Dicty News Electronic Edition Volume 16, number 6 March, 24, 2001 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 DictyBase--http://dictybase.org. ====================== Post-doc available ====================== A postdoctoral position is available at Cornell University to work on a NSF sponsored project on signal transduction networks of eukaryotic cell chemotaxis. We aim at characterizing the signalling pathways involved in chemotaxis towards cAMP in Dictyostelium discoideum by visualising GFP-fused-proteins under very controlled stimuli. This project is a collaboration between the Physics Dept. at Cornell University (with the support of the Nanobiotechnology Center, ) and the Biology Dept. and Physics Dept. at the University of California in San Diego. The nature of the team involved offers a unique opportunity to conduct research in an interdisciplinary environment with researchers from biology, and experimental and theoretical physics. Applications from cell biologists and physicists are welcome. Applications including a curriculum vitae and a publication list should be send to the address below. In addition, applicants should arrange to have two letters of reference send to the same address. E-mail applications are welcome. Cornell University is an Affirmative Action, Equal Opportunity Employer. Please apply to Prof. Eberhard Bodenschatz, Cornell University, LASSP, 618 Clark Hall of Science, Ithaca, NY 14853 (eb22@cornell.edu). ============== Abstracts ============== The Dictyostelium Bcr/Abr-Related Protein DRG regulates both Rac- and Rab-dependent pathways Menno L.W. Knetsch, Nicole Schäfers, Heinz Horstmann, and Dietmar J. Manstein Dept. of Biophysics, Max-Planck-Institute for Medical Research, 69120 Heidelberg, Germany EMBO J. in press Abstract Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterised the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show that DRG-GEF modulates F-actin dynamics and cAMP-induced F-actin formation via Rac1-dependent signalling pathways. DRG's RacE-GAP activity is required for proper cytokinesis to occur. Additionally, we provide evidence that the specificity of DRG is not limited to members of the Rho family of small GTPases. A recombinant DRG-GAP accelerates the GTP-hydrolysis of RabD 30-fold in vitro and our complementation studies show that DRG-GAP activity is required for the RabD-dependent regulation of the contractile vacuole system in Dictyostelium. ----------------------------------------------------------------------------- ADENYLYL CYCLASE A EXPRESSION IS TIP-SPECIFIC IN DICTYOSTELIUM SLUGS AND DIRECTS STATA NUCLEAR TRANSLOCATION AND CUDA GENE EXPRESSION Irene Verkerke-van Wijk2, Masashi Fukuzawa1, Peter N. Devreotes3 and Pauline Schaap1* 1School of Life Sciences, University of Dundee, U.K. 2Department of Biology, University of Leiden, The Netherlands 3Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, USA Developmental Biology, in press. cAMP oscillations, generated by adenylyl cyclase A (ACA), coordinate cell aggregation in Dictyostelium and have also been implicated in organiser function during multicellular development. We used a gene fusion of the ACA promoter with a labile lacZ derivative to study the expression pattern of ACA. During aggregation, most cells expressed ACA, but thereafter expression was lost in all cells except those of the anterior tip. Before aggregation, ACA transcription was strongly upregulated by nanomolar cAMP pulses. Postaggregative transcription was sustained by nanomolar cAMP pulses, but downregulated by a continuous micromolar cAMP stimulus and by the stalk-cell-inducing factor, DIF. Earlier work showed that the transcription factor StatA displays tip-specific nuclear translocation and directs tip-specific expression of the nuclear protein CudA, that is essential for culmination. Both StatA and CudA were present in nuclei throughout the entire slug in an aca null mutant that expresses ACA from the constitutive actin15 promoter. This suggests that the tip-specific expression of ACA directs tip-specific nuclear translocation of StatA and tip-specific expression of CudA. ----------------------------------------------------------------------------- Recruitment of a Myosin Heavy Chain Kinase to Actin-Rich Protrusions in Dictyostelium Authors: Paul A. Steimle*, Shigehiko Yumura†, Graham P. Côté‡, Quint G. Medley**, Mark V. Polyakov *, Brian Leppert*, Thomas T. Egelhoff* *Department of Physiology and Biophysics Case Western Reserve University School of Medicine Cleveland, Ohio 44106-4970 †Department of Biology Yamaguchi University Yamaguchi, Japan 753-8512 ‡Department of Biochemistry Queens University Kingston, Ontario, Canada K7L-3N6 Current Biology, In Press ABSTRACT: Nonmuscle myosin II plays fundamental roles in cell body translocation during migration and is typically depleted or absent from actin-based cell protrusions such as lamellipodia, but the mechanisms preventing myosin II assembly in such structures have not been identified [1-3]. In Dictyostelium discoideum, myosin II filament assembly is controlled primarily through myosin heavy chain (MHC) phosphorylation. The phosphorylation of sites in the myosin tail domain by myosin heavy chain kinase A (MHCK A) drives disassembly of myosin II filaments in vitro and in vivo [4]. To better understand the cellular regulation of MHCK A activity, and thus the regulation of myosin II filament assembly, we studied the in vivo localization of native and green fluorescent protein (GFP)-tagged MHCK A. MHCK A redistributes from the cytosol to the cell cortex in response to stimulation of Dictyostelium cells with chemoattractant in an F-actin dependent manner. During chemotaxis, random migration, and phagocytic/endocytic events, MHCK A is recruited preferentially to actin-rich leading edge extensions. Given the ability of MHCK A to disassemble myosin II filaments, this localization may represent a fundamental mechanism for disassembling myosin II filaments and preventing localized filament assembly at sites of actin-based protrusion. ----------------------------------------------------------------------------- Myosin II-dependent cylindrical protrusions induced by quinine in Dictyostelium - antagonizing effects of actin polymerization at the leading edge Kunito Yoshida and Kei Inouye Department of Botany, Graduate School of Science, Kyoto University Sakyo-ku, Kyoto 606-8502, Japan. J. Cell Sci. in press Summary We found that amoeboid cells of Dictyostelium are induced by a millimolar concentration of quinine to form a rapidly elongating, cylindrical protrusion, which often led to sustained locomotion of the cells. Formation of the protrusion was initiated by fusion of a contractile vacuole with the cell membrane. During protrusion extension, a patch of the contractile vacuole membrane stayed undiffused on the leading edge of the protrusion for over 30 seconds. Protrusion formation was not inhibited by high osmolarity of the external medium (at least up to 400 mosM). On the other hand, mutant cells lacking myosin II (mhc- cells) failed to extend protrusions upon exposure to quinine. When GFP-myosin-expressing cells were exposed to quinine, GFP-myosin was accumulated in the cell periphery forming a layer under the cell membrane, but a newly formed protrusion was initially devoid of a GFP-myosin layer, which gradually formed and extended from the base of the protrusion. F-actin was absent in the leading front of the protrusion during the period of its rapid elongation, and the formation of a layer of F-actin in the front was closely correlated with its slowing-down or retraction. Periodical or continuous detachment of the F-actin layer from the apical membrane of the protrusion, accompanied by a transient increase in the elongation speed at the site of detachment, was observed in some of the protrusions. The detached F-actin layers, which formed a spiral layer of F-actin in the case of continuous detachment, moved in the opposite direction of protrusion elongation. In the presence of both cytochalasin A and quinine, the protrusions formed were not cylindrical but spherical, which swallowed up the entire cellular contents. The estimated bulk flux into the expanding spherical protrusions of such cells was four-times higher than the flux into the elongating cylindrical protrusions of the cells treated with quinine alone. These results indicate that the force responsible for the quinine-induced protrusion is mainly due to contraction of the cell body, which requires normal myosin II functions, while actin polymerization is important in restricting the direction of its expansion. We will discuss about the possible significance of tail contraction in cell movement in the multicellular phase of Dictyostelium development where cell locomotion similar to that induced by quinine is often observed without quinine treatment, and in protrusion elongation in general. ----------------------------------------------------------------------------- Control of cell polarity and chemotaxis by Akt/PKB and PI3 kinase through the regulation of PAKa Chang Y. Chung, Gary Potikyan, and Richard A. Firtel Molecular Cell, in press. We demonstrate that PI3 kinase and protein kinase B (PKB or Akt) control cell polarity and chemotaxis, in part, through the regulation of PAKa, which is required for myosin II assembly. We demonstrate that PI3K and PKB mediate PAKa's subcellular localization, PAKa's activation in response to chemoattractant stimulation, and chemoattractant-mediated myosin II assembly. Mutation of the PKB phosphorylation site in PAKa to Ala blocks PAKa's activation and inhibits PAKa redistribution in response to chemoattractant stimulation, whereas an Asp substitution leads to an activated protein. Addition of the PI3K inhibitor LY294002 results in a rapid loss of cell polarity and the axial distribution of actin, myosin, and PAKa. These results provide a mechanism by which PI3K regulates chemotaxis. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 6]