Dicty News Electronic Edition Volume 14, number 8 April 8, 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" ====================== Position Available ====================== ________________________________________________________________ POSTDOCTORAL POSITION IN SIGNAL TRANSDUCTION LOOMIS LAB - CENTER FOR MOLECULAR GENETICS, UCSD, LA JOLLA, CA ________________________________________________________________ Funding is available to support a Postdoctoral Fellow interested in the molecular mechanisms that coordinate terminal differentition of Dictyostelium. Signaling by SDF-2 from prestalk cells activates the receptor histidine kinase, DhkA, that appears to be directly or indirectly coupled to a MAP kinase cascade involved in the inhibition of the cAMP phosphodiesterase, RegA. The resulting increase in PKA activity stimulates encapsulation of spores. Further studies are aimed at determining whether genes that encode Raf-like protein kinases function in this signal transduction pathway leading to the activation of ERK1 and/or ERK2. One of these genes, splA, has been previously shown to encode a tyrosine kinase essential for spore differentiation. We are using biochemical, molecular genetic and microarray techniques to elucidate the physiological networks. Applicants should indicate their qualifications and interest in the project, a CV, and contact information on individuals willing to support the appliation. The position will be open in the next few months and a start date should be indicated. Applications can be e-mailed to Bill Loomis [ wloomis@UCSD.edu] or sent to: Dr. William F. Loomis Center for Molecular Genetics Division of Cell and Developmental Biology 2310 Bonner Hall UCSD La Jolla, CA 92093- 0368 ============= Abstracts ============= 3D in vivo analysis of Dictyostelium mounds reveals directional sorting of prestalk cells and defines a role for the myosin II regulatory light chain in prestalk cell sorting and tip protrusion. Patricia A. Clow*, Tung-Ling L. Chen†, Rex L. Chisholm†, and James G. McNally1 *Department of Biology, Washington University, Box 1229, St. Louis, Missouri 63130 †Department of Cell and Molecular Biology, Northwestern University Medical School , 303 E. Chicago Ave. Chicago, Illinois 60611 1Laboratory of Receptor Biology and Gene Expression, Division of Basic Sciences , National Cancer Institute, Building 41, Room C615, 41 Library Drive MSC 5055, Bethesda, Maryland 20892-5055 Development, in press ABSTRACT: During cell sorting in Dictyostelium, we observed that GFP-tagged prestalk cells (ecmAO-expressing cells) moved independently and directionally to form a cluster. This is consistent with a chemotaxis model for cell sorting (and not differential adhesion) in which a long-range signal attracts many of the prestalk cells to the site of cluster formation. Surprisingly, the ecmAO prestalk cluster that we observed was initially found at a random location within the mound of this Ax3 strain, defining an intermediate sorting stage not widely reported in Dictyostelium. The cluster then moved en masse to the top of the mound to produce the classic, apical pattern of ecmAO prestalk cells. Migration of the cluster was also directional, suggesting the presence of another long-range guidance cue. Once at the mound apex, the cluster continued moving upward leading to protrusion of the mound's tip. To investigate the role of the cluster in tip protrusion, we examined ecmAO prestalk-cell sorting in a myosin II regulatory light chain (RLC) null in which tips fail to form. In RLC-null mounds, ecmAO prestalk cells formed an initial cluster that began to move to the mound apex, but then arrested as a vertical column that extended from the mound's apex to its base. Mixing experiments with wild-type cells demonstrated that the RLC-null ecmAO prestalk-cell defect is cell autonomous. These observations define a specific mechanism for myosin's function in tip formation, namely a mechanical role in the upward movement of the ecmAO prestalk cluster. The wild type data demonstrate that cell sorting can occur in two steps, suggesting that in this Ax3 strain spatially and temporally distinct cues may guide prestalk cells first to an initial cluster and then later to the tip. ---------------------------------------------------------------------------- Dictyostelium: A model for regulated cell movement during morphogenesis Richard A. Firtel1 and Ruedi Meili In press: Current Opinion in Genetics and Developmental Biology Abstract Dictyostelium has played an important role in unraveling the pathways that control cell movement and chemotaxis. Recent studies have started to elucidate the pathways that control cell sorting, morphogenesis, and the establishment of spatial patterning in this system. In doing so, they provide new insights into how cell movements within a multicellular organism are regulated and the importance of pathways that are similar to those that regulate chemotaxis of cells on two-dimensional surfaces. ---------------------------------------------------------------------------- Title: Involvement of tail domains in regulation of Dictyostelium myosin II. Autors: Xiong Liu1, 2, Kohji Ito1, 3, Randall J. Lee4, and Taro Q. P. Uyeda1 1. Biomolecular Research Group , National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan 2. Present address: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892, USA 3. Present address: Department of Biology, Faculty of Science, Chiba University, Inage, Chiba 263-8522, Japan 4. Department of Medicine and Cardiovascular Research Institute, University of California at San Francisco, San Francisco, CA 94134, USA Biochem. Biophys. Res. Comm., in press Abstract: The actin-dependent ATPase activity of Dictyostelium myosin II filaments is regulated by phosphorylation of the regulatory light chain. Four deletion mutant myosins which lack different parts of subfragment 2 (S2) showed phosphorylation-independent elevations in their activities. Phosphorylation-independent elevation in the activity was also achieved by a double point mutation to replace conserved Glu932 and Glu933 in S2 with Lys. These results suggested that inhibitory interactions involving the head and S2 are required for efficient regulation. Regulation of wild type myosin was not affected by copolymerization with a S2 deletion mutant myosin in the same filaments. Furthermore, the activity linearly correlated with the fraction of phosphorylated molecules in wild type filaments. These latter two results suggest that the inhibitory head-tail interactions are primarily intramolecular. ---------------------------------------------------------------------------- Protein kinases from Dictyostelium discoideum with similarity to LIM-Kinases Wetterauer, B.W. Zoologisches Institut der LMU, Luisenstraße 14, 80333 München email: wetterau@zi.biologie.uni-muenchen.de Abstract: We cloned a protein kinase (DdKinY) from Dictyostelium discoideum by low stringency hybridization using the catalytic domain from DdKinX (Wetterauer et al., 1995) as a probe. Both kinases have low sequence similarity to other protein kinases in the databases. However, phylogenetic analysis showed that both kinases cluster with vertebrate LIM kinases due to homology within the catalytic domain. ---------------------------------------------------------------------------- A protein containing a serine-rich domain with vesicle-fusing properties mediates cell-cycle dependent cytosolic pH regulation Derrick T. Brazill1,2,3, David R. Caprette2, Heather A. Myler1,2, R. Diane Hatton1, Robin R. Ammann1, David F. Lindsey1,4, Debra A. Brock1, and Richard H. Gomer1,2 1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 3 Current address: Department of Biology, Hunter College, 695 Park Avenue, New York, NY 10021 4 Current address: Department of Biological Sciences, Walla Walla College, College Place, WA 99324 J. Biol. Chem., in press Summary Initial differentiation in Dictyostelium involves both asymmetric cell division and a cell cycle-dependent mechanism. We previously identified a gene, rtoA, which when disrupted randomizes the cell cycle-dependent mechanism without affecting either the underlying cell cycle or asymmetric differentiation. We find that in wild-type cells, RtoA levels very during the cell cycle. Cytosolic pH, which normally varies with the cell cycle, is randomized in rtoA cells. The middle 60% of the RtoA protein is 10 tandem repeats of an 11 peptide-long serine-rich motif, which we find has a random coil structure. This domain catalyzes the fusion of phospholipid vesicles in vitro. Conversely, rtoA cells have a defect in the fusion of endocytic vesicles. They also have a decreased exocytosis rate, a decreased pH of endocytic/ exocytic vesicles, and an increased average cytosolic pH. Our data indicate that the serine-rich domain of RtoA can mediate membrane fusion, and that RtoA can increase the rate of vesicle fusion during processing of endoctyic vesicles. We hypothesize that RtoA modulates initial cell type choice by linking vegetative cell physiology to the cell cycle. ---------------------------------------------------------------------------- Neochlamydia hartmannellae gen. nov., sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis Matthias Horn1, Michael Wagner1*, Karl-Dieter Müller3, Ernst N. Schmid3, Thomas R. Fritsche2, Karl-Heinz Schleifer1 and Rolf Michel3 1 Lehrstuhl für Mikrobiologie, Technische Universität München, D-83530 Freising, Germany. 2 Dept. of Laboratory Medicine, University of Washington, Seattle 98195. 3 Central Institute of the Federal Armed Forces Medical Service, D-56065 Koblenz, Germany Microbiology, in press Summary Free living amoebae are increasingly being recognised to serve as vehicles of dispersal for various bacterial human pathogens and as hosts for a variety of obligate bacterial endocytobionts. Several Chlamydia-like Acanthamoeba endocytobionts constituting the newly proposed family Parachlamydiaceae, are of special interest as potential human pathogens. In this study coccoid bacterial endocytobionts of a Hartmannella vermiformis isolate were analysed. Infection of Hartmannella vermiformis with these bacteria resulted in prevention of cyst formation and subsequent host cell lysis. Transfection experiments demonstrated that the parasites were not capable to propagate within other closely related free living amoebae but were able to infect the distantly-related amoebal species Dictyostelium discoideum. Electron microscopy of the parasites revealed typical morphological characteristics of the Chlamydiales, including the existence of a Chlamydia-like life-cycle, but indicated that these endocytobionts in contrast to Chlamydia species do not reside within a vacuole. Comparative 16S rRNA sequence analysis showed that the endocytobiont of Hartmannella vermiformis, classified as Neochlamydia hartmannellae, is affiliated to the family Parachlamydiaceae. Confocal laser scanning microscopy in combination with fluorescence in situ hybridization using rRNA-targeted oligonucleotide probes confirmed the intracellular localisation of the parasites and demonstrated the absence of other bacterial species within the Hartmannella host. These findings extend our knowledge on phylogenetic diversity of the Parachlamydiaceae and demonstrate for the first time that these endocytobionts can naturally develop within amoebae of the genus Hartmannella. ---------------------------------------------------------------------------- A temperature sensitive adenylyl cyclase mutant of Dictyostelium Hitesh Patel, Kunde Guo, Carole Parent, Julian Gross, Peter N. Devreotes and Cornelis J. Weijer EMBO J., in press. Abstract Dictyostelium development starts with the chemotactic aggregation of up to 106 amoebae in response to propagating cAMP waves. cAMP is produced by the aggregation stage adenylyl cyclase (ACA) and cells lacking ACA (aca null) cannot aggregate. Thermo- sensitive mutants of ACA were selected from a population of aca null cells transformed with an library of ACA genes a major segment of which had been amplified by errorprone PCR. One mutant (tsaca2) that can complement the aggregation null phenotype of aca null cells at 22°C but not at 28°C was characterised in detail. The basal catalytic activity of the enzyme in this mutant was rapidly and reversibly inactivated at 28°C. Using this mutant strain we show that cell movement in aggregates and mounds is organised by propagating waves of cAMP. Synergy experiments between wild type and tsaca2 cells, shifted at various stages of development to the restrictive temperature, showed that ACA plays an important role in the control of cell sorting and tip formation. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 8]