Actin Dynamics: from Single Molecules to Competing Cellular Networks

Actin builds an essential part of the cytoskeleton in eukaryotes. Cells utilize the actin cytoskeleton for numerous processes such as endocytosis, motility, and cytokinesis. Multiple actin networks are simultaneously built and maintained in the same crowded cytoplasm. How cells organize and regulate numerous actin networks is an important biological question. Polymerization of the actin cytoskeleton is tightly regulated by activation of assembly factors at specific times and places. Not only is it crucial to understand the molecular mechanisms of how specific actin assembly factors polymerize actin, but it is equally important to understand how actin networks can influence each other on a cell-wide scale. My two projects investigated how actin assembly factors share a common pool of actin monomers in fission yeast and how the actin assembly factors VopL and VopF interact with and polymerize actin filaments. For my first project I investigated whether diverse actin filament networks are homeostatic, whereby actin assembly factor competition for actin monomers is critical to regulate network size and density. The fission yeast Schizosaccharomyces pombe possesses three F-actin networks: Arp2/3 complex-mediated patches, formin For3p-mediated cables, and the formin Cdc12p-mediated contractile ring (Kovar et al. 2011). The amount of actin incorporated into each network is consistent both inter and intra-cellularly, varying less than 50% between structures. The regulation of actin network assembly in cells was thought to be regulated by signaling cascades. If this were true, the systematic depletion of a single actin network should have negligible effects on the other networks. However, in my first project I tested an additional hypothesis that diverse F-actin networks are in competition for actin monomers (G-actin). I discovered that inhibition of the Arp2/3 complex in fission yeast not only eliminates Arp2/3 complex-mediated endocytic actin patches, but due to the newly freed up actin monomer, also induces assembly of ectopic formin-assembled F-actin. Conversely, disruption of the formins increases the density of Arp2/3 complex-mediated actin patches. Disrupting of the flow of monomer by depleting the F-actin severing protein cofilin prevents ectopic actin from forming. Furthermore, modifying actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing the actin concentration favors Arp2/3 complex-mediated actin assembly, whereas decreasing the actin concentration favors formin-mediated contractile rings. Therefore, competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes. My second project focused on the biochemical characterization of two WH2-domain actin assembly factors, VopL and VopF. The WH2 domain is a common actin monomer-binding motif comprising of approximately 17 amino acids and is utilized for multiple different functions within cells like monomer sequestration, assembly factor regulation, as well as a newly identified general class of assembly factors (Dominguez 2016; Paunola et al. 2002). Cells have created multiple assembly factors that utilize the WH2 domain in tandem within the same protein given its high affinity for actin monomers. Multiple organisms have used types of these actin assembly factors for processes such as neural development and cytoplasmic streaming during early embryo development. Conversely, pathogens have mimicked this powerful method of actin assembly for more sinister purposes. VopF and VopL are WH2-domain containing actin assembly factors utilized by the infectious bacteria that cause cholera, Vibrio cholerae and Vibrio parahaemolyticus (Tam et al. 2007; Liverman et al. 2007). Unlike other infectious bacteria that secrete proteins to activate host-cell nucleation factors such as Listeria (ActA) and Shigella (IcsA) to hijack the host-cell's F-actin cytoskeleton for motility, VopF and VopL nucleate the host cell's F-actin directly (Cossart 2000; Tam et al. 2007; Liverman et al. 2007). VopF and VopL are F-actin nucleators and are categorized into the unique group of nucleators such as Spire, Cobl, and Sca2 that rely on tandem G-actin binding WH2 domains for their activity (Chesarone & Goode 2009; Quinlan et al. 2005; Haglund et al. 2010). VopF and VopL are both homodimers, and their domain organization is conserved with 72% protein sequence similarity. From N to C-terminus both have two proline-rich regions, three WH2 domains, followed by a dimerization inducing Vop C-terminal Domain (VCD) that causes a U shaped configuration (Liverman et al. 2007; Tam et al. 2007; Pernier et al. 2013; Namgoong et al. 2011). The F-actin nucleation capabilities of VopF and VopL are undisputed, however recent studies have been in conflict over the exact mechanism employed. Using multi-color TIRF microscopy I have shown that in the presence of actin monomers, VopL and VopF are purely nucleators that associate briefly with the pointed end of actin filaments before dissociating and do not bind to pre-existing filaments. In the absence of monomer VopL and VopF are competent to bind to both the pointed and barbed ends of actin filaments. Additionally VopL and VopF can bind to the barbed end of actin filaments in conditions with saturating profilin with actin monomers. However in the light of the pathogenic nature of Vibrio bacteria, we are more prone to believe that the primary function of VopL and VopF are to be highly efficient nucleators that promote the assembly of unproductive F-actin in the host cell thereby disrupting the G- to F-actin homeostasis.