The clathrin coated vesicle cycle drives synaptic vesicle recycling and is essential for the maintenance of repeated rounds of neurotransmission. Many of the structural components of the clathrin endocytic machinery have been identified, but our understanding of the molecular mechanisms underlying and regulating clathrin-mediated endocytosis is still in its infancy. Moreover, only one enzyme, the GTPase dynamin, has been identified to play a role in clathrin-mediated endocytosis even though these reactions are ATP-dependent. My laboratory has pioneered the development of a set of cell-free assays that together, faithfully and efficiently reconstitute each step in the clathrin-coated vesicle cycle, including coated pit assembly, coated vesicle formation and the sequential uncoating reactions that recycle the coat proteins, freeing the enclosed vesicles. Using these well-characterized assays, we are in a unique position to elucidate the molecular mechanisms that govern synaptic vesicle recycling. The long-term goal of this proposal is to identify the minimal set of cytosolic and peripheral membrane proteins required to reconstitute clathrin-mediated endocytosis and to complete the clathrin-coated vesicle cycle. To meet this objective, we propose the following Specific Aims: 1) To develop a novel assay for coated vesicle budding from highly enriched plasma membranes to enable identification of peripheral and integral membrane components of the endocytic machinery; 2) To determine the hierarchy of events leading to coated pit assembly and endocytic clathrin coated vesicle formation; 3) To identify minimal cytosolic components required for endocytic coated vesicle formation; 4) To identify proteins that regulate the uncoating reaction and to test the role of hsc70, the uncoating ATPase in vivo; and, 5) To develop transient expression systems in primary neurons to demonstrate that protein interactions and mechanisms identified in vitro are required for synaptic vesicle recycling in vivo.

Agency
National Institute of Health (NIH)
Institute
National Institute of Mental Health (NIMH)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37MH061345-04
Application #
6639164
Study Section
Special Emphasis Panel (ZRG1-MDCN-1 (01))
Program Officer
Asanuma, Chiiko
Project Start
2000-06-05
Project End
2005-05-31
Budget Start
2003-06-01
Budget End
2004-05-31
Support Year
4
Fiscal Year
2003
Total Cost
$370,400
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Xiao, Guan-Yu; Mohanakrishnan, Aparna; Schmid, Sandra L (2018) Role for ERK1/2-dependent activation of FCHSD2 in cancer cell-selective regulation of clathrin-mediated endocytosis. Proc Natl Acad Sci U S A 115:E9570-E9579
Kadlecova, Zuzana; Spielman, Stephanie J; Loerke, Dinah et al. (2017) Regulation of clathrin-mediated endocytosis by hierarchical allosteric activation of AP2. J Cell Biol 216:167-179
Bendris, Nawal; Schmid, Sandra L (2017) Endocytosis, Metastasis and Beyond: Multiple Facets of SNX9. Trends Cell Biol 27:189-200
Schmid, Sandra L (2017) Reciprocal regulation of signaling and endocytosis: Implications for the evolving cancer cell. J Cell Biol 216:2623-2632
Bendris, Nawal; Williams, Karla C; Reis, Carlos R et al. (2016) SNX9 promotes metastasis by enhancing cancer cell invasion via differential regulation of RhoGTPases. Mol Biol Cell :
Elkin, Sarah R; Oswald, Nathaniel W; Reed, Dana K et al. (2016) Ikarugamycin: A Natural Product Inhibitor of Clathrin-Mediated Endocytosis. Traffic 17:1139-49
Bendris, Nawal; Stearns, Carrie J S; Reis, Carlos R et al. (2016) Sorting nexin 9 negatively regulates invadopodia formation and function in cancer cells. J Cell Sci 129:2804-16
Reis, Carlos R; Chen, Ping-Hung; Srinivasan, Saipraveen et al. (2015) Crosstalk between Akt/GSK3? signaling and dynamin-1 regulates clathrin-mediated endocytosis. EMBO J 34:2132-46
Aguet, François; Antonescu, Costin N; Mettlen, Marcel et al. (2013) Advances in analysis of low signal-to-noise images link dynamin and AP2 to the functions of an endocytic checkpoint. Dev Cell 26:279-91
Neumann, Sylvia; Schmid, Sandra L (2013) Dual role of BAR domain-containing proteins in regulating vesicle release catalyzed by the GTPase, dynamin-2. J Biol Chem 288:25119-28

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