Dicty News Electronic Edition Volume 18, number 1 January 12, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= A Novel Dictyostelium Gene Encoding Multiple Repeats of Adhesion Inhibitor- Like Domains has Effects on Cell-Cell and Cell-Substrate Adhesion. Timothy R. Varney, Elisabeth Casademunt, Hoa N. Ho, Chere' Petty, Jayne Dolman, and Daphne D. Blumberg* Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Developmental Biology in press Abstract The Dictyostelium protein AmpA (Adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants AmpA is found in association with Anterior-Like Cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in non-nutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells however expression of additional copies of the gene in wild type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitate cell migration during morphogenesis. ----------------------------------------------------------------------------- A Gene Encoding a Novel Anti-Adhesive Protein is Expressed in Growing Cells and Restricted to Anterior-Like Cells During Development of Dictyostelium. Elisabeth Casademunt, Timothy R. Varney, Jayne Dolman, Chere' Petty and Daphne D. Blumberg* Department of Biological Sciences, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Differentiation, in press Abstract The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell- substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicate that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk. ----------------------------------------------------------------------------- Deducing the Origin of Soluble Adenylyl Cyclase, a Gene Lost in Multiple Lineages Jeroen Roelofs and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands Molecular Biology and Evolution, in press The family of eukaryotic adenylyl cyclases consists of a very large group of twelve transmembrane adenylyl cyclases and a very small group of soluble adenylyl cyclase (sAC). Orthologs of human sAC are present in rat, Dictyostelium and bacteria, but absent from the completely sequenced genomes of Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and Saccharomyces cereviciae. sAC consists of two cyclase domains and a long ~1000 amino acid C-terminal (sCKH) region. This sCKH region and one cyclase domain have been found in only four bacterial genes; the sCKH region was also detected in bacterial Lux transcription factors and in complex bacterial and fungal kinases. The phylogenies of the kinase and cyclase domains are identical to the phylogeny of the corresponding sCKH domain, suggesting that the sCKH region fused with the other domains early during evolution in bacteria. The amino acid sequences of sAC proteins yield divergence times from the human lineage for rat and Dictyostelium that are close to the reported divergence times of many other proteins in these species. The combined results suggest that the sCKH region was fused with one cyclase domain in bacteria, and a second cyclase domain was added in bacteria or early eukaryotes. The sAC was retained in a few bacteria and during the entire evolution of the human lineage, but lost independently from many bacteria and in the lineages to plants, yeast, worms and flies. We conclude that within the family of adenylyl cyclases, soluble AC was poorly fixed during evolution while membrane bound AC has expanded to form the subgroups of prevailing adenylyl and guanylyl cyclases. ----------------------------------------------------------------------------- Characterization of Two Unusual Guanylyl cyclases from Dictyostelium Jeroen Roelofs and Peter J.M. Van Haastert GBB, Department of Biochemistry, University of Groningen J. Biol. Chem., in press GCA and sGC encode guanylyl cyclases (GC) in Dictyostelium, and have a topology similar to twelve- transmembrane and soluble adenylyl cyclase, respectively. We demonstrate that all detectable GC activity is lost in a cell line in which both genes have been inactivated. Cell lines with one gene inactivated were used to characterize the other guanylyl cyclase (i.e. GCA in sgc- null cells, and sGC in gca- null cells). Despite the different topologies, the enzymes have many properties in common. In vivo, extracellular cAMP activates both enzymes via a G-protein coupled receptor. In vitro, both enzymes are activated by GTPgS (Ka = 11 and 8 mM for GCA and sGC, respectively); addition of GTPgS leads to a 1.5-fold increase of Vmax and a 3.5-fold increase of the affinity for GTP. Ca2+ inhibits both GCA and sGC with Ki of about 50 and 200 nM, respectively. Other biochemical properties are very different; GCA is mainly expressed during growth and multicellular development, while sGC is mainly expressed during cell aggregation. Folic acid and cAMP activate GCA maximally about 2.5-fold, whereas sGC is activated about 8-fold. Osmotic stress strongly stimulates sGC, but has no effect on GCA activity. Finally, GCA is exclusively membrane bound and mainly active with Mg2+, while sGC is predominantly soluble and more active with Mn2+. ----------------------------------------------------------------------------- A Transcriptional Profile of Multicellular Development in Dictyostelium discoideum Nancy Van Driessche 1,2,10, Chad Shaw 1,10, Mariko Katoh 6,10, Takahiro Morio 6, Richard Sucgang 3, Miroslava Ibarra 1, Hidekazu Kuwayama 6, Tamao Saito 7, Hideko Urushihara 6, Mineko Maeda 8, Ikuo Takeuchi 9, Hiroshi Ochiai 7, William Eaton 5, Jeffrey Tollett 1,4, John Halter 5, Adam Kuspa 1,2,3, Yoshimasa Tanaka 6, and Gad Shaulsky 1,2,11. 1 Department of Molecular and Human Genetics, 2 Graduate Program in Developmental Biology, 3 Department of Biochemistry and Molecular Biology, 4 DNA Array Core Facility, 5 Department of PM&R and Division of Neuroscience, Baylor College of Medicine, Houston TX 77030, USA; 6 Institute of Biological Sciences, University of Tsukuba, Tsukuba, 7 Division of Biological Sciences, Hokkaido University, Sapporo, 8 Department of Biology, Osaka University, Osaka, 9 Novartis Foundation for the Promotion of Science, Takarazuka, Japan. 10 These authors contributed equally to this work Development, in press. SUMMARY A distinct feature of development in the simple eukaryote Dictyostelium discoideum is an aggregative transition from a unicellular to a multicellular phase. Using genome-wide transcriptional analysis we show that this transition is accompanied by a dramatic change in the expression of more than 25% of the genes in the genome. We also show that the transcription patterns of these genes are not sensitive to the strain or the nutritional history, indicating that Dictyostelium development is a robust physiological process that is accompanied by stereotypical transcriptional events. Analysis of the two differentiated cell types, spores and stalk cells, and their precursors revealed a large number of differentially expressed genes as well as unexpected patterns of gene expression that shed new light on the timing and possible mechanisms of cell-type divergence. Our findings provide new perspectives on the complexity of the developmental program and the fraction of the genome that is regulated during development. ----------------------------------------------------------------------------- Cytoplasmic Dynein-Associated Structures Move Bidirectionally in vivo Shuo Ma and Rex L. Chisholm Department of Cell and Molecular Biology, Robert H. Lurie Comprehensive Cancer Center, and Center for Genetic Medicine, Northwestern University Medical School, Chicago, Illinois 60611 J. Cell Sci., in press. Summary Intracellular organelle transport is driven by motors that act upon microtubules or microfilaments. Microtubule-based motors, cytoplasmic dynein and kinesins, are believed to be responsible for retrograde and anterograde transport of intracellular cargo along microtubules. Many vesicles display bidirectional movement, however, the mechanism regulating directionality is unresolved. Directional movement might be accomplished by alternate binding of different motility factors to the cargo. Alternatively, different motors could associate with the same cargo and have their motor activity regulated. While several studies have focused on the behavior of specific types of cargos, little is known about the traffic of the motors themselves and how it correlates with cargo movement. To address this question, we studied cytoplasmic dynein dynamics in living Dictyostelium cells expressing dynein intermediate chain-green fluorescent protein (IC-GFP) fusion in an IC-null background. Dynein-associated structures display fast linear movement along microtubules in both minus-end and plus-end directions, with the velocities similar to that of dynein and kinesin-like motors. In addition, dynein puncta often rapidly reverse direction. Dynein stably associates with cargo moving in both directions as well as with those that rapidly reverse their direction of movement, suggesting that directional movement is not regulated by altering motor- cargo association, but rather by switching activity of motors associated with the cargo. These observations suggest that both plus- and minus-end directed motors associate with a given cargo and that coordinated regulation of motor activities controls vesicle directionality. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 1]