Dicty News Electronic Edition Volume 13, number 1 July 3, 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 ============= Cortexillin I is required for development in Polysphondylium Petra Fey and Edward C. Cox 333 Moffett Laboratories, Department of Molecular Biology, Princeton University, Princeton NJ 08544 Developmental Biology, in press Abstract The actin binding proteins cortexillin I and II play a major role in Dictyostelium cytokinesis where they are found localized to the membranes of the cleavage furrow. Here we report on cortexillin I mutants isolated by gene trapping in Polysphondylium . The original mutation and reconstructed versions of the original, as well as cortexillin I deletions, are unable to form aggregation streams under starvation conditions. The fruiting bodies that do form when cells are grown on bacterial lawns lack the one and two-dimensional symmetries so apparent in wild-type. These two phenotypes and the proposed structural basis for them suggest that cortexillin I functions in chemotaxis and morphogenesis in addition to its role in cytokinesis. ---------------------------------------------------------------------------- A cell-counting factor regulating structure size in Dictyostelium Debra A. Brock and Richard H. Gomer Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 Genes&Development, in press Abstract Developing Dictyostelium cells form large aggregation streams which break up into groups of 0.2 - 1 x 10exp5 cells. Each group then becomes a fruiting body. smlA cells oversecrete an unknown factor which causes aggregation streams to break up into groups of ~5 x 10exp3 cells and thus form very small fruiting bodies. We have purified the counting factor and find that it behaves as a complex of polypeptides with an effective molecular weight of 450 kD. One of the polypeptides is a 40 kD hydrophilic protein we have named countin. Immunofluorescence shows that countin is present in all cells and Western blots indicate that countin is secreted. In transformants with a disrupted countin gene, there is no detectable secretion of counting factor, and the aggregation streams do not break up, resulting in huge (up to 2 x 10exp5 cell) fruiting bodies. Addition of crude countin factor causes the countin cells to form normal size aggregates. Using diffusion calculations, we find that as a general principle such a factor can be used to sense the number of cells in a group. ---------------------------------------------------------------------------- Rearrangement of cortex proteins constitutes an osmoprotective mechanism in Dictyostelium Hans Zischka, Felix Oehme, Tanja Pintsch, Alexander Ott, Heike Keller, Joseph Kellermann and Stephan C. Schuster Max-Planck-Institut für Biochemie, 82152 Martinsried, Germany EMBO Journal, in Press Abstract Dictyostelium responds to hyperosmotic stress of 400 mOsm by a rapid reduction of its cell volume to 50 %. The reduced cell volume is maintained as long as these osmotic conditions prevail. Dictyostelium does not accumulate compatible osmolytes to counteract the osmotic pressure applied. By two dimensional gel electrophoresis we demonstrate that during the osmotic shock the protein pattern remains unaltered in whole cell extracts. However, when cells were fractionated into membrane and cytoskeleton fractions, alterations of specific proteins could be demonstrated. In the crude membrane fraction a 3-fold increase in protein amount was measured upon hyperosmotic stress. In the cytoskeletal fraction the proteins DdLIM and the regulatory myosin light chain (RMLC) were shown to be regulated in the osmotic stress response. The elongation factors eEF1a (ABP50) and eEF1b were found to increase in the cytoskeletal fraction, suggesting a translational arrest upon hyperosmotic stress. Furthermore, the two main components of the cytoskeleton, actin and myosin II, are phosphorylated as a consequence of the osmotic shock with a tyrosine residue as phosphorylation site on actin and specific threonines in the case of the myosin II heavy chain. ---------------------------------------------------------------------------- DAip1, a Dictyostelium Homologue of the Yeast Actin-Interacting Protein 1, Is Involved in Endocytosis, Cytokinesis and Motility Angelika Konzok, Igor Weber, Evelyn Simmeth, Ulrike Hacker, Markus Maniak*, and Annette Müller-Taubenberger Max-Planck-Institut für Biochemie, D-82152 Martinsried, Germany, and *MRC Laboratory for Molecular Cell Biology, University College London WC1E 6BT, UK J. Cell Biol., in press Abstract The 64-kDa protein DAip1 from Dictyostelium contains nine WD40-repeats and is homologous to the actin-interacting protein 1, Aip1p, from Saccharomyces cerevisiae, and to related proteins from Caenorhabditis, Physarum and higher eukaryotes. We show that DAip1 is localized to dynamic regions of the cell cortex that are enriched in filamentous actin: phagocytic cups, macropinosomes, lamellipodia and pseudopodia. In cells expressing GFP-tagged DAip1, the protein rapidly redistributes into newly formed cortical protrusions. Functions of DAip1 in vivo were assessed using null mutants generated by gene replacement, and by overexpressing DAip1. DAip1-null cells are impaired in growth and their rates of fluid phase uptake, phagocytosis and movement are reduced in comparison to wild-type rates. Cytokinesis is prolonged in DAip1-null cells and they tend to become multinucleate. On the basis of similar results of DAip1 overexpression and effects of latrunculin A, we propose a function for DAip1 in the control of actin depolymerization in vivo, probably through interaction with cofilin. Our data suggest that DAip1 plays an important regulatory role in the rapid remodeling of the cortical actin meshwork. ---------------------------------------------------------------------------- A novel Ras interacting protein required for chemotaxis and cAMP signal relay in Dictyostelium. Lee, S., C.A. Parent, R. Insall and R.A. Firtel Mol. Biol. Cell, in press. ABSTRACT We have identified a novel Ras interacting protein from Dictyostelium, RIP3, whose function is required for both chemotaxis and the synthesis and relay of the cAMP chemoattractant signal. rip3 null cells are unable to aggregate and lack receptor activation of adenylyl cyclase but are able, in response to cAMP, to induce aggregation-stage, postaggregative, and cell-type-specific gene expression in suspension culture. In addition, rip3 null cells are unable to properly polarize in a cAMP gradient and chemotaxis is highly impaired. We demonstrate that cAMP stimulation of guanylyl cyclase, which is required for chemotaxis, is reduced ~60% in rip3 null cells. This reduced activation of guanylyl cyclase may account, in part, for the defect in chemotaxis. When cells are pulsed with cAMP for 5 h to mimic the endogenous cAMP oscillations that occur in wild-type strains, the cells will form aggregates, most of which, however, arrest at the mound stage. Unlike the response seen in wild-type strains, the rip3 null cell aggregates that form under these experimental conditions are very small, which is probably due to the rip3 null cell chemotaxis defect. Many of the phenotypes of rip3 null cell, including the inability to activate adenylyl cyclase in response to cAMP and defects in chemotaxis, are very similar to those of strains carrying a disruption of the gene encoding the putative Ras exchange factor AleA. We demonstrate that aleA null cells also exhibit a defect in cAMP-mediated activation of guanylyl cyclase similar to that of rip3 null cells. A double knockout mutant (rip3/aleA null cells) exhibits a further reduction in receptor activation of guanylyl cyclase and these cells display almost no cell polarization or movement in cAMP gradients. As RIP3 preferentially interacts with an activated form of the Dictyostelium Ras protein RasG, which itself is important for cell movement, we propose that RIP3 and AleA are components of a Ras-regulated pathway involved in integrating chemotaxis and signal relay pathways that are essential for aggregation. ---------------------------------------------------------------------------- Biochemical Characterization of a Dictyostelium Myosin II Heavy Chain Phosphatase that Promotes Filament Assembly M.B. Murphy, T.T. Egelhoff Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970. Eur. J. Biochem. in press In Dictyostelium cells, myosin II is found as cytosolic non-assembled monomers and as cytoskeletal bipolar filaments. It is thought that the phosphorylation state of three threonine residues in the tail of myosin II heavy chain regulates the molecular motor's assembly state and localization. Phosphorylation of the myosin heavy chain at threonine residues 1823, 1833, 2029 is responsible for maintaining myosin in the non-assembled state, and subsequent dephosphorylation of these residues is a prerequisite for assembly into the cytoskeleton. We report here the characterization of myosin heavy chain phosphatase activities in Dictyostelium utilizing myosin II phosphorylated by Myosin Heavy Chain Kinase A as a substrate. One of the myosin heavy chain phosphatase activities was identified as protein phosphatase 2A and the purified holoenzyme was composed of a 37 kDa catalytic subunit, a 65 kDa A-subunit, and a 55 kDa B-subunit. The PP2A holoenzyme displays two orders of magnitude more activity towards myosin phosphorylated on the heavy chains than it does towards myosin phosphorylated on the regulatory light chains, consistent with a role in the control of filament assembly. The purified myosin heavy chain phosphatase activity promotes bipolar filament assembly in vitro via dephosphorylation of the myosin heavy chain. This system should provide a valuable model for studying the regulation and localization of PP2A in the context of cytoskeletal reorganization. ---------------------------------------------------------------------------- Molecular Characterization and Immunolocalization of Dictyostelium discoideum Protein Phosphatase 2A M.B. Murphy, S.K. Levi, T.T. Egelhoff Department of Physiology and Biophysics, Case Western Reserve University Cleveland, Ohio 44106-4970 FEBS Letters, in press Protein phosphatase 2A (PP2A) was previously purified from Dictyostelium and biochemically characterized. The purified PP2A holoenzyme was composed of a 37 kDa catalytic "C-subunit", a 65 kDa "A-subunit", and a 55 kDa "B subunit". We report here the characterization of the genes encoding the Dictyostelium PP2A subunits as well as the immunolocalization of the PP2A subunits in Dictyostelium. The cDNAs encoding the B- and C-subunits were isolated from a Dictyostelium library and the deduced amino-acid sequences reveal strong conservation with the mammalian PP2A homologues. Southern blot analysis suggests that each of the PP2A subunit genes are present in a single copy. The PP2A subunits were localized mainly to the cytosol in Dictyostelium cells. However, immunofluorescence confocal microscopy demonstrates that the B-subunit of PP2A is highly enriched in centrosomes, suggesting a potential role for this PP2A regulatory subunit in centrosomal function. ---------------------------------------------------------------------------- [End Dicty News, volume 13, number 1]