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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 localizes to centrioles and basal bodies. (a) Immunofluorescence image of a centrosome in RPE1 cells costained for Pcnt (green) and centriolin (red; bar, 1 μm). (b and c) DIC (b) and immunofluorescence (c) images of a primary cilium (arrow) in RPE1 cell stained for Pcnt (green) and centrioles/primary cilium (GT335, red). Bar, 5 μm for b and c. (d) Immunofluorescence image of a ciliated epithelial cell from mouse trachea showing Pcnt (green) at the base of motile cilia (DIC; bar, 5 μm). (e) Immunogold electron microscopic image of a ciliated cell (as in d) after incubation with antibodies to Pcnt and secondary antibodies bound to 5-nm gold (bar, 250 nm).
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fig1: Pcnt localizes to centrioles and basal bodies. (a) Immunofluorescence image of a centrosome in RPE1 cells costained for Pcnt (green) and centriolin (red; bar, 1 μm). (b and c) DIC (b) and immunofluorescence (c) images of a primary cilium (arrow) in RPE1 cell stained for Pcnt (green) and centrioles/primary cilium (GT335, red). Bar, 5 μm for b and c. (d) Immunofluorescence image of a ciliated epithelial cell from mouse trachea showing Pcnt (green) at the base of motile cilia (DIC; bar, 5 μm). (e) Immunogold electron microscopic image of a ciliated cell (as in d) after incubation with antibodies to Pcnt and secondary antibodies bound to 5-nm gold (bar, 250 nm).

Mentions: In this work, we have studied a larger isoform of Pcnt using specific siRNAs and antibodies unless otherwise noted (Flory and Davis, 2003; Zimmerman et al., 2004). Immunofluorescence imaging demonstrated that Pcnt partially overlapped with centriolin, a protein associated with the mother centriole at centrosomes (Fig. 1 a) (Gromley et al., 2003). In addition, Pcnt associated with both centrioles at the base of primary cilia (Fig. 1, b and c) and motile cilia (Fig. 1 d). Higher resolution immunogold EM demonstrated that Pcnt was on or near the centrioles of motile cilia (Fig. 1 e).


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 localizes to centrioles and basal bodies. (a) Immunofluorescence image of a centrosome in RPE1 cells costained for Pcnt (green) and centriolin (red; bar, 1 μm). (b and c) DIC (b) and immunofluorescence (c) images of a primary cilium (arrow) in RPE1 cell stained for Pcnt (green) and centrioles/primary cilium (GT335, red). Bar, 5 μm for b and c. (d) Immunofluorescence image of a ciliated epithelial cell from mouse trachea showing Pcnt (green) at the base of motile cilia (DIC; bar, 5 μm). (e) Immunogold electron microscopic image of a ciliated cell (as in d) after incubation with antibodies to Pcnt and secondary antibodies bound to 5-nm gold (bar, 250 nm).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172416&req=5

fig1: Pcnt localizes to centrioles and basal bodies. (a) Immunofluorescence image of a centrosome in RPE1 cells costained for Pcnt (green) and centriolin (red; bar, 1 μm). (b and c) DIC (b) and immunofluorescence (c) images of a primary cilium (arrow) in RPE1 cell stained for Pcnt (green) and centrioles/primary cilium (GT335, red). Bar, 5 μm for b and c. (d) Immunofluorescence image of a ciliated epithelial cell from mouse trachea showing Pcnt (green) at the base of motile cilia (DIC; bar, 5 μm). (e) Immunogold electron microscopic image of a ciliated cell (as in d) after incubation with antibodies to Pcnt and secondary antibodies bound to 5-nm gold (bar, 250 nm).
Mentions: In this work, we have studied a larger isoform of Pcnt using specific siRNAs and antibodies unless otherwise noted (Flory and Davis, 2003; Zimmerman et al., 2004). Immunofluorescence imaging demonstrated that Pcnt partially overlapped with centriolin, a protein associated with the mother centriole at centrosomes (Fig. 1 a) (Gromley et al., 2003). In addition, Pcnt associated with both centrioles at the base of primary cilia (Fig. 1, b and c) and motile cilia (Fig. 1 d). Higher resolution immunogold EM demonstrated that Pcnt was on or near the centrioles of motile cilia (Fig. 1 e).

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.

Show MeSH
Related in: MedlinePlus