Dicty News Electronic Edition Volume 15, number 4 August 19, 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" ================ Announcement ================ POSTDOCTORAL POSITIONS MICROARRAY EXPRESSION ANALYSES Funded postdoctoral positions are available for participation in the construction of DNA microarrays and their use in analyses of expression patterns in wild-type and mutant strains of Dictyostelium. We have carried out a pilot project with chips on which 690 genes were arrayed with a Molecular Dynamics robot. We will add >5,000 developmental cDNAs to the chips to extend the analyses. The consequences of mutations affecting networks that regulate PKA (eg. regA, acaA, acrA, dhkA, erkB etc.) will be the initial focus of these studies. Over the next few years we plan on analyzing a large number of morphological mutants to further define dependent sequences during development. Applicants who want to extend their experience in computer assisted manipulation of large data sets are encouraged to contact Bill Loomis by e-mail at wloomis@ucsd.edu or write me at: William F. Loomis Cell and Developmental Biology UCSD La Jolla, CA 92093-0368 ============== Abstracts ============== The Membrane Glycoprotein gp150 is Encoded by the lagC Gene and Mediates Cell-Cell Adhesion by Heterophilic Binding during Dictyostelium Development Jun Wang,*,Ý Liansheng Hou,*,1 Don Awrey,Ý William F. Loomis,§ Richard A. Firtel,§ and Chi-Hung Siu*,Ý,2 From the *Banting and Best Department of Medical Research and ÝDepartment of Biochemistry, University of Toronto, Toronto, Ontario M5G 1L6, Canada; §Section of Cell and Developmental Biology and the Center for Molecular Genetics, University of California at San Diego, La Jolla, CA. Dev. Biol., in press. ABSTRACT gp150 is a membrane glycoprotein, which has been implicated in cell-cell adhesion in the post-aggregation stages of Dictyostelium development. An analysis of its tryptic peptides by mass spectrometry has identified gp150 as the product of the lagC gene, which was previously shown to play a role in morphogenesis and cell-type specification. Antibodies raised against the GST-LagC fusion protein specifically recognized gp150 in wild-type cells and showed that it is missing in lagC-null cells. Immunolocalization studies have confirmed its enrichment in cell-cell contact regions. In mutant cells that lack the aggregation stage-specific cell adhesion molecule gp80, gp150 is expressed precociously. Moreover, these cells acquire EDTA-resistant cell-cell binding during aggregation, suggesting a role for gp150 in this process. Cells in which the genes encoding gp80 and gp150 are both inactivated do not acquire EDTA-resistant cell adhesion during aggregation. Strains transformed with an actin 15::lagC construct express gp150 precociously, but do not show EDTA-resistant adhesion during early development. However, vegetative cells expressing gp150 can be recruited into aggregates of 16-h lagC-null cells. These results, together with those obtained with the cell-to-substratum binding assay, indicate that gp150 mediates cell-cell adhesion via heterophilic interactions with another component that accumulates during the aggregation stage. ---------------------------------------------------------------------------- Mechanism of cAMP-induced H+-efflux of Dictyostelium cells: a role for fatty acids H. Flaadt*, R. Schaloske, D. Malchow Faculty of Biology, University of Konstanz, D-78457 Konstanz, Germany * present address: Differentiation Epitheliale, Ecole Normale Superieure, 46 rue d´ulm, F-75005 Paris, France J. Biosci., in press. Abstract Aggregating Dictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H+ ATPase inhibitor concanamycin A and with the plasma membrane H+ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H+ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H+ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca2+-fluxes involves phospholipase A2 activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca2+-efflux. Here we show that arachidonic acid elicited H+-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca2+-release channel. ---------------------------------------------------------------------------- How well can an amoeba climb? Yoshio Fukui*, Taro Q. P. Uyeda# and Chikako Kitayama#, and Shinya Inoué% *Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611-3008, USA) #Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan, %Marine Biological Laboratory, Woods Hole, MA 02543-1005, USA Proceedings of the National Academy of Sciences, USA, in press. (Supplemental Material will soon become available in PNAS Early Edition at: http://www.pnas.org). Pdf file is also available from http://pubweb.nwu.edu/~yoshifk/fukui.html. Abstract. We report here our efforts to measure the crawling force generated by cells undergoing amoeboid locomotion. In a centrifuge microscope, acceleration was increased until amoebae of Dictyostelium discoideum were "stalled" or no longer able to "climb up." The "apparent weight" of the amoebae at stalling rpm in myosin mutants depended on the presence of myosin II (but not myosins IA and IB) and paralleled the cortical strength of the cells. Surprisingly however, the cell stalled not only in low-density media as expected, but also in media with densities greater than the cell density where the buoyant force should push the amoeba upwards. We find that the leading pseudopod is bent under centrifugal force in all stalled amoebae, suggesting that this pseudopod is very dense indeed. This finding also suggests that directional cell locomotion against resistive forces requires a turgid, forward-pointing pseudopod, most likely sustained by cortical actomyosin II. ---------------------------------------------------------------------------- [End Dicty News, volume 15, number 4]