dictyNews Electronic Edition Volume 39, number 26 September 13 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 ========= Abstracts ========= G17-modified Hammerhead Ribozymes are active in vitro and in vivo Anne Kalweit and Christian Hammann Ribogenetics@Biochemistry Lab, School of Engineering and Science, MoLife Research Center, Jacobs University Bremen, 28759 Bremen, Germany RNA, in press Natural hammerhead ribozymes (HHRz) feature tertiary interactions between hairpin loops or bulges in two of three helices that surround the catalytic core of conserved nucleotides. Their conservation was originally established on minimal versions lacking the tertiary interactions. While those sequence requirements in general also apply to natural versions, we show here differences for the HHRz cleavage site N17. A guanosine at this position strongly impairs cleavage activity in minimal versions, whereas we observe for the G17 variants of four tertiary stabilized HHRz significant cleavage and ligation activity in vitro. Kinetic analyses of these variants revealed reduced rate and extent of cleavage, compared to wild type sequences, while variants with distorted tertiary interactions cleaved at reduced rate, but to the same extent. Contrary to this, G17 variants exhibit similar in vitro ligation activity as compared to the respective wild type motif. To also address the catalytic performance of these motifs in vivo, we have inserted HHRz cassettes in the lacZ gene and tested this beta- galactosidase reporter in Dictyostelium discoideum. In colorimetric assays, we observe differences in the enzymatic activity of beta- galactosidase, which correlate well with the activity of the different HHRz variants in vitro, and which can be unambiguously attributed to ribozyme cleavage by primer extension analysis. Submitted by Christian Hammann [c.hammann@jacobs-university.de] --------------------------------------------------------------------------- Title: Abi is required for modulation and stability of the SCAR/WAVE complex, but not localization or activity. Andrew J. Davidson, Seiji Ura, Peter A. Thomason, Gabriela Kalna & Robert H. Insall Eukaryotic cell, in press The SCAR/WAVE complex drives actin-based protrusion, cell migration and cell separation during cytokinesis. However, the contribution of the individual complex members to the activity of the whole remains a mystery. This is primarily because complex members depend on one another for stability, which limits the scope for experimental manipulation. Several studies suggest that Abi, a relatively small complex member, connects signalling to SCAR/WAVE complex localization and activation through its poly-proline C-terminal tail. We generated a deletion series of the Dictyostelium Abi to investigate its exact role in regulation of the SCAR complex, and identified a minimal fragment that would stabilize the complex. Surprisingly, loss of either the N-terminus of Abi or the C-terminal polyproline tail confers no detectable defect in complex recruitment to the leading edge or formation of pseudopods. A fragment containing approximately 20% of Abi - and none of the sites that couple to known signaling pathways - allowed the SCAR complex to function with normal localisation and kinetics. However, expression of N-terminal Abi deletions exacerbated the cytokinesis defect of the Dictyostelium abi mutant, which was earlier shown to be caused by inappropriate activation of SCAR. This demonstrates - unexpectedly - that Abi does not mediate the SCAR complex's ability to make pseudopods, beyond its role in complex stability. Instead we propose that Abi has a modulatory role when the SCAR complex is activated through other mechanisms. Submitted by Andrew Davidson [a.davidson@beatson.gla.ac.uk] --------------------------------------------------------------------------- Dictyostelium lipid droplets host novel proteins Xiaoli Du1, Caroline Barisch1, Peggy Paschke1, Cornelia Herrfurth3, Oliver Bertinetti2, Nadine Pawolleck1, Heike Otto1, Harald Rźhling1, Ivo Feussner3, Friedrich W. Herberg2, and Markus Maniak1* 1Abt. Zellbiologie, 2Abt. Biochemie, UniversitŠt Kassel, Germany 3Abt. Biochemie der Pflanze, Georg-August-UniversitŠt Gšttingen, Germany Eukaryotic Cell, in press Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general it consists of a hydrophobic core of triglycerides and steryl-esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel proteins components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane. Submitted by Markus Maniak [maniak@uni-kassel.de] --------------------------------------------------------------------------- Identification of pentatricopeptide repeat proteins in the model organism Dictyostelium discoideum. Sam Manna, Jessica Brewster & Christian Barth Department of Microbiology, La Trobe University, VIC 3086, Australia. International Journal of Genomics, vol. 2013, Article ID 586498, doi:10.1155/2013/586498. Pentatricopeptide repeat (PPR) proteins are RNA binding proteins with functions in organelle RNA metabolism. They are found in all eukaryotes, but have been most extensively studied in plants. We report on the identification of 12 PPR-encoding genes in the genome of the protist Dictyostelium discoideum, with potential homologs in other members of the same lineage and some predicted novel functions for the encoded gene products in protists. For one of the gene products, we show that it localises to mitochondria and we also demonstrate that antisense inhibition of its expression leads to slower growth, a phenotype associated with mitochondrial dysfunction. Submitted by Christian Barth [c.barth@latrobe.edu.au] --------------------------------------------------------------------------- Tong Sun, Bohye Kim and Lou W. Kim* Department of Biological Sciences, Florida International University, Miami, FL, 33199, USA Glycogen Synthase Kinase 3 influences cell motility and chemotaxis by regulating PI3K membrane localization in Dictyostelium Develop. Growth Differ., in press Glycogen Synthase Kinase 3 (GSK3) is a multifunctional kinase involved in diverse cellular activities such as metabolism, differentiation, and morphogenesis. Recent studies showed that GSK3 in Dictyostelium affects chemotaxis via TorC2 pathway and Daydreamer. Now we report that GSK3 affects PI3K membrane localization, of which the mechanism has remained to be fully understood in Dictyostelium. The membrane localization domain (LD) of Phosphatidylinositol-3-kinase 1 (PI3K1) is phosphorylated on serine residues in a GSK3 dependent mechanism and PI3K1-LD exhibited biased membrane localization in gsk3 cells compared to the wild type cells. Furthermore, multiple GSK3-phosphorylation consensus sites exist in PI3K1-LD, of which phosphomimetic substitutions restored cAMP induced transient membrane localization of PI3K1-LD in gsk3 cells. Serine to alanine substitution mutants of PI3K1-LD, in contrast, displayed constitutive membrane localization in wild type cells. Biochemical analysis revealed that GSK3 dependent serine phosphorylation of PI3K1-LD is constitutive during the course of cAMP stimulation. Together, these data suggest that GSK3 dependent serine phosphorylation is a prerequisite for chemoattractant cAMP induced PI3K membrane localization. Submitted by Leung Kim [louwkim@icloud.com] ============================================================== [End dictyNews, volume 39, number 26]