Grant: Discovery Grants Program - Individual

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.