Dicty News Electronic Edition Volume 23, number 13 October 15, 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 ============= In vivo analysis of 3-phosphoinositide dynamics during Dictyostelium phagocytosis and chemotaxis Dirk Dormann (1), Gerti Weijer (1), Simon Dowler (2) and Cornelis J. Weijer (1) 1. Division of Cell and Developmental Biology, MSI/WTB Complex, University of Dundee, Dow Street, Dundee DD1 5EH, UK; 2. BioFocus Ltd, Cambridge, UK Journal of Cell Science, in press Phagocytosis and chemotaxis are receptor-mediated processes that require extensive rearrangements of the actin cytoskeleton, and are controlled by lipid second messengers such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2]. We used a panel of Pleckstrin Homology (PH) domains with distinct binding specificities for PI(3,4,5)P3 and PI(3,4)P2 to study the spatiotemporal dynamics of these phosphoinositides in vivo. During phagocytosis and macropinocytosis PI(3,4,5)P3 levels transiently increase at sites of engulfment, followed by a rapid PI(3,4)P2 production round the phagosome/macropinosome upon its internalisation, suggesting that PI(3,4,5)P3 is degraded to PI(3,4)P2. PTEN null mutants, which are defective in phagocytosis, show normal rates of PI(3,4,5)P3 degradation, but unexpectedly an accelerated PI(3,4)P2 degradation. During chemotaxis to cAMP only PI(3,4,5)P3 is formed in the plasma membrane, no PI(3,4)P2 is detectable showing, that all PI(3,4,5)P3 is degraded by PTEN to PI(4,5)P2. We furthermore show that different PI(3,4,5)P3 binding PHdomains give distinct spatial and temporal readouts of the same underlying PI(3,4,5)P3 signal, enabling distinct biological responses to one signal. Submitted by: Dirk Dormann [d.dormann@dundee.ac.uk] ----------------------------------------------------------------------------- Exposure of cells to a cell-number counting factor decreases the activity of glucose-6-phosphatase to decrease intracellular glucose levels in Dictyostelium Wonhee Jang and Richard H. Gomer Howard Hughes Medical Institute and Department of Biochemistry and Cell Biology, MS-140, Rice University, 6100 S. Main Street, Houston, TX 77005-1892 Eukaryotic Cell, in press The development of Dictyostelium discoideum is a model for tissue size regulation as these cells form groups of ~2 x 104 cells. The group size is regulated in part by a negative feedback pathway mediated by a secreted multi-polypeptide complex called counting factor (CF). CF signal transduction involves CF decreasing intracellular glucose levels. A component of CF, countin, has the bioactivity of the entire CF complex, and an 8-minute exposure of cells to recombinant countin decreases intracellular glucose levels. To understand how CF regulates intracellular glucose, we examined the effect of CF on enzymes involved in glucose metabolism. Exposure of cells to CF has little effect on amylase or glycogen phosphorylase, enzymes involved in glucose production from glycogen. Glucokinase activity (the first specific step of glycolysis) is inhibited by high levels of CF but is not affected by an 8-minute exposure to countin. The second enzyme specific for glycolysis, phosphofructokinase, is not regulated by CF. There are two corresponding enzymes in the gluconeogenesis pathway, fructose-1,6-bisphosphatase and glucose-6-phosphatase. The first is not regulated by CF or countin, whereas glucose-6-phosphatase is regulated by both CF and an 8-minute exposure to countin. The countin-induced changes in the Km and Vmax of glucose-6-phosphatase cause a decrease in glucose production that can account for the countin-induced decrease in intracellular glucose levels. It thus appears that part of the CF signal transduction pathway involves inhibiting the activity of glucose-6-phosphatase, decreasing intracellular glucose levels and affecting the levels of other metabolites, to regulate group size. Submitted by: Richard Gomer [richard@rice.edu] ----------------------------------------------------------------------------- Cellular distribution and functions of wild-type and constitutively activated Dictyostelium PakB Marc de la Roche, Amjad Mahasneh, Sheu-Fen Lee, Francisco Rivero* and Graham P. C™tŽ¤ Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L 3N6; *Zentrum fźr Biochemie, Medizinische FakultŠt, UniversitŠt zu Kšln, Joseph-Stelzmann-Strasse 52, 50931 Kšln, Germany Molecular Biology of the Cell, In Press Dictyostelium PakB, previously termed myosin I heavy chain kinase, is a member of the p21-activated kinase (PAK) family. Two-hybrid assays showed that PakB interacts with Dictyostelium Rac1a/b/c, RacA (a RhoBTB protein), RacB, RacC and RacF1. Wild-type PakB displayed a cytosolic distribution with a modest enrichment at the leading edge of migrating cells and at macropinocytic and phagocytic cups, sites consistent with a role in activating myosin I. PakB fused at the N-terminus to GFP was proteolyzed in cells, resulting in removal of the catalytic domain. C-terminal truncated PakB and activated PakB lacking the p21-binding domain strongly localized to the cell cortex, to macropinocytic cups, to the posterior of migrating cells and to the cleavage furrow of dividing cells. These data indicate that in its open, active state, the N-terminus of PakB forms a tight association with cortical actin filaments. PakB-null cells displayed no significant behavioral defects, but cells expressing activated PakB were unable to complete cytokinesis when grown in suspension and exhibited increased rates of phagocytosis and pinocytosis. Submitted by: Graham Cote [coteg@post.queensu.ca] ============================================================================== [End Dicty News, volume 23, number 13]