dictyNews Electronic Edition Volume 30, number 13 April 25, 2008 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. ========= Abstracts ========= Molecular and functional characterization of a Rho GDP dissociation i nhibitor in the filamentous fungus Tuber borchii Michele Menotta(1), Antonella Amicucci(1), Giorgio Basili(1), Emanuela Polidori(2), Vilberto Stocchi(1), Francisco Rivero(3) 1Istituto di Chimica Biologica “G. Fornaini”, Università degli Studi di Urbino, Urbino (PU) Italy. 2Istituto di Ricerca sull’Attività Motoria, Università degli Studi di Urbino, Urbino (PU) Italy. 3Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany, and The Hull York Medical School and Department of Biological Sciences, University of Hull. Hull, United Kingdom. BMC Microbiology, in press Background. Small GTPases of the Rho family function as tightly regulated molecular switches that govern important cellular functions in eukaryotes. Several families of regulatory proteins control their activation cycle and subcellular localization. Members of the guanine nucleotide dissociation inhibitor (GDI) family sequester Rho GTPases from the plasma membrane and keep them in an inactive form. Results. We report on the characterization the RhoGDI homolog of Tuber borchii Vittad., an ascomycetous ectomycorrhizal fungus. The Tbgdi gene is present in two copies in the T. borchii genome. The predicted amino acid sequence shows high similarity to other known RhoGDIs. Real time PCR analyses revealed an increased expression of Tbgdi during the phase preparative to the symbiosis instauration, in particular after stimulation with root exudates extracts, that correlates with expression of Tbcdc42. In a translocation assay TbRhoGDI was able to solubilize TbCdc42 from membranes. Surprisingly, TbRhoGDI appeared not to interact with S. cerevisiae Cdc42, precluding the use of yeast as a surrogate model for functional studies. To study the role of TbRhoGDI we performed complementation experiments using a RhoGDI null strain of Dictyostelium discoideum, a model organism where the roles of Rho signaling pathways are well established. For comparison, complementation with mammalian RhoGDI1 and LyGDI was also studied in the null strain. Although interacting with Rac1 isoforms, TbRhoGDI was not able to revert the defects of the D. discoideum RhoGDI null strain, but displayed an additional negative effect on the cAMP-stimulated actin polymerization response. Conclusions. T. borchii expresses a functional RhoGDI homolog that appears as an important modulator of cytoskeleton reorganization during polarized apical growth that antecedes symbiosis instauration. The specificity of TbRhoGDI actions was underscored by its inability to elicit a growth defect in S. cerevisiae or to compensate the loss of a D. discoideum RhoGDI. Knowledge of the cell signaling at the basis of cytoskeleton reorganization of ectomycorrhizal fungi is essential for improvements in the production of mycorrhized plant seedlings used in timberland extension programs and fruit body production. Submitted by: Francisco Rivero [francisco.rivero@uni-koeln.de] -------------------------------------------------------------------------------- Widespread duplications in the genomes of laboratory stocks of Dictyostelium discoideum Gareth Bloomfield, Yoshimasa Tanaka, Jason Skelton, Alasdair Ivens & Robert R. Kay MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 OQH, UK Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK Accepted, Genome Biology. Background: Duplications of stretches of the genome are an important source of individual genetic variation, but their unrecognized presence in laboratory organisms would be a confounding variable for genetic analysis. Results: We report here that duplications of 15 kb or more are common in the genome of the social amoeba Dictyostelium discoideum. Most stocks of the axenic ‘workhorse’ strains Ax2 and Ax3/4 obtained from different laboratories can be expected to carry different duplications. The auxotrophic strains DH1 and JH10 also bear previously unreported duplications. Strain Ax3/4 is known to carry a large duplication on chromosome 2 and this structure shows evidence of continuing instability; we find a further variable duplication on chromosome 5. These duplications are lacking in Ax2, which has instead a small duplication on chromosome 1. Stocks of the type isolate NC4 are similarly variable, though we have identified some approximating the assumed ancestral genotype. More recent wild-type isolates are almost without duplications, but we can identify small deletions or regions of high divergence, possibly reflecting responses to local selective pressures. Duplications are scattered through most of the genome, and can be stable enough to reconstruct genealogies spanning decades of the history of the NC4 lineage. The expression level of many duplicated genes is increased with dosage, but for others it appears that some form of dosage compensation occurs. Conclusions: The genetic variation described here must underlie some of the phenotypic variation observed between strains from different laboratories. We suggest courses of action to alleviate the problem. Submitted by: Gareth Bloomfield [garethb@mrc-lmb.cam.ac.uk] -------------------------------------------------------------------------------- Changing directions in the study of chemotaxis Robert R. Kay, Paul Langridge1, David Traynor and Oliver Hoeller Nature Reviews of Molecular Cell Biology, in press Chemotaxis — guided movement of cells in chemical gradients — probably first emerged in our single-celled ancestors and is recognisably similar in neutrophils and amoebae today. Chemotaxis enables immune cells to reach sites of infection, allows wounds to heal and is crucial for forming embryonic patterns; its manipulation may help alleviate disease states, including the metastasis of cancer cells. We discuss recent results concerning how cells orientate in chemotactic gradients and the role of PIP3, what produces the force for projecting pseudopodia, and a new role for the endocytic cycle in movement. Submitted by: Rob Kay [rrk@mrc-lmb.cam.ac.uk] -------------------------------------------------------------------------------- Persistent Cell Motion in the Absence of External Signals: A Search Strategy for Eukaryotic Cells Liang Li(1), Simon F. Nørrelykke(2), and Edward C. Cox(2) 1 Department of Physics, Princeton University, Princeton, New Jersey, USA 2 Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA PLoS One, in press Background Eukaryotic cells are large enough to detect signals and then orient to them by differentiating the signal strength across the length and breadth of the cell. Amoebae, fibroblasts, neutrophils and growth cones all behave in this way. Little is known however about cell motion and searching behavior in the absence of a signal. Is individual cell motion best characterized as a random walk? Do individual cells have a search strategy when they are beyond the range of the signal they would otherwise move toward? Here we ask if single, isolated, Dictyostelium and Polysphondylium amoebae bias their motion in the absence of external cues. Methodology We placed single well-isolated Dictyostelium and Polysphondylium cells on a nutrient-free agar surface and followed them at 10 sec intervals for ~10 hr, then analyzed their motion with respect to velocity, turning angle, persistence length, and persistence time, comparing the results to the expectation for a variety of different types of random motion. Conclusions We find that amoeboid behavior is well described by a special kind of random motion: Amoebae show a long persistence time (~10 min) beyond which they start to lose their direction; they move forward in a zig-zag manner; and they make turns every 1-2 min on average. They bias their motion by remembering the last turn and turning away from it. Interpreting the motion as consisting of runs and turns, the duration of a run and the amplitude of a turn are both found to be exponentially distributed. We show that this behavior greatly improves their chances of finding a target relative to performing a random walk. We believe that other eukaryotic cells may employ a strategy similar to Dictyostelium when seeking conditions or signal sources not yet within range of their detection system. Submitted by: Liang Li [liangl@princeton.edu] -------------------------------------------------------------------------------- Disruption of Four Kinesin Genes in Dictyostelium. D.K. Nag, I. Tikhonenko, I. Soga, and M.P. Koonce Accepted, BMC Cell Biology 2008, 9:21. Background Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans. Results We performed individual disruptions of the kinesin genes, kif4, kif8, kif10, and kif11. None of the motors encoded by these genes are essential for development or viability of Dictyostelium. Removal of Kif4 (kinesin-7; CENP-E family) significantly impairs the rate of cell growth and, when combined with a previously characterized dynein inhibition, results in dramatic defects in mitotic spindle assembly. Kif8 (kinesin-4; chromokinesin family) and Kif10 (kinesin-8; Kip3 family) appear to cooperate with dynein to organize the interphase radial microtubule array. Conclusions The results reported here extend the number of kinesin gene disruptions in Dictyostelium, to now total 10, among the 13 isoforms. None of these motors, individually, are required for short-term viability. In contrast, homologs of at least six of the 10 kinesins are considered essential in humans. Our work underscores the functional redundancy of motor isoforms in basal organisms while highlighting motor specificity in more complex metazoans. Since motor disruption in Dictyostelium can readily be combined with other motility insults and stresses, this organism offers an excellent system to investigate functional interactions among the kinesin motor family. Submitted by: Michael Koonce [koonce@wadsworth.org] ============================================================== [End dictyNews, volume 30, number 13]