Dicty News Electronic Edition Volume 12, number 9 April 24, 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 ============== A Cell’s Sense of Direction Dept. Biological Chemistry, Johns Hopkins School of Medicine, Baltimore MD 21205 C.A. Parent and P.N. Devreotes Science, In press. In eukaryotic cells directional sensing is mediated by heterotrimeric guanine nucleotide binding protein (G protein)-linked signaling pathways. In D. discoideum amoebae and mammalian leukocytes, the receptors and G-protein subunits are uniformly distributed around the cell perimeter. Chemoattractants induce the transient appearance of binding sites for several pleckstrin homology domain-containing proteins on the inner face of the membrane. In gradients of attractant these sites are persistently present on the side of the cell facing the higher concentration, even in the absence of a functional actin cytoskeleton or cell movement. Thus, the cell senses direction by spatially regulating the activity of the signal transducution pathway. ---------------------------------------------------------------------------- Green fluorescent protein in the visualization of particle uptake and fluid-phase endocytosis. Markus Maniak was: Max-Planck-Institut fuer Biochemie, Martinsried, Germany now: MRC-Laboratory for Molecular Cell Biology, London, England Meth. Enzymol. 302 March 1999. There is no abstract or summary to this contribution. The chapter mainly describes details how we image endocytosis in Dictyostelium using GFP fusion-proteins. For those who may be interested, there is a colour figure that uses the combination of FITC and TRITC dextrans to visualise the acidification of individual macropinosomes after internalisation. The result is, that the pH inside a macropinosome does not change over the first 40 seconds after its formation (when it is still thought to be surrounded by a dense cytoskeletal coat) then it becomes acidic within less than 10 seconds. ---------------------------------------------------------------------------- SDF-2 induction of terminal differentiation in Dictyostelium is mediated by the membrane-spanning sensor kinase DhkA Nancy Wang, Fredrik Söderbom, Christophe Anjard, Gad Shaulsky and William F. Loomis Center for Molecular Genetics, Department of Biology University of California San Diego, La Jolla, CA 92093 Molecular and Cellular Biology (in press) Abstract SDF-2 is a peptide released by prestalk cells during culmination that stimulates prespore cells to encapsulate. Genetic evidence indicates that the response is dependent on the dhkA gene. This gene encodes a member of the histidine kinase family of genes that functions in two-component signal transduction pathways. The sequence of the N-terminal half of DhkA predicts two hydrophobic domains separated by a 310 amino acid loop that could bind a ligand. By inserting MYC6 epitopes into DhkA we were able to show that the loop is extracellular while the catalytic domain is cytoplasmic. Cells expressing the MYC epitope in the extracellular domain of DhkA were found to respond only if induced with 100 fold higher levels of SDF-2 than required to induce dhkA+ cells; however, they could be induced to sporulate by addition of antibodies specific to the MYC epitope. To examine the enzymatic activity of DhkA we purified the catalytic domain following expression in bacteria and observed incorporation of labelled phosphate from ATP consistent with histidine autophosphorylation. Site-directed mutagenesis of histidine1395 to glutamine in the catalytic domain blocked autophosphorylation. Furthermore, genetic analyses showed that histidine1395 and the relay aspartate2075 of DhkA are both critical to its function but that another histidine kinase, DhkB, can partially compensate for the lack of DhkA activity. Sporulation is drastically reduced in double mutants lacking both DhkA and DhkB. Suppressor studies indicate that the cAMP phosphodiesterase, RegA, and the cAMP dependent protein kinase, PKA, act downstream of DhkA. ---------------------------------------------------------------------------- [End Dicty News, volume 12, number 9]