The dynamic assembly and disassembly of actin filaments underlies diverse processes including cell migrations during development and phagocytosis of invading microorganisms by inunune cells. Mechanisms that control actin dynamics in vivo are largely unknown. A central unsolved question about the actin filament nucleating Arp2/3 complex concerns roles for ATPs bound to Arp2 and Arp3. Based on studies of actin, nucleotide states could influence Arp2/3 complex activation, branching and de-branching activities, and interactions with regulatory factors. Because Arp2 and Arp3 are closely related to actin, and because actin nucleotide pocket mutants have provided valuable insights into cytoskeletal dynamics, similar mutants will be constructed in Arp2 and Arp3. Effects of these mutants will be tested in vivo and in in vitro tests for nucleation activity, interactions with different activators, and branching and debranching reactions. To elucidate more fully the in vivo functional relationships and regulatory networks that regulate the actin cytoskeleton in vivo, the large collection of existing yeast actin cytoskeleton mutants created in the Drubin laboratory will be used in functional genomics studies. Systematic, genome-wide synthetic lethal and suppressor screens using recently developed procedures designed to achieve comprehensive genome coverage will be employed to uncover these complex relationships. Finally, having successfully translated studies of actin-mediated endocytosis in budding yeast to mammalian cells, studies of yeast actin cytoskeleton dynamics will now be translated to the problem of tumor cell invasion in mammalian cells. Cytoskeletal constituents of invadopodia responsible for tumor cell invasion will be systematically identified, then RNAi and dominant negative mutants will be used to knock down expression of these proteins. In total, studies proposed here will increase our understanding of fundamental mechanisms for regulating actin cytoskeleton dynamics, and principles derived from studies of yeast actin dynamics will be used to develop a deeper understanding of the molecular mechanisms responsible for mammalian tumor cell invasion. Novel therapeutic targets may be identified.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042759-17
Application #
6916398
Study Section
Special Emphasis Panel (ZRG1-CDF-4 (02))
Program Officer
Deatherage, James F
Project Start
1989-07-01
Project End
2006-06-30
Budget Start
2005-07-01
Budget End
2006-06-30
Support Year
17
Fiscal Year
2005
Total Cost
$285,797
Indirect Cost
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
Lu, Rebecca; Drubin, David G; Sun, Yidi (2016) Clathrin-mediated endocytosis in budding yeast at a glance. J Cell Sci 129:1531-6
Miao, Yansong; Han, Xuemei; Zheng, Liangzhen et al. (2016) Fimbrin phosphorylation by metaphase Cdk1 regulates actin cable dynamics in budding yeast. Nat Commun 7:11265
Sun, Yidi; Leong, Nicole T; Wong, Tiffany et al. (2015) A Pan1/End3/Sla1 complex links Arp2/3-mediated actin assembly to sites of clathrin-mediated endocytosis. Mol Biol Cell 26:3841-56
Lewellyn, Eric B; Pedersen, Ross T A; Hong, Jessica et al. (2015) An Engineered Minimal WASP-Myosin Fusion Protein Reveals Essential Functions for Endocytosis. Dev Cell 35:281-94
Cortesio, Christa L; Lewellyn, Eric B; Drubin, David G (2015) Control of lipid organization and actin assembly during clathrin-mediated endocytosis by the cytoplasmic tail of the rhomboid protein Rbd2. Mol Biol Cell 26:1509-22
Michelot, Alphée; Drubin, David G (2014) Dissecting principles governing actin assembly using yeast extracts. Methods Enzymol 540:381-97
Weinberg, Jasper S; Drubin, David G (2014) Regulation of clathrin-mediated endocytosis by dynamic ubiquitination and deubiquitination. Curr Biol 24:951-9
Michelot, Alphée; 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
Miao, Yansong; Wong, Catherine C L; Mennella, Vito et al. (2013) Cell-cycle regulation of formin-mediated actin cable assembly. Proc Natl Acad Sci U S A 110:E4446-55
Weinberg, Jasper; Drubin, David G (2012) Clathrin-mediated endocytosis in budding yeast. Trends Cell Biol 22:1-13

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