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精确的适应性体液免疫是必不可少的。这包括激活和抑制BCR信号效应物的募集，例如信号看门人65 kDa的含SH 2结构域的白细胞蛋白（SLP 65）和关键的负调节物SH 2结构域的含肌醇5-磷酸酶（SHIP），分别到质膜上的BCR位点。然而，允许这些快速和定向易位的详细机制迄今尚未完全理解。我们发表的报告和我们在准备本提案时产生的进一步数据提供了几条证据，表明多蛋白复合物的相分离可能是一种通用机制，能够将激活和抑制效应蛋白整合到BCR信号级联中。结果表明，85 kDa的Cbl相互作用蛋白（CIN 85）与其相互作用蛋白SLP 65和SHIP形成高分子蛋白质复合物。这些相互作用的多价性提供了静息B细胞胞质溶胶中液-液相分离的基础，这对于SLP 65募集和BCR信号转导是必不可少的。与先前描述的相分离的信号传导复合物相反，SLP 65和CIN 85需要与囊泡缔合以形成生理浓度的液滴。此外，SLP 65/CIN 85液滴不仅依赖于与囊泡的结合，而且还可以从囊泡定位转移到BCR。因此，我们观察到，通过抑制肌球蛋白马达蛋白或微管形成来干扰囊泡运输会减弱BCR信号转导的效率，基于我们的研究结果，我们假设囊泡及其运输过程包括细胞骨架动力学是BCR信号转导的新参与者，并促进正向和负向信号调节因子向BCR的导航。囊泡的这种作用将代表BCR信号传导的新范例。该项目的主要目标是通过探索SLP 65和SHIP从囊泡相关簇中招募到BCR的细节来证实这一概念。这些BCR效应物是基于我们已发表的和初步的工作选择的，并且因为它们在BCR信号转导中的关键功能，这被包括我们自己的出版物在内的许多报告所证实。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，我们将结合联合收割机各种成像，细胞生物学，生物化学和质谱技术。

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.