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

由B细胞抗原受体(BCR)诱导的信号的精确时空协调对于准确的适应性体液免疫是必不可少的。这包括激活和抑制BCR信号效应物，如信号门卫SH2结构域的65 kDa白细胞蛋白(SLP65)和关键的负调控因子SH2结构域包含的肌醇5-磷酸酶(SHIP)，分别募集到质膜上BCR的位置。然而，到目前为止，允许这些快速和定向易位的详细机制还不完全清楚。我们发表的报告和我们在准备这一提议过程中产生的进一步数据提供了几条证据，证明多蛋白复合体的相分离可能是一种一般机制，使激活和抑制效应蛋白都能够整合到BCR信号级联中。我们可以证明85 kDa的Cbl相互作用蛋白(CIN85)与其相互作用蛋白SLP65和SHIP形成更高分子的蛋白质复合体。这些相互作用的多价性为静息B细胞胞浆中液-液分离提供了基础，而液-液分离是SLP65募集和BCR信号转导所必需的。与先前描述的相分离的信号复合体不同，SLP65和CIN85需要在生理浓度下与小泡结合形成液滴。此外，SLP65/CIN85液滴不仅取决于与囊泡的结合，还可以从这种囊泡定位转移到BCR。我们一致地观察到，通过抑制肌球蛋白马达蛋白或微管的形成来干扰囊泡的运输会削弱BCR信号转导的效率。基于我们的发现，我们假设囊泡及其运输过程包括细胞骨架动力学是BCR信号转导中的新角色，并促进正负信号调节因子向BCR的导航。囊泡的这种作用将代表BCR信号传递的新范式。拟议项目的主要目标是通过探索SLP65的细节和从与囊泡相关的集群向BCR招募船舶来证实这一概念。这些BCR效应器是根据我们已发表的和初步的工作选择的，因为它们在BCR信号转导中起着关键作用，这一点得到了包括我们自己的出版物在内的许多报告的证实。特别是，我们将评估囊泡运输事件在招聘过程中的影响。为此，我们将结合各种成像、细胞生物学、生化和质谱学技术。

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.