Dicty News Electronic Edition Volume 14, number 5 February 26, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" ============= Abstracts ============= Myosin II-independent cytokinesis in Dictyostelium: its mechanism and implications. Taro Q. P. Uyeda1, Chikako Kitayama1 and Shigehiko Yumura2 1: Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan 2: Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan. Cell Structure and Function, in press Abstract Similar to higher animal cells, ameba cells of the cellular slime mold Dictyostelium discoideum form contractile rings containing filaments of myosin II during mitosis, and it is generally believed that contraction of these rings bisects the cells. In suspension, mutant cells lacking the single myosin II heavy chain gene cannot carry out cytokinesis, become large and multinucleate, and eventually lyze, supporting the idea that myosin II plays critical roles in cytokinesis. These mutant cells are however viable on substrates. Analyses of these mutant cells on substrates revealed that, in addition to "classic" cytokinesis which depends on myosin II ("cytokinesis A"), Dictyostelium has two distinct, novel methods of cytokinesis, 1) attachment-assisted mitotic cleavage employed by myosin II null cells on substrates ("cytokinesis B"), and 2) cytofission, a cell cycle-independent division of adherent cells ("cytokinesis C"). Cytokinesis A, B, and C lose their function such as accuracy and robustness, and demand fewer protein factors in this order. Cytokinesis B is of particular importance for future studies. Similar to cytokinesis A, cytokinesis B involves formation of a cleavage furrow in the equatorial region, and it may be a primitive but basic mechanism of efficiently bisecting a cell in a cell cycle-coupled manner. Analysis of large, multinucleate myosin II null cells suggested that interactions between astral microtubules and cortices positively induce polar protrusive activities. A model is proposed to explain how such polar activities drive cytokinesis B, and how cytokinesis B is coordinated with cytokinesis A in wild type cells. ---------------------------------------------------------------------------- Molecular biological approaches to study myosin functions in cytokinesis of Dictyostelium. Taro Q. P. Uyeda1, and Shigehiko Yumura2 1: Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan 2: Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan. Microscopy Research Technique, in press Abstract The cellular slime mold Dictyostelium discoideum is amenable to biochemical, cell biological and molecular genetic analyses, and offers a unique opportunity for multifaceted approaches to dissect the mechanism of cytokinesis. One of the important questions that are currently under investigation using Dictyostelium is to understand how cleavage furrows or contractile rings are assembled in the equatorial region. Contractile rings consist of a number of components including parallel filaments of actin and myosin II. Phenotypic analyses and in vivo localization studies of cells expressing mutant myosin IIs have demonstrated that myosin II's transport to and localization at the equatorial region does not require regulation by phosphorylation of myosin II, specific amino acid sequences of myosin II, or the motor activity of myosin II. Rather, the transport appears to depend on a myosin II-independent flow of cortical cytoskeleton. What drives the flow of cortical cytoskeleton is still elusive. However, a growing number of mutants that affect assembly of contractile rings have been accumulated. Analyses of these mutations, identification of more cytokinesis-specific genes, and information deriving from other experimental systems, should allow us to understand the mechanism of contractile ring formation and other aspects of cytokinesis. ---------------------------------------------------------------------------- Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells Chang Y. Chung, Susan Lee, Celia Briscoe, Charlene Ellsworth, and Richard A. Firtel Proc. Natl. Acad. Sci. USA, in press. ABSTRACT We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin towards a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly towards the cAMP source, Rac1B61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis. ---------------------------------------------------------------------------- Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (Soluble NSF-Attachment Proteins) families in Dictyostelium discoideum Marianne Weidenhaupt1*, Franz Bruckert1**, Mathilde Louwagie2, Jerome Garin2 and Michel Satre1 1 DBMS/BBSI (UMR5092 CNRS) and 2 DBMS/CP, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 09, France *Present address: LIM/IBS, 41 rue Jules Horowitz, 38027 Grenoble Cedex 01, France **corresponding author. E-mail: fbruckert@cea.fr Eur. J. Biochem., in press. The soluble NSF-Attachment Proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta- and gamma-subfamilies. SNAPs act as adapters between NSF (N-ethylmaleimide Sensitive Factor), a hexameric ATPase, and membrane SNARE proteins (SNAP REceptors). Within the NSF/SNAP/SNARE complex, they contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain, overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilised membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36), that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents for the first time the wide distribution of the gamma-subtype. Altogether, these results suggest a particular role for gamma-SNAP, possibly distinct from that of alpha-SNAP. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 5]