CSM News Electronic Edition Volume 7, number 13 November 9, 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" ========== Abstract ========== Stimulation by DIF causes an increase of intracellular Ca++ in Dictyostelium discoideum M. Azhar 1, P.K. Kennady 2, G. Pande 2 and Vidyanand Nanjundiah 1* 1Developmental Biology and Genetics Laboratory, Indian Institute of Science, Bangalore-560012, India. 2Centre for Cellular and Molecular Biology, Hyderabad 5000 07, India. Exp. Cell Res., in press. ABSTRACT Using fluorescence-activated cell sorting (FACS), we have studied the effect of the differentiation-inducing factor DIF on cellular Ca2+ in Dictyostelium discoideum. We have shown previously that freshly starved or post-aggregation amoebae are heterogenous with respect to the amounts of cellular Ca2+ that they contain; the L or 'low Ca2+' class exhibits a prespore tendency and the H or 'high Ca2+' class exhibits a prestalk tendency [Azhar et al., Exp Cell Res. 227, 344-351, 1996] Upon adding DIF there is an approximately two-fold increase in the relative fraction of amoebae falling in the H class within two minutes. A major part of the increase is caused by Ca2+ influx from the extracellular medium. Therefore a rise in the level of cellular Ca2+ is an early step in the signal transduction pathway following stimulation by DIF. Also, in parallel with cellular heterogeneity in respect of Ca2+ content, there is also a heterogeneity in the response to DIF. The main result of this study is that the two classes persist after stimulation with 10nM DIF-1 but the proportions of cells belonging to them change significantly. With t1.5 amoebae, the H ('high Ca2+') class increases in relative size from an average of 18.47% to 34.36% within 2 minutes. In the case of t9 amoebae, about 12.44% fall in the H class and DIF-1 application raises this to 23.07% (on average) after 2 minutes. The nearly two-fold elevation in the H:L ratio caused by DIF-1 remains stable until 15 minutes in all experiments (long-term monitoring was not attempted). Functional heterogeneity, indicating a future prestalk or prespore tendency, is a pervasive feature of the life cycle of D. discoideum. The response to DIF-1 likewise mirrors a qualitative pre-aggregative difference between the two classes of amoebae. ------------------------------------------------------------------- Fluid-phase uptake by macropinocytosis in Dictyostelium Ulrike Hacker, Richard Albrecht and Markus Maniak Max-Planck-Institut fŸr Biochemie, D-82152 Martinsried J. Cell Science, in press. Abstract To study fluid-phase endocytosis in living cells and its relationship to changes in the cell cortex, we have used a green-fluorescent protein (GFP)-tagged version of coronin, an actin-associated protein that localises to dynamic regions of the Dictyostelium cell cortex. In the confocal microscope, internalisation of fluorescently labelled dextran as a fluid-phase marker can be recorded simultaneously with the recruitment of the coronin-GFP fusion-protein from the cytoplasm of the phagocyte. At crown-shaped surface protrusions, extracellular medium is taken up into vesicles with an average diameter of 1.6 um, which is significantly larger than the 0.1 um diameter of clathrin-coated pinosomes. The observed frequency of macropinosome formation can account for a large portion, if not all, of the fluid-phase uptake. The redistribution of coronin-GFP strongly resembles cytoskeletal rearrangements during phagocytosis. Scanning-electron micrographs indicate that crown-shaped cell-surface extensions can undergo shape changes, without a particle bound, that are similar to shape changes that occur during phagocytosis. In quantitative assays, the uptake of particles and fluid are about equally dependent on F-actin and coronin. --------------------------------------------------------------------- Isoforms of gp138, a cell-fusion related protein in Dictyostelium discoideum Kazuhiro Aiba*, Hui Fang*, Nobuyuki Yamaguchi*, Yoshimasa Tanaka,* and Hideko Urushihara*,3 *Institute of biological sciences, University of Tsukuba, Tsukuba, Ibaraki 305 JAPAN J. Biochem., in press. SUMMARY Sexual development of Dictyostelium discoideum is a unique and usefull system for the study of sexual phenomena. We have been studying molecular mechanisms of sexual cell fusion in D. discoideum and have identified several cell-surface proteins involved in the sexual cell fusion. One of the proteins, gp138, was identified as a target molecule for fusion-blocking antibodies, and two genes for gp138, GP138A and GP138B, were cloned. The participation of gp138 in the sexual cell fusion was confirmed by antisense RNA mutagenesis, but it is unclear which of the genes encode gp138. Moreover, the presence of a third gene for gp138 was indicated by gene disruption. In the present study, we generated strains of D. discoideum overexpressing either GP138A or GP138B to investigate the products of these genes. The transformants overexpressing GP138A and GP138B overproduced glycoproteins with the molecular mass of 135 kDa and 130 kDa, respectively. Although their molecular masses were different from that of gp138, the results of peptide mapping and amino acid sequencing showed that they were related proteins, suggesting that proteins encoded by GP138A and GP138B are isoforms of gp138 protein. --------------------------------------------------------------------- Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development Sharon A. Louis, George B. Spiegelman and Gerald Weeks Department of Microbiology and Immunology and Department of Medical Genetics, University of British Columbia, Vancouver, B. C. V6T 1Z3, Canada Mol. Biol. Cell, in press. SUMMARY It has been previously demonstrated that the expression of an activated rasD gene in wild type Dictyostelium cells results in formation of aggregates with multi-tips instead of the normal single tips, and a block in further development (Reymond et al., 1986, Nature, 323: 340-343). In an attempt to better understand the role of activated RasD development, we examined cell type specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell specific genes ecmA and tagB was markedly enhanced while the expression of the prespore cell specific gene cotC was reduced to very low levels. When the fate of cells in the multi-tipped aggregate was monitored using an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed. --------------------------------------------------------------------- [End CSM News, volume 7, number 13]