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Abstract

The actin cytoskeleton is a large, complex, dynamic network that is responsible for a myriad of cellular processes, including, polarity, endocytosis, motility, cytokinesis, and force response. Distinct filamentous actin (F-actin) networks assemble from one crowded cytoplasm. The ability to build several F-actin networks simultaneously with distinct architectures, dynamics, location, and timing is a topic that has drawn a lot of interest. Each of these F-actin networks associate with a set of actin binding proteins (ABPs). How ABPs sort and and contribute to the properties of the F-actin networks is a big question in cell biology. Our lab has done extensive research on different ABPs and the roles in corresponding F-actin networks. Our lab does use fission yeast for cell level analysis of F-actin networks. However, a large part of our research is minimal reconstitution of F-actin networks in vitro to directly investigate actin binding proteins. My contribution to the following projects is the minimal reconstitution biochemistry experiments. A relatively new topic in our lab is mechanosensitive ABPs, and I reconstitute actomyosin networks to study mechanosensitive ABP localization. LIM domain proteins have been studied by previous labs. There are over 70 mammalian LIM domain proteins, and several have been shown to associate with the actin cytoskeleton. In fact, many of them are involved in mechanotransduction pathways and associate with the F-actin networks in a force-dependent manner. Here, we studied the mechanism of mechanosensitive LIM domain protein recruitment with both in vivo assays and in vitro reconstitution assays. LIM domain proteins associate with force responsive F-actin networks: focal adhesions, adhesion junctions, stress fibers, and contractile ring. The LIM domain containing region (LCR) is essential for localization, and the LCR is hypothesized to bind directly to a force induced conformation of F-actin. Our lab and the Alushin lab made progress in understanding the LIM domain mechanosensitive mechanism, but there is still a lot left to be learned. Another ABP discussed here is Arp2/3 complex. Arp2/3 complex is a seven component protein that binds to actin filaments and nucleates branches. Arp2/3 complex networks are important for force generation, like the leading edge of motile cells or the endocytic patches. Arp2/3 complex has been studied extensively by many labs. However, the seven component structure has made fluorescent labeling difficult. We recently labeled the fission yeast Arp2/3 complex and have successfully visualized Arps/3 complex at the branch sites. We can now study the Arp2/3 complex-mediated branch formation pathway in greater detail and better understand the mechanism involved.

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