The focus of our research plan is to determine the fundamental biochemical mechanisms that govern how cells differentially coordinate actin filament assembly for a wide range of tasks. It has been recently established that formins promote actin assembly that drives diverse cellular processes such as division, polarization and migration. The hallmark features of formin proteins are two internal formin homology 1 and 2 domains (FH1FH2) that are flanked by regulatory domains. FH1FH2 domains cooperate to assemble actin filaments by a conceptually novel, but poorly understood, mechanism. Eukaryotic cells contain multiple formin isoforms utilized for different processes, but the mechanistic basis for functional specificity is not clear. Differential regulation, activation of each formin isoform at the right time and place, is probably required but it has not been tested. Additionally, I discovered that the rate of actin assembly varies significantly between diverse formins, suggesting that different cellular processes require different actin filament elongation rates. The unicellular fission yeast Schizosaccharomyces pombe is a superb model system for investigating formin biology because it is amenable to a wide-range of experimental strategies and because it contains three formins that are each required for a specific cellular process. I am initially focusing on the fundamentally important fission yeast cytokinesis formin Cdc12p. I plan to utilize a combination of genetics, cell biology and in vitro real-time observation of the assembly of individual actin filaments by evanescent wave fluorescence microscopy to address my hypothesis that both differential regulation and the specific actin filament elongation rate are functionally important. I will initially address three specific aims.
Aim I : to elucidate the molecular parameters that determine the specific elongation rate of Cdc12p.
Aim II : to determine the consequences for cytokinesis of altering the elongation rate of Cdc12p.
Aim III : to elucidate the mechanism(s) of regulation of Cdc12p. Relevance: Formins assemble the actin filaments necessary for basic cellular processes such as the contractile ring during cell division and filopodial protrusions at the leading edge of motile cells. Cell division and motility are required for normal mammalian development, but then become unregulated in malignant tumors. By utilizing a combination of experimental approaches to study fission yeast formins rapid progress can be made, and given the similarity of formin biology between animal and fission yeast cells these studies will lay the groundwork for establishing their general mechanisms in animals.
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