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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 interacts with proteins involved in cilia assembly and function. (a) Pooled IFT fractions from a sucrose gradient from mouse testes were applied to an FPLC column and fractions were loaded on SDS gels and probed with Pcnt antibodies or IFT88 antibody. (b) Pooled peak centrosome fractions from sucrose gradients (+) containing γ-tubulin, Pcnt, and IFT88 as indicated and pooled noncentrosome fractions (−). (c, top) Pcnt NH2- and COOH- terminal antibodies (PcN, PcC) independently immunoprecipitated endogenous IFT88 from lysates of ciliated 488 cells. IgG, nonimmune rabbit IgG, lysates (Lys) showing IFT88 at right. Pcnt immunoprecipitation confirmed (right). (c, middle) PcC/N immunoprecipitated IFT88 from ciliated RPE1 cells, as did antibodies to IFT88 and IFT57 but not rabbit IgG. (c, bottom) PcN and PcC pull down a GST–GFP–IFT20 fusion protein from a cell line stably overexpressing the protein, as does a glutathione column (IFT20), but not nonimmune IgG. Blots were probed with anti-GFP antibodies, immunoprecipitation with Pcnt (right); IB, immunoblot antibody. (d) PcN immunoprecipitated PC2 from mitotic RPE1 cells, whereas beads alone did not (Bd). Pcnt immunoprecipitation confirmed by immunoblot (Pcnt B). (e) PC2 antibody, but not rabbit IgG, immunoprecipitated IFT57 from ciliated 488 cells. IFT57, top band. Antibody heavy chain, bottom band.
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fig5: Pcnt interacts with proteins involved in cilia assembly and function. (a) Pooled IFT fractions from a sucrose gradient from mouse testes were applied to an FPLC column and fractions were loaded on SDS gels and probed with Pcnt antibodies or IFT88 antibody. (b) Pooled peak centrosome fractions from sucrose gradients (+) containing γ-tubulin, Pcnt, and IFT88 as indicated and pooled noncentrosome fractions (−). (c, top) Pcnt NH2- and COOH- terminal antibodies (PcN, PcC) independently immunoprecipitated endogenous IFT88 from lysates of ciliated 488 cells. IgG, nonimmune rabbit IgG, lysates (Lys) showing IFT88 at right. Pcnt immunoprecipitation confirmed (right). (c, middle) PcC/N immunoprecipitated IFT88 from ciliated RPE1 cells, as did antibodies to IFT88 and IFT57 but not rabbit IgG. (c, bottom) PcN and PcC pull down a GST–GFP–IFT20 fusion protein from a cell line stably overexpressing the protein, as does a glutathione column (IFT20), but not nonimmune IgG. Blots were probed with anti-GFP antibodies, immunoprecipitation with Pcnt (right); IB, immunoblot antibody. (d) PcN immunoprecipitated PC2 from mitotic RPE1 cells, whereas beads alone did not (Bd). Pcnt immunoprecipitation confirmed by immunoblot (Pcnt B). (e) PC2 antibody, but not rabbit IgG, immunoprecipitated IFT57 from ciliated 488 cells. IFT57, top band. Antibody heavy chain, bottom band.

Mentions: Previous reports showed that IFT protein complexes and Pcnt complexes had similar S values on sucrose gradients (17–18S; Dictenberg et al., 1998; Pazour et al., 2002a). To determine whether Pcnt interacted with IFT proteins, we isolated IFT complexes by a multistep procedure (San Agustin and Witman, 2001) and found that Pcnt A and B cofractionated with IFT88 in the final gel filtration column (Fig. 5 a). Based on recent data showing that IFT71 is present on centrosomes and spindle poles (Iomini et al., 2004), we analyzed centrosome preparations (Doxsey et al., 1994) for the presence of IFT proteins. IFT88 (Fig. 5 b) and other IFT proteins (unpublished data) were present in pooled fractions containing centrosome proteins (γ-tubulin, Pcnt), but not in pooled fractions lacking centrosomes. Moreover, our immunofluorescence imaging showed that IFT20, 57, 88, and PC2 were abundant at centrosomes in interphase cells and spindle poles during mitosis in RPE1 cells (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 interacts with proteins involved in cilia assembly and function. (a) Pooled IFT fractions from a sucrose gradient from mouse testes were applied to an FPLC column and fractions were loaded on SDS gels and probed with Pcnt antibodies or IFT88 antibody. (b) Pooled peak centrosome fractions from sucrose gradients (+) containing γ-tubulin, Pcnt, and IFT88 as indicated and pooled noncentrosome fractions (−). (c, top) Pcnt NH2- and COOH- terminal antibodies (PcN, PcC) independently immunoprecipitated endogenous IFT88 from lysates of ciliated 488 cells. IgG, nonimmune rabbit IgG, lysates (Lys) showing IFT88 at right. Pcnt immunoprecipitation confirmed (right). (c, middle) PcC/N immunoprecipitated IFT88 from ciliated RPE1 cells, as did antibodies to IFT88 and IFT57 but not rabbit IgG. (c, bottom) PcN and PcC pull down a GST–GFP–IFT20 fusion protein from a cell line stably overexpressing the protein, as does a glutathione column (IFT20), but not nonimmune IgG. Blots were probed with anti-GFP antibodies, immunoprecipitation with Pcnt (right); IB, immunoblot antibody. (d) PcN immunoprecipitated PC2 from mitotic RPE1 cells, whereas beads alone did not (Bd). Pcnt immunoprecipitation confirmed by immunoblot (Pcnt B). (e) PC2 antibody, but not rabbit IgG, immunoprecipitated IFT57 from ciliated 488 cells. IFT57, top band. Antibody heavy chain, bottom band.
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fig5: Pcnt interacts with proteins involved in cilia assembly and function. (a) Pooled IFT fractions from a sucrose gradient from mouse testes were applied to an FPLC column and fractions were loaded on SDS gels and probed with Pcnt antibodies or IFT88 antibody. (b) Pooled peak centrosome fractions from sucrose gradients (+) containing γ-tubulin, Pcnt, and IFT88 as indicated and pooled noncentrosome fractions (−). (c, top) Pcnt NH2- and COOH- terminal antibodies (PcN, PcC) independently immunoprecipitated endogenous IFT88 from lysates of ciliated 488 cells. IgG, nonimmune rabbit IgG, lysates (Lys) showing IFT88 at right. Pcnt immunoprecipitation confirmed (right). (c, middle) PcC/N immunoprecipitated IFT88 from ciliated RPE1 cells, as did antibodies to IFT88 and IFT57 but not rabbit IgG. (c, bottom) PcN and PcC pull down a GST–GFP–IFT20 fusion protein from a cell line stably overexpressing the protein, as does a glutathione column (IFT20), but not nonimmune IgG. Blots were probed with anti-GFP antibodies, immunoprecipitation with Pcnt (right); IB, immunoblot antibody. (d) PcN immunoprecipitated PC2 from mitotic RPE1 cells, whereas beads alone did not (Bd). Pcnt immunoprecipitation confirmed by immunoblot (Pcnt B). (e) PC2 antibody, but not rabbit IgG, immunoprecipitated IFT57 from ciliated 488 cells. IFT57, top band. Antibody heavy chain, bottom band.
Mentions: Previous reports showed that IFT protein complexes and Pcnt complexes had similar S values on sucrose gradients (17–18S; Dictenberg et al., 1998; Pazour et al., 2002a). To determine whether Pcnt interacted with IFT proteins, we isolated IFT complexes by a multistep procedure (San Agustin and Witman, 2001) and found that Pcnt A and B cofractionated with IFT88 in the final gel filtration column (Fig. 5 a). Based on recent data showing that IFT71 is present on centrosomes and spindle poles (Iomini et al., 2004), we analyzed centrosome preparations (Doxsey et al., 1994) for the presence of IFT proteins. IFT88 (Fig. 5 b) and other IFT proteins (unpublished data) were present in pooled fractions containing centrosome proteins (γ-tubulin, Pcnt), but not in pooled fractions lacking centrosomes. Moreover, our immunofluorescence imaging showed that IFT20, 57, 88, and PC2 were abundant at centrosomes in interphase cells and spindle poles during mitosis in RPE1 cells (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.

Show MeSH
Related in: MedlinePlus