Fig. 5. PCM-1 granules and pericentrin. (A) A GFP fusion protein with a truncated XPCM-1 (aa 745-1128) was transiently overexpressed in A6 cells, in which several large aggregates were formed (arrows). Cells were fixed and double stained with rabbit anti-pericentrin pAb and mouse anti--tubulin mAb, which were detected with rhodamine-conjugated anti-rabbit IgG antibody and Cy5-conjugated anti-mouse IgG antibody, respectively. Both endogenous pericentrin and -tubulin were recruited to the large aggregates of XPCM-1 mutant (arrows). In the absence of anti-pericentrin pAb or mouse anti--tubulin mAb, pericentrin or -tubulin signals, respectively, disappeared from the large aggregates of XPCM-1 mutant, indicating that these signals were not artifacts caused by the GFP emission in additional wavelengths (data not shown). Notice that the amount of -tubulin at the centrosomes in the transfectants (double arrowheads) was significantly smaller than that in the surrounding parental cells (arrowheads). Bar, 10 µm. (B) A6 cells with or without the large aggregates of a GFP fusion protein with a truncated XPCM-1 (aa 745-1128) were incubated in a medium containing 0.4 µM nocodazole for 1 hour to depolymerize the microtubules, washed with fresh medium twice, incubated in fresh medium for 5 minutes to repolymerize the microtubules and then double stained with anti--tubulin mAb (red) and DAPI (blue). A large number of microtubules elongated from the centrosomes in A6 cells without large aggregates (arrowhead), whereas, in A6 cells bearing large aggregates, only a small number of microtubules were associated with the centrosomes. Bar, 10 µm. (C) Mouse CSG cells were triple stained with anti-mPCM-1 pAb (red), anti-mouse pericentrin mAb (green) and DAPI (blue), and observed by sectioning microscopy. Images were obtained at 0.2-µm intervals on the z axis and deconvolved with Delta Vision software. Left panels represent an integrated image of 23 sections. Most pericentrin was concentrated in the pericentriolar region (arrows), but the rest was scattered in the cytoplasm as granules. By contrast, most of the PCM-1 granules were distributed throughout the cytoplasm. The 21st section of the left boxed area and the 23rd section of the right boxed area (in the left panel) are enlarged in the right panels. Pericentrin granules (green) appeared to be scattered in the cytoplasm as distinct granules from PCM-1 granules (red), and these two types of granules were frequently associated with each other in a granule-to-granule manner (yellow). Bar, 10 µm.

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