Autophagy Promotes Primary Ciliogenesis By Removing OFD1 From Centriolar Satellites

The primary cilium is an "antenna-like" organelle that present on almost every vertebrate cell, which transduce extracellular signals that are essential for normal development and tissue homeostasis. Mutations disrupting primary cilia cause misregulated signaling pathways and many human genetic disease termed as "ciliopathy". Mislocalization or loss of essential centrosomal/ciliary proteins have been found underlie the structural or functional defects of primary cilia. However, the molecular mechanisms of primary ciliogenesis and signaling pathways that regulated by primary cilia not well understood. In our study, we proved that autophagy promotes primary ciliogenesis by degrading OFD1 from centriolar satellites. To dissect the detailed mechanism of primary ciliogenesis and expolore the cellular processes in which autophagy functions, we made several breakthroughs in this study described as below:(1) To identify new substrates of autophagy or potential adaptor proteins, we used the most abundant and well characterized mammalian autophagomal marker LC3 as a bait to carry out the tandem affinity purification. By which we identifyied many centriolar satellites proteins including PCM1, OFD1 and Cep131. PCM1 has shown more strong interaction with LC3 and its homologies;(2) By immunoprecipitation, we proved that PCM, OFD1 and LC3 form a same complex in vivo. PCM1 is required for the recruitment of OFD1 to centriolar satellites and it bridges the interaction between OFD1 and LC3;(3) OFD1 is a bona fide substrate of autophagy, which is specifically degraded by autophagy upon serum starvation;(4) Serum starvation induce the colocolization of PCM1 and LC3, and promote the degradation of OFD1 from centriolar satellites;(5) OFD1 at centriolar satellites has a faster turnover rate compare to OFD1 at distal end of centrioles;(6) Compare to Atg5-/- MEFs, Atg5+/+ MEFs form more and longer primary cilia which is accompanied by much more efficient recruitment of BBS4 into the primary cilia. Similar cell-cycle independent defects of primary ciliogenesis was also observed in Atg3-/- MEFs and in CQ-treated Atg5+/+ MEFs, strongly suggesting that autophagy positively regulates primary ciliogensis, while OFD1 accumulation at centriolar satellites underlie defects of primary ciliogenesis;(7) shRNA mediated OFDl knockdown more efficiently target OFD1 at centriolar satellites, and promotes primary ciliogenesis in both cycling MEFs and transformed breast cancer MCF7 cells, suggesting a general suppressing role of OFD1 at centriolar satellites.To summarize, our study suggest autophagy promotes primary ciliogenesis by degrading OFD1 from centriolar satellites, these findings define a newly recognized role of autophagy in organelle biogenesis and further suggesting that dissecting the regulatory mechanisms of OFD1 will provide insight into these functions and potentially offer new therapeutic tools for treatment of ciliopathies and cancers.