dictyNews Electronic Edition Volume 35, number 9 October 8, 2010 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 ========= Coupling Mechanism of a GPCR and a Heterotrimeric G Protein During Chemoattractant Gradient Sensing in Dictyostelium Xuehua Xu,1*† Tobias Meckel,1*‡ Joseph A. Brzostowski,2 Jianshe Yan, 1 Martin Meier-Schellersheim,3 Tian Jin1† 1Chemotaxis Signal Section, National Institutes of Health, Rockville, MD 20852, USA. 2Laboratory of Immunogenetics Imaging Facility, Laboratory of Immunogenetics, National Institutes of Health, Rockville, MD 20852, USA. 3Program in Systems Immunology and Infectious Disease Modeling, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852, USA. *These authors contributed equally to this work. †To whom correspondence should be addressed. E-mail: tjin@niaid.nih.gov ‡Present address: Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 3-5, D-64287 Darmstadt, Germany. Science Signaling, Volume 3 Issue 141 ra71 The coupling of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors (GPCRs) with G proteins is fundamental for GPCR signaling; however, the mechanism of coupling is still debated. Moreover, how the proposed mechanisms affect the dynamics of downstream signaling remains unclear. Here, through experiments involving fluorescence recovery after photobleaching and single-molecule imaging, we directly measured the mobilities of cyclic adenosine monophosphate (cAMP) receptor 1 (cAR1), a chemoattractant receptor, and a G protein bg subunit in live cells. We found that cAR1 diffused more slowly in the plasma membrane than did Gbg. Upon binding of ligand to the receptor, the mobility of cAR1 was unchanged, whereas the speed of a fraction of the faster-moving Gbg subunits decreased. Our measurements showed that cAR1 was relatively immobile and Gbg diffused freely, suggesting that chemoattractant-bound cAR1 transiently interacted with G proteins. Using models of possible coupling mechanisms, we computed the temporal kinetics of G protein activation. Our fluorescence resonance energy transfer imaging data showed that fully activated cAR1 induced the sustained dissociation of G protein a and bg subunits, which indicated that ligand-bound cAR1 activated G proteins continuously. Finally, simulations indicated that a high-affinity coupling of ligand-bound receptors and G proteins was essential for cAR1 to translate extracellular gradient signals into directional cellular responses.We suggest that chemoattractant receptors use a ligand-induced coupling rather than a precoupled mechanism to control the activation of G proteins during chemotaxis. Submitted by Xuehua Xu [XXU@niaid.nih.gov] -------------------------------------------------------------------------------- 14-3-3 coordinates microtubules, Rac, and myosin II to control cell mechanics and cytokinesis Qiongqiong Zhou, Yee-Seir Kee, Christopher C. Poirier, Christine Jelinek, Jonathan Osborne, Srikanth Divi, Alexandra Surcel, Marie E. Will, Ulrike S. Eggert, Annette Müller-Taubenberger, Pablo A. Iglesias, Robert J. Cotter, and Douglas N. Robinson Curr. Biol., In press Background: During cytokinesis, regulatory signals are presumed to emanate from the mitotic spindle. However, what these signals are and how they lead to the spatiotemporal changes in the cortex structure, mechanics, and regional contractility are not well understood in any system. Results: To investigate pathways that link the microtubule network to the cortical changes that promote cytokinesis, we used chemical genetics in Dictyostelium to identify genetic suppressors of nocodazole, a microtubule depolymerizer. We identified 14-3-3 and found that it is enriched in the cortex, helps maintain steady state microtubule length, contributes to normal cortical tension, modulates actin wave formation, and controls the symmetry and kinetics of cleavage furrow contractility during cytokinesis. Furthermore, 14-3-3 acts downstream of a Rac small GTPase (RacE), associates with myosin II heavy chain and is needed to promote myosin II bipolar thick filament remodeling. Conclusion: 14-3-3 connects microtubules, Rac and myosin II to control several aspects of cortical dynamics, mechanics, and cytokinesis cell shape change. Further, 14-3-3 interacts directly with myosin II heavy chain to promote bipolar thick filament r emodeling and distribution. Overall, 14-3-3 appears to integrate several critical cytoskeletal elements that drive two important processes – cytokinesis shape change and cell mechanics. Submitted by Doug Robinson [dnr@jhmi.edu] -------------------------------------------------------------------------------- The Dictyostelium discoideum acaA gene is transcribed from alternative promoters during aggregation and multicellular development. Maria Galardi-Castilla, Ane Garciandía, Teresa Suarez and Leandro Sastre PloS one, in press Background Extracellular cAMP is a key extracellular signaling molecule that regulates aggregation, cell differentiation and morphogenesis during multi-cellular development of the social amoeba Dictyostelium discoideum. This molecule is produced by three different adenylyl cyclases, encoded by the genes acaA, acrA and acgA, expressed at different stages of development and in different structures. Methodology/Principal findings This article describes the characterization of the promoter region of the acaA gene, showing that it is transcribed from three different alternative promoters. The distal promoter, promoter 1, is active during the aggregation process while the more proximal promoters are active in tip-organiser andposterior regions of the structures. A DNA fragment containing the three promoters drove expression to these same regions and similar results were obtained by in situ hybridization. Analyses of mRNA expression by quantitative RT-PCR with specific primers for each of the three transcripts also demonstrated their different temporal patterns of expression. Conclusions/Significance The existence of an aggregation-specific promoter can be associated with the use of cAMP as chemo-attractant molecule, which is specific for some Dictyostelium species. Expression at late developmental stages indicates that adenylyl cyclase A might play a more important role in post-aggregative development than previously considered. Submitted by Leandro Sastre [lsastre@iib.uam.es] -------------------------------------------------------------------------------- Role of Magnesium and a Phagosomal P-type ATPase in Intracellular Bacterial Killing Emmanuelle Lelong, Anna Marchetti, Aurélie Guého, Wanessa C. Lima, Natascha Sattler, Maëlle Molmeret, Monica Hagedorn, Thierry Soldati and Pierre Cosson Cellular Microbiology, in press Bacterial ingestion and killing by phagocytic cells are essential processes to protect the human body from infectious microorganisms. However, only few proteins implicated in intracellular bacterial killing have been identified to date. We used Dictyostelium discoideum, a phagocytic bacterial predator, to study intracellular killing. In a random genetic screen we identified Kil2, a type V P-ATPase as an essential element for efficient intracellular killing of Klebsiella pneumoniae bacteria. Interestingly, kil2 knockout cells still killed efficiently several other species of bacteria, and did not show enhanced susceptibility to Mycobacterium marinum intracellular replication. Kil2 is present in the phagosomal membrane, and its structure suggests that it pumps cations into the phagosomal lumen. he killing defect of kil2 knockout cells was rescued by the addition of magnesium ions, suggesting that Kil2 may function as a magnesium pump. In agreement with this, kil2 mutant cells exhibited a specific defect for growth at high concentrations of magnesium. Phagosomal protease activity was lower in kil2 mutant cells than in wild-type cells, a phenotype reversed by the addition of magnesium to the medium. Kil2 may act as a magnesium pump maintaining magnesium concentration in phagosomes, thus ensuring optimal activity of phagosomal proteases and efficient killing of bacteria. Submitted by Emmanuelle Lelong [emmanuelle.lelong@unige.ch] ============================================================== [End dictyNews, volume 35, number 9]