In response to activation by WASP-family proteins, the Arp2/3 complex nucleates new actin filaments from the sides of preexisting filaments. The Arp2/3-activating (VCA) region of WASP-family proteins binds both the Arp2/3 complex and an actin monomer and the Arp2 and Arp3 subunits of the Arp2/3 complex bind ATP. We show that Arp2 hydrolyzes ATP rapidly---with no detectable lag---upon nucleation of a new actin filament. Filamentous actin and VCA together do not stimulate ATP hydrolysis on the Arp2/3 complex, nor do monomeric and filamentous actin in the absence of VCA. Actin monomers bound to the marine macrolide Latrunculin B do not polymerize, but in the presence of phalloidin-stabilized actin filaments and VCA, they stimulate rapid ATP hydrolysis on Arp2. These data suggest that ATP hydrolysis on the Arp2/3 complex is stimulated by interaction with a single actin monomer and that the interaction is coordinated by VCA. We show that capping of filament pointed ends by the Arp2/3 complex (which occurs even in the absence of VCA) also stimulates rapid ATP hydrolysis on Arp2, identifying the actin monomer that stimulates ATP hydrolysis as the first monomer at the pointed end of the daughter filament. We conclude that WASP-family VCA domains activate the Arp2/3 complex by driving its interaction with a single conventional actin monomer to form an Arp2-Arp3-actin nucleus. This actin monomer becomes the first monomer of the new daughter filament.

Bacterial Actin-Like Proteins (ALPs) participate in many biologically, clinically and commercially important processes, including segregation of low-copy plasmids. To better understand the biochemical principles underlying plasmid DNA segregation we purified a recently discovered plasmid-segregating ALP, Alp7A, and studied its structure and self-assembly in vitro. Monomeric Alp7A has a uniquely low affinity for nucleotides, binding ATP 1,000,000-fold more weakly than eukaryotic actin. The atomic structure of Alp7A (solved to 2.4 A resolution by x-ray crystallography) revealed that the low nucleotide affinity correlates with a unique nucleotide conformation. Alp7A has a low rate of spontaneous nucleation and, at low concentrations (<11 µM), forms ephemeral and highly dynamic polymers. At higher concentrations these ephemeral polymers are stabilized by lateral association, forming large bundles which contain antiparallel filaments. The accessory factor, Alp7R, dramatically increases the nucleation rate of Alp7A filaments and decreases their propensity to bundle, making them behave much more like ParM filaments. These nucleation and stabilization activities of Alp7R occur both in vitro and in vivo and do not require DNA, neither alp7C or non-specific DNA.

In eukaryotic cells, actin networks are built and dynamically reorganized during cell polarization and morphogenesis. The formation of different actin network architectures depends on whether filaments are allowed to elongate persistently or are capped shortly after nucleation. One class of proteins regulating the actin filament length distribution in cells are the barbed end associated proteins, which can terminate or accelerate actin polymerization. Depending on the activity and abundance of these proteins, cells construct actin networks that are either highly branched (lamellipodia) or a densely bundled (filopodia). Regulating the transition between these two types of actin networks, is the ubiquitously expressed Ena/VASP protein family. To determine how Ena/VASP proteins regulate actin assembly I developed a way of visualizing single VASP tetramers interacting with single actin filaments in vitro. Visualization of single VASP tetramers revealed several novel mechanisms controlling lateral F-actin interactions, barbed end association, and processivity. Providing an additional level of regulation, I identified a membrane tethered actin binding protein, Lamellipodin, that can modulate Ena/VASP barbed end processivity by simultaneously interacting with filamentous actin. Together these results provide a mechanistic framework for understanding how Ena/VASP proteins regulate actin polymerization and network architecture in vivo.

Tropomyosin (TM) is an actin binding protein that has been implicated in a variety of developmental and biological processes including neuronal morphogenesis, cell transformation, and cell motility (Gunning et al., 2008; Michelot and Drubin, 2011). The precise roles of TM in these processes has remained murky, largely because of the great diversity of TM isoforms; human cells for example express up to forty TM isoforms from four genes, most of which are poorly characterized (Gunning et al., 2005). In Drosophila there are two TM genes (Tm1 and Tm2) to which various functions have been attributed, but splicing complexity and nomenclature changes have inhibited identifying specific TM isoforms and their cellular roles (Bautch and Storti, 1983; Erdélyi et al., 1995). Here, we have carefully identified and characterized three nonmuscle TM isoforms expressed in Drosophila S2 cells. We found that all three TM isoforms have both overlapping and distinct functions that are cell cycle specific, including localizations and functions at the lamellum, Golgi, and cortex during interphase, and the cortex, kinetochores, and spindle apparatus during mitosis. Using GFP-tagged TM chimeras, we have also identified regions of individual TM isoforms that are responsible for the localization and functional differences. Together, these experiments have led to functional insights that, combined with previously known functions of tropomyosin isoforms, allow us to propose a model of how different nonmuscle TM isoforms function together in S2 cells throughout the cell cycle. Our results implicate Drosophila TM in cell cycle control, chromosome segregation, cellular contractility, and Golgi morphogenesis.