CSM News Electronic Edition Volume 2, number 19 May 28, 1994 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to CSM-News@worms.cmsbio.nwu.edu. Back issues of CSM-News, the CSM Reference database and other useful information is available by anonymous ftp from worms.cmsbio.nwu.edu [165.124.233.50], via Gopher at the same address, or by World Wide Web through www.nwu.edu. ---------- ABSTRACTS ---------- UNIVERSAL SIGNALS CONTROL SLIME MOLD STALK FORMATION Saskia van Es, Bart W. Nieuwenhuijsen, Francois Lenouvel, Esther M. van Deursen and Pauline Schaap. Cell Biology Unit, Institute of Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. Proc. Natl. Acad. Sci., in press The primitive slime mold Dictyostelium minutum does not display oscillations during aggregation, cannot form migrating slugs and does not form a prestalk/prespore pattern, which are all characteristic for development of its advanced relative D.discoideum. We used D.minutum to investigate whether slime molds share common mechanisms controling development. In D.discoideum, the morphogen DIF (Differentiation Inducing Factor) can induce stalk cell differentiation in vitro. However, stalk formation in vivo is supposedly triggered by local depletion of DIF antagonists as ammonia or cAMP. A homologue of the D.discoideum stalk gene ecmB was cloned in D.minutum, which encodes a 3.4 kb mRNA, while its deduced amino acid sequence shows repeats of 24 amino acids, that are characteristic for the D.discoideum ecmB gene. Remarkably, DIF effectively induces expression of the D.minutum ecmB gene and ammonia inhibits its expression. D.discoideum cells were transformed with a construct of the D.minutum ecmB promoter fused to the lacZ reporter gene and showed expression in the stalk, but not in the upper and lower cup of the fruiting body, which also express the D.discoideum ecmB gene. In D.discoideum, the D.minutum ecmB and the ecmB promoter are similarly activated by DIF and repressed by both cAMP and ammonia, suggesting that additional signaling is required for ecmB expression in upper and lower cup cells. Our data indicate that not only the extracellular signals controling stalk formation remained highly conserved during slime mold evolution, but also their intracellular signaling cascades including gene regulatory proteins. ---------------------------------------------------------------- EXTRACELLULAR cAMP CAN RESTORE DEVELOPMENT IN DICTYOSTELIUM CELLS LACKING ONE, BUT NOT TWO SUBTYPES OF EARLY cAMP RECEPTORS (cARs). Evidence for involvement of cAR1 in aggregative gene expression Ron D.M. Soede (1), Robert H. Insall (2), Peter N. Devreotes (2), Pauline Schaap (1) (1) Cell Biology Unit, Institute of Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL Leiden, The Netherlands. (2) Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. Development, in press SUMMARY Extracellular cAMP induces expression of several classes of developmentally regulated genes in Dictyostelium. Four highly homologous surface cAMP receptors (cARs) were identified earlier, but involvement of specific cARs in gene regulation has not been clarified. Cells lacking the chemotactic receptor, cAR1, neither aggregate nor express developmentally regulated genes. Expression of aggregative genes is in wild-type cells induced by nanomolar cAMP pulses and repressed by persistent micromolar cAMP stimuli, that induce expression of prespore and prestalk-enriched genes during the postaggregative stages of development. We show here that in cell lines carrying a cAR1 gene disruption, nanomolar pulses cannot induce aggregative gene expression. Remarkably, micromolar cAMP can induce expression of aggregative genes in car1- cells as well as expression of prespore and prestalk-enriched genes, and furthermore restores their ability to form normal slugs and fruiting bodies. These data indicate that cAR1 mediates aggregative but not postaggregative gene expression and morphogenesis, and suggest that after gene disruption, its function is partially taken over by a lower affinity receptor that is not subjected to desensitization. The absence of another early cAMP receptor, cAR3, does not affect development. However, in a car1-/car3- double mutant, cAMP stimulation cannot restore any developmental gene expression anymore, indicating that cAR3 may have substituted for cAR1 in car1- cell lines. ------------------------------------------------------------------ ASSOCIATION OF THE DICTYOSTELIUM 30,000 DALTON ACTIN BUNDLING PROTEIN WITH CONTACT REGIONS Marcus Fechheimer (1), Hilary M. Ingalls (2), Ruth Furukawa (1), and Elizabeth J. Luna (2,3) 1 Department of Zoology, University of Georgia, Athens, GA, 2 Department of Biology, Princeton University, Princeton, NJ, 3 Worcester Foundation for Experimental Biology, Shrewsbury, MA. J. of Cell Sci., in press. Summary "Contact regions" are plasma membrane domains derived from areas of intercellular contact between aggregating Dictyostelium amebae (Ingalls, et al., 1986, PNAS 83, 4779). Purified contact regions contain a prominent actin-binding protein with an Mr of 34,000. Immunoblotting with monoclonal antibodies identifies this polypeptide as a 34,000-dalton actin-bundling protein (known as 30-kDa protein), previously shown to be enriched in filopodia (Fechheimer, 1987, J. Cell Biol. 104, 1539). About four times more 30-kDa protein by mass is associated with contact regions than is found in total plasma membranes isolated from aggregating cells. In agreement with these observations, immunostaining of the 30-kDa protein in aggregating cells reveals a prominent localization along the plasma membrane at sites of intercellular contact. By contrast, alpha-actinin does not appear to be significantly enriched at sites of cell to cell contact. Binding experiments using purified plasma membranes, actin, and 30-kDa protein indicate that the 30-kDa protein associates with the plasma membrane primarily through interactions with actin filaments. Calcium ions are known to decrease the interaction of actin with 30-kDa protein in solution. Surprisingly, membrane-associated complexes of actin and the 30-kDa protein are much less sensitive to dissociation by micromolar levels of free calcium ions than are complexes in solutions lacking membranes. These results suggest that the interaction of the 30-kDa protein with F-actin at regions of cell-cell contact may be less sensitive to disruption by free calcium ions than elsewhere in the cell cortex. The positively-cooperative assembly of stable complexes of actin and the 30-kDa protein at contact regions may be an important factor in the organization of both the cortex and these membrane domains specialized for intercellular adhesion. -------------------------------------------------------------------- [End CSM News, volume 2, number 19]