Thursday, December 26, 2024
12/26/2024
Project Summary Reorganization of the actin cytoskeleton supports many dynamic cell biological processes, such as cell migration, cytokinesis, vesicle trafficking, and morphogenesis. Although actin spontaneously polymerizes and depolymerizes by itself, the dynamic properties of actin need to be controlled by actin-regulatory proteins in cells. Among many known actin-regulatory proteins, cyclase-associated protein (CAP) regulates multiple key processes of actin dynamics. CAP is conserved among eukaryotes and has been known as an actin-monomer binding protein for many years. However, recent studies have identified important functions of CAP, which are critical to promote rapid actin turnover. CAP promotes disassembly of actin filaments from the pointed ends in the presence of actin-depolymerizing factor (ADF)/cofilin. Spontaneous actin depolymerization from the pointed ends is very slow and rate-limiting in actin filament turnover. The synergy between CAP and ADF/cofilin can enhance actin depolymerization in vitro to a range that can explain rapid actin turnover in cells. However, the precise mechanism by which CAP and ADF/cofilin induce actin depolymerization remains unclear, partly because structural information of a CAP oligomer is limited. CAP oligomerizes under physiological conditions, and oligomerization enhances its activity. We have recently obtained evidence that CAP is tetrameric, and our structural model of the CAP tetramer allowed us to hypothesize a functional link between the tetrameric configuration of CAP and processive actin depolymerizing activity. CAP also binds to the side of actin filaments and synergizes with ADF/cofilin to promote filament severing. We hypothesize that this is due to the effect of CAP on the helical structures of actin filaments, which then causes partial dissociation of ADF/cofilin from the filaments. In addition to the in vitro studies, recent reports of human mutations in the CAP (CAP2) gene have linked impairment of CAP to human diseases in the heart and skeletal muscle involving abnormalities in the actin cytoskeletal organization. We will use the nematode Caenorhabditis elegans as a relevant model organism to study roles of CAP in the assembly of sarcomeric actin filaments in muscle cells in vivo. We proposes three aims: (1) to determine functional significance of CAP tetramerization for actin filament disassembly, (2) to determine the effects of CAP on the helical structures of F-actin, and (3) to determine in vivo roles of CAP in actin cytoskeletal integrity in the nematode Caenorhabditis elegans. We will employ a combination of in vitro and in vivo approaches to gain new insight in the function of CAP in actin cytoskeletal regulation.
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