Dicty News Electronic Edition Volume 12, number 11 May 22, 1999 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 ============= HIGH cAMP IN SPORES OF DICTYOSTELIUM DISCOIDEUM: ASSOCIATION WITH SPORE DORMANCY AND INHIBITION OF GERMINATION Kiran J. Virdy1, Todd W. Sands1, Susan H. Kopko1, Saskia van Es2, Marcel Meima2, Pauline Schaap2 and David A. Cotter1* 1Department of Biological Sciences, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada, N9B 3P4, and 2Institute of Molecular Plant Sciences, Cell Biology Section, Clusius Laboratory, Wassenaarseweg 64, 2333 AL Leiden, Netherlands. Microbiology (in press) SUMMARY Signalling mechanisms involving cAMP have a well documented role in the coordination of multicellular development and differentiation leading to spore formation in the social amoeba, Dictyostelium discoideum. We have examined cAMP's involvement in the poorly understood developmental stages of spore dormancy and germination. Dormant spores contained up to 11-fold more cAMP than nascent amoebae. These Spore cAMP levels were not constant, but typically under went a surge at 14-18 d when spores acquired the ability to germinate spontaneously. The high cAMP levels decreased only during successful spore germination, i.e., emergence of nascent amoebae. The temporal pattern of cAMP decrease was complex and unique to the method of spore activation, supporting our hypothesis that exogenously (e.g. heat) activated and autoactivated spores germinate by different mechanisms. During heat-induced activation, transcription of acg (a gene encoding adenylyl cyclase associated with germination) correlated well with spore cAMP content. Young wild-type spores, incapable of spontaneous germination, maintained a uniformly high cAMP level, and spore cAMP levels also remained high if germination was inhibited. When activated spores were deactivated by applying increased osmotic pressure, cAMP concentrations rose and ultimately levelled off at the high levels typical of dormant spores. The correlation between high cAMP and failure to germinate was evident as well when autoactivation was inhibited by the cAMP analogue, 8-bromo- cAMP. Also, spores from a strain (HTY217) with unrestrained protein kinase A activity were incapable of spontaneous germination. Overall, our experiments provide evidence for continued cAMP signalling in spores up to 18 d after sporulation, and for linkages between elevated cAMP spore deactivation and inhibition of spontaneous germination. --------------------------------------------------------------------------------- Ammonium Phosphate in Sori of Dictyostelium discoideum Promotes Spore Dormancy Through Stimulation of the Osmosensor ACG David A. Cotter1, Andrew J. Dunbar2, Stanley D. Buconjic1, and John F. Wheldrake2 'Department of Biological Sciences, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada, N9B 3P4, and 2School of Biological Sciences, The Flinders University of South, Australia, Bedford Park, GPO Box 2100, Adelaide 5001, Australia Microbiology (in press) ABSTRACT The sori of Dictyostelium discoideum (strains SG1, SG2, NC4 and V12) contained greater than 100 mM ammonium phosphate. Glutamine synthetase (GS) which could remove ammonia from the sorus, was not present in 2 day old dormant spores but enzyme activity returned to vegetative levels after spore germination. Based on mRNA blotting, the activity of this enzyme in germinating spores appeared to be transcriptionally controlled. At the same time that (GS) activity was increasing, ammonia was released from germinating spores. Exogenous ammonium ions at a concentration of 28 mM did not block germination nor modulate GS activity in nascent amoebae. It was concluded that the transcription and translation of GS is not environmentally regulated but is an integral part of the germination process preparing nascent amoebae for vegetative growth. An exogenous concentration of 69 mM ammonium phosphate could maintain dormancy in spores of strains SG1 and SG2 for at least a week in the absence of any other inhibitory component from the sori. The inhibition was reversible at any time by either dilution or by washing the spores free of the ammonium ion. Spores of strain acg- were not inhibited by 100 mM ammonium phosphate. A model is presented in which GS in prespore cells serves as a sink for ammonia to allow the osmotically sensitive adenylyl cyclase aggregation protein (ACA) to activate protein kinase A (PKA) to induce fruiting body formation. After fruiting body formation is complete, the decline in GS and ACA activities in developing spores is offset by their replacement with the osmotically and ammonia-stimulated adenylyl cyclase osmosensor for germination (ACG). Ammonia and discadenine may act as separate signals to synergistically activate PKA by stimulating ACG activity while inhibiting cyclic AMP phosphodiestrase activity in fully dormant spores. ----------------------------------------------------------------------------------- Starvation promotes Dictyostelium development by relieving PufA inhibition of PKA translation through the YakA kinase pathway. Glaucia Mendes Souza Aline Maria da Silva and Adam Kuspa Dept. Bioquímica, Instituto de Química, Universidade de São Paulo, Brazil 05508-900, Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine, Houston, Texas, USA, 77030 Development, in press SUMMARY When nutrients are depleted, Dictyostelium cells undergo cell cycle arrest and initiate a developmental program that ensures survival. The YakA protein kinase governs this transition by regulating the cell cycle, repressing growth- phase genes and inducing developmental genes. YakA mutants have a shortened cell cycle and do not initiate development. A suppressor of yakA that reverses most of the developmental defects of yakA-cells, but none of their growth defects was identified. The inactivated gene, pufA, encodes a member of the Puf protein family of translational regulators. Upon starvation, pufA-cells develop precociously and overexpress developmentally important proteins, including the catalytic subunit of cAMP-dependent protein kinase, PKA-C. Gel mobility-shift assays using a 200-base segment of PKA-C’s mRNA as a probe reveals a complex with wild-type cell extracts, but not with pufA-cell extracts, suggesting the presence of a potential PufA recognition element in the PKA-C mRNA. PKA-C protein levels are low at the times of development when this complex is detectable, whereas when the complex is undetectable PKA-C levels are high. There is also an inverse relationship between PufA and PKA-C protein levels at all times of development in every mutant tested. Furthermore, expression of the putative PufA recognition elements in wild-type cells causes precocious aggregation and PKA-C overexpression, phenocopying a pufA mutation. Finally, YakA function is required for the decline of PufA protein and mRNA levels in the first 4 hours of development. We propose that PufA is a translational regulator that directly controls PKA-C synthesis and that YakA regulates the initiation of development by inhibiting the expression of PufA. Our work also suggests that Puf protein translational regulation evolved prior to the radiation of metazoan species. --------------------------------------------------------------------------------------- Role of myosin II tail sequences in its function and localization at the cleavage furrow in Dictyostelium Shu Shi1, Randall J. Lee2, Janine M. LeBlanc-Straceski3 and Taro Q. P. Uyeda1 1Biomolecular Research Group, National Institute for Advanced Interdisciplinary Research, Tsukuba, Ibaraki 305-8562, Japan; 2Department of Medicine and Cardiovascular Research Institute, University of California at San Francisco, CA 94143-4354, USA; 3Department of Biology, Merrimack College, North Andover, MA 01845, USA J. Cell Sci. (in press) SUMMARY Cytoplasmic myosin II accumulates in the cleavage furrow and provides the force for cytokinesis in animal and amoeboid cells. One model proposes that a specific domain in the myosin II tail is responsible for its localization, possibly by interacting with a factor concentrated in the equatorial region. To test this possibility, we have expressed myosins carrying mutations in the tail domain in a strain of Dictyostelium cells from which the endogenous myosin heavy chain gene has been deleted. The mutations used in this study include four internal tail deletions: My824-941, My943-1464, My943-1194 and My1156-1464. Contrary to the prediction of the hypothesis, immunofluorescence staining demonstrated that all mutant myosins were able to move toward the furrow region. Chimeric myosins, which consisted of a Dictyostelium myosin head and chicken skeletal myosin tail, also efficiently localized to the cleavage furrow. All these deletion and chimeric mutant myosins, except for My943-1464, the largest deletion mutant, were able to support cytokinesis in suspension. Our data suggests that there is no single specific domain in the tail of Dictyostelium myosin II that is required for its functioning at and localization to the cleavage furrow. -------------------------------------------------------------------------------------------- Scanning the available Dictyostelium discoideum proteome for O-linked GlcNAc glycosylation sites using neural networks. Ramneek Gupta (1), Eva Jung (2), Andrew A Gooley (2), Keith L Williams (2), Soren Brunak (1) and Jan E Hansen (1). 1 Center for Biological Sequence Analysis, Dept of Biotechnology Technical University of Denmark, Bldg 208, DK-2800 Lyngby, Denmark; 2 School of Biological Sciences, Macquerie University, Sydney, 2109 NSW, Australia. Glycobiology, in press. Abstract: Dictyostelium discoideum has been suggested as a eukaryotic model organism for glycobiology studies. Presently, the characteristics of acceptor sites for the N- acetylglucosaminyl-transferases in Dictyostelium discoideum, which link GlcNAc in an alpha linkage to hydroxyl residues, are largely unknown. This motivates the development of a species specific method for prediction of O-linked GlcNAc glycosylation sites in secreted and membrane proteins of D. discoideum. The method presented here employs a jury of artificial neural networks. These networks were trained to recognise the sequence context and protein surface accessibility in 39 experimentally determined O-(alpha)-GlcNAc sites found in D. discoideum glycoproteins expressed in vivo. Cross- validation of the data revealed a correlation in which 97% of the glycosylated and non-glycosylated sites were correctly identified. Based on the currently limited data set, an abundant periodicity of two (positions -3, -1, +1, +3, etc.) in Proline residues alternating with hydroxyl amino acids was observed upstream and downstream of the acceptor site. This was a consequence of the spacing of the glycosylated residues themselves which were peculiarly found to be situated only at even positions with respect to each other, indicating that these may be located within beta-strands. The method has been used for a rapid and ranked scan of the fraction of the Dictyostelium proteome available in public databases, remarkably 25-30% of which were predicted glycosylated. The scan revealed acceptor sites in several proteins known experimentally to be O-glycosylated at unmapped sites. The available proteome was classified into functional and cellular compartments to study any preferential patterns of glycosylation. A sequence based prediction server for GlcNAc O-glycosylations in D. discoideum proteins has been made available through the WWW at http://www.cbs.dtu.dk/services/DictyOGlyc/ and via E-mail to DictyOGlyc@cbs.dtu.dk. ---------------------------------------------------------------------------------------------- Myosin I Contributes to the Generation of Resting Cortical Tension. Jianwu Dai*#, H. Ping Ting-BeallÝ, Robert M. HochmuthÝ, Micheal P. Sheetz*, and Margaret A. Titus*@ *Department of Cell Biology, Duke University Medical Center Durham, NC 27710; ÝDepartment of Mechanical Engineering and Materials Sciences, Duke University, Durham, NC 27708 @ author for all correspondence at present address: Department of Genetics, Cell Biology, and Development, University of Minnesota, 4-102 Owre Hall 321 Church St. S.E., Minneapolis, MN 55455 BIOPHYSICAL JOURNAL, in press ABSTRACT The amoeboid myosin Is are required for cellular cortical functions such as pseudopod formation and macropinocytosis, as demonstrated by the finding that Dictyostelium cells lacking one or more of these actin-based motors are defective in these processes. Alterations in these processes are concomitant with changes in the actin-filled cortex of various Dictyostelium myosin I mutants. Given that the amoeboid myosin Is possess both actin and membrane binding domains, the mutant phenotypes could be due to alterations in the generation and/or regulation of cell cortical tension. This has been directly tested by analyzing mutant Dictyostelium that either lack or overexpress various myosin Is using micropipette aspiration techniques. Dictyostelium cells lacking only one myosin I have normal levels of cortical tension. However, myosin I double mutants have significantly reduced (50%) cortical tension, and those that mildly overexpress an amoeboid myosin I exhibit increased cortical tension. Treatment of either type of mutant with the lectin concanavalin A (ConA) that cross-links surface receptors, results in significant increases in cortical tension, suggesting that the contractile activity of these myosin Is is not controlled by this stimulus. These results demonstrate that myosin Is work co-operatively to contribute substantially to the generation of resting cortical tension that is required for efficient cell migration and macropinocytosis. ------------------------------------------------------------------------------------- Evidence that the Dictyostelium Dd-STATa protein is a repressor that regulates commitment to stalk cell differentiation and is also required for efficient chemotaxis Sudhasri Mohanty*, Keith A. Jermyn*, Anne Early, Takefumi Kawata, Laurence Aubry, Adriano Ceccarelli, Pauline Schaap, Jeffrey G. Williams and Richard A. Firtel DEVELOPMENT, in press SUMMARY Dd-STATa is a structural and functional homologue of the metazoan STAT (Signal Transducer and Activator of Transcription) proteins. We show that Dd-STATa null cells exhibit several distinct developmental phenotypes. The aggregation of Dd-STATa null cells is delayed and they chemotax slowly to a cAMP source, suggesting a role for Dd-STATa in these early processes. In Dd-STATa null strains, slug-like structures are formed but they have an aberrant pattern of gene expression. In such slugs, ecmB/lacZ, a marker that is normally specific for cells on the stalk cell differentiation pathway, is expressed throughout the prestalk region. Stalk cell differentiation in Dictyostelium has been proposed to be under negative control, mediated by repressor elements present in the promoters of stalk cell-specific genes. Dd-STATa binds these repressor elements in vitro and the ectopic expression of ecmB/lacZ in the null strain provides in vivo evidence that Dd-STATa is the repressor protein that regulates commitment to stalk cell differentiation. Dd-STATa null cells display aberrant behavior in a monolayer assay wherein stalk cell differentiation is induced using the stalk cell morphogen DIF. The ecmB gene, a general marker for stalk cell differentiation, is greatly over-induced by DIF in Dd-STATa null cells. Also, Dd-STATa null cells are hyper-sensitive to DIF for expression of ST/lacZ, a marker for the earliest stages in the differentiation of one of the stalk cell sub-types. We suggest that both these manifestations of DIF hyper- sensitivity in the null strain result from the balance between activation and repression of the promoter elements being tipped in favor of activation when the repressor is absent. Paradoxically, although Dd-STATa null cells are hypersensitive to the inducing effects of DIF and readily form stalk cells in monolayer assay, the Dd-STATa null cells show little or no terminal stalk cell differentiation within the slug. Dd-STATa null slugs remain developmentally arrested for several days before forming very small spore masses supported by a column of apparently undifferentiated cells. Thus, complete stalk cell differentiation appears to require at least two events: a commitment step whereby the repression exerted by Dd-STATa is lifted and a second step that is blocked in a Dd-STATa null organism. This latter step may involve extracellular cAMP, a known repressor of stalk cell differentiation, because Dd-STATa null cells are abnormally sensitive to the inhibitory effects of extracellular cAMP. --------------------------------------------------------------------------------------------- FINGERPRINTING OF ADENYLYL CYCLASE ACTIVITIES DURING DICTYOSTELIUM DEVELOPMENT INDICATES A DOMINANT ROLE FOR ADENYLYL CYCLASE B IN TERMINAL DIFFERENTIATION Marcel E. Meima and Pauline Schaap Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden Developmental Biology, in press ABSTRACT Activation of cAMP dependent protein kinase (PKA) triggers terminal differentiation in Dictyostelium, without an obvious requirement for the G-protein coupled adenylyl cyclase, ACA, or the osmosensory adenylyl cyclase ACG. A third adenylyl cyclase, ACB, was recently detected in rapidly developing mutants. The specific characteristics of ACA, ACG and ACB were used to determine their respective activities during development of wild-type cells. ACA was highly active during aggregation, with negligible activity in the slug stage. ACG activity was not present at significant levels until mature spores had formed. ACB activity increased strongly after slugs had formed with optimal activity at early fruiting body formation. The same high activity was observed in slugs of ACG null mutants and ACA null mutants that overexpress PKA (acaA/PKA), indicating that it was not due to either ACA or ACG. The detection of high adenylyl cyclase activity in acaA/PKA null mutants contradicts earlier conclusions (Wang, B. and Kuspa, A. 1997. Science 277, 251-254) that these mutants can develop into fruiting bodies in the complete absence of cAMP. In contrast to slugs of null mutants for the intracellular cAMP-phosphodiesterase REGA, where both intact cells and lysates show ACB activity, wild-type slugs only show activity in lysates. This indicates that cAMP accumulation by ACB in living cells is controlled by REGA. REGA inhibition and PKA overexpression both cause precocious terminal differentiation. The developmental regulation of ACB and its relationship to REGA suggest that ACB activates PKA and induces terminal differentiation. ------------------------------------------------------------------------------------------------- [End Dicty News, volume 12, number 11]