dictyNews Electronic Edition Volume 42, number 9 March 18, 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 ========= CP91 is a component of the Dictyostelium centrosome involved in centrosome biogenesis Sascha Putzler; Irene Meyer; Ralph Gräf Eur. J. Cell Biol, in press The Dictyostelium centrosome is a model for acentriolar centrosomes and it consists of a three-layered core structure surrounded by a corona harboring microtubule nucleation complexes. Its core structure duplicates once per cell cycle at the G2/M transition. Through proteomic analysis of isolated centrosomes we have identified CP91, a 91- kDa coiled coil protein that was localized at the centrosomal core structure. While GFP-CP91 showed almost no mobility in FRAP experiments during interphase, both GFP-CP91 and endogenous CP91 dissociated during mitosis and were absent from spindle poles from late prophase to anaphase. Since this behavior correlates with the disappearance of the central layer upon centrosome duplication, CP91 is a putative component of this layer. When expressed as GFP-fusions, CP91 fragments corresponding to the central coiled coil domain and the preceding N- terminal part (GFP-CP91cc and GFP-CP91N, respectively) also localized to the centrosome but did not show the mitotic redistribution of the full length protein suggesting a regulatory role of the C-terminal domain. Expression of all GFP-fusion proteins suppressed expression of endogenous CP91 and elicited supernumerary centrosomes. This was also very prominent upon depletion of CP91 by RNAi. Additionally, CP91-RNAi cells exhibited heavily increased ploidy due to severe defects in chromosome segregation along with increased cell size and defects in the abscission process during cytokinesis. Our results indicate that CP91 is a central centrosomal core component required for centrosomal integrity, proper centrosome biogenesis and, independently, for abscission during cytokinesis. submitted by: Ralph Gräf [rgraef@uni-potsdam.de] ——————————————————————————————————————— Src1 is a protein of the inner nuclear membrane interacting with the Dictyostelium lamin NE81 Petros Batsios, Xiang Ren, Otto Baumann, Denis A. Larochelle, and Ralph Gräf* Cells, in press The nuclear envelope (NE) consists of the outer and inner nuclear membrane (INM), whereby the latter is bound to the nuclear lamina. Src1 is a Dictyostelium homologue of the helix-extension-helix family of proteins, which also includes the human lamin-binding protein MAN1. Both endogenous Src1 and GFP-Src1 localized to the NE during the entire cell cycle. Immuno electron microscopy and light microscopy after differential detergent treatment indicated that Src1 resides in the INM. FRAP experiments with GFP-Src1 cells suggested that at least a fraction of the protein could be stably engaged in forming the nuclear lamina together with the Dictyostelium lamin NE81. Both a BioID proximity assay and mis-localization of soluble, truncated mRFP-Src1 at cytosolic clusters consisting of an intentionally mis-localized mutant of GFP-NE81 confirmed an interaction of Src1 and NE81. Expression GFP-Src11-646, a fragment C-terminally truncated after the first transmembrane domain, disrupted interaction of nuclear membranes with the nuclear lamina, as cells formed protrusions of the NE that were dependent on cytoskeletal pulling forces. Protrusions were dependent on intact microtubules but not actin filaments. Our results indicate that Src1 is required for integrity of the NE and highlight Dictyostelium as a promising model for the evolution of nuclear architecture. submitted by: Ralph Gräf [rgraef@uni-potsdam.de] ——————————————————————————————————————— A High-Throughput, Multi-Cell Phenotype Assay for the Identification of Novel Inhibitors of Chemotaxis/Migration Xin-Hua Liao1,2, Netra Pal Meena2, Noel Southall3, Lunhua Liu4, Manju Swaroop3, Arina Li Zhang1, Jan Jian Xiang1, Carole A. Parent4, Wei Zheng3 & Alan R. Kimmel2 1Institute for Translational Medicine, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian 350108, China. 2Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, The National Institutes of Health, Bethesda, MD 20892, USA. 3Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, The National Institutes of Health, Bethesda, MD 20892, USA. 4Laboratory of Cellular and Molecular Biology, National Cancer Institute, The National Institutes of Health, Bethesda, MD 20892, USA. Scientific Reports, 2016 Chemotaxis and cell migration are fundamental, universal eukaryotic processes essential for biological functions such as embryogenesis, immunity, cell renewal, and wound healing, as well as for pathogenesis of many diseases including cancer metastasis and chronic inflammation. To identify novel chemotaxis inhibitors as probes for mechanistic studies and leads for development of new therapeutics,we developed a unique, unbiased phenotypic chemotaxis-dependent Dictyostelium aggregationassay for high-throughput screening using rapid, laser-scanning cytometry. Under defined conditions,individual Dictyostelium secrete chemoattractants, migrate, and aggregate. Chemotaxis is quantifiedby laser-scanning cytometry with a GFP marker expressed only in cells after chemotaxis/multi- cellaggregation. We applied the assay to screen 1,280 known compounds in a 1536-well plate format and identified two chemotaxis inhibitors. The chemotaxis inhibitory activities of both compounds wereconfirmed in both Dictyostelium and in human neutrophils in a directed EZ-TAXIscan chemotaxis assay.The compounds were also shown to inhibit migration of two human cancer cell lines in monolayerscratch assays. This test screen demonstrated that the miniaturized assay is extremely suited for highthroughputscreening of very large libraries of small molecules to identify novel classes of chemotaxis/migratory inhibitors for drug development and research tools for targeting chemotactic pathwaysuniversal to humans and other systems. submitted by: Xin-Hua Liao [xinhualiao@foxmail.com] ——————————————————————————————————————— The GATA transcription factor gene gtaG is required for terminal differentiation in Dictyostelium Mariko Katoh-Kurasawa*, Balaji Santhanam and Gad Shaulsky* J. Cell. Sci, in press The GATA transcription factor GtaG is conserved in Dictyostelids and essential for terminal differentiation in Dictyostelium discoideum, but its function is not well understood. Here we show that gtaG is expressed in prestalk cells at the anterior region of fingers and in the extending stalk during culmination. The gtaG– phenotype is cell-autonomous in prestalk cells and non-cell-autonomous in prespore cells. Transcriptome analyses reveal that GtaG regulates prestalk gene expression during cell differentiation before culmination and is required for progression into culmination. GtaG-dependent genes include genetic suppressors of the Dd-STATa-defective phenotype as well as Dd-STATa target-genes, including extra cellular matrix genes. We show that GtaG may be involved in the production of two culmination-signaling molecules, cyclic di-GMP and the spore differentiation factor SDF-1 and that addition of c-di-GMP rescues the gtaG– culmination and spore formation deficiencies. We propose that GtaG is a regulator of terminal differentiation that functions in concert with Dd-STATa and controls culmination through regulating c-di-GMP and SDF-1 production in prestalk cells. submitted by: Mariko Katoh-Kurasawa [mkatoh@bcm.edu] ============================================================== [End dictyNews, volume 42, number 9]