Navigation of effector proteins for B cell activation requires vesicle trafficking and cytoskeleton dynamics

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The precise spatio-temporal orchestration of signals induced by the B cell antigen receptor (BCR) is essential for accurate adaptive humoral immunity. This includes the recruitment of activating and inhibiting BCR signal effectors, such as the signal gatekeeper SH2 domain-containing leukocyte protein of 65kDa (SLP65) and the pivotal negative regulator SH2 domain-containing inositol 5-phosphatase (SHIP), respectively, to the site of BCRs at the plasma membrane. However, the detailed mechanisms that allow for these rapid and directed translocations are not fully understood to date. Our published reports and further data that we generated in preparation of this proposal provide several lines of evidence that phase-separation of multi-protein complexes may be a general mechanism, enabling the integration of both, activating and inhibitory effector proteins into the BCR signaling cascade. We could show that Cbl-interacting protein of 85kDa (CIN85) nucleates higher molecular protein complexes with its interacting proteins SLP65 and SHIP. The multivalency of these interactions provides the basis of liquid-liquid phase separation in the cytosol of resting B cells, which is essential for SLP65 recruitment and BCR signal transduction. In contrast to previously described phase-separated signaling complexes, SLP65 and CIN85 need the association with vesicles to form droplets at physiological concentrations. Moreover, the SLP65/CIN85 droplets not only depend on the association with vesicles, but can also translocate to the BCR from this vesicular localization. Consistently, we observed that interference with vesicle trafficking by inhibiting myosin motor proteins or microtubule formation attenuates the efficiency of BCR signal transduction.Based on our findings we hypothesize that vesicles and their transport processes including cytoskeleton dynamics are novel players in BCR signal transduction and promote the navigation of positive and negative signal regulators to the BCR. Such a role of vesicles would represent a new paradigm in BCR signaling. The main objective of the proposed project is to corroborate this concept by exploring the details of SLP65 and SHIP recruitment from vesicle-associated clusters to the BCR. These BCR effectors were chosen based on our published and preliminary work, and because of their pivotal function in BCR signal transduction, which is confirmed by a number of reports including our own publications. In particular, we will assess the impact of vesicle transport events in the recruitment processes. For this purpose we will combine various imaging, cell biology, biochemical, and mass spectrometry techniques.