dictyNews Electronic Edition Volume 39, number 32 November 2, 2013 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 dictyBase has a job opening! http://dictybase.org/dictybase_jobs.html ========= Abstracts ========= The Diaphanous-related formin dDia1 is required for highly directional phototaxis and formation of properly sized fruiting bodies in Dictyostelium Moritz Winterhoff (1), Alexander Junemann (1), Benjamin Nordholz (1), Jšrn Linkner (1), Michael Schleicher (2), and Jan Faix (1) (1) Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg Stra§e 1, 30625 Hannover, Germany; (2) Institute for Anatomy and Cell Biology, Ludwig-Maximilians-University, 80336 MŸnchen, Germany EJCB, in press Diaphanous-related formins (DRFs) act as downstream effectors of Rho family GTPases and drive the formation and elongation of linear actin filaments in various cellular processes. Here we analyzed the DRF dDia1 from Dictyostelium cells. The biochemical characterization of recombinant dDia1-FH1FH2 by bulk polymerization assays and single filament TIRF microscopy revealed that dDia1 is a rather weak nucleator. Addition of any of the three Dictyostelium profilin isoforms, however, markedly accelerated formin-mediated actin filament barbed end elongation in TIRF assays. Interestingly, filament elongation was significantly faster in presence of DdPFN I (profilin I) when compared to the other two isoforms, suggesting selectivity of dDia1 for DdPFN I. Additionally, we frequently observed dissociation of the formin from growing barbed ends. These findings are consistent with dilution-induced depolymerization assays in presence of dDia1-FH1FH2 showing that dDia1 is a weak capper in comparison with heterodimeric capping protein. To study the physiological role of this formin, we created cell lines lacking dDia1 or overexpressing GFP-tagged dDia1. Of note, constitutively active dDia1 accumulated homogenously in the entire pseudopod suggesting that it controls microfilament architecture to regulate cell migration. Comparison of wild type and dDia1-null cells in random cell migration and chemotaxis towards a cAMP gradient revealed no major differences. By contrast, phototaxis of dDia1-deficient cells during the multicellular stage was markedly impaired. While wild type slugs moved with high directionality towards the light source, the trails of dDia1-null slugs displayed a characteristic V-shaped profile and deviated in angles between 50¡-60¡ from the path of the incident light. Possibly in conjunction with this defect, dDia1-null cells also formed substantially smaller fruiting bodies. These findings demonstrate dDia1 to be critically involved in collective cell migration during terminal differentiation. Submitted by Jan Faix [faix.Jan@mh-hannover.de] --------------------------------------------------------------------------- A Dictyostelium cellobiohydrolase orthologue that affects developmental timing Mizuho Kunii 1, Mami Yasuno, Yuki Shindo and Takefumi Kawata * Department of Biology, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan 1 Present address: Department of Biological Production, Graduate School of Agriculture, Tokyo University of Agriculture and Technology, 3-8-1 Harumi-cho, Fuchu, Tokyo 183-8538, Japan Development Genes and Evolution, in press Dictyostelium discoideum is a facultative multicellular amoebozoan with cellulose in the stalk and spore coat of its fruiting body as well as in the extracellular matrix of the migrating slug. The organism also harbors a number of cellulase genes. One of them, cbhA, was identified as a candidate cellobiohydrolase gene based on the strong homology of its predicted protein product to fungal cellobiohydrolase I (CBHI). Expression of the cbhA was developmentally regulated, with strong expression in the spores of the mature fruiting body. However, a weak but detectable level of expression was observed in the extracellular matrix at the mound - tipped finger stages, in prestalk O cells, and in the slime sheath of the migrating slug - late culminant stages. A null mutant of the cbhA showed almost normal morphology. However, the developmental timing of the mutant was delayed by 2 - 4 hours. When a c-Myc epitope-tagged CbhA was expressed, it was secreted into the culture medium and was able to bind crystalline cellulose. The CbhA-myc protein was glycosylated, as demonstrated by its ability to bind succinyl concanavalin A-agarose. Moreover, conditioned medium from the cbhA-mycoe strain displayed 4-methylumbelliferyl beta-D-cellobioside (4-MUC) digesting activity in Zymograms in which conditioned medium was examined via native- polyacrylamide gel electrophoresis or spotted on an agar plate containing 4-MUC, one of the substrates of cellobiohydrolase. Taken together, these findings indicate that Dictyostelium CbhA is an orthologue of CBH I that is required for a normal rate of development. Submitted by Tekefumi Kawata [tkawata@bio.sci.toho-u.ac.jp] ============================================================== [End dictyNews, volume 39, number 32]