Dicty News Electronic Edition Volume 11, number 4 August 22, 1998 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@nwu.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at the Dictyostelium Web Page "http://dicty.cmb.nwu.edu/dicty/dicty.html" =========== Abstracts =========== Overexpression of a Novel Rho-family GTPase, RacC, Induces Unusual Actin-based Structures and Positively Affects phagocytosis in Dictyostelium discoideum David J. Seastone (1) , Eunkyung Lee (2), John Bush (3), David Knecht (2), and James Cardelli(1,4) (1)Department of Microbiology and Immunology and (4) The Feist-Weiller Cancer Center, Louisiana State University Medical Center, Shreveport, LA 71130. (2) Department of Molecular and Cell Biology University of Connecticut. Storrs, CT 06269. (3)Department of Biology, University of Arkansas, Little Rock AR 72204. Mol. Biol. Cell, in press Abstract Rho family proteins have been implicated in regulating a variety of cellular processes including actin cytoskeleton organization, endocytosis, cell cycle, and gene expression. In this study, we analyzed the function of a novel D. discoideum Rho family protein (RacC). A cell-line was generated that conditionally overexpressed wild-type RacC 3-4 fold relative to endogenous RacC. Light microscopy and SEM indicated that the morphology of the RacC overexpressing cells (RacC WT(+) cells) was significantly altered compared to control cells. In contrast to the cortical F-actin distribution normally observed, RacC WT(+) cells displayed unusual dorsal and peripheral F-actin rich surface blebs (petalopodia; for flower-like). Furthermore, phagocytosis in the RacC WT(+) cells was induced three-fold relative to control Ax2 cells, while fluid-phase pinocytosis was reduced three-fold, primarily as the result of an inhibition of macropinocytosis. Efflux of fluid phase markers was also reduced in the RacC WT(+) cells, suggesting that RacC may regulate post-internalization steps along the endo-lysosomal pathway. Treatment of cells with wortmannin and LY294002 (PI 3-kinase inhibitors) prevented the RacC-induced morphological changes but did not affect phagocytosis, suggesting that petalopodia are probably not required for RacC-induced phagocytosis. In contrast, inactivating diacylglycerol- binding motif-containing proteins by treating cells with the drug calphostin C completely inhibited phagocytosis in control and RacC WT(+) cells. These results suggest that RacC plays a role in actin cytoskeleton organization and phagocytosis in Dictyostelium. ------------------------------------------------------------------------- G PROTEIN SIGNALING EVENTS ARE ACTIVATED AT THE LEADING EDGE OF CHEMOTACTIC CELLS Carole A. Parent, Brenda J. Blacklock, Wendy M. Froehlich, Douglas B. Murphy, and Peter N. Devreotes Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore MD 21205 Cell, In Press Abstract Directional sensing by eucaryotic cells does not require polarization of chemoattractant receptors. The translocation of the PH-domain containing protein CRAC in D. discoideum to binding sites on the inner face of the plasma membrane reflects activation of the G protein-linked signaling system. Increments in chemoattractant elicit a uniform response around the cell periphery. Yet when cells are exposed to a gradient the activation occurs selectively at the stimulated edge, even in immobilized cells. We propose that such localized activation, transmitted by the recruitment of cytosolic proteins, may be a general mechanism for gradient sensing by G protein-linked chemotactic systems including those involving chemotactic cytokines in leukocytes. ------------------------------------------------------------------------- Transcription of the Dictyostelium glycogen phosphorylase-2 gene is induced by three large promoter domains Reyna Favis¥, Ian McCaffery*, Gretchen Ehrenkaufer# and Charles L. RutherfordÆ Dept. of Biology, Virginia Polytechnic Institute and State University Blacksburg, VA 24061-0406 USA Developmental Genetics, in press ABSTRACT The promoter of the Dictyostelium glycogen phosporylase-2 (gp2) gene possesses a profound AT-bias, typical of promoters in this organism. To understand how Dictyostelium achieves specificity during transcriptional regulation under the constraint of this highly biased nucleotide composition, we have documented the changes in chromatin structure associated with developmental induction of gp2 gene expression. DNase I hypersensitive analyses indicated the presence of several developmentally regulated nuclease-sensitive sites located upstream of the start codon: two strong sites at approximately -250 bp and -350 bp and three substantially weaker sites at -290 bp, -445 bp and -505 bp. In vitro footprint analyses using nuclear extracts derived from several stages of development (corresponding to varying levels of gp2 expression) revealed three large regions of occupation that were developmentally regulated and corresponded to these nuclease-sensitive sites: -227 to -294 bp (domain 1), -327 to -383 bp (domain 2) and -416 to -534 bp (domain 3). The presence and the extent of the three regulatory domains was confirmed by in vivo footprint analyses spanning the same developmental time points. Southwestern analyses using probes encompassing these footprints demonstrated that probes corresponding to domains 1 and 3 both interacted with 83 and 77 kDa peptides. The domain 3 probe also interacted with a 92 kDa peptide, while only a 62 kDa peptide is recognized by the domain 2 probe. In all cases, peptides capable of binding these probes were found in nuclear extracts derived from differentiated cells and not in undifferentiated cell nuclear extract. Using nuclear extract from differentiated cells and probes corresponding to the three domains, gel mobility shift analyses detected ladders of retarded bands for both domains 1 and 3 and three major retarded bands for domain 2. These results suggest that specificity in transcriptional activation in the AT-rich promoters of Dictyostelium may be achieved by requiring multiple protein-DNA and/or protein-protein interactions to occur before induction can proceed. ------------------------------------------------------------------------- THE IQGAP-RELATED PROTEIN DGAP1 INTERACTS WITH RAC AND IS INVOLVED IN THE MODULATION OF THE F-ACTIN CYTOSKELETON AND CONTROL OF CELL MOTILITY Jan Faix 1, Chris Clougherty1, Angelika Konzok1, Ursula Mintert1, John Murphy1, Richard Albrecht1, Bettina Mühlbauer1, and Jürgen Kuhlmann2 Max-Planck-Institut fuer Biochemie, D-82152 Martinsried, Germany1, Max-Planck-Institut für molekulare Physiologie, D-44026 Dortmund, Germany2 J.Cell Sci., in press Abstract DGAP1 of D. discoideum is a cell cortex associated 95 kDa protein that shows homology to both RasGTPase-activating proteins (RasGAPs) and RasGAP- related proteins. When tested for RasGAP activity, recombinant DGAP1 protein did not promote the GTPase activity of human H-Ras or of Dictyostelium RasG in vitro. Instead, DGAP1 bound to Dictyostelium Rac1A and human Rac1, but not to human Cdc42. DGAP1 preferentially interacted with the activated, GTP- bound forms of Rac1 and Rac1A, but did not affect the GTPase activities. Since Rho-type GTPases are implicated in the formation of specific F-actin structures and in the control of cell morphology, the microfilament system of mutants that either lack or overexpress DGAP1 has been analysed. DGAP1-null mutants showed elevated levels of F-actin that was organized in large leading edges, membrane ruffles or numerous large filopods. Expression of actin fused to green fluorescent protein (GFP) was used to monitor the actin dynamics in these cells, and revealed that the F-actin cytoskeleton of DGAP1-null cells was rapidly re-arranged to form ruffles and filopods. Conversely, in DGAP1-overexpressing cells, the formation of cellular projections containing F-actin was largely suppressed. Measurement of cell migration demonstrated that DGAP1 expression is inversely correlated with the speed of cell motility. ------------------------------------------------------------------------- [End Dicty News, volume 11, number 4]