| Summary: | The activation of B cells, a type of lymphocytes whose main function is to produce antibodies during an immune response, is tightly controlled by antigen binding to the cell surface B-cell receptor (BCR). The present thesis investigates the early cellular and molecular events that regulate B cell activation by membrane-bound antigen, which likely represents the predominant form of antigen encountered by B cells in vivo. Upon recognition of such membrane-bound antigen, B cells undergo a spreading and contraction response that serves to aggregate antigen for subsequent extraction and ultimately determines the extent of B-cell activation. Using planar lipid bilayers and advanced microscopy in combination with genetic and pharmacological approaches, I have characterised the key events and the molecular mechanism of this cellular response. I show that B-cell spreading is propagated through successive rounds of phosphotyrosine signalling and actin polymerization, proportionally to antigen density and affinity. Downstream of the BCR, I identify Lyn, Syk, phospholipase C g2 (PLCγ2) and Vav as the key intracellular players involved in this process. I have visualized, with high definition, the rapid recruitment of signalling molecules such as PLCγ2 to antigen micro-clusters formed across the contact site and demonstrate that they are incorporated into spatially defined ‘microsignalosomes’. The assembly of these micro-signalosomes is sequential and highly coordinated and the presence and correct functioning of all component parts is required for the spreading response to proceed. Results also show that PLCγ2 and Vav cooperate from within micro-signalosomes and they further synergize with the co-receptor CD19 to amplify the spreading response. Finally, the thesis addresses the functioning of cytoskeletal effector pathways downstream of the BCR and I identify a partial role for Rap and Wiskott Aldrich syndrom protein (WASp) in the spreading response, as well as their critical requirement for inside-out integrin activation and immunological synapse (IS) formation.
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