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Tubulin isotypes of C. elegans harness the mechanosensitivity of the lattice for microtubule luminal accessibility

Nature Physics volume 21, pages 1420–1430 (2025)Cite this article

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Abstract

Microtubules are hollow cylindrical cytoskeletal polymers of laterally associated protofilaments that contain head-to-tail aligned ɑ/β-tubulin heterodimers. Although the exposed microtubule exterior is readily accessible to proteins, the mechanism governing the accessibility of the confined microtubule lumen to luminal particles remains unknown. Here we show that certain tubulin family proteins (isotypes) facilitate luminal accessibility because of the mechanical properties and lateral interactions that they confer to the microtubules. We characterized the microtubules reconstituted from defined compositions of Caenorhabditis elegans tubulin isotypes. These tubulin isotypes form microtubules with comparable protofilament numbers but different luminal accessibility. We further revealed the role of tubulin isotypes in regulating the strength of inter-protofilament lateral interactions, which determines luminal accessibility through the mechanosensitivity of reversible protofilament separation. Deformation of the microtubule lattice, which generates stresses exceeding the strength of the lateral interactions, creates gaps between adjacent protofilaments, enhancing the accessibility of the lumen. Together, our findings uncovered the tubulin isotype-dependent mechanical plasticity that confers force sensitivity to the microtubule lattice and modulates the energy barrier for luminal proteins to access the lumen.

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Fig. 1: Differences in luminal accessibility of MEC-12/MEC-7 and TBA-2/TBB-2 microtubules for ATAT-2.
Fig. 2: Direct characterization of microtubule mechanical plasticity.
Fig. 3: Analyses of microtubule force–strain curves.
Fig. 4: The 3D finite element model of microtubule mechanical plasticity.
Fig. 5: Microtubule lattice deformation regulates the luminal accessibility.

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Data availability

Data generated in this study are available from the corresponding authors upon request.

Code availability

The codes for quantifying ATAT-2 dissociation rate on curved microtubules are available via GitHub at https://github.com/JacksonYe61/code-for-measure-time-laps-intensity-of-curved-microtubule-in-Fiji.

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Acknowledgements

S.-C.T. acknowledges support from the Research Grants Council of Hong Kong under the General Research Fund (grant nos. 17122822 and 17118421; principal investigator S.-C.T.) and the Collaborative Research Fund (grant no. C7064-22GF; project coordinator S.-C.T.). Y.L. acknowledges support from the Research Grants Council of Hong Kong under the General Research Fund (grant no. 17210520), the Health@InnoHK programme of the Innovation and Technology Commission of the Hong Kong SAR Government and the National Natural Science Foundation of China (grant no. 12272332). We thank T. M. Kapoor (Rockefeller University) for comments on this paper; J. Guo, M. Chen and H. Zhu (Imaging and Flow Cytometry Core, Center for PanorOmic Sciences, LKS Faculty of Medicine, University of Hong Kong) for support with fluorescence microscopes; Biological Cryo-EM Center at the Hong Kong University of Science and Technology (HKUST) for sample screening, grid preparation and data collection; and Y. Zhang for technical support. A donation from the Lo Kwee Seong Foundation generously supports the HKUST Cryo-EM Center.

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Author notes

  1. These authors contributed equally: Yucheng Ye, Zheng Hao.

Authors and Affiliations

  1. School of Biomedical Sciences, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China

    Yucheng Ye, Jingyi Luo & Shih-Chieh Ti

  2. Department of Mechanical Engineering, Faculty of Engineering, The University of Hong Kong, Hong Kong SAR, China

    Zheng Hao & Yuan Lin

  3. Advanced Biomedical Instrumentation Centre, Hong Kong Science Park, Hong Kong SAR, China

    Zheng Hao & Yuan Lin

  4. Division of Life Science, The Hong Kong University of Science and Technology, Hong Kong SAR, China

    Wai Hei Lam & Yuanliang Zhai

  5. Department of Chemistry, Faculty of Science, The University of Hong Kong, Hong Kong SAR, China

    Zheng Liu & Xiang David Li

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Contributions

Y.Y., Z.H., Y.L. and S.-C.T. conceptualized the study. Y.Y. and J.L. generated the protein materials used in this study. Y.Y. performed and analysed the optical tweezer experiments. Y.Y. and J.L. performed and analysed the TIRF microscopy experiments. Y.Y. performed and analysed the TIRF-based single-molecule experiments. Y.Y., Z.H., Y.L. and S.-C.T. developed the 3D finite element model of the anisotropic microtubule lattice. W.H.L. and Y.Z. collected and analysed the cryo-EM data. Z.L. and X.D.L. characterized the proteins by means of mass spectrometry. Y.L. and S.-C.T. supervised the study. Y.Y., Z.H., Y.L. and S.-C.T. wrote the paper.

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Correspondence to Yuan Lin or Shih-Chieh Ti.

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Extended data

Extended Data Fig. 1 Purification and characterization of recombinant proteins.

(a) Reversed-phase LC-MS spectra of purified TBA-2/TBB-2. See Luo et al. for the LC-MS spectra of purified MEC-12/MEC-730. (b) Representative Coomassie blue-stained SDS-PAGE, immunoblot analyses, and rhodamine fluorescence imaging of purified recombinant proteins. See Luo et al. for the Coomassie blue-stained SDS-PAGE, immunoblot analyses, and rhodamine fluorescence imaging of purified MEC-12/MEC-730. We performed Coomassie blue-stained SDS–PAGE analysis for every batch of protein that we purified. (c and d) Representative TIRF microscopy images showing the incorporation of FITC-labeled tubulin (green) into defects of the rhodamine-labeled TBA-2/TBB-2 (c) or MEC-12/MEC-7 (d) microtubule lattice (red) before and after the treatment of MBP-tagged human katanin P60. Scale bars: 5 µm. Similar results were observed in two independent experiments. (e) Representative cryo-EM images of GMPCPP-stabilized TBA-2/TBB-2 (top) and MEC-12/MEC-7 (bottom) microtubules showing no visible breakage or defects. Similar results were observed in 596 (TBA-2/TBB-2) and 1040 (MEC-12/MEC-7) micrographs from one cryo-EM data collection. Scale bars: 50 nm. (f and g) The FITC fluorescence intensity on per micrometer GMPCPP-stabilized TBA-2/TBB-2 microtubules (0 nM katanin: 1143 ± 374 (a.u.), n=88; 10 nM katanin: 3732 ± 1688 (a.u.), n=90; 50 nM katanin: 4780 ± 1934 (a.u.), n=78, mean ± SD are indicated) (f) and MEC-12/MEC-7 microtubules (0 nM katanin: 1515 ± 678 (a.u.), n=71; 10 nM katanin: 3077 ± 1232 (a.u.), n=73; 50 nM katanin: 5968 ± 2329 (a.u.), n=70, mean ± SD are indicated) (g) treated with different concentrations of MBP-tagged human katanin P60. The mean and SD are based on analyzed microtubules pooled from two independent experiments. The P values were calculated using a two-tailed unpaired Student’s t-test.

Extended Data Fig. 2 The distribution of ATAT-2 binding on TBA-2/TBB-2 and MEC-12/MEC-7 microtubules.

(a and b) Representative time-lapse images showing ATAT-2-GFP (green) binding to and dissociation from GMPCPP-stabilized TBA-2/TBB-2 (a) and MEC-12/MEC-7 (b) microtubules (red). The slow-dissociating GFP fluorescence signal at the microtubule ends and the shaft is indicated by white arrows and arrowheads, respectively. Scale bars: 3 µm. Similar results were observed in at least three independent experiments. (c) Heatmaps of the time-dependent decrease of the ATAT-2-GFP fluorescence intensity on a GMPCPP-stabilized TBA-2/TBB-2 or MEC-12/MEC-7 microtubule ~30 sec after perfusing the TIRF chamber with the ATAT-2-free buffer. The arrows indicate the positions of microtubule ends. Similar results were observed in at least three independent experiments. (d) The decay of the normalized GFP-tagged ATAT-2 fluorescence intensity on GMPCPP-stabilized TBA-2/TBB-2 (black, n=87) or MEC-12/MEC-7 (green, n=82) microtubules upon perfusion with the ATAT-2-free buffer. The GFP fluorescence intensity was normalized against that from the first image of the time-lapse started ~30 sec after perfusing the TIRF chamber with the ATAT-2-free buffer. The solid lines show the single exponential decay fitted to the data points (mean ± SEM) from three independent experiments. The lifetimes (τ) are indicated. (e) The ‘averaged’ normalized rhodamine fluorescence intensity at the end region of TBA-2/TBB-2 (n=72) and MEC-12/MEC-7 (n=67) microtubules from at least three independent experiments. The lengths of the microtubule end regions are indicated. (f) Schematic of the end and shaft regions of a microtubule. (g) The portions of the slow-dissociating ATAT-2-GFP on different regions of microtubules at ~30 sec after the TIRF chamber was perfused with the ATAT-2-free buffer. The mean and SD are based on analyzing TBA-2/TBB-2 (n=147) and MEC-12/MEC-7 (n=123) microtubules from at least three independent experiments. (h) The landing frequency of ATAT-2-GFP on TBA-2/TBB-2 (black bar, n=3838) and MEC-12/MEC-7 (green bar, n=2827) microtubules. Assuming a Poisson distribution, the SEM of the landing frequency was calculated as (observed frequency)0.5/ (imaging time × total microtubule length)0.5. The mean and SEM are based on data from at least three independent experiments. The P values were calculated using a two-tailed unpaired Student’s t-test. (i) The exponential decay of the numbers of detectable ATAT-2-GFP molecules (n=1130) on the coverslip surface under the imaging conditions of the single-molecule assays. The data are from two independent experiments. The measured data (black dots) were fitted with a single exponential distribution (red line) to determine the photobleaching rate (kb). (j) Distribution of the duration of ATAT-2 single molecules on GMPCPP-stabilized TBA-2/TBB-2 (black, n=3838) and MEC-12/MEC-7 (green, n=2827) microtubules from at least three independent experiments. tTBA-2/TBB-2 and tMEC-12/MEC-7 are the average duration (mean ± SD).

Extended Data Fig. 3 Microtubules can sustain cycles of compression without fatigue.

(a-d) The compression (blue) and relaxation (red) force-strain curves (a and c) and the strains with maximum energy absorption efficiency (b and d) of one GMPCPP-stabilized TBA-2/TBB-2 (a and b) or MEC-12/MEC-7 (c and d) microtubule after compression-relaxation cycles. Similar results were observed in at least three independent experiments. (e and f) Schematic of the line scan analysis (e) and the corresponding intensity profiles (f) of TBA-2/TBB-2 microtubule cross-sections (green lines) before compression and at the maximum strain in the optical trap experiments. (g) Intensity profiles of the cross-section line scans from another seven TBA-2/TBB-2 microtubules at the maximum strain. (h and i) Schematic of the line scan analysis (h) and the corresponding intensity profiles (i) of MEC-12/MEC-7 microtubule cross-sections (green lines) before compression and at the maximum strain in the optical trap experiments. (j) Intensity profiles of the cross-section line scans from another seven MEC-12/MEC-7 microtubules at the maximum strain. The background threshold (red dashed lines) was determined from the mean (µ, blue dashed lines) and standard deviation (σ) of the fluorescence intensity of a 40-pixel x 40-pixel area (red squares). We used the fluorescence intensity half-heights (gray dashed line) to determine the width of peaks from microtubule cross-section line scans. The peak width of microtubules before compression was indicated (blue double arrow). All eight GMPCPP-stabilized TBA-2/TBB-2 microtubules remain intact at the maximum strain as (i) the fluorescence intensities are larger than the background threshold (red dashed lines) and (ii) the peak widths of microtubules at the maximum strain are larger than 90% of the width before compression (blue double arrows). All eight GMPCPP-stabilized MEC-12/MEC-7 microtubules are splayed at the maximum strain as the fluorescence intensities are lower than the background threshold (red dashed lines). Scale bars: 3 µm. The data are from at least three independent experiments.

Extended Data Fig. 4 Representative tensile force-strain curves of microtubules.

(a-f) Representative tensile force-strain curves of GMPCPP-stabilized TBA-2/TBB-2 (a-c) and MEC-12/MEC-7 (d-f) microtubules with different lengths, showing the disconnection and re-connection of NeutrAvidin-coated beads on microtubules. Similar results were observed in at least three independent experiments.

Extended Data Fig. 5 The 3D finite element method-based computational model of microtubules.

(a) Rotation (or orientation change) of protofilament j will lead to non-zero displacement δi, j in its local coordinate system (upper row). The resulting moment Mi, j restraining the relative rotation between two protofilaments is effectively the same as that by a rotational spring (lower row). p is the initial distance between the long axes of two neighboring protofilaments. θ is the angle of rotation of protofilament j. (b and c) Simulation results of the cross-sectional flattening and protofilament separation of TBA-2/TBB-2 (b) and MEC-12/MEC-7 (c) microtubules. The heatmap indicates the maximum normal stress loaded on each protofilament in the microtubule lattice. (d) The largest protofilament separation distance in the simulated TBA-2/TBB-2 (black) and MEC12/MEC-7 (green) microtubules under compressive strains from 0 % to 50 % with 5% intervals. (e) The enlarged view of (d) highlights the <10 nm protofilament separation in TBA-2/TBB-2 microtubules at the examined strains.

Extended Data Fig. 6 The luminal accessibility of GMPCPP-stabilized TBA-2/TBB-2 microtubules increases with compressive strains.

Heatmaps showing the initial binding and the dissociation of GFP-tagged ATAT-2 over time on GMPCPP-stabilized TBA-2/TBB-2 microtubules experiencing 0% to ~44% compressive strain (n = 16 microtubules from at least three independent experiments).

Extended Data Fig. 7 The amount of luminal ATAT-2-GFP on GMPCPP-stabilized MEC-12/MEC-7 microtubules decreases with compressive strains.

Heatmaps showing the initial binding and the decrease of GFP-tagged ATAT-2 over time on GMPCPP-stabilized MEC-12/MEC-7 microtubules experiencing 0% to ~44% compressive strain (n = 10 microtubules from at least three independent experiments).

Extended Data Fig. 8 The luminal accessibility of GDPstateTBA-2/TBB-2 microtubules increases with compressive strains.

Heatmaps showing the initial binding and the dissociation of GFP-tagged ATAT-2 over time on GDP-state TBA-2/TBB-2 microtubules experiencing 0% to ~44% compressive strain (n = 11 microtubules from at least three independent experiments).

Extended Data Fig. 9 Examining the lattice mechanical plasticity by bending microtubules proximal to the coverslip surface.

(a) Representative kymographs showing the rapid depolymerization of GDP-state MEC-12/MEC-7 microtubules after the GTP-bound MEC-17/MEC-7 tubulin was replaced with the buffer containing 20 µM GMPCPP-bound MEC-12/MEC-7 tubulin. Microtubule seeds and dynamic microtubule extensions were labeled with 3% and 14% rhodamine-labeled tubulin, respectively. Vertical scale bars: 5 µm; horizontal scale bars: 10 s. Similar results were observed in at least three independent experiments. (b and c) Schematic of the line scan analysis (b) and the corresponding intensity profiles (c) of TBA-2/TBB-2 microtubule cross-sections (green lines) before compression and at the maximum strain in the surface bending experiments. (d and e) Schematic of the line scan analysis (d) and the corresponding intensity profiles (e) of MEC-12/MEC-7 microtubule cross-sections (green lines) before compression and at the maximum strain in the surface bending experiments. (f and g) Representative intensity profiles cross-section line scans from two GMPCPP-stabilized TBA-2/TBB-2 (f) and MEC-12/MEC-7 (g) microtubules under compression in the surface bending experiments. The threshold of the background signal (red dashed line), the fluorescence intensity half-height of a microtubule (gray dashed line), and the peak width of microtubules before compression (blue double arrow) are indicated. (h) The percentage of splayed and non-splayed GMPCPP-stabilized MEC-12/MEC-7 and TBA-2/TBB-2 microtubule extensions while being bent on the coverslip. Data are from at least three independent experiments.

Extended Data Fig. 10 The sequence-structure analysis of the interacting interface between ATAT-2 and microtubules.

(a and b) Primary sequence alignment of MEC-12 and TBA-2 (a) and MEC-7 and TBB-2 (b). The residues of MEC-12 and MEC-7 that interact with ATAT-2 are highlighted in bold font. The ATAT-2 interacting residues divergent between these tubulin isotypes are italicized and underlined. (c) The ATAT-2 interacting residues are mapped onto the luminal surface of MEC-12/MEC-7 microtubules (PDB 8Y9F). The interacting residues that are identical and divergent between MEC-12/TBA-2 and MEC-7/TBB-2 are highlighted in pink and orange, respectively. The taxane-binding pockets and the α-tubulin H1-S2 loop (the K40 loop) are indicated.

Supplementary information

Reporting Summary (download PDF )

Supplementary Video 1 (download AVI )

A GMPCPP-stabilized TBA-2/TBB-2 microtubule undergoes cycles of compression and relaxation. Scale bar, 5 µm.

Supplementary Video 2 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 3 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 4 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 5 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 6 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 7 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 8 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 9 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation. The cross-sectional line scans of each MEC-12/MEC-7 microtubule at the maximum strain are shown in Extended Data Fig. 3. Scale bar, 5 µm.

Supplementary Video 10 (download AVI )

A GMPCPP-stabilized TBA-2/TBB-2 microtubule under tensile forces. Scale bar, 5 µm.

Supplementary Video 11 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule under tensile forces. Scale bar, 5 µm.

Supplementary Video 12 (download AVI )

A GMPCPP-stabilized TBA-2/TBB-2 microtubule undergoes cycles of compression and relaxation when displaced ~100 nm from the coverslip surface. Scale bar, 5 µm.

Supplementary Video 13 (download AVI )

A GMPCPP-stabilized MEC-12/MEC-7 microtubule undergoes cycles of compression and relaxation when displaced ~100 nm from the coverslip surface. Scale bar, 5 µm.

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Ye, Y., Hao, Z., Luo, J. et al. Tubulin isotypes of C. elegans harness the mechanosensitivity of the lattice for microtubule luminal accessibility. Nat. Phys. 21, 1420–1430 (2025). https://doi.org/10.1038/s41567-025-02983-w

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