dictyNews Electronic Edition Volume 42, number 29 December 9, 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 ========= Actin Binding Domain of Filamin Distinguishes Posterior from Anterior Actin Filaments in Migrating Dictyostelium Cells Keitaro Shibata, Akira Nagasaki, Hiroyuki Adachi, Taro Q.P. Uyeda Biophysics and Physicobiology, in press Actin filaments in different parts of a cell interact with specific actin binding proteins (ABPs) and perform different functions in a spatially regulated manner. However, the mechanisms of those spatially-defined interactions have not been fully elucidated. If the structures of actin filaments differ in different parts of a cell, as suggested by previous in vitro structural studies, ABPs may distinguish these structural differences and interact with specific actin filaments in the cell. To test this hypothesis, we followed the translocation of the actin binding domain of filamin (ABDFLN) fused with photoswitchable fluorescent protein (mKikGR) in polarized Dictyostelium cells. When ABDFLN-mKikGR was photoswitched in the middle of a polarized cell, photoswitched ABDFLN-mKikGR rapidly translocated to the rear of the cell, even though actin filaments were abundant in the front. The speed of translocation (>3 µm/s) was much faster than that of the retrograde flow of cortical actin filaments. Rapid translocation of ABDFLN-mKikGR to the rear occurred normally in cells lacking GAPA, the only protein, other than actin, known to bind ABDFLN. We suggest that ABDFLN recognizes a certain feature of actin filaments in the rear of the cell and selectively binds to them, contributing to the posterior localization of filamin. submitted by: Taro Uyeda [t-uyeda@waseda.jp] ——————————————————————————————————————— A novel HECT Ubiquitin Ligase Regulating Chemotaxis and Development in Dictyostelium discoideum Barbara Pergolizzi, Enrico Bracco and Salvatore Bozzaro J Cell Sci, in press Cyclic AMP binding to G protein-coupled receptors orchestrates chemotaxis and development in Dictyostelium. By activating the RasC-TORC2-AKT/PKB module, cAMP regulates cell polarization during chemotaxis. TORC2 also mediates GPCR- dependent stimulation of adenylyl cyclase A (ACA), enhancing cAMP relay and developmental gene expression. Thus, mutants defective in the TORC2 Pia/Rictor subunit are impaired in chemotaxis and development. Near-saturation mutagenesis of a Pia/Rictor mutant by random gene disruption led to selection of two suppressor mutants, in which spontaneous chemotaxis and development were restored. PKB phosphorylation and chemotactic cell polarization were rescued, whereas Pia/Rictor- dependent ACA stimulation was not restored but bypassed, leading to cAMP- dependent developmental gene expression. Knocking out the gene encoding the adenylylcyclase B (ACB) in the parental strain showed ACB to be essential for this process. The gene tagged in the suppressor mutants encodes a novel HECT ubiquitin ligase, homologous to mammalian HERC1, but harbouring a pleckstrin homology domain. Expression of the isolated HECTwt, but not HECTC5185S, domain was sufficient to reconstitute the parental phenotype. The novel ubiquitin ligase appears to regulate cell sensitivity to cAMP signalling and TORC2-dependent PKB phosphorylation. submitted by: Salvo Bozzaro [salvatore.bozzaro@unito.it] ——————————————————————————————————————— Actin-Interacting Protein 1 Contributes to Intranuclear Rod Assembly in Dictyostelium discoideum Hellen C. Ishikawa-Ankerhold, Wioleta Daszkiewicz, Michael Schleicher, and Annette Müller-Taubenberger Scientific Reports, in press Intranuclear rods are aggregates consisting of actin and cofilin that are formed in the nucleus in consequence of chemical or mechanical stress conditions. The formation of rods is implicated in a variety of pathological conditions, such as certain myopathies and some neurological disorders. It is still not well understood what exactly triggers the formation of intranuclear rods, whether other proteins are involved, and what the underlying mechanisms of rod assembly or disassembly are. In this study, Dictyostelium discoideum was used to examine appearance, stages of assembly, composition, stability, and dismantling of rods. Our data show that intranuclear rods, in addition to actin and cofilin, are composed of a distinct set of other proteins comprising actin-interacting protein 1 (Aip1), coronin (CorA), filactin (Fia), and the 34 kDa actin-bundling protein B (AbpB). A finely tuned spatio-temporal pattern of protein recruitment was found during formation of rods. Aip1 is important for the final state of rod compaction indicating that Aip1 plays a major role in shaping the intranuclear rods. In the absence of both Aip1 and CorA, rods are not formed in the nucleus, suggesting that a sufficient supply of monomeric actin is a prerequisite for rod formation. submitted by: Annette Müller-Taubenberger [amueller@lrz.uni-muenchen.de] ——————————————————————————————————————— The long non-coding RNA transcriptome of Dictyostelium discoideum development Rafael D. Rosengarten, Balaji Santhanam, Janez Kokosar and Gad Shaulsky Baylor College of Medicine, Houston, TX, USA Accepted for publication in G3: Genes, Genomes, Genetics Dictyostelium discoideum live in the soil as single cells, engulfing bacteria and growing vegetatively. Upon starvation, tens of thousands of amoebae enter a developmental program that includes aggregation, multicellular differentiation, and sporulation. Major shifts across the protein-coding transcriptome accompany these developmental changes. However, no study has presented a global survey of long non-coding RNAs in D. discoideum. To characterize the antisense and long intergenic non-coding RNA transcriptome, we analyzed previously published developmental time course samples using an RNA-sequencing library preparation method that selectively depletes ribosomal RNAs. We detected the accumulation of transcripts for 9,833 protein-coding messenger RNAs, 621 long intergenic non- coding RNAs and 162 putative antisense RNAs. The non-coding RNAs were interspersed throughout the genome, and were distinct in expression level, length and nucleotide composition. The non-coding transcriptome displayed a temporal profile similar to the coding transcriptome, with stages of gradual change interspersed with larger leaps. The transcription profiles of some non-coding RNAs were strongly correlated with known differentially expressed coding RNAs, hinting at a functional role for these molecules during development. Examining the mitochondrial transcriptome, we modeled two novel antisense transcripts. We applied yet another ribosomal depletion method to a subset of the samples to better retain tRNA transcripts. We observed polymorphisms in tRNA anticodons that suggested a post-transcriptional means by which D. discoideum compensates for codons missing in the genomic complement of tRNAs. We concluded that the prevalence and characteristics of long non-coding RNAs indicate these molecules are relevant to the progression of molecular and cellular phenotypes during development. submitted by: Gad Shaulsky [gadi@bcm.edu] ============================================================== [End dictyNews, volume 42, number 29]