Dicty News Electronic Edition Volume 22, number 15 June 11, 2004 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 Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Distribution of alkaline phosphatase in vegetative Dictyostelium cells in relation to the contractile vacuole complex Margaret Clarke and Lucinda Maddera Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, 73104 Eur. J. Cell Biol., in press. (a special issue in honor of John Heuser) The structure of the contractile vacuole complex of Dictyostelium discoideum has long been a subject of controversy. A model that originated from the work of John Heuser and colleagues described this osmoregulatory organelle as an interconnected array of tubules and cisternae whose membranes are densely populated with vacuolar proton pumps. A conflicting model described this same organelle as bipartite, consisting of a pump-rich spongiome and a pump-free bladder, the latter membranes being identified by their alkaline phosphatase activity. In the present study we have employed an antiserum specific for Dictyostelium alkaline phosphatase to examine the distribution of this enzyme in vegetative cells. The antiserum labels puncta, probably vesicles, that lie at or near the plasma membrane and are sometimes, but only rarely, enriched near contractile vacuole membranes. We conclude that alkaline phosphatase is not a suitable marker for contractile vacuole membranes. We discuss these results in relation to the two models of contractile vacuole structure and suggest that all data are consistent with the first model. Submitted by: Margaret Clarke [clarkem@omrf.ouhsc.edu] ----------------------------------------------------------------------------- The XMAP215-family protein DdCP224 is required for cortical interactions of microtubules Andrea Hestermann and Ralph Graf* A.-Butenandt-Institut/Zellbiologie, Ludwig-Maximilians-UniversitŠt MŸnchen, Schillerstr. 42, D-80336 MŸnchen, Germany BMC Cell Biology 2004, 5:24, in press Background: Interactions of peripheral microtubule tips with the cell cortex are of crucial importance for nuclear migration, spindle orientation, centrosome positioning and directional cell movement. Microtubule plus end binding proteins are thought to mediate interactions of microtubule tips with cortical actin and membrane proteins in a dynein-dependent manner. XMAP215-family proteins are main regulators of microtubule plus end dynamics but so far they have not been implicated in the interactions of microtubule tips with the cell cortex. Results: Here we show that overexpression of an N-terminal fragment of DdCP224, the Dictyostelium XMAP215 homologue, caused a collapse of the radial microtubule cytoskeleton, whereby microtubules lost contact with the cell cortex and were dragged behind like a comet tail of an unusually motile centrosome. This phenotype was indistinguishable from mutants overexpressing fragments of the dynein heavy chain or intermediate chain. Moreover, it was accompanied by dispersal of the Golgi apparatus and reduced cortical localization of the dynein heavy chain indicating a disrupted dynein/dynactin interaction. The interference of DdCP224 with cortical dynein function is strongly supported by the observations that DdCP224 and its N-terminal fragment colocalize with dynein and coimmunoprecipitate with dynein and dynactin. Conclusions: Our data show that XMAP215-like proteins are required for the interaction of microtubule plus ends with the cell cortex in interphase cells and strongly suggest that this function is mediated by dynein. Submitted by: Ralph Graef [rgraef@lrz.uni-muenchen.de] ----------------------------------------------------------------------------- Live cell spinning disk microscopy RALPH GRAF1*, JENS RIETDORF2 AND TIMO ZIMMERMANN2 1) A.-Butenandt-Institut / Zellbiologie Ludwig-Maximilians-UniversitŠt MŸnchen Schillerstr. 42 80336 MŸnchen, Germany 2) Advanced Light Microscopy Facility European Molecular Biology Laboratory Meyerhofstr. 1 69117 Heidelberg, Germany Advances in Biochemical Engineering/Biotechnology, in press Special Volume: Microscopic Techniques Jens Rietdorf (Ed.) In vivo microscopy of dynamic processes in cells and organisms requires very fast and sensitive acquisition methods. Confocal laser scanning microscopy is inherently speed-limited by the requirement of beam scanning movements. In contrast to single beam scanning systems, the parallelized approach of multi beam scanning is much faster. Spinning disk confocal microscopes are therefore very suited for fast in vivo imaging. The principles of spinning disk microscopy will be explained in this chapter and a thorough comparison of the performance of single beam and multi beam scanning systems is made and illustrated with an example of in vivo imaging in Dictyostelium discoideum. Submitted by: Ralph Graef [rgraef@lrz.uni-muenchen.de] ----------------------------------------------------------------------------- Molecular and functional analysis of the Dictyostelium centrosome Ralph Graf, Christine Daunderer and Irene Schulz Adolf-Butenandt-Institut / Zellbiologie, Schillerstr. 42, D-80336 MŸnchen, Germany Int. Rev. Cytol., in press The centrosome is a non-membranous, nucleus-associated organelle which functions not only as the main microtubule-organizing center but also as a cell cycle control unit. How the ~100 different proteins that make up a centrosome contribute to centrosome function is still largely unknown. Considerable progress in the understanding of centrosomal functions can be expected from comparative cell biology of morphologically different centrosomal structures fulfilling conserved functions. Dictyostelium is an alternative model organism for centrosome research in addition to yeast and animal cells. With the elucidation of the morphological changes and dynamics of centrosome duplication, the establishment of a centrosome isolation protocol, and the identification of many centrosomal components there is a solid basis for understanding the biogenesis and function of this fascinating organelle. Here we give an overview of the prospective protein inventory of the Dictyostelium centrosome based on database searches. Moreover, we focus on the comparative cell biology of known components of the Dictyostelium centrosome including the g-tubulin complex and the homologues of centrin, Nek2, XMAP215 and EB1. Submitted by: Ralph Graef [rgraef@lrz.uni-muenchen.de] ============================================================================== [End Dicty News, volume 22, number 15]