Regulation of immune cell vesicular traffic and exocytosis by Rho GTPases

我的研究项目是Rho GTPases对细胞内囊泡运输的调节。Rho蛋白，如Rho、Rac和Cdc42，属于ras-GTPase超家族。它们作为分子开关，在非活性（gdp结合）和活性（gtp结合）状态之间循环，控制许多细胞过程。我们的假设是Rho gtpase在免疫细胞促炎过程中起关键作用。该建议的重点是Rho GTPases如何控制促炎介质的释放，通过胞吐作用，从肥大细胞。肥大细胞是组织内的粒细胞免疫细胞。由于它们在环境界面的组织中定位，它们被认为是免疫系统的哨兵，起着关键的保护作用。肥大细胞是研究胞吐作用基本机制的良好模型细胞。它们有几个已知配体的表面受体，可以用来刺激细胞。静息细胞和受刺激细胞之间的转换是由膜近端src激酶激活的，该激酶将信号传递给几个下游途径，最终导致颗粒胞外分泌。在这里，我们主要研究肥大细胞中的Rho信号，因为该途径中的几种蛋白质主要或仅在免疫细胞中表达。长期目标：确定调节免疫细胞促炎过程的Rho信号通路。短期目标：目的1)鉴定促炎Rho GEFs Rho GEFs是一种激活因子，通过Rho信号将上游激活信号（如Fc受体连接）转化为下游促炎过程，如颗粒胞分泌。我们将描述肥大细胞中免疫细胞特异性GEFs遗传破坏的影响，以确定胞外分泌所需的Rho GEFs。目的2)确定RhoGDI在肥大细胞胞外分泌中的作用，RhoGDI被认为是通过结合和隔离在细胞质中抑制Rho蛋白。然而，我们的研究表明它们是调节Rho信号向激活位点的空间传递的促进剂。肥大细胞大量表达RhoGDI1和RhoGDI2，它们可能具有不同的功能，因为我们已经证明它们存在差异定位。我们将研究在肥大细胞活化过程中Rho蛋白/RhoGDI复合物的形成和解离机制。为了描述肥大细胞分泌颗粒胞吐的下游机制，我们将使用活细胞成像来描述肌动蛋白在胞吐中的作用。我们有活细胞探针，可用于评估Rac信号在f -肌动蛋白重塑、颗粒动员和颗粒内容物释放中的作用。意义：我们的研究结果将定义调控特定膜运输步骤的Rho信号因子。生化分析将使我们能够将功能角色归因于已确定的因素，活细胞成像将显示它们如何影响囊泡交通。总的来说，这些发现可能普遍适用于许多细胞类型的胞吐作用。

My research program studies the regulation of intracellular vesicular traffic by Rho GTPases. Rho proteins, such as Rho, Rac and Cdc42, belong to the ras-GTPase superfamily. They function as molecular switches, cycling between inactive (GDP-bound) and active (GTP-bound) states, to control numerous cellular processes. Our hypothesis is that Rho GTPases serve a pivotal role in immune cell pro-inflammatory processes. This proposal is focused on how Rho GTPases control the release of pro-inflammatory mediators, via exocytosis, from mast cells. Mast cells are tissue-resident granulocytic immune cells. Because of their positioning in tissues at environmental interfaces they are deemed sentinels of the immune system and play a key protective role. Mast cells are particularly good model cells to study the basic mechanism of exocytosis. They have several surface receptors with known ligands that can be used to stimulate cells. The transition between resting and stimulated cells is activated by membrane proximal src kinases that relay signals to several downstream pathways culminating in granule exocytosis. Here we have focused our study on Rho signaling in mast cells since several proteins in this pathway are predominantly or exclusively expressed in immune cells. Long-term goal:  to define the Rho signaling pathway(s) that regulate immune cell pro-inflammatory processes. Short term goals: Aim 1) To identify pro-inflammatory Rho GEFs Rho GEFs are activators that transduce upstream activation signals (eg Fc receptor ligation) into downstream pro-inflammatory process like granule exocytosis via Rho signaling. We will characterize the effect of genetic disruption of immune-cell specific GEFs in mast cells to identify Rho GEFs needed for exocytosis. Aim 2) To define the role of RhoGDI in mast cell exocytosis RhoGDIs are thought to be inhibitors of Rho proteins via binding and sequestration in the cytosol. However, our studies implicate them as facilitators of Rho signaling that regulate spatial delivery to sites of activation. Mast cells abundantly express RhoGDI1 and RhoGDI2, which likely have distinct functions since we have shown that they differentially localize. We will examine Rho protein/RhoGDI complex formation and disassociation mechanisms during mast cell activation. Aim 3) To characterize downstream mechanisms of mast cell secretory granule exocytosis We will use live-cell imaging to characterize the role of actin in exocytosis. We have live-cell probes that can be used to evaluate the role of Rac signaling for F-actin remodeling, granule mobilization and release of granule contents. Significance: Our results will define Rho signaling factors that regulate specific membrane trafficking steps. Biochemical analyses will allow us to ascribe functional roles to identified factors and live-cell imaging will show how they impact vesicular traffic. Overall, these discoveries are likely to be universally applicable to exocytosis in many cell types.