Fission yeast Myo2: Molecular organization and diffusion in the cytoplasm.

Myosin-II is required for the assembly and constriction of cytokinetic contractile rings in fungi and animals. We used electron microscopy, fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS) to characterize the physical properties of Myo2 from fission yeast Schizosaccharomyces pombe. By electron microscopy, Myo2 has two heads and a coiled-coiled tail like myosin-II from other species. The first 65 nm of the tail is a stiff rod, followed by a flexible, less-ordered region up to 30 nm long. Myo2 sediments as a 7 S molecule in high salt, but aggregates rather than forming minifilaments at lower salt concentrations; this is unaffected by heavy chain phosphorylation. We used FRAP and FCS to observe the dynamics of Myo2 in live S. pombe cells and in cell extracts at different salt concentrations; both show that Myo2 with an N-terminal mEGFP tag has a diffusion coefficient of ∼ 3 µm2 s-1 in the cytoplasm of live cells during interphase and mitosis. Photon counting histogram analysis of the FCS data confirmed that Myo2 diffuses as doubled-headed molecules in the cytoplasm. FCS measurements on diluted cell extracts showed that mEGFP-Myo2 has a diffusion coefficient of ∼ 30 µm2 s-1 in 50 to 400 mM KCl concentrations.

High-speed superresolution imaging of the proteins in fission yeast clathrin-mediated endocytic actin patches.

To internalize nutrients and cell surface receptors via clathrin-mediated endocytosis, cells assemble at least 50 proteins, including clathrin, clathrin-interacting proteins, actin filaments, and actin binding proteins, in a highly ordered and regulated manner. The molecular mechanism by which actin filament polymerization deforms the cell membrane is unknown, largely due to lack of knowledge about the organization of the regulatory proteins and actin filaments. We used high-speed superresolution localization microscopy of live fission yeast cells to improve the spatial resolution to ∼35 nm with 1-s temporal resolution. The nucleation promoting factors Wsp1p (WASp) and Myo1p (myosin-I) define two independent pathways that recruit Arp2/3 complex, which assembles two zones of actin filaments. Myo1p concentrates at the site of endocytosis and initiates a zone of actin filaments assembled by Arp2/3 complex. Wsp1p appears simultaneously at this site but subsequently moves away from the cell surface as it stimulates Arp2/3 complex to assemble a second zone of actin filaments. Cells lacking either nucleation-promoting factor assemble only one, stationary, zone of actin filaments. These observations support our two-zone hypothesis to explain endocytic tubule elongation and vesicle scission in fission yeast.

The fission yeast cytokinetic contractile ring regulates septum shape and closure.

During cytokinesis, fission yeast and other fungi and bacteria grow a septum that divides the cell in two. In fission yeast closure of the circular septum hole by the ß-glucan synthases (Bgs) and other glucan synthases in the plasma membrane is tightly coupled to constriction of an actomyosin contractile ring attached to the membrane. It is unknown how septum growth is coordinated over scales of several microns to maintain septum circularity. Here, we documented the shapes of ingrowing septum edges by measuring the roughness of the edges, a measure of the deviation from circularity. The roughness was small, with spatial correlations indicative of spatially coordinated growth. We hypothesized that Bgs-mediated septum growth is mechanosensitive and coupled to contractile ring tension. A mathematical model showed that ring tension then generates almost circular septum edges by adjusting growth rates in a curvature-dependent fashion. The model reproduced experimental roughness statistics and showed that septum synthesis sets the mean closure rate. Our results suggest that the fission yeast cytokinetic ring tension does not set the constriction rate but regulates septum closure by suppressing roughness produced by inherently stochastic molecular growth processes.

A role for F-BAR protein Rga7p during cytokinesis in S. pombe.

F-BAR proteins are known to participate in cytokinesis, but their mechanisms are not well understood. Here we investigated Rga7p, an Schizosaccharomyces pombe F-BAR protein with a RhoGAP domain. Localization of Rga7p to the cytokinetic cleavage furrow depends on its F-BAR domain, actin filaments, the formins Cdc12p and For3p, and the presence of a contractile ring. Rga7p is not required for the constriction of the contractile ring but does participate in the transport of a ß-glucan synthetase (Bgs4p) from the late Golgi compartments to plasma membrane that is adjacent to the contractile ring. Cells without Rga7p moved Bgs4p normally from the poles to the Golgi complex near to the cell center, but Bgs4p then moved slowly from the late Golgi compartments to the cleavage site. The late arrival and lower than normal numbers of Bgs4p result in septal defects late in cytokinesis, and in the lysis of separating cells, similar to that in cells with mutations in the cwg1(+) gene (which encodes Bgs4p).

Contractile ring stability in S. pombe depends on F-BAR protein Cdc15p and Bgs1p transport from the Golgi complex.

Cdc15p is known to contribute to cytokinesis in fission yeast; however, the protein is not required to assemble the contractile ring of actin and myosin, but it helps to anchor the ring to the plasma membrane. Cdc15p has a lipid-binding F-BAR domain, suggesting that it provides a physical link between the plasma membrane and contractile ring proteins. However, we find that a more important function of Cdc15p during cytokinesis is to help deliver a transmembrane enzyme, Bgs1p (also called Cps1p), from the Golgi apparatus to the plasma membrane, where it appears to anchor the contractile ring. Bgs1p synthesizes the cell wall in the cleavage furrow, but its enzyme activity is not required to anchor the contractile ring. We estimate that ∼ 2,000 Bgs1p molecules are required to anchor the ring. Without Bgs1p anchors, contractile rings slide along the plasma membrane, a phenomenon that depends on an unconventional type II myosin called Myp2p.

Distinct roles for F-BAR proteins Cdc15p and Bzz1p in actin polymerization at sites of endocytosis in fission yeast.

BACKGROUND: Genetic analyses of budding and fission yeast identified >50 proteins that assemble at sites of clathrin-mediated endocytosis in structures called actin patches. These proteins include clathrin, clathrin-interacting proteins, actin binding proteins, and peripheral membrane proteins such as F-BAR proteins. Many questions remain regarding the interactions of these proteins, particularly the participation of F-BAR proteins in the assembly of actin filaments. RESULTS: Our microscopic and genetic interaction experiments on fission yeast show that F-BAR proteins Cdc15p and Bzz1p accumulate in two distinct zones on invaginating membrane tubules and interact with Myo1p and Wsp1p, nucleation-promoting factors for Arp2/3 complex. The two F-BAR proteins peak prior to movement of the actin patch and their accumulation in actin patches depends on the nucleation-promoting factors. At their peak local concentrations, we estimated the stoichiometries of the proteins in actin patches to be one Bzz1p per two Wsp1p and one Cdc15p per Myo1p. Purified Bzz1p has two SH3 domains that interact with Wsp1p and stimulate actin polymerization by Arp2/3 complex. Cells lacking either Cdc15p or Bzz1p assemble 3- to 5-fold less actin in patches (in spite of normal levels of Wsp1p, Myo1p, and Arp2/3 complex), and patches move shorter distances from the plasma membrane. CONCLUSION: We propose that during clathrin-mediated endocytosis, F-BAR proteins interact with nucleation-promoting factors to stimulate Arp2/3 complex in two different zones along the invaginating tubule. We further propose that polymerization of actin filaments in these two zones contributes to membrane scission.

The Ste20-like kinase SvkA of Dictyostelium discoideum is essential for late stages of cytokinesis.

The genome of the social amoeba Dictyostelium discoideum encodes approximately 285 kinases, which represents approximately 2.6% of the total genome and suggests a signaling complexity similar to that of yeasts and humans. The behavior of D. discoideum as an amoeba and during development relies heavily on fast rearrangements of the actin cytoskeleton. Here, we describe the knockout phenotype of the svkA gene encoding severin kinase, a homolog of the human MST3, MST4 and YSK1 kinases. SvkA-knockout cells show drastic defects in cytokinesis, development and directed slug movement. The defect in cytokinesis is most prominent, leading to multinucleated cells sometimes with >30 nuclei. The defect arises from the frequent inability of svkA-knockout cells to maintain symmetry during formation of the cleavage furrow and to sever the last cytosolic connection. We demonstrate that GFP-SvkA is enriched at the centrosome and localizes to the midzone during the final stage of cell division. This distribution is mediated by the C-terminal half of the kinase, whereas a rescue of the phenotypic changes requires the active N-terminal kinase domain as well. The data suggest that SvkA is part of a regulatory pathway from the centrosome to the midzone, thus regulating the completion of cell division.

Profilin isoforms in Dictyostelium discoideum.

Eukaryotic cells contain a large number of actin binding proteins of different functions, locations and concentrations. They bind either to monomeric actin (G-actin) or to actin filaments (F-actin) and thus regulate the dynamic rearrangement of the actin cytoskeleton. The Dictyostelium discoideum genome harbors representatives of all G-actin binding proteins including actobindin, twinfilin, and profilin. A phylogenetic analysis of all profilins suggests that two distinguishable groups emerged very early in evolution and comprise either vertebrate and viral profilins or profilins from all other organisms. The newly discovered profilin III isoform in D. discoideum shows all functions that are typical for a profilin. However, the concentration of the third isoform in wild type cells reaches only about 0.5% of total profilin. In a yeast-2-hybrid assay profilin III was found to bind specifically to the proline-rich region of the cytoskeleton-associated vasodilator-stimulated phosphoprotein (VASP). Immunolocalization studies showed similar to VASP the profilin III isoform in filopodia and an enrichment at their tips. Cells lacking the profilin III isoform show defects in cell motility during chemotaxis. The low abundance and the specific interaction with VASP argue against a significant actin sequestering function of the profilin III isoform.

Characterization of the Ste20-like kinase Krs1 of Dictyostelium discoideum.

Ste20-like kinases constitute a ubiquitous and expanding group of serine/threonine kinases, homologous to Ste20 in Saccharomyces cerevisiae. The social amoeba Dictyostelium discoideum contains at least 17 members of this kinase family, 13 from the germinal center kinase (GCK) subgroup and 4 p21-activated kinases (PAK). Here, we describe the kinase Krs1 which is encoded by the gene krsA, and phylogenetic analysis groups it into subfamily GCK-II together with human MST2 and MST1 or Hippo from Drosophila melanogaster. Significant similarities are found especially in the catalytic domain and in a short regulatory region (SARAH) which is thought to be important for protein/protein interactions. Northern blot analysis showed a single krsA transcript throughout development with an upregulation at 12h after the onset of starvation. The protein levels as detected with anti-Krs1 polyclonal antibodies revealed a similar pattern. Gel filtration experiments suggested that AX2 wild-type cells harbored multimeric forms of Krs1. In vitro phosphorylation assays with recombinant protein showed that the kinase exhibits autophosphorylation and accepts myelin basic protein and D. discoideum severin as substrates. A series of C-terminal deletions of Krs1 indicated that the regulatory domain in the C-terminal half contains inhibitory elements, and highlighted the importance of two predicted alpha-helices following subdomain XI of the classical catalytic domain. GFP-Krs1-overexpressing wild-type cells showed an enrichment of the kinase in the cortex, and motility of these cells during aggregation was reduced. Krs1 knockout strains exhibited only subtle differences to wild-type cells which suggests a certain redundancy among Ste20-like kinases in D. discoideum.

The bundling activity of vasodilator-stimulated phosphoprotein is required for filopodium formation.

Filopodia are highly dynamic finger-like cell protrusions filled with parallel bundles of actin filaments. Previously we have shown that Diaphanous-related formin dDia2 is involved in the formation of filopodia. Another key player for the formation of filopodia across many species is vasodilator-stimulated phosphoprotein (VASP). It has been proposed that the essential role of VASP for formation of filopodia is its competition with capping proteins for filament barbed-end interaction. To better understand the function of VASP in filopodium formation, we analyzed the in vitro and in vivo properties of Dictyostelium VASP (DdVASP) and extended our findings to human VASP. Recombinant VASP from both species nucleated and bundled actin filaments, but did not compete with capping proteins or block depolymerization from barbed ends. Together with the finding that DdVASP binds to the FH2 domain of dDia2, these data indicate that the crucial role of VASP in filopodium formation is different from uncapping of actin filaments. To identify the activity of DdVASP required in this process, rescue experiments of DdVASP-null cells with mutant DdVASP constructs were performed. Only WT DdVASP, but not a mutant lacking the F-actin bundling activity, could rescue the ability of these cells to form WT-like filopodia. Our data suggest that DdVASP is complexed with dDia2 in filopodial tips and support formin-mediated filament elongation by bundling nascent actin filaments.

The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia.

Formins have important roles in the nucleation of actin and the formation of linear actin filaments, but their role in filopodium formation has remained elusive. Dictyostelium discoideum Diaphanous-related formin dDia2 is enriched at the tips of filopodia and interacts with profilin II and Rac1. An FH1FH2 fragment of dDia2 nucleated actin polymerization and removed capping protein from capped filament ends. Genetic studies showed that dDia2 is important for cell migration as well as the formation, elongation and maintenance of filopodia. Here we provide evidence that dDia2 specifically controls filopodial dynamics by regulating actin turnover at the barbed ends of actin filaments.