The Rap GTPases regulate B cell polarity and morphology by controlling the localization of Scribble and the activation of cofilin

Naïve resting B cells circulate throughout the body and enter secondary lymphoid organs (SLOs) in response to chemical cues known as chemokines. Within SLOs, B cells scan for foreign antigens, and antigen-induced clustering of the B cell receptor (BCR) initiates B cell activation. This clustering is enhanced through the formation of an immune synapse between the B cell and antigen presenting cell, which greatly amplifies BCR signaling and B cell activation. We have previously shown that activation of the Rap GTPases is important for the cytoskeletal changes that underlie B cell spreading, migration, and immune synapse formation. We have also shown that stimulating B-lymphocytes with a model particulate antigen, anti-BCR-coated polystyrene beads, causes cells to polarize and form actin-rich cups at sites of cell:bead contacts, and that the formation of these cups enhance B cell activation. Although a number of downstream targets of activated Rap (Rap-GTP) have been identified, the mechanisms by which Rap-GTP promotes cell polarization, actin polymerization, reorganization of the actin cytoskeleton, and changes in cell morphology are not completely understood. The establishment of cell polarity often involves complexes of evolutionarily conserved polarity proteins. One such polarity complex includes the Scribble protein. In this thesis, I show that Rap-GTP is important for generating an asymmetric distribution of Scribble when B cells encounter particulate antigens and form cups. The ability of leukocytes to assume a polarized morphology also requires that the cortical F-actin cytoskeleton be disassembled so that new F-actin filaments that contribute to the formation of a leading edge, F-actin-rich cup, or immune synapse can be assembled. Since cofilin plays a major role in severing and depolymerizing F-actin filaments, I tested the hypothesis that the Rap GTPases regulate changes in cell shape by controlling the activation of cofilin, a process that requires its dephosphorylation by the Slingshot phosphatase. I show that BCR clustering leads to cofilin dephosphorylation, and hence activation, which is dependent on activation of the Rap GTPases. This suggests that an early step underlying BCR-induced changes in B cell morphology that involve reorganization of the actin cytoskeleton is the Rap-dependent activation of the actin-severing protein cofilin.Science, Faculty ofMicrobiology and Immunology, Department ofGraduat