The cytoskeleton is a highly dynamic structure that is capable of rapid assembly and disassembly. Actin, one of the primary building blocks of the cytoskeleton, can exist in either monomeric or polymeric form. Using a highly conserved set of approximately 20 to 30 proteins, cells attain remarkable spatial and temporal control of the nucleation of actin filaments, their elongation and their eventual disassembly into monomeric proteins. Formins have recently emerged as important nucleators of actin assembly. After inducing nucleation, formins remain attached to the growing end of a filament and thereafter guide insertion of new subunits. In budding yeast, formins are localized at the bud tip and bud neck and from there they direct the assembly of actin cables which are highly dynamic bundles of actin filaments. Actin cables play an important role in establishing cell polarity and cell morphogenesis. The goal of this mentored quantitative research development award is twofold. From a research perspective, it will provide new insight into mechanisms by which formins accelerate growth rates of actin filaments.
The specific aims of the proposal are to (1) use single molecule fluorescence imaging to elucidate the mechanism by which the FH1 domain of formins enhances the polymerization rate of actin filaments and (2) to use yeast extracts to reconstitute the formation of dynamic actin cables and measure the dependence of actin turnover on various regulatory proteins using advanced optical microscopy. These two processes will be described with quantitative models which will be rigorously tested against experimental data. From a training perspective, the PI of this proposal will gain valuable hands on knowledge in techniques of molecular biology and biochemistry by working in the laboratory of his mentor (Bruce Goode). Additionally, the PI will also acquire formal knowledge of molecular biology and biochemistry by taking graduate level courses and through the attendance of seminars and journal clubs. In this proposal we will use budding yeast S. cerevisiae as a model organism. The majority of actin associated proteins that regulate the assembly of actin structures in yeast have direct counterparts in mammalian cells, with strikingly similar functions. Therefore, the knowledge gained from studies of yeast has direct relevance to our understanding of normal human cell biology and how mutations of various actin regulatory proteins give rise to diseases such as cancer.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Mentored Quantitative Research Career Development Award (K25)
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Macromolecular Structure and Function C Study Section (MSFC)
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Gindhart, Joseph G
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Brandeis University
Schools of Arts and Sciences
United States
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Sanchez, Timothy; Dogic, Zvonimir (2013) Engineering oscillating microtubule bundles. Methods Enzymol 524:205-24
Sanchez, Timothy; Welch, David; Nicastro, Daniela et al. (2011) Cilia-like beating of active microtubule bundles. Science 333:456-9