Dicty News Electronic Edition Volume 16, number 11 May 26, 2001 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 ============== Ammonia Differentially Suppresses the cAMP Chemotaxis of Anterior-Like Cells and Prestalk Cells in Dictyostelium discoideum Ira N. Feit, Erika J. Medynski, and Michael J. Rothrock Department of Biology, Franklin and Marshall College, Lancaster, PA 17603 Journal of Biosciences, In Press A drop assay for chemotaxis to cAMP confirms that both anterior-like cells (ALC) and prestalk cells (pst cells) respond to cAMP gradients. We present evidence that the chemotactic response of both ALC and pst cells is suppressed by ammonia, but a higher concentration of ammonia is required to suppress the response in pst cells. We also confirm that ALC show a chemotactic response to cAMP when moving on a substratum of prespore cells in isolated slug posteriors incubated under oxygen. ALC chemotaxis on a prespore cell substratum is suppressed by the same concentration of ammonia that suppresses ALC chemotaxis on the agar substratum of the drop assays. Chemotaxis suppression is mediated by the unprotonated (NH3) species of ammonia. The observed suppression, by ammonia, of ALC chemotaxis to cAMP supports our earlier hypothesis that ammonia is the tip-produced suppressor of such chemotaxis. We discuss implications of ammonia sensitivity of pst cells and ALC with regard to the movement and localization of ALC and pst cells in the slug and to the roles played by ALC in fruiting body formation. In addition, we suggest that a progressive decrease in sensitivity to ammonia is an important part of the maturation of ALC into pst cells. ----------------------------------------------------------------------------- Sequential Activities of PI 3-Kinase, PKB/Akt and Rab7 during Macropinosome Formation in Dictyostelium Adam Rupper1, Kyung Lee3, David Knecht3 and James Cardelli1,2 1Department of Microbiology and Immunology 2Feist/Weiller Cancer Center LSU Health Sciences Center Shreveport, LA 71130 318-675-5756 jcarde@lsuhsc.edu 3Department of Molecular and Cellular Biology University of Connecticut Storrs, CT 06269-3125 Molecular Biology of the Cell, in press Abstract Macropinocytosis plays an important role in the internalization of antigens by dendritic cells, and is the route of entry for many bacterial pathogens; however, little is known about the molecular mechanisms that regulate the formation or maturation of macropinosomes. Like dendritic cells, Dictyostelium amoebae are active in macropinocytosis, and a variety of proteins have been identified that contribute to this process. As described here, microscopic analysis of null mutants have revealed that the Class I PI 3-kinases, PIK1 and PIK2, and the downstream effector PKB/AKT are important in regulating completion of macropinocytosis. Although actin-rich membrane protrusions form in these cell-lines, they recede without forming macropinosomes. Imaging of cells expressing GFP fused to the pleckstrin homology domain (PH) of PKB indicates that D3 phosphoinositides are enriched in the forming macropinocytic cup, and remain associated with newly formed macropinosomes for less than one minute. A fusion protein, consisting of GFP fused to an F-actin binding domain, overlaps with GFP-PHPKB in the timing of association with forming macropinosomes. Although macropinocytosis is reduced in cells expressing dominant negative Rab7, microscopic imaging studies reveal that GFP-Rab7 associates only with formed macropinosomes at approximately the time that F-actin and D3 phosphoinositide levels decrease. These results support a model in which F-actin modulating proteins and vesicle trafficking proteins coordinately regulate the formation and maturation of macropinosomes. ----------------------------------------------------------------------------- A cell number-counting factor regulates group size in Dictyostelium by differentially modulating cAMP-induced cAMP and cGMP pulse sizes Lei Tang, Robin Amman, Tong Gao, and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 Journal of Biological Chemistry, in press; ms available at http://www.jbc.org/cgi/reprint/M102205200v1 Abstract A secreted counting factor (CF), regulates the size of Dictyostelium discoideum fruiting bodies in part by regulating cell-cell adhesion. Aggregation and the expression of adhesion molecules is mediated by relayed pulses of cAMP. Cells also respond to cAMP with a short cGMP pulse. We find that CF slowly down-regulates the cAMP-induced cGMP pulse by inhibiting guanylyl cyclase activity. A one-minute exposure of cells to purified CF increases the cAMP-induced cAMP pulse. CF does not affect the cAMP receptor or its interaction with its associated G proteins, or the translocation of the cytosolic regulator of adenylyl cyclase (CRAC) to the membrane in response to cAMP. Pulsing streaming wild-type cells with a high concentration of cAMP results in the formation of small groups, while reducing cAMP pulse size with exogenous cAMP phosphodiesterase during stream formation causes cells to form large groups. Altering the extracellular cAMP pulse size does not phenocopy the effects of CF on the cAMP-induced cGMP pulse size or cell-cell adhesion, indicating that CF does not regulate cGMP pulses and adhesion via CF's effects on cAMP pulses. The results suggest that regulating either cell-cell adhesion, the cGMP, or the cAMP pulse size can control group size, and that CF regulates all three of these independently. ----------------------------------------------------------------------------- On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts Ulrich Hentschel, Ilse Zuendorf, Theodor Dingermann and Thomas Winckler Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M. (Biozentrum), Frankfurt am Main, Germany Analytical Biochemistry, in press Abstract: At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analysed proteins, it could also lead to false interpretations. We analysed the subcellular distribution of endogenous and plasmid-borne proteins derived from the retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fractionation experiments the proteins encoded by TRE5-A open reading frame 1 (ORF1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extraction of isolated nuclei did not considerably release the TRE5-A proteins. Instead, the TRE5-A proteins were strongly enriched in a fraction that contained the chromosomal DNA after removal of most cytoskeletal and histone proteins. These observations implied that ORF1p and ENp were both attached to chromatin in vivo, but this conclusion was disproved by the expression of genetic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggregates of native protein in the cytoplasm of D. discoideum cells. ----------------------------------------------------------------------------- Supriya Srinivasan*, Mathew TrainiÝ, Ben HerbertÝ, Diane SextonÝ, Jenny HarryÝ, Hannah Alexander*, Keith L. WilliamsÝ and Stephen Alexander* * Division of Biological Sciences, 303 Tucker Hall, University of Missouri, Columbia, MO 65211, USA Ý Proteome Systems Limited, 1/35-41 Waterloo Road, North Ryde, Sydney, NSW 2113, Australia Proteomics, In Press SUMMARY Secretion of spore coat proteins from the prespore secretory vesicles (PSVs) in Dictyostelium discoideum is a signal mediated event that underlies terminal cell differentiation, and represents an important case of developmentally regulated secretion. In order to study the biochemical mechanisms that govern the regulated fusion of the PSVs with the plasma membrane and the subsequent secretion of their cargo, we purified this organelle from prespore cells. Analysis of protein extracts of highly purified PSVs indicated that, in addition to the cargo of structural spore coat proteins, many more proteins are associated with the PSVs. Their identification is paramount to the understanding of the mechanism of regulated secretion in this system. In this study we have taken the first comprehensive proteomic approach to the analysis of an entire - previously uncharacterized - organelle, with the goal of identifying the major proteins associated with the PSVs. We show that in addition to the structural spore coat proteins, the PSVs contain the enzymes needed for proper spore coat assembly (thioredoxin 2 and 3), regulatory proteins which we predict receive and transduce the developmental signal for secretion (rab7 GTPase, PI-3 kinase, NDP kinase and the calcium binding proteins calfumirin-1 and calreticulin) as well as proteins that interact with the cytoskeleton to mediate movement of the PSVs to the plasma membrane (actin binding proteins coactosin and profilin 1). In addition, the results suggest that proteins can play multiple roles in the cell, and that protein function can be dictated in part by subcellular localization. The identification of the PSV proteins is allowing us to develop testable hypotheses about the roles of these proteins within the functional context of developmentally regulated secretion. ----------------------------------------------------------------------------- A Rab11-like GTPase Associates with and Regulates the Structure and Function of the Contractile Vacuole System in Dictyostelium. Edward Harris*, Kunito Yoshida, James Cardelli* and John Bush+ +Department of Biology University of Arkansas at Little Rock Little Rock, AR 72204 * Department of Microbiology and Immunology LSU Health Sciences Center Shreveport, LA 71130. + Corresponding author: JOURNAL OF CELL SCIENCE, in press ABSTRACT Screening of a cDNA library revealed the existence of a Dictyostelium cDNA encoding a protein 80% identical at the amino acid level to mammalian Rab11. Subcellular fractionation and immunofluorescence studies revealed that DdRab11 was exclusively associated with the ATPase proton pump-rich contractile vacuole membrane system, consisting of a reticular network and bladder-like vacuoles. Video microscopy of cells expressing GFP-DdRab11 revealed that this Rab was associated with contractile vacuolar bladders undergoing formation, fusion, and expulsion of water. The association of DdRab11 with contractile vacuole membranes was disrupted when cells were exposed to either hypo-osmotic conditions or an inhibitor of the ATPase proton pump. Cells over-expressing a dominant negative form of DdRab11 were analyzed biochemically and microscopically to measure changes in the structure and function of the contractile vacuole system. Compared to wild-type cells, the dominant negative DdRab11 expressing cells contained a more extensive contractile vacuole network and abnormally enlarged contractile vacuole bladders, most likely the result of defects in membrane trafficking. Also, the mutant cells enlarged, detached from surfaces, and contained large vacuoles when exposed to water, suggesting a functional defect in osmotic regulation. No changes were observed in mutant cells in the rate of fluid phase internalization or release, suggesting the DdRab11-mediated membrane trafficking defects were not general in nature. Surprisingly, the rate of phagocytosis was increased in the dominant negative DdRab11 expressing cells as compared to control cells. Our results are consistent with a role for DdRab11 in regulating membrane traffic to maintain the normal morphology and function of the contractile vacuole. ----------------------------------------------------------------------------- [End Dicty News, volume 16, number 11]