Self-repair protects microtubules from destruction by molecular motors

Abstract

Microtubule instability stems from the low energy of tubulin dimer interactions, which sets the growing polymer close to its disassembly conditions. Molecular motors use ATP hydrolysis to produce mechanical work and move on microtubules. This raises the possibility that the mechanical work produced by walking motors can break dimer interactions and trigger microtubule disassembly. We tested this hypothesis by studying the interplay between microtubules and moving molecular motors in vitro. Our results show that molecular motors can remove tubulin dimers from the lattice and rapidly destroy microtubules. We also found that dimer removal by motors was compensated for by the insertion of free tubulin dimers into the microtubule lattice. This self-repair mechanism allows microtubules to survive the damage induced by molecular motors as they move along their tracks. Our study reveals the existence of coupling between the motion of molecular motors and the renewal of the microtubule lattice.

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Fig. 1: Gliding assays destroy non-stabilized microtubules.
Fig. 2: Motility assays destroy non-stabilized microtubules.
Fig. 3: Single dyneins can damage the microtubule lattice.
Fig. 4: Free tubulin dimers prevent microtubule destruction by kinesin and dynein.
Fig. 5: Molecular motors enhance tubulin exchange in the microtubule lattice.

Data availability

Raw data are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

This project benefited from several funding sources: European Research Council (ERC) Advanced 741773 (AAA) to L.B., ERC Consolidator 771599 (ICEBERG) to M.T.; Agence Nationale de la Recherche (ANR)-18-CE13-0001 to K.J. and M.T., Howard Hughes Medical Institute to S.L.R.-P and IRTELIS PhD programme from the CEA to S.T. Our imaging platform is supported by the Laboratory of Excellence Grenoble Alliance for Integrated Structural & Cell Biology (LabEX GRAL) (ANR-10-LABX-49-01) and the University Grenoble Alpes graduate school (Ecoles Universitaires de Recherche, CBH-EUR-GS, ANR-17-EURE-0003). We thank S. Diez (TU Dresden) for interesting discussions and sharing reagents.

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Affiliations

Authors

Contributions

S.T. performed all experiments with motility assays; D.I. performed all experiments with gliding assays; J.G. purified and labelled tubulin and assisted S.T. and D.I. for all experiments; Z.M.H., M.E.D. and S.L.R.-P. purified dyneins; D.P., E.D. and C.L. discussed the founding hypothesis of the project; C.A. and L.S. performed preliminary experiments; K.J. analysed data; and L.B. and M.T. designed and directed the project.

Corresponding authors

Correspondence to Laurent Blanchoin or Manuel Théry.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Methods, references, Figs. 1–6, Tables 1–14 and video legends 1–12.

Reporting Summary

Supplementary Video 1

Gliding assay of stabilized microtubules. Taxol-treated (left) and GMPCPP-microtubules (right), labelled with 20% fluorescent tubulin, gliding on a layer of kinesin-1. Images were taken with a ×20 objective in epifluorescence microscopy. Acquisition frame rate, 1 image every 5 seconds over 15 minutes. Display, 25 images s–1. Scale bar, 100 µm.

Supplementary Video 2

Gliding assay of non-stabilized capped GDP-microtubules. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, gliding on a layer of kinesin-1. Images were taken with a ×20 objective in epifluorescence microscopy. Acquisition frame rate, 1 image every 5 seconds over 15 minutes. Display, 25 images s–1. Scale bar, 100 µm.

Supplementary Video 3

Microtubule breakage during gliding. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, gliding on a layer of kinesin-1. Microtubules were automatically detected and tracked in order to reposition the visualization field so as to keep the moving microtubule at the centre of the field. Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 5 seconds over 15 minutes. Display, 5 images s–1. Scale bar, 20 µm.

Supplementary Video 4

Microtubule breakage upon kinesin motor walk. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of kinesins (10 nM Klp2). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 3 seconds over 3 minutes. Display, 5 images s–1. Scale bar, 10 µm.

Supplementary Video 5

Microtubule breakage versus uncapping upon dynein motor walk. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of kinesins (1 nM Klp2). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 3 seconds over 3 minutes. Display, 3 images s–1. Scale bar, 10 µm.

Supplementary Video 6

Single dynein motors walking on a capped microtubule. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of dyneins (50 pM). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 3 seconds over 2 minutes. Display, 10 images s–1. Scale bar, 10 µm.

Supplementary Video 7

Microtubule breakage upon dynein motor walk. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of dyneins (50 pM). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 3 seconds over 3 minutes. Display, 5 images s–1. Scale bar, 10 µm.

Supplementary Video 8

Free tubulin dimers protect microtubules during gliding assay on kinesins. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, gliding on a layer of kinesin-1 in the presence of unlabelled free tubulin dimers. Images were taken with a ×20 objective in epifluorescence microscopy. Acquisition frame rate, 1 image every 5 seconds over 15 minutes. Display, 25 images s–1. Scale bar, 100 µm.

Supplementary Video 9

Free tubulin dimers protect microtubules during gliding assay on dyneins. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, gliding on a layer of dyneins in the presence of unlabelled free tubulin dimers. Images were taken with a ×20 objective in epifluorescence microscopy. Acquisition frame rate, 1 image every 5 seconds over 15 minutes. Display, 25 images s–1. Scale bar, 100 µm.

Supplementary Video 10

Free tubulin dimers protect microtubules during motility assay. Capped GDP-microtubules, labelled with 20% fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of kinesins (10 nM Klp2) in the absence (left) or presence (right) of free tubulin dimers (14 µM). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every 2 minutes over 1 hour. Display, 5 images s–1. Scale bar, 10 µm.

Supplementary Video 11

Free tubulin dimers incorporate along microtubules during gliding assay. Capped GDP-microtubules, with the shaft labelled with 3% red fluorescent tubulin and the stable ends labelled with 20% red fluorescent tubulin (middle column), gliding on a layer of kinesins (kinesin-1) in the presence of unlabelled fluorescent free tubulin dimers after a 30-minute-long exposure to 14 µM free green fluorescent tubulin dimers (left column). Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every second over 30 seconds. Display, 5 images s–1. Scale bar, 20 µm.

Supplementary Video 12

Free tubulin dimers incorporate along microtubules during motility assay. Capped GDP-microtubules, labelled with 3% red fluorescent tubulin, were assembled from micropatterned seeds and served as tracks for the motility of kinesins (10 nM Klp2) in the presence of green free tubulin dimers (14 µM) that were further replaced by unlabelled dimers prior to imaging. Images were taken with a ×63 objective in TIRF microscopy with ×1.5 magnifier. Acquisition frame rate, 1 image every second over 30 seconds. Display, 5 images s–1. Scale bar, 10 µm.

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Triclin, S., Inoue, D., Gaillard, J. et al. Self-repair protects microtubules from destruction by molecular motors. Nat. Mater. (2021). https://doi.org/10.1038/s41563-020-00905-0

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