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Figure 1. Specificity of pAbs. a, Rabbit antiâXenopus PCM-1 (XPCM-1) pAb. Total cell lysate of cultured Xenopus A6 cells (A6 Cell Lysate) and Xenopus egg extract (Egg Extract), and purified GST fusion protein with the COOH-terminal portion of XPCM-1 produced in E. coli (GST-n1) were separated by SDS-PAGE (aâc; CBB staining), followed by immunoblotting with antiâXPCM-1 pAb (dâf). This affinity-purified pAb specifically recognized â¼230 kD XPCM-1 in d and e, as well as GST fusion protein in f. b, Rabbit antiâmouse PCM-1 (mPCM-1) pAb. Total cell lysate of cultured mouse Eph4 cells (Eph4 Cell Lysate) and purified GST fusion protein with the COOH-terminal portion of mPCM-1 produced in E. coli (GST-mPCM-1) were separated by SDS-PAGE (a and b; CBB staining), followed by immunoblotting with antiâmPCM-1 pAb (c and d). This affinity-purified pAb specifically recognized â¼230 kD mPCM-1 in c, as well as GST fusion protein in d.
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Figure 2. Subcellular localization of XPCM-1 in A6 cells. aâc, Double immunofluorescence staining of A6 cells with antiâXPCM-1 pAb (a) and antiâγ-tubulin mAb (b). The merged image (c) revealed that most of the XPCM-1âpositive granular structures were concentrated on and/or around γ-tubulinâpositive centrosomes. Small numbers of XPCM-1âpositive granular structures were observed scattered in the cytoplasm. Bar, 10 μm. d, Localization of XPCM-1 at centriolar satellites. When A6 cells were treated with Triton X-100 and labeled with antiâXPCM-1 pAb, numerous electron dense granules (arrows) around the centrosome (asterisk) were specifically labeled at the electron microscopic level. Some granules appeared to be associated with MTs. Note that pale granules (arrowheads) with similar diameter were not labeled. e, Ultrastructure of centrosomes of A6 cells in situ. A6 cells were processed for ultrathin EM without Triton X-100 treatment. Note the so-called centriolar satellites (arrows) and pale granules (arrowheads) around the centrosome (asterisk). Bar, 200 nm.
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Figure 3. Movement of GFP-tagged centriolar satellites in live A6 cells. aâc, GMX-A6 cells, A6 transfectants stably expressing GFP fusion protein with the middle portion of XPCM-1 (GFP-MX), were stained red with antiâXPCM-1 pAb. Since this pAb did not recognize GFP-MX, it represented the localization of endogenous XPCM-1 (b). Most of the green fluorescence from expressed GFP-MX (a) was colocalized with endogenous XPCM-1 (b and c). dâg, Image series of GMX-A6 cells showing the movement of GFP-tagged centriolar satellites. Numbers at the bottom right indicate the time lapse in seconds. Arrows show the movement of granules toward the centrosome (asterisk), and arrowheads indicate granules moving toward the cell periphery. When the granules moved quickly during 1-s exposure of the CCD camera, they appeared tube-like in shape (arrow in f). In g, the paths of movement of two granules during 30 s were traced, which were represented in 2-s intervals beginning with the coolest colors (purple) and proceeding to the hottest colors (red). Bars, 10 μm.
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Figure 4. In vitro motility of GFP-tagged centriolar satellites. a, Time-lapse observation of the movement of a centriolar satellite (green) in a reconstituted aster (red). The boxed area in the large panel is magnified in the small panels. Numbers at the bottom left in the small panels indicate the time lapse in seconds. GFP-tagged centriolar satellite (arrowheads) moved along a MT toward centrosomes where other granules had already accumulated. On the way to the centrosome, this granule changed MTs (arrows), and finally reached the centrosome. Bar, 10 μm. A QuickTime movie is available at http://www.jcb.org/cgi/content/147/5/969/F4/DC1. b, Immunoelectron microscopy of centriolar satellites accumulating around centrosomes in the in vitro reconstituted asters. Electron-dense granular structures, â¼80â90 nm in diameter, which were specifically labeled with antiâXPCM-1 pAb (10-nm gold particles), were accumulated around fibrous materials of centrosomes. These XPCM-1âcontaining granules were not delineated by membranes. Bar, 200 nm.
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Figure 5. Inhibition of the accumulation of GFP-tagged centriolar satellites around centrosomes in vitro. A reconstituted aster (red) was incubated with GFP-tagged centriolar satellites (green) under the same condition as Fig. 4 a. Without additional reagents, numerous granules were accumulated around the centrosome during 10-min incubation (Control). AMP-PNP at 2 mM, but not 100 μM, significantly suppressed the accumulation of granules. 10 μM vanadate, as well as antidynein intermediate chain mAb (m70.1), also completely suppressed the accumulation. When the accumulation was suppressed, the movement of individual granules itself was always affected. Bar, 10 μm. b, The number of centriolar satellites, which were accumulated around centrosomes during 10-min incubation, were counted per individual centrosomes. Asterisks, F-test showed significant inhibition (P < 0.001).
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Figure 6. Subcellular distribution of mouse PCM-1 (mPCM-1) in mouse nasal respiratory epithelium. When cryosections â¼0.5-μm thick of ciliated epithelium were immunofluorescently stained with antiâmPCM-1 pAb (a, red in c), mPCM-1 signal was detected at the apical cytoplasm of epithelial cells (c; a composite with the phase-contrast image). Arrows, cilia. At four days after irritation of the nasal epithelia with 1% aqueous ZnSO4 in situ, cilia were completely removed from their apical surface, and the PCM-1 signal at the apical cytoplasm was markedly elevated (b, red in d). d, a composite with the phase-contrast image. Bar, 10 μm.
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Figure 7. Localization of mPCM-1 in nasal respiratory epithelial cells at four days after exposure to distilled water (aâc) or irritation with 1% aqueous ZnSO4 (dâf). a, Conventional ultrathin EM. Electron-dense spherical granules (arrowheads), â¼70â100 nm in diameter, which were morphologically indistinguishable from centriolar satellites, were scattered close to ciliary basal bodies (asterisks). Open arrows, microtubules. b, Preembedding immunoelectron microscopy. Nasal epithelial tissues were treated with 0.5% Triton X-100, fixed with glutaraldehyde, then labeled with antiâmPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). c, Postembedding immunoelectron microscopy. Ultrathin cryosections of nasal epithelial cells were labeled with antiâmPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). d, Conventional ultrathin EM. Cilia were completely removed, and at the apical cytoplasm numerous fibrous granules (arrowheads), as well as deuterosomes (arrows), appeared. e, Preembedding immunoelectron microscopy. Samples were treated with 0.5% Triton X-100, fixed with glutaraldehyde, then labeled with antiâmPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were heavily labeled. Both centriolar and acentriolar pathways for centriolar replication were observed (see details in the text). f, Postembedding immunoelectron microscopy. Ultrathin cryosections were labeled with antiâmPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were specifically labeled. Bars, 200 nm.
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