Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome (2020)
Figure 8

Figure 8 Peroxisomes directly contribute to the cholesterol supply into ciliary membranes

(A) Quiescent 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.

(B) Quantification 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, representatively.

(C) Model 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.

Data Availability

Raw data and CRISPR/Cas9-mediated knock-out cells associated with the figures will be made available on a reasonable request

Acknowledgments

We thank Drs. Shinji Yokoyama, Tetsurou Handa, Tomoko Mogami, Hideshi Kawakami, Akihito Inoko, Futoshi Suizu, Toshihiko Iwanaga, Keiji Suzuki, Yuki Kobayashi, Kenichi Suzuki and Kazumasa Fujita for critical reading of the manuscript and helpful discussions. We thank Dr. Satoshi Tashiro, Yasunori Horikoshi, and Junichi Kakimura for help in the 3D-SIM observations. We also thank Ms. Yukiji Tonouchi and Junko Murakami, and Mrs. Yoshinori Masatsuna and Akihiro Fukumitsu for their assistance. This work was supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.M. and S.M.: 15H01216, 15K15017, 18H04979, and 18H02624); Grant-in-Aid for Scientific Research from the Ministry of Health, Labour and Welfare (to S.M.); the Center of World Intelligence Projects for Nuclear S&T and Human Resource Development from the Japan Science and Technology Agency (to S.M.); AMED-PRIME from the Japan Agency for Medical Research and Development, AMED (to T.M.: JP18gm5910011h0004); research grants from the Naito Foundation (to S.M. and T.M.); the Japan Agency for Medical Research and Development, AMED (to T.M.); Ono Medical Research Foundation (to T.M.); and Takeda Science Foundation (to T.M.).

Author contributions

T.M., K.H., T.I., AH.I, S.N.A., and H.O. performed experiments and analyzed data. T.M. conceived the study. AH.I, Y.S., T.Y., and S.M. supervised the study. T.M., K.H., T.I., AH.I and S.M. wrote the paper. All authors interpreted data.

Conflict of interest

The authors declare no competing interests.