When sending chemical messages, cells release transmitters or hormones by Ca-triggered exocytosis. During exocytosis, the membrane of a secretory vesicle fuses with the plasma membrane. Fusion is mediated by three SNARE proteins and the proteins Munc18 and Synaptotagmin, and regulated by accessory proteins including Munc13, CAPS, complexin, granulophilin and rabphilin. Partial or complete structures exist for the SNAREs, for Munc18 and complexin, and our biochemical and structural knowledge about all the above proteins is substantial. In live cells, exocytosis has been explored exclusively by assays that, in one way or another, report membrane fusion. Foremost among them is electrophysiology. However, electrophysiology cannot directly report steps that precede fusion. In absence of direct assay, it is still unknown how the proteins mentioned above interact with each other or with secretory vesicles in living cells. We plan to use high-resolution light microscopy to observe single secretory granules in endocrine cells, as well as the exocytosis-related proteins recruited and released by them. We focus on how secretory vesicles dock at the plasma membrane. Single docking sites are observed simultaneously with the docking vesicle and its exocytosis. We use a microscope that has been calibrated by single-molecule imaging in terms of molecules per granule or molecules per cell surface are. If necessary, imaging is combined with electrophysiology. The goal is to understand the molecular steps in docking, and to determine which proteins form the molecular bridge connecting the membranes of a granule with that of the cell when docking has occurred. This work will provide a basic understanding of the role of each of the proteins of interest. More broadly, the work will explore an important and novel avenue to explore the action of proteins in living cells, at the level of single organelles and single proteins. Because most messages between cells are sent by exocytosis, exocytosis is an important cellular process. When exocytosis is defective in pancreatic beta cells then diabetes is the result. When exocytosis is defective or altered in neurons, then so is the basic mechanism of neural communication in the brain, and cognitive malfunctions result. Through increasing the basic mechanisms of exocytosis and its control, this work will be helpful in finding treatment strategies for diseases where cells fail to send messages.
Most messages between cells are sent by exocytosis. Exocytosis requires a cascade of protein interactions. When any member of this cascade is deficient, impaired or inappropriate exocytosis will result. For instance, defective exocytosis in pancreatic beta can cause diabetes, and defective exocytosis in neurons will disrupt the basic mechanism of neural communication in the brain. By high resolution light microscopy in live cells, this project seeks to understand how individual proteins in the cascade contribute to the mechanism of exocytosis.
|Zhao, Ying; Fang, Qinghua; Herbst, Adam Drew et al. (2013) Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells. Proc Natl Acad Sci U S A 110:14249-54|
|Varlamov, Oleg (2012) Detection of SNARE complexes with FRET using the tetracysteine system. Biotechniques 52:103-8|
|Knowles, M K; Barg, S; Wan, L et al. (2010) Single secretory granules of live cells recruit syntaxin-1 and synaptosomal associated protein 25 (SNAP-25) in large copy numbers. Proc Natl Acad Sci U S A 107:20810-5|
|Barg, S; Knowles, M K; Chen, X et al. (2010) Syntaxin clusters assemble reversibly at sites of secretory granules in live cells. Proc Natl Acad Sci U S A 107:20804-9|
|Felmy, Felix (2009) Actin and dynamin recruitment and the lack thereof at exo- and endocytotic sites in PC12 cells. Pflugers Arch 458:403-17|
|Chen, Xi; Barg, Sebastian; Almers, Wolfhard (2008) Release of the styryl dyes from single synaptic vesicles in hippocampal neurons. J Neurosci 28:1894-903|
|Coggins, Michael R; Grabner, Chad P; Almers, Wolfhard et al. (2007) Stimulated exocytosis of endosomes in goldfish retinal bipolar neurons. J Physiol 584:853-65|
|Felmy, Felix (2007) Modulation of cargo release from dense core granules by size and actin network. Traffic 8:983-97|
|Wan, Lei; Almers, Wolfhard; Chen, Wenbiao (2005) Two ribeye genes in teleosts: the role of Ribeye in ribbon formation and bipolar cell development. J Neurosci 25:941-9|
|Taraska, Justin W; Almers, Wolfhard (2004) Bilayers merge even when exocytosis is transient. Proc Natl Acad Sci U S A 101:8780-5|
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