dictyNews Electronic Edition Volume 39, number 2 January 18, 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 ========= Asymmetric PTEN Distribution Regulated by Spatial Heterogeneity in Membrane-Binding State Transitions Satomi Matsuoka, Tatsuo Shibata, Masahiro Ueda PLOS Computational Biology, in press The molecular mechanisms that underlie asymmetric PTEN distribution at the posterior of polarized motile cells and regulate anterior pseudopod formation were addressed by novel single-molecule tracking analysis. Heterogeneity in the lateral mobility of PTEN on a membrane indicated the existence of three membrane-binding states with different diffusion coefficients and membrane-binding lifetimes. The stochastic state transition kinetics of PTEN among these three states were suggested to be regulated spatially along the cell polarity such that only the stable binding state is selectively suppressed at the anterior membrane to cause local PTEN depletion. By incorporating experimentally observed kinetic parameters into a simple mathematical model, the asymmetric PTEN distribution can be explained quantitatively to illustrate the regulatory mechanisms for cellular asymmetry based on an essential causal link between individual stochastic reactions and stable localizations of the ensemble. Submitted by Satomi Matsuoka [matsuoka@phys1.med.osaka-u.ac.jp] --------------------------------------------------------------------------- Dictyostelium ACAP-A is an ArfGAP involved in cytokinesis, cell migration and actin cytoskeleton dynamics Marco Dias, Cedric Blanc, Nelcy Thazar-Poulot, Sabrina Ben Larbi, Pierre Cosson, and Francois Letourneur Journal of Cell Science, in press ACAPs and ASAPs are Arf-GTPase-activating proteins with BAR, PH, GAP and ankyrin repeat domains and are known to regulate vesicular traffic and actin cytoskeleton dynamics in mammalian cells. The amoeba Dictyostelium has only two proteins with this domain organization instead of six in human, enabling a more precise functional analysis. Genetic invalidation of acapA, resulted in multinucleated cells with cytokinesis defects. Mutant acapA- cells were hardly motile and their multicellular development was significantly delayed. In addition, formation of filopodial protrusions was deficient in these cells. Conversely, re-expression of ACAP-A-GFP resulted in numerous and long filopodia-like protrusions. Mutagenesis studies showed that ACAP-A actin remodeling function was dependent on its ability to activate its substrate, the small GTPase ArfA. Likewise, the expression of a constitutively active ArfA¥GTP mutant in wild-type cells led to a significant reduction of filopodia length. Together our data support a role for ACAP-A in the control of the actin cytoskeleton organization and dynamics through an ArfA-dependent mechanism. Submitted by Francois Letourneur [francois.letourneur@univ-lyon1.fr] --------------------------------------------------------------------------- The precision with which single cells of Dictyostelium discoideum can locate a source of cyclic AMP. Abha Chopra(1) and Vidyanand Nanjundiah(2) (1)Institute for Immunology and Infectious Diseases, Murdoch University, Perth, WA 6150, Australia; (2)Department of Molecular Reproduction and Genetics, Indian Institute of Science, Bangalore 560012, India) Chaos, Solitons & Fractals, accepted When a starved wild-type amoeba of Dictyostelium discoideum is stimulated by cyclic AMP emanating from a nearby point source, it responds by putting out a hollow balloon-like membrane extension followed by a pseudopod. The effect of the stimulus is to influence the position where either of these protrusions is made on the cell rather than to cause them to be made. Because the pseudopod forms perpendicular to the cell surface, the site of formation is a measure of the precision with which a cell can locate the cAMP source. Cells beyond 1hr of starvation respond non-randomly with a precision that improves steadily thereafter. A cell that is starved for 1-2hrs can locate the source accurately 43% of the time; the corresponding figure for a cell starved for 6-7hrs is 87%. The response always displays scatter; and population-level heterogeneity reflects stochasticity in the behaviour of a single cell. From the angular distribution of the response its maximum information content is estimated to be 2 to 3 bits. In summary, we quantitatively demonstrate the stochastic nature of the directional response and the increase in its accuracy over time. Submitted by Vidyanand Nanjundiah [vidya@ces.iisc.ernet.in] --------------------------------------------------------------------------- Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL. Hykollari A, Balog CI, Rendic; D, Braulke T, Wilson IB, Paschinger K. J Proteome Res. 2013 Jan 15. [Epub ahead of print] The HL241 mutant strain of the cellular slime mould Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimised analytical workflow, considering that mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed phase HPLC and analysed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core alpha1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate and sulphate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism. Submitted by Iain Wilson [iain.wilson@boku.ac.at] --------------------------------------------------------------------------- Dictyostelium development shows a novel pattern of evolutionary conservation Xianjun Tian, Joan E. Strassmann and David C. Queller Molecular Biology and Evolution, in press Von BaerÕs law states that early stages of animal development are the most conserved. More recent evidence supports a modified "hourglass" pattern in which an early but somewhat later stage is most conserved. Both patterns have been explained by the relative complexity of either temporal or spatial interactions; the greatest conservation and lowest evolvability occur at the time of the most complex interactions, because these cause larger effects that are harder for selection to alter. This general kind of explanation might apply universally across independent multicellular systems, as supported by the recent finding of the hourglass pattern in plants. We use RNA-seq expression data from the development of the slime mold Dictyostelium to demonstrate that it does not follow either of the two canonical patterns but instead tends to show the strongest conservation and weakest evolvability late in development. We propose that this is consistent with a version of the spatial constraints model, modified for organisms that never achieve a high degree of developmental modularity. Submitted by David Queller [queller@biology2.wustl.edu] --------------------------------------------------------------------------- A non-mitotic CENP-E homolog in Dictyostelium discoideum with slow motor activity Suleyman Kosem (1,3), Zeynep Okten (1,3), Thi-Hieu Ho (1), Gudrun Trommler (1), Michael P. Koonce (2), Matthias Samereier (1), Annette Mueller-Taubenberger (1) (1) Institute for Anatomy and Cell Biology, Ludwig Maximilian University of Munich, Schillerstr. 42, 80336 Munich, Germany (2) 2Division of Translational Medicine, Wadsworth Center, Empire State Plaza, Albany, New York 12201-0509, USA (3) Present address: Physics Department E22, Technical University Munich, James-Franck-Str. 1, 85748 Garching, Germany Biochem. Biophys. Res. Comm., in press Kinesins are ATP-dependent molecular motors that mediate unidirectional intracellular transport along microtubules. Dictyostelium discoideum has 13 different kinesin isoforms including two members of the kinesin-7 family, Kif4 and Kif11. While Kif4 is structurally and functionally related to centromere-associated CENP-E proteins involved in the transport of chromosomes to the poles during mitosis, the function of the unusually short CENP-E variant Kif11 is unclear. Here we show that orthologs of short CENP-E variants are present in plants and fungi, and analyze functional properties of the Dictyostelium CENP-E version, Kif11. Gene knockout mutants reveal that Kif11 is not required for mitosis or development. Imaging of GFP-labeled Kif11 expressing Dictyostelium cells indicates that Kif11 is a plus-end directed motor that accumulates at microtubule plus ends. By multiple motor gliding assays, we show that Kif11 moves with an average velocity of 38 nm per second, thus defining Kif11 as a very slow motor. The activity of the Kif11 motor appears to be modulated via interactions with the non-catalytic tail region. Our work highlights a subclass of kinesin-7-like motors that function outside of a role in mitosis. Submitted by [Annette Mueller-Taubenberger [amueller@lrz.uni-muenchen.de] --------------------------------------------------------------------------- Temporal and non-permanent division of labor during sorocarp formation in the social amoeba Acytostelium subglobosum Kurato Mohri, Yu Kiyota, Hidekazu Kuwayama and Hideko Urushihara* Developmental Biology, in press Somatic cell differentiation is crucial for the development of multicellular organisms. While the development of a fruiting body in Dictyostelium discoideum represents a simple model of this process with separation of stalk cells from the spore lineage, that of Acytostelium subglobosum is not accompanied by cell type separation. This species produces acellular stalks and, seemingly, all aggregated amoebae become spores; however, it possesses homologs for the stalk-cell marker genes of D. discoideum. In this study, we analyzed the spatio-temporal expression of A. subglobosum orthologs for D. discoideum stalk- or spore-lineage markers to clarify the developmental process of A. subglobosum. We first found that the prespore vesicles, which contained spore coat proteins, started to accumulate in the tip region and were observed in the entire sorogen throughout later development, confirming that all A. subglobosum cells became spores. The expression of a stalk-lineage gene ortholog, As-ecmA, started at the mound stage and was prominent in the protruding sorogen. Although two spore- lineage gene orthologs, As-cotD1 and -cotD2, were likewise detected shortly after cell aggregation and increased in intensity until tip formation, their expression diminished in the protruding sorogen. Double-fluorescence staining of these prestalk and prespore marker genes revealed that the expression of these marker genes was mutually exclusive and that expression switching occurred in the early tip. Our results indicate that A. subglobosum cells become committed to the spore lineage first, and then, while keeping this commitment intact, participate in stalk formation. Instead of the permanent division of labor observed in D. discoideum, A. subglobosum produces fruiting bodies by all cells contributing to the formation of the stalk as well as forming spores. Submitted by Hideko Urushihara [hideko@biol.tsukuba.ac.jp] ============================================================== [End dictyNews, volume 39, number 2]