Elucidating the role of Dictyostelium discoideum SadA protein in cell-substrate adhesion

s for Talks Overexpression of the Dictyostelium discoideum paxB gene interferes with normal cell-cell cohesion, cell-substrate adhesion, and cell sorting M. Berenice Duran, Derrick Brazill M. Berenice Duran The development of the eukaryote Dictyostelium discoideum displays many of the features of animal embryogenesis such as regulated cell-cell adhesion and morphogenesis. As in animals, Dictyostelium cell adhesion molecules have a mechanical function. As a result, these molecules may interact with the signal-transduction processes governing development. One such mammalian protein is the focal adhesion molecule Paxillin. Paxillin functions as a docking site on the plasma membrane for signaling and structural proteins. To gain a better understanding of the regulation of paxillin, we are studying the Dictyostelium discoideum orthologue paxB. Using the Tet-off system we created an inducible overexpressor line of paxB. PaxB overexpressing cells (PaxBOE) exhibit an increase in cell-cell clumping, both in non-nutrient buffer and in HL-5. This clustering behavior is indicative of a possible cell-cell cohesion activity. In contrast, PaxBOE cells are less adhesive to the substratum when starved in non-nutrient buffer, suggesting a possible role in cell-substrate adhesion. PaxBOE cells aggregate to form mounds, but subsequent morphogenesis is blocked. However, addition of 20% wild type cells is able to rescue fruiting body formation. This suggests a non-cell autonomous role for paxB. In these chimeras, wild-type cells are predominantly localized to the anterior one-third of the slug, and the middle section of the spore mass. In addition, fewer PaxBOE cells were found in the spore mass. Taken together this suggests paxB plays a role in cell differentiation. We propose that the PaxB protein is required for proper cell-cell cohesion, cell-substrate adhesion, cell sorting and development passed the mound stage. Department of Biological Sciences, Center for the Study of Gene Structure and Function, Hunter College, City University of New York 695 Park Avenue, New York, NY 10021 I : 1 Cell Adhesion on 9/18/2006 from 8:30 to 9:20. Chaired by Adam Kuspa. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA Elucidating the role of Dictyostelium discoideum SadA protein in cell-substrate adhesion Anthony S. Kowal, Geraldine V.Amargo, Rex L. Chisholm Anthony S. Kowal How cells interact with substrates such as food particles or the surfaces along which they migrate, is a very important question in cell and cancer biology. The social amoeba Dictyostelium discoideum has proven to be a useful model in which to address these questions, due in part to the ease of genetic manipulation. One of the proteins found to be important for cell-substrate adhesion is SadA. SadA is a predicted 9-pass transmembrane protein, resides in the plasma membrane, and includes features present in other well characterized mammalian proteins known to have a role in substrate adhesion. SadA mutant cells are deficient in phagocytosis, cell-substrate adhesion, mitosis and exhibit disorganized actin cytoskeletons. To begin to understand how SadA mediates substrate adhesion, we focused in on SadA’s C-terminal cytoplasmic tail. The 32 amino-acid long tail contains a number of serine residues which are highly predicted to be phosphorylated, suggesting that this may be a region for protein-protein interactions. We began by mutating S943, S944, and S950 to either alanine (to mimic a non-phosphorylated state) or glutamic acid (to mimic a phosphorylated state), and attempted to rescue SadA null cells. Both constructs were capable of rescuing the SadA null phenotype equally well, suggesting that these sites are not important for SadA function. We are now focusing our efforts on S924,S925, and S940,S941 to determine if these residues are important for SadA function. Our studies with Dictyostelium will help us to understand substrate adhesion in an evolutionary less complex eukaryote. Northwestern University, Chicago, IL I : 2 Cell Adhesion on 9/18/2006 from 8:30 to 9:20. Chaired by Adam Kuspa. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA Microsatellite evolution; mutation rates and variability David Queller, Clea Scala, Sara Middlemist, Ryan McMullen, Marcus Kronforst, Natasha Mehdiabadi, Katie Stephens, Prince Buzombo, Joan Strassmann David Queller The genome paper showed D. discoideum to be very rich in microsatellite repeats, constituting over 10% of the genome. Most surprising was the large number of trinucleotide repeats inside genes that coded for long stretches of amino acids in proteins. For example, over 2000 genes had runs of 20 or more glutamines or asparagines, and several other amino acids, usually small polar ones, were commonly represented. Why does D. discoideum have so many coding repeats? Do they play some functional role, or are simply the result of the forces of mutation and drift that drive microsatellite evolution in non-coding regions? To begin to address these questions, we studied both their mutational rates, and their variability in natural populations. We first hypothesized that unusually high microsatellite mutation rates might drive microsatellite expansion. Mutations that add or delete repeats are thought to occur by slippage during replication. We estimated mutation rates for 52 loci via a mutation accumulation experiment with 90 lines taken through about 1000 cell generations and 70 single cell bottlenecks that fix mutations. The estimated slippage mutation rate was actually lower than for most organisms: 6.37 x 10. For loci with fewer than 40 repeats, the rate was even lower: 8.49 x 10. Therefore, high mutation rates do not drive the extraordinary abundance of microsatellites. Variability in natural populations might provide insight into selection on these loci. We predicted that, if selection acts strongly on these repeats, alleles should be distributed rather tightly around an optimal number. We first examined natural variation in detail at three loci that each had 2 or more long amino acid repeats: dimA, atg1, and yakA. Repeat number quite variable in all repeat regions suggesting that selection does not act strongly on repeat number. To get a more powerful signal of selection, we compared 50 pairs of repeat loci, one member of each pair inside a coding region and the other nearby but in a non-coding region. Pairs were matched for repeat number and motif. We expected selection to make the repeats less variable in coding regions, but there was no significant difference between coding and non-coding regions, indicating that selection is not acting strongly on these coding sequences, or that it acting equally strongly in coding and non-coding regions. The extreme AT-richness of the D. discoideum genome may have played an important role. The reduced sequence complexity in AT-rich (or CG-rich) genomes means that more small repeat sequences will be formed by random substitutions, which are then subject to slippage mutation and drift. It is possible that D. discoideum has evolved its low slippage mutation rates to help protect against an unusually high input of microsatellite repeats. Rice University, Houston, TX II : 1 Genome Analysis on 9/18/2006 from 9:20 to 12:10. Chaired by Ludwig Eichinger. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA Copy number variation between Dictyostelium discoideum strains Gareth Bloomfield, Jason Skelton, Yoshi Tanaka, Al Ivens, and Robert Kay Robert Kay We have found that duplications typically of many kilobases in length are common amongst laboratory strains of Dictyostelium, and could underlie much of the phenotypic differences observed between them. The genome sequence of Ax4 (and Ax3, and the strains derived from them) famously contains a single large inverted duplication of approximately 750 kilobases on chromosome 2. Previous genetic studies had also found evidence for copy number variation (CNV) in other strains. We have used array comparative genomic hybridisation (array CGH) to survey the genomes of various laboratory strains and wild isolates. Large duplications are widespread among laboratory strains, at diverse, apparently random sites in the genome. Strains affected, apart from Ax4, include Ax2, DH1 and NC4 itself. In contrast, most recent wild isolates appear to lack these large rearrangements, but provide evidence of polymorphic or deleted loci. Smaller scale copy number polymorphisms are also found, and can be informative in tracking lines of descent in the history of laboratory strains. Strikingly, the increased gene-dosage of duplicated genes causes a roughly corresponding increase in RNA levels, potentially leading to functional, phenotypic alterations in affected strains. CNV may also make gene disruption problematic if two copies instead of one have to be targeted. We have identified lines that are free of detectable duplications; the use of such strains (as well as careful maintenance of stocks) should facilitate comparison of results obtained in different laboratories. Sanger Centre and MRC Laboratory of Molecular Biology, Cambridge, UK II : 2 Genome Analysis on 9/18/2006 from 9:20 to 12:10. Chaired by Ludwig Eichinger. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA DNA Methylation and epigenetic gene silencing in Dictyostelium. Branimira Borisova, Blaga Popova, Balint Foeldesi, Markus Kaller, Xiaoxiao Zhang, Manu Dubin, Christian Hammann and Wolfgang Nellen Wolfgang Nellen With the detection of DNA methylation Dictyostelium has become an interesting model system to study chromatin remodelling. Similar to Drosophila, dnmA is the only DNA methyltransferase gene in the genome and responsible for a low but highly specific level of asymmetric C-methylation. The in vivo function of Dnmt2-like methyltransferases is controversially discussed [1, 2] but it now appears clear that they contribute to silencing of retroelements [3] and probably to the regulation of other protein coding genes. The question how specific asymmetric C residues are recognized and (de novo) methylated is still open. We provide evidence that DNA methylation is linked to the RNA interference pathway but that different mechanisms regulate silencing or downregulation of different genomic loci. RNA interference is believed to be a self reinforcing gene silencing machinery that amplifies the initial dsRNA signal by RdRPs and eventually leads to degradation of specific mRNAs. We have shown that RNAi is stringently controlled and that silencing depends on a minimal threshold level of transcribed dsRNA. Surprisingly, primary siRNAs are rapidly lost and only secondary siRNAs (RdRP products) are detected by Northern blot [4]. The function of Argonaute proteins, the RNase EriA and the RNA helicase HelF on RNAi regulation will be discussed1. Goll, M.G., et al., Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science, 2006. 311(5759): p. 395-8. 2. Jeltsch, A., W. Nellen, and F. Lyko, Two substrates are better than one: dual specificities for Dnmt2 methyltransferases. Trends Biochem Sci, 2006. 3. Kuhlmann, M., et al., Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi. Nucleic Acids Res, 2005. 33(19): p. 6405-17. 4. Popova, B., et al., HelF, a putative RNA helicase acts as a nuclear suppressor of RNAi but not antisense mediated gene silencing. Nucleic Acids Res, 2006. 34(3): p. 773-84. Dept. Genetics, Kassel University, 34132 Kassel, Germany II : 3 Genome Analysis on 9/18/2006 from 9:20 to 12:10. Chaired by Ludwig Eichinger. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA Process engineering high throughput restriction enzyme insertional mutagenesis in Dictyostelium Jie Song, Chris Dinh, Adam Kuspa, and Richard Sucgang Richard Sucgang Large scale systematic mutagenesis is an essential tool for the functional characterization of any completely sequenced genome, and this is no different for Dictyostelium discoideum. We have been concentrating our efforts on high-throughput insertional mutagenesis through restriction enzyme mediated integration (REMI), and have constructed an efficient pipeline for producing and cataloging mutants. We have been distributing knockout plasmids to our colleagues from this collection through our website (http://dictygenome.org) for the last few years. We will describe the functional aspects of the pipeline design, which codifies the different outcomes of REMI, and abstracts away many sources of human error. Mining the data from the production workflow has unearthed evidence of mutational hotspots in REMI. Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA II : 4 Genome Analysis on 9/18/2006 from 9:20 to 12:10. Chaired by Ludwig Eichinger. Dicty2006, Sept 17-22, 2006, Santa Fe, New Mexico, USA