dictyNews Electronic Edition Volume 31, number 12 October 18, 2008 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. Upon publication of your paper, please send strains and plamids to  the Dicty Stock Center. For more information see  http://dictybase.org/StockCenter/Deposit.html. Back issues of dictyNews, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ========= Abstracts ========= Dictyostelium discoideum Paxillin Regulates Actin-Based Processes M. Berenice Duran, Asif Rahman, Max Colten and Derrick Brazill Department of Biological Sciences, Center for the Study of Gene Structure and Function Hunter College of the City University of New York, New York, NY 10021 Protist, in press Paxillin is a key player in integrating the actin cytoskeleton with adhesion, and thus is essential to numerous cellular processes including proliferation, differentiation and migration in animal cells.  PaxB, the Dictyostelium discoideum orthologue of paxillin, has been shown to be important for adhesion and development, much like its mammalian counterpart.  Here, we use the overproduction of PaxB to gain better insight into its role in regulating the actin cytoskeleton and adhesion.  We find that PaxB overexpressing (PaxBOE) cells can aggregate and form mounds normally, but are blocked in subsequent development.  This arrest can be rescued by addition of wild-type cells, indicating a non-cell autonomous role for PaxB.   PaxBOE cells also have  alterations in several actin-based processes, including adhesion, endocytosis, motility and chemotaxis.  PaxBOE cells exhibit an EDTA-sensitive increase in cell-cell cohesion, suggesting that PaxB-mediated adhesion is Ca2+ or Mg2+ dependent.  Interestingly, cells overexpressing paxB are less adhesive to the substratum.  In addition, PaxBOE cells display decreased motility under starved conditions, decreased endocytosis, and are unable to efficiently chemotax up a folate gradient.   Taken together, the data suggest that proper expression of PaxB is vital for the regulation of development and actin-dependent processes. Submitted by: Derrick Brazill [Brazill@GENECTR.HUNTER.CUNY.EDU] -------------------------------------------------------------------------------- Regulation of Rap1 Activity is Required for Differential Adhesion, Cell Type Patterning and Morphogenesis in Dictyostelium Katie Parkinson1, Parvin Bolourani2, David Traynor3, Nicola L. Aldren1, Robert R. Kay3, Gerald Weeks2 and Christopher R.L. Thompson1* 1Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester, M13 9PT 2Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3 3MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH *Author for correspondence (christopher.thompson@manchester.ac.uk) Journal of Cell Science, in press Regulated cell adhesion and motility play important roles during growth, development and tissue homeostasis.  Consequently great efforts have been made to identify genes that control these processes.  One candidate is Rap1, as it has been implicated in the regulation of adhesion and motility in cell culture.  In order to further study the role of Rap1 during multicellular development, we have generated a mutant in a potential Rap1 GTPase activating protein (RapGAPB) in Dictyostelium.  rapGAPB- cells have increased levels of active Rap1 compared to wild type cells, indicating that RapGAPB regulates Rap1 activity.  Furthermore, rapGAPB- cells exhibit hallmark phenotypes of other known mutants with hyperactivated Rap1, including increased substrate adhesion and abnormal F-actin distribution.  However, unlike these other mutants, rapGAPB- cells do not exhibit impaired motility or chemotaxis, indicating that RapGAPB may only regulate specific roles of Rap1.   Importantly, we also found that RapGAPB regulates Rap1 activity during multicellular development and is required for normal morphogenesis.   Firstly, streams of aggregating rapGAPB- cells break up as a result of decreased cell-cell adhesion.  Secondly, rapGAPB- cells exhibit cell autonomous defects in prestalk cell patterning.  Using cell type specific markers, we demonstrate that RapGAPB is required for the correct sorting behaviour of different cell types.  Finally, we show that inactivation of RapGAPB affects prestalk and prespore cell adhesion.  We therefore propose that a possible mechanism for RapGAPB regulated cell sorting is through differential adhesion. Submitted by: Chris Thompson [christopher.thompson@manchester.ac.uk] -------------------------------------------------------------------------------- Microarray phenotyping places cyclase associated protein CAP at the crossroad of signaling pathways reorganizing the actin cytoskeleton in Dictyostelium Hameeda Sultana(1), Girish Neelakanta(1), Ludwig Eichinger(1), Francisco Rivero(1,2), Angelika A. Noegel(1) 1Center for Biochemistry, Medical Faculty, Center for Molecular Medicine Cologne (CMMC) and Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, 50931 Köln, Germany. 2The Hull York Medical School and Department of Biological Sciences, University of Hull, Hull HU6 7RX, UK Exp. Cell Res., in press Large-scale gene expression analysis has been applied recently to uncover groups of genes that are co-regulated in particular processes. Here we undertake such an analysis on CAP, a protein that participates in the regulation of the actin cytoskeleton and in cAMP signaling in Dictyostelium. Microarray analysis revealed that loss of CAP altered the expression of many cytoskeletal components. One of these, the Rho GDP-dissociation inhibitor RhoGDI1, was analyzed further. RhoGDI1 null cells expressed lower amounts of CAP, which failed to accumulate predominantly at the cell cortex. To further position CAP in the corresponding signal transduction pathways we studied CAP localization and cellular functioning in mutants that have defects in several signaling components. CAP showed correct localization and dynamics in all analyzed strains except in mutants with deficient cAMP dependent protein kinase A activity, where CAP preferentially accumulated in crown shaped structures. Ectopic expression of CAP improved the efficiency of phagocytosis in Gbeta-deficient cells and restored the pinocytosis, morphology and actin distribution defects in a PI3 kinase double mutant (pi3k1/2 null). Our results show that CAP acts at multiple crossroads and links signaling pathways to the actin cytoskeleton either by physical interaction with cytoskeletal components or through regulation of their gene expression. Submitted by: Francisco Rivero [f.rivero-crespo@hull.ac.uk] -------------------------------------------------------------------------------- Spatiotemporal Regulation of Ras Activity Provides Directional Sensing Sheng Zhang1, Pascale G. Charest1, and Richard A. Firtel 1. Co-first authors Section of Cell and Developmental Biology Division of Biological Sciences Center for Molecular Genetics University of California, San Diego 9500 Gilman Drive La Jolla, California 92093-0380 Curr. Biol., in press Cells’ ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown [1]. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects [2]. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2 [2–5]. To investigate the role of Ras in directional sensing, we studied the effect of its misregulation by using cells with disrupted RasGAP activity. We identified an ortholog of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. We show that disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, we show that tight regulation of Ras is important for gradient sensing. Together, our findings suggest that Ras is part of the cell’s compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing. Submitted by: Rick Firtel [rafirtel@ucsd.edu] ============================================================== [End dictyNews, volume 31, number 12]