Function of Scampi in EXO/Endocytosis in Neuroendocrine Cells

Zhang, Jie, Department of Cell Biology, University of Virginia
Castle, J., Department of Cell Biology, University of Virginia

Neuroendocrine cells release catecholamines, peptide hormones and proteins in response to physiological stimulation by regulated exocytosis. During regulated exocytosis, membrane-bounded dense core vesicles (DCVs) containing stored secretory products and the plasma membrane are brought into close contact and rearranged to create a fusion pore, through which the contents are expelled to the extracellular space in part or in full. Following release, membrane is recovered directly (transient fusion) or indirectly (full fusion) thereby maintaining cell surface area. The sustained mode of transient fusion, which has been designated cavicapture, has emerged as a major mechanism in neuroendocrine/endocrine cells. Sub-quantal release through regulation of pore closure may be a physiologically relevant mechanism for controlling qualitative and quantitative information transfer during intracellular signaling. While much has been learned about the molecular events that lead to formation of a fusion pore, much less is known about the events that follow the initiation of fusion, including expansion and ultimately reclosure of the fusion pore. Studies presented in this thesis provide new insight concerning the mechanisms by which exocytotic fusion pores are regulated during exocytosis and cavicapture in neuroendocrine cells. In particular the focus is on the role of members of the secretory carrier membrane protein (SCAMP) family. These very conserved transmembrane proteins are ubiquitously present on postGolgi membrane trafficking compartments in eukaryotic cells. To pursue possible function of SCAMPs 1 & 2 in coupled exo/endocytosis, I engaged in electrophysiology and different microscopy. Amperometry studies revealed that both SCAMPs 1 and 2 are implicated in opening, expanding and/or stabilizing fusion pores, with SCAMP2 functioning more upstream including events preceding fusion pore opening. Further by using two-photon microscopy and a post-fusion labeling method, opening and closing of fusion pores during exocytosis were detected and analyzed. Herein, I report that SCAMP1 deficiency causes a pronounced delay in pore closure. Subsequently, extended pore opening in SCAMP1-deficient cells increases the level of compound exocytosis. However this feature of SCAMP1 in fusion pore closure is highly selective and not shared by SCAMP2. Thus SCAMPs 1&2 seem to collaborate to regulate the fusion pore during exocytosis in a co-operative yet non-redundant manner. DEDICATION AND ACKNOWLEDGEMENTS today. This dissertation is dedicated to my parents. Their love helps me become who I am My most sincere appreciation goes to my mentor Dr. David Castle, for his guidance, unconditional help and invaluable friendship. In him I see tremendous passion for science and what a serious scientist should be like. Acknowledgements are also given to my committee members and my friends and colleagues in the lab, with whom I shared a great time in my life.

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PHD (Doctor of Philosophy)
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