A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Harriet A.J. Saunders; Cyntha M. van den Berg; Robin A. Hoogebeen; Donna Schweizer; Kelly E. Stecker; Ronald Roepman; Stuart C. Howes; Anna Akhmanova

doi:10.1038/s41594-025-01483-y

A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

Harriet A.J. Saunders, Cyntha M. van den Berg, Robin A. Hoogebeen, Donna Schweizer, Kelly E. Stecker, Ronald Roepman, Stuart C. Howes, Anna Akhmanova^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

Original language	English
Journal	Nature Structural and Molecular Biology
DOIs	https://doi.org/10.1038/s41594-025-01483-y
Publication status	E-pub ahead of print - 24 Jan 2025

Bibliographical note

Publisher Copyright:
© The Author(s) 2025.

Funding

We thank members of the A.A. and S.C.H. labs for insightful discussions. We also thank V. Volkov (Queen Mary, University of London) for advice on cryo-ET sample preparation and V. Volkov and N. Gudimchuk (Lomonosov Moscow State University) for help with cryo-ET analysis. Electron imaging was performed at the Utrecht University Electron Microscopy Center. We thank I. C. Schneijdenberg and M. Bergmeijer for cryo-ET microscopy support. This work was supported by the European Research Council (synergy grant 609822 to A.A.), the Netherlands Organization for Health Research and Development (ZonMw) (grant 09120012110085 to R.R. and A.A.), Dutch Research Council (NWO) (grant OCENW.XL21.XL21.048 to A.A. and S.C.H.) and European Molecular Biology Organization long-term fellowship (AFTL 74-2022 to H.A.J.S.).

Funders	Funder number
Long-Term Fellowship
European Research Council	609822
ZonMw	09120012110085
Nederlandse Organisatie voor Wetenschappelijk Onderzoek	OCENW.XL21.XL21.048
European Molecular Biology Organization	AFTL 74-2022

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Saunders, H. A. J., van den Berg, C. M., Hoogebeen, R. A., Schweizer, D., Stecker, K. E., Roepman, R., Howes, S. C., & Akhmanova, A. (2025). A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth. Nature Structural and Molecular Biology. Advance online publication. https://doi.org/10.1038/s41594-025-01483-y

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abstract = "Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.",

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AU - Schweizer, Donna

AU - Stecker, Kelly E.

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AU - Howes, Stuart C.

AU - Akhmanova, Anna

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AB - Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome. We show that CEP104, a protein with a tubulin-binding TOG domain, and its luminal partner CSPP1 inhibit microtubule growth and shortening. Another TOG-domain protein, TOGARAM1, overcomes growth inhibition imposed by CEP104 and CSPP1. CCDC66 and ARMC9 do not affect microtubule dynamics but act as scaffolds for their partners. Cryo-electron tomography demonstrated that, together, ciliary tip module members form plus-end-specific cork-like structures that reduce protofilament flaring. The combined effect of these proteins is very slow processive microtubule elongation, which recapitulates axonemal dynamics in cells.

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A network of interacting ciliary tip proteins with opposing activities imparts slow and processive microtubule growth

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