Vladimir Sirotkin

Assembly of the cytokinetic contractile ring from a broad band of nodes in fission yeast

We observed live fission yeast expressing pairs of functional fluorescent fusion proteins to test the popular model that the cytokinetic contractile ring assembles from a single myosin II progenitor or a Cdc12p-Cdc15p spot. Under our conditions, the anillin-like protein Mid1p establishes a broad band of small dots or nodes in the cortex near the nucleus. These nodes mature by the addition of conventional myosin II (Myo2p, Cdc4p, and Rlc1p), IQGAP (Rng2p), pombe Cdc15 homology protein (Cdc15p), and formin (Cdc12p). The nodes coalesce laterally into a compact ring when Cdc12p and profilin Cdc3p stimulate actin polymerization. We did not observe assembly of contractile rings by extension of a leading cable from a single spot or progenitor. Arp2/3 complex and its activators accumulate in patches near the contractile ring early in anaphase B, but are not concentrated in the contractile ring and are not required for assembly of the contractile ring. Their absence delays late steps in cytokinesis, including septum formation and cell separation.

Interactions of WASp, myosin-I, and verprolin with Arp2/3 complex during actin patch assembly in fission yeast

Yeast actin patches are dynamic structures that form at the sites of cell growth and are thought to play a role in endocytosis. We used biochemical analysis and live cell imaging to investigate actin patch assembly in fission yeast Schizosaccharomyces pombe. Patch assembly proceeds via two parallel pathways: one dependent on WASp Wsp1p and verprolin Vrp1p converges with another dependent on class 1 myosin Myo1p to activate the actin-related protein 2/3 (Arp2/3) complex. Wsp1p activates Arp2/3 complex via a conventional mechanism, resulting in branched filaments. Myo1p is a weaker Arp2/3 complex activator that makes unstable branches and is enhanced by verprolin. During patch assembly in vivo, Wsp1p and Vrp1p arrive first independent of Myo1p. Arp2/3 complex associates with nascent activator patches over 6–9 s while remaining stationary. After reaching a maximum concentration, Arp2/3 complex patches move centripetally as activator proteins dissociate. Genetic dependencies of patch formation suggest that patch formation involves cross talk between Myo1p and Wsp1p/Vrp1p pathways.

Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization.

Profilin Regulates F-Actin Network Homeostasis by Favoring Formin over Arp2/3 Complex

Fission yeast cells use Arp2/3 complex and formin to assemble diverse filamentous actin (F-actin) networks within a common cytoplasm for endocytosis, division, and polarization. Although these homeostatic F-actin networks are usually investigated separately, competition for a limited pool of actin monomers (G-actin) helps to regulate their size and density. However, the mechanism by which G-actin is correctly distributed between rival F-actin networks is not clear. Using a combination of cell biological approaches and in vitro reconstitution of competition between actin assembly factors, we found that the small G-actin binding protein profilin directly inhibits Arp2/3 complex-mediated actin assembly. Profilin is therefore required for formin to compete effectively with excess Arp2/3 complex for limited G-actin and to assemble F-actin for contractile ring formation in dividing cells.

Homeostatic Actin Cytoskeleton Networks Are Regulated by Assembly Factor Competition for Monomers

Controlling the quantity and size of organelles through competition for a limited supply of components is quickly emerging as an important cellular regulatory mechanism. Cells assemble diverse actin filament (F-actin) networks for fundamental processes including division, motility, and polarization. F-actin polymerization is tightly regulated by activation of assembly factors such as the Arp2/3 complex and formins at specific times and places. We directly tested an additional hypothesis that diverse F-actin networks are in homeostasis, whereby competition for actin monomers (G-actin) is critical for regulating F-actin network size. Here we show that inhibition of Arp2/3 complex in the fission yeast Schizosaccharomyces pombe not only depletes Arp2/3-complex-mediated endocytic actin patches, but also induces a dramatic excess of formin-assembled F-actin. Conversely, disruption of formin increases the density of Arp2/3-complex-mediated patches. Furthermore, modification of actin levels significantly perturbs the fission yeast actin cytoskeleton. Increasing actin favors Arp2/3-complex-mediated actin assembly, whereas decreasing actin favors formin-mediated contractile rings. Therefore, the specific actin concentration in a cell is critical, and competition for G-actin helps regulate the proper amount of F-actin assembly for diverse processes.

Mathematical Modeling of Endocytic Actin Patch Kinetics in Fission Yeast: Disassembly Requires Release of Actin Filament Fragments

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization.

Three's company: the fission yeast actin cytoskeleton

How the actin cytoskeleton assembles into different structures to drive diverse cellular processes is a fundamental cell biological question. In addition to orchestrating the appropriate combination of regulators and actin-binding proteins, different actin-based structures must insulate themselves from one another to maintain specificity within a crowded cytoplasm. Actin specification is particularly challenging in complex eukaryotes where a multitude of protein isoforms and actin structures operate within the same cell. Fission yeast Schizosaccharomyces pombe possesses a single actin isoform that functions in three distinct structures throughout the cell cycle. In this review we explore recent studies in fission yeast that help unravel how different actin structures operate in cells.

Actin Filament Bundling by Fimbrin Is Important for Endocytosis, Cytokinesis, and Polarization in Fission Yeast

Through the coordinated action of diverse actin-binding proteins, cells simultaneously assemble actin filaments with distinct architectures and dynamics to drive different processes. Actin filament cross-linking proteins organize filaments into higher order networks, although the requirement of cross-linking activity in cells has largely been assumed rather than directly tested. Fission yeast Schizosaccharomyces pombe assembles actin into three discrete structures: endocytic actin patches, polarizing actin cables, and the cytokinetic contractile ring. The fission yeast filament cross-linker fimbrin Fim1 primarily localizes to Arp2/3 complex-nucleated branched filaments of the actin patch and by a lesser amount to bundles of linear antiparallel filaments in the contractile ring. It is unclear whether Fim1 associates with bundles of parallel filaments in actin cables. We previously discovered that a principal role of Fim1 is to control localization of tropomyosin Cdc8, thereby facilitating cofilin-mediated filament turnover. Therefore, we hypothesized that the bundling ability of Fim1 is dispensable for actin patches but is important for the contractile ring and possibly actin cables. By directly visualizing actin filament assembly using total internal reflection fluorescence microscopy, we determined that Fim1 bundles filaments in both parallel and antiparallel orientations and efficiently bundles Arp2/3 complex-branched filaments in the absence but not the presence of actin capping protein. Examination of cells exclusively expressing a truncated version of Fim1 that can bind but not bundle actin filaments revealed that bundling activity of Fim1 is in fact important for all three actin structures. Therefore, fimbrin Fim1 has diverse roles as both a filament “gatekeeper” and as a filament cross-linker.

Diverse protective roles of the actin cytoskeleton during oxidative stress

Actin oxidation is known to result in changes in cytoskeleton organization and dynamics. Actin oxidation is clinically relevant since it occurs in the erythrocytes of sickle cell patients and may be the direct cause of the lack of morphological plasticity observed in irreversibly sickled red blood cells (ISCs). During episodes of crisis, ISCs accumulate C284‐C373 intramolecularly disulfide bonded actin, which reduces actin filament dynamics. Actin cysteines 284 and 373 (285 and 374 in yeast) are conserved, suggesting that they play an important functional role. We have been investigating the physiological roles of these cysteines using the model eukaryote Saccharomyces cerevisiae in response to oxidative stress load. During acute oxidative stress, all of the F‐actin in wild‐type cells collapses into a few puncta that we call oxidation‐induced actin bodies (OABs). In contrast, during acute oxidative stress the actin cytoskeleton in Cys‐to‐Ala actin mutants remains polarized longer, OABs are slower to form, and the cells recover more slowly than wild‐type cells, suggesting that the OABs play a protective role. Live cell imaging revealed that OABs are large, immobile structures that contain actin‐binding proteins and that can form by the fusion of actin cortical patches. We propose that actins C285 and C374 may help to protect the cell from oxidative stress arising from normal oxidative metabolism and contribute to the cells general adaptive response to oxidative stress.

UCS Protein Rng3p Is Essential for Myosin-II Motor Activity during Cytokinesis in Fission Yeast

UCS proteins have been proposed to operate as co-chaperones that work with Hsp90 in the de novo folding of myosin motors. The fission yeast UCS protein Rng3p is essential for actomyosin ring assembly and cytokinesis. Here we investigated the role of Rng3p in fission yeast myosin-II (Myo2p) motor activity. Myo2p isolated from an arrested rng3-65 mutant was capable of binding actin, yet lacked stability and activity based on its expression levels and inactivity in ATPase and actin filament gliding assays. Myo2p isolated from a myo2-E1 mutant (a mutant hyper-sensitive to perturbation of Rng3p function) showed similar behavior in the same assays and exhibited an altered motor conformation based on limited proteolysis experiments. We propose that Rng3p is not required for the folding of motors per se, but instead works to ensure the activity of intrinsically unstable myosin-II motors. Rng3p is specific to conventional myosin-II and the actomyosin ring, and is not required for unconventional myosin motor function at other actin structures. However, artificial destabilization of myosin-I motors at endocytic actin patches (using a myo1-E1 mutant) led to recruitment of Rng3p to patches. Thus, while Rng3p is specific to myosin-II, UCS proteins are adaptable and can respond to changes in the stability of other myosin motors.