CSM News Electronic Edition Volume 4, number 5 February 11, 1995 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 the url "http://worms.cmsbio.nwu.edu/dicty.html". =========== Abstracts =========== An Actin-related Protein from Dictyostelium discoideum is Developmentally Regulated and Associated with Mitochondria. Irene Murgia, Sutherland K. Maciver and Piero Morandini MRC Laboratory of Molecular Biology, Hills Road CB2 2QH, Cambridge, UK Tel: +44-1223-402203. Fax: +44-1223-213556 FEBS Letter, in press Summary An actin-related protein (ACLA) has been identified in the cellular slime mould Dictyostelium discoideum. The complete cDNA sequence indicates that within the actin superfamily, it belongs to the ARP3 family of actin-related proteins together with Arp66B from Drosophila melanogaster, Actin2 from Bos taurus, act2 from Schizosaccharomyces pombe and possibly act2 from Caenorhabditis elegans. The ACLA mRNA is regulated during development, showing a maximum between 2 and 4 hr after starvation. The protein has been expressed in E. coli and antibodies raised against it. Immunofluorescence microscopy shows that ACLA protein co-localises with mitochondria; the protein co-purifies with Dictyostelium mitochondria. --------------------------------------------------------------------- The Role of Ca2+ During Spore Germination in Dictyostelium: Autoactivation is Mediated by the Mobilization of Ca2+ While Amoebal Emergence Requires Entry of External Ca2+ Michael A. Lydan and David A. Cotter Department of Biological Sciences, University of Windsor, Windsor, Ontario N9B 3P4, CANADA, Tel: (519) 253-4232 ext. 2701 Fax: (519) 971-3609 J. Cell Sci., in press. Summary One of the developmental pathways used by the social amoeba Dictyostelium discoideum produces dormant spores. As with any temporary resistant stage, these spores must be able to rapidly germinate in response to positive environmental stimuli. One such stimulus is the autoactivator, an endogenous, diffusible molecule that is secreted by spores. Previous work has shown that three phases of germination: autoactivation, spore swelling and amoebal emergence require the activity of the Ca2+-dependent, regulatory protein calmodulin, implicating Ca2+ as an essential cation during germination. In this study we used a pharmacological approach coupled with the direct measurement of Ca2+ levels in germinating spore populations by atomic adsorption to examine Ca2+-dependent signal transduction during spore activation and germination in D. discoideum. Inhibitors of both phospholipase C and of internal Ca2+ release inhibited autoactivation while exogenously added Ins(1,4,5)P3, acted synergistically with the autoactivator. The antagonists specifically affected spore activation as mediated by the autoactivator since neither had any effect on heat activated spores. In contrast, La3+, an inhibitor of Ca2+ uptake, had little or no effect on either autoactivation or the swelling of autoactivated spores. However, an inhibition of Ca2+ influx by La3+ inhibited both the swelling of heat activated spores as well as amoebal emergence following each of autoactivation or heat activation. Ca2+ levels change in the spore population during germination. During activation and swelling, Ca2+ efflux occurs from the spores. Both of the activating stimuli used here, the autoactivator and heat, caused this Ca2+ efflux. The efflux is reversed during emergence when there is a net Ca2+ uptake by the spores and cells from the medium. Together these data provide the first evidence that autoactivation is mediated by Ca2+-dependent signal transduction, leading to Ca2+ efflux, and that the late event of germination, amoebal emergence requires Ca2+ uptake to proceed. The data also suggest that the responses of the spore to the each of autoactivator and heat, i.e. Ca2+ movements and germination, are mediated by different mechanisms. -------------------------------------------------------------------------- IN DICTYOSTELIUM DISCOIDEUM INOSITOL 1,3,4,5-TETRAKISPHOSPHATE IS DEPHOSPHORYLATED BY A 3-PHOSPHATASE AND A 1-PHOSPHATASE Peter van Dijken, Aleida A. Lammers and Peter J.M. van Haastert1 Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Biochem. J., in press. Abstract The degradation of Ins(1,3,4,5)P4 in Dictyostelium was investigated using a mixture of [3H]Ins(1,3,4,5)P4 and [3- 32P]Ins(1,3,4,5)P4. After incubation of this mixture with a Dictyostelium homogenate the 32P/3H ratio found in the InsP3 products was reduced to 24% of the ratio in the substrate. 32P labeled inorganic phosphate was found as well, whereas hardly any InsP2 was detected. This indicates that Ins(1,3,4,5)P4 is mainly degraded by a 3-phosphatase. The other enzyme was characterized by identification of the [32P]-labeled InsP3 isomer. This isomer did not co-elute with Ins(1,3,4)P3, indicating that no 5-phosphatase was present in Dictyostelium. The [32P]-labeled InsP3 could be oxidized using NaIO4. The only InsP3 isomer that has these characteristics is Ins(3,4,5)P3, indicating 1-phosphatase activity. The 1-phosphatase appeared to be dependent on MgCl2, whereas the 3-phosphatase was still active in the absence of MgCl2. An analogue of Ins(1,3,4,5)P4 with a thiophosphate substitution at the 1-position, was found to be almost completely resistant to hydrolysis by the 1- phosphatase, but was degraded by the 3-phosphatase. -------------------------------------------------------------------- GENETIC AND PHYSIOLOGIC MODULATION OF THE PRESTARVATION RESPONSE IN DICTYOSTELIUM DISCOIDEUM Virginia Burdine and Margaret Clarke Program in Molecular and Cell Biology, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104 Mol. Biol. Cell, in press. ABSTRACT Throughout vegetative growth, Dictyostelium amoebae secrete an autocrine factor, prestarvation factor or PSF, which accumulates in proportion to cell density. During late exponential growth, PSF induces the expression of several genes whose products are needed for cAMP signaling and cell aggregation. Among these genes are discoidin-I and the 2.4-kb transcript of cyclic nucleotide phosphodiesterase (PDE). We have identified several parameters that modulate expression of one or both of these prestarvation response genes; all effects were monitored in cells growing exponentially on bacteria. Under these conditions, axenic mutants produce higher levels of PSF activity than wild-type cells. Consistent with the high PSF levels, the 2.4-kb PDE transcript is more abundant in axenic strains than wild-type cells at the same cell density. In contrast, the density-dependent induction of discoidin-I is greatly delayed in axenic strains, occurring only at the very end of exponential growth. Analysis of axenic strains of independent origin suggested that this negative effect on discoidin-I expression is attributable to the axenic mutations themselves. The effects of two environmental factors that inhibit the prestarvation response (the bacteria upon which the cells feed, and a bacterial product, folic acid) were also analyzed. We found that folate does not account for the inhibitory effect of bacteria. Cells deficient in the G-protein beta subunit, which is thought to be common to all heterotrimeric G-proteins in Dictyostelium, respond to PSF in the same manner as G-beta-plus cells, and this response is inhibited by bacteria. However, folate has no inhibitory effect on G-beta-minus cells, indicating that folate inhibition is mediated by a heterotrimeric G-protein. In cells lacking the catalytic subunit of protein kinase A, the prestarvation response is severely impaired, but about 3% of the pka- cells manifest an appar ently normal density-dependent induction of discoidin-I. This behavior and the heterogeneity of the prestarvation response in wild-type cells lead us to speculate that protein kinase A may not be required for PSF signal transduction per se, but rather ma y render the cells responsive to PSF. Based on analysis of adenylyl cyclase mutants (aca-), the effect of protein kinase A is not cAMP-dependent. ------------------------------------------------------------------- [End CSM-News, volume 4, issue 5]