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Taxanes convert regions of perturbed microtubule growth into rescue sites

Nature Materials volume 19, pages 355–365 (2020)Cite this article

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Abstract

Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.

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Fig. 1: Taxol and its fluorescent derivatives induce formation of stable rescue sites in microtubules.
Fig. 2: Taxane-site binding compounds accumulate at growing microtubule ends and perturb microtubule growth and depolymerization.
Fig. 3: Formation of accumulations of taxane-site ligands is controlled by microtubule dynamics.
Fig. 4: Analysis of the kinetics of Fchitax-3 accumulations.
Fig. 5: Analysis of the nucleotide dependence of Fchitax-3 accumulations.
Fig. 6: Fchitax-3 promotes long, sheet-like microtubule defects and generates sites of tubulin incorporation.

Data availability

All data that support the conclusions are available from the authors on request, and/or also available in the Supplementary Information.

Code availability

All MATLAB and Mathematica notebooks used for computations, together with the raw data, are available online at https://doi.org/10.6084/m9.figshare.7520033 and https://github.com/RuddiRodriguez/Analysis-of-MT-plus-end-fluctuations.

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Acknowledgements

We thank G. Fernando Díaz for calf brain supply, and the staff of beamline BL11-NCD-SWEET (ALBA) for their support with the X-ray fibre diffraction experiments. We thank S. Kamimura (Chuo University) for kindly providing the share-flow device employed for fibre diffraction experiments. This work was supported by the European Research Council Synergy (grant no. 609822) and the Netherlands Organization for Scientific Research CW ECHO (grant no. 711.015.005 to A.A.), by a Biotechnology and Biological Sciences Research Council grant (no. BB/N018176/1 to C.A.M.), by an EMBO Long Term Fellowship to R.R.-G., by the CAMS Innovation Fund for Medical Sciences (grant no. 2016-I2M-1-010 to W.-S.F.), by grants from MINECO/FEDER (no. BFU2016-75319-R to J.F.D.) and by COST Action (no. CM1407 to J.F.D. and K.-H.A.). M.O.S is supported by a grant from the Swiss National Science Foundation (no. 31003A_166608).

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Authors and Affiliations

  1. Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, the Netherlands

    Ankit Rai, Eugene A. Katrukha, Ruddi Rodríguez-García, Lukas C. Kapitein & Anna Akhmanova

  2. Institute of Structural and Molecular Biology, Birkbeck, University of London, London, UK

    Tianyang Liu & Carolyn A. Moores

  3. Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zürich, Zurich, Switzerland

    Simon Glauser & Karl-Heinz Altmann

  4. Chemical and Physical Biology, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain

    Juan Estévez-Gallego & J. Fernando Díaz

  5. State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Beijing, China

    Wei-Shuo Fang

  6. Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Villigen, Switzerland

    Michel O. Steinmetz

  7. University of Basel, Biozentrum, Basel, Switzerland

    Michel O. Steinmetz

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Contributions

A.R. designed and performed experiments, analysed data and wrote the paper. T.L. and C.A.M. designed and performed cryo-EM experiments and analysed data. E.A.K. analysed data and performed the modelling. J.E.-G. performed X-ray fibre diffraction experiments. R.R.-G. analysed microtubule tip fluctuation data. W.-S.F. synthesized Fchitax-3 and Flutax-2. S.G. and K.-H.A. synthesized Alexa488-epothilone B. L.C.K., J.F.D. and M.O.S. contributed to the design of the experiments and analysis of the data and models. A.A. designed experiments, coordinated the project and wrote the paper.

Corresponding author

Correspondence to Anna Akhmanova.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Video 1 and 2 legends.

Reporting Summary

Supplementary Video 1

Fchitax-3 accumulation at the growing microtubule plus-end The movie illustrates the formation of an Fchitax-3 accumulation close to the growing microtubule plus-end, as depicted in Fig. 2a. The experiment was performed in the presence of tubulin (15 μM), mCherry-EB3 (20 nM) and Fchitax-3 (100 nM). The movie consists of 177 frames acquired with a 2-s interval between frames and an exposure time of 100 ms. Scale bar, 2 μm. The movie is representative of more than five independent experiments

Supplementary Video 2

Laser-severing experiment showing that Fchitax-3 accumulation zone stabilizes microtubule lattice The movie starts immediately after ablation of the Fchitax-3 accumulation area with a 532-nm laser, as shown in Fig. 2h. After ablation of the growing microtubule at Fchitax-3 accumulation, both newly generated ends survived and began growing again. The experiment was performed in the presence of tubulin (15 μM, supplemented with 3% rhodamine-tubulin), mCherry-EB3 (20 nM) and Fchitax-3 (100 nM). The movie consists of 750 frames acquired in stream acquisition mode with an exposure time of 100 ms. Scale bar, 2 μm. The movie is representative of five independent experiments

NMR Data 1

1H-NMR

NMR Data 2

C13-NMR

Supplementary Data 1

An Excel sheet with the numerical data on the quantification of occurrence of stable rescue sites, intensity measurement of single molecules of Fchitax-3, photobleaching time traces and intensity measurement of Fchitax-3 at stable rescue sites.

Supplementary Data 2

An Excel sheet with the numerical data on the analysis of the time intervals between the appearances of two consecutive accumulations, analysis of duration, length and frequency of Fchitax-3 accumulations at plus and minus ends of microtubules and quantification of microtubule growth rates.

Supplementary Data 3

An Excel sheet with the numerical data on the quantification of microtubule growth rates, catastrophe frequencies and accumulation frequencies, accumulation length and intensity profiles showing the reduction in the EB3 signal.

Supplementary Data 4

An Excel sheet with the numerical data on the quantification of characteristic photobleaching traces, decay times, comparison of the best fits for the models, dependence of initial values and tubulin states, kinetics of tubulin states, numerically solved FRAP curves and fluorescence intensities.

Supplementary Data 5

An Excel sheet with the numerical data on the fiber diffraction analysis of microtubules during different assembly conditions in the presence of Taxol.

Supplementary Data 6

An Excel sheet with the numerical data on the quantification of CAMSAP3 binding near Fchitax-3 accumulations, fluorescence intensity profiles for fluorescence recovery after photobleaching and distribution of the Fchitax-3 accumulations.

Source data

Source Data Fig. 1

An Excel sheet with the numerical data on the quantification of Fchitax-3 and Flutax-2 intensity on GDP lattice and on stable rescue sites and frequency of the occurrence of stable rescue sites in vitro and in cells.

Source Data Fig. 2

An Excel sheet with the numerical data on the quantification of fluctuations of EB3 fluorescence intensities, microtubule growth rates and microtubule survival after the ablation.

Source Data Fig. 3

An Excel sheet with the numerical data on the quantification of Fchitax-3 accumulation frequencies and time plots of the normalized maximum intensity of fitted EB3 comets and the normalized area under the curve (AUC) of fitted Fchitax-3 intensities.

Source Data Fig. 4

An Excel sheet with the numerical data for the intensity time traces of Fchitax-3, best fits to a single profile using Michaelis-Menten or the autocatalysis model, analysis of rate constants, intensity time traces for the FRAP analysis of Fchitax-3 accumulation and modeling of FRAP curves.

Source Data Fig. 5

An Excel sheet with the numerical data for Fchitax-3 fluorescence intensity profiles and quantifications of the normalized value of fluorescence intensities in different conditions and the rate constant of photobleaching.

Source Data Fig. 6.

An Excel sheet with the numerical data on the quantification of cryo-EM defect analysis, transverse microtubule tip fluctuations, fluorescence intensity profiles and quantifications showing CAMSAP3 intensity, fluorescence intensity profiles and quantification of tubulin recovery after FRAP and quantification of EB3 fluorescence.

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Rai, A., Liu, T., Glauser, S. et al. Taxanes convert regions of perturbed microtubule growth into rescue sites. Nat. Mater. 19, 355–365 (2020). https://doi.org/10.1038/s41563-019-0546-6

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