Abstract
The transition zone is a specialized compartment found at the base of cilia, adjacent to the centriole distal end, where axonemal microtubules are heavily crosslinked to the surrounding membrane to form a barrier that gates the ciliary compartment. A number of ciliopathy molecules have been found to associate with the transition zone, but factors that directly recognize axonemal microtubules to specify transition zone assembly at the cilia base remain unclear. Here, through quantitative centrosome proteomics, we identify an axoneme-associated protein, CEP162 (KIAA1009), tethered specifically at centriole distal ends to promote transition zone assembly. CEP162 interacts with core transition zone components, and mediates their association with microtubules. Loss of CEP162 arrests ciliogenesis at the stage of transition zone assembly. Abolishing its centriolar tethering, however, allows CEP162 to stay on the growing end of the axoneme and ectopically assemble transition zone components at cilia tips. This generates extra-long cilia with strikingly swollen tips that actively release ciliary contents into the extracellular environment. CEP162 is thus an axoneme-recognition protein pre-tethered at centriole distal ends before ciliogenesis to promote and restrict transition zone formation specifically at the cilia base.
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Acknowledgements
We are grateful to K. Anderson and her laboratory at Memorial Sloan-Kettering Cancer Center, USA, for reagents and antibodies. We thank L. Gunther, G. Perumal and F. Macaluso at the Analytical Imaging Center of Albert Einstein College of Medicine for assistance with transmission and scanning electron microscopy; K. Uryu at Rockefeller university and N. Lampen at MSKCC for assisting with the usage of electron microscopes; F. Foley at SUNY Upstate and D. Gutierrez at MSKCC for technical assistance and zebrafish management; and A. Hall, Z. Bao and C. Haynes at MSKCC for comments on the manuscript. This work was supported by the National Institutes of Health grants HL095690 to J.D.A. and GM088253 to M-F.B.T.
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W-J.W. and M-F.B.T. designed experiments and analysed data. W-J.W. performed most of the experiments. W-J.W., R.S. and J.M.A. did the quantitative centrosome proteomics. W-J.W. and R.S. prepared purified centrosomes. J.M.A. carried out SILAC mass spectrometry and analysed the data. The zebrafish works were carried out by H.G.T. and J.D.M. M.G.G. helped with initial zebrafish experiments. G.S.P., F.P.M. and W-J.W. performed the correlative light and scanning electron microscopy. M-F.B.T. and W-J.W. wrote the manuscript.
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Ciliary contents are actively released from cilia tips modified with CEP162.
RPE1 cells expressing GFP-tagged CEP162tN1C1C2 were serum-starved for 24 h, and imaged by time-lapse fluorescence microscopy (2 min time interval). Images shown in movies were generated from maximum intensity projections of a z-stack. (MOV 697 kb)
Bursts of cilia tips modified by CEP162.
Images were collected and processed from RPE1 cells expressing GFP-tagged CEP162tN1C1C2 as described in Supplementary Video S1. (MOV 578 kb)
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Wang, WJ., Tay, H., Soni, R. et al. CEP162 is an axoneme-recognition protein promoting ciliary transition zone assembly at the cilia base. Nat Cell Biol 15, 591–601 (2013). https://doi.org/10.1038/ncb2739
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DOI: https://doi.org/10.1038/ncb2739
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