Dicty News Electronic Edition Volume 14, number 9 April 22, 2000 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 ============= Dictyostelium RasD is Required for Normal Phototaxis, but not Differentiation Andrew Wilkins, Meenal Khosla, Derek J. Fraser,George B. Spiegelman, Paul R. Fisher, Gerald Weeks and Robert H. Insall in press, Genes & Development Abstract RasD, a Dictyostelium homologue of mammalian Ras, is maximally expressed during the multicellular stage of development. Normal Dictyostelium aggregates are phototactic and thermotactic, moving towards sources of light and heat with great sensitivity. We show that disruption of the gene for rasD causes a near-total loss of phototaxis and thermotaxis in mutant aggregates, without obvious effects on undirected movement. Previous experiments had suggested important roles for RasD in development and cell-type determination. Surprisingly, rasD- cells show no obvious changes in these processes. These cells represent a novel class of phototaxis mutant, and indicate a role for a Ras pathway in the connections between stimuli and coordinated cell movement. ---------------------------------------------------------------------------- An Akt/PKB-related kinase activated via a cAMP-receptor dependent, PI(3) kinase-independent pathway is required for Dictyostelium multicellular development Ruedi Meili, Charlene Ellsworth, and Richard A. Firtel Current Biology, in press. ABSTRACT Background: Dictyostelium Akt/PKB is homologous to mammalian Akt/PKB and is required for cell polarity and proper chemotaxis during early development. Akt/PKB kinase activity is activated in response to chemoattractants in neutrophils and in Dictyostelium by the chemoattractant cAMP functioning via a G protein-, PI3 kinase-dependent pathway. Dictyostelium contains several kinase structurally related to Akt/PKB, one of which, PKBR-1, is investigated here for its role in cell polarity, movement, and cellular morphogenesis during development. Results: PKBR-1 has a kinase and C-terminal domain related to those of Akt/PKB but no PH domain. Instead, it has an N-terminal myristoylation site, which is required for PKBR-1's constitutive membrane localization. Like Akt/PKB, PKBR-1 is activated by cAMP through a G protein-dependent pathway but which does not require PI3K, probably due to PKBR-1's constitutive membrane localization, supported by experiments demonstrating the requirement of PKBR-1's membrane association for activation and in vivo function. PKBR-1 protein is found in all cells through early development but is then restricted to the apical cells in developing aggregates, which are thought to control morphogenesis. PKBR-1 null cells arrest development at the mound stage and are defective in morphogenesis and multicellular development. These phenotypes are complemented Akt/PKB, suggesting functional overlap between PKBR-1 and Akt/PKB. Akt/PKB:PKBR-1 double knockout cells exhibit growth defects and show stronger chemotaxis and cell polarity defects than Akt/PKB null cells. Conclusions: Our results expand the previously known functions of Akt/PKB family members in cell movement and morphogenesis during multicellular development. The data suggest that Akt/PKB and PKBR-1 have overlapping effectors and biological function: Akt/PKB, functions predominantly during aggregation to control cell polarity and chemotaxis while PKBR-1 is required for morphogenesis during multicellular development. ---------------------------------------------------------------------------- Real-time high-resolution optical sectioning suggests biphasic cytokinetic mechanism in Dictyostelium discoideum. Yoshio Fukui Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611-3008. In Biology of cytokinesis, (ed. Y. Fukui). Microscope Research and Technique, vol. 49, Number 2 (April 2000). In press. Abstract Despite its biological significance, much of the mechanism of cytokinesis is not yet resolved. The problems include: (1) signaling mechanism determining the position of the cleavage furrow, (2) molecular and mechanistic nature of the contractile ring, and (3) the origin of forces responsible for cleavage. Using high-resolution imaging technique, the present study analyzes morphometric changes of cytokinesis in wild type (NC4) Dictyostelium discoideum amoeba. Sample was prepared by the agar-overlay method, creating 3 mm-thick, nearly two-dimensional cells; and high-resolution image was acquired at 16.7 milliseconds’ temporal, 234 nm x, y-, and 100 nm z-axis resolutions. Under this condition, the formation of cleavage furrow initiates at mitotic telophase, and daughter cells separate 18-22 minutes after the furrow initiation. We found that the compression of cells and the room temperature need to be carefully controlled for cytokinesis to proceed in orderly manner. The results demonstrate that the pole-to-pole distance increases by 83% during the initial 5 minutes of cytokinesis, while the distance of equator only decreases by 56%. In contrast, during the subsequent 5 minutes, the pole-to-pole distance only increases by 17%, while the equator distance decreases as much as by 44%. This study indicates that cytokinesis consists of at least two different phases, each of which results from different mechanism. Note: This is available online at http://pubweb.nwu.edu/~yoshifk/fukui.html ---------------------------------------------------------------------------- SPATIALLY REGULATED RECRUITMENT OF CLATHRIN TO THE PLASMA MEMBRANE DURING CAPPING AND CELL TRANSLOCATION Cynthia K. Damer1, and Theresa J. O'Halloran2 1 Department of Biology, Vassar College; 2 Section of Molecular Cell & Developmental Biology The University of Texas at Austin, Austin TX In press, Molecular Biology of the Cell Abstract Clathrin coated vesicles bud from selected cellular membranes to traffic specific intracellular proteins. To study the dynamic properties of clathrin coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) in Dictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile, and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Secondly, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of crosslinked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 9]