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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 localization of XPCM-1 in A6 cells. a–c, Double immunofluorescence staining of A6 cells with anti–XPCM-1 pAb (a) and anti–γ-tubulin mAb (b). The merged image (c) revealed that most of the XPCM-1–positive granular structures were concentrated on and/or around γ-tubulin–positive centrosomes. Small numbers of XPCM-1–positive 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 anti–XPCM-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 2: Subcellular localization of XPCM-1 in A6 cells. a–c, Double immunofluorescence staining of A6 cells with anti–XPCM-1 pAb (a) and anti–γ-tubulin mAb (b). The merged image (c) revealed that most of the XPCM-1–positive granular structures were concentrated on and/or around γ-tubulin–positive centrosomes. Small numbers of XPCM-1–positive 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 anti–XPCM-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.

Mentions: Next, to examine the subcellular localization of XPCM-1 by immunofluorescence microscopy, cultured A6 cells were doubly stained with the anti–XPCM-1 pAb and anti–γ-tubulin mAb (Fig. 2, a–c). The γ-tubulin signal was exclusively detected in centrosomes, whereas XPCM-1 was concentrated on and/or around γ-tubulin–positive centrosomes in large amounts, and was also scattered in the cytoplasm in a punctate manner in small amounts. In metaphase, its concentration around centrosomes became obscure as previously described in HeLa cells (Balczon et al. 1994; data not shown). Interestingly, when these XPCM-1–positive granular structures were examined in Triton X-100–treated A6 cells by immunoelectron microscopy, anti–XPCM-1 pAb specifically labeled electron-dense spherical granules 80–90 nm in diameter located around centrosomes (Fig. 2 d). Some of these granules appeared to be associated with MTs. Conventional EM of A6 cells also identified similar electron-dense granules gathering around centrosomes, which were not surrounded by membranes (Fig. 2 e). Judging from their morphological characteristics, we concluded that these granules were identical to the previously described structures designated as centriolar satellites (Bernhard and De Harven 1960; de-Thé 1964; Berns et al. 1977). As shown in Fig. 2d and Fig. e, pale granules with a similar diameter were also observed around centrioles, but these granules were not labeled with anti–XPCM-1 pAb.


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 localization of XPCM-1 in A6 cells. a–c, Double immunofluorescence staining of A6 cells with anti–XPCM-1 pAb (a) and anti–γ-tubulin mAb (b). The merged image (c) revealed that most of the XPCM-1–positive granular structures were concentrated on and/or around γ-tubulin–positive centrosomes. Small numbers of XPCM-1–positive 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 anti–XPCM-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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169353&req=5

Figure 2: Subcellular localization of XPCM-1 in A6 cells. a–c, Double immunofluorescence staining of A6 cells with anti–XPCM-1 pAb (a) and anti–γ-tubulin mAb (b). The merged image (c) revealed that most of the XPCM-1–positive granular structures were concentrated on and/or around γ-tubulin–positive centrosomes. Small numbers of XPCM-1–positive 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 anti–XPCM-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.
Mentions: Next, to examine the subcellular localization of XPCM-1 by immunofluorescence microscopy, cultured A6 cells were doubly stained with the anti–XPCM-1 pAb and anti–γ-tubulin mAb (Fig. 2, a–c). The γ-tubulin signal was exclusively detected in centrosomes, whereas XPCM-1 was concentrated on and/or around γ-tubulin–positive centrosomes in large amounts, and was also scattered in the cytoplasm in a punctate manner in small amounts. In metaphase, its concentration around centrosomes became obscure as previously described in HeLa cells (Balczon et al. 1994; data not shown). Interestingly, when these XPCM-1–positive granular structures were examined in Triton X-100–treated A6 cells by immunoelectron microscopy, anti–XPCM-1 pAb specifically labeled electron-dense spherical granules 80–90 nm in diameter located around centrosomes (Fig. 2 d). Some of these granules appeared to be associated with MTs. Conventional EM of A6 cells also identified similar electron-dense granules gathering around centrosomes, which were not surrounded by membranes (Fig. 2 e). Judging from their morphological characteristics, we concluded that these granules were identical to the previously described structures designated as centriolar satellites (Bernhard and De Harven 1960; de-Thé 1964; Berns et al. 1977). As shown in Fig. 2d and Fig. e, pale granules with a similar diameter were also observed around centrioles, but these granules were not labeled with anti–XPCM-1 pAb.

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