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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All data that support the conclusions are available from the authors on request, and/or also available in the Supplementary Information.
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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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).
The authors declare no competing interests.
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Supplementary Figs. 1–7 and Video 1 and 2 legends.
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
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
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.
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.
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.
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.
An Excel sheet with the numerical data on the fiber diffraction analysis of microtubules during different assembly conditions in the presence of Taxol.
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.
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.
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.
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.
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.
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.
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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