dictyNews Electronic Edition Volume 35, number 1 July 2, 2010 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 ========= A versatile set of tagged expression vectors to monitor protein localisation and function in Dictyostelium. Manu Dubin, Wolfgang Nellen, Abt. Genetik, Universität Kassel, 34132 Kassel, Germany Gene, in press We describe here a series of vectors for ectopic expression of tagged proteins in Dictyostelium discoideum. These vectors allow the addition of N- or C- terminal tags (GFP, mRFP, 3xFLAG, 3xHA, 6xMYC or TAP) with an optimised polylinker sequence and no additional amino acid residues at the N- or C-terminus of the protein. The expression cassettes were introduced into vectors containing Blasticidin or Geneticin resistance markers and into integrating as well as extrachromosomal plasmids. The vectors are designed as high and low copy versions and thus allow for a limited expression level control. They are also convenient with regard to complementation, co- and super-transformation. Finally the vectors share standardizsed cloning sites, so that a gene of interest can be easily transferred between vectors as experimental requirements evolve. These vectors were used to study the localisation of several putative RNA processing proteins including EriA and DicerB. Submitted by Wolfgang Nellen [nellen@uni-kassel.de] -------------------------------------------------------------------------------- Synergy between two transcription factors directs gene expression in Dictyostelium tip-organiser cells Hong Yu Wang and Jeffrey G. Williams College of Life Sciences, University of Dundee , Dow St., Dundee DD1 5EH, U. K. Int J Dev Biol, in press cotC requires the transcription factor CudA for its expression in the posterior, prespore cells of the slug while the expL7 gene requires CudA for its expression in the anterior, tip-organiser region. In order to identify additional transcription factors that might mediate tip-organiser specific expression, we performed affinity chromatography on slug nuclear extracts. The affinity matrix bore cap-site distal sequences from region A’ of the expL7 promoter; an essential region located upstream of the CudA binding domain. One of the proteins purified was GBF, a zinc finger transcription factor which binds to G-rich elements, known as G boxes, that are present in the promoters of many developmental genes, including cotC. Previous work identified an essential sequence motif within region A’ and we show that this element is a G box, that binds recombinant GBF. Moreover, a G box from within the cotC promoter can substitute for region A’ of expL7 in directing tip-organiser specific expression of expL7. Thus the same two transcription factors, CudA and GBF, seem to co-operate to direct both tip-organiser and prespore gene expression. How then is specificity achieved? Replacing a CudA binding region in the cotC promoter with the CudA binding domain from expL7 strongly represses cotC promoter activity. Hence we suggest that differences in the topology of the multiple CudA half- sites contained within the two different CudA binding regions, coupled with differences in the signalling environment between tip-organiser cells and prespore cells, ensure correct expL7 expression. Submitted by Jeff Williams [j.g.williams@dundee.ac.uk] -------------------------------------------------------------------------------- Analysis of Dictyostelium TACC reveals differential interactions with CP224 and unusual dynamics of Dictyostelium microtubules Matthias Samereier, Otto Baumann, Irene Meyer and Ralph Gräf* University of Potsdam, Institute for Biochemistry and Biology, Dept. of Cell Biology, Karl-Liebknecht-Strasse 24-25, Haus 26, 14476 Potsdam-Golm, Germany Cellular and Molecular Life Science, in press We have localized TACC to the microtubule-nucleating centrosomal corona and to microtubule plus ends. Using RNAi we proved that Dictyostelium TACC promotes microtubule growth during interphase and mitosis. For the first time we show in vivo that both TACC and XMAP215 family proteins can be differentially localized to microtubule plus ends during interphase and mitosis and that TACC is mainly required for recruitment of an XMAP215-family protein to interphase microtubule plus ends but not for recruitment to centrosomes and kinetochores. Moreover, we have now a marker to study dymamics and behavior of microtubule plus ends in living Dictyostelium cells. In a combination of live cell imaging of microtubule plus ends and fluorescence recovery after photobleaching (FRAP) experiments of GFP-alpha-tubulin cells we show that Dictyostelium microtubules are dynamic only in the cell periphery, while they remain stable at the centrosome, which also appears to harbor a dynamic pool of tubulin dimers. Submitted by Ralph Gräf [rgraef@uni-potsdam.de] ------------------------------------------------------------------------------ The exocytic gene secA is required for Dictyostelium cell motility and osmoregulation Roberto Zanchi, Gillian Howard, Mark S. Bretscher and Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK Journal of Cell Science, in press We investigated the link between plasma membrane recycling and cell movement using a fast-acting, temperature sensitive mutant of the Dictyostelium SecA exocytic protein. Strikingly, most mutant cells become almost paralyzed within minutes at the restrictive temperature. However, they can still sense cyclic-AMP gradients and polymerize actin up-gradient, but form only abortive pseudopodia, which cannot expand. They also relay a cyclic-AMP signal normally, suggesting that cyclic-AMP is released by a non-exocytic mechanism. To investigate why SecA is required for motility, we examined membrane trafficking in the mutant. Plasma membrane circulation is rapidly inhibited at the restrictive temperature and the cells acquire a prominent vesicle. Organelle-specific markers show this is an un-discharged contractile vacuole and we find the cells are correspondingly osmo-sensitive. Electron microscopy shows that many smaller vesicles, probably originating from the plasma membrane, also accumulate at the restrictive temperature. Consistent with this, the surface area of mutant cells shrinks. We suggest that SecA mutant cells cannot move at the restrictive temperature because their block in exocytosis results in a net uptake of plasma membrane, reducing its area, and so restricting pseudopodial expansion. This demonstrates the importance of proper surface area regulation in cell movement. Submitted by Rob Kay [rrk@mrc-lmb.cam.ac.uk] ------------------------------------------------------------------------------ The following article has been included because it contains experiments in human cells and Dictyostelium Strumpellin is a Novel VCP Binding Protein linking Hereditary Spastic Paraplegia to Protein Aggregation Diseases. Christoph S. Clemen1),#, Karthikeyan Tangavelou1),#, Karl-Heinz Strucksberg1), Steffen Just2), Linda Gaertner2), Hanna Regus-Leidig3), Maria Stumpf1), Jens Reimann4), Roland Coras5), Reginald O. Morgan6), Maria-Pilar Fernandez6), Andreas Hofmann7), Stefan Müller8), Benedikt Schoser9), Franz-Georg Hanisch8), Wolfgang Rottbauer2,10), Ingmar Blümcke5), Stephan von Hörsten11), Ludwig Eichinger1), Rolf Schröder5) 1) Institute of Biochemistry I, Medical Faculty, University of Cologne, Cologne, Germany 2) Department of Medicine III, University of Heidelberg, Heidelberg, Germany 3) Department of Biology, Animal Physiology, University of Erlangen-Nuremberg, Erlangen, Germany 4) Department of Neurology, University Hospital Bonn, Bonn, Germany 5) Institute of Neuropathology, University Hospital Erlangen, Erlangen, Germany 6) Department of Biochemistry and Molecular Biology, University of Oviedo and University Institute of Biotechnology of Asturias, Oviedo, Spain 7) Structural Chemistry Program, Eskitis Institute for Cell & Molecular Therapies, Griffith University, Brisbane, Australia 8) Institute of Biochemistry II, Medical Faculty, and Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany 9) Friedrich-Baur Institute, Department of Neurology, Ludwig-Maximilians University of Munich, Munich, Germany 10) Department of Medicine II, University of Ulm, Ulm, Germany 11) Franz-Penzoldt-Center, Experimental Therapy, Friedrich-Alexander-University of Erlangen-Nuremberg, Erlangen, Germany # Both authors contributed equally to this work. Brain, in press Mutations of the human VCP gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD). We identified strumpellin as a novel VCP binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate that strumpellin is a ubiquitously expressed protein present in cytosolic and endoplasmic reticulum cell fractions. Over-expression or ablation of wild-type strumpellin caused significantly reduced wound closure velocities in wound healing assays, whereas over-expression of the disease causing strumpellin N471D mutant showed no functional effect. Strumpellin knock-down experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth. Knock-down studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a pre-synaptic localization. We further identified strumpellin in pathological protein aggregates in IBMPFD, various myofibrillar myopathies, and in cortical neurons of a Huntington disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that mutant forms of strumpellin and VCP may have a concerted pathogenic role in various protein aggregate diseases. Submitted by Ludwig Eichinger [ludwig.eichinger@uni-koeln.de] ============================================================== [End dictyNews, volume 35, number 1]