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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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Localization of mPCM-1 in nasal respiratory epithelial cells at four days after exposure to distilled water (a–c) or irritation with 1% aqueous ZnSO4 (d–f). a, Conventional ultrathin EM. Electron-dense spherical granules (arrowheads), ∼70–100 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 anti–mPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). c, Postembedding immunoelectron microscopy. Ultrathin cryosections of nasal epithelial cells were labeled with anti–mPCM-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 anti–mPCM-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 anti–mPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were specifically labeled. Bars, 200 nm.
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Figure 7: Localization of mPCM-1 in nasal respiratory epithelial cells at four days after exposure to distilled water (a–c) or irritation with 1% aqueous ZnSO4 (d–f). a, Conventional ultrathin EM. Electron-dense spherical granules (arrowheads), ∼70–100 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 anti–mPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). c, Postembedding immunoelectron microscopy. Ultrathin cryosections of nasal epithelial cells were labeled with anti–mPCM-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 anti–mPCM-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 anti–mPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were specifically labeled. Bars, 200 nm.

Mentions: Conventional ultrathin EM revealed that in nontreated ciliated cells, electron-dense granules ∼100 nm in diameter were scattered beneath the layer of basal bodies of cilia (Fig. 7 a). Curiously, these granules were morphologically indistinguishable from centriolar satellites. As shown in Fig. 7b and Fig. c, both preembedding and postembedding immunolabeling revealed that these granules were exclusively labeled with anti–mPCM-1 pAb. When cilia were removed from these respiratory epithelia by ZnSO4 treatment, these granules appeared to increase in number and aggregated extensively (Fig. 7 d). In previous reports, these granules were called fibrous granules and were thought to be absent in nonciliogenic cells (Sorokin 1968; Steinman 1968; Anderson and Brenner 1971; Dirksen 1991), but this was not likely. This will be confirmed by the subsequent immunoelectron microscopy. In or close to the aggregation of these granules called fibrogranular area, so-called deuterosomes with multiple replicating procentrioles appeared (Fig. 7 d). These morphological characteristics indicated that synchronized multiple centriolar replication and subsequent ciliogenesis were induced in these cells. Preembedding immunoelectron microscopy revealed that these aggregated fibrous granules, but not deuterosomes, were heavily labeled with anti–mPCM-1 pAb (Fig. 7 e). Since deuterosomes were very large electron-dense structures, it was possible that antibodies cannot access the antigen within deuterosomes. However, postembedding immunolabeling did not detect mPCM-1 within deuterosomes, excluding this possibility (Fig. 7 f). Taken together, we concluded that so-called fibrous granules, which had been intensively examined from the viewpoint of centriolar replication, may be identical to PCM-1–containing centriolar satellites.


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)

Localization of mPCM-1 in nasal respiratory epithelial cells at four days after exposure to distilled water (a–c) or irritation with 1% aqueous ZnSO4 (d–f). a, Conventional ultrathin EM. Electron-dense spherical granules (arrowheads), ∼70–100 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 anti–mPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). c, Postembedding immunoelectron microscopy. Ultrathin cryosections of nasal epithelial cells were labeled with anti–mPCM-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 anti–mPCM-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 anti–mPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were specifically labeled. Bars, 200 nm.
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Figure 7: Localization of mPCM-1 in nasal respiratory epithelial cells at four days after exposure to distilled water (a–c) or irritation with 1% aqueous ZnSO4 (d–f). a, Conventional ultrathin EM. Electron-dense spherical granules (arrowheads), ∼70–100 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 anti–mPCM-1 pAb. The centriolar satellite-like granules were specifically labeled (arrowheads). c, Postembedding immunoelectron microscopy. Ultrathin cryosections of nasal epithelial cells were labeled with anti–mPCM-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 anti–mPCM-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 anti–mPCM-1 pAb. Fibrous granules (arrowheads), but not deuterosomes (arrow), were specifically labeled. Bars, 200 nm.
Mentions: Conventional ultrathin EM revealed that in nontreated ciliated cells, electron-dense granules ∼100 nm in diameter were scattered beneath the layer of basal bodies of cilia (Fig. 7 a). Curiously, these granules were morphologically indistinguishable from centriolar satellites. As shown in Fig. 7b and Fig. c, both preembedding and postembedding immunolabeling revealed that these granules were exclusively labeled with anti–mPCM-1 pAb. When cilia were removed from these respiratory epithelia by ZnSO4 treatment, these granules appeared to increase in number and aggregated extensively (Fig. 7 d). In previous reports, these granules were called fibrous granules and were thought to be absent in nonciliogenic cells (Sorokin 1968; Steinman 1968; Anderson and Brenner 1971; Dirksen 1991), but this was not likely. This will be confirmed by the subsequent immunoelectron microscopy. In or close to the aggregation of these granules called fibrogranular area, so-called deuterosomes with multiple replicating procentrioles appeared (Fig. 7 d). These morphological characteristics indicated that synchronized multiple centriolar replication and subsequent ciliogenesis were induced in these cells. Preembedding immunoelectron microscopy revealed that these aggregated fibrous granules, but not deuterosomes, were heavily labeled with anti–mPCM-1 pAb (Fig. 7 e). Since deuterosomes were very large electron-dense structures, it was possible that antibodies cannot access the antigen within deuterosomes. However, postembedding immunolabeling did not detect mPCM-1 within deuterosomes, excluding this possibility (Fig. 7 f). Taken together, we concluded that so-called fibrous granules, which had been intensively examined from the viewpoint of centriolar replication, may be identical to PCM-1–containing centriolar satellites.

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
Related in: MedlinePlus