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Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly.

Jurczyk A, Gromley A, Redick S, San Agustin J, Witman G, Pazour GJ, Peters DJ, Doxsey S - J. Cell Biol. (2004)

Bottom Line: Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases.Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions.We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotech II, Suite 210, Worcester, MA 01605, USA.

ABSTRACT
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

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Pcnt colocalizes to basal bodies with IFT proteins and PC2, and Pcnt silencing mislocalizes IFT proteins and PC2 from basal bodies and centrosomes. (a–c′′) IFT57 (a–b′′ red) and PC2 (c–c′′, red) are mislocalized from basal bodies in RPE1 cells with reduced Pcnt (b–b′′, arrows; c–c′′, green, small arrows), but not in RPE1 cells treated with lamin siRNAs (a–a′′; bar, 10 μm) or in the cell with control level of Pcnt (c and c′′, bottom). Insets: higher magnification of a′′, b′′, and c′′ as indicated by arrows. DNA, blue.
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fig4: Pcnt colocalizes to basal bodies with IFT proteins and PC2, and Pcnt silencing mislocalizes IFT proteins and PC2 from basal bodies and centrosomes. (a–c′′) IFT57 (a–b′′ red) and PC2 (c–c′′, red) are mislocalized from basal bodies in RPE1 cells with reduced Pcnt (b–b′′, arrows; c–c′′, green, small arrows), but not in RPE1 cells treated with lamin siRNAs (a–a′′; bar, 10 μm) or in the cell with control level of Pcnt (c and c′′, bottom). Insets: higher magnification of a′′, b′′, and c′′ as indicated by arrows. DNA, blue.

Mentions: Next, we addressed the centriolar anchoring mechanism of Pcnt, IFTs, and PC2. We found that Pcnt was dependent on IFT proteins for localization to basal bodies using cells that stably express siRNAs targeting IFT20. These cells showed reduced centriolar IFT20 and lacked primary cilia (Fig. 3, g–g′′ and h) compared with cells of the parent line (Fig. 3, f–f′′ and h). In cells with reduced centriole-associated IFT20, we observed a similar reduction in Pcnt levels (Fig. 3 g, g,′′ and i). In a reciprocal experiment, we found that IFTs and PC2 were dependent on Pcnt for centriole localization. Pcnt localized to both centrioles at the base of cilia, partially colocalized with IFT proteins (Fig. 4 a′′, IFT57) and totally overlapped with PC2 (Fig. 4 c′′). Pcnt silencing reduced the levels of centriolar Pcnt (Fig. 4 b, b′′, c, and c′′; top cell), IFT57 (Fig. 4, b′–b′′), IFT20, IFT88 (unpublished data), and PC2 (Fig. 4, c′–c′′; top cell). In contrast, adjacent nontransfected cells or cells treated with lamin siRNAs had robust staining for IFT57 and PC2 (Fig. 4, a′–a′′ and c′–c′′; bottom cell). These results show that Pcnt and IFTs are codependent in their localization to basal bodies of primary cilia.


Pericentrin forms a complex with intraflagellar transport proteins and polycystin-2 and is required for primary cilia assembly.

Jurczyk A, Gromley A, Redick S, San Agustin J, Witman G, Pazour GJ, Peters DJ, Doxsey S - J. Cell Biol. (2004)

Pcnt colocalizes to basal bodies with IFT proteins and PC2, and Pcnt silencing mislocalizes IFT proteins and PC2 from basal bodies and centrosomes. (a–c′′) IFT57 (a–b′′ red) and PC2 (c–c′′, red) are mislocalized from basal bodies in RPE1 cells with reduced Pcnt (b–b′′, arrows; c–c′′, green, small arrows), but not in RPE1 cells treated with lamin siRNAs (a–a′′; bar, 10 μm) or in the cell with control level of Pcnt (c and c′′, bottom). Insets: higher magnification of a′′, b′′, and c′′ as indicated by arrows. DNA, blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172416&req=5

fig4: Pcnt colocalizes to basal bodies with IFT proteins and PC2, and Pcnt silencing mislocalizes IFT proteins and PC2 from basal bodies and centrosomes. (a–c′′) IFT57 (a–b′′ red) and PC2 (c–c′′, red) are mislocalized from basal bodies in RPE1 cells with reduced Pcnt (b–b′′, arrows; c–c′′, green, small arrows), but not in RPE1 cells treated with lamin siRNAs (a–a′′; bar, 10 μm) or in the cell with control level of Pcnt (c and c′′, bottom). Insets: higher magnification of a′′, b′′, and c′′ as indicated by arrows. DNA, blue.
Mentions: Next, we addressed the centriolar anchoring mechanism of Pcnt, IFTs, and PC2. We found that Pcnt was dependent on IFT proteins for localization to basal bodies using cells that stably express siRNAs targeting IFT20. These cells showed reduced centriolar IFT20 and lacked primary cilia (Fig. 3, g–g′′ and h) compared with cells of the parent line (Fig. 3, f–f′′ and h). In cells with reduced centriole-associated IFT20, we observed a similar reduction in Pcnt levels (Fig. 3 g, g,′′ and i). In a reciprocal experiment, we found that IFTs and PC2 were dependent on Pcnt for centriole localization. Pcnt localized to both centrioles at the base of cilia, partially colocalized with IFT proteins (Fig. 4 a′′, IFT57) and totally overlapped with PC2 (Fig. 4 c′′). Pcnt silencing reduced the levels of centriolar Pcnt (Fig. 4 b, b′′, c, and c′′; top cell), IFT57 (Fig. 4, b′–b′′), IFT20, IFT88 (unpublished data), and PC2 (Fig. 4, c′–c′′; top cell). In contrast, adjacent nontransfected cells or cells treated with lamin siRNAs had robust staining for IFT57 and PC2 (Fig. 4, a′–a′′ and c′–c′′; bottom cell). These results show that Pcnt and IFTs are codependent in their localization to basal bodies of primary cilia.

Bottom Line: Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases.Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions.We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, University of Massachusetts Medical School, 373 Plantation Street, Biotech II, Suite 210, Worcester, MA 01605, USA.

ABSTRACT
Primary cilia are nonmotile microtubule structures that assemble from basal bodies by a process called intraflagellar transport (IFT) and are associated with several human diseases. Here, we show that the centrosome protein pericentrin (Pcnt) colocalizes with IFT proteins to the base of primary and motile cilia. Immunogold electron microscopy demonstrates that Pcnt is on or near basal bodies at the base of cilia. Pcnt depletion by RNA interference disrupts basal body localization of IFT proteins and the cation channel polycystin-2 (PC2), and inhibits primary cilia assembly in human epithelial cells. Conversely, silencing of IFT20 mislocalizes Pcnt from basal bodies and inhibits primary cilia assembly. Pcnt is found in spermatocyte IFT fractions, and IFT proteins are found in isolated centrosome fractions. Pcnt antibodies coimmunoprecipitate IFT proteins and PC2 from several cell lines and tissues. We conclude that Pcnt, IFTs, and PC2 form a complex in vertebrate cells that is required for assembly of primary cilia and possibly motile cilia and flagella.

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