CSM News Electronic Edition Volume 5, number 16 December 2, 1995 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmsbio.nwu.edu/dicty.html" ============== Announcement ============== The December 1995 CSM Email Directory is being distributed at the same time as this issue of the Newsletter. The Directory has not been subjected to a serious pruning since it started. Could everyone, particularly laboratory heads, take a look at the list and let us know if there are entries for people who are no longer likely to be interested in participating in the list (i.e. have left the field). Please sent messages regarding this to "csm-news@worms.cmsbio.nwu.edu". ===================== Positions Available ===================== RESEARCH FELLOW A postdoctoral fellowship funded by the BBSRC for a fixed period of three years is available immediately to work on the identification and characterisation of trans-factors binding to two novel transcriptional control elements located in the promoter of Dictyostelium gene 7E. Applicants should have a PhD and previous experience of recombinant DNA methodology. The salary will be on the RA 1A scale of stlg14, 317 stg - stlg21, 519 stg p.a. according to qualifications and relevant experience. Application forms and further information are available from: Dr B. David Hames Department of Biochemistry & Molecular Biology University of Leeds Leeds LS2 9JT UK Tel: +44-113-233-3145 Fax: +44-113-244-1966 Email: b.d.hames@leeds.ac.uk The closing date for applications is 5 January 1996. =========== Abstracts =========== A cell-cycle phase-associated cell-type choice mechanism monitors the cell cycle rather than using an independent timer Richard H. Gomer and Robin R. Ammann Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892 Developmental Biology, in press Abstract Upon starvation, cells of the simple eukaryote Dictyostelium discoideum aggregate and differentiate into several cell types. Two main cell-types are prestalk and prespore, which later usually become stalk and spore cells. The differentiation is plastic, and several factors can alter cell-type ratios. Two mechanisms have been proposed to regulate the initial differentiation. One hypothesis is that different levels of extracellular factors within the aggregate determine initial cell-type. We and others have proposed that cell type is initially determined by cell-cycle phase at the time of starvation: prestalk cells are derived from cells which, at the time of starvation, happen to be in a roughly two hour long sector of the cell cycle which overlaps S and early G2, and that certain extracellular factors are then used to maintain the proper prestalk: prespore ratio and to control later stages of development such as the prestalk-to-stalk conversion. To examine the relationship between initial cell-type choice and the cell cycle, and how this two hour long sector is generated, we increased the length of S phase by mild treatments of cells with DNA-synthesis inhibitors. When the fraction of the cell cycle occupied by S phase is increased and the cells are then starved, the prestalk: prespore ratio increases. This increase was observed using two markers for prestalk cells, CP2 and ecmA::lacZ. In addition, there is a close correlation between the fraction of the cell cycle occupied by S phase and the prestalk: prespore ratio, irrespective of total cell-cycle length. These results validate the hypothesis that the initial choice of cell-type is determined by cell-cycle phase at the time of starvation, and indicate that the cell-type choice mechanism monitors the cell cycle rather than using an independent two-hour long timer started at the beginning of S phase. -------------------------------------------------------------------------- Analysis of cell movement during the culmination phase of Dictyostelium development Dirk Dormann*, Florian Siegert & Cornelis J. Weijer Zoologisches Institut, Universitaet Muenchen, Luisenstr. 14, 80333 Muenchen, Germany *corresponding author Development, in press Abstract Co-ordinated cell movement of ten-thousands of cells and periodic signals characterise the multicellular development of the cellular slime mould Dictyostelium discoideum. We investigated cell movement by analysing time-lapse video recordings made during the slug stage and the culmination phase of Dictyostelium development. Slugs viewed from the side showed an even, straight forward movement with the tip slightly erected in the air. Slugs, which had migrated for a prolonged period of time either culminated or showed a behaviour best described as abortive culmination. Culmination is initiated by a local aggregation of anterior-like cells at the base of the slug at the prestalk-prespore boundary, where they form a stationary mass of cells. Prespore cells continue to move forward over this stationary pile and, as a result, are lifted into the air. The stationary group of anterior-like cells gets thereby translated to the back of the slug. At this point the slug either falls back on the agar surface or continues culmination. If the slug continues to migrate these cells regain motility, move forward to the prespore-prestalk boundary and form a new pile again. In the case of culmination the neutral red stained cells in the pile move to the back of the slug and form a second signalling centre beside the tip. Both centres are characterised by vigorous rotational cell movement. The cells belonging to the basal centre will form the basal disc and the lower cup in the fruiting body. The upper cup will be formed by the prestalk cells rotating most vigorously at the prestalk-prespore boundary. The remaining neutral red stained anterior-like cells in the prespore zone sort either to the upper or lower organising centre in the fruiting body. ---------------------------------------------------------------------- The initiation of basal disc formation in Dictyostelium discoideum is an early event in culmination Keith Jermyn, David Traynor+ and Jeffrey Williams MRC Laboratory of Molecular Cell Biology and Department of Biology, University College London, Gower St, London WC1E 6BT +Present address, University of Cambridge, Dept of Pharmacology, Tennis Court Rd, Cambridge, CB2 1QJ Development, in press. Summary We have analysed expression of the ecmA and ecmB genes of Dictyostelium by enzymatic double staining using b-galactosidase and b-glucuronidase reporter gene constructs. Cells expressing the ecmA gene first appear as scattered cells at the mound stage of development and we show that this is also true for cells expressing the ecmB gene. During tip formation the ecmA expressing cells move to the apex of the mound, while the ecmB expressing cells accumulate in the base. The ecmB expressing cells constitute part of the basal disc if the culminant is formed in situ but are discarded if a migratory slug is formed. During slug migration they are replaced by a band of ecmB expressing cells, situated in the front half of the prespore zone and tightly apposed to the substratum. When culmination is triggered these cells rapidly move into the back half of the prestalk zone, possibly acting as a point of attachment to the subtratum. Ultimately, they are joined by cells at the back of the slug, the rearguard cells, to form the basal disc. Thus, contrary to previous belief, basal disc formation is initiated very early during culmination and occurs by the forward movement of cells located in the anterior of the prespore zone. -------------------------------------------------------------------- Linking microfilaments to intracellular membranes: the actin-binding and vesicle associated protein comitin exhibits a mannose-specific lectin activity Eva Jung, Paola Fucini, Murray Stewart1, Angelika A. Noegel, Michael Schleicher2 Max-Planck-Institut fuer Biochemie, 82152 Martinsried, FRG, 1 MRC Laoratory of Molecular Biology, Cambridge CB2 2QH, UK, 2 Institut fuer Zellbiologie, Ludwig-Maximilians-Universitaet Muenchen, Schillerstr. 42, 80336 Muenchen, FRG EMBO J., in press Abstract Comitin is a 24 kDa actin-binding protein from Dictyostelium discoideum that is located primarily on Golgi and vesicle membranes. We have probed the molecular basis of comitin's interaction with both actin and membranes using a series of truncation mutants obtained by expressing the appropriate cDNA in E. coli. Comitin dimerizes in solution; its principal actin binding activity is located between residues 90 and 135. The N- terminal 135 "core" residues of comitin contain a three fold sequence repeat that is homologous to several monocotyledon lectins and which retains key residues that determine these lectin's three-dimensional structure and mannose binding. These repeats of comitin appear to mediate its interaction with mannose residues in glycoproteins or glycolipids on the cytoplasmic surface of membrane vesicles from D. discoideum, and comitin can be released from membranes with mannose. Our data indicate that comitin binds to vesicle membranes via mannose residues and, by way of its interaction with actin, links these membranes to the cytoskeleton. -------------------------------------------------------------------- Stress-induced tyrosine phosphorylation of actin in Dictyostelium cells and localization of the phosphorylation site to Tyrosine 53 adjacent to the DNase I binding loop Andreas Jungbluth", Christoph Eckerskorn, Guenther Gerisch, Friedrich Lottspeich, Susanne Stocker and Anton Schweiger Max-Planck-Institut fur Biochemie, D-82152 Martinsried, Germany "corresponding author FEBS Letters, in press. Abstract Actin is known to be phosphorylated at tyrosine, serine, or threonine residues in various cells. In cells of Dictyostelium discoideum, a rise in the tyrosine phosphorylation of actin is observed in response to ATP depletion. An actin fraction rich in phosphotyrosine was obtained by chromatography on the weak anion exchanger MONO-P. Mass spectrometry and amino acid sequencing of protease cleavage products indicated that a single tyrosine residue was phosphorylated. Localization of this residue to position 53 of the actin sequence attributed the modification to a site that is critical for the capability of actin to polymerize. Induction of the tyrosine phosphorylation by heat shock and Cd2+ ions indicates that this modification of actin is implicated in the response of Dictyostelium cells to stress. --------------------------------------------------------------------- Antisense RNA Inhibition of the Putative Vacuolar H+-ATPase Proteolipid of Dictyostelium discoideum Reduces Intracellular Ca2+ Transport and Cell Viability Yanyan Xie, Barrie Coukell and Zoltan Gombos Department of Biology, York University, 4700 Keele St., North York, Ontario, CANADA, M3J 1P3 J. Cell Sci., in press Abstract Transport of Ca2+ via a P-type pump into the contractile vacuole of Dictyostelium discoideum appears to be facilitated by vacuolar proton (V-H+) ATPase activity (Rooney and Gross, Proc. Nat. Acad. Sci. USA 89, 8025-8029, 1992). To investigate the involvement of the V-H+-ATPase in this process using molecular techniques, we cloned a cDNA (vatP) encoding the putative proteolipid subunit of this enzyme. The deduced protein product of this cDNA is composed of 196 amino acids with a calculated Mr of 20, 148 and the primary structure exhibits high amino acid sequence identity with V-H+-ATPase proteolipids from other organisms. vatP is a single-copy gene and it produces one ~900 nt transcript at relatively constant levels during growth and development. Attempts to disrupt the endogenous gene using vatP cDNA were unsuccessful. But, expression of vatP antisense RNA reduced the levels of vatP message and V-H+-ATPase activity by 50% or more. These antisense strains grew and developed slowly, especially under acidic conditions, and the cells seemed to have difficulty forming acidic vesicles. During prolonged cultivation, all of the antisense strains either reverted to a wild-type phenotype or died. Thus in Dictyostelium, unlike yeast, the V-H+-ATPase seems to be indispensable for cell viability. When different antisense strains were analyzed for Ca2+ uptake by the contractile vacuole, they all accumulated less Ca2+ than control transformants. These results are consistent with earlier pharmacological studies which suggested that the V-H+_ATPase functions in intracellular Ca2+ transport in this organism. ------------------------------------------------------------------- Ponticulin Plays A Role In The Positional Stabilization Of Pseudopods Damon C. Shutt, Deborah Wessels, Keith Wagenknecht, Anand Chandrasekhar, Anne Hitt#, Elizabeth J. Luna#, and David R. Soll Department of Biological Sciences University of Iowa Iowa City, Iowa 52242 #Cell Biology Group Worcester Foundation for Biomedical, Shrewsbury, Massachusetts 01545 J. Cell Biology, in press Abstract Ponticulin is a 17-kDa glycoprotein which represents a major high affinity link between the plasma membrane and the cortical actin network of Dictyostelium. To assess the role of ponticulin in pseudopod extension and retraction, the motile behavior of two independently-generated mutants lacking ponticulin was analyzed using computer-assisted two-dimensional and three-dimensional motion analysis systems. More than half of the lateral pseudopods formed off the substratum by ponticulin-minus cells slipped relative to the substratum during extension and retraction. In contrast, all pseudopods formed off the substratum by wild-type cells were positionally fixed in relation to the substratum. Ponticulin-minus cells also formed a greater proportion of both anterior and lateral pseudopods off the substratum and absorbed a greater proportion of lateral pseudopods into the uropod than do wild type cells. In a spatial gradient of cAMP, ponticulin-minus cells were less efficient in tracking the source of chemoattractant. Since ponticulin-minus cells extend and retract pseudopods with the same time course as do wild type cells, these behavioral defects in ponticulin-minus cells appear to be the consequence of pseudopod slippage. These results demonstrate that pseudopods formed off the substratum by wild type cells are positionally fixed in relation to the substratum, that ponticulin is required for positional stabilization, and that the loss of ponticulin and the concomitant loss of positional stability of pseudopods correlate with a decrease in the efficiency of chemotaxis. --------------------------------------------------------------------- [End CSM-News, volume 5, number 16]