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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 silencing inhibits primary cilia formation. (a) Pcnt and lamin protein levels (Western blot) after siRNA as indicated. α-Tubulin or PKC, loading controls. (b) Fluorescence intensity of individual centrosomes (bars) after treatment with siRNAs targeting Pcnt or lamin. Centrosomal Pcnt is reduced to levels below the lowest control levels (lamin) in 87% of cells. (c) Immunofluorescence image of RPE1 cells after Pcnt silencing showing reduced centrosomal Pcnt in one cell (green, arrow) and normal level in the other. γ-Tubulin (red) is not significantly affected. Low (d and e) and high (f and g) magnification immunofluorescence images of cilia and centrioles stained with GT335 after treatment with Pcnt (e and g) or lamin (d and f) siRNAs. Bar in e, 5 μm (for d and e); bar in f, 1 μm (for f and g). DNA, blue. (h) Graph showing percentage of cells that lack cilia after treatment with indicated siRNAs. Bars represent average of three experiments. P value, standard t test. (i–k) Electron micrographs showing centriole structure in cells with reduced Pcnt. Bar in k, 200 nm (for i–k).
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fig2: Pcnt silencing inhibits primary cilia formation. (a) Pcnt and lamin protein levels (Western blot) after siRNA as indicated. α-Tubulin or PKC, loading controls. (b) Fluorescence intensity of individual centrosomes (bars) after treatment with siRNAs targeting Pcnt or lamin. Centrosomal Pcnt is reduced to levels below the lowest control levels (lamin) in 87% of cells. (c) Immunofluorescence image of RPE1 cells after Pcnt silencing showing reduced centrosomal Pcnt in one cell (green, arrow) and normal level in the other. γ-Tubulin (red) is not significantly affected. Low (d and e) and high (f and g) magnification immunofluorescence images of cilia and centrioles stained with GT335 after treatment with Pcnt (e and g) or lamin (d and f) siRNAs. Bar in e, 5 μm (for d and e); bar in f, 1 μm (for f and g). DNA, blue. (h) Graph showing percentage of cells that lack cilia after treatment with indicated siRNAs. Bars represent average of three experiments. P value, standard t test. (i–k) Electron micrographs showing centriole structure in cells with reduced Pcnt. Bar in k, 200 nm (for i–k).

Mentions: To test the role of Pcnt in cilia organization, we depleted protein levels by siRNA. We observed a 75–90% reduction in protein levels and a dramatic reduction in centrosome levels of Pcnt in most cells (Fig. 2, a–c; arrow in c) when compared with cells treated with control siRNAs targeting lamins A/C (Fig. 2, a and b) or cells that did not respond to siRNA treatment (Fig. 2 c, bottom cell). In contrast, centrosome localization of γ-tubulin was only slightly affected under these conditions (Fig. 2 c, top cell). Primary cilia were induced in retinal pigmented epithelial cells (RPE1) treated with siRNAs targeting Pcnt or lamin A/C. Cilia were detected with antibodies to polyglutamylated tubulins (GT335; Gromley et al., 2003) and by differential interference contrast (DIC) microscopy. In most cells treated with siRNAs targeting Pcnt, primary cilia failed to assemble (Fig. 2, e, g, and h), whereas control cells treated with siRNAs targeting lamin or ninein assembled normal full-length primary cilia (Fig. 2, d, f, and h; unpublished data).


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 silencing inhibits primary cilia formation. (a) Pcnt and lamin protein levels (Western blot) after siRNA as indicated. α-Tubulin or PKC, loading controls. (b) Fluorescence intensity of individual centrosomes (bars) after treatment with siRNAs targeting Pcnt or lamin. Centrosomal Pcnt is reduced to levels below the lowest control levels (lamin) in 87% of cells. (c) Immunofluorescence image of RPE1 cells after Pcnt silencing showing reduced centrosomal Pcnt in one cell (green, arrow) and normal level in the other. γ-Tubulin (red) is not significantly affected. Low (d and e) and high (f and g) magnification immunofluorescence images of cilia and centrioles stained with GT335 after treatment with Pcnt (e and g) or lamin (d and f) siRNAs. Bar in e, 5 μm (for d and e); bar in f, 1 μm (for f and g). DNA, blue. (h) Graph showing percentage of cells that lack cilia after treatment with indicated siRNAs. Bars represent average of three experiments. P value, standard t test. (i–k) Electron micrographs showing centriole structure in cells with reduced Pcnt. Bar in k, 200 nm (for i–k).
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fig2: Pcnt silencing inhibits primary cilia formation. (a) Pcnt and lamin protein levels (Western blot) after siRNA as indicated. α-Tubulin or PKC, loading controls. (b) Fluorescence intensity of individual centrosomes (bars) after treatment with siRNAs targeting Pcnt or lamin. Centrosomal Pcnt is reduced to levels below the lowest control levels (lamin) in 87% of cells. (c) Immunofluorescence image of RPE1 cells after Pcnt silencing showing reduced centrosomal Pcnt in one cell (green, arrow) and normal level in the other. γ-Tubulin (red) is not significantly affected. Low (d and e) and high (f and g) magnification immunofluorescence images of cilia and centrioles stained with GT335 after treatment with Pcnt (e and g) or lamin (d and f) siRNAs. Bar in e, 5 μm (for d and e); bar in f, 1 μm (for f and g). DNA, blue. (h) Graph showing percentage of cells that lack cilia after treatment with indicated siRNAs. Bars represent average of three experiments. P value, standard t test. (i–k) Electron micrographs showing centriole structure in cells with reduced Pcnt. Bar in k, 200 nm (for i–k).
Mentions: To test the role of Pcnt in cilia organization, we depleted protein levels by siRNA. We observed a 75–90% reduction in protein levels and a dramatic reduction in centrosome levels of Pcnt in most cells (Fig. 2, a–c; arrow in c) when compared with cells treated with control siRNAs targeting lamins A/C (Fig. 2, a and b) or cells that did not respond to siRNA treatment (Fig. 2 c, bottom cell). In contrast, centrosome localization of γ-tubulin was only slightly affected under these conditions (Fig. 2 c, top cell). Primary cilia were induced in retinal pigmented epithelial cells (RPE1) treated with siRNAs targeting Pcnt or lamin A/C. Cilia were detected with antibodies to polyglutamylated tubulins (GT335; Gromley et al., 2003) and by differential interference contrast (DIC) microscopy. In most cells treated with siRNAs targeting Pcnt, primary cilia failed to assemble (Fig. 2, e, g, and h), whereas control cells treated with siRNAs targeting lamin or ninein assembled normal full-length primary cilia (Fig. 2, d, f, and h; unpublished data).

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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