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Self-eating to remove cilia roadblock ab

a

b

Zaiming Tang , Muyuan Zhu & Qing Zhong a

College of Life Sciences; Zhejiang University; Hangzhou, China

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Center for Autophagy Research; Department of Internal Medicine; University of Texas Southwestern Medical Center; Dallas, TX USA Published online: 12 Dec 2013.

Click for updates To cite this article: Zaiming Tang, Muyuan Zhu & Qing Zhong (2014) Self-eating to remove cilia roadblock, Autophagy, 10:2, 379-381, DOI: 10.4161/auto.27346 To link to this article: http://dx.doi.org/10.4161/auto.27346

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Autophagic Punctum

Autophagic Punctum

Autophagy 10:2, 379–381; February 2014; © 2014 Landes Bioscience

Self-eating to remove cilia roadblock Zaiming Tang,1,2 Muyuan Zhu,1,* and Qing Zhong2,* College of Life Sciences; Zhejiang University; Hangzhou, China; 2Center for Autophagy Research; Department of Internal Medicine; University of Texas Southwestern Medical Center; Dallas, TX USA

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Keywords: autophagy, primary ciliogenesis, centriolar satellites, PCM1, OFD1 Submitted: 11/13/2013 Revised: 11/21/2013 Accepted: 11/26/2013 http://dx.doi.org/10.4161/auto.27346 *Correspondence to: Muyuan Zhu; Email: [email protected]; Qing Zhong; Email: [email protected] Punctum to: Tang Z, Lin MG, Stowe TR, Chen S, Zhu M, Stearns T, Franco B, Zhong Q. Autophagy promotes primary ciliogenesis by removing OFD1 from centriolar satellites. Nature 2013; 502:254–7; PMID:24089205; http://dx.doi. org/10.1038/nature12606

utophagy delivers many proteins and cellular components to the lysosome for degradation via selective or nonselective mechanisms. By controlling the stability of defined protein factors, autophagy might regulate cellular processes in a precise and finely-tuned manner. In this study, we demonstrated that autophagy positively regulates the biogenesis of the primary cilium, an antenna-like organelle that senses the environment and transduces signals. Defects in the function or structure of cilia cause a number of human diseases called “ciliopathies.” We found that the autophagosome membrane anchored protein LC3 interacts with OFD1 (oralfacial-digital syndrome 1) and removes it from the centriolar satellite upon serum starvation to initiate primary cilium biogenesis. OFD1 regulation and primary cilium formation are defective in autophagy-deficient cells, and reducing OFD1 protein levels through RNA interference rescues primary cilium formation. More strikingly, knockdown of OFD1 induces primary cilium formation in unstressed cells as well as in a human breast cancer cell that was previously reported to have lost the ability to form primary cilia. These findings therefore suggest an unexpected link among autophagy, ciliogenesis, ciliopathy, and cancers. The cytosolic protein LC3 is conjugated to phosphatidylethanolamine on the phagophore membrane through an autophagy-specific ubiquitin-like reaction. Given the presumed abundance of phosphatidylethanolamine in the phagophore membrane and the high specificity

determined by the unique autophagy conjugation enzymes, it is not surprising that LC3 becomes the most abundant protein decorating the inner and outer membranes of autophagosomes. This characteristic makes LC3 an ideal candidate to serve as a membrane receptor to select and recruit cargoes for degradation. Indeed, nearly all selective autophagy is mediated by interaction with LC3 directly or indirectly. Thus, defining the LC3 interactome may allow the identification of novel cargoes and/or cargo receptors in the autophagy pathway. We used LC3, and two LC3 mammalian orthologs, GABARAPL2/GATE-16 and GARBARAP, as baits in a tandem affinity purification to search for interacting proteins in human osteosarcoma U2OS cells. We identified several known LC3 interacting partners by mass spectrometry, including SQSTM1/p62, FYCO1, and KEAP1, indicating that our purification was successful. We also found that LC3 interacts with a group of proteins mainly localized to centriolar satellites including PCM1, OFD1, AZI1/CEP131, and MIB1. The interaction between LC3 and these centriolar satellite proteins was further confirmed by co-immunoprecipitation experiments. Among these proteins, only OFD1 was degraded upon serum starvation. This degradation was mediated by autophagy, based on the observation that the serum starvation-induced OFD1 degradation was efficiently suppressed in autophagy-deficient cells. We then examined cilia formation in Atg5+/+ and atg5−/− mouse embryonic fibroblasts (MEFs). Primary ciliogenesis in MEFs is mainly influenced by the status of the cell cycle and can be induced by

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serum starvation (0.5% FBS) for 24 h and by contact inhibition caused by high cell density, both of which result in G0 / G1 arrest and cilia formation. Thus, since high cell density can induce cilia formation, a faster-growing cell line that reaches confluency earlier could grow cilia faster despite a moderate defect in ciliogenesis than a slower-growing cell line with normal ciliogenesis. We compared the percentage of cells with cilia and average cilium length in Atg5+/+ and atg5−/− MEFs. We seeded equal amounts of Atg5+/+ and atg5−/− MEFs on cell dishes for 8–12 h to reach 90% confluency and immediately treated these cells in low-serum (0.5% FBS) medium for 24 h. Under these conditions, primary cilia are efficiently induced in about 50% of Atg5+/+ MEFs with the average length of 5 μm. Although atg5−/− MEFs grow slightly faster and reach confluency earlier, we observed that both the percentage of cells with cilia (about 35%) and the average cilium length (about 3.5 μm) in atg5−/− MEFs are decreased compared with wild-type cells, suggesting that primary ciliogenesis is defective in autophagy-deficient cells. We confirmed this by demonstrating that ciliogenesis is

defective in MEFs lacking another essential autophagy gene, Atg3, as well as in wild-type MEFs in which autophagy is blocked by treatment with a lysosomal inhibitor. We then investigated whether OFD1 accumulation is a causal factor for the ciliogenesis defect in autophagy-deficient cells. OFD1 is partially depleted in autophagy-deficient cells by RNA interference, which seems to remove OFD1 mainly from the centriolar satellite due to a high turnover rate of this OFD1 population. Depletion of OFD1 from centriolar satellites rescues the ciliogenesis defects in atg5−/− MEFs upon serum starvation and, to our surprise, depletion of OFD1 also evokes primary cilia formation in MEFs in serum-rich conditions in both wild-type cells and atg5−/− MEFs. These findings raise the possibility that OFD1 at the centriolar satellite is the main roadblock for ciliogenesis that must be removed before cilia formation (Fig. 1). In addition to MEFs, the roadblock hypothesis also applies to a human breast cancer cell line, MCF7, which has been shown previously to have no cilia formation even after prolonged serum starvation. We

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found that depletion of OFD1 by shRNA in MCF7 cells led to formation of primary cilia in about 20% of the cells with an average length of 4 μm. To demonstrate that these structures are authentic cilia we stained them with cilium markers: acetylated tubulin for the axoneme (backbone of cilium); ARL13B for the ciliary membrane; and IFT88, a transporter in the ciliary compartment. We also used scanning electron microscopy to demonstrate that the cilia emanate from the cell surface. It is still unclear whether these cilia perform normal functions in signal transduction and if the reacquisition of cilia in the breast cancer cells affects cell growth, polarity, and tumorigenicity. Although ciliopathies are very rare diseases, emerging evidence indicates that primary cilia are lost in a variety of human cancer cells/tissues, whereas cilia still form in adjacent normal cell/tissues. It has been proposed that primary ciliogenesis might be a tumor suppressor mechanism and that some, if not all, human cancers have an acquired ciliopathy. The ciliated MCF7 cells will allow us to test the functionality of cilia-associated signaling pathways including the hedgehog,

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Figure  1. A working model of autophagy-regulated ciliogenesis. OFD1 at centriolar satellites functions as a suppressor of primary ciliogenesis by sequestering BBS4. Upon serum starvation, OFD1 is removed from the pericentrosomal compartment by autophagy via the interaction with LC3, which allows BBS4 translocation and cilia induction.

necessary and sufficient for pathogenic features of ciliopathies and may lead to new therapeutic options for ciliopathy patients. Disclosure of Potential Conflicts of Interest

Acknowledgments

We thank Rhea Sumpter for the critical reading of the manuscript. This work is supported by grants (RSG-11-274-01CCG, CA133228) to QZ and a fund by China Scholarship Council to ZT.

No potential conflicts of interest were disclosed.

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WNT, and Hippo pathways and explore the role of cilia in cell growth, polarity, and migration in cultured cells. We will also use this system to examine the impact of cilia on tumorigenesis in mouse models in the near future. In addition, the ability to regrow primary cilia in ciliopathy cells might help to clarify if primary cilia are

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Self-eating to remove cilia roadblock.

Autophagy delivers many proteins and cellular components to the lysosome for degradation via selective or nonselective mechanisms. By controlling the ...
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