Eitzen, Gary - University of Alberta - Department of Cell Biology

研究领域

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 secretion (mast cell/neutrophil degranulation) which occurs during infection and also in an allergic reaction. 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 for in vivo studies and simple to isolate in great quantity and purity for in vitro studies. We have reconstituted vacuole docking and fusion in vitro with isolated vacuoles, ATP, cytosol and physiological salt. This fusion event has three distinct subreactions: priming, docking and fusion. Priming is catalyzed by the Sec18p(NSF)-ATPase which releases Sec17p (aSNAP) and dissociates pre-existing cis-SNARE complexes. Docking occurs in two stages: first the Rab-GTPase, Ypt7p, catalyzes tethering through association with its effector complex, and then trans-SNARE pairs form across membranes. Final fusion requires a calcium flux, triggering the calmodulin-dependent formation of a proteolipid channel between apposed vacuoles. An appealing feature of the vacuole fusion reaction is that a host of proteins and drugs can rapidly be examined and their effects can be assigned to particular subreactions of membrane fusion. Using GTPgS, GDI, GTPase-activator proteins and GTP exchange factors as molecular tools, we can dissect the molecular role of GTPases signaling. Using this approach we found a requirement for two Rho GTPases (Rho1p and Cdc42p) in membrane 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 secretion in inflammatory cells Inflammatory cells (neutrophils, eosinophils, basophils and mast cells) are among the primary haematopoietic effector cells involved in an early immune response, but are also involved in allergic inflammatory responses. During an allergic reaction they contribute to inflammation and tissue damage by degranulating in response to inappropriate activation, resulting in release of mediators including chemoattractants, cytokines, chemokines and cytotoxic enzymes contained in intracellular granules. Degranulation occurs by regulated secretion (membrane fusion) of granules in response to cell activation. Therefore, an effective therapy to modify an allergic response would be one that is directed at downregulation of inflammatory cell secretion. Several key signaling molecules have been implicated in distal receptor signaling pathways leading to exocytosis from granulocytes. The Lacy lab (Dept. of Medicine, UA) has recently shown that the Rac2-GTPase 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 shows limited tissue expression (primarily in granulocytes). Indeed, even the upstream activator of Rac2, Vav1, is expressed predominantly in haematopoietic tissues. Our hypothesis is that inhibition of key regulatory pathways leading to inflammatory cell secretion provides ideal targets for modulation of an allergic response. In collaboration with the Lacy lab we plan to identify signaling pathways downstream of Rac2 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 affect on inflammatory cell function and allergic response using a combination of in vitro assays and mouse model systems.

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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. Myosin-driven peroxisome partitioning in S. cerevisiae. Fagarasanu A, Mast FD, Knoblach B, Jin Y, Brunner MJ, Logan MR, Glover JN, Eitzen GA, Aitchison JD, Weisman LS, Rachubinski RA. J Cell Biol. 2009 Aug 24;186(4):541-54. Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling. Mitchell T, Lo A, Logan MR, Lacy P, Eitzen G. Am J Physiol Cell Physiol. 2008 Nov;295(5):C1354-65. Control of granule exocytosis in neutrophils. Lacy P, Eitzen G. Front Biosci. 2008 May 1;13:5559-70. Review. Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling. Isgandarova S, Jones L, Forsberg D, Loncar A, Dawson J, Tedrick K, Eitzen G. J Biol Chem. 2007 Oct 19;282(42):30466-75. The peroxisomal membrane protein Inp2p is the peroxisome-specific receptor for the myosin V motor Myo2p of Saccharomyces cerevisiae. Fagarasanu A, Fagarasanu M, Eitzen GA, Aitchison JD, Rachubinski RA. Dev Cell. 2006 May;10(5):587-600. Enhanced membrane fusion in sterol-enriched vacuoles bypasses the Vrp1p requirement. Tedrick K, Trischuk T, Lehner R, Eitzen G. Mol Biol Cell. 2004 Oct;15(10):4609-21. Actin remodeling to facilitate membrane fusion. Eitzen G. Biochim Biophys Acta. 2003 Aug 18;1641(2-3):175-81. Review. Remodeling of organelle-bound actin is required for yeast vacuole fusion. Eitzen G, Wang L, Thorngren N, Wickner W. J Cell Biol. 2002 Aug 19;158(4):669-79. Genomic analysis of homotypic vacuole fusion. Seeley ES, Kato M, Margolis N, Wickner W, Eitzen G. Mol Biol Cell. 2002 Mar;13(3):782-94. Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking. Eitzen G, Thorngren N, Wickner W. EMBO J. 2001 Oct 15;20(20):5650-6. Sequential action of two GTPases to promote vacuole docking and fusion. Eitzen G, Will E, Gallwitz D, Haas A, Wickner W. EMBO J. 2000 Dec 15;19(24):6713-20.