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Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis.

Kubo A, Sasaki H, Yuba-Kubo A, Tsukita S, Shiina N - J. Cell Biol. (1999)

Bottom Line: These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization.At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis.These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

View Article: PubMed Central - PubMed

Affiliation: Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan.

ABSTRACT
We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, approximately 70-100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1-containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

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Subcellular distribution of mouse PCM-1 (mPCM-1) in mouse nasal respiratory epithelium. When cryosections ∼0.5-μm thick of ciliated epithelium were immunofluorescently stained with anti–mPCM-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 6: Subcellular distribution of mouse PCM-1 (mPCM-1) in mouse nasal respiratory epithelium. When cryosections ∼0.5-μm thick of ciliated epithelium were immunofluorescently stained with anti–mPCM-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.

Mentions: First, full-length cDNA encoding mouse PCM-1 (mPCM-1) was isolated. Its product encoded a protein of 2,025 aa with significant similarity to hPCM-1 and XPCM-1 (87.3% and 57.2% identity at the amino acid sequence level, respectively; the sequence data are available from GenBank/EMBL/DDBJ under accession number AB029291). Then, using recombinant mPCM-1 produced in E. coli as an antigen, a pAb was generated. This pAb specifically recognized an ∼230-kD band in the total lysate of mouse Eph4 cells on immunoblots (Fig. 1 b) and exclusively labeled centriolar satellites of Eph4 cells at the electron microscopic level (data not shown). Interestingly, when cryosections of mouse nasal respiratory ciliated epithelium were immunofluorescently stained with this pAb, the mPCM-1 signal was specifically detected at their apical cytoplasm in a granular pattern (Fig. 6, a and c). Four days after irritation of the nasal epithelia with 1% aqueous ZnSO4 in situ, cilia were completely removed from their apical surface (Fig. 6 d) and, interestingly, the mPCM-1 signal was markedly elevated at the apical cytoplasm (Fig. 6b and Fig. d). Then, we examined the ZnSO4-induced morphological changes of these ciliated epithelia at the electron microscopic level.


Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis.

Kubo A, Sasaki H, Yuba-Kubo A, Tsukita S, Shiina N - J. Cell Biol. (1999)

Subcellular distribution of mouse PCM-1 (mPCM-1) in mouse nasal respiratory epithelium. When cryosections ∼0.5-μm thick of ciliated epithelium were immunofluorescently stained with anti–mPCM-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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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169353&req=5

Figure 6: Subcellular distribution of mouse PCM-1 (mPCM-1) in mouse nasal respiratory epithelium. When cryosections ∼0.5-μm thick of ciliated epithelium were immunofluorescently stained with anti–mPCM-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.
Mentions: First, full-length cDNA encoding mouse PCM-1 (mPCM-1) was isolated. Its product encoded a protein of 2,025 aa with significant similarity to hPCM-1 and XPCM-1 (87.3% and 57.2% identity at the amino acid sequence level, respectively; the sequence data are available from GenBank/EMBL/DDBJ under accession number AB029291). Then, using recombinant mPCM-1 produced in E. coli as an antigen, a pAb was generated. This pAb specifically recognized an ∼230-kD band in the total lysate of mouse Eph4 cells on immunoblots (Fig. 1 b) and exclusively labeled centriolar satellites of Eph4 cells at the electron microscopic level (data not shown). Interestingly, when cryosections of mouse nasal respiratory ciliated epithelium were immunofluorescently stained with this pAb, the mPCM-1 signal was specifically detected at their apical cytoplasm in a granular pattern (Fig. 6, a and c). Four days after irritation of the nasal epithelia with 1% aqueous ZnSO4 in situ, cilia were completely removed from their apical surface (Fig. 6 d) and, interestingly, the mPCM-1 signal was markedly elevated at the apical cytoplasm (Fig. 6b and Fig. d). Then, we examined the ZnSO4-induced morphological changes of these ciliated epithelia at the electron microscopic level.

Bottom Line: These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization.At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis.These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

View Article: PubMed Central - PubMed

Affiliation: Tsukita Cell Axis Project, Exploratory Research for Advanced Technology, Japan Science and Technology Corporation, Kyoto Research Park, Shimogyo-ku, Kyoto 600-8813, Japan.

ABSTRACT
We identified Xenopus pericentriolar material-1 (PCM-1), which had been reported to constitute pericentriolar material, cloned its cDNA, and generated a specific pAb against this molecule. Immunolabeling revealed that PCM-1 was not a pericentriolar material protein, but a specific component of centriolar satellites, morphologically characterized as electron-dense granules, approximately 70-100 nm in diameter, scattered around centrosomes. Using a GFP fusion protein with PCM-1, we found that PCM-1-containing centriolar satellites moved along microtubules toward their minus ends, i.e., toward centrosomes, in live cells, as well as in vitro reconstituted asters. These findings defined centriolar satellites at the molecular level, and explained their pericentriolar localization. Next, to understand the relationship between centriolar satellites and centriolar replication, we examined the expression and subcellular localization of PCM-1 in ciliated epithelial cells during ciliogenesis. When ciliogenesis was induced in mouse nasal respiratory epithelial cells, PCM-1 immunofluorescence was markedly elevated at the apical cytoplasm. At the electron microscopic level, anti-PCM-1 pAb exclusively labeled fibrous granules, but not deuterosomes, both of which have been suggested to play central roles in centriolar replication in ciliogenesis. These findings suggested that centriolar satellites and fibrous granules are identical novel nonmembranous organelles containing PCM-1, which may play some important role(s) in centriolar replication.

Show MeSH