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
In vitro motility of GFP-tagged centriolar satellites. a, Time-lapse observation of the movement of a centriolar satellite (green) in a reconstituted aster (red). The boxed area in the large panel is magnified in the small panels. Numbers at the bottom left in the small panels indicate the time lapse in seconds. GFP-tagged centriolar satellite (arrowheads) moved along a MT toward centrosomes where other granules had already accumulated. On the way to the centrosome, this granule changed MTs (arrows), and finally reached the centrosome. Bar, 10 μm. A QuickTime movie is available at http://www.jcb.org/cgi/content/147/5/969/F4/DC1. b, Immunoelectron microscopy of centriolar satellites accumulating around centrosomes in the in vitro reconstituted asters. Electron-dense granular structures, ∼80–90 nm in diameter, which were specifically labeled with anti–XPCM-1 pAb (10-nm gold particles), were accumulated around fibrous materials of centrosomes. These XPCM-1–containing granules were not delineated by membranes. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2169353&req=5

Figure 4: In vitro motility of GFP-tagged centriolar satellites. a, Time-lapse observation of the movement of a centriolar satellite (green) in a reconstituted aster (red). The boxed area in the large panel is magnified in the small panels. Numbers at the bottom left in the small panels indicate the time lapse in seconds. GFP-tagged centriolar satellite (arrowheads) moved along a MT toward centrosomes where other granules had already accumulated. On the way to the centrosome, this granule changed MTs (arrows), and finally reached the centrosome. Bar, 10 μm. A QuickTime movie is available at http://www.jcb.org/cgi/content/147/5/969/F4/DC1. b, Immunoelectron microscopy of centriolar satellites accumulating around centrosomes in the in vitro reconstituted asters. Electron-dense granular structures, ∼80–90 nm in diameter, which were specifically labeled with anti–XPCM-1 pAb (10-nm gold particles), were accumulated around fibrous materials of centrosomes. These XPCM-1–containing granules were not delineated by membranes. Bar, 200 nm.

Mentions: A movie file corresponding to Fig. 4 a is available online (http://www.jcb.org/cgi/content/147/5/969/F4/DC1). Time-lapse images collected using DeltaVision were processed with Adobe Photoshop software and converted to a QuickTime movie (JPEG compression) with Adobe Premiere Software. This movie contains the time-lapse images from the first to the last panel in the corresponding figures. Images were recorded for 3 min at 5–6 s intervals.


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)

In vitro motility of GFP-tagged centriolar satellites. a, Time-lapse observation of the movement of a centriolar satellite (green) in a reconstituted aster (red). The boxed area in the large panel is magnified in the small panels. Numbers at the bottom left in the small panels indicate the time lapse in seconds. GFP-tagged centriolar satellite (arrowheads) moved along a MT toward centrosomes where other granules had already accumulated. On the way to the centrosome, this granule changed MTs (arrows), and finally reached the centrosome. Bar, 10 μm. A QuickTime movie is available at http://www.jcb.org/cgi/content/147/5/969/F4/DC1. b, Immunoelectron microscopy of centriolar satellites accumulating around centrosomes in the in vitro reconstituted asters. Electron-dense granular structures, ∼80–90 nm in diameter, which were specifically labeled with anti–XPCM-1 pAb (10-nm gold particles), were accumulated around fibrous materials of centrosomes. These XPCM-1–containing granules were not delineated by membranes. Bar, 200 nm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: In vitro motility of GFP-tagged centriolar satellites. a, Time-lapse observation of the movement of a centriolar satellite (green) in a reconstituted aster (red). The boxed area in the large panel is magnified in the small panels. Numbers at the bottom left in the small panels indicate the time lapse in seconds. GFP-tagged centriolar satellite (arrowheads) moved along a MT toward centrosomes where other granules had already accumulated. On the way to the centrosome, this granule changed MTs (arrows), and finally reached the centrosome. Bar, 10 μm. A QuickTime movie is available at http://www.jcb.org/cgi/content/147/5/969/F4/DC1. b, Immunoelectron microscopy of centriolar satellites accumulating around centrosomes in the in vitro reconstituted asters. Electron-dense granular structures, ∼80–90 nm in diameter, which were specifically labeled with anti–XPCM-1 pAb (10-nm gold particles), were accumulated around fibrous materials of centrosomes. These XPCM-1–containing granules were not delineated by membranes. Bar, 200 nm.
Mentions: A movie file corresponding to Fig. 4 a is available online (http://www.jcb.org/cgi/content/147/5/969/F4/DC1). Time-lapse images collected using DeltaVision were processed with Adobe Photoshop software and converted to a QuickTime movie (JPEG compression) with Adobe Premiere Software. This movie contains the time-lapse images from the first to the last panel in the corresponding figures. Images were recorded for 3 min at 5–6 s intervals.

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