CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

Dipti Rai; Yinlong Song; Shasha Hua; Kelly Stecker; Jooske L Monster; Victor Yin; Riccardo Stucchi; Yixin Xu; Yaqian Zhang; Fangrui Chen; Eugene A Katrukha; Maarten Altelaar; Albert J R Heck; Michal Wieczorek; Kai Jiang; Anna Akhmanova

doi:10.1038/s41556-024-01366-2

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

Dipti Rai
, Yinlong Song
, Shasha Hua
, Kelly Stecker
, Jooske L Monster
, Victor Yin
, Riccardo Stucchi
, Yixin Xu
, Yaqian Zhang
, Fangrui Chen
, Eugene A Katrukha
, Maarten Altelaar
, Albert J R Heck
, Michal Wieczorek
, Kai Jiang^*
, Anna Akhmanova^*

^*Corresponding author for this work

extern
Utrecht University
Institute for Molecular Biology and Biophysics
Wuhan University

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

Abstract

γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

Original language	English
Pages (from-to)	404-420
Number of pages	17
Journal	Nature Cell Biology
Volume	26
Issue number	3
DOIs	https://doi.org/10.1038/s41556-024-01366-2
Publication status	Published - Mar 2024

Bibliographical note

Publisher Copyright:
© The Author(s) 2024.

Funding

Negative-stain EM grid screening was performed with support of the ScopeM imaging center, ETH Zurich. Negative-stain EM data collection was performed with support of the Center for Microscopy and Image Analysis, University of Zurich. The authors gratefully acknowledge L. Carlini for her help in developing the MATLAB analysis scripts used to compare γ-TuRC structural data. The authors acknowledge support from the Dutch Research Council-funded Netherlands Proteomics Centre through the National Roadmap for Large-scale Research Infrastructures programme X-Omics (project 184.034.019) for the MS and MP analysis. This work was supported by the European Research Council Synergy grant 609822, the ZonMW TOP 91216006 programme and the Spinoza Prize to A.A., Swiss National Science Foundation Project Grant (no. 310030_208120) to M.W., and grants from National Natural Science Foundation of China (31871356 and 32070705) and the Fundamental Research Funds for the Central Universities (2042022dx0003 and 2042023kf0212) to K.J.

Funders	Funder number
Dutch Research Council-funded Netherlands Proteomics Centre	184.034.019
European Research Council	609822
European Research Council
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung	310030_208120
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
National Natural Science Foundation of China	31871356, 32070705
National Natural Science Foundation of China
ZonMw	TOP 91216006
ZonMw
Fundamental Research Funds for the Central Universities	2042023kf0212, 2042022dx0003
Fundamental Research Funds for the Central Universities

Keywords

Cytoskeleton/metabolism
Microtubule-Associated Proteins/genetics
Microtubule-Organizing Center/metabolism
Microtubules/metabolism
Tubulin/metabolism

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title = "CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC",

abstract = "γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.",

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note = "Publisher Copyright: {\textcopyright} The Author(s) 2024.",

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month = mar,

doi = "10.1038/s41556-024-01366-2",

language = "English",

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AU - Rai, Dipti

AU - Song, Yinlong

AU - Hua, Shasha

AU - Stecker, Kelly

AU - Monster, Jooske L

AU - Yin, Victor

AU - Stucchi, Riccardo

AU - Xu, Yixin

AU - Zhang, Yaqian

AU - Chen, Fangrui

AU - Katrukha, Eugene A

AU - Altelaar, Maarten

AU - Heck, Albert J R

AU - Wieczorek, Michal

AU - Jiang, Kai

AU - Akhmanova, Anna

PY - 2024/3

Y1 - 2024/3

N2 - γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

AB - γ-Tubulin ring complex (γ-TuRC) is the major microtubule-nucleating factor. After nucleation, microtubules can be released from γ-TuRC and stabilized by other proteins, such as CAMSAPs, but the biochemical cross-talk between minus-end regulation pathways is poorly understood. Here we reconstituted this process in vitro using purified components. We found that all CAMSAPs could bind to the minus ends of γ-TuRC-attached microtubules. CAMSAP2 and CAMSAP3, which decorate and stabilize growing minus ends but not the minus-end tracking protein CAMSAP1, induced microtubule release from γ-TuRC. CDK5RAP2, a γ-TuRC-interactor, and CLASP2, a regulator of microtubule growth, strongly stimulated γ-TuRC-dependent microtubule nucleation, but only CDK5RAP2 suppressed CAMSAP binding to γ-TuRC-anchored minus ends and their release. CDK5RAP2 also improved selectivity of γ-tubulin-containing complexes for 13- rather than 14-protofilament microtubules in microtubule-capping assays. Knockout and overexpression experiments in cells showed that CDK5RAP2 inhibits the formation of CAMSAP2-bound microtubules detached from the microtubule-organizing centre. We conclude that CAMSAPs can release newly nucleated microtubules from γ-TuRC, whereas nucleation-promoting factors can differentially regulate this process.

KW - Cytoskeleton/metabolism

KW - Microtubule-Associated Proteins/genetics

KW - Microtubule-Organizing Center/metabolism

KW - Microtubules/metabolism

KW - Tubulin/metabolism

UR - https://www.scopus.com/pages/publications/85186180897

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DO - 10.1038/s41556-024-01366-2

M3 - Article

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SN - 1465-7392

VL - 26

SP - 404

EP - 420

JO - Nature Cell Biology

JF - Nature Cell Biology

IS - 3

ER -

CAMSAPs and nucleation-promoting factors control microtubule release from γ-TuRC

Abstract

Bibliographical note

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Keywords

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