CSM News Electronic Edition Volume 6, number 16 June 22, 1996 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmb.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmb.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web at the URL "http://worms.cmb.nwu.edu/dicty.html" =========== Abstracts =========== Cortexillins, Major Determinants of Cell Shape and Size, are Actin-Bundling Proteins with a Parallel Coiled-coil Tail Jan Faix, Michel Steinmetz, Heike Boves, Richard A. Kammerer, Friedrich Lottspeich, Ursula Mintert, John Murphy, Alexander Stock, Ueli Aebi, and Guenther Gerisch Cell, in press. Summary Cortexillins I and II of Dictyostelium discoideum constitute a new subfamily of proteins with actin-binding sites of the alpha-actinin/spectrin type. C-terminal halves of these dimeric proteins contain a heptad repeat domain by which the two subunits are joined to form a two-stranded, parallel coiled-coil, giving rise to a 19 nm tail. The N-terminal domains that encompass a consensus actin-binding site are folded into globular heads. Cortexillin-linked actin filaments form preferentially anti-parallel bundles that associate into meshworks. Both cortexillins are enriched in the cortex of locomoting cells, primarily at the anterior and posterior ends. Elimination of the two isoforms by gene disruption gives rise to large, flattened cells with rugged boundaries, portions of them often being connected by thin cytoplasmatic bridges. The double-mutant cells are multinucleate due to a severe impairment of cytokinesis. ---------------------------------------------------------------------- The cell density factor CMF permits cAMP-mediated activation of the chemoattractant receptor cAR1 in Dictyostelium Peter J.M. Van Haastert1, John D. Bishop2, and Richard H. Gomer2 1Department of Biochemistry, University of Groningen, Nijenborgh 4, 9727 AG Groningen, the Netherlands and 2Howard Hughes Medical Institute, Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251-1892, USA Journal of Cell Biology, in press Abstract Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called CMF reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form and the loss of ligand binding which is the first step of ligand induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess about 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a ten-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF-binding; inactivation of the cAR1 gene abolishes both cAMP- and CMF-binding. In wild-type cells, down-regulation of cAR1 with high levels of cAMP also down-regulates CMF-binding, and CMF similarly downregulates cAMP- and CMF-binding. This suggests that the cAMP-binding and CMF-binding are closely linked. Binding of roughly 200 molecules of CMF to starved cells affects the affinity of the majority of the cAMP receptor cAR1 within two minutes, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cAR's. In cells lacking the G-protein b-subunit, cAMP induces a loss of cAMP-binding but not CMF-binding, while CMF induces a reduction of CMF-binding without affecting cAMP-binding, suggesting that the linkage of the cell-density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G protein. -------------------------------------------------------------------- A Ca2+-dependent early functional heterogeneity in amoebae of Dictyostelium discoideum, revealed by flow cytometry. M.Azhar1, P.S. Manogaran2, P. K. Kennady2, G. Pande2, and Vidyanand Nanjundiah1* 1Developmental Biology and Genetics Laboratory, Indian Institute of Science, Bangalore-560012, India. 2Centre for Cellular and Molecular Biology, Hyderabad 500007, India. Exp. Cell Research, in press ABSTRACT When freshly starved amoebae of Dictyostelium discoideum are loaded with the Ca2+-specific dye indo-1/AM and analysed in a fluorescence-activated cell sorter (FACS), they exhibit a quasi- bimodal distribution of fluorescence. This permits a separation of the population into two classes: H or 'high Ca2+-indo-1 fluorescence' and L or 'low Ca2+-indo-1 fluorescence'. Simultaneous monitoring of Ca2+-indo-1 and Ca2+-chlortetracycline fluorescence shows that by and large the same cells tend to have high (or low) levels of both cytoplasmic and sequestered Ca2+. Next we label H cells with tetramethylrhodamine isothioscyanate (TRITC) and mix them in a 1:4 ratio with L cells. In the slugs that result, TRITC fluorescence is confined mainly to the anterior prestalk region. This implies that amoebae with relatively high Ca2+ at the vegetative stage tend to develop into prestalk cells and those with low Ca2+, into prespores. Polysphondylium violaceum, a cellular slime mold that does not possess prestalk and prespore cells, also does not display a Ca2+-dependent heterogeneity at the vegetative stage or in slugs. Finally, confirming earlier findings with the fluorophore fura-2 (Azhar et al., Curr. Sci. 68, 337-342 (1995)), a prestalk- prespore difference in cellular Ca2+ is present in the cells of the slug in vivo. These findings are discussed in the light of the possible roles of Ca2+ for cell differentiation in D. discoideum. ----------------------------------------------------------------- [End CSM-News, volume 6, number 16]