Dicty News Electronic Edition Volume 13, number 6 September 11, 1999 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" ==================== Postdoc Available ==================== NIH Funded Postdoctoral Postition: Structure and Function of the Cytoskeleton Department of Molecular and Cell Biology, University of Connecticut My laboratory is interested in the assembly and dynamics of the actin cytoskeleton of motile cells. The mechanisms by which cells create specific types of actin assemblies to carry out functions such as phagocytosis, macropinocytosis, surface attachment and chemotactic motility are being investigated. Current projects focus on the role of different isoforms of the Rac family of small G proteins in regulating actin assembly, and the role of actin cross-linking proteins and myosin in creating different cytoskeletal structures. The work is being carried out using a combination of biochemical, molecular genetic and advanced imaging techniques in the model organism Dictyostelium discoideum. Candidates should have a PhD in an area of Cell or Molecular Biology and enthusiasm to explore new areas in a multidisciplinary approach. Starting salary will be determined based on experience and potential, and the position is for a minimum of 2 years with renewal contingent on satisfactory performance. The University of Connecticut is located in rural Eastern Connecticut in a safe and family friendly environment. The University is in the middle of a $1 billion building campaign and the lab will be moving into a new facility in the Spring of 2000. The combination of excellent facilities and a rural lifestyle with proximity to Boston (1.5 hours) and New York (2.5 hours) makes this an ideal location for those wish to do cutting edge science in a relaxed environment. Send vita and names and addresses (Email and phone) of 3 references to: Dr. David Knecht Molecular and Cell Biology U-125 University of Connecticut, Storrs, CT 06269 e-mail:knecht@uconnvm.uconn.edu web: http://www.sp.uconn.edu/~mcbstaff/knecht/knecht.html ============= Abstracts ============= LbrA, a protein predicted to have a role in vesicle trafficking, is necessary for normal morphogenesis in Polysphondylium pallidum. Yoshinori Kawabe, Toshiteru Enomoto, Takahiro Morio, Hideko Urushihara, and Yoshimasa Tanaka* Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan Gene, in press Abstract The fruiting body of Polysphondylium pallidum is composed of whorls of branches along the axis of a central stalk. In the course of fruiting body formation, the interval between neighboring whorls, and the number and the spacing of branches in a whorl are highly regulated. In this study, using the REMI (restriction enzyme mediated integration) insertional mutagenesis method, we obtained a mutant (strain M2323) with longer branches than those of the wild-type strain PN500. The sequence analyses revealed the presence of an ORF of 206 aa residues (23 kDa) near the vector insertion site. Disruption of the gene, lbrA (long branch A), by homologous recombination causes the same phenotype as that of M2323. A lbrA transcript is expressed maximally at the early aggregation stage in the parental strain, but not detectable in the REMI mutant. A homology search showed that LbrA is a member of the p24 family proteins, which have been proposed to function as receptors for cargo proteins that are transported by COP I- (coat protein I) and / or COP II-coated vesicles between the endoplasmic reticulum and the Golgi complex. As far as we know, this is the first paper to show that a p24 family member is implicated in morphogenesis. ---------------------------------------------------------------------------- NOVEL CELLULAR TRACKS OF MIGRATING DICTYOSTELIUM CELLS Kazuhiko Uchida and Shigehiko Yumura Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi 7 53-8512, Japan Eur. J. Cell Biol. in press. Summary After Dictyostelium cells were settled on a coverslip and allowed to migrate freely on the surface, they were stained with fluorescently labeled Concanavalin A. Tracks with distinct patterns that consist of dots and short fibers were observed behind the cells. In this study, we refer to these tracks as 'cellular tracks', CTs for short. We characterized the biological effect of CTs on cell behavior and development. CTs decreased the strength of cell-substratum adhesion, increased the velocity of cell migration, but did not affect growth of cells. CTs also promoted cell aggregation. When pre-aggregation cells touched the CTs of other cells, they avoided or orthogonally crossed them, but did not migrate along them. These observations suggest that the CTs of pre-aggregation cells prompts cells to disperse uniformly on substratum and may enable cells to sense cell density. On the other hand, when aggregation-competent cells touched the CTs of other aggregation-competent cells, a half of them migrated along the CTs. Pre-aggregation cells did not migrate along the CTs of aggregation- competent cells. The CTs of aggregation-competent cells may help the cells to aggregate toward the aggregation center. ---------------------------------------------------------------------------- DOMAIN ANALYSIS OF CORTEXILLIN I: ACTIN-BUNDLING, PIP2-BINDING AND THE RESCUE OF CYTOKINESIS Alexander Stock1, Michel O. Steinmetz2, Paul A. Janmey3, Ueli Aebi2, Günther Gerisch1*, Richard A. Kammerer2, Igor Weber1, and Jan Faix1 1 Max-Planck-Institut für Biochemie, D-82152 Martinsried, Am Klopferspitz 18a, Germany, 2 Biozentrum der Universität Basel, CH-4161 Basel, Klingelbergstrasse 70, Switzerland, 3 Brigham & Women's Hospital, Harvard Medical School, 221 Longwood Avenue, Boston, USA *Corresponding author: Günther Gerisch, Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany. Tel.: (49) 89-8578-2326; Fax: (49) 89-8578-3885; e-mail: gerisch@biochem.mpg.de EMBO Journal, in press ABSTRACT: Cortexillins are actin-bundling proteins that form a parallel two-stranded coiled-coil rod. Actin-binding domains of the alpha-actinin/spectrin type are located N-terminal to the rod and unique sequence elements are found in the C-terminal region. Domain analysis in vitro revealed that the N-terminal domains are not responsible for the strong actin-filament bundling activity of cortexillin I. The strongest activity resides in the C-terminal region. Phosphatidylinositol 4,5-bisphosphate (PIP2) suppresses this bundling activity by binding to a C-terminal nonapeptide sequence. These data define a new PIP2-regulated actin-bundling site. In vivo the PIP2-binding motif enhances localization of a C-terminal cortexillin I fragment to the cell cortex and improves the rescue of cytokinesis. This motif is not required, however, for translocation to the cleavage furrow. A model is presented proposing that cortexillin translocation is based on a mitotic cycle of polar actin polymerization and midzone depolymerization. ---------------------------------------------------------------------------- Cell-sorting in aggregates of Dictyostelium discoideum. Alastair Nicol, Wouter-Jan Rappel, Herbert Levine and William F. Loomis *Departments of Physics and Biology, UCSD, La Jolla, CA J. Cell Science (in press) SUMMARY When Dictyostelium cells are induced to develop between a cover-slip and a layer of agarose, they aggregate normally into groups containing up to a thousand cells but are then constrained to form disks only a few cells thick that appear to be equivalent to the three-dimensional mounds formed on top of agarose. Such vertically restricted aggregates frequently develop into elongated motile structures, the flattened equivalent of three dimensional slugs. The advantage of using this system is that the restricted z-dimension enables direct microscopic visualization of most of the cells in the developing structure. We have used time lapse digital fluorescence microscopy of Dictyostelium strains expressing green fluorescent protein (GFP) under the control of either prestalk or prespore specific promoters to follow cell sorting in these flattened mounds. We find that prestalk and prespore cells expressing GFP arise randomly in early aggregates and then rotate rapidly around the disk mixed with the other cell type. After a few hours, the cell types sort out by a process which involves striking changes in relative cell movement. Once sorted, the cell types move independently of each other showing very little heterotypic adhesion. When a group of prestalk cells reaches the edge of the disk, it moves out and is followed by the prespore cell mass. We suggest that sorting may result from cell type specific changes in adhesion and the consequent disruption of movement in the files of cells that are held together by end-to-end adhesion. ---------------------------------------------------------------------------- The contractile vacuole network of Dictyostelium as a distinct organelle: its dynamics visualized by a GFP marker protein Daniela Gabriel, Ulrike Hacker, Jana Koehler, Annette Mueller-Taubenberger, Jean-Marc Schwartz, Monika Westphal and Guenther Gerisch Max-Planck-Institut für Biochemie D-82152 Martinsried, Germany J. Cell Science, in press. Summary The contractile vacuole system is an osmoregulatory organelle composed of cisternae and interconnecting ducts. Large cisternae act as bladders that periodically fuse with the plasma membrane, forming pores to expel water (Heuser et al. (1993) J. Cell Biol. 121, 1311-1327). To visualize the entire network in vivo and to identify constituents of the vacuolar complex in cell fractions, we introduced a specific marker into Dictyostelium cells, GFP-tagged dajumin. The C-terminal, GFP-tagged region of this transmembrane protein is responsible for sorting to the contractile vacuole complex. Dajumin-GFP negligibly associates with the plasma membrane, indicating its retention during discharge of the bladder. Fluorescent labeled cell-surface constituents are efficiently internalized by endocytosis, while no significant cycling through the contractile vacuole is observed. Endosomes loaded with yeast particles or a fluid-phase marker indicate sharp separation of the endocytic pathway from the contractile vacuole compartment. Even after dispersion of the contractile vacuole system during mitosis (Zhu et al. (1993) J. Cell Sci. 104, 1119-1127), dajumin-GFP distinguishes the vesicles from endosomes, and visualizes post-mitotic re-organization of the network around the nucleus. Highly discriminative sorting and membrane fusion mechanisms are proposed to account for the sharp separation of the contractile vacuole and endosomal compartments. Evidence for a similar compartment in other eukaryotic cells is discussed. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 6]