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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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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 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).

Mentions: To identify the motor protein responsible for this in vitro movement of GFP-tagged centriolar satellites, we examined the effects of some inhibitors of motor proteins (Fig. 5). At 10 μM, vanadate abolished the accumulation of centriolar satellites around centrosomes. This finding suggested that dynein was involved, since dynein, but not kinesin, is inhibited by low concentrations of vanadate (10–20 μM; Schroer and Sheetz 1989). AMP-PNP did not affect centriolar satellite accumulation at a concentration of 100 μM, whereas at higher concentrations, such as 2 mM, AMP-PNP showed complete suppression. This again favored the notion that dynein is responsible for the centriolar satellite movement, since 100 μM AMP-PNP inhibits kinesin, but not dynein (2 mM AMP-PNP inhibits both; Schroer and Sheetz 1989). In good agreement with these observations, antidynein intermediate chain mAb (m70.1; 60 μg/ml) completely abolished the accumulation of GFP-tagged centriolar satellites around centrosomes, while control IgG had no effect.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

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

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).
Mentions: To identify the motor protein responsible for this in vitro movement of GFP-tagged centriolar satellites, we examined the effects of some inhibitors of motor proteins (Fig. 5). At 10 μM, vanadate abolished the accumulation of centriolar satellites around centrosomes. This finding suggested that dynein was involved, since dynein, but not kinesin, is inhibited by low concentrations of vanadate (10–20 μM; Schroer and Sheetz 1989). AMP-PNP did not affect centriolar satellite accumulation at a concentration of 100 μM, whereas at higher concentrations, such as 2 mM, AMP-PNP showed complete suppression. This again favored the notion that dynein is responsible for the centriolar satellite movement, since 100 μM AMP-PNP inhibits kinesin, but not dynein (2 mM AMP-PNP inhibits both; Schroer and Sheetz 1989). In good agreement with these observations, antidynein intermediate chain mAb (m70.1; 60 μg/ml) completely abolished the accumulation of GFP-tagged centriolar satellites around centrosomes, while control IgG had no effect.

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