal rabbit anti-mouse Ig? extracellular Ab produced against a 29 amino acid peptide from the Ig? extracellular domain, and the polyclonal rabbit anti-mouse Ig? extracellular Ab produced against a 29 amino acid peptide from the the Ig? extracellular domain, were both gifts from Abeome (Athens, Georgia) and Dr. Richard Meagher and Elizabeth McKinney (University of Georgia, Athens, Georgia).  The polyclonal rabbit anti-mouse phosphotyrosine Ab used for intracellular staining and immunofluorescence was purchased from BD Biosciences.  Mouse anti-?-actin Ab was purchased from Sigma/Aldrich;  80  St. Louis, Missouri.  Goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) was purchased from Invitrogen Life Technologies (Burlington, Ontario) and Goat anti-rabbit-HRP was purchased from Jackson ImmunoResearch Labs, Inc (West Grove, Pennsylvania). The fluoroscein isothiocyanate (FITC)-conjugated affinipure goat anti-mouse IgG Ab (Fc fragment specific) was purchased from Jackson ImmunoResearch Lab, Inc.  The Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) Ab was purchased from Invitrogen Life Technologies.  Rhodamine coupled phalloidin, used to detect actin, and ProLong Gold anti-fade reagent were purchased from Invitrogen Life Technologies.  2.3.2 Cells and culture The J558?m3 and J558.15-25 murine plasmacytoma cell lines were obtained from Dr. Louis Justement (University of Alabama, Birmingham, Alabama; (Justement et al., 1990).  The WEHI-231 murine B cell lymphoma cell line has been previously described (Jackson et al., 2005).  Cells were incubated at 37?C and at 5% CO2 and maintained using Roswell Park Memorial Institute (RPMI) 1640 media containing 4.5 g/L glucose, 2 mM L-glutamine, 110 mg/L sodium pyruvate (Invitrogen Life Technologies), 8.5-10% heat inactivated fetal bovine serum (FBS) (Cancera, Ontario, Canada), 50 units/mL of penicillin and streptomycin sulfate (Invitrogen Life Technologies).      2.3.3 Transfections The pWZL-Blast3-C? plasmid encoding Ig? with a cytoplasmic swap with Ig? was made by Janis Dylke and has been previously described (Dylke et al., 2007).  The pWZL- 81  blast3-?trunc plasmid encoding a cytoplasmic trunctation of Ig? was made by Caren Jang and described in (Jang et al., 2010).  J558 cell lines were transfected with the plasmids encoding C? and ?trunc constructs using Nucleofection. Recipient cells were resuspended with 2 ?g of the appropriate DNA construct and solutions from the Amaxa Nucleofection Kit T (Amaxa Biosystems, Gaithersburg, Maryland) according to the manufacturer?s instructions. The electrical parameters from the G-016 program in the Amaxa Nucleofector Device (Amaxa Biosystems) were used. Drug resistant populations were enriched for BCR-expressing cells using fluorescent activated cell sorting (FACS) with the help of the UBC Flow Cytometry Facility. Cells were resuspended in 0.5 ml PBS with 2% FBS and labeled with 2 ?g Alexa Fluor 633-conjugated goat anti-mouse IgM (? chain specific). Sorting was performed on a BD FACS Aria (BD Biosciences) by Jeffrey Duenas, UBC FACS Facility (University of British Columbia, Vancouver, BC). The Alexa Fluor 633 positive collected cells were then grown in culture.  2.3.4 Western blotting Cells were lysed as described with 0.5-1 ml of cold Triton X-100 lysis buffer (20 mM Tris-HCL pH 8.0, 137 mM NaCl, 1% Triton X-100, 2 mM EDTA, 10% glycerol) and with the addition of protease inhibitors (10 ?g/mL leupeptin, 1 ?g/ml aprotinin, 1 mM pepstatin A, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF) for 20 min on ice (Jackson et al., 2005).  The cells were then centrifugated at 1500 rpm for 10 min at 4?C, supernatants collected and the protein concentration of each sample was calculated using a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnologies, Rockford, Illinois). Lysates were mixed with an appropriate amount of sodium dodecylsulfate (SDS) polyacrylamide gel  82  electrophoresis (SDS-PAGE) reducing sample buffer (62.5 mM Tris-HCl pH 6.8, 4% glycerol, 2.5% SDS, 0.02% bromophenol blue, 100 mM dithiothreitol) and boiled for 5 min and stored at -20?C. The samples were then loaded onto an SDS-PAGE mini-gel and analyzed by Western immunoblotting using standard procedures (Burnette, 1981).    2.3.5 Scanning electron microscopy Samples were prepared for scanning electron microscopy using standard procedures at the UBC Bioimaging Facility.  Cells were placed in 4% paraformaldehyde at room temperature for 20 min, decanted and washed twice with warm wash buffer in a 24 well plate.  The samples were then placed in a 28oC, pH 7.4, 0.1 M, 4 % formaldehyde/ 2.5 % glutaraldehyde solution and fixed in laboratory microwave (Pelco, Redding California, USA) under vacuum for two cycles (2 min each,100 W/off/100 W) without changing fixative.  Samples were washed three times with 0.1 M pH7.4 sodium cacodylate.  Samples were then post-fixed with buffered 1% OsO4; same as the primary fixation.  Samples washed three times with ddH2O and coverslips transferred to a stacked coverslip holder, keeping the samples wet.  Samples were then microwave dehydrated at 28oC through a graded ethanol series (30%, 50%, 70%, 80%, 90%, 95%, and three times at 100%) at atmospheric pressure for 40 s, 270 W each step or at RT for 10 min then critically point dried (Tousimis AutoSamDri 815B) or chemically dried with hexamethyldisilazane (HMDS). Samples were mounted to aluminum SEM stubs with double sided tape and sputter coated (Cressington HR208) with 8 nm gold.  Images were acquired on a Hitachi S4700 FESEM (Field Emission Scanning Electron microscope) (Lin et al., 2008).   83  2.3.6 B cell spreading assay and actin-rich membrane projection quantification Cell spreading assays were performed as described (Lin et al., 2008; Santos-Argumedo, 1997) and graphically represented in Figure 2-1.  Briefly, 12 mm glass coverslips were coated with 40 ?g/ml of goat anti-mouse IgM for 2 hours at 37?C.  J558 plasmacytoma cell lines and WEHI-231 B lymphoma cells were incubated on the coverslips for various amounts of time at 37?C, and then fixed with 4% paraformaldehyde for 20 min at room temperature (RT).   The cells were then permeabilized in PBS containing 0.5% Triton-X 100.  The cells were blocked in PBS containing 3% bovine serum albumin (BSA) for 30 min at RT then incubated with rhodamine (1:40 in PBS plus 3% BSA) for 20 min at RT, rinsed three times in PBS and then mounted onto a slide using ProLong gold anti-fade reagent and sealed using clear acrylic nail polish. Cell contact sites of the cell:coverslip interface were imaged using an Olympus Fluoview1000.  Membrane projections were counted manually from compiled Z-Stacks of 0.5?m step size of 5-50 cells per time point taken using a 60X objective using Olympus FV-Viewer, repeated three times.  The Olympus Fluoview1000 confocal microscope and imaging software used in these procedures and analyses were part of the Life Sciences Institute Imaging Facility at the University of British Columbia.   84   Figure 2-1 Overview of B cell spreading assay from section 2.3.6  2.3.7 Particulate antigen bead assay Particulate antigen bead assay with J558 cell lines was performed as described (Lin et al., 2008; Vidard et al., 1996) and graphically represented in Figure 2-2.  Breifly, 4.5 ?m polystyrene beads (Polyscience, Warrington, PA, USA) were mixed with 40 ?g/ml Goat anti-mouse IgM in 250 ?l PBS for 1 hour at RT.  They were then blocked with 2% BSA for 30 min at RT and re-suspended in quinsaline (25mM sodium Hepes pH 7.4, 125 mMNaCl, 5mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM MgSO4, 1 g/L glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 ?M 2-?ME).   Beads were then added to 50,000-100,000 J558 B cells at a 5:1 ratio (beads:cells) in quinsaline for the required time points in a 37?C water bath.  At the indicated time points, the cells were then fixed in a final concentration of 4% paraformaldehyde for 10 min.  The  85  paraformaldehyde was then quenched in PBS plus 10% BSA and the cells then centrifugated at 1000 rpm for 4 min.   The supernatant was removed and the cells were gently washed three times in PBS containing 3% BSA.  The cells were permeabilized in PBS containing 0.5% Triton X-100 for 10 min.  The supernatant was removed and 450 ?l of a polyacrylamide gel stock solution (7.4 ml dH2O, 1.2 ml 40% acrylamide/BiS (BioRad, Mississauga, ON), 1.2 ml 1.0 M Tris HCL pH 8.0, 100 ?l APS (BioRad, Mississauga, Ontario)) was added to each sample and was subsequently transferred to a 24 well plate (BD Biosciences, Palo Alto, CA).  The cells were allowed to settle to the bottom of the dish for 15 min, when 1 ?l of TEMED (BioRad) was added and the gel was allowed to polymerize for 10 min.  The circular polymerized gel was removed, inverted (cell side up) and placed in a 12 well dish (BD Biosciences) and subsequently blocked in PBS containing 3% BSA for 40 min at RT.  The cells were incubated with 1:500 dilution of anti-phosphotyrosine in PBS containing 3% BSA overnight at 4?C.  The polymerized gel plugs were washed 5 times in PBS containing 3% BSA for five minutes each.  The polymerized gel plugs were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) Ab and Rhodamine Phalloidin for 3 hours at RT.  The polymerized gel plugs were washed five times for five minutes each in cold PBS.  The polymerized gel plugs were then mounted using ProLong Gold anti-fade reagent (Invitrogen) and imaged using an Olympus Fluoview FV1000 confocal microscope.      86   Figure 2-2  Overview of particulate antigen bead assay described in Section 2.3.7  2.3.8 BCR surface expression J558 cells were chilled on ice and incubated with Rabbit anti- Mouse Ig?.  Cells were then fixed with 8% paraformaldehyde and surface BCR was labeled using the secondary antibody FITC-conjugated Goat?Rabbit-IgG.  The FITC positive cells were then sorted and the mean fluorescent intensity (MFI) was calculated using FlowJo 7.2.2 (Tree Star Inc., Ashland, Ore).  BCR positive cells were defined by having FITC fluorescence that was higher than the BCR negative cell line J55815-25.  The average MFI values were then expressed as a percentage of the J558 positive control ?M3.    87  2.4 Results 2.4.1 Expression of the mutant C? and ?trunc BCRs in the J558 B cells In order to address the relative contribution of the cytoplasmic domain of Ig? to BCR-mediated cytoskeletal rearrangements, the J558 plasmacytoma cell line was utilized as a model.  The J558 plasmacytoma has been used previously to assay both the biochemical and structural requirements for BCR signalling (Flaswinkel and Reth, 1994; Tolar et al., 2009).  Here, J558 cells that lack Ig? expression but express the rest of the BCR components were transfected with one of two Ig? mutants.  C? has the cytoplasmic domain of Ig? swapped with Ig? and ?trunc has a cytoplasmic deletion of Ig? (Figure 2-3).    Figure 2-3 Schematic representation of the WT BCR, and the mutant BCRs C? and ?trunc The reconstituted BCR expressed in the J558 cell line consists of an Ig? (red), Ig? (blue), Ig? (green) and Ig? (orange).  The mutant BCR C? consists of WT Ig?, Ig? and Ig?, but a chimeric Ig? with the transmembrane and extracellular domains of Ig? and the cytoplasmic domain of Ig?.  The mutant BCR ?trunc consists of a WT Ig?, Ig? and Ig?, but a cytoplasmic truncated Ig?. Reproduced/adapted with permission from (Machtaler et al., 2011).   88  The expression of the mutant Ig? constructs C? and ?trunc were assessed using standard SDS-PAGE and Western blot analysis.   J558 15-25 cells were previously transfected with Ig? heavy chain, Ig? light chain and express endognenous Ig?, but do not express Ig? (Figure 2-4A).  Expression of the Ig? mutant C? was detected using an Ab against the N-terminus of Ig-? (anti-Ig? N-terminal Ab) and the mutated protein appeared at a molecular weight of 25 kDa (Figure 2-4A).  To ensure that this was not WT Ig?, it was reprobed with a C-terminal specific anti-Ig? antibody.  Because the Ig? mutant C? has a cytoplasmic Ig? domain, it was not detected (Figure 2-4).  Similar results were obtained for ?trunc expression. The Ig? mutant ?trunc was expressed in J558 15-25 (Figure 2-4 B) and ?trunc was detected at 20 kDa using a N-terminal specific anti-Ig? antibody.  Verification that this was in fact a C-terminal truncation was assessed by reprobing with an Ab that was specific for the cytoplasmic tail of Ig?.  Because the Ig? mutant ?trunc lacks a C-terminal doman, there was no detectable band observed (Figure 2-4).  To assess whether the mutant BCRs were able to traffic to the cell surface at similar levels as the WT BCR, cell surface expression was assessed using FACS.   The mutant BCRs expressed similar cell surface levels as the WT BCR (Figure 2-4D).  These data show that successful transfection and cell surface expression of the Ig? mutants C? and ?trunc into the J558 15-25 cell line was achieved, which allowed for the assay of BCR function.        89    Figure 2-4 C? and ?trunc are expressed in J558 15-25 B cells.  A and B) Expression of BCR components in J558 cell lines. Whole cell lysates of various J558 transfected cell lines were separated on a 12% SDS-PAGE polyacrylamide gels and  90  visualized using standard Western blotting procedures. The cytoplasmic tail of Ig? was detected using the anti-mb-1 Ab (specific for the cytoplasmic domain of mouse Ig?) followed by Protein A horse radish peroxidase (PAHRP). Ig? was detected using the anti-Ig? Ab and PAHRP. Ig? was detected using the anti-Ig? Ab and PAHRP. The extracellular domain of Ig? was detected using Abeome 689 rabbit anti-Ig? and a Goat ?Rabbit-HRP secondary Ab.  The extracellular domain of Ig? was detected using Abeome 624 rabbit anti-Ig? and a Goat ?Rabbit-HRP secondary Ab. Versions of A and B were published in (Jang et al., 2010).  C) Legend listing the BCR polypeptides expressed in the various J558 cell lines.  D) BCR expression on the cell surface in J558 cell lines as analyzed by FACS using a fluorescent anti-Ig? Ab.  MFI = mean fluorescence intensity. A and B were reprinted with permission from (Jang et al., 2010).  2.4.2 The Ig? mutants C? and ?trunc are signalling competent As previously described, BCR crosslinking results in phosphorylation of the ITAMs of Ig? and Ig?, which in turn recruit signalling molecules and initiate B cell activation.  Tyrosine phosphorylation (P-Tyr) was thus used as a marker of B cell activation. To assess whether the BCRs containing mutated Ig??? signalling components of the receptor were signalling competent, WT J558?m3 and J558C? and ?trunc were incubated on anti-IgM coated glass coverslips and P-Tyr was visualized using confocal microscopy.  WT BCR expressing J558?m3 cells exhibited a robust P-Tyr response that was localized to the cell:coverslip interface (Figure 2-5A, Top panel).  Swapping the cytoplasmic domain of Ig? with Ig? (C?) resulted in an apparent reduced localized P-Tyr response (Figure 2-5A, middle panel) and there was little to no P-Tyr visible when the cytoplamsic domain of Ig? (?trunc) was deleted (Figure 2-5A, bottom panel).  These results were verified by quantitative FACS analysis after activation of the BCRs with anti-IgM Ab in solution.  WT J558?m3 B cells respond to soluble anti-IgM activation with a robust P-Tyr increase by 10 min (Figure 2-5B).  This response was reduced when the cytoplasmic domain of Ig? was swapped with Ig? (C?) (Figure 2-5B) and there was little to no P-Tyr detectable when the cytoplamsic domain of Ig?  91  (?trunc) was deleted (Figure 2-5B).  These results confirm that a WT BCR is required to initiate maximal BCR-mediated signalling.       92    93  Figure 2-5 BCR signalling competency of J558 ?m3, C? and ?trunc cell lines A) 3D reconstructions of J558?m3, J558C? and J558?trunc cells incubated on anti-IgM coated coverslips to activate the BCR for 0, 5 and 30 min. These images show the location of phosphotyrosine (green: Anti-Ptyr/Alexa 488 Goat anti-rabbit IgG), the F-actin cytoskeleton (red: Rhodamine phalloidin) and the nucleus (blue: DAPI). B) Tyrosine phosphorylation after stimulation with soluble anti-IgM BCR. Cells were stimulated at 37?C for the indicated times with anti-IgM followed by paraformaldehyde fixation and intracellular staining for phosphotyrosine. Data were collected by flow cytometry. A and B are reproduced/adapted with permission from (Machtaler et al., 2011) and (Jang et al., 2010) respectively.   2.4.3 J558 plasmacytoma cells with a reconstituted BCR were able to make signalling dependent membrane protuberances, but were not able to spread radially. Normally, when B cells encounter a membrane bound Ag or an immobilized Ab specific for its BCR that acts as a surrogate Ag, the cell responds by spreading its membrane across the Ag-bearing surface (Fleire et al., 2006; Lin et al., 2008). To assess the role of the cytoplasmic domain of Ig? in BCR-mediated cytoskeletal rearrangements, BCR-mediated spreading was modeled using an anti-BCR (anti-IgM) coated coverslip.  This assay has been previously used to assess BCR-mediated spreading (Lin et al., 2008).  BCR-expressing J558?m3 cells were incubated on anti-IgM coated coverslips for 30 min and spreading was assessed using both confocal and scanning electron microscopy.  WT BCR-expressing J558?m3 cells incubated on anti-BCR coated coverslips for 30 minutes were unable to initiate a radial spreading response (Figure 2-6A) compared to the immature B cell lymphoma cell line WEHI231 (Figure 2-6B).  They were, however, able to extend small protrusions after 30 min incubation on anti-BCR coated surfaces (Figure 2-6A, black arrows).  This was observed using scanning electron microscopy (SEM) (Figure 2-6A top pannels) and by immunofluorescence showing a Z-series of collected stacks of confocal images of cells stained with rhodamine phalloidin to show the actin cytoskeleton (Figure 2-6A bottom pannels).  Similar to WT BCR expressing J558?m3 cells, J558C? and ?trunc  94  cells also did not spread on anti-BCR coated coverslips (Figure 2-6C). These data indicate that although J558 cells with a reconstituted WT BCR are able to initiate normal signalling responses like those reported in the literature (Flaswinkel and Reth, 1994; Tolar et al., 2005) and form BCR-mediated membrane protrusions, they are unable to intitiate radial membrane spreading like that seen in WEHI 231 cells.  Expression of mutated BCRs in the J558 cell system, which result in even less effective signalling, show even poorer formation of cell protrusions after BCR signalling is initiated.         95    96  Figure 2-6 BCR-mediated cell spreading of J558 and WEHI 231 B cells on anti-BCR (anti-IgM) coated coverslips.  A) Scanning electron micrographs (SEM; top panels) and 3D confocal reconstructions (bottom panels) of J558?m3 cells incubated on anti-IgM coated glass coverslips for 0 and 30 min.  Black arrows on the SEM images indicate the membrane projections (Scale bar: 10 ?m).  White arrows on the confocal images point to the regions where the protuberences/projections are extending.  Red = F-actin detected by rhodamine phalloidin.  (Scale bar: 10 ?m).  B) SEM of WEHI 231 cells plated and spreading for 0 or 30 min on anti-IgM coated coverslips (Scale bar: 10 ?m). C) Single Z plane slices of the cell:coverslip contact area of J558?m3 and WEHI 231 cells incubated on anti-IgM coated coverslips for 30 min (Red = F-actin) (Scale bar: 10 ?m).  Reproduced/adapted with permission from (Machtaler et al., 2011)    2.4.4 The cytoplasmic domain of Ig? is required for maximal formation of BCR-mediated membrane protrusions Although J558 B cells with a reconstituted BCR are unable to initiate B cell spreading, they were able to form membrane protrusions in response to BCR signalling.  In order to determine if the C-terminal domain of Ig? was required for the formation of these actin-rich membrane protrusions, J558C? and J558?trunc B cells were incubated on anti-BCR (anti-IgM) coated coverslips and the formation of membrane protrusions was assayed using confocal microscopy.  BCRs containing the Ig? mutant C?, showed a reduced number of membrane projections when incubated on the anti-BCR coverslip over a time course of 60 minutes compared to WT BCR J558?m3 (Figure 2-7A, quantified in B; white arrows are pointing to the protrusions).  There was a further reduction in the number membrane projections when BCRs containing the Ig? mutant ?trunc were incubated on the anti-BCR coverslip (Figure 2-7A, quantified in B).  This is a mutant where the Ig? tail was removed, leaving a BCR with only a single Ig-? tail.  These results indicate that the optimal initiation of BCR-mediated cytoskeletal rearrangements requires both the cytoplasmic domains of Ig?  97  and Ig?, and that the cytoplasmic domains are not equivalent with respect to their ability to initiate rearrangement of the cytoskeleton.     98    99   Figure 2-7 The cytoplasmic domain of Ig? is required for optimal BCR cytoskeletal rearrangements in J558 cells A) Compiled confocal Z-stacks of the F-actin cytoskeleton (red:  rhodamine phalloidin. Nuclei\blue: DAPI) of J558?trunc, J558C? and J558?m3 cells incubated on anti-IgM coated glass coverslips for the indicated time course from 0 to 60 min.  Arrows highlight the membrane projections. (Scale bar: 10 ?m). B) Quantification of the number of membrane projections on J558?trunc, J558C? and J558?m3 cells (mean of triplicate experiments, error bars indicate standard error, * indicates that p < 0.05, brackets indicate the comparisons being made). Quantification as described in the Materials and Methods. Reproduced/adapted with permission from (Machtaler et al., 2011)  2.4.5 The cytoplasmic domain of Ig? is required for an optimal response to a particulate antigen As previously described, B cells within secondary lymphoid tissues are rapidly activated by binding to particulate antigen.  In order to address whether the cytoplasmic domain of Ig? was necessary for responding to a particulate antigen, 4.5 ?m latex beads were  100  coated with an anti-IgM antibody, mimicking a particulate antigen, and this was used to activate the BCR on the various transfected J558 cell lines. This assay, similar to the spreading assay, uses an immobilized antibody as the BCR crosslinker, however the difference is that the bead assay concentrates the antibody into a small locus, assaying the B cells ability to polarize to the site of antigen contact and mimicking the biological response to a particulate antigen as well as being similar to an early immune synapse (Lin et al., 2008).  This assay also allows for the visualization of activation markers (P-Tyr) to the site of BCR crosslinking.  When incubated with the anti-IgM coated beads, J558?m3 cells respond by accumulating F-actin at the sites adjacent to the cell:bead contact site and accumulate P-Tyr at the cell:bead interface (Figure 2-8 left panel, bead position is indicated by the star).  When J558 cells were transfected with C? there was a reduction in the F-actin and P-Tyr accumulation at the cell:bead interface (Figure 2-8 middle panel).  When the J558 cells were transfected with ?trunc, there was a further reduction in F-actin and P-Tyr accumulation at the cell:bead interface (Figure 2-8 right panel).  These data show that the cytoplasmic domain of Ig? is: a) not sufficient to reconstitute complete BCR-mediated cytoskeletal rearrangements when antigen is localized to a single locus and b) the cytoplasmic domain of Ig? is not sufficient to reconstitute maximal P-Tyr accumulation at the cell:bead interface.      101    Figure 2-8 Assays for binding to particulate Ab reveal that the formation of actin-rich cup is reduced when the cytoplasmic domain of Ig? is swapped with Ig? or deleted.    J558?M3, J558C?, and J558?trunc were incubated with anti-IgM coated beads for 3, 5, 15 and 30 minutes.  The cells were then fixed in 4% paraformaldehyde and then embedded in a polyacrylamide gel matrix.  P-Tyr (green) was detected using a rabbit ?-mouse P-Tyr Ab and  102  visualized using a goat ?-rabbit IgG conjugated to the Alexa-488 fluorochrome. Actin (red) was visualized using Rhodamine-Phalloidin. The star represents the position of the bead. (Scale bar: 10 ?m)  2.5 Discussion In this chapter, the role of the cytoplasmic domains of Ig? and Ig? in BCR-mediated cytoskeletal rearrangements is examined and discussed.  Using a gain-of-function approach, mutated BCRs containing two Ig? tails or a single Ig? tail were expressed in the terminally differentiated J558 plasmacytoma cell line and the resulting transfected stable cell lines subsequently assayed for their ability to initiate BCR-mediated cytoskeletal rearrangements using a variety of assays that are commonly used in the lab.  Here, a WT BCR expressing J558 B cell was unable to initiate BCR-mediated spreading on an anti-IgM coated coverslip, but was able to initiate the formation of actin-rich protuberances.  The effective formation of these actin-rich protuberances was dependent on the presence of both the C-terminal domains of Ig? and Ig?, and the Ig? cytoplasmic domain is not equivalent to that of Ig? in BCR-mediated reorganization of the cytoskeleton.  The C-terminal domain of Ig? was also required for an efficient response to a particulate antigen and this response was diminished when the cytoplasmic domain of Ig? was swapped with Ig? or deleted. The J558 plasmacytoma model has previously been used to assay both the biochemical and structural requirements for BCR signalling and clustering, however the structural requirement of the Ig?/? tail in cytoskeletal rearrangements have yet to be clearly established (Flaswinkel and Reth, 1994; Tolar et al., 2009).  Expression of a WT BCR in J558 cells was sufficient to initate a phosphotyrosine response when incubated on an anti-IgM coated coverslip, but was unable to initiate B cell spreading.  This inability for a reconstituted BCR  103  to initiate BCR-mediated spreading is likely due to the developmental stage of J558 plasmacytoma cells.   Transition from a mature B cell into a plasma cell results in the upregulation of the protein Blimp-1 which inhibits the expression of the cell surface proteins Ig? and CD19, as well as intracellular proteins including Pax-5 (which is required for high levels of BLNK expression) (Bartholdy and Matthias, 2004; LeBien and Tedder, 2008; Oracki et al., 2010).  CD19 has been previously identified as important for B cell spreading (Depoil et al., 2008), however it is possible that there are other proteins that are downregulated during the developmental process which may be important for intiating B cell spreading.  Because of this, a gain-of-function approach using the J558 plasmacytoma model may prove to be an invaluable tool for understanding the molecular components required for BCR-mediated spreading. Although J558 B cells were unable to initiate B cell spreading in response to immobilized anti-IgM, they were able to initiate changes in the cytoskeleton in the form of F-actin rich membrane protrusions that were dependent on the presence of both cytoplasmic domains of Ig? and Ig?.  This differential requirement for both the cytoplasmic domains of Ig? and Ig? is consistent with previous studies that have investigated the role of Ig? and Ig? in B cell development and internalization.  Due to an inability to express a pro-BCR on the cell surface, mice with deletions in either Ig? or Ig? result in an inability to move past the pro-B cell stage (Gong and Nussenzweig, 1996; Pelanda et al., 2002).  However, mice with a cytoplasmic truncation in Ig? show a reduction in immature B cells by 80% and a reduction in mature B cells by 99% (Reichlin et al., 2001).  In contrast to this, mice with a similar truncation in Ig? result in normal numbers of immature B cells, but a decrease in mature B cells and cell surface expression of the BCR (Reichlin et al., 2001).  These results suggest  104  that there are differences in the cytoplasmic tails of Ig? and Ig? to initate signals required for B cell development.  There are also differences in the requirement for the cytoplasmic domains of Ig? and Ig? in BCR internalization, a process which is also dependent on the F-actin cytoskeleton (Jang et al., 2010).  Consistent with the results observed in this chapter, a truncation of Ig? (J558?trunc) showed a severe reduction in BCR-mediated internalization.  Though both the cytoplasmic domains of Ig? and Ig? contain internalization signals, Ig? is the most dominant in this process due the presence of a 4 amino acid motif within the ITAM (Jang et al., 2010).  When this motif was expressed in Ig?, it was sufficient to rescue aberrant internalization.  These results infer that not only is B cell development dependent on expression of a WT Ig?/? heterodimer, but receptor-mediated internalization is as well. The most likely explanation for the reduction in BCR-mediated cytoskeletal rearrangements when the cytoplasmic domain of Ig? was swapped with Ig? or deleted was the loss of the non-ITAM tyrosine 204 of Ig?.  This residue has been implicated in the recruitment and subsequent phosphorylation of the adaptor protein BLNK (Engels et al., 2001; Pike and Ratcliffe, 2005).  As previously described, the adaptor protein BLNK links PLC?2 and calcium signalling to the BCR.  An inability to activate this protein complex may result in defects in the activation of downstream signalling pathways required for rearrangement of the cytoskeleton, specifically activation of the GTPase Rap1.  Rap1 has been previously shown to regulate BCR-mediated spreading, response to a particulate antigen and immune synapse formation (Lin et al., 2008).  Our lab has been able to show that J558 B cells expressing a WT BCR only weakly activate Rap1 after activation with soluble anti-IgM, however there was an observable decrease in Rap1 activation in J558C? cells, and little to no Rap1 activated in J558?trunc cells (data not shown: Kate choi, unpublished results).  These  105  data support our findings that there was a reduction in BCR-mediated membrane projections and response to a particulate antigen when the cytoplasmic domain of Ig? was swapped with Ig?, and a further reduction when the cytoplasmic domain of Ig? was deleted.  Due to the minimal activation of Rap1 in J558 cells with a WT BCR, it is likely that one or more members in this pathway are either missing or downregulated in this cell line.  The formation of membrane projections, or blebs, have been previously described.  In an alternative model to F-actin mediated forward protrusive forces, a different model based on observations of Dictyostelium 2D migration has been proposed in which membrane protrusions may push forward the plasma membrane (L?mmermann and Sixt, 2009; Langridge and Kay, 2007; Yoshida and Soldati, 2006).  In this model, a protruding bleb is free of actin filaments and protrudes forward and can adhere to the substrate.  As the actin-free bleb ages, it becomes coated in F-actin where it is possible that the retrograde forces pull the cell body forward into the bleb (L?mmermann and Sixt, 2009).  Though this description does resemble the BCR-induced membrane blebs observed in J558 cells, it is likely not the same structure.  Because BCR signalling results in a migratory stop signal (Cyster, 2010), the production of a migratory structure in response to BCR signalling is unlikely.  Here is it more likely that the BCR-mediated membrane projections are the result of an inability to co-ordinate a spreading response due to missing signalling components or adaptor proteins rather than a previously described cellular structure. Interestingly, a new role for Ig? has recently been identified with respect to resting BCR mobility on the surface of B cells.  Using TIRF microscopy, it was shown that the cortical F-actin cytoskeleton, in conjunction with the ERM family protein ezrin, restrict the movement of the BCR, keeping it confined to areas of low F-actin (Treanor et al., 2010).   106  Here, the cytoplasmic domain of Ig? was determined to be important for restricting the movement of the BCR across these areas of high F-actin/ezrin.  These results indicate that there is also a differential requirement for the cytoplasmic domains of Ig? and Ig? with respect to interaction with the cortical actin cytoskeleton.   These data indicate that the cytoplasmic domain of Ig? is required for BCR-mediated cytoskeletal rearrangements, and that the cytoplasmic domain of Ig? is not equivalent in its ability to initiate this response.  These results also indicate that because the J558 plasmacytoma cell line is able to initate BCR-mediated signalling and rearrangement of the cytoskeleton, but not initiate B cell spreading, it is an ideal gain-of-function model to study the signalling components required for B cell spreading.  In subsequent chapters, this cell line was used to identify and characterize a novel role in BCR-mediated B cell spreading for the gap junction protein connexin43 (Cx43).          107  3 The characterization of the GJ protein Cx43 expressed in B lymphocytes and its role in BCR-mediated membrane spreading and B cell adhesion 3.1 Synopsis In the previous chapter we present evidence that expression and subsequent activation of the BCR in the terminally differentiated plasmacytoma cell line J558 was able to initiate rearrangements of the cytoskeleton; however, these signalling responses were unable to reconstitute radial cell spreading that is normally seen when B lymphoma cell lines are similarly activated.    In  this chapter, we show that the gap junction protein connexin43 (Cx43) is important for cell spreading and  may represent a family of proteins that contain important motifs, structures and functions essential for this cellular process.  Normally terminally differentiated plasmacytoma cell lines, including J558, do not express the gap junction protein Cx43, although it is widely expressed in mammalian cells, including lymphocytes. Recently, Cx43 has been shown to regulate cell adhesion and migration in neurons and glioma cells, leading us to propose similar functions for Cx43 in lymphocytes.  In this chapter we show that Cx43 influences BCR, LFA-1 and CXCL12 mediated activation of the Rap1 GTPase as well as having effects on cell adhesion and cell spreading.  Using a shRNA knockdown of Cx43 in WEHI 231 B lymphoma cells we show that Cx43 is necessary for sustained Rap1 activation and BCR-mediated spreading.  To determine the domains of Cx43 that are important for this effect, Cx43-null J558?m3 Plasmacytoma cells (expressing a WT IgM BCR) were transfected with WT Cx43-GFP or a C-terminal truncated Cx43 (Cx43?T-GFP).  Expression of WT Cx43-GFP, but not Cx43?T-GFP, was sufficient to restore sustained, BCR-mediated Rap1 activation and cell spreading.  Cx43, and specifically the C-terminal domain, was also important for LFA-1 and CXCL12 mediated Rap1  108  activation, spreading and adhesion to an endothelial cell monolayer.  These data show that Cx43 plays an important and previously unreported role in B cell processes that are essential to normal B cell development and immune responses. In the following chapter, this thesis will continue to explore the importance of Cx43 in motility, directed migration and transendothelial migration.  3.2 Introduction The antigen receptor on B lymphocytes (BCR) mediates the uptake of antigen for presentation to T lymphocytes and initiates signals that promote B cell growth and differentiation (Fairfax et al., 2008; Rodr?guez-Pinto, 2005). One of the key cellular responses that results from BCR activation by membrane-bound Ag is the initiation of actin-dependent cell spreading, a cellular process that is a key mechanism for B cell activation (Fleire et al., 2006; Lin et al., 2008).  This membrane spreading is thought to promote Ag gathering and maximize formation of BCR microclusters and an immune synapse, the formation of which lowers threshold for B cell activation (Fleire et al., 2006). The immunological synapse is where cell:cell contact occurs between the Ag presenting cell and the B cell, resulting in B cell activation and differentiation into Ab-secreting plasma cells (Harwood and Batista, 2008).   The gap junction (GJ) protein connexin43 (Cx43) is expressed in many cell types including hematopoetic cells (Bermudez-Fajardo et al., 2007; Laird, 2006; Oviedo-Orta et al., 2002).  Cx43 is a 43 kDa membrane protein that spans the membrane four times.  Its topology includes an intracellular amino-terminal domain, an intracellular carboxyl-terminal  109  domain, two extracellular loops, and a single intracellular cytoplasmic loop (Solan and Lampe, 2009).  Cx43 monomers combine to form hexameric hemichannels called connexons that can associate when at the plasma membrane, with connexons on adjacent cells to form GJs that allow the intercellular passage of ions and small molecules (generally less than 1 kDa) such as Ca++, ATP and cAMP (Laird, 2006).  Along with its role in cell-cell communication, Cx43 also regulates glioma and neural cell motility and migration (Bates et al., 2007; Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  Knockdown of Cx43 protein levels using shRNA constructs or by using a neuronal progenitor specific Cx43 conditional knockout in the developing cortex of mouse embryos results in reduced neuronal migration (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007). Although Cx43 is widely accepted as important for neuronal migration, the role of the C-terminal domain of Cx43 in this process remains debated.   In 2007, Elias et al used in utero cotransfection into a developing rat brain of Cx43 shRNA and a C-terminal truncated Cx43 to show that the extracellular domain, and not the C-terminal domain of Cx43 was required for proper neuronal migration (Elias et al., 2007).  In contrast to this report Bates et al., 2007 showed, using the Cx43-low expressing C6 glioma cell line, that over-expression of a C-terminal tail truncated Cx43 has a dominant negative effect on motility compared to cells where WT Cx43 was over-expressed instead (Bates et al., 2007).  In agreement with the results of Bates etal, Cina et al., 2009 used in utero electroporation of a C-terminal truncated Cx43 in a neuron-progenitor specific Cx43 conditional knockout background to show that the C-terminal domain is required for neuronal migration to the cortical plate (Cina et al., 2009).  In 2010, Elias et al. showed, using in utero cotransfection into the developing rat brain, of Cx43 shRNA and a C-terminal truncated Cx43, that the C-terminal domain of Cx43 was, in  110  fact, required for the tangenital to radial migratory switch of developing interneurons (Elias et al., 2010).  Although there is evidence for the C-terminal domain of Cx43 being required for neuronal migration, the molecular mechanism by which Cx43 influences the cytoskeleton remains unknown. Although Cx43 is expressed in B cells (Oviedo-orta et al., 2000), its function is not understood.  Cx43 knockout mice die perinatally from hypoxia due to malformation of the heart (Reaume et al., 1995), making assessment of the adult immune system difficult. However, heterozygote Cx43 mice do exhibit defects in T and B lymphocyte development (Montecino-Rodriguez and Dorshkind, 2001).  It is not clear however if the defect is intrinsic to the B cell, due to defects in other cell types that interact with them. Given the role of Cx43 in cell migration and morphology in neural cells, we proposed that Cx43 was involved in similar cytoskeletal-mediated responses in B-lymphocytes.   By using a combination of loss- and gain-of-function approaches, we show in this chapter that Cx43 expression is necessary and sufficient for BCR-mediated changes in activation of the Rap1 GTPase, a master regulator of B cell cytoskeletal reorganization, adhesion and migration (Lin et al., 2008; McLeod et al., 2002; McLeod et al., 2004). Cx43 was also important for LFA-1 and CXCL12 mediated Rap1 activation and spreading.  This increase in LFA-1 mediated spreading correlated with an increase in B cell adhesion to an endothelial cell monolayer.  These results demonstrate that Cx43 is a novel regulator of B cell morphology and cytoskeletal changes in response to BCR signalling.   111  3.3 Materials and methods  3.3.1 Plasmids Expression vectors encoding cDNAs for Rat wild type Cx43 (NAP2-Cx43GFP) and Cx43 lacking the C-terminal domain (NAP2-Cx43?244-382GFP) have been described (Bates et al., 2007; Mao et al., 2000) and were obtained from Dr. Christian Naus, Dept of Cellular and Physiological Sciences, Life Sciences Institute, UBC. Both vectors contained genes for GFP fused in-frame to the C-terminal end. The empty vector encoding only GFP (NAP2) is described in (Mao et al., 2000).  The expression vectors encoding the truncated Ig? protein (?trunc) and the Ig?/? chimeric protein (C?) have been described in chapter 2 (Dylke et al., 2007; Jang et al., 2010).   3.3.2 Antibodies Polyclonal goat anti-mouse IgM (?-chain specific) and polyclonal anti-IgG (?-chain specific) were from Jackson ImmunoResearch Labs  (West Grove, PA).  Polyclonal rabbit anti-Cx43, which recognizes an epitope in the C-terminal domain (amino acids 363-382), was from Sigma-Aldrich (Saint Louis, MO).  The monoclonal Ab (mAb) Cx43NT1, which recognizes the Cx43 N-terminal region (amino acids 1-20) was purchased from the Fred Hutchinson Cancer Research Institute (Seattle, WA).   Monoclonal mouse anti-human transferrin receptor was from Invitrogen Life Technologies (Carlsbad, CA).  The rat-anti-mouse LAMP1 monoclonal Ab (1D4B) was from the Developmental Studies Hybridoma Bank (Iowa City, IA).  Mouse anti-?-actin was from Sigma-Aldrich.  The rabbit anti-Rap1A/1B was from Cell Signalling Technology (Danvers, MA).  Goat anti-mouse IgG  112  conjugated to horseradish peroxidase (HRP) was from Invitrogen Canada Inc (Burlington, Ontario), goat anti-rabbit IgG-HRP was from Jackson ImmunoResearch Labs and the Alexa Fluor 647-conjugated goat anti-mouse IgG and Alexa Fluor 568-conjugated goat anti-rat IgG were from Invitrogen Molecular Probes.  Rhodamine-coupled phalloidin was from Invitrogen Life Technologies.  ER-tracker dye and CellTrackerGreen (CMFDA) were from Invitrogen and used as per manufacturers instructions.  The rat anti-LFA-1 mAb TIB213 was from ATCC (Manassas, VA).  3.3.3 Cells and cell growth  The J558 cell and WEHI 231 lines were described in section 2.3.2.  The A20 mouse B lymphoma, the MPC11 murine plasmacytoma and the mouse bEND.3 endothelial cell lines were obtained from ATCC.  The murine plasmacytoma 5TGM1 (Oyajobi et al., 2003) was a gift from Dr. B.O. Oyajobi (Univ. Texas, San Antonio,Tx).  Murine splenic B cells were isolated using a B cell selection kit (StemCell Technologies, Vancouver, Canada) as per the manufacturer?s instructions. Cells were cultured in high glucose (4.5 g/L) RPMI-1640 (all B cell lines) or DMEM (bEND.3) supplemented with 2 mM L-glutamine, 110 mg/L sodium pyruvate, 10% heat-inactivated FBS, 50 units/mL of pen/strep.  3.3.4 Transfection and retroviral transduction BCR mutants were expressed as described in section 2.3.3.   Retroviral transduction of WEHI 231 cells was performed as described in (Krebs et al., 1999).  To express Cx43 in B cell lines, supernatants of 293-GPG cells containing the  113  retrovirally packaged NAP2-Cx43GFP, NAP2-Cx43?244-382GFP or NAP2-GFP plasmids (Bates et al., 2007) were incubated with 5 x 105 WEHI 231 or J558?m3 cells overnight at 37?C.  GFP-positive cells were isolated by FACS.   Retroviral vectors encoding Cx43 shRNA constructs 1 (specific for the third transmembrane domain)  and 2 (specific for the cytoplasmic loop), as well as the corresponding scrambled sequence, have been described previously (Shao et al., 2005) and were used for retroviral transduction of  WEHI 231 cells.    3.3.5 B cell stimulation and preparation of cell extracts  Cells were washed with PBS, resuspended at 5 x 106 cells/ml in modified Hepes-buffered saline (25 mM sodium Hepes pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM, 1 g/L glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 ?M 2-ME), and stimulated with 20 ?g/ml of goat anti-mouse IgM, 5 ?g/ml anti LFA-1 or 100 nM CXCL12 (R&D systems, Minneapolis, MN). Reactions were stopped by adding 1 ml cold PBS.  Cells were then lysed in cold lysis buffer (PBS, 1% Triton-X 100, 1% IGEPAL (Sigma-Aldrich), 50 mM CaCl2) (Troxell et al., 1999) containing protease inhibitors (10 ?g/ml leupeptin, 1 ?g/ml aprotinin, 1 mM pepstatin A, 1 mM Na3VO4, 1 mM PMSF).  To extract Cx43, cells were lysed using 200 ?l of lysis buffer, sonicated for 10 seconds, and incubated at 37?C for 30 min before being analyzed by SDS-PAGE.   Dephosphorylation of Cx43 was performed as follows.  WEHI 231 cells were resuspended to 1x107 cells per timepoint, in 500 ?l quinsaline.  Cells were stimulated for 0, 5, 10, 15, 30, 60, and 120 min with 20 ?g/ml of goat anti-mouse IgM at 37oC.  Reactions were stopped using 500 ?l cold PBS.  Cells were centrifuged at 1500 RPM at 4oC for 2 min,  114  supernatants were removed and lysed as previously described.  Protein concentrations of the cell lysates were determined using a BCA assay (Thermo Scientific Pierce, Rockford, Illinois).  For each timepoint, lysates were split into two sets containing 30 ?g of protein each, one subjected to dephosphorylation and the other used as an undigested control.  Lysates used as a control were suspended in 1X NEBuffer (New England BioLabs Inc, Ipswich, Massachusetts).  Dephosphorylation of the remaining lysates was carried out using 1X NEBuffer and 30 units of Calf Intestinal Alkaline Phosphatase (CIP) (New England BioLabs Inc, Ipswich, Massachusetts) as per the manufacturer?s instructions.  Lysates were incubated for 60 min at 37oC and subsequently suspended in 1X SDS-PAGE reducing sample buffer and incubated for 30 min at 37oC.  3.3.6 Rap1 activation assay Rap1 activation assays were performed as described by McLeod et al. (McLeod et al., 1998).   Briefly, a RalGDS-GST fusion protein bound to Gluthathione-Sepharose 4B beads was used to pull down the active form of Rap1, which was then detected on a western blot using an anti-Rap1A/B Ab (Cell Signalling Technologies).  Total Rap1 was detected by separating 30 ?l of lysate from the portion used for the RalGDS prior to the pulldown, per time point, and by running a second SDS-PAGE.  This was used to monitor total Rap1 and ensure equal loading of all lanes.   115  3.3.7 Cell spreading assay Cell spreading assays using a fixed time course were performed as described in section 2.3.6 and an overview diagram is described in Figure 3-1.    Cell contact sites at the cell:coverslip interface were imaged on an Olympus FlowView1000 confocal microscope (LSI Imaging). The contact area between the cell and coverslip of the spreading cells was quantified using ImagePro version 6.2 (LSI Imaging) and the mean contact area was calculated.  Spread cells were defined as having a contact area that extended as wide as the cell diameter and a radial ring of F-actin (~30% of cells by 30 min in fixed samples).  In the event that no cells fit this critera, all cell contact areas were measured.  Membrane projections were counted manually from compiled Z-Stacks of 0.5 ?m step size images of 5-50 cells per time point (60x objective) using Olympus FV-Viewer.  To assess cell spreading in real time, J558?m3 or WEHI 231 cells were labelled with CellTracker Green CMFDA and splenic B cells were labeled with CellMask Orange (Invitrogen Life Technologies) as per the manufacturer?s instructions.  The cells were deposited into glass bottom microwell dishes (MatTek, Ashland, MA) that were coated with goat anti-mouse IgM, and placed in a 37?C incubation chamber mounted on the microscope stage.  The cell:coverslip interface was imaged every 20 seconds for 20 min.  Cell contact sites of individual spreading cells were quantified at each timepoint using Image Pro version 6.2.  In this assay, spreading cells were defined as remaining in one stationary position and having a contact area that changes in shape and size.   116   Figure 3-1 Overview of B cell spreading assays described in Section 3.3.7  3.3.8 Scanning electron microscopy (SEM) The cell spreading assay was performed as previously described (Lin et al., 2008).  Samples were prepared for SEM as described in Section 2.3.5.   3.3.9 B cell adhesion to bEND.3 cells An overview diagram describing B cell adhesion to bEND.3 cells is located in Figure 3-2.   Glass coverslips were coated with 1 ?g/ml bovine fibronectin in PBS for 1 hour at RT before plating bEND.3 endothelial cells.  After 4-7 days, the bEND.3 cells were activated  117  overnight with 10 ng/ml TNF-? (eBioscience, San Diego, CA).  J558?m3 and 5TGM1 plasmacytoma cells (4 x 105) were pre-activated for 1 hr with 100 ng/ml CXCL12, labeled with CellTracker Green CMFDA, and then allowed to adhere to the endothelial cell layers.  After 1 hr at 37?C, non-adherent cells were gently removed by washing and the remaining cells were fixed for 10 min with 4% paraformaldehyde and stained as described for B cell spreading assays. The cells were imaged using a 10X objective and the number of CMFDA-positive cells per field was determined using Image Pro version 6.2 software.   Three-dimensional reconstructions were generated using Olympus 3D FluoView Version 1.7 reconstruction software (LSI Imaging).  118   Figure 3-2 Overview of B cell adhesion to bEND.3 cells described in Section 3.3.9  3.3.10 Statistics A comparison of means was assessed using a student?s unpaired, two-tailed t test.   119  3.4 Results 3.4.1 Cx43 is expressed in immature and mature B cells and is a target of BCR signalling The expression of Cx43 in B cell lines from different stages in development was determined using standard Western blotting and immunofluorescence microscopy procedures.  Cx43 was expressed in the membrane IgM+ WEHI 231 immature murine B cell line and in the membrane IgG+ A20 mature murine B cell line but not in J558?m3, 5TGM1 or MPC11 plasmacytoma cell lines (Figure 3-3A).  On western blots, the broad bands observed were consistent with published reports of differentially phosphorylated populations of Cx43 (Lampe et al., 2000).  Immunofluorescence microscopy with Abs specific for Cx43showed strong cell surface staining in the B cell lines as well as normal splenic B cells, (Figure 3-3B, white arrows).  There was also an accumulation of Cx43 in intracellular aggregates (Figure 3-3B, yellow arrows).      120    121   Figure 3-3 Cx43 is expressed in B cells and is a target of BCR signalling.  A) Expression of Cx43 in B lymphoma (WEHI 231, A20) and plasmacytoma (J558?m3, 5TGM1 and MPC11) cell lines as determined by immunoblotting with an Ab specific for the C-terminal tail of Cx43.  Day 18 mouse embryo brain lysate was used as a positive control.  Stripping the blot and re-probing for actin indicated equal loading of the lanes.   B) Localization of endogenously expressed Cx43 (green) using immunofluorescence microscopy. Cx43 expression by and localization in WEHI 231 B cells, mature splenic B cells and A20 B cells was detected by anti-Cx43 Ab specific for the C-terminal tail, followed by a fluorescently tagged secondary Ab.  Actin was detected using rhodamine-phalloidin (red). Cx43 was localized at the plasma membrane (white arrows) and in an intracellular aggregate (yellow arrows).  Scale bar: 10 ?m.    122  C) BCR signalling leads to phosphorylation of Cx43.  Cell lysates were prepared from WEHI 231 B cells after crosslinking of the BCR with anti-IgM for the indicated times.  The lysates were then divided into two equal portions, one was treated with calf intestinal phosphatase (CIP) and one was not.  The lysates were separated by SDS-PAGE and Cx43 was detected using an Ab specific for the cytoplasmic tail.  Stripping the blots and re-probing for actin indicated equal loading of the lanes.    Representative experiment of three similar, independent replicates.  Reproduced/adapted with permission from (Machtaler et al., 2011).  Clustering the BCR on WEHI 231 cells caused an apparent increase in the molecular weight of Cx43 on SDS-PAGE gels (Figure 3-3C).  This change was first seen at 5 min after cross-linking, persisted for 15 min, and started to decline at 30 min (Figure 3-3C, upper panels).  This BCR-induced bandshift was likely due to phosphorylation as treatment of cell lysate with calf intestinal phosphatase prior to SDS-PAGE reduced the amount of the higher molecular weight bands (Figure 3-3C, lower panels). The carboxyl-terminal domain of Cx43 contains multiple phosphorylation sites that have been implicated in regulating Cx43 function and internalization (Lampe and Lau, 2004).  The Cx43 bandshift in this B cell line is consistent with the findings that Cx43 is phosphorylated in other cell types in response to growth factor receptor signalling and activation with phorbol esters (Lampe et al., 2000; Loo et al., 1995; S?ez et al., 1997; Warn-Cramer et al., 1996).  3.4.2 Cx43 is required for sustained activation of Rap1 GTPase Activation of the Rap1 GTPase is critical for BCR-induced cytoskeletal reorganization, cell spreading, integrin activation and immune synapse formation, as well as for chemokine-induced migration (Lin et al., 2008; McLeod et al., 2002; McLeod et al., 2004).  Because Cx43 has been implicated in neural and glial cell migration and adhesion, processes regulated by Rap1, we hypothesized that Cx43 contributes to BCR-induced Rap1  123  activation.  To test this, we used shRNA-encoding vectors (Bates et al., 2007; Shao et al., 2005) to knock down the expression of Cx43 in WEHI 231 cells.  Cx43 shRNA construct 2 (specific for the third transmembrane domain), as well the combination of shRNA constructs 1 (specific for the cytoplasmic loop) and 2 reduced Cx43 protein levels (Figure 3-4A).  Importantly, knockdown of Cx43 in WEHI 231cells did not lead to a change in the cell surface expression of the BCR, the chemokine receptor CXCR4 or the levels of ?1 integrins (data not shown: Kate Choi, unpublished results).  Compared to WEHI 231 cells transfected with a nonsense shRNA construct, cells transfected with shRNA 1 + 2 constructs or shRNA 2 alone were unable to effectively sustain BCR-mediated Rap1 activation (Figure 3-4B).  Similarily, we have observed that a knockdown of Cx43 in the mature B cell line A20 also caused a decrease in the ability of BCR signalling to sustain the activation of Rap1 (data not shown).  Thus this effect on Rap1 is something observable in at least two B lymphoma cell lines and not a peculiarity of the WEHI 231 immature B cell line.          124    Figure 3-4 Reduction of Cx43 expression alters the ability of B cells to sustain activation of the Rap1 GTPase.    A) Cx43 protein levels in WEHI 231 cells transfected with a nonsense Cx43 shRNA, Cx43 shRNA construct 1 (targeted to the cytoplasmic loop) and  Cx43 shRNA construct 2 (targeted to the third transmembrane domain of Cx43) together or Cx43 shRNA 2 alone, as assessed by immunoblotting. Anti-actin was used as a loading control.   B)  Rap1 activation in WEHI 231 cells transfected with Cx43 nonsense, Cx43 shRNA 1 + 2 or Cx43 shRNA 2.  Cells were stimulated for the indicated times with anti-IgM and activated Rap1 was precipitated using a GST-RalGDS fusion protein and total Rap1 detected by blotting with anti-Rap1 Abs. Representative experiment of three similar, independent replicates. Reproduced/adapted with permission from (Machtaler et al., 2011)   125  3.4.3 Cx43 expression is necessary for BCR-mediated B cell spreading   The process of BCR-mediated B cell spreading is dependent on the activation of Rap1 (Lin et al., 2008; McLeod et al., 1998).  Because knocking down the level of Cx43 in WEHI 231 cells resulted in a reduction in sustained activation of Rap1, we hypothesized that Cx43 was important for BCR-induced B cell spreading.  To test this, real time imaging was used to assess the ability of WEHI 231 cells to spread on coverslips coated with immobilized anti-IgM Abs that activate the BCR.  Compared to WEHI 231 cells transfected with a scrambled shRNA construct, cells transfected with shRNA 1 +2 exhibited significantly reduced cell spreading (Figure 3-5A, lower frames; quantified in Figure 3-5B, red line).  In addition to undergoing decreased spreading, WEHI 231 cells transfected with Cx43 shRNA 1 + 2 cells spread non-uniformly and sent out multiple, dynamic membrane projections (Figure 3-5A, lower frames, white arrows indicate dynamic projections), as opposed to the uniform radial spreading exhibited by the cells transfected with the nonsense vector (Figure 3-5A, upper frames).  Consistent with the data from knockdown WEHI 231 lymphoma cells, splenic B cells from Cx43 heterozygous knockout (Cx43+/-) C57/BL6 mice, which express reduced amounts of Cx43 compared to wild type splenic B cells (Figure 3-6A), exhibited a reduced ability to spread on anti-IgM-coated coverslips when compared with wild type splenic B cells from a normal littermate (Figure 3-6A; quantified in Figure 3-6B).   Since the reduction in Cx43 expression has significant effects on B cell spreading we hypothesized that over expression would enhance BCR-mediated spreading. To test this, WEHI 231 B cells were transfected with Cx43-GFP and their ability to spread on an immobilized anti-IgM coated coverslips were compared with non-transfected WEHI 231 cells. Consistent with the knockdown data, we found that increasing the amount of Cx43 in  126  WEHI 231 cells significantly increased the degree to which these cells spread (Figure 3-7A, quantified in Figure 3-7B).  Taken together, these data show that Cx43 is important for BCR-induced B cell spreading.            127    128  Figure 3-5 Decreased Cx43 expression impairs BCR-induced spreading in WEHI 231 B cells.  A) Real-time spreading of WEHI 231 cells transfected with Cx43 nonsense shRNA or Cx43 shRNA constructs 1 + 2. Cells were labeled with CellTracker Green CMFDA and plated on anti-IgM coated coverslips for 20 min at 37?C.  A single confocal scan was taken at the cell:coverslip interface every 20 s.  The cells were pseudo-colored using the Olympus software in order to visualize fine membrane projections. Yellow equates to the highest pixel intensity and green is the lowest pixel intensity. Representative images are shown from 0 to 9 min at 1 min intervals.  White arrows indicate dynamic membrane projections (Scale bar: 10 ?m).  B) The mean contact area of each time point between the cell and the substrate (pooled data from 3 independent experiments, n = 36 individual cells for nonsense shRNA transfected cells, n = 49 individual cells for shRNA 1+2 transfected cells). Reproduced/adapted with permission from (Machtaler et al., 2011)   129    130  Figure 3-6 Decreased Cx43 expression impairs BCR-induced spreading in splenic B cells from Cx43 heterozygous mice. A) Cx43 protein levels in splenic B cells from WT and Cx43+/- mice.  Cx43 protein levels were assessed by immunoblotting. Anti-actin was used as a loading control. B and C) Real time analysis of the spreading of WT splenic B cells and Cx43+/- B cells on immobilized anti-IgM was performed as in A except that the cells were labeled with the membrane dye CellMask Orange. For each time point, the mean contact area of each time point between the cell and the substrate is shown in D (pooled data from 3 independent experiments, * indicates that p < 0.05, n = 87 individual cells for WT splenic B cells, n = 89 individual cells for Cx43+/- splenic B cells).  Reproduced/adapted with permission from (Machtaler et al., 2011)   131     132  Figure 3-7 Expression of Cx43-GFP enhances BCR-mediated B cell spreading in WEHI 231 B cells A) WEHI231 and WEHI231.Cx43-GFP cells were labelled with CellTracker Green CMFDA and placed onto anti-IgM coated coverslips for 20min at 37?C.  One single confocal scan was taken at the cell:coverslip interface every 20 seconds for the duration of the time course.  The cells were pseudocoloured using the Olympus software where yellow equates to the highest pixel intensity and green equates to the lowest pixel intensity (Scale bar: 10?m). B)  Quantification of the contact area of WEHI231 and WEHI231.Cx43-GFP cell lines spreading on anti-Ig? coated coverslips (representative quantification of a single triplicate experiment).   3.4.4 Wild type Cx43 is sufficient to confer BCR-mediated radial cell spreading and enhance Rap1 activation in J558 plasmacytoma cells: The C-terminal tail of Cx43 is required for this response. J558 plasmacytoma cells, stably transfected with Ig? and Ig? so that they express a functional BCR on the cell surface (J558?m3), have been used to assay both the biochemical and structural requirements for BCR signalling (Flaswinkel and Reth, 1994; Tolar et al., 2009).  As previously described in section 2.4.3, J558?m3 cells do not spread radially like WEHI 231 cells but extend small, actin-rich protrusions after 30 min incubation on anti-IgM coated coverslips.  This indicates that J558?m3 cells have the capacity to reorganize their cytoskeleton into membrane projections in a BCR-dependent manner, but are unable to sustain a BCR-mediated B cell spreading response. Loss-of-function experiments in WEHI 231 cells showed that Cx43 is required for both sustained BCR-mediated Rap1 activation and for maximal spreading (Figure 3-4 and Figure 3-5).  Since J558?m3 cells, which do not express Cx43, exhibit low, transient and variable BCR-mediated Rap1 activation, we hypothesized that expression of Cx43-GFP in J558?m3 cells would be sufficient to confertheir ability to sustain BCR-mediated Rap1  133  activation and spreading.  Transfection of wild type Cx43-GFP, Cx43?T-GFP (a C-terminal truncation of Cx43) or a GFP encoding vector alone (cartoons representing what these proteins encoded by these constructs would look like are shown in Figure 3-8A) resulted in their expression in the WT BCR expression J558 cells (Figure 3-8B and C) and did not alter cell surface BCR levels as determined by FACS (data not shown) or the expression of CXCR4 and ?2 integrins (data not shown: Kate Choi, unpublished results).  Consistent with immunofluorescence data of Cx43 localization in B cell lines, transfected Cx43-GFP was localized at the plasma membrane and within a large intracellular aggregate (Figure 3-8C).  TIRF microscopy confirmed the presence of Cx43-GFP and Cx43?T-GFP at the plasma membrane in J558 cells (Figure 3-8D). The large intracellular aggregate co-localized with the endoplasmic reticulum (Figure 3-9, top pannel) and with early endosomal compartments (Figure 3-9 middle pannel) but not with lysosomes (Figure 3-9 bottom pannel).         134   Figure 3-8 Endogenous Cx43, Cx43-GFP and Cx43?T-GFP expression and localization in B cells. A) Schematic representation of the proposed protein product of the Cx43-GFP and the carboxyl-terminal tail truncation of Cx43 (Cx43?T-GFP) constructs. B) Expression of wild type Cx43-GFP (~65 kDa) and a Cx43?T-GFP (~50 kDa) in transfected B cell lines was detected by immunoblotting.  Cx43 was detected using an anti-Cx43 antibody specific for the N-terminus of Cx43.  Filters were stripped and re-probed with anti-actin Ab to show equal loading of all lanes.  C) Localization of Cx43-GFP and Cx43?T-GFP in transfected J558?m3 and WEHI 231 cells was detected using confocal microscopy (GFP: green) (Scale bar: 10  135  ?m).  D) Cell surface localization of Cx43-GFP (left) and Cx43?T-GFP (right) in J558 transfected B cells spreading on anti-IgM coated coverslipsfor 30 min using TIRF-microscopy (Scale bar: 10 ?m). Reproduced/adapted with permission from (Machtaler et al., 2011).   Figure 3-9 Cx43-GFP localization in J558?m3 B cells The Cx43-GFP intracellular aggregate was determined to partially co-localize with the endoplasmic reticulum in live cells (detected by ER-Tracker, top pannel), as well as partially co-localizing with endosomes in fixed cells (identified with anti-transferring receptor (TfnR) Abs, middle pannel) and to a much lesser extent in lysosomes in fixed cells (detected by anti-LAMP-1 Abs, bottom pannel).  DIC images of the cells are shown on the right. (Scale bar: 10 ?m). Reproduced/adapted with permission from (Machtaler et al., 2011)  After BCR activation using a soluble anti-BCR ab, there was a minimal, transient increase in the activation of Rap1 in J558?m3.GFP cells (Figure 3-10A).  Expression of Cx43-GFP in J558?m3 cells resulted in increased Rap1 activation after BCR stimulation that  136  was sustained for at least 30 min (Figure 3-10A).  Expression of Cx43-GFP in J558?m3 cells also resulted in an increase in spreading on immobilized anti-IgM (Figure 3-10B, center panels; quantified in Figure 3-10C, compare green line with blue line, Figure 3-11A, B center panel  and quantified in C). This increase in spreading was determined to be dependent on a wild type BCR as transfection of Cx43-GFP into the BCR mutant cell lines J558C? and J558?trunc were unable to initiate a spreading response (Figure 3-12A; quantified in Figure 3-12B). This is consistent with WEHI 231 data showing that Cx43 is important for both sustained Rap1 activation and spreading.   137    138  Figure 3-10 Wild type Cx43-GFP but not the C-terminal tail truncated Cx43?T-GFP supports sustained activation of the Rap1 GTPase and B cell spreading. A) J558?m3 B cells transfected with GFP-alone, wild type Cx43-GFP or C-terminal tail truncated Cx43?T-GFP were stimulated for the indicated times with an anti-IgM antibody.  Rap1 activation was assessed as described previously.   B and C) Real-time spreading of J558?m3-GFP, J558?m3-Cx43-GFP and J558?m3-Cx43?T-GFP cells on immobilized anti-IgM Ab.  The cells were labelled with CellTracker Green CMFDA, plated on anti-IgM coated coverslips and imaged for 20 min at 37?C.  One single confocal scan was taken at the cell:coverslip interface every 20 seconds for the duration of the time course.  The cells were pseudo-coloured using the Olympus software in order to visualize fine membrane projections.  Yellow equates to the highest pixel intensity and green equates to the lowest pixel intensity.  Representative images are shown at 0, 5, 10 and 20 min, where the left panels represent the pseudo-coloured CMFDA and the right panels include the DIC overlay (Scale bar: 10 ?m).   C) Quantification of the mean contact area of each time point of J558?m3-GFP, J558?m3-Cx43-GFP and J558?m3-Cx43?T-GFP expressing cell lines spreading on anti-IgM coated coverslips (mean of four experiments, * indicates that p < 0.05, brackets indicate the comparisons being made, n = 45 individual cells for GFP transfected cells, n = 44 individual cells for Cx43-GFP transfected cells, n = 44 for Cx43?T-GFP transfected cells).  Reproduced/adapted with permission from (Machtaler et al., 2011)    139    140  Figure 3-11 WT Cx43-GFP expression is sufficient to restore/enhance BCR-mediated spreading.   A) Scanning electron micrographs of J558?m3 cells (top two panels) and J558?m3-Cx43-GFP cells (bottom two panels) plated on anti-IgM coated coverslips (Scale bar: 10 ?m).   B) Single confocal slices of the cell:coverslip interface of J558?m3 cells expressing GFP-alone, Cx43-GFP or C-terminal tail truncated Cx43?T-GFP  spreading on anti-IgM coated coverslips over a time course from 0 to 60 min.  (Green: GFP, Red: F-actin, Scale bar: 10 ?m).   C) Quantification of the mean contact area of J558?m3-GFP, J558?m3-Cx43-GFP and J558?m3-Cx43?T-GFP cells spreading on anti-IgM coated coverslips (mean of triplicate experiments, error bars indicate standard error, * indicates that p < 0.05, brackets indicate the comparisons being made). Reproduced/adapted with permission from (Machtaler et al., 2011)    141    142  Figure 3-12 Wild type Cx43-GFP expression is not sufficient to initiate BCR-mediated cell spreading of J558?m3 cells expressing mutated BCRs.   A) In BCR mutants containing defective Ig?/??s (?trunc and C?; described in Chapter 2), Cx43 expression was not sufficient to conferthe BCR-mediated cell spreading response. Single confocal slices at the cell:coverslip interface of J558?trunc (top two panels), J558C? (middle two panels) and wild type J558?m3 cells (bottom two panels) transfected with Cx43-GFP.  Cells incubated on anti-IgM coated coverslips for 30 min and the relative size of the contact area compared.  (Green: GFP, Red: F-actin, Scale Bar: 10 ?m).   B) Quantification of the mean contact area of J558?trunc.Cx43-GFP, J558C?.Cx43-GFP and J558?m3.Cx43-GFP cells (mean of three experiments, * indicates that p < 0.05, brackets indicate the comparisons being made, n = 88 individual cells for J558?trunc.Cx43-GFP cells, n = 177 individual cells for J558C?.Cx43-GFP cells, n = 109 for J558?m3.Cx43-GFP cells).  Reproduced/adapted with permission from (Machtaler et al., 2011)  The carboxyl-terminal tail of Cx43 contains a proline-rich domain, two tyrosines and ten serine residues, some of which are phosphorylated in other cell types.  This domain has been shown to be important for the motility of C6 glioma tumors and neuronal cells (Bates et al., 2007; Cina et al., 2009).  To determine if the carboxyl-terminal tail of Cx43 is important for BCR-mediated Rap1 activation, J558?m3 cells were transfected with a carboxyl-terminal tail truncation of Cx43-GFP that had amino acids 244-382 removed (Cx43?T-GFP) (Figure 3-8A and B). This mutated form of Cx43 was the same version used for the study in C6 glioma cells (Bates et al., 2007) mentioned above.  Unlike the full-length Cx43-GFP, the mutant form lacking the C-terminal cytoplasmic domain did not confer upon J558?m3 cells the ability to sustain Rap1 activation.  Instead, there was a transient increase in Rap1 activation at 5 min after receptor stimulation that was not sustained (Figure 3-10A, rightmost lanes).  Consistent with this finding, the mutant form of Cx43, lacking the C-terminal cytoplasmic domain, did not result in a sustained spreading response (Figure 3-10B, rightmost panels; quantified in Figure 3-10C, Figure 3-11A, B rightmost panel and quantified  143  in C).  Interestingly, J558?m3-Cx43?T-GFP cells showed an initial increase in B cell spreading from 0 to 5 minutes, followed by a slight decline (Figure 3-10C, red line).  The brief increase in dynamic, asymmetrical spreading observed coincides in time with the in Rap1 activation at this time point (Figure 3-10A).  Similar results were obtained when examining J558?m3-Cx43?T-GFP spreading using fixed samples (Figure 3-11B and C).  These data suggest that Cx43 has two important functions in mediating signalling responses critical for BCR-mediated Rap1 activation and spreading: a C-terminal domain independent function which is required for initiation of Rap1 activation and spreading, and a C-terminal domain dependent function, which is required for sustained Rap1 activation and spreading.  3.4.5 Cx43-GFP enhances LFA-1 mediated Rap1 activation and spreading The process of B cell spreading is important during interactions with Ag presenting cells and during the process of transendothelial migration (TEM) through vascular endothelial cells, where it is required for maximal adhesion prior to extravasation (Batista et al., 2007; Ley et al., 2007).  Like BCR-mediated spreading, LFA-1 induced spreading is also dependent on Rap1 activation (Lin et al., 2008).  Because BCR-mediated sustained Rap1 activation and spreading was dependent on Cx43 expression, we hypothesized that LFA-1 mediated Rap1 activation and spreading was also influenced by Cx43 expression.  LFA-1 signalling was initiated by incubating J558?m3 cells transfected with GFP, Cx43-GFP and Cx43?T-GFP with the TIB213 anti-LFA-1 mAb.  LFA-1 clustering led to a minimal increase in Rap1 activation in J558?m3-GFP cells, but to a bigger and more sustained activation in J558?m3-Cx43-GFP cells (Figure 3-13A).  Truncation of Cx43 in J558?m3-Cx?T-GFP cells  144  led to an increase in Rap1 activation by 5 min, however, consistent with the results after BCR signalling, the increase in Rap1 activation was not sustained (Figure 3-13A).    145    146  Figure 3-13 Cx43-GFP expression enhances LFA-1 induced Rap1 activation and cell spreading.   A) J558?m3-GFP, J558?m3.Cx43-GFP and J558?m3.Cx43?T-GFP cells were stimulated for the indicated times with soluble anti-LFA-1 and Rap1 activation was assessed as in Figure 2.  Representative western blots of 3 independent experiments.  B) B cell spreading on anti-LFA-1.  Single confocal slice taken at the cell:coverslip interface of J558?m3 B cells spreading on anti-LFA-1 coated glass coverslips (Red: F-actin, scale bar: 10 ?m).  C) Mean contact area of B cell spreading on anti-LFA-1 coverslips.  The mean contact area of cells from three independent experiments was measured by quantifying the contact area between the cell and coverslip using ImagePro software (error bars represent standard error of the mean* indicates that p < 0.05, brackets indicate the comparisons being made, n = 232 individual cells for GFP transfected cells, n = 197 individual cells for Cx43-GFP transfected cells, n = 136 for Cx43?T-GFP transfected cells).  Reproduced/adapted with permission from (Machtaler et al., 2011)  Because LFA-1 induced spreading is dependent on Rap1 activation, we assessed whether Cx43 expression also alters the LFA-1 mediated spreading response.  J558?m3-GFP, Cx43-GFP and Cx43?T-GFP were incubated on an anti-LFA-1 coated coverslip for 3 hours, then they were fixed, stained and the contact area with the coverslip measured.  J558?m3-GFP cells, similar to the results from the anti-BCR spreading assay, showed a minimal amount of spreading, whereas the cell:coverslip contact area of Cx43-GFP transfected J558?m3 cells was two-fold larger (Figure 3-13B; quantified in C).  There was a slight increase in contact area of J558?m3-Cx43?T-GFP cells compared to GFP alone, but the contact area was still less than that of full length Cx43-GFP transfected cells (Figure 3-13B and quantified in C).  These results indicate that Cx43, and specifically the C-terminal domain, is required for optimal LFA-1 mediated Rap1 activation and cell spreading.   147  3.4.6 Cx43-GFP expression increases the adhesion of B cells to bEND.3 endothelial cells Transendothelial migration (TEM) like that seen when lymphocytes extravasate from blood vessels, is a complex, multi-step process that requires initial arrest and spreading of B cells on top of the endothelial cells (Ley et al., 2007).  Initially, B cells come into contact with endothelial cells via selectin-mediated capture and rolling.  During this process, B cells are exposed to integrin ligands and chemokines on the surface of activated endothelial cells, which work in concert, triggering both outside-in and inside-out activation of integrins like LFA-1, resulting in firm adhesion, arrest and spreading (Ley et al., 2007).  This adhesion of B cells to endothelial cells is both integrin and Rap1 dependent (Lin et al., 2009; McLeod et al., 2004).  LFA-1 binding of ICAM-1 induces a conformational change in LFA-1, resulting in outside-in signalling and Rap1 activation. Chemokine signalling (eg. CXCL12/CXCR4) leads to Rap1 activation that is required for optimal LFA-1 activation (Freeman et al., 2010; McLeod et al., 2004; Shattil et al., 2010).  Because chemokine-mediated Rap1 activation is important for integrin activation and adhesion, the ability of J558?m3-GFP cells, Cx43-GFP expressing cells, as well as mutated Cx43?T-GFP expressing cells to activate Rap1 in response to the chemokine CXCL12 was assessed.   CXCL12 induced small, transient Rap1 activation in J558?m3-GFP cells.  This was greatly enhanced by expressing WT Cx43-GFP, but not by Cx43?T-GFP, which only supports transient Rap activation (Figure 3-14A).     148    149    150  Figure 3-14 Cx43-GFP expression enhances chemokine mediated Rap1 activation and B cell adhesion to bEND.3 endothelial cells.   A) J558?m3-GFP, J558?m3-Cx43-GFP and J558?m3-Cx43?T-GFP cells were stimulated for the indicated times with 100 nM CXCL12 before assaying for Rap1 activation.  Representative western blots of 3 independent experiments.  B) Immunofluorescence of Cx43 expression in bEND.3 endothelial cells (compiled Z-stack, Green: Cx43, Red: F-actin, Scale bar: 50 ?m).   C) Quantification of adhesion of transfected plasmacytoma cells to the bEND.3 monolayer  (representative quantification of a single triplicate experiment, error bars indicate standard deviation, * indicates that p < 0.05, brackets indicate the comparisons being made).   D) Representative images showing adhesion of transfected plasmacytoma cells to bEND.3 by immunofluorescence. Top panels show transfected J558?m3 plasmacytoma cells while the bottom panels show transfected 5TGM1 plasmacytoma cells. Top, single confocal images of the contact points between CellTracker Green CMFDA labeled cells and TNF?-activated bEND.3 endothelial cells.  Below the first row of images are 3D reconstructions of the J558 cell lines adhering to the bEND3 monolayer.   (Green: CMFDA labeled B cells, Red: F-actin, Scale bars: 20 ?m).  Lower panels show transfected 5TGM1 plasmacytoma cells expressing the same Cx43 constructs as the J558?m3 cells.  Shown are single confocal images of the contact points between CellTracker Green CMFDA labeled cells and TNF?-activated bEND.3 endothelial cells. Only wild type Cx43 expression leads to large contact areas between the plasmacytoma cells and the endothelial cell layer. Reproduced/adapted with permission from (Machtaler et al., 2011)  Cx43-GFP expression in J558?m3 cells enhanced cell spreading on immobilized anti-LFA-1, resulted in more sustained LFA-1 mediated Rap1 activation as well as sustained CXCL12 mediated Rap1 activation, processes involved in B cell adhesion to endothelial cells. Therefore, we hypothesized that Cx43-GFP, and specifically the carboxyl -terminal domain of Cx43, would be required for strong adhesion to an endothelial cell monolayer. To achieve maximal adhesion, the J558?m3 cells were pre-treated with CXCL12 prior to being added to a monolayer of TNF? activated bEND.3 endothelial cells, which normally express Cx43 (Figure 3-14B).  J558?m3-GFP cells adhere to the activated bEND.3 cells, but there was an increase in both the number of cells adhering to the monolayer and the size of the contact area between the B cell and endothelial cell when Cx43-GFP was expressed (Figure  151  3-14 C and D).  When the transfected cell line expressing the carboxyl-terminal tail truncated form of Cx43, J558?m3-Cx43?T-GFP, was similarly assayed, there was both an increase in adhesion and the size of the contact area between the B cell and endothelial cell compared with the GFP?alone transfected cells (J558?m3-GFP cells), but not to the same extent as cells expressing wild type Cx43-GFP (Figure 3-14C and D).  The relative size of the footprint/contact area for Cx43-GFP expressing cells appears larger than in GFP-only cells (J558?m3 transfected cells, Figure 3-14D, top panels).  To determine if this increase in adhesion was specific only for the J558?m3 plasmacytoma cell line, another Cx43-negative plasmacytoma cell line, 5TGM1 (Figure 3-3A), was transfected with the same Cx43-GFP constructs (Figure 3-8B).  Similarly, 5TGM1-Cx43-GFP cells showed both an increase in adhesion (Figure 3-14C, right set of columns) and in the size of the contacts with bEND.3 cells (Figure 3-14D, lower panels) compared to cells expressing GFP alone.  Expression of the carboxyl-terminal tail truncation of Cx43 (5TGM1-Cx43?T-GFP cells) failed to increase adhesion compared to WT Cx43-GFP (Figure 3-14C, D).  These results suggest that Cx43, and specifically the carboxyl-terminal domain, mediates cellular responses that are important for B cell adhesion to endothelial cells.   3.5 Discussion In this chapter, by using both loss- and gain- of function approaches we show that the GJ protein Cx43 plays a previously unappreciated role in B lymphocyte spreading and adhesion. Cx43 becomes phosphorylated after BCR signalling, enhances signals required for sustained BCR- mediated activation of the Rap1 GTPase and cell spreading, as well as  152  LFA-1 mediated Rap1 activation and spreading. Our results also show that the carboxyl-terminal tail of Cx43 is required for BCR, LFA-1 and CXCL12 mediated Rap1 activation, spreading and adhesion to endothelial cells. Immature and mature B lymphoma cell lines and normal splenic B cells, but not plasmacytoma tumor cell lines express Cx43.  This differing expression pattern over a developmental time course is similar to that seen in neurons.  In neuronal progenitors, which migrate during development, Cx43 levels are high.  In contrast, Cx43 levels are low in later stages of differentiation where migration is no longer necessary (Rozental et al., 2000).  In support of this, developing neurons require Cx43 expression to correctly migrate along radial glial cells in the cortex (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007) indicating that Cx43 is important for normal neuronal development.  Interestingly, Cx43 heterozygote mice show a decrease in the number of IgM positive B cells, supporting that Cx43 expression may play a role in normal B cell developmental processes (Montecino-Rodriguez and Dorshkind, 2001). This is consistent with the idea that a developing B cell interacts with stromal and endothelial cells during development, possibly with the assistance of connexin proteins.  However, when B cells undergo differentiation into the plasma cell stage, Cx43 expression is reduced. The reported reduction in B cell numbers seen in Cx43+\- mice, however, could be due to roles of Cx43 levels in the bone marrow stromal cells or other cells that affect B cell development.  A B cell specific knockout mouse would be required to resolve this issue. In B lymphocytes, both cell:cell contact and cytoskeletal rearrangements are required for optimal adhesion, spreading, immune synapse formation and for cell migration.  Although these processes are regulated by different receptors, they have the same fundamental requirements of being facilitated by changes in the actin cytoskeletal  153  architecture.  BCR-mediated spreading results from the activation of cytosolic signalling proteins, many of which are also involved in Cx43 regulation.  After BCR cross-linking, the ITAMs of Ig? and Ig? become phosphorylated by Src-family kinases leading to the recruitment of the Syk tyrosine kinase and the well-characterized recruitment and activation of the enzymes PLC?2, PI3K and the activation of the Ras/Rac/Rap pathways (Burkhardt et al., 1991; Engels et al., 2001; Gold et al., 1992; Gold et al., 1990; Ishiai et al., 1999; Lin et al., 2008; McLeod et al., 1998; Rowley et al., 1995a; Yamanashi et al., 1992).   The cytoplasmic domain of Cx43 is also modified by phosphorylation. It has been shown to be phosphorylated by c-Src, MAP kinases and PKC, resulting in changes to the state of the pore and life cycle/ turnover of the GJ complexes on the plasma membrane (Lampe et al., 2000; Loo et al., 1995; S?ez et al., 1997; Warn-Cramer et al., 1996).  BCR activation does alter the apparent molecular weight of Cx43 in B cells; however the residues that are modified in lymphocytes, as well as any functional consequences of the individual residues, have yet to be determined and are beyond the scope of this thesis.   A likely possibility for how Cx43 could be involved in the regulation of the B cell cytoskeleton is that it is being used as an adaptor/scaffold protein, providing sites for protein interactions with regions of its cytoplasmic tail required for BCR signalling. There are several reports of proteins that are ??associated? with Cx43 summarized below in Table 3-1 and the Cx43 interacting proteome in C6 glioma cells is being determined by the Naus lab (unpublished).     154  Table 3-1 Potential connexin-cytoskeleton interacting proteins.  A summary of previously identified proteins related to the cytoskeleton which may interact with Cx43.  Reprinted with permission from (Olk et al., 2009)   In J558?m3 cells, expression of Cx43 may allow these cells to activate normal B cell signalling pathways which are no longer required due their developmental stage, resulting in their ability to activate Rap1 and initiate B cell spreading.  When a truncated form of Cx43 (Cx43?T-GFP) was expressed, there was less BCR-mediated cell spreading, suggesting that this region of Cx43 may be the site of interaction with proteins that regulate the arrangements  155  of the actin cytoskeleton.  We have shown that Cx43 expression does have a dramatic effect on the activation of Rap1, which is essential for B lymphocyte spreading and immune synapse formation (Lin et al., 2008; McLeod et al., 2004).  Because Cx43 has no intrinsic enzymatic activity, it is likely that it is acting as an adaptor/scaffolding protein, recruiting proteins involved in the regulation of Rap1.  This function is partially abrogated when the C-terminal domain is deleted, resulting in the inability to maintain Rap1 activation and spreading in J558 cells.  This also suggests that there may be another domain within Cx43 that can act to enhance Rap1 activation, although the C-terminal domain is required for sustained Rap1 activation and spreading.   Cx43 may also act to stabilize the actin cytoskeleton by directly connecting to it, providing a stable platform from where spreading can be initiated.  In astrocytes, Cx43 interacts with the scaffolding protein drebrin (Butkevich et al., 2004),  which interacts with the cytoplasmic tail of Cx43 and stabilizes gap junctions by binding to the F-actin cytoskeleton.  Though B cells have not been shown to express drebrin, they do express other members of the actin binding protein (ABP) family drebrin-like proteins, including mABP-1.  These adaptors may link Cx43 to the actin cytoskeleton in B-lymphocytes.  It is also possible that Cx43 may facilitate changes in the cytoskeleton by passing molecules through its pore via a hemichannel or bona fide GJ.   It has previously been reported that B cells can form functional Cx43 GJs with follicular dendritic cells within germinal centers (Krenacs et al., 1997).  In our GOF system, Cx43-GFP and Cx43?T-GFP are likely present at the surface as a hexamer, as opposed to a monomer, where they are able to mediate its effects on Rap1 activation, B cell spreading and adhesion. The assembly of Cx43 into a hexamer (or connexon) has been reported to occur in the trans-golgi network  156  prior to trafficking to the surface (Musil and Goodenough, 1993).  Both Cx43-GFP and the C-terminal truncated Cx43-GFP that we are using were previously expressed in C6 glioma cells, were able to traffic to the membrane and were communication competent as determined by dye preloading techniques (Bates et al., 2007).  These results imply that there is no gross defect in hexamer formation and trafficking using these fluorescent-tagged Cx43 constructs.   Because our in vitro spreading assays test B cell spreading independent of GJ communication, this is likely not the mechanism of how Cx43 is involved in regulation of the B cell cytoskeleton.  We cannot rule out the possibility that Cx43 may be mediating these effects via a hemichannel-mediated mechanism; however, our results clearly indicate that the majority of Cx43?s contribution to cytoskeletal rearrangement is mediated though its C-terminal domain. We also show that both LFA-1 and CXCL12 mediated Rap1 activation, spreading and adhesion to endothelial cells is enhanced by Cx43 expression.  B lymphocytes bind to the endothelium by engaging two major integrins, VLA-4 and LFA-1 (Ley et al., 2007).  VLA-4 is required for slow rolling of B cells on the endothelium and LFA-1 is required for firm adhesion in preparation for transendothelial migration, however a possible role for Cx43 in B cell adhesion has not been investigated.  In developing neurons in the brain, Cx43 has been shown to be required for migration from the ventricular zone to the cortical plate where it was proposed that gap junction coupling is mediating adhesion of neurons to the radial glial cells, facilitating normal migration, however, there are conflicting results for the role of the C-terminal domain (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  Our results show that the C-terminal domain of Cx43 enhances both LFA-1 and CXCL12 mediated Rap1 activation.  We did, however, observe a transient increase in Rap1 activation, spreading and  157  adhesion when the C-terminal domain was truncated.  Because the small increase in LFA-1 mediated Rap1 activation and spreading was independent of gap junctional coupling, it is possible that there is another domain within Cx43 besides the C-terminal tail that is responsible for mediating this effect. One possible interpretation of the observed increase in adhesion of B cells expressing Cx43?T-GFP compared to GFP alone is that it is mediated by gap junction coupling.  Truncation of Cx43 in Xenopus oocytes has previously been show to form gap junctions that are resistant to uncoupling (Homma et al., 1998), and coupling has been proposed as the mechanism of Cx43?s effect on neuronal migration (Elias et al., 2007).  Though this effect cannot be completely ruled out, the effect of Cx43 coupling on lymphocyte adhesion is likely negligible compared to integrin-mediated adhesion as blockage of integrin activity results in gross defects in adhesion and homing (Abram and Lowell, 2009; Berlin-Rufenach et al., 1999; Lo et al., 2003). Because of this, the more likely possibility in our system is that Cx43 is regulating integrin-mediated functions, resulting in the observed changes in adhesion to endothelial cells.  In conclusion, in this chapter we have shown that Cx43 is important for BCR, LFA-1 and CXCL12 mediated Rap1 activation, resulting in enhancement of B cell spreading and adhesion.  This is the first reported data linking Cx43 to Rap1 activation, a key regulator of spreading, adhesion and migration.  This link may explain the role of Cx43 in other cell types, and its emerging role as a regulator of neuronal and glial migration. Cx43?s role in modulating BCR, integrin and chemokine-induced Rap1 activation and cytoskeletal rearrangement also makes it likely that Cx43 will play a key role in B cell development, trafficking and activation.  In the next chapter we show that Cx43 is not only involved in B  158  cell spreading and adhesion, but is an important regulator of B cell migration and associated biological processes.             159  4 The role of the GJ protein Cx43 in B cell motility, migration and transendothelial migration 4.1 Synopsis  In the previous chapter we show that the gap junction protein Cx43 is necessary for Rap1 activation, spreading and adhesion.  Since these processes are involved in B cell migration, the aim of this chapter is to determine if Cx43 is necessary for cell movement and migration.  Here, using a loss-of-function approach similar to the previous chapter, we show that knockdown of Cx43 protein levels leads to a significant decrease in B cell motility, directed migration toward a chemokine and the ability to migrate through an endothelial monolayer (transendothelial migration).    We also show that, similar to the gain-of-function results from the previous chapter, that the knockdown of Cx43 leads to a decrease in sustained integrin (VLA-4) and chemokine (CXCL12) mediated Rap1 activation.  Consistent with the previous chapter, these results indicate that Cx43 is an important regulator of B cell motility and directed migration.  4.2 Introduction  The regulation of B cell migration is essential for B cell development and for the immune response.   In order to develop into mature B cells, the majority of immature B cells have to migrate from the bone marrow to the spleen, where they come in contact with signals that direct their further development (Pillai and Cariappa, 2009).  Once in the spleen, or other secondary lymphoid organs, there is choreographed migration to different niches within that organ which not only directs further development, but also the interaction with Ag (Pillai and  160  Cariappa, 2009).  The regulation of these movements is determined by the interaction between gradients of chemokines and the receptors located on the B lymphocytes (Cyster, 2010).  Within the bone marrow, B cell precursors are retained due to their interaction with the chemokine CXCL12 (Cyster, 2003).  Bone marrow stromal cells produce CXCL12, which signals pro- and pre-B cells to stay in contact with the stromal cells, and may also act by directly stimulating growth (Cyster, 2003).  As the B cell precursors express the IgM form of the BCR on their surface, they downregulate their responsiveness to CXCL12, allowing them to home along a chemokine gradient to the spleen (Cyster, 2003, 2010).  In order to move from one organ to another, the developing B cells need to be able to transit into and out of the blood stream.  This is accomplished through the biological process known as transendothelial migration (TEM) (Ley et al., 2007).  When the migrating B cells come in contact with an endothelial barrier that stands between it and its destination, the B cell undergoes a well characterized series of events which involve tethering and rolling which are mediated by selectins, activation by chemokines on the surface of the endothelial cell, integrin-mediated arrest, spreading, crawling and ultimately transmigration (Ley et al., 2007). Along with its role in cell-cell communication, the gap junction protein Cx43 has also been identified as playing an important role in glioma and neuronal cell motility and migration (Bates et al., 2007; Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  Knockdown of Cx43 expression by in utero transfection of Cx43 shRNA, or using a neuron-progenitor specific conditional knockout resulted in decreased neuronal migration, however the role of the different domains of Cx43 in this process remains unclear (Cina et al., 2009; Elias et al., 2010; Elias et al., 2007).  Cx43 has previously been studied in B cell  161  transendothelial migration, where it has been shown that B cells and endothelial cells form functional gap junctions (Oviedo-Orta et al., 2002).  Using an endothelial monolayer loaded with a cytosolic dye which is GJ permeable, B cells accumulated the dye as they passed through the monolayer; however, blocking the GJ pore, severely limiting GJ intercellular communication, had no effect on TEM (Oviedo-Orta et al., 2002).   In the previous chapter, we show that Cx43 plays an important role in the sustained activation of BCR-mediated Rap1 GTPase and spreading, as well as integrin-mediated Rap1 activation, cell spreading and adhesion to an endothelial monolayer.  Given that Cx43 plays an important role in neuronal and glial cell migration, and that B cells form functional GJs between themselves and endothelial cells during the process of TEM, we hypothesized that Cx43 plays an important role in B cell migration.  Using a combination of microscopy and loss-of-function assays, we provide evidence in this chapter that Cx43 is linked to the cytoskeleton in B cells, and that it is necessary for B cell motility, CXCL12-mediated Rap1 activation, directed migration and transendothelial migration.  These results demonstrate that Cx43 is also an important regulator of B cell migration.  4.3 Methods and materials 4.3.1 Plasmids Expression vectors encoding cDNA for Rat wild type Cx43 (NAP2-Cx43GFP) has been described in section 3.3.1.   Retroviral vectors encoding Cx43 shRNA constructs 1 and 2, as well as the corresponding scrambled sequence, have been described previously in  162  section 3.3.4.   The expression vector encoding actin-GFP (C1EGFP) was obtained from Dr. M. Gold, (University of British Columbia, Vancouver BC).  4.3.2 Antibodies and inhibitors Polyclonal rabbit anti-Cx43, which recognizes an epitope in the C-terminal domain (amino acids 363-382), was from Sigma-Aldrich and was described in the previous chapter.  Mouse anti-?-actin and rabbit anti-ZO-1 were from Sigma-Aldrich.  The rabbit anti-Rap1A/1B was from Cell Signalling Technology.  Goat anti-mouse IgG conjugated to horseradish peroxidase (HRP) was from Invitrogen Canada Inc, goat anti-rabbit IgG-HRP was from Jackson ImmunoResearch Labs and the Alexa Fluor 488-conjugated goat anti-Rabbit IgG was from Invitrogen Molecular Probes.  Rhodamine-coupled phalloidin was from Invitrogen Life Technologies.  DMSO was from MP Biomedical, LLC (Solon, OH).  Cytochalasin D was from Enzo Life Sciences International, Inc. (Plymouth Meeting, PA).  4.3.3 Cells and growth conditions The WEHI 231 B lymphoma, the mouse brain endothelioma bEND.3 and the mouse lymphoid endothelial SVEC4-10 cell lines were obtained from ATCC.  Cells were cultured in high glucose (4.5 g/L)  RPMI-1640 (WEHI 231) or DMEM (bEND.3 and SVEC4-10) supplemented with 2 mM L-glutamine, 110 mg/L sodium pyruvate , 10% heat-inactivated FBS, 50 units/mL of pen/strep. Retroviral transduction of WEHI 231 cells was performed as in section 3.3.4.    163  4.3.4 B cell stimulation and preparation of cell extracts  Cells were washed with PBS, resuspended at 5 x 106 cells/ml in modified Hepes-buffered saline (25 mM sodium Hepes pH 7.4, 125 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM Na2HPO4, 0.5 mM, 1 g/L glucose, 2 mM glutamine, 1 mM sodium pyruvate, 50 ?M 2-ME), and stimulated with 100 nM CXCL12 (R&D systems, Minneapolis, MN). Reactions were stopped by adding 1 ml cold PBS.  Cells were then lysed in cold lysis buffer (PBS, 1% Triton-X 100, 1% IGEPAL (Sigma-Aldrich), 50 mM CaCl2) (Troxell et al., 1999) containing protease inhibitors (10 ?g/ml leupeptin, 1 ?g/ml aprotinin, 1 mM pepstatin A, 1 mM Na3VO4, 1 mM PMSF).  To extract Cx43, cells were lysed using 200 ?l of lysis buffer, sonicated for 10 seconds, and incubated at 37?C for 30 min before being analyzed by SDS-PAGE.   Dephosphorylation of Cx43 was performed as follows.  WEHI 231 cells were resuspended to 1x107 cells in 500 ?l quinsaline per timepoint.  Cells were stimulated for 0, 5, 10, 15, 30, 60, and 120 min 100 nM CXCL12 at 37oC.  Reactions were stopped using 500 ?l cold PBS.  Cells were centrifuged at 1500 RPM at 4oC for 2 min, supernatants were removed and were lysed as previously described.  Protein concentration of the cell lysates was determined using a BCA assay (Thermo Scientific Pierce, Rockford, Illinois).  For each timepoint, lysates were split into two sets containing 30 ?g of protein each, one subjected to dephosphorylation and the other used as a control.  Lysates used as a control were suspended in 1X NEBuffer (New England BioLabs Inc, Ipswich, Massachusetts).  Dephosphorylation of the remaining lysates was carried out using 1X NEBuffer and 30 units of Calf Intestinal Alkaline Phosphatase (CIP) (New England BioLabs Inc, Ipswich, Massachusetts) as per  164  manufacturer?s instructions.  Lysates were incubated for 60 min at 37oC and subsequently suspended in 1X SDS-PAGE reducing sample buffer and incubated for 30 min at 37oC.  4.3.5 Rap1 activation assay Rap1 activation assays were performed as described in section 3.3.6.     4.3.6 Mobility of Cx43-GFP and actin-GFP in transfected WEHI 231 B cells using fluorescence recovery after photobleaching (FRAP). Cx43-GFP transduced WEHI 231 B cells were incubated on glass bottom microwell dishes (MatTek, Ashland, MA) that were coated with 10 ?g/ml fibronectin overnight at 37?C, 5% CO2 in complete media.  The following day, FRAP was assessed using an Olympus FV 1000 confocal microscope (LSI Imaging).  A circular ROI was drawn on the cell periphery and fluorescent intensity was determined every 10 seconds.  The average of the first two intervals was used as the ?pre-bleach? intensity.  The ROI was photobleached using the tornado setting, and the fluorescent intensity at this point was used as the ?bleached? intensity.  The fluorescent recovery in this ROI was determined using the formula ([intensity at a given timepoint]-[bleached intensity])/([pre-bleach] ? [bleached]).  Addition of 10 ?M Cytochalasin D or an equivalent volume of DMSO was done 10 min prior to FRAP.  FRAP was quantified and visualized using Microsoft Excel.   WEHI 231 B cells were transfected with actin-GFP using an AMAXA nucleoporator as per the manufacturers instructions.  Eighteen hours post transfection, the transfected WEHI 231 B cells were incubated for 3 hours at 37?C, 5% CO2 in complete  165  media on glass bottom microwell dishes (MatTek) that were coated with 10 ?g/ml fibronectin.  In actin-GFP postitive cells, FRAP was assessed using an Olympus FV 1000 confocal microscope.  A circular ROI was drawn on the cell periphery and fluorescence intensity was determined every 5 seconds.  The average of the first two intervals was used as the ?pre-bleach? intensity.  The ROI was photobleached using the tornado setting, and the fluorescence intensity at this point was used as the ?bleached? intensity.  The fluorescence recovery in this ROI was determined using the formula ([intensity at a given timepoint]-[bleached intensity])/([pre-bleach] ? [bleached]).   FRAP was quantified and visualized using Microsoft Excel.   4.3.7 TIRF microscopy  Colocalization of Cx43-GFP and F-actin was accomplished using TIRF microscopy.  Cx43-GFP transduced WEHI 231 B cells were incubated overnight at 37?C, 5% CO2 in complete media on glass bottom microwell dishes (MatTek, Ashland, MA) that were coated with 10 ?g/ml fibronectin.  The following day the cells were fixed with 4% paraformaldehyde and permeablized in PBS containing 0.5% Triton-X 100 for 10 min at room temperature (RT).   The samples were blocked with PBS containing 3% BSA for 30 min at RT then incubated with rhodamine-phalloidin (1:40 in PBS plus 3% BSA) for 20 min at RT, rinsed three times in PBS and then left in PBS for imaging.  TIRF was accomplished using a Zeiss spinning disk confocal microscope and the Slidebook version 5.0 software (both courtesy of LSI Imaging).      166  4.3.8 Cell motility assays  An overview of the cell motility assay is presented in Figure 4-11.  Glass coverslips (18 mm) were coated for 3 h at RT or overnight at 4?C with 10 ?g/ml bovine fibronectin (Invitrogen) in 1 X PBS.  The coverslips were washed and coated with fluorescent blue fluospheres (Invitrogen).  B cells were added to the coverslip and incubated for 18 hr at 37?C, 5% CO2 in complete media, after which the cells were fixed in 4% paraformaldehyde for 20 min. F-actin was visualized using rhodamine-phalloidin.  Individual cell tracks were observed using a confocal microscope with a 20X objective and the area of the beads that were cleared were quantified using ImagePro software.  Figure 4-1 Overview of B cell motility assay described in Section 4.3.8 Motile B cell is depicted in Red, the blue represents the blue flurospheres and the black area represents the cleared bead path.  167  4.3.9 B cell migration  An overview of the transwell migration assay is presented in Figure 4-2.   Transwell inserts (5 ?m pore size) (BD Biosciences, Mississauga, ON) were coated with 2 ?g/ml bovine fibronectin (Sigma) for 3 hours at RT or overnight at 4?C.  Filters were washed with 1X PBS, and placed in a 24 well dish (BD Biosciences) with 600 ?l chemotaxis buffer (RPMI 1640, 10 mM HEPES, 0.5% BSA and 2% FBS).  For cell migration, WEHI 231 B cells were added to the top chamber, diluted to 5x105 cells in 100 ?l chemotaxis buffer with no chemokine in the bottom chamber, or 100 nM CXCL12 in the bottom chamber (for total number of cells, 5x105 cells were added to a well with no transwell chamber).  The cells were incubated for 4 hours at 37?C, 5% CO2.  The cells from the bottom chamber were then collected, centrifugated at 1500 RPM for 5 min and resuspended in 500 ?l FACS buffer (1X PBS with 10% FBS).  The percentage of cells migrating across the transwell into the bottom chamber was determined using a FACS LSRII (UBC Flow Cytometry Facility).  Cells were counted for 1 min and the different migration conditions were compared to the total number of cells where percent migration = ([number of cells counted from migration condition]/[number of cells counted from total well]) X 100.   168   Figure 4-2 Overview of transwell migration assay described in Section 4.3.9  4.3.10 Transendothelial migration of WEHI 231 B cells  Transendothelial Migration (TEM) of WEHI 231 B cells was determined using a modified version of the migration assay previously described (Figure 4-3).  bEND.3 or SVEC4-10 cells were grown in 10 ng/ml TNF-? (eBioscience, San Diego, CA) as a single monolayer on top of an 8.0 ?m pore transwell filter (BD Biosciences).  The transwell inserts with the endothelial monolayer were added to wells of a 24 well dish with each well containing 600 ?l of complete DMEM, with or without 50 nM CXCL12 in the bottom chamber.  5x105 WEHI 231 B cells resuspended in 100 ?l DMEM were added to the top chamber.  The cells were incubated for 24 hours at 37?C, 10 % CO2.  The percentage of the cells that migrated into the bottom chamber was determined as described for the migration assay.  169   Figure 4-3 Overview of transendothelial migration assay described in Section 4.3.10  4.3.11 Immunofluorescence  Non-TIRF immunofluorescence images were taken using standard protocols on an Olympus FV1000 confocal microscope.  Primary and secondary Ab pairs were as follows: Rabbit anti-ZO-1: Goat anti-Rabbit conjugated to Alexafluor488; Rabbit anti-Cx43: Goat anti-Rabbit conjugated to Alexafluor488.   The F-actin cytoskeleton was visualized using Rhodamine phalloidin.  4.3.12 Statistics A comparison of means was assessed using a student?s unpaired, two-tailed t test.    170  4.4 Results 4.4.1 Cx43 expression is necessary for maximal VLA-4-mediated Rap1 activation and migration on fibronectin   Lymphocyte motility is an important process in migration and normal B cell trafficking within lymphoid organs (Cahalan and Parker, 2008).  Because we observed that B cell spreading and Rap1 activation were dependent on the expression of Cx43, we hypothesized that Cx43 was also necessary for B cell motility.  To show that Cx43 is involved in B cell motility, Cx43 levels in WEHI 231 B cells were knocked down using shRNA.  Transduction of WEHI 231 cells with Cx43 shRNA construct 2 (specific for the cytoplasmic loop), as well the combination of shRNA constructs 1 (specific for the third transmembrane domain) and 2 led to a reduction in Cx43 protein levels compared to nonsense shRNA transfected cells (Figure 4-4A).    The process of cell motility on an extracellular matrix has been previously shown to be dependent on the activation of Rap1 (Freeman et al., 2010).  To assess whether Cx43 was required for normal B cell motility, we first assayed the ability of WEHI 231 B cells transfected with Cx43 shRNA to activate Rap1 in response to outside-in signalling through the fibronectin-binding VLA-4 integrin.    VLA-4 clustering using a soluble anti-?1 integrin led to sustained Rap1 activation in WEHI 231 Cx43 nonsense transfected cells after 5 min (Figure 4-4B, left-most series).   Compared to WEHI 231 cells transfected with the nonsense shRNA construct, cells transfected with shRNA 2 alone or with shRNA 1 + 2 constructs were unable to effectively sustain VLA-4-mediated Rap1 activation (Figure 4-4B, middle and rightmost series respectively).  These cells were then assayed for their ability to move on a fibronectin-coated glass coverslip over 18 hours.  To measure the degree of motility, the  171  fibronectin-coated coverslips were also coated with fluorescent-blue fluospheres.  As the cells move along the fibronectin, they push away the fluospheres leaving a cleared path that can then be measured.  The mean area of the cleared path of WEHI 231 Cx43 nonsense cells was 1470 ?m2 where as WEHI 231 Cx43 shRNA 2 and shRNA 1 + 2 were significantly lower at 608 ?m2 and 730 ?m2 respectively (Figure 4-4C and quantified in D).  This change in motility was not due to a change in actin polymerization/turnover (Figure 4-5).  Actin-GFP was expressed in Cx43-nonsense and shRNA construct 2 transfected WEHI 231 B cells and actin-GFP turnover was assessed using FRAP (Figure 4-5A).   After incubation on fibronectin-coated coverslips for 3 hours, there was no significant change in FRAP of actin-GFP between the nonsense shRNA and Cx43 shRNA construct 2 transduced cells (Figure 4-5B).  These results indicate that Cx43 is required for efficient B cell motility, but not for F-actin turnover.   172    173   Figure 4-4 Motility of WEHI 231 B cells decreases after Cx43 expression is knocked down. A) Expression of Cx43 in WEHI 231 cells after transduction with nonsense shRNA, Cx43 shRNA construct 2 or Cx43 shRNA constructs 1 + 2.  B) Rap1 activation in WEHI 231 cells transfected with Cx43 nonsense, Cx43 shRNA 1 + 2 or Cx43 shRNA 2.  Cells were  174  stimulated for the indicated times with anti-?1 and activated Rap1 was precipitated using a GST-RalGDS fusion protein and total Rap1 detected by blotting with anti-Rap1 Abs. Representative experiment of three similar, independent replicates. C) WEHI 231 cells were incubated on 10 ?g/ml fibronectin-coated glass coverslips covered in fluospheres (blue).  After 18 hours, the coverslips were fixed and the cytoskeleton of the B cells was visualized with rhodamine phalloidin (red).  The motility tracks are identified as the bead-free zones (black).  D) Quantification of the area cleared of beads by the WEHI 231 B cells (pooled data from three independent experiments,   error bars represent standard error of the mean, * denotes P < 0.05).  175   Figure 4-5 Knockdown of Cx43 does not alter actin-GFP dynamics A) Actin-GFP (green) transfected nonsense (left) or Cx43 shRNA construct 2 (right) WEHI 231 B cells were incubated on a 10 ?g/ml fibronectin coated glass coverslip for 3 hours prior to FRAP (scalebar = 10 ?m). Circle indicates the area photobleached.  The panels on the right of each large image show the post-bleached area at the indicated timepoints.     176  B)  Quantification of fluorescent recovery after photobleaching (pooled data from 3 independent replicates, n = 79 for Cx43 nonsense, n = 82 for Cx43 shRNA construct 2, error bars represent standard error of the mean, there was no statistical difference between the two groups).  4.4.2 Cx43 is necessary for CXCL12 activation of Rap1 and migration B lymphocytes respond to the chemokine CXCL12 by activating Rap1 GTPase and moving along a chemokine gradient (McLeod et al., 2002). Because Cx43 plays an important role in B cell motility, we hypothesized that it was also required for chemokine-induced migration. The initiation of signalling by CXCL12 caused an increase in the molecular weight of Cx43 in WEHI 231 B cells on an SDS-PAGE gel (Figure 4-6A, left panels), similar to what we observed in Chapter 3 after BCR signalling.  This change in molecular weight was first seen 5 minutes after addition of CXCL12, and persisted for 15 minutes where it began to decline.  The bandshift was likely due to phosphorylation as treatment of the cell lysate with CIP prior to SDS-PAGE reduced the amount of higher molecular weight bands (Figure 4-6A, right panels).   This indicates that Cx43 is likely a downstream target of CXCL12 (chemokine)/CXCR4 (chemokine receptor) signalling.    The activation of Rap1 GTPase is critical for chemokine-induced migration in B lymphocytes (McLeod et al., 2002).  To determine if Cx43 plays a role in CXCL12-induced Rap1 activation, WEHI 231 B cells transduced with nonsense shRNA, Cx43 shRNA construct 2 or a combination or Cx43 constructs 1 and 2 were activated with 100 nM CXCL12 and activated Rap1 was assayed for.  Compared to WEHI 231 cells transfected with nonsense shRNA, cells transfected with shRNA 1 + 2 constructs and shRNA 2 alone exhibited an inability to sustain Rap1 activation (Figure 4-6B).  These results show that Cx43 is required for efficient and sustained CXCL12 mediated activation of Rap1.  177  Binding of the chemokine CXCL12 to its receptor, CXCR4, induces a strong migratory response in WEHI 231 B cells in a Rap1 dependent manner (McLeod et al., 2002).   To determine if Cx43 plays a role in B cell migration, WEHI 231 B cells transduced with nonsense shRNA, Cx43 shRNA construct 2 or a combination of Cx43 shRNA constructs 1 and 2 were assayed for their ability to move across a 5.0 ?m pore transwell filters coated with 2 ?g/ml of fibronectin towards a CXCL12 gradient (100 nM).  Without the addition of CXCL12 to the bottom chamber of the transwell, there was negligible migration after 4 hours of all three cell lines (Figure 3C).  When a CXCL12 gradient was established, there was a twofold decrease in migration across the transwell of both of the Cx43 knockdown cell lines compared to the nonsense shRNA transduced WEHI 231 cells after 4 hours (Figure 4-6C).  These results indicated that Cx43 is required for efficient migration toward a CXCL12 gradient.  178    179   Figure 4-6 Cx43 is a target of chemokine signalling and important for expression in CXCL12 mediated Rap1 activation and cell migration of WEHI 231 B cells  A) CXCL12 signalling leads to phosphorylation of Cx43.  Cell lysates were prepared from WEHI 231 B cells after incubation with CXCL12 (100 nM) for the indicated times.  The  180  lysates were then divided into two equal portions, one was treated with calf intestinal phosphatase (CIP) (right panels) and one was not (left panels).  The lysates were separated by SDS-PAGE and analyzed by western blotting.  Cx43 was detected using an Ab specific for the Cx43 cytoplasmic tail.  Re-probing bots with anti-Actin Abs was used as a loading control.  Representative experiment of three similar, independent experiments.   B) Rap1 activation in WEHI 231 Cx43 nonsense, WEHI 231 Cx43 shRNA 1 + 2 and WEHI 231 Cx43 shRNA 2 cell lines after CXCL12 activation.  Cells were stimulated for the indicated times with CXCL12 (100 nM) and activated Rap (Rap1-GTP) was captured using a GST-RalGDS fusion protein. Representative experiment of three similar, independent replicates.  C) Transwell migration assay of WEHI 231 B cells transduced with nonsense shRNA, Cx43 shRNA construct 2 and Cx43 shRNA construct 1 + 2 towards CXCL12.  5 X 105 WEHI 231 cells were loaded into the top chamber of a transwell insert with no CXCL12 in the bottom chamber or with 100 nM in the bottom chamber.  The percentage of the total input cells that migrated to the bottom chamber after 4 hours was expressed as the number of cells in the bottom chamber/the total number of cells added (cells added with no transwell insert) (pooled data from four independent experiments,  error bars represent standard error of the mean, * denotes P < 0.05).  4.4.3 Cx43 is important for transendothelial migration TEM is an important biological process for B cells migrating from one tissue to another, however the role of Cx43 in this process is not completely understood (Ley et al., 2007).  It has been previously shown that blockage of the Cx43 pore using a mimetic peptide has no effect on B cell TEM, however the importance of regions of Cx43 other then the pore in this process was not addressed (Oviedo-Orta et al., 2002).  In order to assess whether Cx43 is required for TEM, a modified transwell migration assay was used similar to the one described in (Lin et al., 2009).  Briefly, endothelial monolayers were grown to confluency on 8.0 ?m pore sized transwell filters and the cells were activated overnight with TNF?.  WEHI 231 B cells transduced with nonsnense shRNA, Cx43 shRNA construct 2 or a combination of Cx43 shRNA constructs 1 and 2 were added to the top chamber and CXCL12 was added to the bottom chamber.  After 18 hours, the number of WEHI 231 B cells that migrated though  181  the monolayer and filter, into the bottom chamber was quantified. When bEND.3 cells are grown to a monolayer, they have a long, spindle-shaped morphology, form tight junctions as determined by ZO-1 staining, and have an accumulation of Cx43 at perinuclear regions as well as cell:cell contacts (Figure 4-7A). When transendothelial migration across a bEND.3 monolayer was assessed, there was a two-fold decrease in the number of cells that migrated across the endothelium toward CXCL12 when Cx43 protein levels were knocked down (Figure 4-7B).  To ensure that this was not specific to bEND.3 endothelial cells, transendothelial migration across another endothelial cell line, SVEC4-10, was assessed.  These cells, when grown to a monolayer, have a cuboidal morphology, large tight junctions as determined by ZO-1 staining, and a Cx43 distribution similar to that of bEND.3 cells (Figure 4-7C), although the perinuclear staining is less obvious in the image shown.  When TEM across these cells was assessed, there was a 2-3 fold decrease when Cx43 expression was knocked down (Figure 4-7D).  The overall decrease in the number of WEHI 231 cells migrating across the SVEC4-10 monolayer compared to the bEND.3 monolayer was likely due to the cell morphology (a smaller edge:cell area ratio) and the strength of the tight junctions (SVEC4-10 cells having a much thicker/denser/brighter ZO-1 staining compared to bEND.3 cells).  These results indicate that Cx43 is important for B cell TEM.  182    183    184  Figure 4-7 Knockdown of Cx43 expression in WEHI 231 B cells results in a decrease in transendothelial migration (TEM) across two different endothelial cell layers.  A) Immunofluorescence staining of a bEND.3 monolayer showing ZO-1 (green, top left pannel), Cx43 (green, top right pannel) and the F-actin cytoskeleton (red) (Scale bar: 10 ?m).  B) TEM of WEHI 231 B cells transduced with nonsense shRNA, Cx43 shRNA construct 2 or Cx43 shRNA constructs 1 and 2 through a bEND.3 monolayer.  A monolayer of TNF? activated bEND.3 endothelial cells was grown on top of the filter of a transwell insert and 5 x 105 WEHI 231 cells were loaded into the top chamber with no CXCL12 in the bottom chamber or 50 nM in the bottom chamber.  The percent of cells that migrated through the monolayer to the bottom chamber after 18 hours is expressed as the number of cells in the bottom chamber/the total number of cells added (cells added with no transwell insert) (pooled data from six independent experiments, error bars represent standard error of the mean, * denotes P < 0.05).    C) Immunofluorescence staining of a SVEC4-10 monolayer showing ZO-1, Cx43 (labeled as in A above) and the F-actin cytoskeleton (red) (Scale bar: 10 ?m).   D) TEM of WEHI 231 B cells through a SVEC4-10 monolayer.  A monolayer of TNF? activated SVEC4-10 endothelial cells was grown on top of the filter of a transwell insert and 5 x 105 WEHI 231 cells were loaded into the top chamber with no CXCL12 in the bottom chamber or 50 nM in the bottom chamber.  The percent of cells that migrated through the monolayer to the bottom chamber after 18 hours is expressed as the number of cells in the bottom chamber/the total number of cells added (cells added with no transwell insert) (pooled data from four independent experiments, error bars represent standard error of the mean, * denotes P < 0.05).    4.4.4 Cx43 membrane localization is determined by the F-actin cytoskeleton The gap junction protein Cx43 has been previously linked to the F-actin cytoskeleton in multiple cell types including astrocytes, tenocytes and epithelial cells (Butkevich et al., 2004; Vitale et al., 2009; Wall et al., 2007).  To determine if Cx43 is interacting with the F-actin cytoskeleton in B lymphocytes, the colocalization of Cx43-GFP and F-actin was assessed using TIRF microscopy.  WEHI 231 B cells transduced with Cx43-GFP were incubated on10 ?g/ml fibronectin coated glass coverslips for 24 hours and subsequently stained and imaged.  Cx43-GFP accumulated at the periphery of the spreading cell, along with F-actin, where there was a strong co-localization between the two (Figure 4-8A).  To  185  determine if there was a direct link between Cx43-GFP and the F-actin cytoskeleton, the mobility of Cx43-GFP was assessed with and without disruption of the cytoskeleton using FRAP.  WEHI 231 B cells were transduced with Cx43-GFP and FRAP was assessed at the periphery of cells incubated on fibronectin-coated coverslips for 24 hours.  Cx43-GFP at the periphery showed a maximal mean recovery after 170 seconds, with an approximate 50% immobile fraction (Figure 4-8B, top).  After treatment with 10 ?M cytochalasin D, there was a decrease in the mobility of Cx43-GFP at the periphery of the cell, with an approximate 62% immobile fraction (Figure 4-8B, bottom).  There was, however, a change in the localization of Cx43-GFP after disruption of the cytoskeleton.  Cx43-GFP redistributed to large, membrane aggregates which were not located at the periphery of the cell after cytoskeletal disruption (Figure 4-8B, bottom).  These results suggest that there is a functional association between the F-actin cytoskeleton and Cx43-GFP, and the membrane localization of Cx43 is determined by the F-actin cytoskeleton.    186   Figure 4-8 Mobility of Cx43-GFP decreases after cytochalasin D disruption of the cytoskeleton.   A) Colocalization of F-actin (red) and Cx43-GFP (green) by TIRF microscopy.  WEHI 231.Cx43-GFP cells were incubated on glass coverslips coated with 10 ?g/ml of fibronectin for 24 hours before imaging (Scale bar: 10 ?m).  B)  FRAP of Cx43-GFP (green) transfected WEHI 231 cells incubated on 10 ?g/ml of fibronectin for 24 hours treated with DMSO alone (top) or cytochalasin D (bottom) (Scale bar: 10 ?m).  Circle indicates the area photobleached. C)  Quantification of fluorescent recovery after photobleaching (pooled data from 3 independent replicates, n = 45 for DMSO, n = 53 for cytochalasin D, error bars represent standard error of the mean, * denotes P < 0.05).   187  4.5 Discussion In this study, we show that the GJ protein Cx43 plays an important role in B cell motility, migration and transendothelial migration.  Using a loss-of-function approach we show that Cx43 is required for sustained VLA-4 and CXCL12-mediated Rap1 activation as well as both motility on fibronectin-coated coverslips and CXCL12-mediated Rap1 activation and migration across a transwell filter.  Consistent with these results,  transendothelial migration across an endothelial monolayer was also impaired when Cx43 protein levels are knocked down.   We also show, using a fluorescently-tagged Cx43 that the membrane distribution of Cx43 is determined by the F-actin cytoskeleton and that disruption of the F-actin cytoskeleton results in a redistribution of Cx43-GFP from the periphery of a spreading cell to large membrane aggregates. These results indicate that Cx43 is an important regulator of B cell migration.  B cell migration is a very complex process which is regulated by the response to chemokines, interaction with integrin ligands on other cells and within basement membranes, and intracellular signalling cascades (Ley et al., 2007; Shulman et al., 2009).  In order to address if Cx43 is involved in B cell migration, we divided migration into three basic elements: the ability to move (motility), the ability to move towards a chemokine (migration) and the ability to migrate through an endothelial monolayer (TEM) and asked whether Cx43 plays a role in that process.   B cell motility is an important component of B cell immune responses.  The ability of a B cell to move laterally along endothelial cells in order to find sites permissive for TEM as well as movement around and within lymphoid tissues is essential to normal B cell functioning (Allen et al., 2007b; Cahalan and Parker, 2008; Ley et al., 2007; Miller et al.,  188  2002).  Here, using a loss-of-function approach, we observed a defect in the ability to sustain Rap1 activation after initiating outside-in signalling.  These results are similar to our previous findings describing the importance of Cx43 expression to LFA-1-mediated Rap1 activation and adhesion using the gain-of-function J558 B cell plasmacytoma cell line (section 3.4.5).  In accordance with these results, a decrease in integrin-mediated B cell motility when Cx43 was knocked down was observed.  A similar phenotype was observed in other studies assaying the role of Cx43 in neuronal migration (Cina, 2007; Cina et al., 2009; Elias et al., 2010; Elias et al., 2007) with one exception, that using this approach, the change in motility is independent of gap junctional cell to cell coupling.  Because the reduction in motility occurs in the absence of a basement cell layer, it is likely that Cx43 is involved in the signalling pathways leading to Rap1 activation and cell motility, as opposed to directly facilitating motility via passage of small molecules through GJ intercellular communication or by physically coupling GJ.  B cell motility on endothelial cells requires the integration of outside-in integrin signalling and chemokine-mediated inside-out signalling resulting in high affinity integrin activation and cytoskeletal remodeling (Shulman et al., 2009). We show that Cx43 is important for integrin-mediated B cell motility, however whether it is required for integrating chemokine signals is not known. To assess whether Cx43 is required for integrating chemokine signals, we assayed the response of WEHI 231 cells to the chemokine CXCL12.  The chemokine CXCL12 binds to the CXCR4 receptor on B cells where it activates kinases and Rap1 GTPase resulting in the activation of integrins and polarization of the cell towards the signal (Abram and Lowell, 2009; Kinashi, 2005) .  When we assayed whether Cx43 was affected by CXCL12 signalling, we observed that it became phosphorylated after activation  189  with CXCL12.  The cytoplasmic domain of Cx43 is highly regulated by phosphorylation via c-Src, MAP kinases and PKC, resulting in changes to the state of the pore and life cycle/ turnover (Lampe et al., 2000; Loo et al., 1995; S?ez et al., 1997; Warn-Cramer et al., 1996). Though it is not known which of these kinases are required for the CXCL12-mediated phosphorylation of Cx43, it is likely that it is involved in the signalling pathway.   Activation of the Rap1 GTPase is required for normal B cell migration and cytoskeletal rearrangements in response to both chemokine and integrin-mediated signals (Lin et al., 2008; McLeod et al., 2002; Tse et al., 2009).   Because we observed that integrin-mediated Rap1 activation and motility was reduced after knockdown of Cx43 and that Cx43 was a target of CXCL12 signalling, we hypothesized that Cx43 was involved in CXCL12-mediated Rap1 activation.   Knockdown of Cx43 expression dramatically reduced the CXCL12-mediated activation of Rap1, indicating that it is important for Rap1 activation.  Because Cx43 has no intrinsic enzymatic activity, it is likely that it is acting as an adaptor/scaffolding protein, recruiting proteins involved in the regulation of Rap1.  Since activated Rap1 is required for normal B cell migration towards CXCL12 (McLeod et al., 2002), we assessed whether knockdown of Cx43 had a similar effect.  There was a significant decrease in migration through a fibronectin-coated transwell insert towards a CXCL12 gradient, indicating that it is important for normal B cell migration.  The most characterized role of the gap junction protein Cx43 is in forming channels between cells.  Though it has previously been shown that B cells and endothelial cells form functional gap junctions during the process of transendothelial migration, blockage of the pore had no effect on it (Oviedo-Orta et al., 2002).  What has not been investigated is if the level of Cx43 protein expression was important for this process.  Because we observed that  190  Cx43 was required for B cell motility and migration, we hypothesized that it was also important in TEM.  When we assayed the effect of Cx43 knockdown on B cells ability to undergo TEM though an endothelial monolayer, we observed that there was a significant decrease in their ability to traverse across.  The most probable hypothesis here is that Cx43 is acting as a component in the signalling pathways required for TEM, likely Rap1 GTPase activation. This is consistent with the finding that Rap1 inactivation significantly inhibits TEM of B cells through an endothelial monolayer (Lin et al., 2009).  It is also possible that in conjunction with the activation of Rap1, that there is another function for Cx43 in B cell TEM.  When assaying the role of Cx43 in neuronal migration, Elias et al. observed the Cx43 was required for cortical cell adhesion and that there was a colocalization of Cx43 and actin, suggesting that Cx43 was mediating adhesion though interaction with the F-actin cytoskeleton (Elias et al., 2007).  We observed a similar co-localization between Cx43 and F-actin using TIRF microscopy and have previously found that the C-terminal domain of Cx43 is important for integrin-mediated spreading and adhesion to endothelial cells (section 3.4.5 and section 3.4.6).  It is possible that Cx43 may be both enhancing adhesion to the endothelium and provide a platform for the integration of signals required for B cell motility, migration and TEM. One potential mechanism for how Cx43 may be mediating the observed effects on B cell migration is by opposing the retrograde force generated when polymerizing F-actin drives the lamellopodia forward.  In a normal non-lymphoid migrating cell, the retrograde force gererated when polymerizing F-actin at the leading edge pushes the membrane forward is opposed by integrin-mediated focal adhesions to the ECM (Giannone et al., 2007).  Lymphocytes, however, do not form focal adhesions, but have a diffuse distribution of  191  integrins linking the ECM to the F-actin cytoskeleton (Smith et al., 2007).  It is possible in lymphocytes, Cx43 coupling to the F-actin cytoskeleton at sites of actin polymerization may aid integrins in opposing this retrograde force, allowing for lamellapodia formation and migration to occur.   To address whether Cx43 may be interacting with the F-actin cytoskeleton in B cells, we first used TIRF microscopy to determine if they were in the same location.  Cx43 has been observed in the same location as F-actin where it is thought to be interacting with F-actin via interaction with ZO-1 and Drebrin (Butkevich et al., 2004; Giepmans and Moolenaar, 1998).  Though B cells do not express ZO-1 or Drebrin, there was co-localization of Cx43-GFP and F-actin in the periphery of B cells spreading on fibronectin.  This region of a spreading/moving cell is important for generating the force required to drive the membrane forward (Giannone et al., 2007). When the cytoskeleton was disrupted using cytochalasin D, there was a change in the localization and mobility of Cx43-GFP.  Cx43-GFP moved from the cell periphery where there was a large mobile fraction and accumulated in large membrane aggregates with a lower membrane mobility, indicating that its localization is determined by the F-actin cytoskeleton.  A change in membrane mobility of Cx43 has been described before in rat mammary tumour cells (Simek et al., 2009).  Here, Cx43-GFP was highly mobile in non-junctional areas and decreased in mobility when part of a gap junction.  These data suggest that Cx43 is actively recruited to sites of actin polymerization and uncoupling it from the cytoskeleton leads to self-aggregation and a decrease in membrane mobility. In summary, these findings highlight a novel role for Cx43 in lymphocyte biology.  We show using a model independent of gap junction coupling, that Cx43 is necessary for B cell motility and migration.  We also show that both sustained VLA-4 and CXCL12-mediated  192  Rap1 activation require Cx43 expression, suggesting that Cx43 may be acting as a signalling platform.  Using a modified transwell assay, we show that Cx43 is necessary for B cell TEM.  Finally we show that the F-actin cytoskeleton is important for the membrane distribution of Cx43, and that ablation of it results in Cx43 clustering and a decrease in membrane mobility.  These results suggest that Cx43 may be an important regulator of B cell biological processes which involve reorganization of the F-actin cytoskeleton, and may play an important role in immune system development and function.              193  5 Concluding chapter 5.1 Summary of main findings In this thesis, we provide the first evidence that the gap junction protein Cx43 plays an essential role in the regulation of B cell morphology and signalling.  We show that Cx43 is a novel regulator of the Rap1 GTPase, the master regulator of B cell morphology, mediated by BCR, integrin and CXCR4 signalling.  We also show that Cx43 is required for B cell spreading, adhesion, migration and transendothelial migration and determined the C-terminal domain of Cx43 was important for these effects.  This thesis is the first report to describe a function for Cx43 in regulating B cell morphology and provides a mechanism, the activation of Rap1 GTPase, for its action.  This work provides the basis for further studies on the function of Cx43 in the immune system.  Chapter 2: To determine which domains of the BCR are important for BCR-mediated cytoskeletal rearrangements The cytoplasmic domains of Ig? and Ig? each contain an ITAM that becomes phosphorylated after BCR activation.  Though similar in structure, they both have unique domains that have differential requirements for B cell development and BCR internalization.  The goal of this chapter was to assess whether the cytoplasmic domain of Ig? was necessary for BCR-mediated rearrangement of the cytoskeleton.  Using the J558 plasmacytoma gain-of-function tissue culture model system, two mutated BCRs were reconstituted.  One contained a BCR with two Ig? tails (C?: made by expressing with wild type Ig?, a chimeric Ig? with a cytoplasmic Ig? tail).  The second mutant contained a single Ig? tail (?trunc: made  194  by expressing wild type Ig? and Ig? with a cytoplasmic truncation).  These two mutated BCRs were expressed in J558 cells and compared to cells expressing a WT BCR (J558?m3cells).  We showed that J558 B cells with a WT reconstituted BCR were unable to initiate BCR-mediated B cell spreading, but were able to extend F-actin rich membrane protuberances.  The formation of these protuberances was dependent on the WT BCR, as there was a reduction in protuberance formation when the cytoplasmic domain of Ig? was swapped with Ig?, and a further reduction when the cytoplasmic domain of Ig? was deleted.  The importance of this chapter was twofold: it was the first report to identifiy that the cytoplasmic domain Ig? is necessary for BCR-mediated rearrangements of the cytoskeleton and second, it identified that although the terminally differentitated B cell line J558 with a reconstituted WT BCR was able to initiate signals and rearrangements of the cytoskeleton, it was not able to initiate BCR-mediated radial cell spreading.  This allowed us to use this cell line for a gain-of-function approach, and this led to the identification of a novel regulator of B cell spreading and cytoskeletal rearrangements, Cx43.  These results will be discussed below.  Chapter 3: The characterization of the GJ protein Cx43 expressed in B lymphocytes and its role in BCR-mediated membrane spreading and B cell adhesion The aim of chapter 3 was to determine if Cx43 was necessary (important) for B cell spreading and identify the potential molecular mechanism that led to the changes in the cytoskeleton.  The expression of Cx43 was characterized in immature and mature B cell lines as well as in terminally differentiated B cell lines. Cx43 was found to be a target of BCR  195  signalling.  Using a loss-of-function approach, we were able to show that Cx43 knockdown cells were unable to sustain BCR-mediated Rap1 activation and radial cell spreading.  Using a gain-of-function approach, Cx43-GFP was expressed in the J558?m3 plasmacytoma cell line and it was sufficient to increase BCR, LFA1 and CXCL12 Rap1 activation, radial cell spreading and cell adhesion to an endothelial monolayer.  Expressing a C-terminal truncated version of Cx43 failed to conferthese responses, thus pointing to the importance of the cytoplasmic tail of Cx43 in these effects.   These results show for the first time that Cx43 is an important regulator of Rap1 activation, spreading and adhesion in B cells.    Chapter 4: The role of the GJ protein Cx43 in B cell motility, migration and transendothelial migration.   The aim of chapter 4 was to determine if Cx43 was necessary for B cell migration and the associated biological processes of motility and transendothelial migration.  Using a loss-of-function approach and knocking down Cx43 expression, there was a decrease in sustained VLA-4 and CXCL12-mediated Rap1 activation, as well as a decrease in cell motility, CXCL12-mediated directed migration and transendothelial migration through two different endothelial monolayers.  Using FRAP of Cx43-GFP transfected WEHI 231 B cells, the membrane distribution of Cx43 was shown to be determined by the organization of the F-actin cytoskeleton, and ablation of the cytoskeleton led to Cx43 aggregation.  This chapter also contained the first results showing that Cx43 is an important regulator of motility and directed cell migration in B cells.  These results are consistent with the findings in Chapter 3   196  that Cx43 is required for B cell spreading and adhesion, showing that Cx43 is a fundamental regulator of multiple biological processes that require regulation of the cytoskeleton.   5.2 General discussion 5.2.1 How might Cx43 regulate morphology? One of the most important conclusions from this thesis was that the gap junction protein Cx43 is necessary for the regulation of B cell morphology, likely via the sustained activation of Rap1 GTPase.  Although this thesis provides ample evidence that Cx43 is required for Rap1 activation, the proteins that act upstream of Rap1in this pathway remain unknown.  A potential mechanism as to how Cx43 may be regulating Rap1 activation comes from the gap junction literature and the finding that the actin-binding protein drebrin interacts with Cx43 (Butkevich et al., 2004).  Drebrin is a member of the actin binding protein family and it is thought to bind to the tail of Cx43, coupling it to the F-actin cytoskeleton and stabilizing gap junction plaques.  Although B cells to not express drebrin, they do express a drebrin family member known as HIP55 (also known as mABP-1 or SH3P7).  Drebrin and HIP55 share high sequence homology and this is a strong candidate for a Cx43 interacting protein in lymphocytes.  In T cells, HIP55 is recruited to the immune synapse and T cells from HIP55 knockout mice show decreased cytokine production and impaired up-regulation of activation markers induced by TCR stimulation, though through an unknown mechanism (Han et al., 2005; Le Bras et al., 2004).   Along with drebrin, the actin binding protein cortactin has also been shown to co-localize with Cx43 (Vitale et al., 2009).  Lymphocytes do not express cortactin, but they do  197  express the hematopoietic-specific homolog of cortactin, HS1 (Kitamura et al., 1989; Schuuring et al., 1998).  Knockdown of HS1 in T cells leads to an inability to effectively initiate TCR-mediated cell spreading; a response similar to the results obtained in chapter 3 after Cx43 knockdown (Gomez et al., 2006).  HS1 has also been implicated as a regulator of chemotaxis in NK cells, likely through its regulation of the GTPases Rac1 and Cdc42 and the GEF Vav1 (Butler et al., 2008).  Interestingly, in B cells HS1 has been shown to interact with HIP55 where it is thought to play a role in BCR-mediated cytoskeletal rearrangements, though the mechanism remains unknown (Muzio et al., 2007).  The recruitment of the HS1/HIP55 complex to the C-terminal domain of Cx43 could provide a potential mechanism of how Cx43 may be linked to the F-actin cytoskeleton and how it may recruit proteins (likely GEFs) involved in Rap1 activation.  An overview of this model is described in Figure 5-1.   198   Figure 5-1 Overview model of the role Cx43 plays in regulation of the B cell cytoskeleton A) Extracellular signals generated through integrins, GPCRs or the BCR result in activation of signalling cascades and the B) activation of kinases.  These kinases result in the phosphorylation of the C-terminal tail of Cx43 which can act as docking sites or expose protein interaction domains.  These domains may allow for the recruitment of C) adaptor proteins (possibly HS1 or HIP55) which can interact with the cytoskeleton and/or allow for D) the sustained activation of the GTPase Rap1.  Sustained Rap1 activation results in activation of signalling cascades and regulation of B cell morphology.   Incorporating these findings we propose a molecular model for how Cx43 is able to regulate B cell morphology (Figure 5-2).  Initial signalling, (i.e. through the BCR) leads to a localized uncoupling of ezrin from the cortical actin cytoskeleton along with activation of the actin depolymerizing machinery (Batista et al., 2010; Hao and August, 2005; Treanor et al., 2010).  Untethering of membrane ?corrals? which constrain BCR mobility results in the formation of signalling microclusters which activate downstream signalling pathways and phosphorylation of Cx43 (Figure 5-2 A-II).  Cx43 can then recruit adaptor proteins, possibly  199  HS1 and/or HIP55, to its C-terminal tail which interact with the actin cytoskeleton and recruit proteins responsible for sustained activation of Rap1.  Actin polymerization is initiated from the free barbed ends of the newly severed actin filaments, resulting in a polarized outward force that pushes the plasma membrane forward and drives membrane spreading (Figure 5-2 A-III).  Interaction of Cx43 with the cytoskeleton facilitates stabilization of the F-actin cytoskeleton at these sites of force generation and co-ordinated membrane spreading.  When Cx43 is mutated or absent, initation of signalling still results in ezrin decoupling and actin depolymerization (Figure 5-2 B-II); however, there is a defect in the recruitment of adaptor proteins (HS1 and/or HIP55) to the signalling sites.  This results in an uncoordinated spreading response due to inability to sustain Rap1 activation and short, transient membrane protuberances due to a destabilization of actin polymerization (Figure 5-2 B-III).    Alternatively, Cx43 may be constitutively interacting with the cytoskeleton through an adaptor protein (possibly HIP-55) where it would localize to areas of the plasma membrane rich in F-actin.  After the initiation of BCR signalling, actin depolymerization would untether Cx43 from the cytoskeleton, leading to an aggregation of Cx43 in regions of the plasma membrane which are still in contact with the F-actin cytoskeleton (likely areas rich in newly severed actin filaments).  In these areas, BCR-activated HS1 could be recruited to the C-terminal domain of Cx43 through an interaction with HIP-55, where it can then bind and stabilize the F-actin cytoskeleton and rectuit GEF?s which are required for sustained Rap1 activation.  From this site, actin polymerization can drive the plasma membrane forward leading to a co-ordinated B cell spreading response.  200    201    202  Figure 5-2 Proposed molecular mechanism of how Cx43 regulates B cell morphology A)  When Cx43 is present.  A-I) Cell surface proteins (BCR, Cx43) are confined within membrane corrals defined by coupling of membrane-bound ezrin (blue) to the cortical F-actin cytoskeleton (red).  A-II) Signalling results in uncoupling of ezrin, the formation of signalling microclusters and localized depolymerization of F-actin.  A-III) Adaptor proteins which couple Cx43 to the cytoskeleton (orange) as well as proteins involved in sustained Rap1 activation (brown) are recruited to the C-terminal domain of Cx43, allowing for sustained and coordinated membrane spreading (as seen in SEM).   B) When Cx43 is mutated or absent.  B-I) Cell surface proteins (BCR, mutated Cx43?T) are confined within membrane corrals defined by coupling of membrane-bound ezrin to the cortical F-actin cytoskeleton.  B-II) Signalling results in uncoupling of ezrin, the formation of signalling microclusters and localized depolymerization of F-actin.  B-III) Inability to recruit proteins to Cx43 which are required for sustained Rap1 activation and stabilization of F-actin polymerization results in formation of uncoordinated and transient membrane protuberances (as seen in SEM).  5.2.2 Could there be a role for Cx43-mediated GJIC in B-lymphocytes? Within the immune system, a function for Cx43 has been indentified in dendritic cells and macrophages where it can provide a mechanism for antigen cross-presentation. This presents an important function for Cx43 in these cells, but it does not take into account the role of gap junction proteins in non-professional APCs, i.e. T cells and B cells.   The work from this thesis identifies that Cx43 is an important regulator of B cell morphology, but it does not take into consideration the role of the pore and GJIC.   GJIC between B cells and follicular dendritic cells using a dye transfer assay was first observed in 1997, sparking the idea that it was an important form of signalling within the immune system (Krenacs et al., 1997).  It has since been reported to occur between B cells and endotheilial cells (Oviedo-Orta et al., 2002), macrophages and dendritic cells (Mendoza-Naranjo et al., 2007) and between T cells and B cells,T cells and macrophages and T cells and dendritic cells (Bermudez-Fajardo et al., 2007; Elgueta et al., 2009; Oviedo-Orta et al., 2001) though the  203  function of this communication, remains to be better clarified.  The work from this thesis may provide an interesting idea as to how GJIC may function in the immune system.  One important assumption that is apparent within the reports that identify GJIC between T cells/B cells and APCs is that it is assumed that information is moving only one way.  These dye coupling experiments have been done by loading the APC with the GJ permeable dye and then measuring the transfer to the adjacent immune cell, but are not assessed in the reverse direction.  This bias is also prevalent in the immunology field with respect to the APC.  It is generally assumed that there is little to no signalling that occurs within the APC when presenting Ag to B or T cells.  This assumption is apparent when modeling the immune synapse formation between B cell and an Ag-bearing lipid bilayer.  Here both Ag and integrin ligands are abundant and easily accessible, whereas in vivo the ligands on an APC may be much more limiting.  Therefore, not only is a mechanism necessary for Ag accumulation necessary for activation (spreading), but a mechanism to maximize adhesion may also be required. One potential function for GJIC between an APC and B/T cell could be to flux calcium to the APC.  The binding of MCHII bound Ag from the APC to the BCR/TCR leads to an increase in intracellular calcium in the B/T cell through well characterized pathways, resulting in Rap1 activation and changes in cell morphology.  One important Rap1 GEF is CalDAG-GEF1 which is dependent on calcium flux for Rap1 activation (Gold, 2002).  It is possible that an influx of calcium into the APC through the GJ pore may result in the activation of CalDAG-GEF1, or a similar GEF.  The activated GEFs may be recruited to the tail of Cx43 within the APC where it could activate Rap1, resulting in a localized activation of integrins on the surface of the APC.  This increase in integrin activation could aid in  204  adhesion between the APC and T/B cell, resulting in a stabilized immune synapse.  It has been shown that blockage of the Cx43 pore during the coupling of T cells and DCs resulted in a decrease in activation (Elgueta et al., 2009).  In this context, it is possible that this decrease in activation is due to an inability to sustain a stable immune synapse.    A similar mechanism may also be involved in B cell adhesion to the endothelium and subsequent TEM.  It has been previously shown that GJIC between B cells and endothelial cells during TEM does occur; however, there was again a bias to the observed dye transfer .  Here endothelial cells were loaded with a GJ permeable dye and transfer was measured  in the B cells (Oviedo-Orta et al., 2002).  When the pore was blocked, there was no decrease in TEM observed.  Similar to the TEM results in chapter 4, neither assay was done under flow conditions, which would also test the strength of adhesion to the endothelium.  It is possible that both lymphocyte adhesion to the endothelium and subsequent endotheilial cell-mediated adhesion to the lymphocytes are both required under the flow conditions seen in vivo.  This may also be a potential mechanism to explain why J558?m3 B cells expressing a C-terminal truncated Cx43-GFP showed a significant increase in adhesion to the endothelium.  Truncation of Cx43-GFP does not block GJIC in other cell types, and it is possible that Cx43-mediated Rap1 activation may be intact in the endothelial cells and impared in the J558?m3 cells, leading to the minimal increase in adhesion observed in Chapter 3 compared to the wild type Cx43.     205  5.2.3 Is coupling necessary for gap junction protein functions? Another question that this thesis addresses is whether coupling is necessary for function. The larger body of work within the GJ field has focused on its role as a pore that regulates the transfer of material between adjacent cells.  As such, the importance of gap junctions as hemichannels remains debated (Scemes et al., 2009).  One problem with the study of Cx43 hemichannels has to with its trafficking to the cell surface   When Cx43 is expressed on the cell surface, it is rapidly incorportated into GJ plaques between adjacent cells (Simek et al., 2009).  Therefore, the study of hemichannels could differ depending on whether the cell is solitary or part of a group of cells. The study of GJs in an immune cell model, which are often found in the absence of stable cell:cell contacts, allows for the study of GJ proteins that are both not found in junctions and in their hemichannel state.  The work from chapters 3 and 4 would suggest that Cx43 plays a very important function in lymphocytes that is independent of GJIC.      5.2.4 Cx43 as a regulator of Rap1 GTPase in non-lymphoid cells? The work from this thesis highlights an additional way that Cx43 functions in a non-immune cells.  Rap1 is ubiquitously expressed and is a key regulator of cell adhesion and migration.  This thesis identifies Cx43 as a key regulator of sustained Rap1 activation, likely as an adaptor which recruits proteins that modulate Rap1 function.  How can this be important to non-immune cells?    Cx43 has recently been identified as an important regulator of neuronal migration, however the mechanism remains debated.  The first report to identify Cx43 as a regulator of  206  neuronal migration was that of Elias et al (Elias et al., 2007).  The main conclusion from this study was that gap junction coupling was required for neuronal migration during development, however the work from this thesis may provide an alternative interpretation for the results.  In that study, knockdown of Cx43 was achieved using in utero transfection of developing neurons.  While knockdown leads to a significant decrease in migration, the actual efficiency of the transfection was not addressed.  In chapters 3 and 4, using shRNA knockdown of Cx43 was effective at reducing Cx43 expression, there was not a 100% decrease in expression and there was still some Cx43 protein expressed.  We, like Elias et al, were able to measure a decrease in function after knockdown, but in our opinion, attempting a rescue with Cx43 mutants may not be not be a good idea because endogenous Cx43 is still present.  In the Elias et al rescue experiments, they were able to measure a complete rescue of both channel mutated and C-terminal truncated mutants; however, since the ratio of mutated to WT connexins in their model was not assessed, and whether they are forming mutant:WT heteromeric connexons, their conclusions are of concern.  They also presented evidence that an extracellular mutation of Cx43 that interferes with GJ coupling resulted in an inability to rescue the migration defect seen in the Cx43-knockdown neurons, leading them to conclude that coupling is the mechanism for how Cx43 regulates migration.  The problem with this assumption is that they did not determine if this mutant traffics normally to the cell surface, and is expressed at the surface at levels similar to the other mutants.  Our lab, in an attempt to study the role of the extracellular cysteine residues in GJ-mediated adhesion, observed that mutations of the extracellular cysteine residues dramatically affected the ability of Cx43 to traffic to the cell surface.  If there is a defect in trafficking of this mutant, then it could behave like a dominant negative and greatly reduced protein levels at  207  the surface.   Taking into consideration the work from this thesis, the defect observed when Cx43 expression is knocked down in neuronal cells could also affect the ability to activate Rap1.  Rap1 is required for both migration and polarization.  Interfering with the ability to activate Rap1 would likely result in very similar defects in neuronal migration observed when Cx43 is knocked down.  This idea could fit in well with the work from Cina et al, who, using a neuron-specific Cx43 knockout mouse observed that the C-terminal domain of Cx43 was required for migration of developing neurons (Cina et al., 2009).  Because Cina et al used GOF rescue experiments on a genetic knockout background, endogenous Cx43 would likely not be incorporated into the connexons.  They conclude that the migration defect observed when Cx43 is either deleted or truncated is likely due to an inability to interact with scaffolding proteins.  The work from this thesis would suggest that this may be due to an inability to activate Rap1.  5.2.5 Could Cx43 be a novel regulator of B cell cancers? The role of Cx43 in regulating B cell morphology may have implications for B cell tumor biology.  In astrocyte tumors, Cx43 expression has been shown to be inversely related to tumor grade (Peiyu et al., 2004).  Interestingly, it has been shown that there is an inverse relationship between tumor motility and Cx43 expression in glioma tumour cells (McDonough et al., 1999).  Here, tumour sphereoids were created in vitro and tumour cell migration away from the spheroid was measured.  Using this model, cells expressing the lowest levels of Cx43 were found to have moved the furthest from the spheroid.  The authors described this as an increase in motility, however an equally plausible explanation is that this result was due to a decrease in cell:cell adhesion and that the decrease in cell:cell adhesion  208  resulted in a higher probability that the low Cx43 expressing cells would separate from the tumour and disseminate.    In support of this alternative explanation, Cx43 expression has been shown to increase glioma cell adhesion in C6 glioma cell lines (Lin et al., 2002).  These results suggest that in gliomas, a decrease in Cx43 expression leads to tumour cells that are less adhesive, more motile and associated with a higher tumor grade.  Similarly, in the embryonic carcinoma cell line P19, epithelial-to-mesenchymal transition (EMT) was correlated with repression of Cx43 by Snail1.  Snail1 repression resulted in a mesenchyme-to-epithelium transition and increased Cx43 levels (Boer et al., 2007).  It will be interesting to see if Cx43 expression levels have a similar effect on B cell tumors.    5.2.6 Contributions This thesis addressed many different aspects of the role of the BCR and Cx43 in B cell morphology, as such many novel contributions were made: Chapter 2 demonstrated that the cytoplasmic domain of Ig? was required for optimal BCR-mediated rearrangement of the cytoskeleton.  Though the cytoplasmic domain of Ig? was shown to be important in this, this study also highlighted that the terminally differentiated B cell line J558 was missing compontents, besides the BCR, that are required for B cell spreading.  This established the J558 plasmacytoma cell line as an important gain-of-function system to study cytoskeletal rearrangements that has previously not been used in the literature. Chapter 3 demonstrated that the gap junction protein Cx43 was required for Rap1 activation, spreading and adhesion in B lymphocytes.  209  1) This is the first report showing that Cx43 is necessary for regulation of B cell morphology in response to BCR, integrin and chemokine signalling.   2) This study highlighted lymphocytes as a powerful tool to study the function of Cx43 in the absence of gap junction coupling, limiting the potential mechanism to either hemichannel function or Cx43-mediated signalling. 3) This was the first study to link Cx43 to the activation of the Rap1 GTPase, the master regulator of B cell morphology.  The importance of this finding cannot be overstated.  Rap1 is ubiquitously expressed and Cx43-mediated activation represents a potential mechanism in all cell types expressing Cx43, and is likely the mechanism for the newer functions emerging for Cx43 including neuronal migration and adhesion.   4) The sustained activation of Rap1 and spreading are dependent on the C-terminal domain of Cx43.  There is disagreement within the Cx43 field as to the role of the C-terminal tail in neuronal migration.  These results suggest that the C-terminal domain is important for biological processes, including migration, that are dependent on the rearrangement of the cytoskeleton.   Chapter 4 demonstrated that Cx43 was necessary for B cell motility, migration and transendothelial migration.   1) This identified Cx43 as an important regulator of B cell migration that was previously unknown, identifying a new class of proteins that are important for B cell migration. 2) It had previously been shown that gap junctional intercellular communication was not important for transendothelial migration of B cells (Oviedo-Orta et al., 2002).  We show that Cx43 is important for TEM, likely via Rap1 mediated mechanism.  These  210  findings challenge the importance of gap junction coupling to neuronal migration, providing an alternative mechanism to explain these results. 3) Using a variety of microscopic techniques, we show that in B lymphocytes the distribution of Cx43 is determined by the F-actin cytoskeleton.    5.2.7 Strengths and limitations Both strengths and limitations to the work presented in this thesis are as follows: Strengths: ? Both the WEHI231 and J558 in vitro model allowed for a detailed cell biology approach using microscopy, biochemistry and functional assays to assess the role of Cx43 in B cells. ? We paired loss-of-function with gain-of function approaches to show that Cx43 was necessary for sustained B cell Rap1 activation, spreading and adhesion. ? We validated our loss-of-function pheonotye using splenic B cells from Cx43+/- mice, both of which showed the same phenotype. ? In our gain-of-function system we were able to use a B cell that does not express Cx43.  Previously in the Cx43 literature, gain-of-function approaches were used where Cx43 expression was knocked down with siRNA or low Cx43 expressing cell lines were used prior to transfection with WT or mutant Cx43 (Bates et al., 2007; Elias et al., 2010; Elias et al., 2007).  Because WT Cx43 is still expressed in these systems, it is possible that the mutant phenotype is masked or diminished.  211  ? Our study of Cx43 in lymphocytes allowed for the identification of roles for Cx43 that are independent of gap junction coupling. ? This work provides a new model to study the role of GJ proteins in migration across epithelial/endothelial barriers from the point of view of the lymphocyte ? Lymphocytes provide an excellent model for studying the emerging role of Cx43 in migration due to their rapid migration and the sensitive, pre-established approaches for identifying subtle effects. Limitations ? We were unable to pair Ig? mutants with the Ig? mutants assessed in chapter 2.  Expression of Ig? is developmentally regulated, however expression of Ig? is not.  Because of this, endogenous Ig? was expressed in our J558 gain-of-function system and we were unable to introduce similar Ig? mutants and assess the effect of two or a single Ig? tail on BCR mediated cytoskeletal rearrangement. ? We did not utilize an in vivo model to assess the role of Cx43 in B lymphocytes.  Though it would be ideal to study the role of B cell spreading, adhesion and migration in vivo, there is currently no B cell specific Cx43 knockout mouse model.  Also, there is currently no way to accurately measure B cell spreading in vivo because the technology does not exist.   ? We assessed B cell spreading using an immobilized antibody on a glass coverslip as opposed to on a planar lipid bilayer where the BCR ligand is embedded in a phospholipid bilayer and is infinitely mobile.  Though B cell spreading on a planar lipid bilayer is much closer to what would be seen in vivo, both are artificial systems.  Assessing B cell spreading using an immobilized antibody on a glass coverslip is  212  much less problematic and allowed for an increased number of cells to be analyzed; however, it only assesses the early events of B cell spreading and does not assess the contraction phase that occurs after spreading has stopped. ? B cell adhesion to endothelial cells and transendotheilal migration was not assessed under flow conditions.  Normally B cells moving to secondary lymphoid organs within the circulatory system attach to the endothelium under flow conditions prior to diapedeisis.  Our model does not incorporate flow; however, it would still accurately model the conditions seen within the sinusus surrounding the spenic white pulp. ? B cell motility was assessed in 2D and not 3D.  B cells routinely move in 3D within secondary lymphoid organs and motility in 2D is not as common and is mainly restricted to the surface of endothelial cells searching for sites perimisive to TEM.  Though not ideal, the measurement of B cell motility in 2D does allow for accurate quatification assessing whether the capacity to move is altered when Cx43 expression is augmented.  5.2.8 Future directions This thesis highlights a novel and important role for Cx43 in regulating B cell morphology.  As such, there are great implications to this work in the areas of B cell immune system regulation, development and B cell tumours. 5.2.8.1 What is the molecular mechanism by which Cx43 regulated Rap1 activation in B cells? In chapters 3 and 4 we identified that Cx43 was required for sustained Rap1 activation in response to BCR, integrin and chemokine signalling; however, the mechanism  213  remains unknown.   Because Cx43 has no intrinsic enzymatic activity, and deletion of the tail resulted in a significant decrease in Rap1 activation, it is likely that Cx43 is acting as a scaffolding protein that recruits proteins involved in Rap1 activation.   The most straight forward approach for determining what is interacting with the tail of Cx43 is to use co-immunoprecipitation; however, there is a problem with using this approach for Cx43.  Cx43 has a differential detergent solubility depending on which residues are phosphorylated.  Though this approach has been used in the literature before, the detergents permissive for co-immunoprecipitation pull out only a small population of Cx43 (i.e. the ER population) and bias the interpretation of the results.  Therefore, a FRET-based approach using a candidate protein (HIP-55-YFP or HS1-YFP and Cx43-CFP) would be the best way to identify protein-protein interactions.  Once a poteintial candidate is identified, a mutational analysis of Cx43 could be used to identify the interaction motif.    5.2.8.2 B cell development As previously described, the processes of adhesion and migration dramatically affect where B cells can move during development and which cells they can interact with.  Interfering with these signals results in an iniability to receive the proper developmental cue?s and affects the ability to go down specific developmental paths.   Because migration and adhesion are important processes to B cell development, our results from chapters 2 and 3 would suggest that Cx43 may play a significant role. In support of this idea, a recent report identified Cx43 as an important regulator of HSCs within their bone marrow niche (Schajnovitz et al., 2011).  Here, gap junction coupling between stromal cells was shown to be important for the production of CXCL12 on their surface and maintaining interaction with  214  HSCs.  When the gap junctional communication was interrupted, there was a dramatic decrease in the number of HSCs that were able to adhere to the stromal cells.  This may provide a mechanism for HSC retainment within their niches, but more importantly it shows that Cx43 does play a role in hematopoeisis.    The role of Cx43 in B cell development could be assessed using a B cell specific Cx43 mouse knockout model.  Since Ig? is one of the earliest B cell specific markers expressed, a cre-lox system by which crossing mb1-cre (mb-1 being the gene encoding Ig?) with a gja1-lox (gja1 being the gene encoding Cx43) could be used to create a B cell specific mouse knockout.  These mice could be compared to the WT mice with respect to B cell subsets and localization of B cell populations.   A likely first candidate population that could be examined would be MZ B cells.  As described in chapter 1, these cells are dependent on integrin-mediated adhesion and S1P signalling to remain within the MZ surrounding the white pulp of the spleen.  Since Cx43 was implicated as important for B cell adhesion, loss of Cx43 would likely result in a decrease in the number of MZ B cells present within the splenic white pulp.  5.2.8.3 Regulation of the immune response The regulation of B cell morphology dictates where in the body B cells are located, what they can interact with and how the signals they recive are integrated.   One of the most important conclusions from this thesis was that the gap junction protein Cx43 is necessary for the regulation of B cell morphology.  The results from Chapters 3 and 4 imply that Cx43 is required for biological processes that are essential for a normal immune response.  As  215  previously described, B cell spreading and adhesion are important processes during the initial identification and response to a membrane-bound antigen.  The process of spreading increases the formation of BCR microclusters and signalling required to activate the B cell, where as adhesion is required to lower the threshold of B cell activation by increasing the duration of attachment between the B cell and APC (Carrasco et al., 2004; Fleire et al., 2006).  Because we show in chapter 3 that Cx43 is required for these biological processes, it is likely that B cells with Cx43 protein deficiencies would be defective in their ability to become activated following antigenic challenege.  This hypothesis could be tested by challenging B cell specific Cx43-knockout mice with a pathogen (either bacteria or virus) and assessing the progression of the infection.  An important consideration for this would be how knockout of Cx43 affects B cell development.  If knockout of Cx43 results in a complete loss of B cells, then the ability to make antibodies would be severely compromised and would have to be taken into consideration when assessing the results. Along with spreading and adhesion, B cell migration is also important to the immune response.  After B cells bind antigen within the B cell follicle, they quickly migrate towards the B cell:T cell interface where they present the processed antigen to T cells (Cyster, 2010).  Because we show in chapter 4 that Cx43 is necessary for B cell migration, it is possible that there will be defects in antigen presentation to T cells due to an inability of the activated B cells to migrate towards the T cell zone.  This hypothesis could be tested by assessing the localization of follicular B cells before and after antigenic challenge using in vivo two-photon microscopy with subsequent analysis of T cell development after activation.   216  5.2.8.4 The role of Cx43 in B cell cancers Based on the results from chapters 3 and 4, an important question that has yet to be asked is if Cx43 expression affects B cell tumour invasiveness.  Using an A20 B cell tumour homing model similar to what was utilized in (Lin et al., 2009) using Cx43 shRNA knocked-down cells, B cell tumour invasiveness could be assessed.  Our results from Chapters 3 and 4 would suggest that A20 B cells with low expression of Cx43 would result in low invasiveness and fewer tumours.  Changes in Cx43 levels may have an effect on how quickly B cell cancers progresses and the ability of tumor cells to metastasize to new areas of the body.  This could potentially be used as a diagnostic marker for tumour invasivness.  5.3 Conclusion Though the regulation of B cell morphology is essential for normal immune system function, much is still unknown about the proteins that are involved.  The work in this thesis showed that the cytoplasmic domain of Ig? is important for BCR-mediated rearrangement of the cytoskeleton; however, reconstitution of a WT BCR in the terminally differentiated B cell line J558 was insufficient for restoring the ability to initiate B cell spreading.  This thesis also showed that the gap junction protein Cx43 was a novel regulator of B cell spreading in response to BCR and integrin ligands, and is necessary for sustained BCR, integrin and chemokine-mediated activation of Rap1 GTPase.  Further, it was shown that Cx43 was required for B cell adhesion to an endothelial monolayer, motility, migration and TEM.  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