Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages
- Academia Sinica, Taiwan, Republic of China
- National Yang-Ming University, Taiwan, Republic of China
- Institute of Cancer Research, United Kingdom
- Memorial Sloan Kettering Cancer Center, United States
- National Taiwan University, Taiwan, Republic of China
Abstract
Subdistal appendages (sDAPs) are centriolar elements observed proximal to the distal appendages (DAPs) in vertebrates. Despite their obvious presence, structural and functional understanding of sDAPs remains elusive. Here, by combining super-resolved localization analysis and CRISPR-Cas9 genetic perturbation, we find that, although DAPs and sDAPs are primarily responsible for distinct functions in ciliogenesis and microtubule anchoring respectively, the presence of one element actually affects the positioning of the other. Specifically, we find dual layers of both ODF2 and CEP89, where their localizations are differentially regulated by DAP and sDAP integrity. DAP depletion relaxes longitudinal occupancy of sDAP protein ninein to cover the DAP region, implying a role of DAPs in sDAP positioning. Removing sDAPs alter the distal border of centrosomal γ-tubulins, illustrating a new role of sDAPs. Together, our results provide an architectural framework of sDAPs to shed light on functional understanding, surprisingly revealing the coupling between DAPs and sDAPs.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Meng-Fu Bryan Tsou
Funding
Ministry of Science and Technology, Taiwan (107-2112-M-001-037)
- Weng Man Chong
- Tzu-Yuan Chiu
- Ting-Jui Chang
- You-Pi Liu
- T Tony Yang
- Jung-Chi Liao
Ministry of Science and Technology, Taiwan (107-2313-B-001-009)
- Weng Man Chong
- Tzu-Yuan Chiu
- Ting-Jui Chang
- You-Pi Liu
- T Tony Yang
- Jung-Chi Liao
Academia Sinica (2317-1040300)
- Weng Man Chong
- Tzu-Yuan Chiu
- Ting-Jui Chang
- You-Pi Liu
- T Tony Yang
- Jung-Chi Liao
Ministry of Science and Technology, Taiwan (108-2313-B-010-001)
- Won-Jing Wang
Ministry of Science and Technology, Taiwan (108-2628-B-010-007)
- Won-Jing Wang
Ministry of Science and Technology, Taiwan (108-2638-B-010-001 -MY2)
- Won-Jing Wang
National Institutes of Health (GM088253)
- Meng-Fu Bryan Tsou
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2020, Chong et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Super-resolution microscopy reveals coupling between mammalian centriole subdistal appendages and distal appendages
eLife 9:e53580.
https://doi.org/10.7554/eLife.53580
Further reading
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- Cell Biology
Distal appendages are nine-fold symmetric blade-like structures attached to the distal end of the mother centriole. These structures are critical for formation of the primary cilium, by regulating at least four critical steps: ciliary vesicle recruitment, recruitment and initiation of intraflagellar transport (IFT), and removal of CP110. While specific proteins that localize to the distal appendages have been identified, how exactly each protein functions to achieve the multiple roles of the distal appendages is poorly understood. Here we comprehensively analyze known and newly discovered distal appendage proteins (CEP83, SCLT1, CEP164, TTBK2, FBF1, CEP89, KIZ, ANKRD26, PIDD1, LRRC45, NCS1, CEP15) for their precise localization, order of recruitment, and their roles in each step of cilia formation. Using CRISPR-Cas9 knockouts, we show that the order of the recruitment of the distal appendage proteins is highly interconnected and a more complex hierarchy. Our analysis highlights two protein modules, CEP83-SCLT1 and CEP164-TTBK2, as critical for structural assembly of distal appendages. Functional assays revealed that CEP89 selectively functions in RAB34+ ciliary vesicle recruitment, while deletion of the integral components, CEP83-SCLT1-CEP164-TTBK2, severely compromised all four steps of cilium formation. Collectively, our analyses provide a more comprehensive view of the organization and the function of the distal appendage, paving the way for molecular understanding of ciliary assembly.
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- Cell Biology
The primary cilium is a microtubule-based organelle that cycles through assembly and disassembly. In many cell types, formation of the cilium is initiated by recruitment of ciliary vesicles to the distal appendage of the mother centriole. However, the distal appendage mechanism that directly captures ciliary vesicles is yet to be identified. In an accompanying paper, we show that the distal appendage protein, CEP89, is important for the ciliary vesicle recruitment, but not for other steps of cilium formation (Tomoharu Kanie, Love, Fisher, Gustavsson, & Jackson, 2023). The lack of a membrane binding motif in CEP89 suggests that it may indirectly recruit ciliary vesicles via another binding partner. Here, we identify Neuronal Calcium Sensor-1 (NCS1) as a stoichiometric interactor of CEP89. NCS1 localizes to the position between CEP89 and a ciliary vesicle marker, RAB34, at the distal appendage. This localization was completely abolished in CEP89 knockouts, suggesting that CEP89 recruits NCS1 to the distal appendage. Similarly to CEP89 knockouts, ciliary vesicle recruitment as well as subsequent cilium formation was perturbed in NCS1 knockout cells. The ability of NCS1 to recruit the ciliary vesicle is dependent on its myristoylation motif and NCS1 knockout cells expressing a myristoylation defective mutant failed to rescue the vesicle recruitment defect despite localizing properly to the centriole. In sum, our analysis reveals the first known mechanism for how the distal appendage recruits the ciliary vesicles.
