dictyNews Electronic Edition Volume 26, number 2 January 13, 2006 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 ============= Cleavage with phospholipase of the lipid anchor in the cell adhesion molecule, csA, from Dictyostelium discoideum Motonobu Yoshida*, Naoya Sakuragi, Ken Kondo, Eiji Tanesaka Department of Agriculture, Kinki University, Nakamachi, Nara 631-8505, Japan Comp. Biochem. Physiol., in press A cell adhesion molecule, 80-kDa csA, is involved in EDTA-resistant cell contact at the aggregation stage of Dictyostelium discoideum. A 31-kDa csA was isolated from the 80-kDa csA by treatment with Achromobacter protease I. Results from thin-layer chromatography and MALDI-TOF MS analysis indicated that the 31-kDa csA contains ceramide as a component of glycosylphosphatidyl-inositol (GPI). Comparison between the 80-kDa csA and the 31-kDa csA treated with phosphatidylinositol-specific phospholipase C (PI-PLC) or GPI-specific phospholipase D (GPI-PLD) was carried out. Our results indicated that the GPI-anchor of the 31-kDa csA was more sensitive to PI-PLC treatment than that of the 80-kDa csA, and that the anchor in both was easily cleaved by GPI-PLD treatment. The results of the 80-kDa csA and the 31-kDa csA treated with sphingomyelinase were almost the same as those with PI-PLC treatment. In the presence of 1,10-phenanthroline, a GPI-PLD inhibitor, development of Dictyostelium was markedly inhibited, suggesting that GPI-PLD is functional in developmental regulation. Submitted by: Motonobu Yoshida [yoshida_m@nara.kindai.ac.jp] ----------------------------------------------------------------------------- A secondary disruption of the lmpA gene encoding a large membrane protein allows aggregation defective Dictyostelium rasC- cells to form multicellular structures Meenal Khosla1, Paul Kriebel2, Carole A. Parent2, George B. Spiegelman1 and Gerald Weeks1 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada and 2The Laboratory of Cellular and Molecular Biology, National Cancer Institute, NIH, MD, USA Corresponding author; G. Weeks Tel: 604-822-6649. Fax: 604-822-6041 Mailing address: 2350 Health Sciences Mall., Vancouver, BC, V6T 1Z3, Canada. Email: gerwee@interchange.ubc.ca Developmental Biology, in press The disruption of the gene encoding the Dictyostelium Ras sub-family protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC- strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (lmpA) encoded a novel large membrane protein that was designated Lmp1. Although the rasC-/lmpA- cells progressed through early development, they did not form aggregation streams on a plastic surface under submerged starvation conditions. Phosphorylation of PKB in response to cAMP, which is significantly reduced in rasC- cells, remained low in the rasC-/lmpA- cells. However, in spite of this low PKB phosphorylation, the rasC-/lmpA- cells underwent efficient chemotaxis to cAMP in a spatial gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC- cells, was restored in the rasC-/lmpA- strain, but cAMP relay in these cells was not apparent. These data indicate that, although the rasC-/lmpA- cells were capable of associating to form multicellular structures, normal aggregative cell signaling was clearly not restored. Disruption of the lmpA gene in a wildtype background resulted in cells that exhibited a slight defect in aggregation and a more substantial defect in late development. These results indicate that in addition to the role played by Lmp1 in aggregation, it is also involved in late development. Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca] ----------------------------------------------------------------------------- A secondary disruption of the lmpA gene encoding a large membrane protein allows aggregation defective Dictyostelium rasC- cells to form multicellular structures Meenal Khosla1, Paul Kriebel2, Carole A. Parent2, George B. Spiegelman1 and Gerald Weeks1 1Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada and 2The Laboratory of Cellular and Molecular Biology, National Cancer Institute, NIH, MD, USA Corresponding author; G. Weeks Tel: 604-822-6649. Fax: 604-822-6041 Mailing address: 2350 Health Sciences Mall., Vancouver, BC, V6T 1Z3, Canada. Email: gerwee@interchange.ubc.ca Developmental Biology, in press The disruption of the gene encoding the Dictyostelium Ras sub-family protein, RasC, results in a strain that does not aggregate and has defects in both cAMP signal relay and cAMP chemotaxis. Disruption of a second gene in the rasC- strain by Restriction Enzyme Mediated Integration produced cells that were capable of forming multicellular structures in plaques on bacterial lawns. The disrupted gene (lmpA) encoded a novel large membrane protein that was designated Lmp1. Although the rasC-/lmpA- cells progressed through early development, they did not form aggregation streams on a plastic surface under submerged starvation conditions. Phosphorylation of PKB in response to cAMP, which is significantly reduced in rasC- cells, remained low in the rasC-/lmpA- cells. However, in spite of this low PKB phosphorylation, the rasC-/lmpA- cells underwent efficient chemotaxis to cAMP in a spatial gradient. Cyclic AMP accumulation, which was greatly reduced in the rasC- cells, was restored in the rasC-/lmpA- strain, but cAMP relay in these cells was not apparent. These data indicate that, although the rasC-/lmpA- cells were capable of associating to form multicellular structures, normal aggregative cell signaling was clearly not restored. Disruption of the lmpA gene in a wildtype background resulted in cells that exhibited a slight defect in aggregation and a more substantial defect in late development. These results indicate that in addition to the role played by Lmp1 in aggregation, it is also involved in late development. Submitted by: Gerald Weeks [gerwee@interchange.ubc.ca] ----------------------------------------------------------------------------- HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing. Blagovesta Popova1, Markus Kuhlmann1 Andrea Hinas2, Fredrik Söderbom2 and Wolfgang Nellen1* 1Abt. Genetik, Universität Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany 2Department of Molecular Biology, Biomedical Center, Swedish University of Agricultural Sciences, Box 590, S-75124 Uppsala, Sweden *corresponding author: Wolfgang Nellen Abt. Genetik, Universität Kassel, Heinrich-Plett-Str. 40, D-34132 Kassel, Germany Nucleic Acids Res. in press We have identified a putative RNA helicase from Dictyostelium that is closely related to drh1, the “dicer-related-helicase” from C. elegans and that also has significant similarity to proteins from vertebrates and plants. GFP-tagged HelF protein was localized in speckles in the nucleus. Disruption of the helF gene resulted in a mutant morphology in late development. When transformed with RNAi constructs, HelF- cells displayed enhanced RNA interference on four tested genes. One gene that could not be knocked down in the wild type background was efficiently silenced in the mutant. Furthermore, the efficiency of silencing in the wild type was dramatically improved when helF was disrupted in a secondary transformation. Silencing efficiency depended on transcription levels of hairpin RNA and the threshold was dramatically reduced in HelF- cells. However, the amount of siRNA did not depend on hairpin transcription. HelF is thus a natural nuclear suppressor of RNA interference. In contrast, no improvement of gene silencing was observed when mutant cells were challenged with corresponding antisense constructs. This indicates that RNAi and antisense have distinct requirements even though they may share parts of their pathways. Submitted by: Wolfgang Nellen [nellen@uni-kassel.de] ----------------------------------------------------------------------------- Distinct roles of PI(3,4,5)P3 during chemoattractant signaling in Dictyostelium: A quantitative in vivo analysis by inhibition of PI3-kinase Harriët M. Loovers, Marten Postma, Ineke Keizer-Gunnink, Yi Elaine Huang, Peter N. Devreotes, and Peter J.M. van Haastert Department of Molecular Cell Biology, University of Groningen, Kerklaan 30, 9751NN Haren, the Netherlands Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., 114 WBSB, Baltimore, MD 21205 Molecular Biology of the Cell, in press The role of PI(3,4,5)P3 in Dictyostelium signal transduction and chemotaxis was investigated using the PI3-kinase inhibitor LY294002 and pi3k-null cells. The increase of PI(3,4,5)P3 levels after stimulation with the chemoattractant cAMP was blocked >95% by 60 ?M LY294002 with half-maximal effect at 5 ?M. This correlated well with the inhibition of the membrane translocation of the PH-domain protein, PHcracGFP. LY294002 did not reduce cAMP-mediated cGMP production, but significantly reduced the cAMP response up to 75% in wild type and completely in pi3k-null cells. LY294002-treated cells were round, not elongated as control cells. Interestingly, cAMP induced a time and dose-dependent recovery of cell elongation. These elongated LY294002-treated wild type and pi3k-null cells exhibited chemotactic orientation towards cAMP that is statistically identical to chemotactic orientation of control cells. In control cells, PHcrac-GFP and F-actin colocalize upon cAMP stimulation. However, inhibition of PI3-kinases does not affect the first phase of the actin polymerization at a wide range of chemoattractant concentrations. Our data show that severe inhibition of cAMP-mediated PI(3,4,5)P3 accumulation leads to inhibition of cAMP relay, cell elongation and cell aggregation, but has no detectable effect on chemotactic orientation, provided that cAMP had sufficient time to induce cell elongation. Submitted by: Peter van Haastert [P.J.M.van.haastert@rug.nl] ============================================================================== [End dictyNews, volume 26, number 2]