Dicty News Electronic Edition Volume 15, number 12 December 30, 2000 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 the Dictyostelium Web Page "http://dicty.cmb.northwestern.edu/dicty" ========================================== A HAPPY AND PRODUCTIVE NEW YEAR TO ALL ========================================== ============== Abstracts ============== Interaction of gdt1 and protein kinase A (PKA) in the growth-differentiation-transition in Dictyostelium. Changjiang Zeng1, 3, Christophe Anjard1, 4, Gerd Primpke2, Birgit Wetterauer2 , Sandra Wille1 and Wolfgang Nellen1* *corresponding author e-mail: nellen@hrz.uni-kassel.de phone: ++49 (0561) 804 4805 fax: ++49 (0561) 804 4800 accepted «Differentiation» Abstract The gdt1 gene is a negative regulator of the growth-differentiation-transition (GDT) in Dictyostelium. gdt1- cells express the GDT marker discoidin earlier and at higher levels and prematurely enter the differentiation pathway. Protein kinase A is a positive regulator of the GDT and is required for multicellular development. Disruption of the PKA catalytic subunit or overexpression of a constitutively active mutant of the regulatory subunit results in cells which do not form multicellular aggregates and which show strongly reduced levels of discoidin. We have created PKA-/gdt1- double mutants and show that these display high levels of discoidin expression but no aggregation, suggesting that gdt1 may be a downstream target of PKA in a branched signalling cascade initiating differentiation. Data obtained with the PKA inhibitor H89 support these result: in wild type cells H89 inhibits discoidin expression while in gdt1- mutants there is no obvious effect. However, since PKA-/gdt1- cells display less discoidin expression than the single gdt1 mutant, we propose that PKA and gdt1 are in two parallel interacting pathways. To get insight into the mechanism how PKA may block gdt1, we have tested two putative PKA phosphorylation sites in the protein and find that one of them is efficiently phosphorylated by PKA in vitro. A model for the interplay between PKA and gdt1 during the growth-differentiation-transition is discussed. ----------------------------------------------------------------------------- The Histidine Kinases of Dictyostelium. Christophe Anjard and William F. Loomis Center for Molecular Genetics, Division of Biology, University of California San Diego, La Jolla, CA 92093 Chapter in "Histidine Kinases in Signal Transduction" edited by R. Dutta and M. Inouye. Acad. Press, San Diego (in press). Summary At least 15 members of the histidine kinase family can be recognized in the genomic sequences of Dictyostelium discoideum. The predicted products of each of these genes have well conserved catalytic and receiver domains although two do not appear to be active kinases since they lack the histidine in the H motif that is autophosphorylated and one, DokA, appears to function predominantly as a phosphatase. One member of the family, DhkD, is a double histidine kinase with two catalytic and two receiver domains. Alignment of this large family with the Sln1 histidine kinase of yeast extends the sequence profile that characterizes eukaryotic histidine kinases. Biochemical studies of the first member of this family to be discovered, DhkA, have shown that it is a transmembrane receptor kinase that autophosphorylates and relays the phosphate to the receiver aspartate when dimerized. Genetic studies on dhkA, dhkB, dhkC and dokA have indicated their roles in cellular and developmental processes. It is likely that DhkC relays phosphate to the N-terminal receiver domain of the cAMP phosphodiesterase RegA through the H2 domain of RdeA. Phosphorylation of the receiver domain of RegA activates it. DhkA and DhkB appear to inhibit RegA activity by indirectly activating the MAP kinase ERK2. When the carboxy terminal region of RegA is phosphorylated, the phosphodiesterase activity is inhibited. Histidine kinases may also activate the late adenylyl kinase, ACR. Thus these histidine kinases seem to be focused on regulating cAMP to modulate the activity of the cAMP dependent protein kinase, PKA. ----------------------------------------------------------------------------- Identification of Clathrin-Adaptor Medium Chains in Dictyostelium discoideum: Differential Expression during Development Benoît de Chassey, Annick Dubois, Yaya Lefkir and François Letourneur* Institut de Biologie et Chimie des Protéines, UMR5086 - CNRS / Université Lyon I- France *corresponding author Institut de Biologie et Chimie des Protéines, UMR5086 - CNRS / Université Lyon I 7, Passage du Vercors 69367 LYON cedex 07 - France phone: 0033 4 72 72 26 81 Fax: 0033 4 72 72 26 01 e.mail: f.letourneur@ibcp.fr "Gene" in Press ABSTRACT Clathrin-adaptor complexes (APs) are vesicle coat components that participate in cargo selectivity and transport vesicle formation. Here we cloned and characterized apm1, apm3 and apm4 cDNAs encoding AP medium chains (µ) in D. discoideum. Amino acid comparison suggested that predicted proteins were homologous to known µ1, µ3 and µ4 subunits of mammalian APs as they shared 69, 51, and 26% identity with mouse µ1A, human µ3A and human µ4 respectively. In all chains, amino acids residues predicted to interact with tyrosine based sorting signals were conserved. Southern blot analysis indicated only one copy of each gene in D. discoideum genome. Expression of apm1 and apm3 mRNAs stayed relatively constant during vegetative growth and throughout development. In contrast, apm4 was poorly expressed in amoebae but became well detectable by RT-PCR upon cell differentiation. This regulated expression of coat proteins enlightens the importance of intracellular membrane transport vesicles during development in D. discoideum and strengthens this attractive model organism for studying the function of coat complexes in vivo. ----------------------------------------------------------------------------- Real-time visualization of intracellular hydrodynamics in single living cells Eric O. Potma, Wim P. de Boeij, Peter J.M. van Haastert* and Douwe A. Wiersma Ultrafast Laser and Spectroscopy Laboratory, Materials Science Centre, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands. *Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands. PNAS USA in press Abstract Intracellular water concentrations in single living cells were visualized by nonlinear coherent anti-Stokes Raman scattering (CARS) microscopy. In combination with isotopic exchange measurements CARS microscopy allowed the real-time observation of transient intracellular hydrodynamics at a high spatial resolution. Studies of the hydrodynamics in the microorganism Dictyostelium discoideum indicated the presence of a microscopic region near the plasma membrane where the mobility of water molecules is severely restricted. Modeling the transient hydrodynamics eventuated in the determination of cell-specific cytosolic diffusion and plasma membrane permeability constants. Our experiments demonstrate that CARS microscopy offers an invaluable tool for probing single cell water dynamics. ----------------------------------------------------------------------------- Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients Saskia van Es1, Deborah Wessels2, David R. Soll2, Jane Borleis1 and Peter N. Devreotes*1 J. Cell.Biol. in press Abstract We have identified a novel gene, Tortoise (TorA) that is required for the efficient chemotaxis of D. discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH-domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, while overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-GFP is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension. ----------------------------------------------------------------------------- [End Dicty News, volume 15, number 12]