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Microtubules tune mechanosensitive cell responses

Abstract

Mechanotransduction is a process by which cells sense the mechanical properties of their surrounding environment and adapt accordingly to perform cellular functions such as adhesion, migration and differentiation. Integrin-mediated focal adhesions are major sites of mechanotransduction and their connection with the actomyosin network is crucial for mechanosensing as well as for the generation and transmission of forces onto the substrate. Despite having emerged as major regulators of cell adhesion and migration, the contribution of microtubules to mechanotransduction still remains elusive. Here, we show that talin- and actomyosin-dependent mechanosensing of substrate rigidity controls microtubule acetylation (a tubulin post-translational modification) by promoting the recruitment of α-tubulin acetyltransferase 1 (αTAT1) to focal adhesions. Microtubule acetylation tunes the mechanosensitivity of focal adhesions and Yes-associated protein (YAP) translocation. Microtubule acetylation, in turn, promotes the release of the guanine nucleotide exchange factor GEF-H1 from microtubules to activate RhoA, actomyosin contractility and traction forces. Our results reveal a fundamental crosstalk between microtubules and actin in mechanotransduction that contributes to mechanosensitive cell adhesion and migration.

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Fig. 1: Integrin-mediated signalling and substrate rigidity regulate microtubule acetylation.
Fig. 2: Microtubule acetylation tunes cell mechanosensitivity.
Fig. 3: Microtubule acetylation reorganizes the actomyosin and intermediate filament networks.
Fig. 4: Microtubule acetylation promotes traction force generation and RhoA activation.
Fig. 5: Microtubule acetylation promotes the release of GEF-H1 into the cytosol.
Fig. 6: Microtubule acetylation is required for mechanosensitive migration.

Data availability

The data supporting the findings of this study are available within the manuscript. The mass spectrometry proteomics data have been deposited with the ProteomeXchange Consortium via the PRIDE (refs. 52,53) partner repository with the dataset identifier PXD015871. Source data are provided with this paper. Other raw data generated during this study are available on reasonable request.

Code availability

Codes for focal adhesion distribution and traction forces are available from the authors upon request.

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Acknowledgements

This work was supported by the La Ligue contre le cancer (S-CR17017), the Centre National de la Recherche Scientifique and Institut Pasteur. S.S. is funded by an ITN PolarNet Marie Curie grant and the Fondation pour la Recherche Médicale (FDT201904007930) and was enrolled at the Ecole Doctorale FIRE Frontières de l’Innovation en Recherche et Éducation (Frontières du Vivant FdV – Programme Bettencourt), CRI and the Université de Paris. We thank A. Akhmanova and Y.-C. Ammon for providing the GFP-talin construct, C. Leduc for the FA distribution macro and discussions, B. Bance, F. Coumailleau and O. Destaing for discussions, N. M. Rafiq for fixation protocols and discussions, and E. van Bodegraven, F. Peglion and S. Sivaranjani for critical reading of the manuscript and discussions.

Author information

Authors and Affiliations

Authors

Contributions

S.S. designed and performed the experiments, analysed and interpreted the results, and wrote the paper; B.V. assisted in the set-up and analysis of TFM experiments, and helped with data interpretation and discussions; V.R. performed immunoprecipitation and pull-down experiments; A.J.F. carried out a part of the experiments using HUVECs; C.D.P. helped with experimental techniques and discussions; B.B. optimized the GFP nanobody and IP conditions used for mass spectrometry sample preparation and helped set up the chemical wound assay; F.D. carried out the MS experimental work; D.L. supervised the MS experiments and data analysis; S.V. performed the PREM experiments; A.B. helped with data interpretation and discussions; M.T. provided ideas and assisted data interpretation and discussions; S.E.-M. supervised the project, interpreted the results and wrote the paper.

Corresponding author

Correspondence to Sandrine Etienne-Manneville.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Materials thanks Martin Humphries, Guillaume Montagnac and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Substrate rigidity regulates αTAT1- dependent microtubule acetylation.

a, b, (a. i) Schematic and (a. ii, b. i) western blots showing (a) acetylation and (b) αTAT1 levels using two distinct sets of siRNAs targeting αTAT1 (siαTAT1-1 and siαTAT1-2), and Tubacin (an inhibitor of HDAC6 (deacetylase)); (a. iii, a. iv) ratio of the acetylated tubulin or (b. ii) αTAT1 intensity over GAPDH intensity, normalized to the values observed for the respective controls. c, Migrating astrocytes transfected with siCtl, (c. i) siαTAT1-1 and (c. ii) siαTAT1-2, or treated with Tubacin prior to wounding, showing acetylated tubulin and α-tubulin. d, Astrocytes plated on PAA gels of different rigidities and treated with Tubacin; ratio of the intensities of Acetylated tubulin over total Tubulin of each cell; n = 49 for 1.26 kPa WT, 51 for 1.26 kPa Tubacin, 72 for 48 kPa WT, 66 for 48 kPa Tubacin. e, siCtl or WT astrocytes plated on crossbow-shaped micropatterned hydrogels of different rigidities, (e. i) showing Acetylated tubulin and α-tubulin; (e. ii) ratio of the intensities of Acetylated tubulin over total Tubulin of each cell; n = 86 for 2 kPa and 103 for 48 kPa. f, Astrocytes plated on PAA gels of different rigidities, (f. i) stained with Detyrosinated tubulin and α-tubulin; (f. ii) ratio of Detyrosinated tubulin over total tubulin intensities in each cell; n = 39 for 2 kPa, 31 for 9 kPa and 40 for 48 kPa. g, (g. i) Western blots showing the levels of Detyrosinated tubulin and GAPDH in astrocytes plated on PAA gels of different substrate rigidities; (g. ii) ratio of the intensities of Detyrosinated tubulin over GAPDH normalized to the values observed for 2 kPa. Scale bar (c, e, f): 10 μm; Number of independent experiments = 4 (for a - siαTAT1), 6 (for a - Tubacin, c, g), 3 (for b), 5 (for e), 2 (for f). In box-and-whisker plots, box extends from the 25th to 75th percentile, whiskers show minimum and maximum values, and the line within the box represents the median; Statistical tests: one-way ANOVA followed by Tukey’s multiple comparison’s test or Holm-Šídák’s multiple comparisons test (a - siαTAT1, b, d, e, g) or Paired Student’s t-test (for a - Tubacin).

Source data

Extended Data Fig. 2 Integrin signaling controls microtubule acetylation and αTAT1 localization.

a, b, Astrocytes were treated with (a) RGD control or RGD peptides, (b) solvant (Ctl) or MnCl2, and (a. i, b. i) stained for acetylated tubulin, α-tubulin, paxillin or DAPI; (a. ii, b. ii) ratio of the intensities of Acetylated tubulin over total Tubulin; n = 146 for RGD control, 99 for RGD peptide, 116 for Ctl and 105 for MnCl2. c, d, Astrocytes were transfected with siCtl or siβ1 integrin; (c. i) Western blots showing levels of β1 integrin, Acetylated tubulin and GAPDH; (c. ii, iii) ratio of intensities of β1 integrin or Acetylated tubulin over GAPDH, normalized to the levels in siCtl respectively; (d. i) stained for Acetylated tubulin, α-tubulin and DAPI; (d. ii) ratio of the intensities of Acetylated tubulin over total Tubulin; n = 122 for siCtl and 117 for siβ1 integrin. e, Astrocytes were treated with Ctl, Src kin 1 or PF-562271; (e. i) stained for Acetylated tubulin, α-tubulin and Paxillin; (e. ii) ratio of the intensities of Acetylated tubulin over total Tubulin intensity; n = 78 for Ctl, 70 for Src kin 1, 79 for PF-562271. f, Astrocytes were plated on 48 kPa gels and treated with Ctl or Src kin 1. (f. i) Western blots showing the levels of Acetylated tubulin and GAPDH; (f. ii) ratio of the intensities of Acetylated tubulin over GAPDH normalized to the Ctl. g, Volcano plot analysis showing fold changes (GFP-αTAT1/GFP-Ctl) of the quantified proteins with threshold of >3 peptides, minimum absolute fold change of 1.5 (green lines) and maximum adjusted p-value of 0.05 (red line). Enriched protein interactors related to GO:0005925 focal adhesion (red boxes; ratio = 1.98 and p = 7.89 ×10−5). External plots show proteins with peptides identified only in one sample type (left in GFP-Ctl and right in GFP-αTAT1). h, i, Normalized Talin interaction in (h) GST-pulldown and (i) GFP-immunoprecipitations. j, Western blots (j. i) showing the levels of Talin and GAPDH in astrocytes transfected with siCtl and siTalin; (j. ii) ratio of the intensities of Talin over GAPDH normalized to the Ctl. k, l, Astrocytes showing (k) GFP-αTAT1 localization by epifluorescence, on microtubules, (l) by TIRF, at FAs, in cells treated with or without nocodazole. Scale bar (a, b, d, e, k, l): 10 μm; Number of independent experiments = 3 (for ad, hj), 2 (for e), 4 (for f, g), 5 (for l). In box-and-whisker plots, the box extends from the 25th to the 75th percentile, whiskers show the minimum and maximum values, and the line within the box represents the median; Statistical tests: Student’s t-test (for aj).

Source data

Extended Data Fig. 3 Microtubule acetylation controls focal adhesion distribution.

a, HUVECs plated on different substrate rigidities and (a. i) stained for Acetylated tubulin (insets shown) and α-Tubulin; (a. ii) ratio of the intensities of Acetylated tubulin over total Tubulin of each cell; n = 46 for 2 kPa WT and 70 for 48 kPa. b, HUVECs treated with Niltubacin (Ctl) or Tubacin, and stained for Acetylated tubulin (insets shown), α-tubulin and Paxillin. c, Western blots showing (c. i) Acetylated and α-tubulin levels in HUVECs treated with DMSO (Ctl) and Tubacin; (c. ii) ratio of intensities of Acetylated tubulin over α-tubulin. d, Schematic representation of the different cell regions used to quantify FA density (Fig. 2d–f). e, f, Graphs show FA density (number of FAs/μm2) in different regions of astrocytes transfected with siCtl or siαTAT1-2, or treated with Niltubacin or Tubacin, and plated on 1.26 kPa or 48 kPa substrates. g, h, Graphs show FA density (number of FAs/μm2) in different regions of astrocytes transfected with siCtl or siαTAT1-1, and plated on 1.26 kPa or 48 kPa substrates. i. siCtl or siαTAT1-2-transfected astrocytes plated on crossbow-shaped micropatterned polyacrylamide gels of 40 kPa or 2 kPa, (i. i) stained with Paxillin and α-tubulin; (i. ii) FA density (number of FAs/μm2) within 8-16 μm layer of the cell, or (i. iii) in different regions of astrocytes transfected with siCtl or siαTAT1-2, and plated on 2 kPa or 40 kPa substrates. Scale bar (a, b, i): 10 μm. Number of independent experiments = 2 (for a), 3 (for b, ei). In box-and-whisker plots, the box extends from the 25th to the 75th percentile, the whiskers show the minimum and maximum values, and the line within the box represents the median. Statistical tests: Paired Student’s t-test (for c), one-way ANOVA followed by Tukey’s multiple comparisons test (for ei).

Source data

Extended Data Fig. 4 Microtubule acetylation promotes acto-myosin contractility.

a, Astrocytes transfected with siCtl or siαTAT1-1, and (a. i) stained for Actin, Paxillin and Myosin IIa; (a. ii) percentage of siCtl or siαTAT1-transfected astrocytes with transverse interjunctional actin arcs; n = 191 for siCtl and 125 for siαTAT1-1; Scale bar: 10 μm. Number of independent experiments = 3; Statistical test: Paired Student’s t-test (two-tailed).

Extended Data Fig. 5 Microtubule acetylation controls GEF-H1 association with microtubules.

a, Migrating astrocytes transfected with siCtl, siαTAT1-1 and siαTAT1-1 treated with Tubacin, (a. i) stained for Acetylated tubulin, α-tubulin and GEF-H1; (a. ii) percentage of GEF-H1 colocalized with microtubules; n = 119 for siCtl, 131 for siαTAT1-1, 103 for siαTAT1-1 + Tubacin. b, Astrocytes transfected with siCtl and siαTAT1-2, and plated on 2 kPa PAA gels, stained for GEF-H1 and α-tubulin. c, Ultrastructural organization of a focal adhesion at the leading edge of a control astrocyte. Platinum replica electron microscopy (PREM) high magnification view of a focal adhesion on the cytoplasmic surface of the leading edge in siCtl unroofed astrocytes. Microtubules are both colored in purple and indicated by white arrowheads. d, Traction forces in astrocytes plated on micropatterned polyacrylamide gels of different rigidities; traction forces for cells on different rigidities; Values for 2 kPa and 48 kPa are from experiments shown in Fig. 4a, b, pooled along with the values for 50 kPa, 91.8 kPa and 121 kPa; n = 136 for 2 kPa Niltubacin, 163 for 48 kPa siCtl, 31 for 50 kPa WT, 33 for 91.8 kPa WT and 30 for 121 kPa WT. In box-and-whisker plots, the box extends from the 25th to the 75th percentile, the whiskers show the minimum and maximum values, and the line within the box represents the median. Scale bar (a, b): 10 μm, (c): 200 nm; Number of independent experiments = 3 (for a, b, d - 2 kPa Niltubacin and 48 kPa siCtl), 2 (for d - 50 kPa, 91.8 kPa and 121 kPa). Statistical tests: One-way ANOVA followed by Tukey’s multiple comparison’s test (for a).

Supplementary information

Reporting Summary

Supplementary Video 1

GFP-αTAT1 localization at FAs upon nocodazole treatment. Cells were transfected with GFP-αTAT1 and mCherry-vinculin. First, images were acquired every 2 min for 15 min. Then, 1 μM nocodazole was added and images were acquired every 2 min for 1 h. Acquisitions were performed with a Nikon Eclipse Ti-E epifluorescence inverted microscope equipped with a pco.edge sCMOS camera, Metamorph software and using a ×60 1.49 NA oil objective. Cells were maintained under 5% CO2 at 37 °C in normal astrocyte medium during acquisition. Scale bar, 20 μm.

Supplementary Video 2

GFP-αTAT1 localization at FAs upon Y-27632 treatment. Cells were transfected with GFP-αTAT1 and mCherry-vinculin. First, images were acquired every 2 min for 15 min. Then, 10 μM Y-27632 was added and images were acquired every 2 min for 1 h. Acquisitions were performed with a Nikon Eclipse Ti-E epifluorescence inverted microscope equipped with a pco.edge sCMOS camera, Metamorph software and using a ×60 1.49 NA oil objective. Cells were maintained under 5% CO2 at 37 °C in normal astrocyte medium during acquisition. Scale bar, 20 μm.

Supplementary Video 3

Chemical wound set-up. Cells were plated on polyacrylamide hydrogels of different rigidities and grown into a monolayer for 1–2 days. A chemical wound was induced using a microinjector needle, with 0.05 M NaOH. Once the chemical had been microdropped onto the surface of the monolayer, a circular wound was created. Dead cells and debris were washed out and cells were allowed to migrate for 12 h. The video was acquired using a ×10 objective on a Leica DMI 6000B microscope equipped with the Leica software. Cells were maintained under 5% CO2 at 37 °C in normal astrocyte medium during acquisition.

Supplementary Video 4

Astrocytes migrate faster on stiff substrates. Wild-type cells were plated on polyacrylamide hydrogels of different rigidities and grown into a monolayer for 1–2 days. After creating a chemical wound, cells were allowed to migrate towards the wound. Images were acquired every 15 min for 12 h using a Zeiss Axiovert 200M with a dry objective of ×10 0.45 NA and a pco.edge sCMOS camera. Cells were maintained under 5% CO2 at 37 °C in normal astrocyte medium during acquisition. Scale bar, 25 μm.

Supplementary Video 5

αTAT1 regulates mechanosensitive cell migration. Cells transfected with siCtl or siαTAT1 were plated on polyacrylamide hydrogels of different rigidities and grown into a monolayer for 2–3 days. After creating a chemical wound, cells were allowed to migrate towards the wound. Images were acquired every 15 min for 12 h using a Zeiss Axiovert 200M with a dry objective ×10 0.45 NA and a pco.edge sCMOS camera. Cells were maintained under 5% CO2 at 37 °C in normal astrocyte medium during acquisition. Scale bar, 25 μm.

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Unprocessed western blots of Fig. 4.

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Unprocessed western blots of Fig. 5.

Source Data Extended Data Fig. 1

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Source Data Extended Data Fig. 2

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Source Data Extended Data Fig. 3

Unprocessed western blots of Extended Data Fig. 3.

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Seetharaman, S., Vianay, B., Roca, V. et al. Microtubules tune mechanosensitive cell responses. Nat. Mater. 21, 366–377 (2022). https://doi.org/10.1038/s41563-021-01108-x

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