Dicty News Electronic Edition Volume 24, number 6 March 11, 2005 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Computer-Assisted Reconstruction and Analysis of Filopod Formation and the Role of Myosin II Heavy Chain Phosphorylation in Dictyostelium Paul J. Heid, Jeremy Geiger, Deborah Wessels, Edward Voss and David R. Soll W.M. Keck Dynamic Image Analysis Facility, Department of Biological Sciences, The University of Iowa, Iowa City, IA 52242 J Cell Science, in press To investigate the role played by filopodia in the motility and chemotaxis of amoeboid cells, a computer-assisted 3D reconstruction and motion analysis system, DIAS 4.0, has been developed. Reconstruction at short time intervals of Dictyostelium amoebae migrating in buffer or in response to chemotactic signals revealed 1) that the great majority of filopodia form on pseudopodia, not on the cell body; 2) that filopodia on the cell body originate primarily on pseudopodia and relocate; and 3) that filopodia on the uropod are longer and more stable than those located on other portions of the cell. When adjusting direction through lateral pseudopod formation in a spatial gradient of chemoattractant, the temporal and spatial dynamics of lateral pseudopodia suggest that filopodia may be involved in stabilizing pseudopodia on the substratum while the decision is being made by a cell either to turn into a pseudopodium formed in the correct direction (up the gradient) or to retract a pseudopodium formed in the wrong direction (down the gradient). Experiments in which amoebae were treated with high concentrations of chemoattractant further revealed that receptor occupancy plays a role both in filopod formation and retraction. Since phosphorylation-dephosphorylation of myosinII heavy chain (MHC) plays a role in lateral pseudopod formation, turning and chemotaxis, the temporal and spatial dynamics of filopod formation were analyzed in MHC phosphorylation mutants. These latter studies revealed that MHC phosphorylation-dephosphorylation plays a role in the regulation of filopod formation during cell migration in buffer and during chemotaxis. The computer-assisted technology described here for reconstructing filopodia at short time intervals in living cells, therefore, provides a new tool for investigating the role filopodia play in the motility and chemotaxis of amoeboid cells. Submitted by: Deborah Wessels [deborah-wessels@uiowa.edu] ----------------------------------------------------------------------------- Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae Hao Lu and Margaret Clarke (Program in Molecular, Cell, and Developmental Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73121) Cellular Microbiology, in press The natural hosts of the bacterial pathogen Legionella pneumophila are amoebae and protozoa. In these hosts, as in human macrophages, the pathogen enters the cell through phagocytosis, then rapidly modifies the phagosome to create a compartment that supports its replication. We have examined L. pneumophila entry and behavior during early stages of the infection of Dictyostelium discoideum amoebae. Bacteria were labeled with a red fluorescent marker, and selected proteins and organelles in the host were labeled with GFP, allowing the dynamics and interactions of L. pneumophila- containing phagosomes to be tracked in living cells. These studies demonstrated that entry of L. pneumophila is an actin-mediated process, that the actin-binding protein coronin surrounds the nascent phagosome but dissociates immediately after internalization, that ER membrane is not incorporated into a phagosome during uptake, that the newly internalized phagosome is rapidly transported about the cell on microtubules, that association of ER markers with the phagosome occurs in two steps that correlate with distinct changes in phagosome movement, and that the vacuolar H(+)-ATPase does not associate with mature replication vacuoles. These studies have clarified certain aspects of the infection process and provided new insights into the dynamic interactions between the pathogen and its host. Submitted by: Margaret Clarke [clarkem@omrf.ouhsc.edu] ----------------------------------------------------------------------------- Manifestations of multicellularity - Dictyostelium reports Jeffrey G. Williams*, Angelika A. Noegel+ and Ludwig Eichinger+ *School of Life Sciences University of Dundee MSI/WTB Complex Dow Street Dundee DD1 5EH U. K. + Centre for Biochemistry and Centre for Molecular Medicine Cologne Medical Faculty, University of Cologne Joseph-Stelzmann-Str. 52 50931 Cologne Germany TIGS Ms, in press Foreword The Dictyostelium genome sequence, the first complete amoebozoan sequence to be released, has revealed many important evolutionary insights; notable among these is the high degree of overlap between metazoan and Dictyostelium proteomes. Abstract The recent release of the Dictyostelium genome sequence is important because Dictyostelium has become a much-favoured model system for cell and developmental biologists. The sequence has revealed a remarkably high total number of app. 12,500 genes, only a thousand fewer than are encoded by Drosophila. Previous protein sequence comparisons suggested that Dictyostelium is evolutionary closer to animals and fungi than to plants and the global protein sequence comparison, now made possible by the genome sequence, confirms this. This review focuses on several classes of proteins that are shared by Dictyostelium and animals: a highly sophisticated array of microfilament components, a large family of G protein-coupled receptors and a diverse set of SH2 domain-containing proteins. The presence of these proteins strengthens the case for a relatively close relationship with animals and extends the range of problems that can be addressed using Dictyostelium as a model organism Submitted by: Jeff Williams [j.g.williams@dundee.ac.uk] ----------------------------------------------------------------------------- cAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum Daniel F. Lusche, Karen Bezares-Roder, Kathrin Happle, Christina Schlatterer Faculty for Biology, University of Konstanz, 78457 Konstanz, Germany BMC Cell Biology, in press Abstract Background: Differentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i. Results: We analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced. Conclusions: We conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance. Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de] ----------------------------------------------------------------------------- Ca2+-regulation in the absaence of the iplA gene product in Dictyostelium discoideum Ralph H Schaloske 1, Daniel F Lusche 2, Karen Bezares-Roder 2, Kathrin Happle 2, Dieter Malchow 2 and Christina Schlatterer 2 1 Department of Chemistry and Biochemistry, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0601, USA 2 Faculty for Biology, University of Konstanz, 78457 Konstanz, Germany BMC Cell Biology, in press Abstract Background Stimulation of Dictyostelium discoideum with cAMP evokes an elevation of the cytosolic free Ca2+ concentration ([Ca2+]i). The [Ca2+]i-change is composed of liberation of stored Ca2+ and extracellular Ca2+-entry. The significance of the [Ca2+]i-transient for chemotaxis is under debate. Abolition of chemotactic orientation and migration by Ca2+-buffers in the cytosol indicates that a [Ca2+]i-increase is required for chemotaxis. Yet, the iplA- mutant disrupted in a gene bearing similarity to IP3-receptors of higher eukaryotes aggregates despite the absence of a cAMP-induced [Ca2+]i-transient which favours the view that [Ca2+]i-changes are insignificant for chemotaxis. Results We investigated Ca2+-fluxes and the effect of their disturbance on chemotaxis and development of iplA- cells. Differentiation was altered as compared to wild type amoebae and sensitive towards manipulation of the level of stored Ca2+. Chemotaxis was impaired when [Ca2+]i-transients were suppressed by the presence of a Ca2+-chelator in the cytosol of the cells. Analysis of ion fluxes revealed that capacitative Ca2+-entry was fully operative in the mutant. In suspensions of intact and permeabilized cells cAMP elicited extracellular Ca2+-influx and liberation of stored Ca2+, respectively, yet to a lesser extent than in wild type. In suspensions of partially purified storage vesicles ATP-induced Ca2+-uptake and Ca2+-release activated by fatty acids or Ca2+-ATPase inhibitors were similar to wild type. Mn2+-quenching of fura2 fluorescence allows to study Ca2+-influx indirectly and revealed that the responsiveness of mutant cells was shifted to higher concentrations: roughly 100 times more Mn2+ was necessary to observe agonist-induced Mn2+-influx. cAMP evoked a [Ca2+]i-elevation when stores were strongly loaded with Ca2+, again with a similar shift in sensitivity in the mutant. In addition, basal [Ca2+]i was significantly lower in iplA- than in wild type amoebae. Conclusions These results support the view that [Ca2+]i-transients are essential for chemotaxis and differentiation. Moreover, capacitative and agonist-activated ion fluxes are regulated by separate pathways that are mediated either by two types of channels in the plasma membrane or by distinct mechanisms coupling Ca2+-release from stores to Ca2+-entry in Dictyostelium. The iplA- strain retains the capacitative Ca2+-entry pathway and an impaired agonist-activated pathway that operates with reduced efficiency or at higher ionic pressure. Submitted by: Christina Schlatterer [Christina.Schlatterer@uni-konstanz.de] ============================================================================== [End Dicty News, volume 24, number 6]