Regulation of immune cell vesicular traffic and exocytosis by Rho GTPases

我的研究项目是研究Rho GTP酶对细胞内囊泡运输的调节。Rho蛋白，如Rho、Rac和CDC42，属于ras-GTP酶超家族。它们起着分子开关的作用，在不活跃(GDP结合)和活跃(GTP结合)状态之间循环，控制许多细胞过程。我们的假设是，Rho GTP酶在免疫细胞促炎过程中起着关键作用。这项建议的重点是Rho GTP酶如何通过胞吐作用控制肥大细胞释放促炎介质。肥大细胞是驻留在组织中的粒细胞免疫细胞。由于它们位于环境界面的组织中，它们被认为是免疫系统的哨兵，发挥着关键的保护作用。肥大细胞是研究胞吐作用基本机制的良好模型细胞。它们有几个具有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，它将信号传递到几个下游通路，最终导致颗粒胞吐。在这里，我们的研究重点是肥大细胞中的Rho信号，因为这一途径中的几种蛋白质主要或唯一地在免疫细胞中表达。长期目标：明确调节免疫细胞促炎过程的Rho信号通路(S)。短期目标：目的1)鉴定促炎因子Rho GEF是一种激活剂，通过Rho信号将上游的激活信号(如Fc受体连接)传导到下游的促炎过程，如颗粒胞吐。我们将表征肥大细胞中免疫细胞特异性GEF的遗传破坏的影响，以确定胞吐所需的Rho GEF。目的2)明确RhoGDI在肥大细胞胞吐中的作用，RhoGDI被认为是通过结合和隔离在胞浆中抑制Rho蛋白的。然而，我们的研究表明，它们是Rho信号的促进者，调节激活部位的空间传递。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有不同的定位。我们将研究肥大细胞激活过程中Rho蛋白/RhoGDI复合体的形成和解离机制。目的3)为了研究肥大细胞分泌颗粒胞吐作用的下游机制，我们将利用活细胞成像技术研究肌动蛋白在胞吐作用中的作用。我们有活细胞探针，可以用来评估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：我们的结果将定义调节特定膜转运步骤的Rho信号因子。生化分析将使我们能够将功能作用归因于已识别的因素，而活细胞成像将显示它们如何影响囊泡运输。总体而言，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.