UCSF

Actin is a supremely conserved cytoskeletal protein whose homologs can be found across eukaryotes, archaea, and bacteria. Polymerized into micrometer-scale filaments, actin networks organize within the cell to accomplish several critical functions such as motility, division, and mechanosensing. To perform these functions, the actin cytoskeleton must adopt a variety of morphologies with spatial precision, which is all controlled by a vast menagerie of actin-regulating proteins. The Mullins Lab is broadly interested in delineating the structure-function relationships of these proteins in the context of cellular and molecular biology. A key tool for studying the structure and function of actin and actin-regulators is cryo-electron microscopy (cryo-EM), which enables near-atomic resolution visualization of in vitro and in vivo systems of proteins in hydrated environments. In this dissertation, we will explore the unique insights gained into several actin cytoskeleton systems through single particle cryo-EM, a result of years of collaborative work with the talented cell biologists and biochemists within the lab. In Chapter 1, I characterize the binding posture of novel Drosophila tropomyosins on actin filaments, present the world's first fly actin polymer structure, and report my attempts to engineer a fiducial onto tropomyosin. In Chapter 2, I elucidate the binding sites of single-stranded DNA to the Arp2/3 complex which compete with the VCA of nucleation promoting factors. In Chapter 3, I present my work in investigating the cytoskeletal elements of non-eukaryotic organisms: SegA from Sulfolobus, Alp7A from Bacillus subtilis, and Ta0583 from Thermoplasma acidophilum.