Dicty News Electronic Edition Volume 18, number 3 February 23, 2002 Please submit abstracts of your papers as soon as they have been accepted for publication by sending them to dicty@northwestern.edu. Back issues of Dicty-News, the Dicty Reference database and other useful information is available at DictyBase--http://dictybase.org. ============= Abstracts ============= Phosphorylation Of The Myosin Regulatory Light Chain Plays A Role In Motility and Polarity During Dictyostelium Chemotaxis Hui Zhang(a), Deborah Wessels(a), Petra Fey(b), Karla Daniels(a), Rex L. Chisholm(b) and David R. Soll(a) (a)Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242 (b)Department of Cell and Molecular Biology Northwestern University Medical School, Chicago, IL 60611 J. Cell Sci., in press The myosin regulatory light chain (RLC) of Dictyostelium discoideum is phosphorylated at a single serine site in response to chemoattractant. To investigate the role of the phosphorylation of RLC in both motility and chemotaxis, mutants were generated in which the single phosphorylatable serine was replaced with a nonphosphorylatable alanine. Several independent clones expressing the mutant RLC in the RLC null mutant, mlcR-, were obtained. These S13A mutants were subjected to high resolution computer- assisted motion analysis to assess the basic motile behavior of cells in the absence of a chemotatic signal, and the chemotactic responsiveness of cells to the spatial, temporal and concentration components of natural cAMP waves. In the absence of a cAMP signal, mutant cells formed lateral pseudopods less frequently and crawled faster than wild type cells. In a spatial gradient of cAMP, mutant cells chemotaxed more efficiently than wild type cells. In the front of simulated temporal and natural waves of cAMP, mutant cells responded normally by suppressing lateral pseudopod formation. However, at the peak and in the back of either wave, mutant cells did not lose cellular polarity like wild type cells. Since depolarization at the peak and in the descending phase of the natural wave is necessary for efficient chemotaxis, this deficiency resulted in a decrease in the capacity of S13A mutant cells to track natural cAMP waves relayed by wild type cells, and in the fragmentation of streams late in mutant cell aggregation. These results reveal a regulatory pathway induced by the peak and back of the chemotactic wave that alters RLC phosphorylation and leads to cellular depolarization. We suggest that depolarization requires myosin II rearrangement in the cortex facilitated by RLC phosphorylation, which increases myosin motor function. ----------------------------------------------------------------------------- Element analysis of the Polysphondylium pallidum gp64 promoter Naohisa Takaoka1, Masashi Fukuzawa2, Atsushi Kato1, Tamao Saito1 and Hiroshi Ochiai1 1. Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, 060-0810 Japan and 2. Wellcome Trust Building, Department of Anatomy and Physiology, University of Dundee, Dow Street, Dundee, DD1 4HN, UK Biochim. Biophys. Acta, in press. gp64 mRNA in Polysphondylium pallidum is expressed extensively during vegetative growth, and begins to rapidly decrease at the onset of development. To examine this unique regulation, 5 deletion analysis of the gp64 promoter was undertaken, and two growth-phase activated elements have been found: a food-dependent, upstream regulatory region (FUR, -222 to 170) and a vegetatively activated, downstream region (VAD, -110 to 63). Here we concentrate our analysis on an A1 and A2 sequences in the FUR region: A1 consists of a GATTTTTTTA sequence called a corresponding sequence and A2 consists of the direct repeat TTTGTTGTG. The cells carrying a combined construct of A1 and A2 acted synergistically in a reporter activity. A point mutation analysis in A1 indicates that a G residue is required for the activation of A1. From analyses of promoter regulation in a liquid or a solid medium, the promoter activity of the cells fed on bacteria in A-medium (axenic medium for Polysphondylium) or grown in A-medium alone was only one-fourth of that of the cells fed on bacteria. ----------------------------------------------------------------------------- Quantitation of the distribution and flux of myosin-II during cytokinesis. Douglas N. Robinson#, Guy Cavet*, Hans M. Warrick and James A. Spudich Departments of Biochemistry and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305-5307 Email: Douglas.Robinson@jhu.edu; jspudich@cmgm.stanford.edu #Current address: Department of Cell Biology, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Baltimore, MD 21205 *Current address: Rosetta Inpharmatics, 12040 115th Ave NE, Kirkland, WA 98034. These two authors contributed equally to this work. BMC Cell Biology 3:4, 2002. Background: During cytokinesis, the cell's equator contracts against the cell's global stiffness. Identifying the biochemical basis for these mechanical parameters is essential for understanding how cells divide. To achieve this goal, the distribution and flux of the cell division machinery must be quantified. Here we report the first quantitative analysis of the distribution and flux of myosin-II, an essential element of the contractile ring. Results: The fluxes of myosin-II in the furrow cortex, the polar cortex, and the cytoplasm were examined using ratio imaging of GFP fusion proteins expressed in Dictyostelium. The peak concentration of GFP-myosin-II in the furrow cortex is 1.8-fold higher than in the polar cortex and 2.0-fold higher than in the cytoplasm. The myosin-II in the furrow cortex, however, represents only 10% of the total cellular myosin-II. An estimate of the minimal amount of this motor needed to produce the required force for cell cleavage fits well with this 10% value. The cell may, therefore, regulate the amount of myosin-II sent to the furrow cortex in accordance with the amount needed there. Quantitation of the distribution and flux of a mutant myosin-II that is defective in phosphorylation-dependent thick filament disassembly confirms that heavy chain phosphorylation regulates normal recruitment to the furrow cortex. Conclusion: The analysis indicates that myosin-II flux through the cleavage furrow cortex is regulated by thick filament phosphorylation. Further, the amount of myosin-II observed in the furrow cortex is in close agreement with the amount predicted to be required from a simple theoretical analysis. ----------------------------------------------------------------------------- Outside-In Signaling of Cellulose Synthesis by a Spore Coat Protein in Dictyostelium Christopher M. West, Ping Zhang, Aiko C. McGlynn and Lee Kaplan Dept. of Anatomy and Cell Biology, College of Medicine, University of Florida, Gainesville, FL 32610-0235 USA Eukaryotic Cell, in press. ABSTRACT The spore coat of Dictyostelium is formed de novo from proteins secreted from vesicles and cellulose synthesized across the plasma membrane as differentiating spores rise up the stalk. The mechanism by which these events are coordinated is not understood. In the course of experiments designed to test the function of the inner layer coat protein SP85 (PsB), expression of a specific partial length fragment was found to interrupt coat assembly after protein secretion and prior to cellulose synthesis in 85% of the cells. This fragment consisted of SP85's N-terminal domain, containing prespore vesicle targeting information, and its Cys-rich C1-domain. The effect of the NC1 fusion was non-cell autonomous in interstrain chimeras, suggesting it acted at the cell surface. SP85-null spores presented an opposite phenotype in which spores differentiated prematurely before reaching the top of the stalk, and cellulose was slightly overproduced in a disorganized fashion. A similar though less severe phenotype occurred when a fusion of the N- and C2-domains was expressed. In a double mutant, absence of SP85 was epistatic to NC1- expression suggesting that NC1 inhibited SP85 function. Together, these results suggest the existence of an outside-in signaling pathway that constitutes a checkpoint to ensure that cellulose synthesis does not occur until coat proteins are properly organized at the cell surface and stalk formation is complete. Checkpoint execution is proposed to be regulated by SP85, which is in turn under the influence of contacts with other coat proteins that are competed by the presence of NC1 and NC2. ----------------------------------------------------------------------------- Ralph Grf Adolf-Butenandt-Institut / Zellbiologie, Universitt Mnchen, Schillerstr. 42, D-80336 Mnchen, Germany J. Cell Sci. in press Dictyostelium Nek2 (DdNek2) is the first structural and functional non- vertebrate homologue of human Nek2, a NIMA-related serine/threonine kinase required for centrosome splitting in early mitosis. DdNek2 shares 43% overall amino-acid identity with its human counterpart and even 54% within the catalytic domain. Both proteins can be subdivided in an N-terminal catalytic domain, a leucine zipper, and a C-terminal domain. Kinase assays with bacterially expressed DdNek2 and C-terminal deletion mutants revealed that catalytic activity requires the presence of the leucine zipper and that autophosphorylation occurs at the C-terminus. Microscopic analyses with DdNek2 antibodies and expression of a GFP-DdNek2 fusion protein in Dictyostelium showed that DdNek2 is a permanent centrosomal resident and suggested that it is a component of the centrosome core. The GFP-DdNek2 overexpressing mutants frequently exhibit supernumerary microtubule- organizing centers (MTOCs). This phenotype did not require catalytic activity because it was also observed in cells expressing inactive GFP-K33R. However, it was caused by overexpression of the C-terminal domain since it also occurred in GFP-mutants expressing only the C-terminus or a leucine zipper/C-terminus construct but not in those mutants expressing only the catalytic domain or a catalytic domain/leucine zipper construct. These results suggest that DdNek2 is involved in the formation of MTOCs. Furthermore, the localization of the GFP-fusion proteins revealed two independent centrosomal targeting domains of DdNek2, one within the catalytic or leucine zipper domain and one in the C-terminal domain. ----------------------------------------------------------------------------- Liu, T., Mirschberger, C., Chooback, L., Arana, Q., Dal Sacco, Z., MacWilliams, H., and Clarke, M. Altered expression of the 100-kDa subunit of the Dictyostelium vacuolar proton pump impairs enzyme assembly, endocytic function, and cytosolic pH regulation. J. Cell Sci., in press. The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100-kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria, the level of the 100-kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase, became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially-grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, suggesting that the V-ATPase also plays a role in cytosolic pH regulation. ----------------------------------------------------------------------------- Transfer RNA gene-targeted retrotransposition of Dictyostelium TRE5-A into a chromosomal UMP synthase gene trap Peter Beck, Theodor Dingermann and Thomas Winckler Institut fuer Pharmazeutische Biologie, Universitaet Frankfurt/M. (Biozentrum), Frankfurt am Main, Germany J. Mol. Biol., in press The genome of the eukaryotic microorganism Dictyostelium discoideum hosts a family of seven non-long terminal repeat retrotransposons (TREs) which show remarkable insertion preferences near tRNA genes. We developed an in vivo assay to detect tRNA gene-targeted retrotransposition of endogenous TREs in a reporter strain of D. discoideum. A tRNA gene positioned within an artificial intron was placed into the D. discoideum UMP synthase gene. This construct was inserted into the D. discoideum genome and presented as a landmark for de novo TRE insertions. We show that the tRNA gene-tagged UMP synthase gene was frequently disrupted by de novo insertions of endogenous TRE5-A copies, thus rendering the resulting mutants resistent to 5-fluoro orotic acid selection. Approx. 96% of all isolated 5-FOA-resistent clones contained TRE5-A insertions, whereas the remaining 4% resulted from transposition-independent mutations. The inserted TRE5-As showed complex structural variations and were found about 50 bp upstream of the reporter tRNA gene similar to previously analysed genomic copies of TRE5-A. No integration by other members of the TRE family was observed. We found that only 51% of the de novo insertions were derived from autonomous TRE5-A.1 copies. The remaining 49% of new insertions were due to TRE5-A.2 elements, which lack the proteins required for reverse transcription and integration, but retained functional promoter sequences. ----------------------------------------------------------------------------- [End Dicty News, volume 18, number 3]