CEP290 interacts with the centriolar satellite component PCM-1 and is required for Rab8 localization to the primary cilium

Abstract

Joubert syndrome (JS) is a developmental brain disorder characterized by cerebellar vermis hypoplasia, abnormal eye movement, ataxia and mental retardation. Mutations in CEP290 mutations are responsible for the cerebello-oculo-renal subtype of JS that includes kidney cysts and retinal degeneration, two phenotypes commonly linked to ciliopathies. CEP290 mutations are also associated with Meckel-Gruber syndrome and Bardet-Biedl syndrome (BBS). Here we demonstrate that CEP290 interacts with a centriolar satellite protein PCM-1, which is implicated in BBS4 function. CEP290 binds to PCM-1 and localizes to centriolar satellites in a PCM-1- and microtubule-dependent manner. The depletion of CEP290 disrupts subcellular distribution and protein complex formation of PCM-1. In accord with PCM-1's role in microtubule organization, CEP290 knockdown causes the disorganization of the cytoplasmic microtubule network. Moreover, we show that both CEP290 and PCM-1 are required for ciliogenesis and are involved in the ciliary targeting of Rab8, a small GTPase shown to collaborate with BBS protein complex to promote ciliogenesis. Our results suggest that PCM-1 is a potential mediator that may link CEP290 with BBS proteins in common molecular pathways.

Figure 1.

CEP290 localizes to centriolar satellites and is associated with PCM-1. (A) Double-immunofluorescent localization of CEP290 in hTERT-RPE cells. Anti-CEP290 antibody labels granular structures scattered around the centrosome. CEP290 is adjacent to, but not overlapping with, pericentrin and γ-tubulin. CEP290 only partially overlaps with centrin. CEP290;Ac-tub double-labeling shows that ciliated cells exhibit similar CEP290 immunostaining patterns. GFP-tagged CEP290, which recapitulated endogenous CEP290 localization at lower expression levels, extensively co-localizes with centriolar satellite marker PCM-1. Bottom panels are higher magnification images of an individual optical section captured by a ×100 objective. (B) HEK293T cells were co-transfected with GFP–PCM-1 and either mCherry–CEP290 or mCherry plasmid. Cell lysates were immunoprecipitated with anti-mCherry antibody and then immunoblotted with anti-GFP antibody. GFP–PCM-1 was co-immunoprecipitated with mCherry–CEP290. (C) PCM-1 knockdown was assessed by immunoblotting and immunofluorescence staining 3 days after PCM-1 siRNA transfection. (D) hTERT-RPE cells were transfected with siRNAs for 3 days and stained with anti-CEP290 antibody. Depletion of PCM-1 leads to the reduction of the number of CEP290 granules scattered around the centrosome. Right panels are higher magnification view of the boxed areas in the left panels. Cell nuclei were stained with Hoechst. Scale bars: (A) 5 µm; (C) 15 µm; (D) 20 µm.

Figure 2.

CEP290 moves along microtubules to the centrosome. (A) Cultured hTERT-RPE cells were treated with 20 µm nocodazole for 2 h and double-stained with anti-CEP290 and anti-γ-tubulin antibodies. Nocodazole treatment causes substantial decrease in granular CEP290 staining detected around the centrosome, suggesting that the recruitment of CEP290 to centriolar satellites requires intact microtubules. (B) Time-lapse observation of the movement of GFP-CEP290 along mCherry-labeled microtubules in live IMCD-3 cells. The boxed area in the left panel is magnified in the small panels. Numbers indicate the time lapse in seconds. Scale bars: (A) 10 µm; (B) 5 µm.

Figure 3.

CEP290 affects the localization and complex formation of PCM-1. (A) CEP290 knockdown was assessed by immunoblotting and immunofluorescence staining 3 days after CEP290 siRNA transfection. (B) hTERT-RPE cells were stained with anti-PCM-1 antibody 3 days after siRNA transfection. CEP290 knockdown causes the concentric accumulation of PCM-1, depleting widely dispersed PCM-1 pool. Arrow indicates a cell showing widely dispersed PCM-1 granules without apparent centrosomal concentration. Line drawings mark cell outline. (C) Histogram quantifying cells without noticeable concentration of PCM-1 granules around the centrosome as shown in (B) (arrow). More than 700 cells from three independent experiments were counted for each bar by two investigators (one of them was blind to the experiment). Error bars represent SD. Mitotic cells were excluded from the counting. (D) hTERT-RPE cells were stained with anti-PCM-1;anti-pericentrin antibodies 15 h after transfection with either GFP or GFP-CEP290 plasmid DNA. CEP290 overexpression facilitates the dispersion of centrosomal pool of PCM-1 granules. (E) Histogram quantifying the result shown in (D). More than 170 cells expressing transgenes from two independent experiments were counted for each bar by two investigators (one of them was blind to the experiment). (F) Histogram showing integrated pixel density of anti-PCM-1 immunofluorescence around the centrosome (7 µm²). Each bar is mean value (arbitrary unit) of 18 transgene-positive cells. (G) Cells were treated with nocodazole and cytochalasin to disassemble cytoskeleton 3 days after siRNA transfection and lysed in 0.5% NP-40 and 150 mm NaCl. Low-speed (1500 g) supernatants were fractionated on discontinuous sucrose gradients. PCM-1 and BBS4 proteins from CEP290-depleted lysates fail to migrate into higher density fractions, indicating that they form smaller protein complexes in the absence of CEP290. Scale bar: 20 µm.

Figure 4.

Microtubule organization and ciliogenesis require CEP290 function. (A) The depletion of CEP290 from hTERT-RPE cells causes a collapse of microtubule networks, which is accompanied by an apparent decrease in cell size. (B–C) hTERT-RPE cells were shifted from 10% serum to serum-free medium 24 h after transfection with the indicated siRNAs and cultured for additional 60 h before fixation. Ciliated cells were identified by anti-Ac-tub antibody staining. Basal body was labeled by anti-γ-tub antibody. The depletion of either CEP290 or PCM-1 inhibits ciliogenesis. (C) Histogram quantifying the data shown in (B). More than 200 cells from two independent experiments were counted for each bar. Error bars represent SD. (D) Representative images of hTERT-RPE cells stained with the indicated antibodies after CEP290 siRNA transfection and serum starvation. Both ninein and pericentrin staining of CEP290-depleted cells were indistinguishable from those of control cells. Scale bars: (A) 10 µm; (B and D) 20 µm.

Figure 5.

Both CEP290 and PCM-1 are involved in the regulation of Rab8 translocation into the primary cilium. (A) hTERT-RPE cells were shifted from 10% serum to serum-free medium 24 h after siRNA transfection, cultured for 24 h, and then re-transfected with Rab8-GFP for 36 h. Cells were triple-labeled with Rab8-GFP, anti-Glu-tub antibody (a ciliary marker) and anti-CEP290/PCM-1. (B) Histogram quantifying the data shown in (A). Ciliated Rab8-GFP+ cells that show substantial reduction of CEP290 or PCM-1 were compared with ciliated Rab8-GFP+ control cells. One hundred and fifteen control cells, 50 CEP290-depleted cells and 48 PCM-1-depleted cells from two independent experiments were counted for each bar. Error bars represent SD. The depletion of either CEP290 or PCM-1 interferes with Rab8-GFP translocation into the primary cilium. Scale bar: (A) 5 µm.