Small GTPase signal transduction and the regulation of membrane fusion
Membrane fusion is an important process in our bodies, controlling numerous physiological functions. One that is of particular interest to my lab is the requirement of membrane fusion to release mediators of an immune response. We study how small GTPases of the Rab and Rho family control membrane fusion events. We have two active projects: the first uses a model system (yeast vacuole fusion) to dissect the membrane fusion mechanism biochemically; the second is to apply our mechanistic studies in yeast to lymphocyte activation and exocytosis (e.g. mast cell and neutrophil degranulation) which occurs during infection and allergic reactions.
Project I: Mechanism of membrane fusion using a yeast vacuole model system
The yeast vacuole (lysosome) is an attractive organelle for studying the membrane fusion mechanism since it is easily visualized by microscopy and simple to isolate in large quantities for in vitro studies. We have reconstituted membrane docking and fusion in vitro with isolated vacuoles, ATP, cytosol and physiological salt. An appealing feature of the vacuole fusion reaction is that a host of proteins and drugs can be rapidly examined and their effects can be assigned to particular subreactions of membrane fusion. This project will use GTPgS, GDI, GTPase-activator proteins and GTP exchange factors as molecular tools to dissect the role of GTPases signaling during membrane fusion. Using this approach we found a requirement for two Rho GTPases (Rho1p and Cdc42p) in yeast vacuole fusion. We have shown that Rho proteins along with Rabs are part of a novel GTPase cascade that coordinately regulates vacuole docking and fusion.
Project II: Rac signal transduction and control of inflammatory cells exocytosis
Innate inflammatory cells such as neutrophils, eosinophils, basophils and mast cells are the primary hematopoietic effector cells involved in an early immune response. These cells are also known as granulocytes and have numerous cytoplasmic granules that package potent pro-inflammatory mediators. During infection or an allergic reaction they contribute to inflammation and tissue damage by degranulating in response to activation, resulting in release of mediators including chemoattractants, cytokines, chemokines and cytotoxic enzymes contained in intracellular granules. Degranulation occurs by regulated exocytosis (granule-plasma membrane fusion) of granules in response to cell activation. Therefore, an effective anti-inflammatory therapy or to modify an allergic response would be one that is directed at downregulation of inflammatory cell exocytosis.
Several key signaling molecules have been implicated in the regulation of exocytosis from granulocytes. The Rho GTPase, Rac2, and specific SNAREs are critical signaling elements responsible for triggering granule exocytosis in granulocytes. Rac2 and its downstream signaling pathway associated with SNARE-mediated exocytosis represent ideal targets for allergic response modulation; particularly since Rac2 is primarily in granulocytes. Indeed, even the upstream activator of Rac2, Vav1, is expressed predominantly in hematopoietic tissues. Our hypothesis is that inhibition of key regulatory pathways leading to inflammatory cell exocytosis provides ideal targets for modulation of an allergic response. We plan to identify signaling pathways downstream of Rho GTPases leading to SNARE-triggered membrane fusion using a proteomics approach. Future goals are to antagonistically target Rac2, its signaling pathway including the upstream activator Vav1, and downstream SNAREs. We will evaluate the specific effect on inflammatory cell function and allergic response using a combination of in vitro assays and mouse model systems.
The effect of Rho drugs on mast cell activation and degranulation. Sheshachalam A, Baier A, Eitzen G. J Leukoc Biol. 2017 Jul;102(1):71-81
Yeast translation elongation factor-1A binds vacuole-localized Rho1p to facilitate membrane integrity through F-actin remodeling. Bodman JA, Yang Y, Logan MR, Eitzen G. J Biol Chem. 2015 Feb 20;290(8):4705-16.
Granule protein processing and regulated secretion in neutrophils. Sheshachalam A, Srivastava N, Mitchell T, Lacy P, Eitzen G. Front Immunol. 2014 Sep 19;5:448.
Rac1 and Rac2 control distinct events during antigen-stimulated mast cell exocytosis. Baier A, Ndoh VN, Lacy P, Eitzen G. J Leukoc Biol. 2014 May;95(5):763-774.
Analysis of Rho GTPase activation in Saccharomyces cerevisiae. Eitzen G, Logan MR. Methods Mol Biol. 2012;827:369-80.
Proteomic analysis of secretagogue-stimulated neutrophils implicates a role for actin and actin-interacting proteins in Rac2-mediated granule exocytosis. Eitzen G, Lo AN, Mitchell T, Kim JD, Chao DV, Lacy P. Proteome Sci. 2011 Nov 14;9:70.
Functional analysis of RhoGDI inhibitory activity on vacuole membrane fusion. Logan MR, Jones L, Forsberg D, Bodman A, Baier A, Eitzen G. Biochem J. 2011 Mar 15;434(3):445-57.
Natamycin inhibits vacuole fusion at the priming phase via a specific interaction with ergosterol. te Welscher YM, Jones L, van Leeuwen MR, Dijksterhuis J, de Kruijff B, Eitzen G, Breukink E. Antimicrob Agents Chemother. 2010 Jun;54(6):2618-25.
Cdc42p and Rho1p are sequentially activated and mechanistically linked to vacuole membrane fusion. Logan MR, Jones L, Eitzen G. Biochem Biophys Res Commun. 2010 Mar 26;394(1):64-9.
Cdc42p is activated during vacuole membrane fusion in a sterol-dependent subreaction of priming. Jones L, Tedrick K, Baier A, Logan MR, Eitzen G. J Biol Chem. 2010 Feb 12;285(7):4298-306.