Dicty News Electronic Edition Volume 23, number 4 July 30, 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 ============= Dictyostelium calcium-binding protein 4a interacts with NumA, a BRCT-domain protein that regulates nuclear number Michael A. Myre & Danton H. OâDay* Department of Biology, University of Toronto at Mississauga, Mississauga, ON. Canada Biochemical and Biophysical Research Communications, in press Nucleomorphin from Dictyostelium discoideum is a nuclear calmodulin binding protein that is a member of the BRCT-domain containing cell cycle checkpoint proteins. Two differentially expressed isoforms, NumA and NumB, share an extensive acidic domain (DEED) that when deleted produces highly multinucleated cells. We performed a yeast two-hybrid screen of a Dictyostelium cDNA library using NumA as bait. Here we show that nucleomorphin interacts with calcium binding protein 4a (CBP4a) in a Ca2+-dependent manner. Further deletion analysis suggests this interaction requires residues found within the DEED domain. NumA and CBP4a mRNAs are expressed at the same stages of development. CBP4a belongs to a large family of Dictyostelium CBPs, for which no cellular or developmental functions had previously been determined. Since the interaction of CBP4a with NumA requires the DEED domain, this suggests that CBP4a may respond to Ca2+-signalling through modulating factors that might function in concert to regulate nuclear number. Submitted by: M. Myre" [mmyre@utm.utoronto.ca] ----------------------------------------------------------------------------- Rac regulation of chemotaxis and morphogenesis in Dictyostelium Kyung Chan Park1, Francisco Rivero2, Ruedi Meili1, Susan Lee1, Fabio Apone1,3, and Richard A. Firtel1,4 1Section of Cell and Developmental Biology, Division of Biological Sciences Center for Molecular Genetics, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0634 2Zentrum fźr Biochemie der Medizinischen FakultŠt, UniversitŠt zu Kšln Joseph-Stelzmann-Strasse 52 50931 Kšln, Germany EMBO J, in press Chemotaxis requires localized F-actin polymerization at the site of the plasma membrane closest to the chemoattractant source, a process controlled by Rac/Cdc42 GTPases. We identify Dictyostelium RacB as an essential mediator of this process. RacB is activated upon chemoattractant stimulation, exhibiting biphasic kinetics paralleling F-actin polymerization. racB null cells have strong chemotaxis and morphogenesis defects and a severely reduced chemoattractant-mediated F-actin polymerization and PAKc activation. RacB activation is partly controlled by the PI3K pathway. pi3k1/2 null cells and wild-type cells treated with LY294002 exhibit a significant reduced second peak of RacB activation and that is linked to pseudopod extension, whereas a PTEN hypomorph exhibits elevated RacB activation. We identify a RacGEF, RacGEF1, which has a specificity for RacB in vitro. racgef1 null cells exhibit reduced RacB activation and cells expressing mutant RacGEF1 proteins display chemotaxis and morphogenesis defects. RacGEF1 localizes to sites of F-actin polymerization. Inhibition of this localization reduces RacB activation, suggesting a feedback loop from RacB via F-actin polymerization to RacGEF1. Our findings provide a critical linkage between chemoattractant stimulation, F-actin polymerization, and chemotaxis in Dictyostelium. Submitted by: Rick Firtel [rafirtel@ucsd.edu] ----------------------------------------------------------------------------- A cell number counting factor regulates Akt/ PKB to regulate group size in Dictyostelium Tong Gao1, David Knecht2, Lei Tang3, R. Diane Hatton1, and Richard H. Gomer1,3 1HHMI/ 3Rice University, Houston, TX 77005-1892 and 2University of Connecticut, Storrs, Connecticut 06269-3125 Eukaryotic Cell, in press Little is known about how individual cells can organize themselves to form structures of a given size. During development, Dictyostelium discoideum aggregates in dendritic streams and forms groups of ~20,000 cells. D. discoideum regulates group size by secreting and simultaneously sensing a multi-protein complex called counting factor (CF). If there are too many cells in a stream, the associated high concentration of CF will decrease cell-cell adhesion and increase cell motility, causing aggregation streams to break up. The pulses of cAMP that mediate aggregation cause a transient translocation of the Akt/PKB kinase to the leading edge of the plasma membrane and a concomitant activation of the kinase activity, which in turn stimulates motility. We found that countinř cells (which lack bioactive CF) and wild-type cells starved in the presence of anti-countin antibodies (which block CF activity) showed a decreased level of cAMP-stimulated Akt/PKB membrane translocation and kinase activity compared to parental wild type cells. Recombinant countin has the bioactivity of CF, and a 1-minute treatment of cells with recombinant countin potentiated Akt/PKB translocation to membranes and Akt/PKB activity. Western blots of total cell lysates indicated that countin does not affect the total level of Akt/PKB. Fluorescence microscopy of cells expressing an Akt/PKB PH-GFP fusion protein indicated that recombinant countin and anti-countin antibodies do not obviously alter the distribution of Akt/PKB PH-GFP when it translocates to the membrane. Our data indicate that CF increases motility by potentiating the cAMP-stimulated activation and translocation of Akt/PKB. Submitted by: Richard Gomer [richard@rice.edu] ============================================================================== [End Dicty News, volume 23, number 4]