Dicty News Electronic Edition Volume 9, number 13 November 15, 1997 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 =========== Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II. Dmitrii I. LEVITSKY, Michael A. PONOMAREV, Michael A. GEEVES, Valery L. SHNYROV and Dietmar J. MANSTEIN Europ. J. Biochem., in press Absract: The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry (DSC). These fragments containing 754 or 761 amino acid residues (M754 and M761, respectively) correspond to the globular motor portion of the myosin head that contains ATP- and actin-binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761: the maximum of the thermal transition occurred at 41.7oC for M754 and at 45.6oC for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755-761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2-3.5oC shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlF4-) causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of BeFx was less distinct than that of AlF4- or Vi. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by DSC in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head. ------------------------------------------------------------------------- Signal transduction pathways leading to spore differentiation in Dictyostelium discoideum. Christophe Anjard1, Changjiang Zeng1, William F. Loomis2. and Wolfgang Nellen1* 1 Department of Genetics Kassel University, 34132 Kassel, Heinrich-Plett-Str. 40, Germany. 2 Center for Molecular Genetics, Department of Biology, UCSD, La Jolla, CA 92093, USA Dev. Biol., in press. Summary: Cells that overexpress PKA as a consequence of carrying multiple copies of the gene encoding the catalytic subunit can be induced to sporulate when developing as single cells. A peptide phosphorylated by PKA, termed SDF-1, has recently been shown to stimulate this process (Anjard et al., 1997). Several genes have been implicated in a signal transduction pathway by which prestalk cells induce encapsulation of prespore cells during terminal differentiation including a prestalk specific putative membrane protease (TagC) and a two-component system consisting of a receptor-histidine kinase (DhkA) and a response regulator with cAMP phosphodiesterase activity (RegA). To determine whether SDF-1 uses this pathway, strains carrying null mutations in the pertinent genes were transformed with a pkaC plasmid such that they can overexpress PKA. Since these mutant strains all sporulated efficiently when SDF-1 was added, it appears that other gene products mediate the response. However, we found that regA- mutant cells release a distinct factor, SDF-2, that rapidly induces encapsulation of test cells overexpressing pkaC. Since cells in which tagC is disrupted do not form SDF-2 and cells in which dhkA is disrupted do not respond to SDF-2, this peptide appears to use the two-component system that regulates PKA activity. SDF-2 is a small peptide released by prestalk cells in a manner dependent on TagC. It appears to acts on prespore cells through the DhkA receptor to inhibit the cAMP phosphodiesterase of RegA thereby activating PKA via cAMP. The process of induction by SDF-2 can be shown to be distinct from that by SDF-1. SDF-2 appears to stimulate prestalk cells to release additional SDF-2 by acting through a signal transduction pathway that also involves DhkA, RegA, and PKA. Based on these results we present a model for the signal transduction cascade regulating spore differen- tiation. ------------------------------------------------------------------------- Dictyostelium discoideum Protein Disulfide Isomerase, an Endoplasmic Reticulum Resident Enzyme Lacking a KDEL-type Retrieval Signal. Jean Monnat (1), Ulrike Hacker (2), Heidrun Geissler (1), Robert Rauchenberger (2), Eva M. Neuhaus (1), Markus Maniak (2), and Thierry Soldati (1) (1) Department of Molecular Cell Research, Max-Planck-Institute for Medical Research, Jahnstrasse 29, D-69120 Heidelberg, Germany (2) Department of Cell Biology, Max-Planck-Institute for Biochemistry, D-82152 Martinsried, Germany FEBS Letters, in press Abstract The primary activity of protein disulfide isomerase (PDI), a multifunctional resident of the endoplasmic reticulum (ER), is the isomerization of disulfide bridges during protein folding. We isolated a cDNA encoding Dictyostelium discoideum PDI (Dd-PDI). Phylogenetic analyses and basic biochemical properties indicate that it belongs to a subfamily called P5, many members of which differ from the classical PDIs by many aspects. They lack an intervening inactive thioredoxin module, a C-terminal acidic domain involved in Ca2+ binding and a KDEL-type retrieval signal. Despite the absence of this motif, the ER is the steady state location of Dd-PDI, suggesting the existence of an alternative retention mechanism for P5-related enzymes. ------------------------------------------------------------------------- Three-dimensional Patterns and Redistribution of Myosin II and Actin in Mitotic Dictyostelium Cells Ralph Neujahr, Christina Heizer, Richard Albrecht, Maria Ecke, Jean-Marc Schwartz, Igor Weber, and Guenther Gerisch Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany J. Cell Biology, in press. Abstract. Myosin II is not essential for cytokinesis in cells of Dictyostelium discoideum that are anchored on a substrate (Neujahr, R., C. Heizer, and G. Gerisch. 1997. J. Cell Sci. 110:123-137), in contrast to its importance for cell division in suspension (DeLozanne, A., and J.A. Spudich. 1987. Science 236:1086-1091. Knecht, D.A., and W.F. Loomis. 1987. Science 236:1081-1085.). These differences have prompted us to investigate the three-dimensional distribution of myosin II in cells dividing under one of three conditions: (1) in shaken suspension, (2) in a fluid layer on a solid substrate surface, and (3) under mechanical stress applied by compressing the cells. Under the first and second conditions tested above, myosin II does not form patterns that suggest a contractile ring is established in the furrow. Most of the myosin II is concentrated in the regions that flank the furrow on both sides towards the poles of the dividing cell. It is only when cells are compressed, that myosin II extensively accumulates in the cleavage furrow, as has been previously described (Fukui, Y., T.J. Lynch, H. Brzeska, and E.D. Korn. 1989. Nature 341: 328-331), i.e. this massive accumulation is a response to the mechanical stress. Evidence is provided that the stress associated translocation of myosin II to the cell cortex is a result of the dephosphorylation of its heavy chains. F-actin is localized in the dividing cells in a distinctly different pattern from that of myosin II. The F-actin is shown to accumulate primarily in protrusions at the two poles that ultimately form the leading edges of the daughter cells. This distribution changes dynamically as visualized in living cells with a GFP-actin fusion. ------------------------------------------------------------------------- A new collection of thermosensitive endocytosis mutants in the cellular slime mold Dictyostelium discoideum. Arnaud Labrousse* and Michel Satre Laboratoire de Biochimie et Biophysique des Systemes Integres, UMR 314 CEA-CNRS, CEA-Grenoble, France. *present address: Department of Biological Chemistry, UCLA-School of Medicine, Los Angeles, CA 90095-1737 Journal of Eukaryotic Microbiology, in press Abstract: We used a photoactivatable fluid-phase marker to isolate a new collection of thermosensitive endocytosis mutants in the cellular slime mold Dictyostelium discoideum. All the strains were thermosensitive for growth on bacteria or axenic medium at 27¡ C. Initial rates of endocytosis rapidly decreased upon incubation at the restrictive temperature, but surprisingly most of the strains showed a transient recovery of activity with prolonged exposure to 27¡ C. Endocytosis and exocytosis activities were uncoupled for some of the cell lines at 27¡ C whereas the others had to be shifted to 29¡ C. Further molecular analysis of these mutants could lead to the discovery of new proteins involved in endocytosis and its regulation. ------------------------------------------------------------------------- On the Role of Myosin-II in Cytokinesis: Division of Dictyostelium Cells under Adhesive and Nonadhesive conditions Ji-Hong Zang, Guy Cavet, James H. Sabry, Peter Wagner, Sheri L. Moores, and James A. Spudich Molecular Biology of the Cell, in press. Abstract: We have investigated the role of myosin in cytokinesis in Dictyostelium cells by examining cells under both adhesive and nonadhesive conditions. On an adhesive surface, both wild type and myosin null cells undergo the normal processes of mitotic rounding, cell elongation, polar ruffling, furrow ingression, and separation of daughter cells. When cells are denied adhesion through culturing in suspension or on a hydrophobic surface, wild type cells undergo these same processes. However, cells lacking myosin round up and polar ruffle, but fail to elongate, furrow, or divide. These differences show that cell division can be driven by two mechanisms which we term Cytokinesis A, which requires myosin, and Cytokinesis B, which is cell adhesion dependent. We have used these approaches to examine cells expressing a myosin whose two light chain binding sites were deleted (DBLCBS-myosin). Although this myosin is a slower motor and has constitutively high activity due to the abolition of regulation by light chain phosphorylation, cells expressing DBLCBS- myosin were previously shown to divide in suspension (Uyeda et al., 1996) . However, we suspected their behavior during cytokinesis to be different from wild type cells given the large alteration in their myosin. Surprisingly, DBLCBS-myosin undergoes relatively normal spatial and temporal changes in localization during mitosis. Furthermore, the rate of furrow progression in cells expressing a DBLCBS-myosin is similar to that in wild type cells. ----------------------------------------------------------------------- The Homeobox-Containing Gene Wariai Regulates Anterior-Posterior Patterning and Cell-Type Homeostasis in Dictyostelium DEVELOPMENT, in press Zhihua Han and Richard A. Firtel1 Department of Biology, Center for Molecular Genetics, University of California, San Diego, La Jolla, CA 92093-0634 SUMMARY We have identified a Dictyostelium gene, Wariai (Wri), that encodes a protein with a homeobox and seven ankyrin repeats; both domains are required for function. A null mutation results in a more than doubling of the size of the prestalk O (pstO) compartment, one of the anterior prestalk compartments lying along the anterior /posterior axis of the migrating slug. There is a concomitant decrease in the more posterior prespore domain and no change in the more anterior prestalk A (pstA) and prestalk AB (pstAB) domains. wri null cells also have a morphological defect consistent with an increase in the pstO cell population. Wri itself is preferentially expressed in the pstA but not the pstO compartment, raising the possibility that Wri regulation of pstO compartment size is non-autonomous. Analysis of chimeric organisms is consistent with this model. Development in Dictyostelium is highly regulative, with cells within the prestalk and prespore populations being able to transdifferentiate into other cells to maintain proper cell-type proportioning. Our results suggest that Wri controls cell-type proportioning, possibly by functioning as a negative regulator of a pathway mediating pstO cell differentiation and controlling the mechanism of homeostasis regulating the size of one or more of the cell-type compartments. Our results also suggest that homeobox gene regulation of anterior/posterior axis patterning may have evolved prior to the evolution of metazoans. ------------------------------------------------------------------------- [End Dicty News, volume 9, number 13]