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Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity

Nature Cell Biology volume 18, pages 540–548 (2016)Cite this article

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Abstract

Cell function depends on tissue rigidity, which cells probe by applying and transmitting forces to their extracellular matrix, and then transducing them into biochemical signals. Here we show that in response to matrix rigidity and density, force transmission and transduction are explained by the mechanical properties of the actin–talin–integrin–fibronectin clutch. We demonstrate that force transmission is regulated by a dynamic clutch mechanism, which unveils its fundamental biphasic force/rigidity relationship on talin depletion. Force transduction is triggered by talin unfolding above a stiffness threshold. Below this threshold, integrins unbind and release force before talin can unfold. Above the threshold, talin unfolds and binds to vinculin, leading to adhesion growth and YAP nuclear translocation. Matrix density, myosin contractility, integrin ligation and talin mechanical stability differently and nonlinearly regulate both force transmission and the transduction threshold. In all cases, coupling of talin unfolding dynamics to a theoretical clutch model quantitatively predicts cell response.

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Figure 1: Talin sets a rigidity threshold that triggers increased force transmission, adhesion maturation and YAP nuclear translocation.
Figure 2: Talin depletion affects β3 ligation, FAK phosphorylation and stress fibre formation only above the stiffness threshold.
Figure 3: The balance between clutch unbinding and talin unfolding predicts the force/rigidity curves and the rigidity threshold for mechanotransduction.
Figure 4: The rigidity threshold requires an intact integrin–cytoskeletal link mediated by full-length talin.
Figure 5: The rigidity threshold is mediated by talin unfolding under force and subsequent vinculin binding.
Figure 6: The elements of the molecular clutch tune force transmission and the rigidity threshold required for talin unfolding and YAP nuclear translocation.

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Acknowledgements

We acknowledge support from the Spanish Ministry for Economy and Competitiveness (BFU2011-23111, BFU2012-38146 and BFU2014-52586-REDT), a Career Integration Grant within the seventh European Community Framework Programme (PCIG10-GA-2011-303848), the European Research Council (Grant Agreements 242993 and 240487), the Generalitat de Catalunya, Fundació La Caixa, Fundació la Marató de TV3 (project 20133330), and the National Institutes of Health (US NIH R01AI044902). A.E.-A., R.O. and C.P.-G. were supported respectively by a Juan de la Cierva Fellowship (Spanish Ministry of Economy and Competitiveness), a FI fellowship (Generalitat de Catalunya), and the fundació ‘La Caixa’. We thank R. Sunyer, J. Alcaraz, E. Bazellières, F. Rico, S. Garcia-Manyes, N. Bate, N. Berrow and the members of the P.R.-C. and X.T. laboratories for technical assistance and discussions.

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Authors and Affiliations

  1. Institute for Bioengineering of Catalonia, Barcelona 08028, Spain

    Alberto Elosegui-Artola, Roger Oria, Anita Kosmalska, Carlos Pérez-González, Natalia Castro, Xavier Trepat & Pere Roca-Cusachs

  2. University of Barcelona, Barcelona 08028, Spain

    Roger Oria, Anita Kosmalska, Carlos Pérez-González, Xavier Trepat & Pere Roca-Cusachs

  3. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    Yunfeng Chen & Cheng Zhu

  4. Parker H. Petit Institute for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    Yunfeng Chen & Cheng Zhu

  5. Coulter Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA

    Cheng Zhu

  6. Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona 08010, Spain

    Xavier Trepat

  7. Centro de Investigación Biomédica en Red en Bioingeniería, Biomateriales y Nanomedicina, Madrid 28029, Spain

    Xavier Trepat

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  1. Alberto Elosegui-Artola
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Contributions

A.E.-A. and P.R.-C. conceived the study, A.E.-A., C.Z., X.T. and P.R.-C. designed the experiments, A.E.-A., R.O., Y.C., A.K., C.P.-G. and N.C. performed the experiments, P.R.-C. carried out the theoretical modelling, and A.E.-A. and P.R.-C. wrote the paper.

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Correspondence to Pere Roca-Cusachs.

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Integrated supplementary information

Supplementary Figure 4 Cell adhesion to fibronectin-coated gels is mediated by αvβ3 and α5β1 integrins.

(a) Images showing control and Talin 2 shRNA cells on 29 kPa fibronectin-coated polyacrylamide gels with or without blocking integrin α5β1 (using 10 μg ml−1 of BMB5 antibody), αvβ3 (using 0.5 mM of the specific Gpen peptide) or both. Scale bar is 50 μm. (b) Corresponding quantification of the percentage of spread cells (from left to right, n = 12, 12, 11, 11, 20, 11, 10, 12 fields of view)(∗∗∗, p ≤ 0.001, two-way Anova). Data show 1 out of 2 independent experiments. Blocking both integrins abolished cell adhesion almost completely.

Supplementary Figure 5 Talin1 Head L325R expression progressively reduces force transmission above but not below the rigidity threshold.

Traction forces exerted by control cells on 5 kPa gels (blue) and 29 kPa gels (red) as a function of the efficiency of transfection with Talin1 Head L325R. Values are compared to mean forces of untransfected control cells (left) and talin shRNA cells (right). Note that in Fig. 1, talin 1 Head L325R data represent averages for well transfected cells only. Dotted lines represent sigmoidal fits to the data. (Control: 5 kPa, n = 35 cells; 29 kPa, n = 12 cells. Control + Talin 1 Head: 5 kPa, n = 37 cells; 29 kPa, n = 42 cells. Talin 2 shRNA: 5 kPa, n = 34 cells; 29 kPa, n = 29 cells). Data show 1 out of 3 independent experiments.

Supplementary Figure 6 Further quantifications of integrin and pFAK in adhesions.

(a) quantification of integrin density from staining images of ligand bound β3 for Control cells (red, n = 20, 31, 24, 33, 20, 29 fields respectively for increasing rigidity measured in 8–10 cells) and Talin 2 shRNA cells (blue, n = 20, 21, 25, 29, 24, 29 fields measured in 8–9 cells). Data show 1 out of 3 independent experiments. Integrin densities were significantly different between control and depleted cells only above 5 kPa (P < 0.001, two-way Anova). (b) Quantification of the percentage of cell spreading area covered by pFAK-positive adhesions from staining images of Control cells (red. n = 11, 10, 17, 17, 17, 15 cells respectively for increasing rigidity) and Talin 2 shRNA cells (blue, n = 11, 10, 10, 12, 10, 10 cells) as a function of substrate stiffness. Data show 1 out of 3 independent experiments. Significant differences were observed only above 5 kPa (P = 0.039, two-way Anova).

Supplementary Figure 7 Dependence of cell area and myosin phosphorylation on substrate stiffness.

(a) Quantification of cell area in response to substrate stiffness for control and talin 2 shRNA cells (Control: n = 17, 12, 35, 42, 42, 12 cells respectively for increasing stiffness; Talin 2 shRNA: n = 10, 11, 34, 23, 25, 29 cells). Data show 1 out of 14 independent experiments. Talin depletion did not have a significant effect (two-way Anova). (b) For cells plated on gels of the indicated stiffness, representative western blots of talin, GAPDH as loading control, phosphorylated myosin light chain and total myosin light chain for Control and Talin 2 shRNA cells. (c) Corresponding quantification of the phosphorylated/total myosin light chain ratio (pooled from n = 3 independent experiments). No significant differences were found (two-way Anova). (d) Representative western blots of phosphorylated myosin light chain and total myosin light chain for wild-type MEF cells. (e) Corresponding quantification of the phosphorylated/total myosin light chain ratio (pooled from n = 3 independent experiments). No significant differences were found, suggesting that myosin phosphorylation is not significantly affected in MEF cells regardless of talin (one-way Anova).

Supplementary Figure 8 Further analyses on the effects of vinculin fragments.

(a) Average forces in response to substrate stiffness for cells transfected with Talin 2 shRNA + VD1 (red, n = 10, 13, 11, 11, 13, 10 cells, respectively for increasing stiffness) and Talin 2 shRNA + VD1 A501 (blue, n = 11, 11, 12, 11, 11, 10 cells). No significant differences were found between transfections (two-way Anova). Data show 1 out of 3 independent experiments. (b) Quantification of Nuclear/Cytosolic YAP ratio for the same conditions as in (a) (Talin 2 shRNA + VD1:n = 26, 20, 30, 29, 32, 32 cells respectively for increasing stiffness; Talin 2 shRNA + VD1 A501: n = 22, 21, 21, 23, 23, 24 cells). No significant differences were found between transfections (two-way Anova). Data show 1 out of 3 independent experiments. (c) Quantification of Nuclear/Cytosolic YAP ratio for control cells transfected with VD1 (red) and or VD1 A501 (blue) as a function of transfection efficiency (measured as the relative intensity of EGFP fluorescence) on 29 kPa polyacrylamide gels (Control + VD1:n = 59 cells; Control + VD1 A501: n = 49 cells). Data show 1 out of 3 independent experiments. Dashed lines are a sigmoidal fit to the experimental results for each condition. Further confirming the blocking role of VD1, increasing transfection efficiencies progressively decreased nuclear localization of YAP. In contrast, increasing efficiencies of transfection with VD1 A501 had no effect.

Supplementary Figure 9 Fibronectin coating densities.

Resulting fibronectin coating densities on the surface of polyacrylamide gels of 5 and 29 kPa coated with solutions containing 1, 10, or 100 μg ml−1 of fibronectin. n = 6 gels in all cases except 100 μg ml−1–29 kPa (5 gels). Data pooled from two independent experiments.

Supplementary Figure 10 Unprocessed versions of the western blots shown in Supplementary Fig. 4.

(a,b) blots corresponding to panel b in Supplementary Fig. 4. (c) blots corresponding to panel d in Supplementary Fig. 4. All measured bands corresponded to the molecular weights of the different proteins as detailed by antibody providers: talin (225–235 kDa), GADPH (36 kDa), and MLC (18 kDa). Note that blots do not show the entire molecular weight spectrum because membranes were cut before antibody incubation to incubate each band only with the relevant antibody.

Supplementary Table 1 Model parameters.
Full size table
Supplementary Table 2 Polyacrylamide gel rigidities measured with AFM.
Full size table
Supplementary Table 3 Statistical details of Fig. 6 panels.
Full size table

Supplementary information

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Supplementary Information (PDF 1057 kb)

41556_2016_BFncb3336_MOESM8_ESM.avi

Time-lapse of control cells transfected with lifeact-GFP and plated on fibronectin-coated substrates of increasing Young’s modulus (2-5-11-14-29 kPa from left to right). Scale bar is 20 μm. (AVI 1996 kb)

41556_2016_BFncb3336_MOESM9_ESM.avi

Time-lapse of talin 2 shRNA cells transfected with lifeact-GFP and plated on fibronectin-coated substrates of increasing Young’s modulus (2-5-11-14-29 kPa from left to right). Scale bar is 20 μm. (AVI 1733 kb)

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Elosegui-Artola, A., Oria, R., Chen, Y. et al. Mechanical regulation of a molecular clutch defines force transmission and transduction in response to matrix rigidity. Nat Cell Biol 18, 540–548 (2016). https://doi.org/10.1038/ncb3336

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