Limits...
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.

Show MeSH
Specificity of pAbs. a, Rabbit anti–Xenopus 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 (a–c; CBB staining), followed by immunoblotting with anti–XPCM-1 pAb (d–f). This affinity-purified pAb specifically recognized ∼230 kD XPCM-1 in d and e, as well as GST fusion protein in f. b, Rabbit anti–mouse 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 anti–mPCM-1 pAb (c and d). This affinity-purified pAb specifically recognized ∼230 kD mPCM-1 in c, as well as GST fusion protein in d.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169353&req=5

Figure 1: Specificity of pAbs. a, Rabbit anti–Xenopus 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 (a–c; CBB staining), followed by immunoblotting with anti–XPCM-1 pAb (d–f). This affinity-purified pAb specifically recognized ∼230 kD XPCM-1 in d and e, as well as GST fusion protein in f. b, Rabbit anti–mouse 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 anti–mPCM-1 pAb (c and d). This affinity-purified pAb specifically recognized ∼230 kD mPCM-1 in c, as well as GST fusion protein in d.

Mentions: To analyze the molecular components of centrosomes, we raised mAbs against pericentriolar material isolated from Xenopus egg extracts. Since one of these mAbs, W8C3, stained centrosomes of cultured Xenopus epithelial A6 cells, we isolated a full-length cDNA encoding its antigen by screening a Xenopus embryo λZAP II cDNA expression library. DNA sequencing revealed that its product encoded a protein of 2,031 aa with significant similarity to human PCM-1 (hPCM-1; 56.8% identity at the amino acid sequence level), indicating that W8C3 recognized a Xenopus homologue of PCM-1 (XPCM-1; sequence data are available from GenBank/EMBL/DDBJ under accession number AB025414). As this mAb showed some cross-reactivity with α-tubulin, we then raised a pAb against recombinant XPCM-1 produced in E. coli. As shown on immunoblots, this pAb specifically recognized a 230-kD band in A6 cell lysates, as well as Xenopus egg extracts (Fig. 1 a). Judging from the molecular mass of human PCM-1 (∼230 kD) and from the reactivity of this pAb with recombinant Xenopus PCM-1, we concluded that this pAb specifically recognized XPCM-1.


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)

Specificity of pAbs. a, Rabbit anti–Xenopus 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 (a–c; CBB staining), followed by immunoblotting with anti–XPCM-1 pAb (d–f). This affinity-purified pAb specifically recognized ∼230 kD XPCM-1 in d and e, as well as GST fusion protein in f. b, Rabbit anti–mouse 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 anti–mPCM-1 pAb (c and d). This affinity-purified pAb specifically recognized ∼230 kD mPCM-1 in c, as well as GST fusion protein in d.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Specificity of pAbs. a, Rabbit anti–Xenopus 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 (a–c; CBB staining), followed by immunoblotting with anti–XPCM-1 pAb (d–f). This affinity-purified pAb specifically recognized ∼230 kD XPCM-1 in d and e, as well as GST fusion protein in f. b, Rabbit anti–mouse 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 anti–mPCM-1 pAb (c and d). This affinity-purified pAb specifically recognized ∼230 kD mPCM-1 in c, as well as GST fusion protein in d.
Mentions: To analyze the molecular components of centrosomes, we raised mAbs against pericentriolar material isolated from Xenopus egg extracts. Since one of these mAbs, W8C3, stained centrosomes of cultured Xenopus epithelial A6 cells, we isolated a full-length cDNA encoding its antigen by screening a Xenopus embryo λZAP II cDNA expression library. DNA sequencing revealed that its product encoded a protein of 2,031 aa with significant similarity to human PCM-1 (hPCM-1; 56.8% identity at the amino acid sequence level), indicating that W8C3 recognized a Xenopus homologue of PCM-1 (XPCM-1; sequence data are available from GenBank/EMBL/DDBJ under accession number AB025414). As this mAb showed some cross-reactivity with α-tubulin, we then raised a pAb against recombinant XPCM-1 produced in E. coli. As shown on immunoblots, this pAb specifically recognized a 230-kD band in A6 cell lysates, as well as Xenopus egg extracts (Fig. 1 a). Judging from the molecular mass of human PCM-1 (∼230 kD) and from the reactivity of this pAb with recombinant Xenopus PCM-1, we concluded that this pAb specifically recognized XPCM-1.

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