Autophagy Promotes Primary Ciliogenesis by Removing OFD1 from Centriolar Satellites

LETTER doi:10.1038/nature12606

Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites

Zaiming Tang1,2,3,4, Mary Grace Lin3, Timothy Richard Stowe5, She Chen6, Muyuan Zhu4, Tim Stearns5, Brunella Franco7,8 & Qing Zhong1,2,3

The primary cilium is a microtubule-based organelle that func- and BBS4 protein levels were not altered by serum starvation or in tions in sensory and signalling pathways. Defects in ciliogenesis Atg52/2 MEFs (Fig. 1g). The messenger RNA levels of OFD1 were not can lead to a group of genetic syndromes known as ciliopathies1–3. significantly changed upon serum starvation in Atg51/1 and Atg52/2 However, the regulatory mechanisms of primary ciliogenesis in MEFs (Extended Data Fig. 1c), indicating that OFD1 protein level normal and cancer cells are incompletely understood. Here we reduction upon serum starvation is through protein degradation demonstrate that autophagic degradation of a ciliopathy protein, rather than transcriptional downregulation. Blocking autophagic flux OFD1 (oral-facial-digital syndrome 1), at centriolar satellites pro- by lysosomal inhibitors bafilomycin A1 (Baf) or chloroquine resulted motes primary cilium biogenesis. Autophagy is a catabolic path- in increased OFD1 accumulation upon serum starvation (Fig. 1h). way in which cytosol, damaged organelles and protein aggregates Taken together, these data indicate that OFD1 is degraded via the are engulfed in autophagosomes and delivered to lysosomes for autophagy–lysosome pathway upon serum starvation. destruction4. We show that the population of OFD1 at the centrio- OFD1 is the gene underlying the human disease oral-facial-digital lar satellites is rapidly degraded by autophagy upon serum star- syndrome type 1 (OFD1), an X-linked ciliopathy characterized by vation. In autophagy-deficient Atg5 or Atg3 null mouse embryonic morphological abnormalities and renal cysts, as well as Joubert syn- fibroblasts, OFD1 accumulates at centriolar satellites, leading to drome and Simpson–Golabi–Behmel syndrome type 213–18. OFD1 fewer and shorter primary cilia and a defective recruitment of localizes to the distal ends of centrioles and is necessary for distal BBS4 (Bardet–Biedl syndrome 4) to cilia. These defects are fully appendage formation, IFT88 recruitment and primary cilium forma- rescued by OFD1 partial knockdown that reduces the population tion18,19. OFD1 also localizes to centriolar satellites, interacting with of OFD1 at centriolar satellites. More strikingly, OFD1 depletion proteins associated with human ciliary disease, PCM1, CEP290 and at centriolar satellites promotes cilia formation in both cycling BBS420. However, the function of this OFD1 population remains cells and transformed breast cancer MCF7 cells that normally do unclear. not form cilia. This work reveals that removal of OFD1 by autop- The centriolar satellite localization of OFD1 is determined by hagy at centriolar satellites represents a general mechanism to PCM1, because OFD1 was lost from satellites when PCM1 was promote ciliogenesis in mammalian cells. These findings define a depleted (Extended Data Fig. 2a, b). LC3 partially colocalized with newly recognized role of autophagy in organelle biogenesis. PCM1 upon serum starvation in a majority of U2OS cells expression During autophagy, the membrane anchored LC3 (microtubule- Myc-LC3, but rarely in unstressed cells (Extended Data Fig. 2c, d). LC3 associated protein 1 light chain 3, also known as MAP1LC3B) interacts also partially colocalized with endogenous OFD1 when lysosome with cargo and cargo-adaptor proteins, recruiting cargoes to the activity is blocked by Baf or chloroquine treatment (Extended Data autophagosome for subsequent degradation upon fusion of the autop- Fig. 3a, b). This colocalization was limited to centriolar satellites, as hagosome with the lysosome5–11. We carried out a tandem-affinity LC3 did not colocalize with the centriole marker c-tubulin (Extended purification using tagged LC3 as bait to search for its interacting Data Fig. 3c, d). proteins (Fig. 1a). In addition to known LC3-interacting proteins OFD1 was present at both centrioles and centriolar satellites in (MAP1B, FYCO1, p62 (also known as SQSTM1) and KEAP112), we untreated retinal pigment epithelial (RPE) cells. Remarkably, the cen- identified a set of centriolar satellite proteins, including PCM1, OFD1 triolar satellite pool of OFD1 was much reduced upon serum star- and CEP131 (also known as AZI1), that had not previously been vation, whereas the population of OFD1 at centrioles remained shown to associate with LC3. PCM1 was also pulled down by LC3 unchanged (Fig. 2a). This serum starvation-induced OFD1 degrada- orthologues, GATE16 (also known as GABARAPL2) and GABARAP tion from centriolar satellites was blocked in Atg51/1 MEFs treated (Extended Data Fig. 1a). PCM1, OFD1 and LC3 co-immunoprecipitated with the lysosome inhibitor chloroquine (Extended Data Fig. 4a, b). with each other, indicating that they are in the same complex (Fig. 1b–d). Notably, PCM1 protein levels are not controlled by autophagy and the PCM1 probably enhances the interaction between LC3 and OFD1, as centriolar satellite distribution of PCM1 is not altered upon serum the OFD1–LC3 interaction is compromised in PCM1-depleted cells starvation (Fig. 1g and Extended Data Fig. 4c–e), indicating that the (Fig. 1e, f). autophagic degradation is specific to OFD1 at centriolar satellites Depletion of PCM1 by RNA interference had no significant effect rather than centriolar satellites as a whole. This notion is further sup- on autophagy activity as determined by LC3 lipidation and p62 degra- ported by our observation that OFD1 remained at centriolar satellites dation (Extended Data Fig. 1b). We then examined if any of these upon serum starvation in Atg52/2 MEFs but was lost from centriolar centriolar satellite proteins is an autophagic substrate. OFD1 protein satellites in Atg51/1 MEFs (Fig. 2b). levels were reduced by serum starvation and this reduction was com- The loss of OFD1 from centriolar satellites was significantly faster promised in autophagy-deficient Atg52/2 mouse embryonic fibro- than the loss of OFD1 from centrioles, and the rate of loss of OFD1 in blasts (MEFs) compared to Atg51/1 MEFs, whereas PCM1, IFT88 Atg52/2 MEFs was slower than that in Atg51/1 MEFs (Extended Data

1Center for Autophagy Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA. 3Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, USA. 4State Key Laboratory of Plant Physiology and Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou 310058, China. 5Department of Biology, Stanford University, Stanford, California 94305, USA. 6National Institute of Biological Sciences, Beijing 102206, China. 7Medical Genetics Services, Department of Translational Medical Sciences, Federico II University, Via Pietro Castellino 111, Naples, Italy. 8Telethon Institute of Genetics and Medicine (TIGEM), Via Pietro Castellino 111, Naples, Italy.

abcdFig. 5a). The centriolar satellite pool of OFD1 in Atg51/1 cells was lost EBSS: ––+ within 6 h of serum starvation, whereas this pool of OFD1 in Atg52/2 KDa MAP1B PCM1* cells remained stable even after 24 h of serum starvation (Extended 240 FYCO1 CEP131* Input lgG IP LC3 IP Input lgG IP PCM1 IP Input lgG IP HA IP Data Fig. 5b). These data confirm that OFD1 at centriolar satellites 140 OFD1* OFD1 PCM1 HA–PCM1 has a faster turnover rate than OFD1 at centrioles, and this serum- 100 LC3 LC3 Flag–OFD1 70 KEAP1 starvation-induced accelerated degradation is controlled by autophagy. p62 PCM1 50 –+shRNA We next sought to understand how OFD1 regulation might affect ef ciliogenesis. We observed that the percentage of Atg52/2 cells that 35 PCM1 1/1 –+shRNA form a primary cilium, as compared to Atg5 cells, was significantly Input lgG IP LC3 IP Input lgG IP LC3 IP 25 PCM1 Flag–OFD1 reduced and the cilia that did form were shorter (Fig. 3a). This differ- 20 LC3 Flag–OFD1 ence was not due to cell cycle regulation (Extended Data Fig. 6a). 15 Tubulin LC3 Lysosome inhibition also compromised primary ciliogenesis in 1/1 IP efficiency: 1 0.53 Atg5 MEFs (Extended Data Fig. 6b–d). The defective ciliogenesis phenotypes were not limited to Atg52/2 MEFs, as similar phenotypes gh Serum- were also observed in MEFs lacking another essential autophagy gene, Untreated starved Atg3 (Extended Data Fig. 6e–g). Atg5 +/+ –/– +/+ –/– SS –– ––+ +++ We next investigated if the ciliary recruitment of BBS4, a critical OFD1 Baf –+ ––– –+ – CQ ––+–– ––+ 21 O/T: 1 1.3 0.5 1.5 event for ciliogenesis , is affected in autophagy-deficient cells. We MG132 –– –++ –– – 2/2 PCM1 observed that BBS4 accumulated at centriolar satellites in Atg5 OFD1 1/1 IFT88 MEFs; more than 50% of cilia had detectable BBS4 in Atg5 O/T: 1 1 1 2 0.7 1.8 4.6 0.8 MEFs, whereas only about 10% of cilia were positive for BBS4 in BBS4 p62 Atg52/2 MEFs (Extended Data Fig. 7). These data indicate that p62 LC3-I BBS4 recruitment to cilia is also defective in autophagy-deficient cells. LC3-II LC3-I If OFD1 accumulation at centriolar satellites is responsible for the LC3-II 2 2 Tubulin ciliary defects in Atg5 / MEFs, we would expect that depletion of Tubulin OFD1 by RNA interference might rescue these defects. In Atg52/2 Figure 1 | OFD1 is an autophagic substrate. a, Silver staining of LC3 MEFs stably expressing OFD1 short hairpin RNA (shRNA), with complexes purified from U2OS cells expressing ZZ–Flag–LC3 in normal medium or Earle’s balanced salt solution (EBSS) for 2 h. Asterisks mark about 50% efficiency of overall depletion of OFD1 (Extended Data centriolar satellite proteins. b–d, Co-immunoprecipitation (IP) of OFD1 Fig. 8a), OFD1 remained at centrioles but was lost from centriolar with LC3, LC3 with PCM1, or OFD1 with PCM1 in HEK293T cells. HA, satellites (Extended Data Fig. 8b-d). Primary cilium formation upon haemagglutinin. e, Western blotting analysis of PCM1 and OFD1 protein serum starvation was fully restored in these cells; nearly 70% of cells levels in control or PCM1 knockdown (KD) HEK293T cells. f, Co- formed a cilium and the length of these cilia was comparable to those immunoprecipitation of OFD1 with LC3 in control or PCM1 KD HEK293T formed in Atg51/1 MEFs (Fig. 3b). Hence, the centriolar satellite pool cells. IP efficiency is calculated as the ratio of immunoprecipitated OFD1/LC3. of OFD1 indeed plays a key role in suppressing primary ciliogenesis in g, h, Western blotting analysis of protein levels of indicated proteins in MEFs Atg52/2 MEFs. Notably, with OFD1 knockdown, nearly 45% of the with indicated genotypes in normal medium or subjected to 24 h serum Atg52/2 cells formed cilia even without serum starvation (Fig. 3b), starvation (SS), 50 nM bafilomycin A1 (Baf), 20 mM chloroquine (CQ) or 1 mM indicating that OFD1 degradation is likely to be required for serum- MG132. Quantified OFD1 level was normalized with b-tubulin. Similar results were observed in three independent experiments. starvation-induced primary ciliogenesis. This notion is supported by

a Untreated Zoomed Serum-starved Zoomed *** 100 Ac-Tubulin OFD1 80 5 μm 5 μm 60

γ 20

-Tubulin satellite OFD1 (%) OFD1 Cells with centriolar 0 Un SS 5 μm 5 μm

b OFD1 Ac-Tubulin Merged+DNA Zoomed 100 ***

80 Atg5+/+ 10 μm 5 μm 60 40

–/– 20

Atg5 satellite OFD1 (%) Cells with centriolar 10 μm μ 0 5 m Atg5+/+ Atg5–/– Figure 2 | Autophagy specifically degrades OFD1 at centriolar satellites OFD1 and acetylated tubulin in Atg51/1 and Atg52/2 MEFs subjected to 24 h upon serum starvation. a, Representative confocal images of endogenous serum starvation. a, b, Data shown represent mean 6 s.d. percentage of cells OFD1 puncta with cilium marker acetylated tubulin (Ac-tubulin), or centriole with centriolar satellite OFD1 for 100 cells per well in triplicate samples. marker c-tubulin in hTERT-RPE1 cells in normal medium (Un) or subjected to ***P , 0.001, two-tailed unpaired student’s t-test. Similar results were 24 h serum starvation (SS). b, Representative confocal images of endogenous observed in three independent experiments.

a Untreated Serum-starved Zoomed b Atg5+/+ Atg5–/– Atg5–/–, OFD1 KD DAPI Ac-Tubulin

Atg5+/+ Un

10 μm 10 μm 5 μm 10 μm 10 μm 10 μm

Atg5–/– SS

10 μm 10 μm 5 μm 10 μm 10 μm 10 μm

*** Untreated Starvation 60 80 Atg5+/+ *** *** *** 7 *** *** 6 70 50 Atg5–/– 6 5 60 40 5 4 50 30 4 3 40 20 3 2 30 10 20 2 Cells with cilia (%) 1 Cilia length ( μ m) Cilia length ( μ m)

Cells with cilia (%) 1 0 0 10 Untreated Serum- Atg5+/+ Atg5–/– 0 0 starved Atg5+/+ Atg5–/– Atg5–/–, Atg5+/+ Atg5–/– Atg5–/–, OFD1 KD OFD1 KD Figure 3 | Autophagy promotes primary cilia biogenesis by regulating length of the cilia for 500 cells or 100 cells per well, respectively, in triplicate OFD1 levels. a, b, Representative confocal images of cilium marker acetylated samples. ***P , 0.001, two-tailed unpaired student’s t-test. Similar results tubulin from MEFs with indicated genotype subjected to 24 h serum starvation. were observed in three independent experiments. Un, untreated; SS, Data shown represent mean 6 s.d. percentage of cells with primary cilia or serum-starved. the observation that primary cilia formed efficiently even in the cycling depletion efficiency (Fig. 4a). Similar to OFD1 knockdown MEFs, Atg51/1 MEFs depleted of OFD1 without serum starvation (Extended the centriolar satellite population of OFD1 in C19 cells was markedly Data Fig. 9a). The presence of cilia in MEFs was further confirmed by reduced, whereas the centriole pool remained stable (Extended Data the positive staining of the cilium marker ARL13B (Extended Data Fig. 9b), Fig. 10a–c). Remarkably, primary cilia formed in about 20% C19 cells which specifically decorates the ciliary membrane22. upon serum starvation, but were completely absent in parental MCF7 Primary cilia are formed in normal breast epithelial cells, but not cells (Fig. 4b). The length of these cilia ranged from 2 to 6 mm detected in breast cancer cell lines such as MCF723. Restoration of (Fig. 4c,d). As expected for normal primary cilia, these cilia extended primary cilia in human cancer cells might be beneficial to reduce from basal bodies and were positive for ARL13B, acetylated-tubulin malignancy, because cilia are required for proper functions of several and the intraflagellar transport protein IFT88 (Fig. 4e and Extended signalling pathways (hedgehog etc.) that have crucial roles in many Data Fig. 10d–f). Finally these cilia could be directly visualized under types of cancers24. We further tested if OFD1 reduction could restore the scanning electron microscope (Fig. 4f). Thus, by manipulating the primary ciliogenesis in MCF7 cells. We generated an OFD1 stable expression of one protein, OFD1, it is possible to reverse the cilia- knockdown clone (C19) in MCF7 cells, which showed 44% OFD1 defective phenotype of a transformed breast cancer cell line. Primary

abcd 25 6 MCF7 MCF7 OFD1 KD (C19) Parental *** C19 MCF7 parental 20 5 OFD1 C19 4 15 3 O/T: 1 0.56 10 2 μ μ μ μ Tubulin 5 1 2 m2 m2 m2 m Cilia length ( μ m)

Cilia percentage (%) Cilia percentage 0 0 γ MCF7 Un SS 48 72 (h) Ac-tubulin -tubulin

ef MCF7 SS OFD1 KD Atg5+/+ Un OFD1 KD C19 γ-tubulin ARL13B

5 μm 2 μm μ 5 kV ×7,500 5 kV ×7,500 2 m

Figure 4 | Forced OFD1 reduction promotes ciliogenesis in human breast 72 h serum starvation. d, e, Representative confocal images of primary cilia with cancer MCF7 cells. a, Western blotting analysis of OFD1 protein levels in variable length formed in MCF7 or C19 cells subject to 72 h serum starvation. control or OFD1 knockdown MCF7 (C-19) cells, quantified OFD1 level was Data shown represent 100 cells per well in triplicate samples. f, Scanning normalized with b-tubulin. b, Quantification of percentage of cells with electron microscope analysis of primary cilia (marked by arrows) formed in primary cilia in MCF7 or C19 cells subjected to 72 h serum starvation. Data C-19 cells subject to 72 h serum starvation and cycling Atg51/1 MEFs in shown represent mean 6 s.d. percentage of cells with primary cilia for 500 cells normal medium for 24 h. Similar results were observed in three independent per well in triplicate samples. ***P , 0.001, two-tailed unpaired student’s experiments. t-test. c, Quantification of length of primary cilia in C19 cells subject to 48 h or

256 | NATURE | VOL 502 | 10 OCTOBER 2013 ©2013 Macmillan Publishers Limited. All rights reserved LETTER RESEARCH ciliogenesis remains inefficient in C19 cells, indicating that addition 11. Kirkin, V. et al. A role for NBR1 in autophagosomal degradation of ubiquitinated substrates. Mol. Cell 33, 505–516 (2009). factors are required to reach full capacity. Nevertheless, these data 12. Fan, W. et al. Keap1 facilitates p62-mediated ubiquitin aggregate clearance via confirm that OFD1 at centriolar satellites functions as a crucial sup- autophagy. Autophagy 6, 614–621 (2010). pressor of primary ciliogenesis in human cancer cells. 13. Feather, S. A., Woolf, A. S., Donnai, D., Malcolm, S. & Winter, R. M. The oral-facial- digital syndrome type 1 (OFD1), a cause of polycystic kidney disease and We show here that autophagy is linked to primary ciliogenesis, by associated malformations, maps to Xp22.2-Xp22.3. Hum. Mol. Genet. 6, controlling the degradation of OFD1 at centriolar satellites. We pro- 1163–1167 (1997). pose that autophagy deficiency serves as a potential underlying mech- 14. Coene, K. L. et al. OFD1 is mutated in X-linked Joubert syndrome and interacts with anism of ciliopathies, and that autophagy modulation might provide a LCA5-encoded lebercilin. Am. J. Hum. Genet. 85, 465–481 (2009). 15. Field, M. et al. Expanding the molecular basis and phenotypic spectrum of X-linked novel means of ciliopathy treatment. We demonstrate that OFD1 at Joubert syndrome associated with OFD1 mutations. Eur. J. Hum. Genet. 20, centriolar satellites has a crucial role in suppressing primary ciliogen- 806–809 (2012). esis, whereas OFD1 at centrioles has been shown to be essential for 16. Zullo, A. et al. Kidney-specific inactivation of Ofd1 leads to renal cystic disease associated with upregulation of the mTOR pathway. Hum. Mol. Genet. 19, primary ciliogenesis. Removing OFD1 from centriolar satellites pro- 2792–2803 (2010). motes ciliogenesis in autophagy-deficient cells, wild-type MEFs with- 17. Ferrante, M. I. et al. Identification of the gene for oral-facial-digital type I syndrome. out serum starvation, and human breast cancer MCF7 cells that Am. J. Hum. Genet. 68, 569–576 (2001). normally completely lack cilia, suggesting a general role of OFD1 in 18. Ferrante, M. I. et al. Oral-facial-digital type I protein is required for primary cilia formation and left-right axis specification. Nature Genet. 38, 112–117 (2006). suppressing ciliogenesis. Primary ciliogenesis is defective in human 19. Singla, V., Romaguera-Ros, M., Garcia-Verdugo, J. M. & Reiter, J. F. Ofd1, a human breast and pancreatic cancer cells, but activated in the corresponding disease gene, regulates the length and distal structure of centrioles. Dev. Cell 18, normal tissues/cells23,25. The contribution of primary cilium function 410–424 (2010). 26,27 20. Lopes, C. A. et al. Centriolar satellites are assembly points for proteins implicated in to tumorigenesis is complex ; however, our results suggest that dis- human ciliopathies, including oral-facial-digital syndrome 1. J. Cell Sci. 124, secting the regulatory mechanisms of OFD1 will provide insight into 600–612 (2011). these functions and potentially offer new therapeutic tools for treat- 21. Nachury, M. V. et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell 129, 1201–1213 (2007). ment of ciliopathies and cancers. 22. Cantagrel, V. et al. Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome. Am. J. Hum. Genet. 83, 170–179 (2008). METHODS SUMMARY 23. Yuan, K. et al. Primary cilia are decreased in breast cancer: analysis of a collection Primary cilia biogenesis assay. Cells were incubated in either fresh normal med- of human breast cancer cell lines and tissues. J. Histochem. Cytochem. 58, ium (10% FBS, 1% P/S DMEM) or serum-starved medium (0.5% FBS, 1% P/S 857–870 (2010). 24. Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during DMEM) for 24 h. Cells were seeded at different numbers to ensure that they vertebrate development. Nature Rev. Genet. 11, 331–344 (2010). reached the same confluency 1 day after treatment. 25. Seeley, E. S., Carriere, C., Goetze, T., Longnecker, D. S. & Korc, M. Pancreatic cancer Tandem affinity purification of LC3 complexes. This protocol was adapted and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary from previously described methods12,28,29. Cell lysates from inducible cell lines cilia. Cancer Res. 69, 422–430 (2009). expressing ZZ–Flag–LC3 treated with 10 ng ml21 doxycycline for one day were 26. Wong, S. Y. et al. Primary cilia can both mediate and suppress Hedgehog pathway- purified through immunoglobulin G (IgG) beads followed by tobacco etch virus dependent tumorigenesis. Nature Med. 15, 1055–1061 (2009). 27. Han, Y. G. et al. Dual and opposing roles of primary cilia in medulloblastoma (TEV) protease cleavage and anti-Flag M2 affinity gel. All LC3-interacting pro- development. Nature Med. 15, 1062–1065 (2009). teins were identified by mass spectrometry analysis. 28. Sun, Q. et al. Identification of Barkor as a mammalian autophagy-specific factor for Beclin 1 and class III phosphatidylinositol 3-kinase. Proc. Natl Acad. Sci. USA 105, Online Content Any additional Methods, Extended Data display items and Source 19211–19216 (2008). Data are available in the online version of the paper; references unique to these 29. Chen, D. et al. A mammalian autophagosome maturation mechanism mediated by sections appear only in the online paper. TECPR1 and the Atg12-Atg5 conjugate. Mol. Cell 45, 629–641 (2012).

Received 19 October 2012; accepted 29 August 2013. Acknowledgements We thank D. Moughon and W. Fan for initial purification of LC3 Published online 2 October 2013. complex; N. Mizushima for Atg51/1 and Atg52/2 MEFs and M. Komatsu for Atg31/1 and Atg32/2 MEFs; H. Zhou for helpful discussions and quantitative RT–PCR data 1. Sang, L. et al. Mapping the NPHP-JBTS-MKS protein network reveals ciliopathy analysis; A. Kodani and J. F. Reiter for reagents, helpful discussions and technical disease genes and pathways. Cell 145, 513–528 (2011). assistance; and B. Levine for helpful discussions and critical reading of the manuscript. 2. Hildebrandt, F., Benzing, T. & Katsanis, N. Ciliopathies. N. Engl. J. Med. 364, The electron microscopy studies were performed by C. Miller and V. Gattone II at the 1533–1543 (2011). Indiana University School of Medicine Electron Microscopy Center that is supported by 3. Nigg, E. A. & Raff, J. W. Centrioles, centrosomes, and cilia in health and disease. Cell the Polycystic Kidney Disease Foundation. This work was supported by grants from the 139, 663–678 (2009). American Cancer Society (RSG-11-274-01-CCG) and National Cancer Institute 4. Levine, B. & Klionsky, D. J. Development by self-digestion: molecular mechanisms (CA133228) to Q.Z. and grants from the Italian Telethon Foundation (TGM11CB3) and and biological functions of autophagy. Dev. Cell 6, 463–477 (2004). the EU FP7/2007-2013 Programme (Syscilia grant agreement no. 241955) to B.F. The 5. Thurston, T. L., Ryzhakov, G., Bloor, S., von Muhlinen, N. & Randow, F. The TBK1 work was partially supported by China Scholarship Council to Z.T. adaptor and autophagy receptor NDP52 restricts the proliferation of ubiquitin- coated bacteria. Nature Immunol. 10, 1215–1221 (2009). 6. Johansen, T. & Lamark, T. Selective autophagy mediated by autophagic adapter Author Contributions Z.T., M.G.L. and T.R.S. performed the experiments; S.C. carried proteins. Autophagy 7, 279–296 (2011). out mass spectrometry analysis; M.Z., T.S. and B.F. provided technical and intellectual 7. Kirkin, V., McEwan, D. G., Novak, I. & Dikic, I. A role for ubiquitin in selective support; Z.T. and Q.Z. conceived the project, designed the experiments, analysed the autophagy. Mol. Cell 34, 259–269 (2009). data and wrote the manuscript with the help of all authors. 8. Novak, I. et al. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep. 11, 45–51 (2010). Author Information Reprints and permissions information is available at 9. Orvedahl, A. et al. Image-based genome-wide siRNA screen identifies selective www.nature.com/reprints. The authors declare no competing financial interests. autophagy factors. Nature 480, 113–117 (2011). Readers are welcome to comment on the online version of the paper. Correspondence 10. Zhang, Y. et al. SEPA-1 mediates the specific recognition and degradation of P and requests for materials should be addressed to Q.Z. granule components by autophagy in C. elegans. Cell 136, 308–321 (2009). ([email protected]).

METHODS 1:10,000 dilution of the antibody against LC3 (Sigma) and 1:1,000 dilution of Reagents. Rabbit anti-OFD1 antibody, EGFP–OFD1 and Flag–OFD1 were the antibody against p62 (MBL). described before18,30,31. Another rabbit anti-OFD1 antibody was a gift from J. F. Establishment of stable RNA interference cell lines. Human MCF7 cells or 1/1 2/2 Reiter. Rabbit anti-IFT88 antibody was a gift from B. Yoder. To generate BBS4– Atg5 and Atg5 MEFs were seeded into 12-well dishes at 50% confluency MycHAHis (pTS1686), BBS4 was PCR-amplified, verified by sequencing and before transfection. 24 h after seeding, cells were incubated with normal complete 21 cloned into a pEGFP-N1-derived plasmid (Clontech Laboratories) in which medium containing 5 mgml polybrene (Santa Cruz), and infected overnight GFP was replaced with a Myc–HA–His tag created as BBS4–MycHAHis32. with lentiviral particles carrying short hairpin RNA OFD1 (Santa Cruz). 24 h later, 21 Primary antibodies used for western blotting and immunostaining are rabbit cells were selected with puromycin (Invitrogen) (1 mgml for MCF7 or 10 mg 21 anti-PCM1 (Cell signaling), mouse anti-PCM1 (Sigma), mouse anti-b-tubulin ml for MEFs) until positive clones were identified. Single clones were picked and (Sigma), rabbit anti-p62 (MBL), rabbit anti-LC3 (Sigma), mouse anti-acetylate identified by western blotting or immunofluorescence analysis. tubulin (Sigma), mouse or rabbit anti-c tubulin (Sigma), Rabbit anti-BBS4 (Santa Cell cycle analysis. 106 cells were centrifuged at 485g for 5 min, washed once with Cruz), chloroquine (Sigma), bafilomycin A1 and rapamycin (LC Laboratories), PBS, and pellets were resuspended in 1 ml PBS and fixed overnight in 75% ethanol and MG132 (Alexis). at 220 uC. Fixed cells were washed three times with 10 ml PBS, resuspended in Cell culture and transfection. HEK293T, MCF7 and MEFs were cultured in 200–400 ml PBS with 10 ml RNase (Qiagen) at 37 uC for 30 min in the dark. Cells DMEM with 10% FBS and 1% penicillin and streptomycin in a 5% CO2 incubator were subjected to FACS analysis using DAKO-Cytomation MoFlo High Speed (Thermo). RPE1 cells were cultured in DMEM/Hams F12 with 10% FBS and 1% Sorter at the Flow Cytometry Facility at UC Berkeley. penicillin and streptomycin in a 5% CO2 incubator (Thermo). Lipofectamine 2000 Immunoprecipitation and immunoblot. Transfected cells were lysed in TAP lysis (Invitrogen) was used for mammalian cell transfection. Cells were incubated in buffer on ice for 30 min, and centrifuged at 13,000g for 15 min. For immunopre- OPTI medium (GIBCO 51985091) for 1 h before transfection. 10 mgor5mg plas- cipitation, cell lysates were incubated with primary antibody overnight at 4 uC, and mid was used for each 150-mm or 100-mm tissue culture dish, respectively. U2OS then incubated with IgG beads for 2 h at 4 uC. The beads were washed 5 times with 1/1 2/2 cells stably expressing Myc–LC3 were described before28. Atg5 and Atg5 1 ml of lysis buffer and eluted with elution buffer containing 200 mgml21 33Flag 1/1 2/2 MEFs were gifts from N. Mizushima33. Atg3 and Atg3 MEFs were gifts peptide (Sigma) or resolved with SDS sample buffer. Co-immunoprecipitated from M. Komatsu34. PCM1 shRNA knockdown was carried out using a 19-mer proteins were resolved by SDS–PAGE, transferred to PVDF membranes (Bio- shRNA targeting human PCM1 with the sequence: 59-GTATCACATCTG Rad), and detected with antigen-specific primary antibodies followed by horse- AACTAAA-39 (pTS2063). PCM1 shRNA oligonucleotides were designed using radish peroxidase-conjugated secondary antibodies. pSicoOligomaker 1.5, annealed and subcloned into the lentiviral vector, pSicoR- Tandem affinity purification of LC3 complexes. This protocol is adapted from puro, which confers puromycin resistance. Stable depletion of PCM1 was carried what was described before12,28,29. Stable cell lines expressing ZZ–Flag–LC3, ZZ– out by infection of cells with shRNA-expressing lentiviral particles and selection 21 Flag–GATE16, or ZZ–Flag–GABARAP, upon doxycycline induction were treated with puromycin (3 mgml ); positive clones were screened by western blotting. Stable with 10 ng ml21 doxycycline for one day. The collected cell pellet was washed with depletion of OFD1 was carried out by infection of cells with shRNA-expressing chilled PBS three times and suspended in TAP lysis buffer. Resuspended cell lentiviral particles (sc-91245-V, from Santa Cruz) and selection with puromycin pellets were incubated on ice for 30 min and then gently vortexed for one minute. m 21 m 21 (1 gml or 10 gml ) for MCF7 cells or MEFs, positive clones were screened Homogenates were centrifuged for 20 min at 10,000g. The supernatants were by western blotting. The OFD1 lentiviral particles are a pool of 3 different shRNA transferred to fresh tubes containing 0.8 ml of packed IgG beads followed by gentle plasmids, targeting the OFD1 sequence: 59-GGAUGACUACAUCAUUAGA-39, rotation overnight at 4 uC. Bound protein was eluted using TEV protease cleavage 59-CUACUCAGGUUGCCGAUUU-39,59-GAACGAAGAGAACUAGAAA-39. and further purified by anti-Flag M2 Affinity Gel (Sigma). Bound protein were Primary cilia biogenesis assay. Equal numbers (3 3 105 for serum rich medium eluted with elution buffer containing 200 mgml21 33Flag peptide (Sigma) and and 4.5 3 105 for serum-starved medium) of Atg51/1 MEFS and Atg52/2 MEFs resolved by SDS–PAGE on a 4–12% gradient gel (Invitrogen), then visualized by were seeded into 6-well dishes. After cells were attached on the coverslips for 8 h, silver staining (Invitrogen). Distinct bands were cut and subjected to mass spec- they were incubated in either fresh normal medium (10% FBS, 1% P/S DMEM) or trometry analysis. serum-starved medium (0.5% FBS, 1% P/S DMEM) for 24 h. Cells were seeded at Quantitative RT–PCR. different numbers at beginning to ensure that these cells reach the same confluence Total RNAs were extracted with TRIzol reagent m m 1 day after treatment. Cells were then fixed for immunofluorescence staining. For (Invitrogen), from which 1 g was used for reverse transcription in a 20- l reaction western blotting, cells cultured in 10-cm dishes were serum-starved alone or in system with the RNA PCR (AMV) kit (Promega). Quantitative RT–PCR was combination with 50 nM bafilomycin A1, 20 mM chloroquine or 1 mM MG132 performed with SYBR-Green PCR Mastermix (Applied Biosystems), and amp- treatment for 24 h and lysed with TAP buffer. lification was real-time monitored on a MicorAmp optical 96-Well Reaction Plate Immunofluorescence staining. Cells were treated according to the protocol (Applied Biosystems). The level of transcript abundance relative to reference gene described above with or without serum starvation or compound treatment. (termed DCt) was determined according to the function DCt 5 Ct (test gene) 2 Ct Cells were fixed with cold methanol for five minutes at 220 uC. Cells were then (reference gene). To compare untreated and treated expression levels, the function washed three times with PBS and then blocked with blocking buffer (2.5% BSA 1 DDCt was first determined using the equation DDCt 5 DCt (treatment) 2 DCt 0.1% Triton X-100 in PBS) at room temperature for 1 h. Cells were incubated with (control) where control represented mock-treated cells. The induction ratio of {DDC primary antibodies at 4 uC overnight, washed five times with PBS buffer and then treatment/control was then calculated by the equation 2 t . Gene expression 1/1 incubated with appropriate secondary antibodies conjugated to Alexa 488-Alexa levels were normalized with the Atg5 untreated sample. 549 (Molecular Probes) for 2 h at room temperature. DNA was stained with DAPI. 30. Giorgio, G. et al. Functional characterization of the OFD1 protein reveals a nuclear Slides were examined using a laser scanning confocal microscope (Zeiss LSM 510 localization and physical interaction with subunits of a chromatin remodeling META UV/Vis). complex. Mol. Biol. Cell 18, 4397–4404 (2007). Autophagy analysis. Autophagy was induced by serum starvation or rapamycin 31. Bimonte, S. et al. Ofd1 is required in limb bud patterning and endochondral bone treatment, or blocked by lysosome inhibitors, chloroquine or bafilomycin A1. For development. Dev. Biol. 349, 179–191 (2011). rapamycin (LC Laboratories) treatment, cells were incubated with 500 nM rapa- 32. Stowe, T. R., Wilkinson, C. J., Iqbal, A. & Stearns, T. The centriolar satellite proteins mycin in complete medium for 16 h at 37 uC. To block autophagy flux, 50 nM Cep72 and Cep290 interact and are required for recruitment of BBS proteins to bafilomycin A1 (LC Laboratories) or 20 mM chloroquine (Sigma) was added to the cilium. Mol. Biol. Cell 23, 3322–3335 (2012). 33. Kuma, A. et al. The role of autophagy during the early neonatal starvation period. normal complete medium or serum-starved medium and incubated for 24 h at Nature 432, 1032–1036 (2004). 37 uC. Autophagy activity was assessed using two approaches, LC3-II formation 34. Sou, Y. S. et al. The Atg8 conjugation system is indispensable for proper and p62 degradation. For LC3-II and p62 detection, cell lysates were prepared as development of autophagic isolation membranes in mice. Mol. Biol. Cell 19, described above and subjected to standard western blotting protocols using 4762–4775 (2008).

a b

EBSS: − − + − + − + Rapamycin (μM): 0 0.5 1 2 0 0.5 1 2 KDa Time (h) 0 20 20 4 0 20 20 4 PCM1 PCM1 shRNA − − − − + + + + 240 140 PCM1 100 70 p62 p62 50

Tubulin 35

25 LC3-I 20 LC3 orthologs LC3-II 15 LC3-II/Tubulin: 1 1.4 1.2 1.4 1.5 1.7 2.2 1.8

1.5 Atg5+/+

Atg5−/−

0.5 Relative mRNA levels (fold) Relative mRNA 0 Untreated Serum starved Extended Data Figure 1 | LC3-interacting protein PCM1 is not required for normal medium or subjected to rapamycin treatment; quantified LC3-II level autophagy. a, PCM1 associates with LC3, GATE16 and GABARAP. Silver was normalized with b-tubulin. c, OFD1 messenger RNA levels remain staining of LC3, GATE16 or GABARAP complexes purified from U2OS cells unchanged upon serum starvation. Quantitative analysis of messenger RNA that stably express ZZ–Flag–LC3, ZZ–Flag–GATE16, or ZZ–Flag–GABARAP levels of OFD1 in Atg51/1 and Atg52/2 MEFs in normal medium or subjected in normal medium or subjected to 2 h Earle’s balanced salt solution (EBSS) to 24 h serum starvation. OFD1 mRNA levels were detected by quantitative starvation. Both PCM1 and p62 were identified by mass spectrometry analysis. RT–PCR and plotted after normalization. Similar results were obtained in three b, PCM1 is not required for autophagy. Western blotting analysis of p62, independent experiments. LC3-I/II, PCM1 levels in control or PCM1 shRNA knockdown U2OS cells in

a b

PCM1 OFD1 Merged+DAPI Zoomed

100 *** 90 80 70 60 50 40 30 20

PCM1 KD Control 10 0 Control KD PCM1 KD Cells with OFD1 and PCM1 colocalization at centriolar satellites (%)

c d Myc-LC3 PCM1 Merged+DAPI

60 ***

10 Cells with LC3 containing PCM1 domains (%)

Serum starved Untreated 0 Untreated Serum starved Extended Data Figure 2 | PCM1 is required for OFD1 centriolar satellite serum starvation. Representative confocal images of Myc–LC3 and PCM1 localization. a, Representative confocal images of OFD1 and PCM1 colocalization in U2OS cells expressing Myc–LC3 in normal medium or localization from control or PCM1 knockdown U2OS cells in normal medium. subjected to 24 h serum starvation. Arrows denote colocalized LC3 (green) and Data shown represent 100 cells per well in triplicate samples. b, Quantified PCM1 (red) puncta. Data shown represent 100 cells per well in triplicate percentage of cells with PCM1 positive centriolar satellite OFD1 in a.Data samples. d, Quantified percentage of cells with colocalization of Myc–LC3 and shown represent 100 cells per well in triplicate samples. ***P , 0.001, two- PCM1 in c. ***P , 0.001, two-tailed unpaired student’s t-test. Similar results tailed unpaired student’s t-test. c, LC3 partially colocalizes with PCM1 upon were observed in three independent experiments.

a b Myc-LC3 OFD1 Merged+DAPI

35 30

25 20

Cells with LC3 and OFD1 colocalization (%) 0 CQ Baf Untreated Untreated Baf CQ

c d LC3 γ-tubulin Merged+DAPI

Cells with LC3 and γ -tubulin colocalization (%) Untreated Serum starved Serum starved Untreated

Extended Data Figure 3 | LC3 partially colocalizes with OFD1 but not with colocalization of Myc–LC3 and OFD1 in a. c, LC3 does not colocalize with c-tubulin. a, LC3 colocalizes with OFD1 when the lysosome activity is blocked. centrioles. Representative confocal images of LC3 and c-tubulin colocalization Representative confocal images of Myc–LC3 and OFD1 colocalization in in U2OS cells in normal medium or subjected to 24 h serum starvation. Data U2OS cells that stably express Myc–LC3 in normal medium or subjected to shown represent 100 cells per well in triplicate samples. d, Quantified 2 h 50 nM bafilomycin A1 (Baf) or 100 mM CQ. Data shown represent 100 cells percentage of cells with colocalization of LC3 and c-tubulin in c. Similar results per well in triplicate samples. b, Quantified percentage of cells with were obtained in three independent experiments.

a b

EGFP-OFD1 PCM1 Merged+DAPI

100 *** Serum- 90 starved 80 70 60 50 Serum- 40 30 starved 20 +CQ

Cells with centriolar satellite OFD1 (%) 10 0 SS SS+CQ

c d Ac-tubulin PCM1 Merged+DAPI

100

Cells with centriolar satellite PCM1 (%) 0 Untreated Serum-

Serum starved Untreated starved

e PCM1 OFD1 Merged+DAPI Serum starved Untreated

Extended Data Figure 4 | OFD1 but not PCM1 at centriolar satellites was subjected to 24 h serum starvation. Data shown represent mean 6 s.d. for 200 degraded by autophagy. a, OFD1 accumulates at centriolar satellites in CQ- cells per well in triplicate samples. d, Quantified percentage of cells with PCM1 treated cells. Representative confocal images of EGFP–OFD1 and PCM1 centriolar satellite staining in c. e, OFD1 but not PCM1 is degraded from colocalization in Atg51/1 cells expressing EGFP–OFD1 subjected to 24 h centriolar satellites upon serum starvation. Representative confocal images of serum starvation or 20 mM CQ. b, Quantified percentage of cells with centriolar PCM1 and OFD1 colocalization in Atg51/1 cells in normal medium or satellite OFD1 in a. Data shown represent mean 6 s.d. for 100 cells per well in subjected to 24 h serum starvation. Data shown represent 200 cells per well in triplicate samples. ***P , 0.001, two-tailed unpaired student’s t-test. c, PCM1 triplicated samples. Enlarged images were shown in the left bottom panels. is not degraded upon serum starvation. Representative confocal images of Similar results were obtained in three independent experiments. PCM1 centriolar satellite staining in Atg51/1 cells in normal medium or

a b

120 120

100 100

80 80

60 60

40 40

20 20

Cells with OFD1 at centrioles or at centriolar satellites (%) 0

Cells with OFD1 at centrioles or at centriolar satellites (%) 0 0 1 2 4 6 8 14 h 0 1 2 3 4 5 6 7 8 9 10 11 12 14 16 18 20 22 24 h Time course of cycloheximide treatment Time course of serum starvation

+/+ Atg5+/+ OFD1 at centrioles Atg5+/+ OFD1 at centriolar satellites Atg5 OFD1 at centrioles Atg5+/+ OFD1 at centriolar satellites Atg5−/− OFD1 at centrioles Atg5−/− OFD1 at centriolar satellites Atg5−/− OFD1 at centrioles Atg5−/− OFD1 at centriolar satellites

Extended Data Figure 5 | The turnover rate of centriolar satellite OFD1 is b, Centriolar satellite OFD1 but not centriole OFD1 degrades upon serum faster than OFD1 at centrioles. a, Centriolar satellite OFD1 has a shorter half- starvation. Quantified percentage of cells with OFD1 at centrioles or at life compared to OFD1 at centrioles. Quantified percentage of cells with OFD1 centriolar satellites from Atg51/1 and Atg52/2 MEFs in normal medium or at centrioles or at centriolar satellites from Atg51/1 and Atg52/2 MEFs in subjected to serum starvation with indicated time points. Data shown represent normal medium or subjected to 75 mM cycloheximide (CHX) with indicated 200 cells per well in triplicate samples. Similar results were obtained in three time points. Data shown represent 200 cells per well in triplicate samples. independent experiments.

a b

Atg5+/+ Atg5−/− SS SS+CQ

Atg5+/+ MEF

cd *** 70 *** 6 60 5 μ m) 50 4 40 3 30 2 20 Serum starved Untreated 10 1 Cilia length ( Cells with cilia (%) 0 0 SS SS+CQ SS SS+CQ

g e Serum- f Untreated starved Atg3 +/+ −/− +/+ −/− OFD1 *** 70 *** 5 O/T: 1 1.2 0.4 1.2 Atg3+/+ 60 BBS4 Atg3−/− 4 50 μ m) 3 p62 40 LC3-I 30 2 20 LC3-II 1 10 Cilia length ( Tubulin Cells with cilia (%) 0 0 Untreated Serum- Atg3+/+ Atg3−/− starved

Extended Data Figure 6 | Autophagy regulates primary ciliogenesis in a cell LC3-I/II and BBS4 protein levels in MEFs with indicated genotypes in normal cycle independent manner. a, FACS analysis of Atg51/1 and Atg52/2 MEFs in medium or subjected to 24 h serum starvation; quantified OFD1 levels were normal medium or subjected to 24 h serum starvation. Data shown represent normalized with b-tubulin. f, g, Primary ciliogenesis is also defective in Atg32/2 106 cells per well in triplicate samples. b, Primary ciliogenesis is less efficient MEFs. f, Quantified percentage of cells with primary cilia in Atg31/1 and when the lysosome activity is blocked in MEFs. Representative confocal images Atg32/2 MEFs in normal medium or subjected to 24 h serum starvation. of primary cilia formed in Atg51/1 MEFs subjected to 24 h serum starvation g, Quantified length of primary cilia formed in Atg31/1 and Atg32/2 MEFs as alone or combined with 20 mM CQ treatment. c, Quantified percentage of cells described in f. c, d, f, g, Data shown represent mean 6 s.d. for 100 cells per well with primary cilia in b. d, Quantified length of primary cilia in b. e, Degradation in triplicate samples. ***P , 0.001, two-tailed unpaired student’s t-test. Similar of OFD1 is also blocked in Atg32/2 MEFs. Western blot analysis of OFD1, p62, results were obtained in three independent experiments.

a b

Ac-tubulin Myc-BBS4 Merged+DAPI Zoomed

70 *** +/+ 60 Atg5 50

30 −/− 20 Atg5 10

BBS4 recruitment to cilia (%) 0 Atg5+/+ Atg5−/−

Extended Data Figure 7 | BBS4 recruitment to primary cilia is defective in cilia in Atg51/1 and Atg52/2 MEFs. Data shown represent mean 6 s.d. for 100 Atg52/2 MEFs. a, Representative confocal images of Atg51/1 and Atg52/2 cells per well in triplicate samples. ***P , 0.001, two-tailed unpaired student’s MEFs expressing Myc–BBS4 subjected to 24 h serum starvation. Scale bar 5 mm. t-test. Similar results were obtained in three independent experiments. b, Quantified percentage of cells with Myc–BBS4 translocation into primary

+/+ −/− +/+ −/− *** 100 *** Atg5 Atg5 Atg5 OFD1 KD Atg5 OFD1 KD 80

OFD1 60

O/T: 1 2 0.3 0.9 40

20 Tubulin Cells with centriolar satellite OFD1 (%) 0 Atg5+/+ Atg5−/− Atg5+/+ Atg5−/− OFD1 KD OFD1 KD

c Ac-tubulin OFD1 Merged+DAPI +/+ Atg5 OFD1 KD −/− Atg5 OFD1 KD

d γ-tubulin OFD1 Merged+DAPI +/+ Atg5 OFD1 KD −/− OFD1 KD Atg5

Extended Data Figure 8 | Partial shRNA knockdown of OFD1 leads to well in triplicate samples. ***P , 0.001, two-tailed unpaired student’s t-test. depletion of OFD1 from centriolar satellites in Atg51/1 and Atg52/2 MEFs. c, d, OFD1 was depleted from centriolar satellites but not centrioles in OFD1 a, Western blot analysis of OFD1 in MEFs with indicated genotypes in normal knockdown MEFs. Representative confocal images of OFD1 and axoneme medium. Quantified OFD1 levels were normalized with b-tubulin. KD, marker acetylated tubulin in c or centriole marker c-tubulin in d in MEFs with knockdown. b, Quantified percentage of cells with centriolar satellite OFD1 in indicated genotypes in normal medium. Data shown represent 100 cells per MEFs with indicated genotypes in normal medium. Data shown represent well in triplicate samples. Similar results were obtained in three independent mean 6 s.d. percentage of cells with centriolar satellite OFD1 for 100 cells per experiments.

a b Atg5+/+ Atg5+/+ OFD1 KD Ac-tubulin ARL13B Merged+DAPI

+/+ UN Atg5

−/−

SS Atg5 +/+ OFD1 KD Atg5 WT 70 8 OFD1 KD 60 7 50 μ m) 6 40 5 4 −/− 30 3 20 Atg5 2 OFD1 KD 10 1 Cilia length (

Cells with cilia (%) 0 0 +/+ +/+ Untreated Serum- Atg5 Atg5 starved OFD1 KD Extended Data Figure 9 | Knockdown of OFD1 in wild-type MEFs for 100 cells per well in triplicate samples. b, Representative confocal images of promotes primary ciliogenesis. a, Representative confocal images of primary primary cilia formed in MEFs with indicated genotypes subjected to 24 h serum cilia formed in MEFs with indicated genotypes in normal medium (Un) or starvation. The primary cilia formed are positive for both axoneme marker subjected to 24 h serum starvation (SS). Quantified percentage of cells with acetylated tubulin and ciliary membrane marker ARL13B. Data shown primary cilia and the length of primary cilia from MEFs with indicated represent 100 cells per well in triplicate samples. Similar results were obtained genotypes were shown in the bottom panels. Data shown represent mean 6 s.d. in three independent experiments.

γ Ac-tubulin OFD1 Merged+DAPI -tubulin OFD1 Merged+DAPI MCF7 OFD1 KD MCF7 OFD1 KD

γ-tubulin ARL13B Merged+DAPI *** 80 70 60 50 40 30

20 MCF7 OFD1 KD 10 Cells with centriolar satellite OFD1 (%) 0 MCF7 MCF7 OFD1 KD

e f Ac-tubulin ARL13B Merged+DAPI Ac-tubulin IFT88 Merged+DAPI MCF7 OFD1 KD MCF7 OFD1 KD Extended Data Figure 10 | Partial knockdown OFD1 in MCF7 cells depletes satellite OFD1. Data shown represent mean 6 s.d. for 100 cells per well in OFD1 from centriolar satellites and promotes primary ciliogenesis. triplicate samples. ***P , 0.001, two-tailed unpaired student’s t-test. a, b, OFD1 was depleted from centriolar satellites in OFD1 shRNA knockdown d–f, Primary cilia formed in OFD1 knockdown C19 MCF7 cells are positive for MCF7 cells. a, Representative confocal images of relative localization of OFD1 ciliary markers. Representative confocal images of primary cilia formed in C19 with axoneme marker acetylated tubulin in MCF7 OFD1 knockdown clone subjected to 72 h serum starvation. Cilia were positive for ciliary membrane (C19). Data shown represent 100 cells per well in triplicate samples. marker ARL13B, axoneme marker acetylated tubulin and intraflagellar b, Representative confocal images of OFD1 and centriole marker c-tubulin transport protein IFT88. Similar results were obtained in three independent from C19. Data shown represent 100 cells per well in triplicate samples. experiments. c, Quantified percentage of parental MCF7 and C19 cells with centriolar