Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome (2020)
Figure 5

Figure 5 ORP3 is involved in the peroxisome-mediated cholesterol supply into the ciliary membrane

(A) Schematic of ORP3 structure. ORP3 contains an N-terminal pleckstrin homology (PH) domain that interacts with phospholipids, an FFAT motif that specifically interacts with VAP-A/B, and a highly conserved C-terminal OSBP-homology domain (OHD).

(B) Western blot analysis showing depletion of ORP3 in the ORP3−/− hTERT-RPE1 cell clones. GAPDH served as a loading control.

(C) ORP3+/+ and ORP3−/− hTERT-RPE1 cells incubated for 24 h without serum were immunostained with anti-pericentrin (red) and anti-acetylated tubulin (blue) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 5 μm.

(D) Quantification of the Filipin III intensity at primary cilia from (C). ORP3−/− hTERT-RPE1 cells exhibited a significant reduction of ciliary cholesterol (***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.

(E) Three-dimensional reconstitution of the quiescent G0-phase ORP3+/+ and ORP3−/− hTERT-RPE1 cells immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ORP3 (green), and anti-PMP70 (white) antibodies indicates that ORP3 at the ciliary pocket (arrow and arrowhead) mediates the membrane regions of ciliary pocket and peroxisomes (arrowhead). The scale bars indicate 5 μm.

(F) Quantification of proportion of primary cilia interacting with peroxisomes from (E). Depletion of ORP3 significantly interfered with the spatial interaction between peroxisomes and primary cilia (mean ± s.d.: ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 45-50 cells per experiment).

(G) 3×FLAG-tagged ORP3 and AcGFP1-tagged PEX14, EHD1 or EHD3 were coexpressed in HEK293T cells and then immunoprecipitated from whole-cell lysates using the anti-FLAG antibody. 3×FLAG-tagged ORP3 and AcGFP1-tagged PEX14, EHD1 or EHD3 fragments in the IP fractions and inputs were detected by western blotting.

(H) ORP3−/− hTERT-RPE1 cells were transfected with AcGFP1-tagged ORP3, PH domain deleted-ORP3 mutant (ΔPH), FFAT motif-deleted-ORP3 mutant (ΔFFAT), or OHD domain deleted-ORP3 mutant (ΔOHD) and cultured without serum for 24 h before Filipin-III (green)-mediated cholesterol staining and immunostaining with anti-GFP (red), anti-pericentrin (white), and anti-acetylated-tubulin (blue) antibodies. Arrows represent AcGFP1-tagged ORP3 or the mutants localized to the ciliary pocket. AcGFP1-tagged ORP3ΔPH mutant mis-localized to the ciliary pocket. The scale bars indicate 5 μm.

(I) Quantification of the Filipin III intensity at primary cilia from (H). AcGFP1-tagged ORP3 deletion mutants did not restore the ciliary cholesterol insufficiency in the ORP3−/− hTERT-RPE1 cells (*p<0.05, ***p<0.001: one-way ANOVA with Tukey's HSD, n=3: 40-50 cells per experiment). In the boxplot, medians, 25th/75th percentile and min/max were represented by the central lines, the box limits, and the whiskers/error bars, representatively.