Dicty News Electronic Edition Volume 15, number 11 December 9, 2000 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" Editors note: The following is a draft proposal developed by a committee constituted at the Dundee Dicty 2000 meeting to develop a proposal for genetic nomenclature. The committee is anxious for feedback. To facilitate the widest possible involvement of the community in this important discussion, please send your comments to the email server address: dicty@listserv.it.northwestern.edu. ======================== Postdocs Available ======================== Postdoctoral position A Wellcome Trust funded postdoctoral position is available immediately in the laboratory of Kees Weijer in the Wellcome Trust Biocentre, University of Dundee, UK. The position is available initially for two years to work on the investigation of cAMP signal transduction pathways during chemotactic cell movement in the multicellular stages of Dictyostelium discoideum. The project involves the visualization and analysis of early rapid events in the cAMP signal transduction pathway leading to cAMP generation and chemotactic cell movement. We are investigating the activation of a number of phosphatidyl- inositol-lipid kinases and phosphatases in vivo, by measuring the translocation of GFP tagged PH (Plekstrin Homology) domains with different lipid specificities from the cytosol to the plasma membrane upon chemotactic stimulation of cells at different stages of development. Activation of these pathways are essential steps in the activation of adenylylcylase and localized actin polymerization. This allows us to detect in vivo cell-cell signaling with high spatial and temporal resolution in wildtype and mutant strains. Furthermore the project involves the development and use of FRET (Fluorescence Resonance Energy Transfer) between fusion proteins tagged with different spectral forms of GFP to analyse in-vivo complex formation between components of the cAMP signal transduction pathway. Experience in molecular biology/biochemistry and fluorescence imaging techniques are advantageous. Applicants should submit a CV, a summary of their current research interests and the names and addresses of three referees to c.j.weijer@dundee.ac.uk or to C.J. Weijer, School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee, DD1 5EH, UK ---------------------------------------------------------------------------- POSTDOCTORAL POSITION IN BERLIN ROLE OF CALCINEURIN IN DICTYOSTELIUM A 5 year postdoctoral position is open from January, 2001, in the Microbiology II unit at the Institut fuer Biologie, Freie Universitaet Berlin. The successful candidate will work on the function and regulation of expression of the calcium/calmodulin-dependent protein phosphatase calcineurin in Dictyostelium, and participate in the teaching activities of our group. Applicants should have a strong background in molecular biology and biochemistry. For more information, please contact me. Rupert Mutzel Fachbereich Biologie, Chemie, Pharmazie Institut fuer Biologie - Mikrobiologie Freie Universitaet Berlin Koenigin-Luise-Strasse 12 - 16 14195 Berlin Germany Tel.: +30-838-53116 Fax: +30-838-53118 ============== Abstracts ============== CYTOKINESIS FAILURE IN CLATHRIN-MINUS CELLS IS CAUSED BY CLEAVAGE FURROW INSTABILITY. Noel J. Gerald1, Cynthia K. Damer2, Theresa J. O'Halloran3 and Arturo De Lozanne3 1Dept. of Cell Biology, Duke University Medical Center, Durham, NC 27710, 2Dept. of Biology, Vassar College, Poughkeepsie, NY 12604 and 3Section of Molecular Cell & Developmental Biology and Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712. Cell Motility and the Cytoskeleton, in press ABSTRACT The role of membrane traffic during cell division has only recently begun to be investigated. A growing number of trafficking proteins seem to be involved in the successful completion of cytokinesis. Clathrin was the first trafficking protein to be shown to be essential for cytokinesis in Dictyostelium. Here we investigate the nature of the cytokinesis defect of Dictyostelium clathrin null cells. We found that adherent clathrin null cells do form cleavage furrows but cannot maintain a consistent rate of furrow ingression. Clathrin null cells are completely defective in cytokinesis when placed in suspension. In these conditions the cells develop an abnormal division morphology that consists of two lateral "furrows" on either side of a bulging equatorial region. Cells expressing GFP-myosin II were examined at various stages of cytokinesis. Clathrin null cells show multiple defects in myosin organization and localization that parallel the striking failure in furrow morphology. We postulate that this morphology is the result of contraction at the rear of the presumptive daughter cells in concert with incomplete furrow ingression. ---------------------------------------------------------------------------- Analysis of Skp1 glycosylation and nuclear enrichment in Dictyostelium. Sassi, S., M. Sweetinburgh, J. Erogul, P. Zhang, P. Teng-umnuay & C.M. West Glycobiology, in press. Abstract Skp1 is a subunit of SCF-E3 ubiquitin ligases and other protein complexes in the nucleus and cytoplasm of yeast and mammalian cells. In Dictyostelium, Skp1 is partially modified by an unusual pentasaccharide O-linked to hydroxyproline143. This modification was found to be susceptible to known prolyl hydroxylase inhibitors based on Mr-shift analysis using SDS-PAGE/Western blotting. In addition, Dictyostelium Skp1 consists of 2 genetic isoforms, Skp1A and Skp1B, which differ by a single amino acid and appear to be expressed throughout the life cycle based on RT-PCR. The significance of these structural variations was examined by expressing myc-tagged Skp1s and mutants that lacked the glycosylation site. Gel-based Mr-shift studies showed that Skp1A and Skp1B are both nearly completely glycosylated during growth and early development, and mass spectrometry of glycopeptides showed that they were glycosylated similarly. Skp1 expressed later in prespore cells was not glycosylated, unlike bulk Skp1 persisting from earlier in development, but became glycosylated after return to growth medium. Skp1A and Skp1B were each concentrated in the nucleus and regions of the cytoplasm, based on immunofluorescence localization. However, when Skp1 glycosylation was blocked by mutation, prolyl hydroxylase inhibitors, or expression in prespore cells, Nuclear concentration of Skp1 was not detected. Furthermore, nuclear concentration occurred in a mutant that attached only the core disaccharide to Skp1. Overall, there was no evidence for differential Skp1 isoform expression, glycosylation variants in the bulk Skp1 pool, or for regulation of nuclear localization. However, these studies uncovered evidence that the glycosylation pathway is developmentally regulated and can function posttranslationally, and that core glycosylation is required for Skp1's nuclear concentration. ---------------------------------------------------------------------------- Calcium stores in differentiated Dictyostelium discoideum: prespore cells sequester calcium more efficiently than prestalk cells Christina Schlatterer(1), Paul Walther (2), Martin Müller (3), Kurt Mendgen (1), Karl Zierold (4), Gerd Knoll 1 Faculty of Biology, University of Konstanz, Konstanz, Germany 2 Section Electron Microscopy, University of Ulm, Albert Einstein Allee 11, Ulm, Germany 3 Laboratory for EM I, ETH Zürich, Schmelzbergstr. 7, Zürich, Switzerland 4 Max Planck Institute for Molecular Physiology, Otto Hahn Str. 11, Dortmund, Germany Cell Calcium, in press. Summary Dictyostelium discoideum pseudoplasmodia exhibit a gradient of the cytosolic free Ca 2+-concentration ([Ca2+]i) along their anterior-posterior axis involved in cell-type specific differentiation. [Ca2+]i is high in prestalk and low in prespore cells. We determined the content and localization of calcium and other elements in cryosectioned cells of pseudoplasmodia and fruiting bodies by X-ray microanalysis. Granular stores rich in Ca, Mg and P were identified. Average Ca was higher in prespore than prestalk granules (225 vs 111 mmol/kg dry weight). Total Ca stored in granules was also higher in prespore than prestalk cells. The amount of P and S in granules differed between the two cell types indicating different store composition. In spores mean granular Ca was 120 mmol/kg dry weight. Stalk cells had smaller granules with 360 mmol Ca/kg dry weight. Complementary to microanalysis, vesicular Ca2+-fluxes were studied in fractionated cell homogenates. The rate of Ca2+-uptake was higher in pellet fractions of prespore than prestalk amoebae (4.7 vs 3.4 nmol/min x mg). Ca2+-release was greater in supernatant fractions from prestalk than prespore cells (16.5 vs 7.7 nmol/10 8 cells). In summary, prestalk and prespore cells possess qualitatively different, high-capacity stores containing distinct amounts of Ca and probably being involved in regulation of the anterior-posterior [Ca2+]i-gradient. ---------------------------------------------------------------------------- Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae Thomas Winckler (1), Christine Trautwein (2), Christina Tschepke (1), Christin Neuhduser (1), Ilse Z|ndorf (1), Peter Beck (1), G|nter Vogel (2) and Theodor Dingermann (1) (1) Institut f|r Pharmazeutische Biologie, Universitdt Frankfurt/M. (Biozentrum), D-60439 Frankfurt am Main, Germany and (2) Fachbereich 9 - Chemie, Bergische Universitdt, D-42119 Wuppertal, Germany J. Molecular Biology, in press Abstract The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20% of wildtype CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination. ---------------------------------------------------------------------------- SINGLE-MOLECULE TRACKING OF MYOSINS WITH GENETICALLY ENGINEERED AMPLIFIER DOMAINS Christine Ruff, Marcus Furch, Bernhard Brenner, Dietmar J. Manstein, and Edgar Meyhöfer Molecular and Cellular Physiology, Medical School Hanover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany and Department of Biophysics, Max-Planck-Institute for Medical Research, Jahnstr. 29, 69120 Heidelberg, Germany Nature Strucural Biology (in press) Abstract We combined protein engineering and single-molecule measurements to directly record the step size of a series of myosin constructs with shortened and elongated necks generated by deleting or replacing the natural neck with artificial domains. Our results show a clear linear dependence of the step size on the length of the neck domain and establish that mechanical amplification in the myosin motor is based on a rotation of the neck domain relative to the actin-bound head. For all our constructs, including those with artificial necks, the magnitude of the neck rotation concurrent with the displacement step is about 30°. The engineered step size-change of myosins marks a significant advance in our ability to selectively modify the functional properties of molecular motors. ---------------------------------------------------------------------------- STRUCTURE OF A GENETICALLY ENGINEERED MOLECULAR MOTOR Werner Kliche, Setsuko Fujita-Becker, Martin Kollmar, Dietmar J. Manstein, and F. Jon Kull Department of Biophysics, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany EMBO Journal (in press) Abstract Molecular motors move unidirectionally along polymer tracks, producing movement and force in an ATP-dependent fashion. They achieve this by amplifying small conformational changes in the nucleotide-binding region into force-generating movements of larger protein domains. Here we present the 0.28 nm resolution crystal structure of an artificial actin-based motor. By combining the catalytic domain of myosin II with a 13.0 nm conformational amplifier consisting of repeats 1 and 2 of alpha-actinin, we demonstrate that it is possible to genetically engineer single-polypeptide molecular motors with precisely defined lever arm lengths and specific motile properties. Furthermore, our structure shows the consequences of mutating a conserved salt bridge in the nucleotide-binding region and visualizes the structure of alpha-actinin repeats 1 and 2 to be composed of two rigid, triple-helical bundles linked by an uninterrupted alpha-helix. ---------------------------------------------------------------------------- Maltose-binding protein as a fusion-tag for the localization and purification of cloned proteins in Dictyostelium Ralph Gräf Adolf-Butenandt-Institut / Zellbiologie, Schillerstr. 42, D-80336 München, Germany Anal. Biochem. in press One of the best and most frequently used tags for protein purification from E. coli extracts is maltose binding protein (MBP). MBP fusion proteins exhibit high affinity to amylose allowing purification by amylose-agarose chromatography and elution at low concentrations of maltose. The isolated fusion protein is highly pure and the elution conditions are generally harmless to native proteins and do not involve addition of proteinacious eluting agents. However, the use of MBP as a protein tag was so far mainly restricted to E. coli expression systems and it has never been used in Dictyostelium. Using the Dictyostelium Ser/Thr kinase DdNek2 as an example, this work demonstrates that MBP is an excellent fusion tag not only in the E. coli system but also in Dictyostelium since it allows quick and simple purification of a catalytically active protein from crude cell extracts. Moreover, it is proven to be suitable for protein localization studies with anti-MBP antibodies. Thus, MBP as a protein tag may be useful for other eukaryotic experimetal systems as well. ---------------------------------------------------------------------------- Small GTPases in Dictyostelium: lessons from a social amoeba Andrew Wilkins and Robert H. Insall Trends in Genetics, January 00 Although the process of sequencing the Dictyostelium genome is not complete, it is already producing surprises, including an unexpectedly large number of Ras and Rho small GTPases. Members of these families control a wide variety of cellular processes in eukaryotes, including proliferation, differentiation, cell motility and cell polarity. Comparison of small GTPases from Dictyostelium with those from higher eukaryotes provides an intriguing view of their cellular and evolutionary roles. In particular, although mammalian Ras proteins interact with several signalling pathways, the Dictyostelium pathways appear more linear, with each Ras apparently performing a specific cellular function. ---------------------------------------------------------------------------- Review Dictyostelium discoideum: a model system for differentiation and patterning RICARDO ESCALANTE* and JUAN J. VICENTE Instituto de Investigaciones Biomédicas. C.S.I.C/U.A.M., Madrid, Spain. *Address correspondence to: Ricardo Escalante. Instituto de Investigaciones Biomédicas. C.S.I.C/U.A.M., C/Arturo Duperier, 4, 28029 Madrid, Spain. FAX: +34-91-5854587. e-mail: rescalante@iib.uam.es Int.J.Dev.Biol. (in press) CONTENTS Introduction The morphological development and genome of Dictyostelium discoideum Tools used to study Dictyostelium Biochemistry and Molecular Genetics Transformation Restriction enzyme-mediated integration (REMI) Disruption of genes by homologous recombination Suppressor screening In situ hybridization and lacZ staining for gene expression studies Mixing experiments for detection of subtle phenotypes and extracellular signaling Regulatory pathways in Dictyostelium development Chemotaxis and aggregation Regulation of post-aggregative cell-type differentiation Regulation of cell-type proportion Terminal differentiation ---------------------------------------------------------------------------- Biphasic expression of rnrB in Dictyostelium discoideum suggests a direct relationship between cell cycle control and cell differentiation Harry MacWilliams1*, Pascale Gaudet2, Heike Deichsel1, Claire Bonfils3,4, and Adrian Tsang2,3 1Zoologisches Insitut, Ludwig-Maximilians-Universitat, Luisenstrasse 14, 80333 München 2, Germany 2Department of Chemistry and Biochemistry, Centre for Functional and Structural Genomics and 3Department of Biology, Concordia University, 1455 de Maisonneuve Blvd West, Montréal, Québec H3G 1M8, Canada 4present address: MethylGene Inc., 7220 Frederick-Banting, Ville St-Laurent, Québec H4S 2A1, Canada Accepted, DIFFERENTIATION Abstract Cell differentiation in Dictyostelium is strongly affected by the cell cycle. Cell cycle control is well-understood in other systems, but this has had almost no impact on the study of Dictyostelium cell differentiation, in part because the cell cycle in Dictyostelium is unusual, lacking a G1 phase. Here we describe the cell-cycle regulated expression of rnrB, which codes for the small subunit of ribonucleotide reductase, and is a marker of late G1 in many systems. There appear to be two expression peaks, one in mid-G2 and the other near the G2/M transition. Using Xgal/anti-BrdU double staining, we show that cells in asynchronously growing cultures express in both phases, with a gap between them during which the gene is transcriptionally silent. Cold-synchronized cells show exclusively G2/M expression, while mid-G2 expression is seen in high-density synchronized cells and can also be inferred in cells undergoing synchronization by either method. rnrB expression occurs in other systems shortly after cells pass a point (the "restriction point" or "Start") at which they commit to complete their current cell cycle. We demonstrate a similar commitment point in Dictyostelium and show that this occurs shortly before the mid-G2 rnrB expression peak. The Dictyostelium cell cycle thus appears to include a well-defined although inconspicuous event, between early and mid-G2, with some features which are normally associated with the G1/S transition. Others have described a switch from stalk to spore differentiation preference at about this time. Since Dictyostelium cells switch back from spore to stalk preference approximately at the G2/M rnrB expression maximum, cell differentiation as well as rnrB expression may be regulated directly by fundamental cell cycle control processes. ---------------------------------------------------------------------------- During multicellular migration, myosin II serves a structural role independent of its motor function Xiaoxin Susan Xu#,1, Eunkyung Lee#, Tung-ling Chen*, Edward Kuczmarski†, Rex L. Chisholm*, and David A. Knecht# #Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269, †Department of Physiology and Biophysics, Chicago Medical School, Chicago IL 60064 and *Department of Cell and Molecular Biology, Northwestern University Medical School. Chicago, Illinois 60611 Develop. Biol., in press. ABSTRACT We have shown previously that cells lacking myosin II are impaired in multicellular motility. We now extend these results by determining whether myosin contractile activity function is necessary for normal multicellular motility and shape control. Myosin from mutants lacking the essential (mlcE-) myosin light chain retains the ability to form bipolar filaments that bind actin, but shows no measurable in vitro or in vivo contractile function. This contractile function is necessary for cell shape control since mlcE- cells, like myosin heavy chain null mutants (mhcA-), were defective in their ability to control their three-dimensional shape. When mixed with wild-type cells in chimeric aggregation streams, the mlcE- cells were able to move normally, unlike mhcA- cells which accumulated at the edges of the stream and became distorted by their interactions with wild-type cells. When mhcA- cells were mixed with mlcE- streams, the mhcA- cells were excluded. The normal behavior of the mlcE- cells in this assay suggests thatthe actin binding activity of myosin II, in the absence of motor function, is sufficient to allow movement in this constrained, multicellular environment. We hypothesize that myosin II is a major contributor to cortical integrity, even in the absence of contractile function. ----------------------------------------------------------------------------- Golvesin-GFP Fusions as Distinct Markers for Golgi and Post-Golgi Vesicles in Dictyostelium Cells Natalie Schneider+, Jean-Marc Schwartz+, Michael Becker+, Jana Koehler+, Heinz Schwarz§ and Guenther Gerisch+ + Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany § Max-Planck-Institut fuer Entwicklungsbiologie, D-72076 Tuebingen, Germany Biology of the Cell, in press. Abstract Golvesin is a new protein associated with membranes of the Golgi apparatus and post-Golgi vesicles in Dictyostelium cells. An internal hydrophobic sequence of 24 amino-acid residues is responsible for anchoring golvesin to the membranes of these organelles. In an attempt to visualize organelle dynamics in vivo, we have used specific antibody and other labels to localize golvesin-GFP constructs to different cellular compartments. With a GFP tag at its N-terminus, golvesin shows the same localization as the untagged protein. It is transferred to two post-Golgi compartments, the endosomal and contractile vacuole systems. Endosomes are decorated with GFP-golvesin within less than 10 minutes of their internalization, and keep the label during the acidic phase of the pathway. Blockage of the C-terminus with GFP causes entrapment of the protein in the Golgi apparatus, indicating that a free C-terminus is required for transfer of golvesin to any of the post-Golgi compartments. The C-terminally tagged golvesin proved to be a reliable Golgi marker in Dictyostelium cells, revealing protrusion of Golgi tubules at peak velocities of 3 to 4 mm per second. The fusion protein is retained in Golgi vesicles during mitosis, visualizing Golgi disassembly and reorganization in line with cytokinesis. ----------------------------------------------------------------------------- [End Dicty News, volume 15, number 11]