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DdNek2, the first non-vertebrate homologue of human Nek2, is involved in the formation of microtubule-organizing centers

Ralph Gräf

Adolf-Butenandt-Institut/Zellbiologie, Universität München, Schillerstr. 42, D-80336 München, Germany



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  		Fig. 1. Phylogenetic relationship between DdNek2 and other NIMA-related kinases. (A) Pairwise alignment of the DdNek2 and human Nek2 amino-acid sequences using the ClustalW program. Conserved residues specific for NIMA-related kinases are highlighted by the black boxes and residues common to all serine/threonine kinases appear in light gray boxes. The lysine residue that was replaced by arginine in the catalytically inactive kinases is printed in bold and boxed in gray. The six heptads of the leucine zipper are underlined and the end of the catalytic domain is marked by a slash. (B) Phylogenetic trees derived from multiple alignments of all relevant NIMA-related kinases using the complete DdNek2 coding sequence. The alignments were made with the ClustalW program and the trees calculated with the PHYLIP package, version 3.5c. Numbers in brackets refer to the percentage of amino-acid identity to DdNek2. Abbreviations and accession numbers are: Dictyostelium discoideum DdNek2 (SLD805), Xenopus laevis Xl-Nek2A (Q9W622), Mus musculus Mm-Nek2 (O35942), Homo sapiens Hs-Nek2 (P51955), Drosophila melanogaster Dm-Nek2 (Q9W3N8), Schizosaccharomyces pombe Sp-Nrk (O13839), Saccharomyces cerevisiae Sc-KIN3 (P22209), Neurospora crassa Nc-NIM1 (P48479), Aspergillus nidulans An-NIMA (P11837), Tetrahymena pyriformis TpNrk (O76134) and Trypanosoma brucei Tb-Nrk (Q08942).

 


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  		Fig. 2. (A) An overview of DdNek2 deletion mutants. (B) Catalytic activity of MBP-DdNek2 and its mutants. The substrates were {alpha}/ß-casein, the recombinant C-terminal DdNek2 fragment MBP-{Delta}258-C and BSA (negative control). DdNek2 and its C-terminal deletion constructs were analyzed on the same gel to allow comparison of their catalytic activity. Proteins of each kinase reaction precipitated with TCA were separated by SDS-gel electrophoresis, stained with Coomassie and subjected to autoradiography. The molecular mass (kDa) of standard proteins is indicated on the left and the position of the individual protein bands on the right. The kinase derivatives and substrates used are given at the bottom. Nek refers to DdNek2, K33R to MBP-DdNek2-K33R, Cas to {alpha}/ß-casein, {Delta}258C to MBP-{Delta}258-C, {Delta}315N to MBP-{Delta}315-N and {Delta}267N to MBP-{Delta}267-N.

 


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  		Fig. 3. DdNek2 is a component of isolated centrosomes. (A) Western blot of cytosolic extract (CE; ~2x106 cells) and isolated centrosome (IC; ~5x107 cells) proteins separated on a 12.5% polyacrylamide gel (acrylamide:bisacrylamide, 200:1) and stained with anti-DdNek2 antibodies. (B,C), Immunofluorescence microscopy of isolated centrosomes spun down to coverslips, fixed with methanol and stained with rabbit anti-DdNek2 (B) and mouse anti-DdCP224 antibodies (C). Secondary antibodies were Alexa-568-labeled anti-rabbit IgG and Alexa-488-labeled anti-mouse IgG. Bar, 2 µm.

 


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  		Fig. 4. DdNek2 resides at the centrosome throughout the entire cell cycle. Fluorescence microscopy of Dictyostelium AX2 cells labeled with anti-DdCP224 antibodies (A-A'') and GFP-DdNek2 cells (B-B''). Cells were fixed with methanol. The secondary antibody in (A-A'') was Cy3-labeled anti-mouse IgG. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Bar, 2 µm.

 


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  		Fig. 5. Overview of the phenotypes of GFP-DdNek2 (A-E) and GFP-K33R (F-J) mutants. Immunofluorescence microscopy of cells fixed with methanol and labeled with anti-{gamma}-tubulin (A'-J'). The secondary antibody was Alexa-568-labeled anti-rabbit IgG. Nuclei were stained with DAPI. The main phenotypes shown are supernumerary MTOCs (A,B,F,G,H,E,J), nuclear aberrations (C,H,J), misshapen centrosomes (D,I) and multiple GFP foci (E,J). Bar, 2 µm.

 


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  		Fig. 6. Supernumerary MTOCs possess many features of centrosomes. Confocal (A,D,E,F) and non-confocal (B,C) immunofluorescence microscopy of cells fixed with methanol showing the presence of DdCP224 (A'), the NAB350 antigen (B') and microtubules (C'') at supernumerary MTOCs. The monoclonal antibody used to stain microtubules (shown in yellow for better visibility) in (C') was 2/141 (Gräf et al., 1999Go). The secondary antibody was Cy3-labeled anti-mouse IgG. Nuclei were stained with DAPI (B,C) or with 2 µM TO-PRO3 (A,D,E,F) (Molecular Probes, Leiden, Nederlands) after a 1 hour treatment with 100 µg/ml RNaseA.

(D-D'') shows an enlarged image of the left cell in (A). Images (D,E,F) were processed with the Huygens 2.2 deconvolution program (Bilplane AG, Zürich, Switzerland). The brightest point projections of two (D,F) or three (E) confocal sections, respectively, are shown,. The merged images (D''-F'') are three examples demonstrating that DdCP224 labeling of the centrosomal corona (D'-F') surrounds the structure labeled by GFP-DdNek2 (D-F) in both normal, nucleus-associated and supernumerary MTOCs. This is visualized graphically in (D[UNK]), where the fluorescence intensity of the DdCP224 (red) and GFP-DdNek2 (green) labeling, respectively, was plotted against the distance along a cross-section (arrows in D''). Bar, 2 µm.

 


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  		Fig. 7. Centrosomal localization and phenotypes of GFP-DdNek2 deletion mutants. Two representative examples each are shown for GFP-{Delta}267-N (A,B), GFP-{Delta}315-N (C,D), GFP-{Delta}258-C (E,F) and GFP-{Delta}312-C (G,H) mutants. Cells were fixed with methanol and labeled with anti-{gamma}-tubulin. The secondary antibody was Alexa-568-labeled anti-rabbit IgG. Nuclei were stained with DAPI. Bar, 2 µm.

 

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© The Company of Biologists Ltd 2002