Dicty News Electronic Edition Volume 12, number 3 January 23, 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" ============== Abstracts ============== Mechanism of action of the Rep protein from the Dictyostelium Ddp2 plasmid family Iman M. Shammat and Dennis L. Welker Biology Department, Utah State University, Logan Utah 84322-5305 Plasmid, in press. Summary: The yeast two hybrid system was used to show that the Rep proteins from three members of the Dictyostelium discoideum Ddp2 plasmid family, Ddp2, Ddp5, and Ddp6, form homomultimers but not heteromultimers. The results with deletion mutations suggest that multiple regions of the Rep proteins are involved in the multimerization. Electrophoretic mobility shift assays with heterologously expressed and purified Ddp2 Rep protein showed that it is a DNA binding protein. The nucleosomal organization of Ddp2 and Ddp6 in their inverted repeat and promoter regions was investigated. Analysis of mutants derived from the Ddp6 plasmid revealed that its Rep protein is required for nucleosome positioning (i.e. phasing) to occur in the promoter region. On the other hand, nucleosome positioning in the inverted repeat regions of both plasmids is not dependent on Rep protein but on either a feature of the DNA sequence or the binding of cellular factors, perhaps the Dictyostelium origin recognition complex. Rep protein is likely involved in transcription regulation and control of DNA replication, specifically amplification of plasmid at low copy numbers. The formation of homomultimers may be required for their regulatory activity. ---------------------------------------------------------------------------- The Actin-Associated Protein Coronin in Chemotaxis, Cytokinesis, and Phagocytosis of Dictyostelium discoideum Karmela Barisic, Maria Ecke, Christina Heizer, Markus Maniak, Monika Westphal, Richard Albrecht, and Guenther Gerisch Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany http://www.biochem.mpg.de/gerisch/ Trends in Cell Biology, in press. Background: In the highly motile cells of Dictyostelium discoideum the actin system is rapidly re-organized in response to self-generated or extraneous signals. In this eukaryotic microorganism the actin, together with associated proteins, serves multiple functions providing a basis for cell motility, endocytosis, and mitotic cell division. Coronin is one of the actin-binding proteins that are recruited from the cytoplasm within seconds to locally form membrane-associated complexes together with filamentous actin. --------------------------------------------------------------------- Microtubule-Dependent Golgi Disassembly and Reconstitution During Mitosis in Dictyostelium discoideum Natalie Schneider, Guenther Gerisch, and Jean-Marc Schwartz Max-Planck-Institut fuer Biochemie, D-82152 Martinsried http://www.biochem.mpg.de/gerisch/ Trends in Cell Biology, in press. Background: The Golgi apparatus vesiculates at the beginning of mitosis, and is afterwards reconstituted by assembly of the vesicles close to the centrosome. This is demonstrated here in Dictyostelium cells, which are distinguished from cells of higher eukaryotes by intranuclear mitosis. To visualize Golgi disassembly and re-organization simultaneously with microtubule dynamics, cells were transfected with both golvesin-GFP, which localizes to the Golgi apparatus, and GFP-alpha-tubulin, which visualizes the mitotic apparatus and positions of the centrosomes. At the end of mitosis the centrosomes are rapidly moved around within the cells by guiding microtubules anchored to the cell cortex. --------------------------------------------------------------------- Cytokinesis in Uni- and Multinucleate Myosin II-null Cells of Dictyostelium discoideum Ralph Neujahr, Jana Koehler, John Murphy, Jean-Marc Schwartz, Monika Westphal, and Guenther Gerisch Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany http://www.biochem.mpg.de/gerisch/ Trends in Cell Biology, in press. Background: During mitotic cell division, the segregation of chromosomes along the spindle is coordinated in time and space with the formation of a cleavage furrow. In Dictyostelium, myosin II-null cells are capable of undergoing cytokinesis when they are attached to an adhesive substrate surface. The mitotic division of these myosin II-depleted cells uncovers basic mechanisms of cytokinesis that drive cleavage furrow formation in the absence of the conventional, filamentous myosin. To relate microtubule dynamics to changes in cell shape, confocal fluorescence images of GFP-alpha-tubulin colored in green are superimposed in the movies onto simultaneously recorded phase- contrast images displayed in red. ---------------------------------------------------------------------------- Myosin II-Independent F-Actin Flow Contributes to Cell Locomotion in Dictyostelium Yoshio Fukui*1, Toshiko Kitanishi-Yumura*2, and Shigehiko Yumura*2 *1) Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois, *2) Department of Biology, Faculty of Science, Yamaguchi University, Yamaguchi, Japan. J. Cell Science, in press Summary: While the treadmilling and retrograde flow of F-actin are believed to be responsible for the protrusion of leading edges, little is known about the mechanism that brings the posterior cell body forward. To elucidate the mechanism for the global cell locomotion, we examined the organizational changes of filamentous (F-) actin in live Dictyostelium discoideum. We labeled F-actin with a trace amount of fluorescent phalloidin and analyzed its dynamics in nearly two-dimensional cells by using a sensitive, high- resolution charge-coupled device. We optically resolved a cyclic mode of tightening and loosening of fibrous cortical F-actin and quantitated its flow by measuring temporal and spatial intensity changes. The rate of F-actin flow was evaluated with respect to migration velocity and morphometric changes. In migrating monopodial cells, the cortical F-actin encircling the posterior cell body gradually accumulated into the tail end at a speed of 0.35 ?m/min. We show qualitatively and quantitatively that the F-actin flow is closely associated with cell migration. Similarly, in dividing cells, the cortical F-actin accumulated into the cleavage furrow. Although five times slower than the wild type, the F-actin also flows rearward in migrating mhcA- cells; demonstrating that myosin II (conventional myosin) is not absolutely required for the observed dynamics of F-actin. Yet consistent with reported transportation of ConA-beads (Jay and Elson, Nature 356: 438-440, 1992), the direction of observed F-actin flow in Dictyostelium is conceptually opposite from a barbed-end binding to the plasma membrane. This study suggests that the posterior end of the cell has a unique motif that tugs the cortical actin layer rearward by means of a mechanism independent from myosin II; and this mechanism may be also involved in the cleavage furrow formation. ---------------------------------------------------------------------------- MOUND CELL MOVEMENT AND MORPHOGENESIS IN DICTYOSTELIUM Kathryn A. Kellerman* and James G. McNally# Department of Biology and the Institute for Biomedical Computing, Washington University, St. Louis, MO 63130 Developmental Biology, in press Summary: To examine mechanisms of cell locomotion within a 3-dimensional (3-D) cell mass, we have undertaken a systematic 3-D analysis of individual cell movements in the Dictyostelium mound, the first 3-D structure to form during development of the fruiting body. We used time-lapse deconvolution microscopy to examine two strains whose motion represents endpoints on the spectrum of motile behaviors that we have observed in mounds. In AX-2 mounds, cell motion is slow and trajectories are a combination of random and radial, compared to KAX-3, where motion is five-fold faster and most trajectories are rotational. Although radial or rotational motion was correlated with the optical-density wave patterns present in each strain, we also found small but significant sub-populations of cells that moved differently from the majority, demonstrating that optical-density waves are at best insufficient to explain all motile behavior in mounds. In examining morphogenesis in these strains, we noted that AX-2 mounds tended to culminate directly to a fruiting body, whereas KAX-3 mounds first formed a migratory slug. By altering buffering conditions we could interchange these behaviors, and then found that mound- cell motions also changed accordingly. This demonstrates a correlation between mound cell motion and subsequent development, but it is not obligatory. Chimeric mounds composed of only 10% KAX-3 cells and 90% AX-2 cells exhibited rotational motion suggesting that a diffusible molecule induces rotation, but many of these mounds still culminated directly demonstrating that rotational motion does not always lead to slug migration. Our observations provide a detailed analysis of cell motion for two distinct modes of mound and slug formation in Dictyostelium. ---------------------------------------------------------------------------- A Mutation that separates the RasG signals that regulate development and cytoskeletal function in Dictyostelium T. Zhang, P.J. Rebstein, M. Khosla, J. Cardelli, G. Buczynski, J. Bush, G.B. Spiegelman and G. Weeks Departments of Microbiology and Immunology and Medical Genetics, University of British Columbia, 6174 University Blvd., Vancouver, V6T 1Z3 Exp. Cell Res., accepted ABSTRACT The expression of an activated RasG protein, RasG-G12T, in vegetative cells of Dictyostelium discoideium produced an alteration in cell morphology. Cells underwent a transition between an extensively flattened form that exhibited lateral membrane ruffling to a less flattened form that exhibited prominent dorsal membrane ruffling. These rasG-G12T transformants exhibited a redistribution of F-actin at the cell periphery and did not undergo the rapid contraction upon refeeding that is characteristic of wild type cells. These results suggest a role for RasG in regulating cytoskeletal rearrangement in D. discoideum. We had shown previously that expression of rasG-G12T inhibited starvation induced aggregation (Khosla et al., 1996, Mol Cell Biol, 16, 4156-4162). rasG-G12T genes containing secondary mutations were transformed into cells to test whether the effects of rasG-G12T were transmitted through a single downstream effector. Cells expressing rasG- G12T/T35S or rasG-G12T/Y40C (secondary mutations within the effector domain) exhibited normal morphology and underwent normal aggregation, suggesting that signalling through the effector domain was required for both the morphological and development changes induced by rasG-G12T. In contrast, cells expressing rasG-G12T/T45Q (a secondary mutation in the effector distal flanking domain) exhibited normal aggregation but a morphology indistinguishable from that of rasG-G12T transformants. This result suggests that RasG regulates developmental and cytoskeletal functions by direct interaction with more than one downstream effectors. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 3]