Fig 2: Peroxisomes directly contribute to the cholesterol supply into ciliary membranes AQuiescent G0-phase wild-type, Rab10 -/-, KIFC3 -/-, ORP3 -/-, and EHD3 -/- hTERT-RPE1 cells transfected with PEX3-GFP-FRB (red) and tdTomato-BicD2-FKBP (white) were treated with or without 100 µM rapamycin for 30 min and then immunostained with anti-acetylated-tubulin (blue) antibody. Cholesterol was stained with Filipin III (Green). Arrows indicate the enrichment of ciliary cholesterol. The scale bars represent 5 µm.BQuantification of the Filipin III intensity at primary cilia from (A). Rapamycin-induced peroxisome targeting to the ciliary pocket restored the insufficiency of ciliary cholesterol in the Rab10 -/-and KIFC3 -/-hTERT-RPE1 cells, but not in ORP3 -/-and EHD3 -/- hTERT-RPE1 cells (*P < 0.05, **P < 0.01, ***P < 0.001: one-way ANOVA with Tukey's HSD, n = 3: 35–40 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, respectively.CModel for the molecular machinery for the supply of cholesterol into the ciliary membrane. The Rabin8–Rab10–KIFC3 complex associates peroxisomes with cholesterol to load them on microtubule arrays. KIFC3-mediated microtubule minus-end-directed movement guides peroxisomes to the ciliary pocket for the supply of cholesterol to the ciliary membrane. The ORP3–EHD1–EHD3 complex is involved in cholesterol trafficking at the ciliary pocket. In the pathological context of ZS, insufficient ciliary cholesterol might impair the cilium-related Shh signal competence, causing ciliopathy-related symptoms.

Fig 3: The Rabin8–Rab10–KIFC3 complex is implicated in the peroxisome-mediated supply of cholesterol into the ciliary membrane AWhole-cell lysates from HEK293T cells expressing AcGFP1 or AcGFP1-tagged PEX14 and 3×FLAG-tagged KIFC3 were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.BWhole-cell lysates from HEK293T cells expressing AcGFP1-tagged KIFC3 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.CWhole-cell lysates from HEK293T cells expressing AcGFP1-tagged Pex14 and 3×FLAG-tagged Rab10 or Rab10-Q68L were immunoprecipitated with anti-FLAG antibody and immunoblotted with anti-GFP or anti-FLAG antibody.DWestern blot analysis of the ciliary cholesterol trafficking-associated components in Pex1 +/+ and Pex1 -/- hTERT-RPE1 cells. Total cell lysates were separated to crude peroxisomal (Crude Pex), lysosomal and mitochondrial (Lyso/Mito), and peroxisomal (PEX) fractions. CYPOR, a lysosomal and mitochondrial protein, served as a positive control for the Lyso/Mito fractionation. Total cell lysates were gradually injected at 20 µg and 5 µg into a gel for SDS–PAGE, while equal amounts (5 µg) of protein from each fraction were loaded. Rabin8, Rab10, and KIFC3 were concentrated in the PEX fraction in Pex1 +/+ hTERT-RPE1 cells. CYPOR (cytochrome P450 reductase) is a mitochondrial protein.EQuiescent G0-phase hTERT-RPE1 cells transfected with 3×FLAG-tagged KIFC3 were immunostained with anti-FLAG (green), anti-PEX14 (red), and anti-a-tubulin (white) antibodies. Magnified 3D-constituted images of the boxed regions showing peroxisomes are located on the microtubule arrays via KIFC3 (arrows). The scale bars indicate 5 µm.FWestern blot analysis showing depletion of KIFC3 in the KIFC3 -/- hTERT-RPE1 cell clones. GAPDH served as a loading control.G KIFC3 +/+ and KIFC3 -/- 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). Scale bar, 5 µm.HQuantification of (G) indicating that KIFC3 -/- hTERT-RPE1 cells significantly reduced the ciliary accumulation of 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, respectively.IQuiescent G0-phase KIFC3 +/+ and KIFC3 -/- hTERT-RPE1 cells were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows represent the peroxisomes contacting the ciliary pocket. Scale bar, 5 µm.JQuantification of (I) showing that disruption of the KIFC3 gene significantly interfered with the contact 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).K KIFC3 -/- hTERT-RPE1 cells were transfected with AcGFP1, AcGFP1-tagged KIFC3, rod domain-deleted KIFC3 mutant (?Rod), or motor domain-deleted KIFC3 mutant (?Motor) 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 ciliary localization of cholesterol. The scale bars indicate 5 µm.LQuantification of the Filipin III intensity at primary cilia from (K). AcGFP1-tagged KIFC3 deletion mutants did not restore the ciliary cholesterol insufficiency in the KIFC3 -/- hTERT-RPE1 cells (mean ± s.d.: ***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, respectively. Source data are available online for this figure.