13 research outputs found

    Phenotypical characterization of mutant strains lacking functional DipA.

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    <p><b>(A)</b> Top and bottom view of point-inoculated wild type (WT), <i>dipA</i>* (codon exchange of catalytic core), <i>dipA</i> deletion (Δ<i>dipA</i>) and complementation (Compl.) strains incubated for three days under asexually development inducing conditions. Zoomed view represents binocular images of respective strains with asexual structures (conidiophores, co) and the sexual fruiting bodies (cleistothecia, cl) after seven days. Scale bar: 100 μm. <b>(B)</b> Colony diameter of point-inoculated asexually grown colonies measured for six days. The mean values with standard deviations derived from three independent experiments are shown. <b>(C)</b> Quantification of conidiospores after four days. The mean values with standard deviations from three independent experiments are shown. <b>(D)</b> Diagram illustrates distances between septa. Data derived from analyzing 70 hyphae of each strain. Shown are the mean values with standard deviations. <b>(E)</b> Fluorescence microscopy of hyphae of WT and <i>dipA</i> deletion strain. Membranes/septa were stained with FM4-64. White arrows are highlighting septa. Scale bar: 5 μm.</p

    DenA-DipA cytoplasmatic movements <i>in vivo</i>.

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    <p><b>(A)</b> Fluorescence microscopy of DenA-GFP subpopulations within vegetative hyphae located the protein in the nucleus (N), in the cytoplasm and there with a specific enrichment at septa (S). Control: wild type without GFP. <b>(B)</b> Bimolecular fluorescence studies (BiFC) of DenA (<i>denA</i>::<i>nyfp</i>) and DipA (<i>dipA</i>::<i>cyfp</i>) showed restricted interaction in the cytoplasm, at septa (S) and close to, but not inside nuclei (N). The septal and the nuclear regions are enlarged (white squares; scale bar: 1 μm). Control: strain co-expressing <i>denA</i>::<i>nyfp</i> and <i>cyfp</i>, respectively. <b>(C)</b> Dynamic co-transport of DenA-DipA between nuclei and septa in time lapse of bimolecular fluorescence strain <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> over 170 seconds. White arrows mark a single interaction complex. <b>(D</b>) Time lapse microscopy over 110 seconds of <i>denA</i>::<i>nyfp</i>-<i>dipA</i>::<i>cyfp</i> with stained mitochondria (red) with a white arrow marking single DenA-DipA. Expressed <i>rfp</i>::<i>h2A</i> decorates nuclei, membranes were stained with FM4-64 and mitochondria with MitoTracker. Scale bar: 5 μm.</p

    Comparative analyses of DenA protein levels in Δ<i>dipA</i>, Δ<i>csnG</i> and Δ<i>dipA</i>/Δ<i>csnG</i> double mutant strains.

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    <p><b>(A)</b> DipA destabilizes DenA during illumination which induces asexual development in wild type. Western hybridization showed a stable DenA-GFP in wild type background at vegetative growth (Veg.) and an unstable protein during illumination induced asexual development (Asex). DenA-GFP in a Δ<i>dipA</i> strain is stable under the same conditions. <b>(B)</b> Top and bottom view of illuminated wild type colony (WT) developing asexual spores and <i>dipA</i>, <i>csnG</i> and combined <i>dipA</i>/<i>csnG</i> deletion strains under the same conditions. Equal amount of spores were point-inoculated and incubated for three days during illumination. <b>(C)</b> DenA-GFP protein stability in WT and <i>dipA/csnG</i> deletion background without the potential to produce asexual spores during illumination (Asex.). <b>(D)</b> Diagram of DenA-GFP protein levels in Δ<i>csnG</i>, Δ<i>dipA</i> and <i>dipA</i>/<i>csnG</i> double deletion strain from three independent experiments during vegetative growth (Veg.) and illumination (Asex.) for indicated time points. SDS gels were loaded with equal amounts of protein crude extract. Membranes were treated with GFP-antibody and stained with Ponceau as loading control and the GFP/Ponceau pixel ratio was calculated. The standard deviations are shown. DenA-GFP: 54.5 kDa and free GFP: 25 kDa.</p

    DenA protein levels during fungal development.

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    <p>DenA is stable during vegetative growth but not during illumination corresponding to advanced wild type asexual development. <b>(A)</b> Protein level of DenA-GFP (54.5 kDa) is monitored in presence (<i>csnA</i>, <i>csnB</i>, <i>csnD</i>) or absence (Δ<i>csnA</i>, Δ<i>csnB</i>, Δ<i>csnD</i>) of indicated CSN subunit encoding genes by western hybridization with GFP-antibody. Protein extracts from indicated time points (in hours) of vegetative (Veg.) or illumination induced development (Asex.) were applied. The signal of free GFP (25 kDa) corresponds to DenA degradation. Loading control: Ponceau staining (below). Lower panels show quantification of band intensities of DenA-GFP relative to vegetative growth. <b>(B)</b> Western hybridization with GFP-antibody of DenA-GFP in wild type in comparison to Δ<i>csnC</i>, Δ<i>csnE</i>, Δ<i>csnF</i>, Δ<i>csnG</i> or Δ<i>csnH</i> strains. DenA is present throughout development in respective deletion strains. Quantifications of DenA-GFP protein levels during development relative to vegetative growth are shown in the lower panels.</p

    <i>dipA</i> gene locus and multiple alignment of its deduced metallophosphatase domain.

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    <p><b>(A)</b> Schematic view of the <i>dipA</i> (AN10946) gene locus, transcript (mRNA) and deduced DipA protein. White boxes correspond to three introns (I, II, III). The <u>m</u>etallo<u>p</u>hos<u>p</u>hatase domain (MPP) is highlighted in black and the domain of unknown function (DUF) in grey. <b>(B)</b> Multiple alignment of MPP consensus sequence (cl13995) with DipA from <i>Aspergillus nidulans</i> and related proteins from other organisms including <i>Aspergillus niger</i>, <i>Aspergillus oryzae</i>, <i>Penicillium roqueforti</i>, <i>Neurospora crassa</i>, <i>Ustilago maydis</i> and <i>Schizosaccharomyces pombe</i>. Asterisks: putative active site D51, H73 and D76 residues. Red: high (90%), blue: low (50%) consensus values [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005949#pgen.1005949.ref056" target="_blank">56</a>].</p

    Protein amount of DenA-GFP and amino acid substituted variants during development.

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    <p>Western hybridization with equal amounts of protein extracts of DenA-GFP (54.5 kDa) compared to <b>(A)</b> DenA<sup>S253D</sup>-GFP carrying a negative charge reminiscent of a phosphorylated protein, <b>(B)</b> DenA<sup>S253A</sup>-GFP which cannot be phosphorylated, <b>(C)</b> DenA<sup>S243D-S245D-S253D</sup>-GFP with three negative charges mimicking a triple phosphorylated protein and <b>(D)</b> the corresponding DenA<sup>S243A-S245A-S253A</sup>-GFP which cannot be phosphorylated. Samples were taken from vegetative hyphae (Veg) and at indicated time points (in hours) of illumination which induces asexual development (Asex). Membranes were treated with GFP-antibody to visualize the fusion protein and free GFP (25 kDa). Loading control: Ponceau staining. Lower panels show quantification of band intensities of DenA-GFP and its respective variants relative to vegetative growth.</p

    Changes in deneddylase activity and its consequences for the cellular pool of neddylated proteins and fungal development.

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    <p><b>(A)</b> Western analyses with Nedd8 or Tubulin (reprobed as loading control, lower part) antibodies of vegetative grown mycelia of <i>A</i>. <i>nidulans</i>. Wild type (WT) was compared to mutant strains with altered deneddylase activity either with decreased COP9 signalosome (Δ<i>csnG</i>, Δ<i>csnE</i>) or increased <i>denA</i> (OE <i>denA</i>) and combinations of defective CSN and increased DenA (Δ<i>csnG</i>/OE <i>denA</i>, or Δ<i>csnE</i>/OE <i>denA</i>). Neddylated cullins correspond to ≈ 100 kDa and faster migrating bands are summarized as neddylated non-cullin proteins. <b>(B)</b> Semi-quantitative analyses of Nedd8 signal intensities of three independent experiments of strains shown in (A) normalized to Tubulin signals, including standard deviations. <b>(C)</b> Western hybridization using cullinA (CulA) or <b>(D)</b> cullinC (CulC) antibodies and determination of the ratios (lower panels) of neddylated (CulA-N8 or CulC-N8) in comparison to deneddylated cullins of three independent experiments. <b>(E)</b> Cellular deneddylase activity and fungal development. Equal amount of spores of indicated strains were point-inoculated and grown for four days under illumination which induces asexual development in wild type. Respective strains are shown either on minimal (MM) or stress inducing media (+menadione, +SDS).</p
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