dictyNews Electronic Edition Volume 42, number 14 May 20, 2016 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. Follow dictyBase on twitter: http://twitter.com/dictybase ========= Abstracts ========= Dictyostelium EHD associates with Dynamin and participates in phagosome maturation Aurélie Gueho1, Cristina Bosmani1, Navin Gopaldass2, Virginie Molle3, Thierry Soldati1, and François Letourneur3* 1Department of Biochemistry, University of Geneva, CH-1211 Geneva, Switzerland 2Department of Biochemistry, University of Lausanne, CH-1066 Epalinges, Switzerland 3Laboratoire de Dynamique des Interactions Membranaires Normales et Pathologiques, Université de Montpellier, CNRS, UMR 5235, Place Eugène Bataillon, 34095 Montpellier Cedex 05, France. *Author for correspondence (francois.letourneur@univ-montp2.fr) Journal of Cell Science, in press C-terminal EHDs (Eps15 homology-domain-containing proteins) are newly identified key regulators of endosomal membrane trafficking. Here we show that D. discoideum contains a single EHD protein that localizes to endosomal compartments and newly formed phagosomes. We provide the first evidence that EHD regulates phagosome maturation. Deletion of EHD results in defects in intraphagosomal proteolysis and acidification. These defects are linked to early delivery of lysosomal enzymes and fast retrieval of the vacuolar H+-ATPase in maturing phagosomes. We also demonstrate that EHD physically interacts with DymA. Our results indicate that EHD and DymA can associate independently to endomembranes, and yet they share identical kinetics of phagosome recruitment and release during phagosome maturation. Functional analysis of ehd-, dymA-, and double dymA-/ehd- knock-out strains indicate that DymA and EHD play non-redundant independent functions in phagosome maturation. Finally, we show that the absence of EHD leads to increase tubulation of endosomes, indicating that EHD participates in the scission of endosomal tubules as reported for DymA. submitted by: François Letourneur [francois.letourneur@univ-montp2.fr] ——————————————————————————————————————— Two HAP2-GCS1 homologs responsible for gamete interactions in the cellular slime mold with multiple mating types: Implication for common mechanisms of sexual reproduction shared by plants and protozoa and for male-female differentiation Marina Okamoto, Lixy Yamada, Yukie Fujisaki, Gareth Bloomfield, Kentaro Yoshida, Hidekazu Kuwayama, Hitoshi Sawada, Toshiyuki Mori, and Hideko Urushihara Developmental Biology, in press Fertilization is a central event in sexual reproduction, and understanding its molecular mechanisms has both basic and applicative biological importance. Recent studies have uncovered the molecules that mediate this process in a variety of organisms, making it intriguing to consider conservation and evolution of the mechanisms of sexual reproduction across phyla. The social amoeba Dictyostelium discoideum undergoes sexual maturation and forms gametes under dark and humid conditions. It exhibits three mating types, type-I, -II, and -III, for the heterothallic mating system. Based on proteome analyses of the gamete membranes, we detected expression of two homologs of the plant fertilization protein HAP2-GCS1. When their coding genes were disrupted in type-I and type-II strains, sexual potency was completely lost, whereas disruption in the type-III strain did not affect mating behavior, suggesting that the latter acts as female in complex organisms. Our results demonstrate the highly conserved function of HAP2-GCS1 in gamete interactions and suggest the presence of additional allo-recognition mechanisms in D. discoideum gametes. submitted by: Hideko Urushihara [hideko@biol.tsukuba.ac.jp] ——————————————————————————————————————— Analysis of the microprocessor in Dictyostelium: the role of RbdB, a dsRNA binding protein. Doreen Meier, Janis Kruse, Jann Buttlar, Michael Friedrich, Fides Zenk, Benjamin Boesler, Konrad U. Förstner, Christian Hammann, Wolfgang Nellen. PLoS Genetics, accepted We identified the dsRNA binding protein RbdB as an essential component in miRNA processing in Dictyostelium discoideum. RbdB is a nuclear protein that accumulates, together with Dicer B, in nucleolar foci reminiscent of plant dicing bodies. Disruption of rbdB results in loss of miRNAs and accumulation of primary miRNAs. The phenotype can be rescued by ectopic expression of RbdB thus allowing for a detailed analysis of domain function. The lack of cytoplasmic dsRBD proteins involved in miRNA processing, suggests that both processing steps take place in the nucleus thus resembling the plant pathway. However, we also find features e.g. in the domain structure of Dicer which suggest similarities to animals. Reduction of miRNAs in the rbdB- strain and their increase in the Argonaute A knock out allowed the definition of new miRNAs one of which appears to belong to a new non-canonical class. submitted by: Wolfgang Nellen [nellen@uni-kassel.de] ——————————————————————————————————————— Mechanism and biological role of Dnmt2 in Nucleic Acid Methylation Albert Jeltsch, Ann Ehrenhofer-Murray, Tomasz Jurkowski, Frank Lyko, Gunter Reuter, Serge Ankri, Wolfgang Nellen, Matthias Schaefer & Mark Helm RNA Biology, in press A group of homologous nucleic acid modification enzymes called Dnmt2, Trdmt1, Pmt1, DnmA, and Ehmet in different model organisms catalyse the transfer of a methyl group from the cofactor S-adenosyl-methionine (SAM) to the carbon-5 of cytosine residues. Originally considered as DNA MTases, these enzymes were shown to be tRNA methyltransferases about a decade ago. Between the presumed involvement in DNA modification-related epigenetics, and the recent foray into the RNA modification field, significant progress has characterized Dnmt2-related research. Here, we review this progress in its diverse facets including molecular evolution, structural biology, biochemistry, chemical biology, cell biology and epigenetics. submitted by: Wolfgang Nellen [nellen@uni-kassel.de] ——————————————————————————————————————— Gene discovery by chemical mutagenesis and whole-genome sequencing in Dictyostelium Cheng-Lin Frank Li, Balaji Santhanam, Amanda Nicole Webb, Blaž Zupan,1 and Gad Shaulsky Baylor College of Medicine and University of Ljubljana Genome Research, in press Whole-genome sequencing is a useful approach for identification of chemical-induced lesions, but previous applications involved tedious genetic mapping to pinpoint the causative mutations. We propose that saturation mutagenesis under low mutagenic loads, followed by whole-genome sequencing, should allow direct implication of genes by identifying multiple independent alleles of each relevant gene. We tested the hypothesis by performing three genetic screens with chemical mutagenesis in the social soil amoeba Dictyostelium discoideum. Through genome sequencing, we successfully identified mutant genes with multiple alleles in near-saturation screens, including resistance to intense illumination and strong suppressors of defects in an allorecognition pathway. We tested the causality of the mutations by comparison to published data and by direct complementation tests, finding both dominant and recessive causative mutations. Therefore, our strategy provides a cost- and time-efficient approach to gene discovery by integrating chemical mutagenesis and whole-genome sequencing. The method should be applicable to many microbial systems, and it is expected to revolutionize the field of functional genomics in Dictyostelium by greatly expanding the mutation spectrum relative to other common mutagenesis methods. submitted by: Gad Shaulsky [gadi@bcm.edu] ——————————————————————————————————————— Uses and abuses of macropinocytosis Gareth Bloomfield and Robert R. Kay MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge, CB2 0QH, UK J. Cell Science, in press Macropinocytosis is a means by which eukaryotic cells ingest extracellular liquid and dissolved molecules. It is widely conserved amongst cells that can take on amoeboid form, and therefore appears to be an ancient feature that can be traced back to an early stage of evolution. Recent advances have highlighted how this endocytic process can be subverted during pathology: certain cancer cells use macropinocytosis to feed on extracellular protein, and many viruses and bacteria use it to enter host cells. Prion and prion-like proteins can also spread and propagate from cell to cell via macropinocytosis. Progress is being made towards using macropinocytosis therapeutically, either to deliver drugs to or cause cell death by inducing catastrophically rapid fluid uptake. Mechanistically, the Ras signalling pathway plays a prominent and conserved activating role in amoebae and in mammals; mutant amoebae with abnormally high Ras activity resemble tumour cells in their increased capacity for growth using nutrients ingested by macropinocytosis. This Commentary takes a functional and evolutionary perspective to highlight progress in understanding and utilising macropinocytosis, which is viewed as an ancient feeding process used by single-celled phagotrophs but now put to varied uses by metazoan cells and abused in disease states, including submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk] ============================================================== [End dictyNews, volume 42, number 14]