The actin cytoskeleton is a key component of cell structure necessary for many cellular processes such as cell division and cell motility. Actin exists at concentrations inside cells that favor assembly and pose serious challenges to disassembly;in fact, we currently do not know how the cell is able to disassembly actin filaments under physiological conditions. This proposal aims to understand physiological actin dynamics by identifying and characterizing the factors responsible for the least understood side of the actin cycle: disassembly. Such an understanding would have a major impact on any area of clinical or pre-clinical research where cellular problems are actin-dependent;one such area is nephrology. Genetic analysis has found actin-binding proteins prominent among those proteins responsible for familial kidney disorders: 1-actinin and formins in focal segmental glomerulosclerosis and filamin and other actin bundling protein in polycystic kindney disease. We have identified two previously unappreciated proteins using an activity-based biochemical reconstitution that augment the previously characterized cofilin-, coronin-, and AIP1- mediated actin depolymerization system. Cyclase associated protein (CAP) allows this triple-protein mix to depolymerize a fluorescent actin substrate in the presence of cellular concentrations of polymeric and monomeric actin when alone the triple-mix can only deal with physiological monomeric actin concentrations. We propose to use a range of techniques including fluorescent microscopy of bulk actin arrays and electron and fluorescent microscopy of single actin filaments to better characterize the mechanism by which this reaction is accomplished. Somatic nuclear autoantigenic sperm protein (sNASP) functions similarly to AIP1, though further analysis of this similarity is needed. While AIP1 is a cytoplasmic protein, sNASP is a nuclear protein that we have identified as an actin disassembly factor, and since it is known to bind histones and function in chromatin remodeling it represents a possible link between the cytoskeleton and chromatin structure. We plan to use many of the same techniques to characterize sNASP as we will use to characterize CAP, with additional genetic manipulations to test AIP1 and sNASP redundancy in vivo.
Actin is an essential protein that undergoes a dynamic cycle of assembly and disassembly. Though it is directly or indirectly responsible for many human diseases, actin dynamics in physiologically relevant contexts are simply not understood. This research seeks to understand the factors that are responsible for physiological actin disassembly such that a greater understanding of this fundamental process might impact those diseases its dysfunction is responsible for.
|Normoyle, Kieran P M; Brieher, William M (2012) Cyclase-associated protein (CAP) acts directly on F-actin to accelerate cofilin-mediated actin severing across the range of physiological pH. J Biol Chem 287:35722-32|