13 research outputs found
Generation of IP<sub>7</sub> by Asp1 variants.
No full text<p>(A) Diagrammatic representation of the Asp1 variants used. (B) Western blot analysis using a GST antibody of GST-tagged wild-type Asp1, GST-Asp1<sup>D333A</sup> and GST-Asp1<sup>H397A</sup>. Proteins were purified from <i>E.coli</i>, quantified (Coomassie stained gel is shown in left panel) and equal amounts loaded on a 10% PAGE. The tagged Asp1 variants (arrow) run at the expected size of 139 kDa. (C) Generation of IP<sub>7</sub> by GST-Asp1 variants. 4 µg of the indicated proteins were used in an ATP-dependent enzymatic reaction (16 hrs) and the resulting inositol pyrophosphates were resolved on a 35.5% PAGE and stained with Toluidine Blue <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen.1004586-Loss1" target="_blank">[54]</a>. −, component not present; +, component present. (D) Time-dependent (0–10 hrs in 2 hr. steps) generation of IP<sub>7</sub> by Asp1 and Asp1<sup>H397A</sup>. Assay conditions were as in 1C. (E) Quantification and diagrammatic representation of the IP<sub>7</sub> bands obtained in the assay shown in (D). (F) Determination of K<sub>m</sub> and V<sub>max</sub> values. 2 µg GST-Asp1 and GST-Asp1<sup>H397A</sup> were incubated with varying amounts of substrate for 6 hrs. The IP<sub>7</sub> was detected as in 1C, and quantified using an IP<sub>6</sub> calibration curve (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#s4" target="_blank">Materials and Methods</a>). v, reaction rate. V<sub>max</sub> Asp1: 450.5 nM/min/µg; V<sub>max</sub> Asp1<sup>H397A</sup>: 611.2 nM/min/µg.</p
Model for the regulation of Asp1 kinase function by the C-terminal phosphatase domain.
No full text<p>(A) <i>In vitro</i> IP<sub>7</sub> output of wild-type Asp1 (top), Asp1<sup>H397A</sup> (middle) and Asp1 plus Asp1<sup>365-920</sup> (phosphatase domain only). ++++ - +, high to low IP<sub>7</sub> output. (B) Two possible modes of action are shown. (I) The phosphatase domain could modulate the function of the kinase domain directly leading to reduced inositol pyrophosphate generation. (II) The phosphatase domain has enzymatic activity using the inositol pyrophosphate generated by the kinase domain as a substrate. IPP = inositol pyrophosphate. (C) MT stability correlates directly with intracellular inositol pyrophosphate levels.</p
Asp1 kinase function affects MT organization.
No full text<p>(A) Live cell images of the indicated strains expressing <i>nmt81::gfp-atb2<sup>+</sup></i>. The same imaging and image-processing conditions were used for all strains. Bar, 5 µm. (B) Percentage of MTs depolymerising at a cell end or at the lateral cortex/in the cytoplasm. Wild-type: n = 102, <i>asp1<sup>H397A</sup></i>: n = 218, <i>asp1<sup>D333A</sup></i>: n = 166, <i>asp1</i>Δ: n = 131. ** P<0.005 for <i>asp1<sup>D333A</sup></i> or <i>asp1</i>Δ compared to wild-type as determined using χ<sup>2</sup>-test. (C) MT pausing time (sec) at cell ends in the indicated strains. Overall MT pausing time of these strains is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen-1004586-t001" target="_blank">table 1</a>. We arbitrarily defined the 4 categories to show the variability within this system. Wild-type: n = 100, <i>asp1<sup>H397A</sup></i>: n = 67, <i>asp1<sup>D333A</sup></i>: n = 75. (D) MT pausing time (sec) at cell ends in the <i>asp1<sup>H397A</sup></i> strain transformed with a vector control or expressing pasp1<sup>1-364</sup>. Overall MT pausing time of these strains is shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen-1004586-t001" target="_blank">table 1</a>. Cells were grown in plasmid-selective minimal medium. <i>asp1<sup>H397A</sup></i>+vector: n = 105, <i>asp1<sup>H397A</sup></i>+p<i>asp1<sup>1-364</sup></i>: n = 110. pasp1<sup>1-364</sup> denotes plasmid-borne expression of Asp1<sup>1-364</sup> via the <i>nmt1<sup>+</sup></i> promoter under promoter de-repressing conditions. (E) Live cell images of the <i>nmt81</i>::<i>gfp</i>-<i>atb2</i><sup>+</sup> expressing <i>asp1<sup>H397A</sup></i> strain transformed with the vector control or expressing pasp1<sup>1-364</sup>. Images shown are 10 sec intervals. Asterisks (*) denote MTs touching the cell end. Bar, 5 µm.</p
Function of the Asp1 phosphatase-like domain.
No full text<p>(A) Diagrammatic representation of the Asp1 protein with the conserved phosphatase signature motif (motif: RH(GNA)XR-HD in Asp1 RHADR-HI)(top) and the Asp1 variants used. Top: (B) Generation of IP<sub>7</sub> by GST-Asp1 with varying amounts (2,4,8 µg) of Asp1<sup>365-920</sup>. Enzymatic reaction was carried out as for 1C. −, component not present; +, component present. (C) Serial dilution patch tests (10<sup>4</sup>–10<sup>1</sup> cells) of the <i>asp1</i>Δ (deletion) strain expressing the indicated <i>asp1</i> variants via the <i>nmt1<sup>+</sup></i> promoter. This promoter is repressed in the presence of thiamine and de-repressed in its absence. Cells were grown for 6 days at 25°C on plasmid selective minimal medium without (−) or with (+) TBZ. (D) Serial dilution patch tests (10<sup>5</sup>–10<sup>1</sup> cells) of the <i>asp1</i>Δ strain expressing either <i>asp1<sup>1-364</sup></i>, <i>asp1<sup>365-920</sup></i> or <i>asp1<sup>1-364</sup></i> and <i>asp1<sup>365-920</sup></i>. Cells were grown for 7 days at 25°C on plasmid selective minimal medium without (−) or with (+) TBZ.</p
Loss of <i>Umasp1</i> causes aberrant morphology of and altered MT organization in <i>U. maydis</i> yeast cells.
No full text<p>(A) Diagrammatic representation of growth inhibition test. The radius of growth zone inhibition was determined for the indicated strains on plates with 10 µl TBZ (concentration: 10 mg/ml) at the centre (experiments, n = 3. Error bars show SEM. ***, p<0.001; t-test). Representative examples are shown on the right and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen.1004586.s007" target="_blank">Figure S7C</a> (size bar, 1 cm). (B) Percentage of cells with disturbed cell shape. Bars show the mean of three independent experiments with n>100 cells (error bar shows SEM. *p<0.03, t-test). Representative examples are shown on the right (size bar, 5 µm). (C) Top: Deconvolved fluorescence photomicrographs depicting MT morphology (via expression of GFP-Tub1 (GFP fused to α-tubulin)) of wild-type and <i>Umasp1</i>Δ cells (size bar, 5 µm). Note that due to deconvolution fluorescence for the <i>Umasp1</i>Δ cell appears brighter. Bottom: Corresponding intensity profile showing longitudinal maximum intensity of background subtracted raw images. (D and E) Whisker diagrams showing the number of wild-type and <i>Umasp1</i>Δ MT bundles (D) and their relative intensity (E). Whiskers indicate 90%/10% percentiles (n>49 cells in (D) and n = 10 cells in (E); *** p<0.001 Mann-Whitney test for (D) and (E)). (F) MT growth parameters. Growth of MTs was determined by analyzing the comet-like movement of Pep1-GFP (error bars indicate standard deviation). Only MTs that grew >2 µm were analyzed (n = 225 and 125 for wild-type and <i>Umasp1</i>Δ, respectively). (G) The residence time of dynamic MTs was determined in GFP-Tub1 strains. n = 79 and 96 for wild-type and <i>UMasp1</i>Δ respectively. Error bar indicates standard deviation (unpaired t-test, *** p<0.001).</p
The Vip1 inositol polyphosphate kinase family regulates polarized growth and modulates the microtubule cytoskeleton in fungi.
Get PDFMicrotubules (MTs) are pivotal for numerous eukaryotic processes ranging from cellular morphogenesis, chromosome segregation to intracellular transport. Execution of these tasks requires intricate regulation of MT dynamics. Here, we identify a new regulator of the Schizosaccharomyces pombe MT cytoskeleton: Asp1, a member of the highly conserved Vip1 inositol polyphosphate kinase family. Inositol pyrophosphates generated by Asp1 modulate MT dynamic parameters independent of the central +TIP EB1 and in a dose-dependent and cellular-context-dependent manner. Importantly, our analysis of the in vitro kinase activities of various S. pombe Asp1 variants demonstrated that the C-terminal phosphatase-like domain of the dual domain Vip1 protein negatively affects the inositol pyrophosphate output of the N-terminal kinase domain. These data suggest that the former domain has phosphatase activity. Remarkably, Vip1 regulation of the MT cytoskeleton is a conserved feature, as Vip1-like proteins of the filamentous ascomycete Aspergillus nidulans and the distantly related pathogenic basidiomycete Ustilago maydis also affect the MT cytoskeleton in these organisms. Consistent with the role of interphase MTs in growth zone selection/maintenance, all 3 fungal systems show aspects of aberrant cell morphogenesis. Thus, for the first time we have identified a conserved biological process for inositol pyrophosphates
Loss of UmAsp1 causes defects in filamentous growth.
No full text<p>(A) Edges of colonies of the indicated AB33 derived strains grown on charcoal plates. Aerial hyphae are emanating from the colony. (B) Photomicrographs (DIC) of the indicated strains grown for 8 hrs under filament inducing conditions. Wild-type and UmAsp1-GFP filaments form characteristic empty sections at the basal pole. White arrow: growth zone; white star: yeast cell (bar, 10 µm). (C) Bar diagram showing percentage of filaments exhibiting normal or disturbed growth. Bars show the mean of three independent experiments with n>100 cells (error bar, SEM; ** p = 0.0108). (D) Examples of UmAsp1-GFP yeast cells and filaments (8 hours post induction) (bar, 10 µm). (E) Bar chart showing mean average fluorescence intensity of UmAsp1-GFP in yeast and hyphae (yeast, n = 10 cells and hyphae, n = 7 cells; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen.1004586.s011" target="_blank">Figure S11</a> for example photomicrographs). Error bars indicate standard deviation (*** p<0.001 unpaired t-test). (F) Western blot analysis of protein extracts of strain AB33 Umasp1-GFP after induction (0–8 hrs) of filamentous growth. Tub1 served as a loading control (hpi, hours post induction).</p
Function of Vip1-like protein <i>A. nidulans</i> VlpA.
No full text<p>(A) VlpA has kinase activity. 2 µg GST-VlpA<sup>1-574</sup> or GST-Asp1<sup>1-364</sup> (kinase domain only) were used in an enzymatic reaction as described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004586#pgen.1004586-Loss1" target="_blank">[54]</a>. −, component not present in assay; +, component present in assay. (B) Correlation between GST-VlpA<sup>1-574</sup> protein amount/assay and the amount of IP<sub>7</sub> generated. (C) Colonies of the <i>vlpA</i>-deletion strain Δ<i>vlpA</i> (left), wild-type (middle) and a strain in which GFP-VlpA was expressed under the control of <i>alcA</i> promoter (right). The strains were grown on minimal medium agar with threonine (upper), glycerol (middle) or glucose (bottom) for 2 days at 37°C. (D) The Δ<i>vlpA</i> strain (SCoS94, left) and wild-type strain (right) were grown in minimal medium with glucose overnight at 28°C. Some hyphae showed swelling in the Δ<i>vlpA</i> strain (arrow). Bars, 10 µm. (E) Growth conditions of the GFP-VlpA strain (Scos176) as in (D). GFP-VlpA expressed under native promoter localized predominantly in the cytoplasm with weak nuclear staining (arrows). The wild-type strain photographed under the same condition is shown on the right. Bars, 10 µm. (F) Left panel: position of the second germtube or branch: second germtube opposite first germtube (white), random position (grey) or second hypha branching out of the first hypha (black). Right panel: quantification of the number of germlings with a second germtube in the indicated strains. Spores were grown in minimal medium with glucose overnight. To induce expression of VlpA<sup>1-574</sup>, minimal medium with glycerol was used. N germlings/strain = 100. (G) Number of MT plus-ends appearing from the SPB or septal MTOC during 5 minutes in the wild-type (SSK92, white) and the <i>vlpA</i>-deletion strain (SDO2, grey). At SPB, wild-type: n = 20, Δ<i>vlpA</i>: n = 12, **<i>p</i><0.01 as determined using t-test. At MTOC, wild-type: n = 10, Δ<i>vlpA</i>: n = 9, **<i>p</i><0.01 as determined using t-test. (H) Growth rate of MT in the wild-type (SSK92) and the Δ<i>vlpA</i> strain (SDO2, grey). N = 10 cells for each of the strains, **<i>p</i><0.01 as determined using t-test. (I) Number of MT plus-ends reaching hyphal tips during 1 minute in the wild-type (SSK92) and Δ<i>vlpA</i> strain (SDO2). N = 13 cells for each of the strains, <i>*p</i><0.05 as determined using t-test.</p
Asp1 is required for growth zone selection.
No full text<p>(A) The indicated strains were released from stationary phase by streaking cells on solid YE5S at 25°C and analyzing the cell shape microscopically. Wild-type strain, n = 16; asp1<sup>H397A</sup>, n = 19; asp1<sup>D333A</sup> strain, n = 15. (B) Photomicrograph of a wild-type and <i>asp1<sup>D333A</sup></i> cell that exits stationary phase on solid agar. Bars, 5 µm. […] denotes no change in cell morphology at these time points. (C) The indicated strains were released from stationary phase by inoculating an aliquot with YE5S liquid medium. Phenotype was scored after 7 hrs at 25°C. Wild-type strain, n = 142; <i>asp1<sup>H397A</sup></i>, n = 110; <i>asp1<sup>D333A</sup></i> strain, n = 122 ***p<0.001 <i>asp1<sup>D333A</sup></i> compared to wild-type as determined using χ<sup>2</sup>-test. (D) Photomicrographs of cells in stationary phase (top panels) and after release into YE5S liquid medium (bottom panels). Cells were stained with Calcofluor white. Bar, 5 m.</p
