dictyNews Electronic Edition Volume 32, number 12 May 1, 2009 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. ========= Abstracts ========= Identification and cell cycle-dependent localization of nine novel, genuine centrosomal components in Dictyostelium discoideum   Irene Schulz, Alexander Erle, Ralph Gräf, Anne Krüger, Heiner Lohmeier,  SaschaPutzler, Matthias Samereier, Sebastian Weidenthaler University of Potsdam, Institute for Biochemistry and Biology, Dept. of  Cell BiologyKarl-Liebknecht-Strasse 24-25, Haus 26 14476 Potsdam-Golm, Germany   Cell Motility and the Cytoskeleton: Mechanics and Dynamics of the Cytoskeleton The centrosome is the main microtubule-organizing center and constitutes  the largest protein complex in a eukaryotic cell. The Dictyostelium centrosome  is an established model for acentriolar centrosomes and it consists of a layered  core structure surrounded by a so-called corona, which harbors microtubule  nucleation complexes. We have identified 34 new centrosomal candidate proteins  through mass spectrometrical analysis of the proteome of isolated Dictyostelium  centrosomes. Here we present a characterization of 12 centrosomal candidate  proteins all featuring coiled coil regions and low expression levels, which are the most common attributes of centrosomal proteins. We used GFP fusion proteins  to localize the candidate proteins in whole cells and on microtubule-free, isolated  centrosomes. Thus we were able to identify nine new genuine centrosomal proteins  including a putative orthologue of Cep192, an interaction partner of polo-like  kinase 4 in human centriole biogenesis. In this respect, centrosomal localization  of the only polo-like kinase in Dictyostelium, Plk, is also shown in this work.  Using confocal deconvolution microscopy, four components, CP39,CP55, CP75  and CP91 could be clearly assigned to the so far almost uncharacterized  centrosomal core structure, while CP148 and Cep192 localized to a zone  between that of corona marker and core proteins. Finally, CP103 and CP248  were constituents of the corona. In contrast, NE81 was localized at the nuclear  envelope and three others, an orthologue of the spindle checkpoint component  Mad1, the novel Cenp68, and the centrosomal CP248 were observed at the  centromeres, which are clustered and linked to the centrosome throughout  the entire cell cycle. Submitted by: Irene Schulz [Irene.Schulz@uni-potsdam.de] -------------------------------------------------------------------------------- The Ordered Extension of Pseudopodia by Amoeboid Cells in the Absence  of External Cues. Leonard Bosgraaf, and Peter J.M. Van Haastert PLOS ONE, in press Eukaryotic cells extend pseudopodia for movement. In the absence of  external cues, cells move in random directions, but with a strong element  of persistence that keeps them moving in the same direction Persistence  allows cells to disperse over larger areas and is instrumental to enter  new environments where spatial cues can lead the cell. Here we explore  cell movement by analyzing the direction, size and timing of ~2000  pseudopodia that are extended by Dictyostelium cells. The results show  that pseudpopod are extended perpendicular to the surface curvature at  the place where they emerge. The location of new pseudopods is not random  but highly ordered. Two types of pseudopodia may be formed: frequent splitting  of an existing pseudopod, or the occasional extension of a de novo pseudopod  at regions devoid of recent pseudopod activity. Split-pseudopodia are extended  at ~60 degrees relative to the previous pseudopod, mostly as alternating  Right/Left/Right steps leading to relatively straight zigzag runs. De novo  pseudopodia are extended in nearly random directions thereby interrupting  the zigzag runs. Persistence of cell movement is based on the ratio of split  versus de novo pseudopodia. We identify PLA2 and cGMP signaling  pathways that modulate this ratio of splitting and de novo pseudopodia,  and thereby regulate the dispersal of cells. The observed ordered  extension of pseudopodia in the absence of external cues provides a  fundamental insight into the coordinated movement of cells, and might  form the basis for movement that is directed by internal or external cues.  Submitted by: Peter Van Haastert [p.j.m.van.haastert@rug.nl] -------------------------------------------------------------------------------- Regulation of the formation and trafficking of vesicles from Golgi by PCH  Family Proteins During Chemotaxis S. Lee, J. W. Han#, L. Leeper, J. S. Gruver, C. Y. Chung* Department of Pharmacology, Vanderbilt University Medical Center,  Nashville, TN 37232-6600 BBA-Molecular Cell Research, In press Previous study demonstrated that WASP localizes on vesicles during  Dictyostelium chemotaxis and these vesicles appear to be preferentially  distributed at the leading and trailing edge of migrating cells.  In this  study, we have examined the role of PCH family proteins, Nwk/Bzz1p-like  protein (NLP) and Syndapin-like protein (SLP), in the regulation of the  formation and trafficking of WASP-vesicles during chemotaxis. NLP and SLP  appear to be functionally redundant and deletion of both nlp and slp genes  cause the loss of polararized F-actin organization and significant defects  in chemotaxis. WASP and NLP are colocalized on vesicles and interactions  between two molecules via the SH3 domain of NLP/SLP and the proline-rich  repeats of WASP are required for vesicle formation from Golgi. Microtubules  are required for polarized trafficking of these vesicles as vesicles showing  high directed mobility are absent in cells treated with nocodazole. Our  results suggest that interaction of WASP with NLP/SLP is required for the  formation and trafficking of vesicles from Golgi to the membrane, which  might play a central role in the establishment of cell polarity during  chemotaxis. Submitted by: Chan Chung [chang.chung@vanderbilt.edu] -------------------------------------------------------------------------------- The STE group kinase SepA controls cleavage furrow formation in Dictyostelium    Annette Müller-Taubenberger*, Hellen C. Ishikawa-Ankerhold, Peter M. Kastner,  Emmanuel Burghardt, and Günther Gerisch Cell Motility and the Cytoskeleton, in press. During a REMI screen for proteins regulating cytokinesis in Dictyostelium  discoideum we isolated a mutant forming multinucleate cells. The gene  affected in this mutant encoded a kinase, SepA, which is an ortholog of  Cdc7, a serine-threonine kinase essential for septum formation in  Schizosaccharomyces pombe. Localization of SepA-GFP in live cells and its presence in isolated centrosomes indicated that SepA, like its upstream  regulator Spg1, is associated with centrosomes. Knockout mutants of SepA  showed a severe cytokinesis defect and a delay in development. In  multinucleate SepA-null cells nuclear division proceeded normally and  synchronously. However, often cleavage furrows were either missing or  atypical: they were extremely asymmetric and constriction was impaired.  Cortexillin-I, a marker localizing strictly to the furrow in wild-type cells,  demonstrated that large, crescent-shaped furrows expanded and persisted  long after the spindle regressed and nuclei returned to the interphase state.  Outside the furrow the filamentous actin system of the cell cortex showed  strong ruffling activity. These data suggest that SepA is involved in the  spatial and temporal control system organizing cortical activities in  mitotic and post-mitotic cells.      Submitted by: Annette Müller-Taubenberger [amueller@lrz.uni-muenchen.de] ============================================================== [End dictyNews, volume 32, number 12]