Fig 2: Peroxisomes accompanied by cholesterol move along the microtubules to interact with the ciliary pocket structure AQuiescent G0-phase hTERT-RPE1 cells were immunostained with anti-PEX14 (red), anti-ninein (white), and anti-acetylated-tubulin (blue) antibodies. Cholesterol was stained with Filipin III (Green). Magnified images of the boxed regions showing peroxisome accompanied by cholesterol (arrows). Three-dimensional reconstitution of the same cell indicates that Filipin III stains the membrane regions of ciliary axonemes and peroxisomes. The scale bars indicate 2.5 µm and 1.25 µm in lower- and higher-magnified images, respectively.BQuiescent G0-phase wild-type hTERT-RPE1 cells treated with Cytochalasin-D (200 nM), colcemid (50 nM), or Ciliobrevin-D (10 µM) for 6 h were immunostained with anti-ARL13B (blue), anti-phospho-S473-Akt (red), anti-ninein (green), and anti-PMP70 (white) antibodies. Arrows indicate the peroxisomes interacting with the ciliary pocket. Scale bar, 5 µm.CQuantification of proportion of primary cilia interacting with peroxisomes from (B). Colcemid (50 nM) and Ciliobrevin-D (10 µM) significantly inhibited 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).DQuiescent G0-phase wild-type hTERT-RPE1 cells were treated with Cytochalasin-D (200 nM), Colcemid (50 nM), or Ciliobrevin-D (10 µM) for 6 h and then immunostained with anti-acetylated-tubulin (blue) and anti-pericentrin (green) antibodies. Cholesterol was stained with Filipin III (green). Arrows indicate primary cilia. Scale bar, 2.5 µm.EQuantification of the Filipin III intensity at primary cilia from (D). Colcemid and Ciliobrevin-D interfered with the distribution of cholesterol in the ciliary membrane (***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.FhTERT-RPE1 cells were transfected with AcGFP1-tagged EHD3 and cultured without serum for 24 h before immunostaining with anti-GFP (green), anti-ARL13B (blue), anti-PMP70 (white), and anti-phospho-S473-Akt (red) antibodies. The scale bars represent 2.5 µm.G3×FLAG-tagged EHD1 or EHD3 and AcGFP1-tagged PEX14 were coexpressed in HEK293T cells and then immunoprecipitated from whole-cell lysates using the anti-FLAG antibody. AcGFP1-tagged PEX14 and 3×FLAG-tagged EHD1 or EHD3 fragments in the IP fractions and inputs were detected by Western blotting.HRecombinant GST (0.2 µg) or GST fused to PEX14 proteins (1 µg) and 6×His-EHD3 protein (1 µg) were pulled down using glutathione-Sepharose beads. GST-tagged PEX14 and 6×His-tagged EHD3 proteins in the pull-down fractions and inputs were detected by western blotting.I3D reconstitution of the quiescent G0-phase hTERT-RPE1 cells transfected with AcGFP1-tagged EHD3 (white) immunostained with anti-acetylated-tubulin (blue) and anti-PEX14 (red) antibodies. Cholesterol was stained with Filipin III (green). Cholesterol-containing peroxisome interacted with the ciliary pocket (arrow). The scale bars indicate 2 µm. Source data are available online for this figure.

Fig 3: ARF6-GDP, ACAP2 and Rab35 act upstream of EHD1 to regulate TNTs: (a) Confocal images of cells transfected with shControl + GFP, shEHD1 + GFP-ARF6-T27N. Arrows indicate TNTs. (b) Bar graphs representing the relative percentage of TNT-connected cells described in (a) (shControl + GFP = 100%, shEHD1 + GFP-ARF6-T27N = 83.7 ± 4.4%). (c) Bar graph showing the relative percentage of acceptor cells containing DiD-labelled vesicles from the co-cultures, where donor cells were transfected with shControl + GFP, shEHD1 + GFP-ARF6-T27N (shControl + GFP = 100%, shEHD1 + GFP-ARF6-T27N = 75.1 ± 13.3%). (d) Confocal images of cells transfected with shControl + GFP, shEHD1 + GFP-ACAP2. Arrows indicate TNTs. (e) Bar graphs representing the relative percentage of TNT-connected cells described in (d) (shControl + GFP = 100%, shEHD1 + GFP-ACAP2 = 98.1 + 19.3%). (f) Bar graph showing the relative percentage of acceptor cells containing DiD-labelled vesicles from the co-cultures, where donor cells were transfected with shControl + GFP, shEHD1 + GFP-ACAP2 (shControl + GFP = 100%, shEHD1 + GFP-ACAP2 = 90.8 + 14.6%). (g) Confocal images of cells transfected with shControl + GFP, shEHD1 + GFP- Rab35-WT. Arrows indicate TNTs. (h) Bar graphs representing the relative percentage of TNT-connected cells described in (g) (shControl + GFP = 100%, shEHD1 + GFP-Rab35-WT = 78.3 ± 11.2%). (i) Bar graph showing the relative percentage of acceptor cells containing DiD-labelled vesicles from the co-cultures, where donor cells were transfected with shControl + GFP, shEHD1 + GFP-Rab35-WT (shControl + GFP = 100%, shEHD1 + GFP-Rab35-WT = 83.3 ± 12.4%). All graphs from three independent experiments and show mean ± SEM. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; by unpaired Student’s t-test). All bar graphs were analysed and represented using Graph Pad Prism version 7. Scale bars 10 µm.

Fig 4: EHD1 is required for TNT formation: (a) Confocal images of cells transfected with GFP, GFP-EHD1. Arrows indicate TNTs. (b) Bar graphs representing the relative percentage of TNT-connected cells described in (a) (GFP = 100%, GFP-EHD1 = 150.5 ± 23%). (c) Bar graph showing the relative percentage of acceptor cells containing DiD-labelled vesicles from the co-cultures, where donor cells were transfected with GFP, GFP-EHD1 (GFP = 100%, GFP-EHD1 = 157.6 ± 5.5%). All graphs from three independent experiments and show mean ± SEM. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; by unpaired Student’s t-test); (d) Confocal images of cells transfected with shControl + GFP, shEHD1 + GFP, shEHD1 + GFP-EHD1. Arrows indicate TNTs. (e) Western blot of cells transfected with shRNA non-targeting (shCTL) or targeting EHD1 (shEHD1), showing the expression of EHD1 and a-tubulin as loading control. (f) Bar graphs representing the relative percentage of TNT-connected cells described in (a) (shControl + GFP = 100%, shEHD1 + GFP = 51.7 ± 12.7%, shEHD1 + GFP-EHD1 = 110.5 ± 8.2%). (g) Bar graph showing the relative percentage of acceptor cells containing DiD- labelled vesicles from the co-cultures, where donor cells were transfected with shControl + GFP, shEHD1 + GFP, shEHD1 + GFP-EHD1 (shControl + GFP = 100%, shEHD1 + GFP = 51.5 ± 9.0%, shEHD1 + GFP-EHD1 = 107.9 ± 5.6%). All graphs from at least three independent experiments and shows mean ± SEM. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; by one-way ANOVA with Tukey’s multiple comparison test). All bar graphs were analysed and represented using Graph Pad Prism version 7. Scale bars 10 µm.

Fig 5: ORP3 is involved in the peroxisome-mediated cholesterol supply into the ciliary membrane ASchematic 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).BWestern 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.DQuantification 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, respectively.EThree-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.FQuantification 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).G3×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 (?PH), FFAT motif-deleted-ORP3 mutant (?FFAT), or OHD domain-deleted-ORP3 mutant (?OHD) 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 ORP3?PH mutant mis-localized to the ciliary pocket. The scale bars indicate 5 µm.IQuantification 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, respectively. Source data are available online for this figure.