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., 1999). 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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