Microfilaments are major cytoskeletal elements of all eucaryotic cells. A large number of proteins that associate with actin in higher cells have been identified and characterized, but their in vivo role is frequently poorly understood. The ubiquitious nature of microfilaments, and their altered state in oncogenically transformed cells stresses the importance of studying their biological functions. The time has come to analyze all the microfilament-associated proteins in one cell type and pinpoint the biological role of each protein. For such a project it, is desirable to use a combined biochemical, genetic and cell biological approach. We have therefore chosen the budding yeast, Saccharomyces cerevisiae, as our experimental organism as it is amenable to detailed genetic analysis and contains a single essential actin gene. We have begun to isolate microfilament- associated proteins from this organism, including actin, a trophomysin-like protein, and a putative Ca2+-regulated F-actin severing protein. We propose to characterize these proteins in detail and compare them with their higher cell counterparts to determine both their properties and how useful yeast will be as a model system. We also propose to search for and characterize other microfilament associated proteins in yeast extracts. We then propose to prepare antibodies to each of these proteins to localize them in yeast cells and to isolate their respective genes from a DNA library in the Lambda gt11 expression vector. When the genes have been isolated gene disruption experiments will be used to determine whether the proteins perform essential functions. If these disruptions in haploids result in cell death, we will embark on a project to isolate temperature-sensitive mutations in each of the genes. We will then explore the cell biological consequences of the temperature-sensitive mutations in these cells at the restrictive temperature. This will lead into a future genetic analysis of extragenic suppressors to identify interacting microfilament-associated proteins. By this work, we expect to determine, both biochemically and genetically, whether yeast is a good model system for studying the functional organization of microfilaments. If so, it should set the stage for the thorough analysis of these important cytoskeletal elements.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039066-05
Application #
3295889
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-02-01
Project End
1993-06-30
Budget Start
1992-02-01
Budget End
1993-06-30
Support Year
5
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Shin, Myungjoo; van Leeuwen, Jolanda; Boone, Charles et al. (2018) Yeast Aim21/Tda2 both regulates free actin by reducing barbed end assembly and forms a complex with Cap1/Cap2 to balance actin assembly between patches and cables. Mol Biol Cell 29:923-936
Lwin, Kyaw Myo; Li, Donghao; Bretscher, Anthony (2016) Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo. Mol Biol Cell 27:2450-62
Donovan, Kirk W; Bretscher, Anthony (2015) Head-to-tail regulation is critical for the in vivo function of myosin V. J Cell Biol 209:359-65
Donovan, Kirk W; Bretscher, Anthony (2015) Tracking individual secretory vesicles during exocytosis reveals an ordered and regulated process. J Cell Biol 210:181-9
Xu, Li; Bretscher, Anthony (2014) Rapid glucose depletion immobilizes active myosin V on stabilized actin cables. Curr Biol 24:2471-9
Wayt, Jessica; Bretscher, Anthony (2014) Cordon Bleu serves as a platform at the basal region of microvilli, where it regulates microvillar length through its WH2 domains. Mol Biol Cell 25:2817-27
Viswanatha, Raghuvir; Bretscher, Anthony; Garbett, Damien (2014) Dynamics of ezrin and EBP50 in regulating microvilli on the apical aspect of epithelial cells. Biochem Soc Trans 42:189-94
Bretscher, Anthony (2013) Deconstructing formin-dependent actin cable assembly. Proc Natl Acad Sci U S A 110:18744-5
Chernyakov, Irina; Santiago-Tirado, Felipe; Bretscher, Anthony (2013) Active segregation of yeast mitochondria by Myo2 is essential and mediated by Mmr1 and Ypt11. Curr Biol 23:1818-24
Liu, Wenyu; Santiago-Tirado, Felipe H; Bretscher, Anthony (2012) Yeast formin Bni1p has multiple localization regions that function in polarized growth and spindle orientation. Mol Biol Cell 23:412-22

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