Dicty News Electronic Edition Volume 21, number 11 October 10, 2003 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at dictyBase - http://dictybase.org. ============= Abstracts ============= Macroautophagy is dispensable for intracellular replication of Legionella pneumophila in Dictyostelium discoideum Grant P. Otto, Mary Y. Wu, Margaret Clarke, Hao Lu, O. Roger Anderson, Hubert Hilbi, Howard A. Shuman, and Richard H. Kessin. Accepted, Molecular Microbiology SUMMARY The gram-negative bacterium Legionella pneumophila is a facultative intracellular pathogen of free-living amoebae and mammalian phagocytes. L. pneumophila is engulfed in phagosomes that initially avoid fusion with lysosomes. The phagosome associates with endoplasmic reticulum and mitochondria, and eventually resembles ER. The morphological similarity of the replication vacuole to autophagosomes, and enhanced bacterial replication in response to macroautophagy-inducing starvation, led to the hypothesis that L. pneumophila infection requires macroautophagy. Since L. pneumophila replicates in D. discoideum, and macroautophagy genes have been identified and mutated in D. discoideum, we have taken a genetic and cell biological approach to evaluate the relationship between host macroautophagy and intracellular replication of L. pneumophila. Mutation of the apg1 , apg5, apg6, apg7 and apg8 genes produced typical macroautophagy defects, including reduced bulk protein degradation and cell viability during starvation. We show that L. pneumophila replicates normally in D. discoideum macroautophagy mutants and produces replication vacuoles that are morphologically indistinguishable from those in wild-type D. discoideum. Furthermore, a GFP-tagged marker of autophagosomes, Apg8, does not systematically co-localise with DsRed-labelled L. pneumophila. We conclude that macroautophagy is dispensable for L. pneumophila intracellular replication in D. discoideum. Submitted by: Grant Otto [go25@columbia.edu] ----------------------------------------------------------------------------- A mechanical unfolding intermediate in an actin-crosslinking protein. Ingo Schwaiger, Angelika Kardinal, Michael Schleicher, Angelika Noegel and Matthias Rief Accepted, Nature Structural Biology A large number of F-actin cross-linking proteins consists of two actin-binding domains separated by a rod domain that can vary considerably in length and structure. We used single-molecule force-experiments to investigate the mechanics of the immunoglobulin (Ig) rod domains of filamin from Dictyostelium discoideum (ddFLN). We find that one of the 6 Ig-domains unfolds at lower forces than all other domains and exhibits a stable unfolding intermediate on its mechanical unfolding pathway. Amino acid inserts into various loops of this domain lead to length changes in the single molecule unfolding pattern which allowed us to map the stable core of ~60 amino acids that constitute the unfolding intermediate. Fast refolding in combination with low unfolding forces suggest a potential in vivo role for this domain as a mechanically extensible element within the ddFLN rod. Submitted by: Michael Schleicher [schleicher@lrz.uni-muenchen.de] ----------------------------------------------------------------------------- Disruption of aldehyde reductase increases group size in Dictyostelium Karen Ehrenman1, Gong Yang1, Wan-Pyo Hong1, Tong Gao1, Wonhee Jang2, Debra A. Brock1, R. Diane Hatton1, James D. Shoemaker3, and Richard H. Gomer1,2 1Howard Hughes Medical Institute and 2Department of Biochemistry and Cell Biology, MS-140, Rice University, Houston, TX and 3Metabolic Screening Laboratory, Department of Biochemistry and Molecular Biology, St. Louis University, St. Louis, MO Journal of Biological Chemistry, in press ABSTRACT Developing Dictyostelium cells form structures containing ~20,000 cells. The size regulation mechanism involves a secreted counting factor (CF) repressing cytosolic glucose levels. Glucose or a glucose metabolite affect cell-cell adhesion and motility; these in turn affect whether a group stays together, loses cells, or even breaks up. NADPH-coupled aldehyde reductase reduces a wide variety of aldehydes to the corresponding alcohols, including converting glucose to sorbitol. The levels of this enzyme previously appeared to be regulated by CF. We find that disrupting alrA, the gene encoding aldehyde reductase, results in the loss of alrA mRNA and AlrA protein and a decrease in the ability of cell lysates to reduce both glyceraldehyde and glucose in a NADPH-coupled reaction. Counterintuitively, alrA¯ cells grow normally and have decreased glucose levels compared to parental cells. The alrA¯ cells form long unbroken streams and huge groups. Expression of AlrA in alrA¯ cells causes cells to form normal fruiting bodies, indicating that AlrA affects group size. alrA¯ cells have normal adhesion but a reduced motility, and computer simulations suggest that this could indeed result in the formation of large groups. alrA¯ cells secrete low levels of countin and CF50, two components of CF, and this could partially account for why alrA¯ cells form large groups. alrA¯ cells are responsive to CF and are partially responsive to recombinant countin and CF50, suggesting that disrupting alrA inhibits but does not completely block the CF signal transduction pathway. Gas chromatography/ mass spectroscopy indicates that the concentrations of several metabolites are altered in alrA¯ cells, suggesting that the Dictyostelium aldehyde reductase affects several metabolic pathways in addition to converting glucose to sorbitol. Together, our data suggest that disrupting alrA affects CF secretion, causes many effects on cellular metabolism, and has a major effect on group size. Submitted by: Richard Gomer [richard@rice.edu] ----------------------------------------------------------------------------- Uniform cAMP stimulation of Dictyostelium cells induces localized patches of signal transduction and pseudopodia Marten Postma, Jeroen Roelofs, Joachim Goedhart, Theodorus W.J. Gadella, Antonie J.W.G. Visser, and Peter J.M. Van Haastert Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, the Netherlands Molecular Biology of the Cell, in press ABSTRACT The chemoattractant cAMP induces the translocation of cytosolic PHCrac-GFP to the plasma membrane. PHCrac-GFP is a green fluorescent protein fused to a PH domain that presumably binds to phosphatydylinositol polyphosphates in the membrane. We determined the relative concentration of PHCrac-GFP in the cytosol and at different places along the cell boundary. In cells stimulated homogeneously with 1 microM cAMP we observed two distinct phases of PHCrac-GFP translocation. The first translocation is transient and occurs to nearly the entire boundary of the cell; the response is maximal at 6-8 seconds after stimulation and disappears after about 20 seconds. A second translocation of PHCrac-GFP starts after about 30 seconds and persists as long as cAMP remains present. Translocation during this second response occurs to small patches with radius of about 4-5 µm, each covering about 10 % of the cell surface. Membrane patches of PHCrac-GFP are both temporally and spatially closely associated with pseudopodia, which are extended at about 10 s from the area with a PHCrac-GFP patch. These signaling patches in pseudopodia of homogeneously stimulated cells resemble the single patch of PHCrac-GFP at the leading edge of a cell in a gradient of cAMP, suggesting that PHCrac-GFP is a spatial cue for pseudopod formation also in uniform cAMP. Submitted by: Peter J.M. Van Haastert [P.J.M.van.Haastert@chem.rug.nl] =============================================================================== [End Dicty News, volume 21, number 11]