The nnolecular mechanisnn of clathrin-mediated endocytosis is highly conserved among eukaryotes. The WASP/WIP/myosin module regulates actin assembly to generate forces that invaginate the plasma membrane and pinch off endocytic vesicles. A multidisciplinary approach combining live-cell imaging, yeast genetics and actin biochemistry will be used to investigate this process. Ultimate goals are reconstitution of actin assembly-driven membrane invagination and vesicle scission events on artificial liposomes, and activation and uitrastructural analysis of purified contractile rings. These studies will elucidate functions of individual cytoskeletal proteins and will provide a systems level understanding of the interplay among the proteins of each complex system. The ability to dissect the individual steps of actin-mediated endocytosis in live cells by image analysis, combined with reconstitution of WASP-mediated endocytic actin assembly on microbead surfaces in cell extracts, will make attainable a comprehensive understanding of how actin assembly is initiated, harnessed for vesicle formation, and terminated. Actin assembly in extracts will be reconstituted on membrane surfaces to investigate both the roles of type 1 myosins in driving membrane invagination, and the roles of actin assembly and BAR proteins in driving vesicle scission. Theoretical modeling will generate hypotheses for future experiments. Candidates for the protein factors that regulate and mediate disassembly will be added to our extract system to reconstitute biological disassembly activity. How the same actin subunit assembles into distinct filament-based structures with vastly different organization and associated protein composition will be investigated in the extract system by EM and protein mass spectrometry. Particular attention will be focused upon the myosins, since a different isoform associates with each ofthe three actin structures despite sharing a common, conserved ATPase domain. The first successful isolation of the contractile apparatus from a genetically tractable organism will be exploited for EM uitrastructural analysis of isolated wild-type and mutant structures, and a full investigation ofthe contractile mechanism, its regulation, and the ring disassembly pathway will be investigated.

Public Health Relevance

Actin assembly and function are fundamental to the immune response, cellular morphogenesis including wiring ofthe nervous system, and muscle contraction. Disruption of actin function is implicated in diseases ofthe immune system, cardiovascular system and respiratory system. A better understanding actin function and regulation promises to facilitate development of new therapeutic and prevention measures.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM042759-24
Application #
8293045
Study Section
Special Emphasis Panel (NSS)
Program Officer
Deatherage, James F
Project Start
1989-07-01
Project End
2016-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
24
Fiscal Year
2012
Total Cost
$440,682
Indirect Cost
$149,121
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
124726725
City
Berkeley
State
CA
Country
United States
Zip Code
94704
Michelot, Alphée; Drubin, David G (2014) Dissecting principles governing actin assembly using yeast extracts. Methods Enzymol 540:381-97
Michelot, Alphee; Grassart, Alexandre; Okreglak, Voytek et al. (2013) Actin filament elongation in Arp2/3-derived networks is controlled by three distinct mechanisms. Dev Cell 24:182-95
Michelot, Alphee; Drubin, David G (2011) Building distinct actin filament networks in a common cytoplasm. Curr Biol 21:R560-9
Michelot, Alphee; Costanzo, Michael; Sarkeshik, Ali et al. (2010) Reconstitution and protein composition analysis of endocytic actin patches. Curr Biol 20:1890-9
Okreglak, Voytek; Drubin, David G (2010) Loss of Aip1 reveals a role in maintaining the actin monomer pool and an in vivo oligomer assembly pathway. J Cell Biol 188:769-77
Young, Brian A; Buser, Christopher; Drubin, David G (2010) Isolation and partial purification of the Saccharomyces cerevisiae cytokinetic apparatus. Cytoskeleton (Hoboken) 67:13-22
Barker, Sarah L; Lee, Linda; Pierce, B Daniel et al. (2007) Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis. Mol Biol Cell 18:2893-903
Sun, Yidi; Carroll, Susheela; Kaksonen, Marko et al. (2007) PtdIns(4,5)P2 turnover is required for multiple stages during clathrin- and actin-dependent endocytic internalization. J Cell Biol 177:355-67
Toshima, Jiro; Toshima, Junko Y; Duncan, Mara C et al. (2007) Negative regulation of yeast Eps15-like Arp2/3 complex activator, Pan1p, by the Hip1R-related protein, Sla2p, during endocytosis. Mol Biol Cell 18:658-68
Sun, Yidi; Martin, Adam C; Drubin, David G (2006) Endocytic internalization in budding yeast requires coordinated actin nucleation and myosin motor activity. Dev Cell 11:33-46

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