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First published online 23 January 2003
doi: 10.1242/jcs.00282

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Non-membranous granular organelle consisting of PCM-1: subcellular distribution and cell-cycle-dependent assembly/disassembly

¹ Department of Cell Biology, Kyoto University Faculty of Medicine, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan
² Solution Oriented Research for Science and Technology, Japan Science and Technology Corporation, Yoshida-Konoe, Sakyo-ku, Kyoto 606-8501, Japan

View larger version (88K): [in a new window]   Fig. 1. Widespread occurrence of PCM-1 granules. Cultured mouse L fibroblasts (a), Eph-4 epithelial cells (b) or frozen sections of mouse intestine (c) or kidney (d) were triple stained with anti-mPCM-1 pAb (green), anti--tubulin mAb (red) and anti--tubulin mAb (blue, turned white) (a,b) or anti-mPCM-1 pAb (green), anti--tubulin mAb (red) and DAPI (blue) (c,d). In cultured cells (a,b), some PCM-1 granules were gathered around the centrosomes (arrows) but the others were scattered in the cytoplasm. In intestinal and renal epithelial cells (c,d), PCM-1 granules were concentrated abundantly in the apical region of the cytoplasm (arrows). The liver (e) or the brain (f) were triple stained with anti-mPCM-1 pAb (green), anti-occludin mAb (red) and DAPI (blue) (e) or anti-mPCM-1 pAb (green), Cy3-conjugated anti--tubulin pAb (red) and DAPI (blue) (f). In hepatocytes (e), a fairly small number of PCM-1 granules were detected, which were scattered around the occludin-positive bile canaliculi (arrow) where the centrosomes (and minus ends of microtubules) were located. In nerve cells in the brain (f), PCM-1 granules were scattered over the cytoplasm, showing no significant concentration around the -tubulin-positive centrosomes (arrow). Bars, 10 µm. (g) To clarify the spatial relationship of PCM-1 granules with centrosomes quantitatively, four distinct types of cultured cells were double stained with anti-mPCM-1 pAb and Cy3-conjugated anti--tubulin pAb, and the number of PCM-1 granules inside (red) and outside (black) a radius of 3 µm of the -tubulin-positive centrioles were counted for 16 L cells, 10 Eph4 cells, 18 CSG cells and 8 A6 cells as described in Materials and Methods. Error bars show the s.e.m.

View larger version (60K): [in a new window]   Fig. 2. Formation of large aggregates consisting of PCM-1 deletion mutants. (A) Xenopus A6 cells were transfected with cDNA encoding a GFP fusion protein with a truncated XPCM-1 mutant (aa 745-1273; C). Transfectants expressing small or large amounts of the GFP fusion protein were double stained with anti-XPCM-1 pAb (red) and DAPI (blue). This pAb recognized endogenous XPCM-1 but not the exogenously expressed XPCM-1 mutant. Notice that the GFP-positive large aggregates were formed when the exogenous protein was overexpressed, and that these aggregates recruited endogenous XPCM-1. Bar, 10 µm. (B) Ultrathin section electron microscopy. A6 transfectants overexpressing a GFP fusion protein with a truncated XPCM-1 mutant (aa 745-1273) bore large aggregates, homogenous electron-dense structures (asterisk) (a). Notice that the nucleus, but not these granules, were delineated by membranes (inset). Cells were treated with Triton X-100 and labeled with anti-GFP pAb (b). The surface of the large aggregate (asterisk) was specifically labeled with gold particles (inset). N, nucleus. Bars, (a,b) 500 nm; (inset) 200 nm. (C) The ability of the formation of large aggregates for various deletion mutants of XPCM-1. The constructs represented in color, but not those in black and white, formed large aggregates when overexpressed.

View larger version (78K): [in a new window]   Fig. 3. Self-aggregation of PCM-1. (A) Yeast two-hybrid analyses. The cDNA encoding aa 1-484 or aa 745-1273 of XPCM-1 (p53 as a control) was fused to the DNA-binding domain (DNA-BD) in the pLexA vector and the cDNA encoding aa 1-711 or aa 745-1273 (SV40 large T-antigen as a control) to the activation domain (AD) in the pB42AD vector. DNA-BD and AD constructs were then transformed into yeast. Notice that aa 1-484 and aa 745-1273 bound directly to aa 1-711 and aa 745-1273, respectively. (B) Overexpression of a GST fusion protein with XPCM-1 fragment (aa 745-1271) in insect Sf9 cells. As seen in A6 cells (Fig. 2B), electron-dense large aggregates were formed within the cytoplasm abundantly (a). These Sf9 cells were lysed with 1% Triton X-100 and the lysate was separated by centrifugation. The aggregates were partially isolated as a pellet (b). Bars, 1 µm. (C) Components of the partially isolated aggregates. The pellet (ppt) and supernatant (sup) were separated by SDS-PAGE and stained with Coomassie Brilliant Blue (CBB). The pellet contained only one major band, around 83 kDa (sometimes divided into two bands for an unknown reason), which was identified as the GST-XPCM-1 fragment by immunoblotting with anti-GST mAb (western). Bars represent molecular masses of 203, 116 and 83 kDa from the top. (D) In vitro binding assay. A column consisting of GST fusion proteins with a XPCM-1 fragment (aa 745-1271 and aa 1020-1271) was constructed, to which the whole lysate of Xenopus interphase egg extract was applied. Bound proteins were then eluted (CBB) and immunoblotted with anti-XPCM-1 pAb (western). Arrows indicate the GST fusion proteins. Notice that endogenous full-length XPCM-1 bound to aa 745-1271 but not to aa 1020-1271. Bars represent molecular masses of 203, 116 and 83 kDa from the top.

View larger version (99K): [in a new window]   Fig. 4. Cell-cycle-dependent assembly and disassembly of PCM-1 granules and aggregates. (A) A6 cells were triple stained with anti-XPCM-1 pAb (red), FITC-conjugated anti--tubulin mAb (green) and DAPI (blue). Lower panels represent only PCM-1 signals. At interphase, PCM-1 granules were scattered around the cytoplasm, showing significant concentration around the centrosomes (a). When cells entered the mitotic phase, PCM-1 granules gathered around two divided centrosomes very intensely (b) and then gradually began to disappear (c,d). At the end of the mitotic phase, PCM-1 granules again gradually appeared around the centrosomes (e). Bar, 10 µm. (B) Time-lapse images of the dynamic behavior of the large aggregates of GFP fusion proteins with a truncated XPCM-1 (aa 745-1128; Fig. 2C) during mitosis. Elapsed time is indicated at the top (in minutes). The large aggregates (arrows) decreased in size when cells entered the mitotic phase with concomitant rounding-up and then recovered to the original size at the interphase. Bar, 10 µm.

View larger version (87K): [in a new window]   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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