“Understanding the Role of Coactosin in Actin Dynamics”
A fundamental feature of the actin cytoskeleton is its ability to be rapidly assembled and disassembled in cells by the action of a variety of actin-interacting proteins. One family of proteins that are particularly central to the regulation of actin disassembly is the ADF-H (actin disassembly factor homology) domain proteins, which includes Cofilin, an actin-severing factor, and Twinfilin, which accelerates filament end depolymerization. Coactosin, a small protein composed of a single ADF-H domain, is the least well-studied member of the ADF-H protein family. Although it shares close structural homology with Cofilin and other ADF-H domain proteins, its role in actin dynamics is poorly understood.
Here, I test the effects of Coactosin on its own and in combination with other disassembly factors in-vitro. A fluorescently-labeled Coactosin will be generated as a tool to help us to understand how Coactosin contributes to actin turnover. In order to label Coactosin in a way that is least likely to affect the function of the protein, the endogenous cysteines will be mutated to serines. Based on previously identified binding sites on Cofilin, a cysteine will be introduced into Coactosin where it is least likely to disrupt these key surfaces. These mutations will result in a single cysteine on the Coactosin protein where the Cy3 dye will bind. TIRF microscopy will be used to visualize how Coactosin interacts with actin and other ADF-H domain proteins in order to better understand the mechanisms by which Coactosin affect actin dynamics.