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Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites

Nature volume 502, pages 254–257 (2013)Cite this article

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Abstract

The primary cilium is a microtubule-based organelle that functions in sensory and signalling pathways. Defects in ciliogenesis can lead to a group of genetic syndromes known as ciliopathies1,2,3. However, the regulatory mechanisms of primary ciliogenesis in normal and cancer cells are incompletely understood. Here we demonstrate that autophagic degradation of a ciliopathy protein, OFD1 (oral-facial-digital syndrome 1), at centriolar satellites promotes primary cilium biogenesis. Autophagy is a catabolic pathway in which cytosol, damaged organelles and protein aggregates are engulfed in autophagosomes and delivered to lysosomes for destruction4. We show that the population of OFD1 at the centriolar satellites is rapidly degraded by autophagy upon serum starvation. In autophagy-deficient Atg5 or Atg3 null mouse embryonic fibroblasts, OFD1 accumulates at centriolar satellites, leading to fewer and shorter primary cilia and a defective recruitment of BBS4 (Bardet–Biedl syndrome 4) to cilia. These defects are fully rescued by OFD1 partial knockdown that reduces the population of OFD1 at centriolar satellites. More strikingly, OFD1 depletion at centriolar satellites promotes cilia formation in both cycling cells and transformed breast cancer MCF7 cells that normally do not form cilia. This work reveals that removal of OFD1 by autophagy at centriolar satellites represents a general mechanism to promote ciliogenesis in mammalian cells. These findings define a newly recognized role of autophagy in organelle biogenesis.

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Figure 1: OFD1 is an autophagic substrate.
Figure 2: Autophagy specifically degrades OFD1 at centriolar satellites upon serum starvation.
Figure 3: Autophagy promotes primary cilia biogenesis by regulating OFD1 levels.
Figure 4: Forced OFD1 reduction promotes ciliogenesis in human breast cancer MCF7 cells.

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Acknowledgements

We thank D. Moughon and W. Fan for initial purification of LC3 complex; N. Mizushima for Atg5+/+ and Atg5−/− MEFs and M. Komatsu for Atg3+/+ and Atg3−/− MEFs; H. Zhou for helpful discussions and quantitative RT–PCR data analysis; A. Kodani and J. F. Reiter for reagents, helpful discussions and technical assistance; and B. Levine for helpful discussions and critical reading of the manuscript. The electron microscopy studies were performed by C. Miller and V. Gattone II at the Indiana University School of Medicine Electron Microscopy Center that is supported by the Polycystic Kidney Disease Foundation. This work was supported by grants from the American Cancer Society (RSG-11-274-01-CCG) and National Cancer Institute (CA133228) to Q.Z. and grants from the Italian Telethon Foundation (TGM11CB3) and the EU FP7/2007-2013 Programme (Syscilia grant agreement no. 241955) to B.F. The work was partially supported by China Scholarship Council to Z.T.

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Authors and Affiliations

  1. Department of Internal Medicine, Center for Autophagy Research, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Zaiming Tang & Qing Zhong

  2. Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, 75390, Texas, USA

    Zaiming Tang & Qing Zhong

  3. Division of Biochemistry, Department of Molecular and Cell Biology, Biophysics and Structural Biology, University of California, Berkeley, 94720, California, USA

    Zaiming Tang, Mary Grace Lin & Qing Zhong

  4. State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, 310058, China

    Zaiming Tang & Muyuan Zhu

  5. Department of Biology, Stanford University, Stanford, 94305, California, USA

    Timothy Richard Stowe & Tim Stearns

  6. National Institute of Biological Sciences, Beijing, 102206, China

    She Chen

  7. Department of Translational Medical Sciences, Medical Genetics Services, Federico II University, Via Pietro Castellino 111, Naples, Italy,

    Brunella Franco

  8. Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, Naples, Italy,

    Brunella Franco

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  1. Zaiming Tang
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Contributions

Z.T., M.G.L. and T.R.S. performed the experiments; S.C. carried out mass spectrometry analysis; M.Z., T.S. and B.F. provided technical and intellectual support; Z.T. and Q.Z. conceived the project, designed the experiments, analysed the data and wrote the manuscript with the help of all authors.

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Correspondence to Qing Zhong.

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Extended data figures and tables

Extended Data Figure 1 LC3-interacting protein PCM1 is not required for autophagy.

a, PCM1 associates with LC3, GATE16 and GABARAP. Silver staining of LC3, GATE16 or GABARAP complexes purified from U2OS cells that stably express ZZ–Flag–LC3, ZZ–Flag–GATE16, or ZZ–Flag–GABARAP in normal medium or subjected to 2 h Earle’s balanced salt solution (EBSS) starvation. Both PCM1 and p62 were identified by mass spectrometry analysis. b, PCM1 is not required for autophagy. Western blotting analysis of p62, LC3-I/II, PCM1 levels in control or PCM1 shRNA knockdown U2OS cells in normal medium or subjected to rapamycin treatment; quantified LC3-II level was normalized with β-tubulin. c, OFD1 messenger RNA levels remain unchanged upon serum starvation. Quantitative analysis of messenger RNA levels of OFD1 in Atg5+/+ and Atg5−/− MEFs in normal medium or subjected to 24 h serum starvation. OFD1 mRNA levels were detected by quantitative RT–PCR and plotted after normalization. Similar results were obtained in three independent experiments.

Extended Data Figure 2 PCM1 is required for OFD1 centriolar satellite localization.

a, Representative confocal images of OFD1 and PCM1 localization from control or PCM1 knockdown U2OS cells in normal medium. Data shown represent 100 cells per well in triplicate samples. b, Quantified percentage of cells with PCM1 positive centriolar satellite OFD1 in a. Data shown represent 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. c, LC3 partially colocalizes with PCM1 upon serum starvation. Representative confocal images of Myc–LC3 and PCM1 colocalization in U2OS cells expressing Myc–LC3 in normal medium or subjected to 24 h serum starvation. Arrows denote colocalized LC3 (green) and PCM1 (red) puncta. Data shown represent 100 cells per well in triplicate samples. d, Quantified percentage of cells with colocalization of Myc–LC3 and PCM1 in c. ***P < 0.001, two-tailed unpaired student’s t-test. Similar results were observed in three independent experiments.

Extended Data Figure 3 LC3 partially colocalizes with OFD1 but not with γ-tubulin.

a, LC3 colocalizes with OFD1 when the lysosome activity is blocked. Representative confocal images of Myc–LC3 and OFD1 colocalization in U2OS cells that stably express Myc–LC3 in normal medium or subjected to 2 h 50 nM bafilomycin A1 (Baf) or 100 µM CQ. Data shown represent 100 cells per well in triplicate samples. b, Quantified percentage of cells with colocalization of Myc–LC3 and OFD1 in a. c, LC3 does not colocalize with centrioles. Representative confocal images of LC3 and γ-tubulin colocalization in U2OS cells in normal medium or subjected to 24 h serum starvation. Data shown represent 100 cells per well in triplicate samples. d, Quantified percentage of cells with colocalization of LC3 and γ-tubulin in c. Similar results were obtained in three independent experiments.

Extended Data Figure 4 OFD1 but not PCM1 at centriolar satellites was degraded by autophagy.

a, OFD1 accumulates at centriolar satellites in CQ-treated cells. Representative confocal images of EGFP–OFD1 and PCM1 colocalization in Atg5+/+ cells expressing EGFP–OFD1 subjected to 24 h serum starvation or 20 µM CQ. b, Quantified percentage of cells with centriolar satellite OFD1 in a. Data shown represent mean ± s.d. for 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. c, PCM1 is not degraded upon serum starvation. Representative confocal images of PCM1 centriolar satellite staining in Atg5+/+ cells in normal medium or subjected to 24 h serum starvation. Data shown represent mean ± s.d. for 200 cells per well in triplicate samples. d, Quantified percentage of cells with PCM1 centriolar satellite staining in c. e, OFD1 but not PCM1 is degraded from centriolar satellites upon serum starvation. Representative confocal images of PCM1 and OFD1 colocalization in Atg5+/+ cells in normal medium or subjected to 24 h serum starvation. Data shown represent 200 cells per well in triplicated samples. Enlarged images were shown in the left bottom panels. Similar results were obtained in three independent experiments.

Extended Data Figure 5 The turnover rate of centriolar satellite OFD1 is faster than OFD1 at centrioles.

a, Centriolar satellite OFD1 has a shorter half-life compared to OFD1 at centrioles. Quantified percentage of cells with OFD1 at centrioles or at centriolar satellites from Atg5+/+ and Atg5−/− MEFs in normal medium or subjected to 75 µM cycloheximide (CHX) with indicated time points. Data shown represent 200 cells per well in triplicate samples. b, Centriolar satellite OFD1 but not centriole OFD1 degrades upon serum starvation. Quantified percentage of cells with OFD1 at centrioles or at centriolar satellites from Atg5+/+ and Atg5−/− MEFs in normal medium or subjected to serum starvation with indicated time points. Data shown represent 200 cells per well in triplicate samples. Similar results were obtained in three independent experiments.

Extended Data Figure 6 Autophagy regulates primary ciliogenesis in a cell cycle independent manner.

a, FACS analysis of Atg5+/+ and Atg5−/− MEFs in normal medium or subjected to 24 h serum starvation. Data shown represent 106 cells per well in triplicate samples. b, Primary ciliogenesis is less efficient when the lysosome activity is blocked in MEFs. Representative confocal images of primary cilia formed in Atg5+/+ MEFs subjected to 24 h serum starvation alone or combined with 20 µM CQ treatment. c, Quantified percentage of cells with primary cilia in b. d, Quantified length of primary cilia in b. e, Degradation of OFD1 is also blocked in Atg3−/− MEFs. Western blot analysis of OFD1, p62, LC3-I/II and BBS4 protein levels in MEFs with indicated genotypes in normal medium or subjected to 24 h serum starvation; quantified OFD1 levels were normalized with β-tubulin. f, g, Primary ciliogenesis is also defective in Atg3−/− MEFs. f, Quantified percentage of cells with primary cilia in Atg3+/+ and Atg3−/− MEFs in normal medium or subjected to 24 h serum starvation. g, Quantified length of primary cilia formed in Atg3+/+ and Atg3−/− MEFs as described in f. c, d, f, g, Data shown represent mean ± s.d. for 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. Similar results were obtained in three independent experiments.

Extended Data Figure 7 BBS4 recruitment to primary cilia is defective in Atg5−/− MEFs.

a, Representative confocal images of Atg5+/+ and Atg5−/− MEFs expressing Myc–BBS4 subjected to 24 h serum starvation. Scale bar 5 µm. b, Quantified percentage of cells with Myc–BBS4 translocation into primary cilia in Atg5+/+ and Atg5−/− MEFs. Data shown represent mean ± s.d. for 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. Similar results were obtained in three independent experiments.

Extended Data Figure 8 Partial shRNA knockdown of OFD1 leads to depletion of OFD1 from centriolar satellites in Atg5+/+ and Atg5−/− MEFs.

a, Western blot analysis of OFD1 in MEFs with indicated genotypes in normal medium. Quantified OFD1 levels were normalized with β-tubulin. KD, knockdown. b, Quantified percentage of cells with centriolar satellite OFD1 in MEFs with indicated genotypes in normal medium. Data shown represent mean ± s.d. percentage of cells with centriolar satellite OFD1 for 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. c, d, OFD1 was depleted from centriolar satellites but not centrioles in OFD1 knockdown MEFs. Representative confocal images of OFD1 and axoneme marker acetylated tubulin in c or centriole marker γ-tubulin in d in MEFs with indicated genotypes in normal medium. Data shown represent 100 cells per well in triplicate samples. Similar results were obtained in three independent experiments.

Extended Data Figure 9 Knockdown of OFD1 in wild-type MEFs promotes primary ciliogenesis.

a, Representative confocal images of primary cilia formed in MEFs with indicated genotypes in normal medium (Un) or subjected to 24 h serum starvation (SS). Quantified percentage of cells with primary cilia and the length of primary cilia from MEFs with indicated genotypes were shown in the bottom panels. Data shown represent mean ± s.d. for 100 cells per well in triplicate samples. b, Representative confocal images of primary cilia formed in MEFs with indicated genotypes subjected to 24 h serum starvation. The primary cilia formed are positive for both axoneme marker acetylated tubulin and ciliary membrane marker ARL13B. Data shown represent 100 cells per well in triplicate samples. Similar results were obtained in three independent experiments.

Extended Data Figure 10 Partial knockdown OFD1 in MCF7 cells depletes OFD1 from centriolar satellites and promotes primary ciliogenesis.

a, b, OFD1 was depleted from centriolar satellites in OFD1 shRNA knockdown MCF7 cells. a, Representative confocal images of relative localization of OFD1 with axoneme marker acetylated tubulin in MCF7 OFD1 knockdown clone (C19). Data shown represent 100 cells per well in triplicate samples. b, Representative confocal images of OFD1 and centriole marker γ-tubulin from C19. Data shown represent 100 cells per well in triplicate samples. c, Quantified percentage of parental MCF7 and C19 cells with centriolar satellite OFD1. Data shown represent mean ± s.d. for 100 cells per well in triplicate samples. ***P < 0.001, two-tailed unpaired student’s t-test. d–f, Primary cilia formed in OFD1 knockdown C19 MCF7 cells are positive for ciliary markers. Representative confocal images of primary cilia formed in C19 subjected to 72 h serum starvation. Cilia were positive for ciliary membrane marker ARL13B, axoneme marker acetylated tubulin and intraflagellar transport protein IFT88. Similar results were obtained in three independent experiments.

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Tang, Z., Lin, M., Stowe, T. et al. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 502, 254–257 (2013). https://doi.org/10.1038/nature12606

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