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

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.

Figure 1.

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).

Figure 2.

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).

Figure 3.

Localization of IFT proteins and PC2, and mislocalization of Pcnt in cells with reduced IFT20. (a–d) RPE1 cells stained for IFT57, IFT88, IFT20, and PC2 (green) and for basal bodies/cilia (GT335, red). Bar in d, 1 μm. (e) Pcnt (green) partially colocalizes with IFT20 (red) at the base of motile cilia (seen by DIC) in mouse epithelial cells. DNA, blue. Bar, 5 μm. (f–g′′) Untreated RPE1 cells (f–f′′) or RPE1 cells stably expressing siRNA targeting IFT20 (g–g′′) showing centrosomal levels of IFT20 (f′ and g′), Pcnt (f and g; bar, 5 μm), or merge (f′′ and g′′). Pcnt, red, IFT20, green, DNA, blue at arrows. Insets, enlargements of f′′ and g′′. (h and i) Fluorescence intensity of IFT20 (h) and Pcnt (i) at individual centrosomes (bars) in cells stably expressing IFT20 siRNA or mock, as indicated below graph.

Figure 4.

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.

Figure 5.

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.