dictyNews Electronic Edition Volume 38, number 5 February 17, 2012 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu or by using the form at http://dictybase.org/db/cgi-bin/dictyBase/abstract_submit. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Rab8a regulates the exocyst-mediated kiss-and-run discharge of the Dictyostelium contractile vacuole Miriam Essid, Navin Gopaldass, Kunito Yoshida, Christien Merrifield, and Thierry Soldati Molecular Biology of the Cell, in press Water expulsion by the contractile vacuole in Dictyostelium is carried out by a giant kiss-and-run focal exocytic event during which the two membranes are only transiently connected but do not completely merge. We present a molecular dissection of the GTPase Rab8a and the exocyst complex in tethering of the contractile vacuole to the plasma membrane, fusion and final detachment. Right before discharge, the contractile vacuole bladder sequentially recruits Drainin, a Rab11a-effector, Rab8a, the exocyst complex and LvsA, a protein of the Chediak-Higashi family. Rab8a recruitment precedes the nucleotide-dependent arrival of the exocyst to the bladder by a few seconds. A dominant-negative mutant of Rab8a strongly binds to the exocyst and prevents recruitment to the bladder suggesting that a Rab8a GEF activity is associated with the complex. Absence of Drainin leads to over-tethering and blocks fusion, while expression of constitutively active Rab8a allows fusion but blocks vacuole detachment from the plasma membrane, inducing complete fragmentation of tethered vacuoles. An indistinguishable phenotype is generated in cells lacking LvsA, implicating this protein in post-fusion de-tethering. Interestingly, overexpression of a constitutively active Rab8a mutant reverses the lvsA-null CV phenotype. Submitted by Thierry Soldati [thierry.soldati@unige.ch] -------------------------------------------------------------------------------------- Bestatin inhibits cell growth, division and spore cell differentiation in Dictyostelium Yekaterina Poloz1 , Andrew Catalano1 and Danton H. OĠDay1,2 1Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5 2Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Eukaryotic Cell, In press Bestatin methyl ester (BME) is an inhibitor of Zn2+-binding aminopeptidases that inhibits cell proliferation and induces apoptosis in normal and cancer cells. We have used Dictyostelium as a model organism to study the effects of BME. Only two Zn2+-binding aminopeptidases have been identified in Dictyostelium to date, puromycin sensitive aminopeptidase A and B (PsaA and PsaB). PSA from other organisms is known to regulate cell division and differentiation. Here we showed that PsaA is differentially expressed throughout growth and development of Dictyostelium and its expression is regulated by developmental morphogens. We present evidence that BME specifically interacts with PsaA and inhibits its aminopeptidase activity. Treatment of cells with BME inhibited the rate of cell growth and the frequency of cell division in growing cells and inhibited spore cell differentiation during late development. Overexpression of PsaA-GFP also inhibited spore cell differentiation but did not affect growth. Using chimeras, we have identified that nuclear versus cytoplasmic localization of PsaA affects the choice between stalk or spore cell differentiation pathway. Cells that overexpressed PsaA-GFP (primarily nuclear) differentiated into stalk cells, while cells that overexpressed PsaADeltaNLS2-GFP (cytoplasmic) differentiated into spores. In conclusion, we have identified that BME inhibits cell growth, division and differentiation in Dictyostelium likely through inhibition of PsaA. Submitted by: Danton OĠDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------------- CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum Robert J. Huber1, Andres Suarez1, and Danton H. OĠDay1,2 1Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, Canada M5S 3G5 2Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Mississauga, ON, Canada L5L 1C6 Matrix Biology, in press CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63 kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16 hours of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16 hours of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca2+ release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over- expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in Dictyostelium discoideum. Submitted by: Danton OĠDay [danton.oday@utoronto.ca] -------------------------------------------------------------------------------------- TM9/Phg1 and SadA proteins control surface expression and stability of SibA adhesion molecules in Dictyostelium. Froquet R, le Coadic M, Perrin J, Cherix N, Cornillon S, Cosson P. DŽpartement de Physiologie Cellulaire et Metabolisme, Centre Medical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland Mol Biol Cell, in press TM9 proteins form a family of conserved proteins with nine transmembrane domains essential for cellular adhesion in many biological systems, but their exact role in this process remains unknown. Here we found that in Dictyostelium amoebae, genetic inactivation of the TM9 protein Phg1A dramatically decreases the surface levels of the SibA adhesion molecule. This is due to a decrease in sibA mRNA levels, in SibA protein stability, and in SibA targeting to the cell surface. A similar phenotype was observed in cells devoid of SadA, a protein that does not belong to the TM9 family but also exhibits 9 transmembrane domains and is essential for cellular adhesion. A csA-SibA chimeric protein comprising only the transmembrane and cytosolic domains of SibA and the extracellular domain of csA, a Dictyostelium surface protein, also showed reduced stability and relocalization to endocytic compartments in phg1A knockout cells. These results indicate that TM9 proteins participate in cell adhesion by controlling the levels of adhesion proteins present at the cell surface. Submitted by Pierre Cosson [Pierre.Cosson@unige.ch] ============================================================== [End dictyNews, volume 38, number 5]