Dicty News Electronic Edition Volume 14, number 1 January 8, 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/dicty.html" ============= Abstracts ============= Dissection of functional domains by expression of point mutated profilins in Dictyostelium mutants Soo Sim Lee 1*, Iakowos Karakesisoglou 1§*, Angelika A. Noegel 2, Daniela Rieger 1 and Michael Schleicher 1 1 Adolf-Butenandt-Institut f. Zellbiologie, Ludwig-Maximilians-Universität, Schillerstr. 42, 80336 München, FRG 2 Biochemie I, Med. Fak., Universitaet zu Köln, J. Stelzmann-Str. 52, 50931 Köln, FRG 3 corresponding author * contributed equally to this work § Current address: Howard Hughes Medical Institute, Department of Molecular Genetics, Cell Biology, Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637, USA accepted: Eur. J. Cell Biol. Profilin is an ubiquitous cytoskeletal protein whose function is fundamental to the maintenance of normal cell physiology. By site directed mutagenesis of profilin II from Dictyostelium discoideum the point mutations K114E and W3N were generated by PCR thus changing actin and poly-(L)-proline binding activity respectively. W3N profilin is no longer able to bind to poly-(L)-proline concomitant with a slight reduction in actin binding. The K114E profilin exhibited profound decrease in its ability to interact with actin, whereas binding to poly-(L)-proline was essentially unchanged. Binding to phospholipids was indistinguishable from the wild-type profilin. The in vivo properties of the point mutated profilins were studied by expressing either W3N or K114E in profilin-minus D. discoideum mutants which have defects in the F-actin content, cytokinesis and development (Haugwitz et al., Cell 79:303-314, 1994). Expression of K114E or W3N displayed a reduction in the F-actin content, normal cell morphology, and the transformants were capable of undergoing complete development. Interestingly, only cells that drastically overexpressed W3N could restore the aberrant phenotype, whereas the mutant protein K114E with its fully functional poly-(L)-proline binding and its strongly reduced actin-binding activities rescued the phenotype at low concentrations. Wild-type and both mutated profilins are enriched in phagocytic cups during uptake of yeast particles. These data suggest a) that a functional poly-(L)-proline binding activity is more important for suppression of the mutant phenotype than the G-actin binding activity of profilin, and b) that the enrichment of profilin in highly active phagocytic cups might be independent of either poly-(L)-proline or actin-binding activities. ---------------------------------------------------------------------------- amiB, a novel gene required for the growth/differentiation transition in Dictyostelium Takahide Kon, Hiroyuki Adachi, and Kazuo Sutoh Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan. Genes to Cells in press (Vol. 5, No. 1) Abstract Background: The differentiation programme of Dictyostelium discoideum is initiated by starvation. Nutrient depletion triggers the differentiation of Dictyostelium cells through the transcriptional inactivation of some growth-phase genes as well as through the transcriptional activation of essential genes required for the aggregation of the cells. The adenylyl cyclase (ACA) gene, acaA, is one of the earliest genes expressed following starvation. ACA produces intracellular and extracellular cAMP that drives further differentiation by inducing chemotaxis, developmental gene expression, and morphogenesis of Dictyostelium cells. Although several genes have been identified as being essential for the initiation of differentiation process, such as the transcriptional activation of ACA expression, the molecular mechanisms of the growth/differentiation transition are still remain to be explored. Results: Using insertional mutagenesis, we have isolated a mutant that does not aggregate upon starvation. The disrupted gene, amiB (aggregation minus B), is predicted to encode a novel protein of 298.9 kDa. When starved, amiB- cells produced an undetectable level of cAMP. Analyses of gene expression showed that amiB- cells fail to turn off the expression of one of the growth-phase genes, cprD, and to turn on the expression of ACA following starvation. The ectopic expression of ACA from a constitutive promoter rescued the differentiation and morphogenesis of amiB- mutants. Furthermore, the ectopic expression of a putative transcriptional factor DdMyb2 or a catalytic subunit of cAMP-dependent protein kinase (PKA-C), both of which are suggested to be involved in the ACA expression pathway(s), also rescued the starvation-induced ACA expression and further differentiation of amiB- mutant. Conclusion: These results suggest that AmiB play a role at the start of Dictyostelium differentiation through the induction of ACA expression essential for the cAMP signaling. ---------------------------------------------------------------------------- Evidence that a cell-type-specific efflux pump regulates cell differentiation in Dictyostelium. J. Randall Good and Adam Kuspa Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas, USA, 77030, USA Dev. Biol., in press. Summary We have identified a cellular efflux pump, RhT, with the properties of an MDR transporter- a type of ATP-binding cassette (ABC) transporter whose substrates include small hydrophobic molecules. RhT transports rhodamine 123 (Rh123), and is inhibited by low temperature, energy poisons, and several MDR transport inhibitors, such as verapamil. All vegetative cells have RhT activity, but during development prestalk cells lose RhT activity while prespore cells retain it. We also identified several RhT inhibitors. The most effective inhibitor is the stalk cell-inducing chlorinated alkyl phenone, DIF-1. The RhT inhibitors disrupted development, to varying degrees, and induced stalk cell formation in submerged culture. The inhibitors displayed the same rank order of pharmacological efficacy for stalk cell induction as they did for Rh123 transport inhibition. We also found that cerulenin, a specific inhibitor of DIF-1 biosynthesis [Kay (1998). J. Biol. Chem. 273(5):2669-2675], abolished the induction of stalk cells by each of the RhT inhibitors, and this effect could be reversed by DIF-1. Thus, DIF-1 synthesis appears to be required for the induction of stalk cells by the RhT inhibitors. Since DIF-1 is the most potent inhibitor of RhT activity, and thus a likely transport substrate itself, we propose that RhT inhibitors induce stalk cell differentiation by blocking DIF-1 export, causing DIF-1 to build-up within cells. Our results provide evidence for a prespore-specific efflux pump that regulates cell fate determination, perhaps by regulating the cellular concentration of DIF-1. ---------------------------------------------------------------------------- "Localization of the G-protein beta-gamma Complex in Living Cells during Chemotaxis" Tian Jin, Ning Zhang, Yu Long, Carole A. Parent, Peter N. Devreotes* Science, in press Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, U.S.A. Abstract Gradients of chemoattractants elicit signaling events at the leading edge of a cell even though chemoattractant receptors are uniformly distributed on the cell surface. In highly polarized Dictyostelium discoideum amoebae, membrane-associated beta-gamma subunits of heterotrimeric guanine nucleotide binding proteins (G-proteins) were localized in a shallow anterior-posterior gradient. A uniformly applied chemoattractant generated binding sites for pleckstrin homology (PH) domains on the inner surface of the membrane in a pattern similar to that of the Gbg-subunits. Loss of cell polarity resulted in uniform distribution of both the Gbg-subunits and the sensitivity of PH-domain recruitment. These observations indicate that Gbg-subunits are not sufficiently localized to restrict signaling events to the leading edge but their distribution may determine the relative chemotactic sensitivity of polarized cells. ---------------------------------------------------------------------------- A diffusible factor involved in MAP-kinase ERK2-regulated development of Dictyostelium Mineko Maeda* and Hidekazu Kuwayama Department of Biology, Graduate School of Science, Osaka University 1-16 Machikaneyama-cho, Toyonaka, Osaka 560-0043, JAPAN Dev. Growth Differ. in press ABSTRACT MAP kinase ERK2 is essential for regulation of the intracellular cAMP level in Dictyostelium. The mutant lacking ERK2, erk2-null, is arrested at the pre-aggregation stage, but develops into a fruiting body in a mixed population of wild-type and mutant cells. This fact implies that wild-type cells provide a certain factor that is missing in erk2-null. We clarified that both wild-type strains KAx3 and Ax2 secrete a diffusible factor that enables erk2-null to develop. The fruiting body formed from erk2-null cells was smaller than that formed by the wild-type cells and consisted of a small sorus supported by a slender stalk with a single row of vacuolated stalk cells. The resulting spores were able to germinate and multiply on a bacterial lawn, but they were unable to develop unless the factor was provided. After 8 h of starvation, wild-type cells started to secrete the factor, which had a molecular mass of less than 3-kD and was heat stable. The effect of this factor could not be mimicked by either cAMP or folate. Adenylyl cyclase A and cell surface cAMP receptors cAR1 and cAR3 were all indispensable components for the factor to function. Considering the molecular mass and the mode of action, this factor could be a novel one. Possible targets of this factor are discussed in terms of PKA activation. ---------------------------------------------------------------------------- [End Dicty News, volume 14, number 1]