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Force loading explains spatial sensing of ligands by cells

Abstract

Cells can sense the density and distribution of extracellular matrix (ECM) molecules by means of individual integrin proteins and larger, integrin-containing adhesion complexes within the cell membrane. This spatial sensing drives cellular activity in a variety of normal and pathological contexts1,2. Previous studies of cells on rigid glass surfaces have shown that spatial sensing of ECM ligands takes place at the nanometre scale, with integrin clustering and subsequent formation of focal adhesions impaired when single integrin–ligand bonds are separated by more than a few tens of nanometres3,4,5,6. It has thus been suggested that a crosslinking ‘adaptor’ protein of this size might connect integrins to the actin cytoskeleton, acting as a molecular ruler that senses ligand spacing directly3,7,8,9. Here, we develop gels whose rigidity and nanometre-scale distribution of ECM ligands can be controlled and altered. We find that increasing the spacing between ligands promotes the growth of focal adhesions on low-rigidity substrates, but leads to adhesion collapse on more-rigid substrates. Furthermore, disordering the ligand distribution drastically increases adhesion growth, but reduces the rigidity threshold for adhesion collapse. The growth and collapse of focal adhesions are mirrored by, respectively, the nuclear or cytosolic localization of the transcriptional regulator protein YAP. We explain these findings not through direct sensing of ligand spacing, but by using an expanded computational molecular-clutch model10,11, in which individual integrin–ECM bonds—the molecular clutches—respond to force loading by recruiting extra integrins, up to a maximum value. This generates more clutches, redistributing the overall force among them, and reducing the force loading per clutch. At high rigidity and high ligand spacing, maximum recruitment is reached, preventing further force redistribution and leading to adhesion collapse. Measurements of cellular traction forces and actin flow speeds support our model. Our results provide a general framework for how cells sense spatial and physical information at the nanoscale, precisely tuning the range of conditions at which they form adhesions and activate transcriptional regulation.

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Figure 1: Increasing ligand spacing promotes the growth of focal-adhesion complexes on intermediate-rigidity substrates, and collapse on high rigidities.
Figure 2: A molecular-clutch model explains cellular response to ligand spacing.
Figure 3: Ligand disorder promotes adhesion growth, as predicted by the molecular-clutch model.
Figure 4: Myosin contractility regulates adhesion growth according to model predictions.

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References

  1. Lu, P., Weaver, V. M. & Werb, Z. The extracellular matrix: a dynamic niche in cancer progression. J. Cell Biol. 196, 395–406 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Daley, W. P., Peters, S. B. & Larsen, M. Extracellular matrix dynamics in development and regenerative medicine. J. Cell Sci. 121, 255–264 (2008)

    CAS  PubMed  Google Scholar 

  3. Arnold, M. et al. Activation of integrin function by nanopatterned adhesive interfaces. ChemPhysChem 5, 383–388 (2004)

    CAS  PubMed  Google Scholar 

  4. Cavalcanti-Adam, E. A. et al. Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly. Eur. J. Cell Biol. 85, 219–224 (2006)

    CAS  PubMed  Google Scholar 

  5. Altrock, E., Muth, C. A., Klein, G., Spatz, J. P. & Lee-Thedieck, C. The significance of integrin ligand nanopatterning on lipid raft clustering in hematopoietic stem cells. Biomaterials 33, 3107–3118 (2012)

    CAS  PubMed  Google Scholar 

  6. Amschler, K., Erpenbeck, L., Kruss, S. & Schon, M. P. Nanoscale integrin ligand patterns determine melanoma cell behavior. ACS Nano 8, 9113–9125 (2014)

    CAS  PubMed  Google Scholar 

  7. Cavalcanti-Adam, E. A. et al. Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands. Biophys. J. 92, 2964–2974 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Huang, J. et al. Impact of order and disorder in RGD nanopatterns on cell adhesion. Nano Lett. 9, 1111–1116 (2009)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  9. Schvartzman, M. et al. Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level. Nano Lett. 11, 1306–1312 (2011)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  10. Elosegui-Artola, A. 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)

    CAS  PubMed  Google Scholar 

  11. Elosegui-Artola, A. et al. Rigidity sensing and adaptation through regulation of integrin types. Nat. Mater. 13, 631–637 (2014)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  12. Plotnikov, S. V., Pasapera, A. M., Sabass, B. & Waterman, C. M. Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151, 1513–1527 (2012)

    CAS  PubMed  Google Scholar 

  13. Lohmüller, T. et al. Nanopatterning by block copolymer micelle nanolithography and bioinspired applications. Biointerphases 6, MR1–MR12 (2011)

    PubMed  Google Scholar 

  14. Ahmed, E. M. Hydrogel: preparation, characterization, and applications: a review. J. Adv. Res. 6, 105–121 (2015)

    CAS  PubMed  Google Scholar 

  15. Hersel, U., Dahmen, C. & Kessler, H. RGD modified polymers: biomaterials for stimulated cell adhesion and beyond. Biomaterials 24, 4385–4415 (2003)

    CAS  PubMed  Google Scholar 

  16. Lawson, C. et al. FAK promotes recruitment of talin to nascent adhesions to control cell motility. J. Cell Biol. 196, 223–232 (2012); erratum 196, 387 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Emsley, J., Knight, C. G., Farndale, R. W., Barnes, M. J. & Liddington, R. C. Structural basis of collagen recognition by integrin alpha2beta1. Cell 101, 47–56 (2000)

    CAS  PubMed  Google Scholar 

  18. Dupont, S. et al. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183 (2011)

    CAS  PubMed  Google Scholar 

  19. de Beer, A. G. et al. Force-induced destabilization of focal adhesions at defined integrin spacings on nanostructured surfaces. Phys. Rev. E 81, 051914 (2010)

    ADS  Google Scholar 

  20. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Wolfenson, H., Bershadsky, A., Henis, Y. I. & Geiger, B. Actomyosin-generated tension controls the molecular kinetics of focal adhesions. J. Cell Sci. 124, 1425–1432 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Bangasser, B. L. & Odde, D. J. Master equation-based analysis of a motor-clutch model for cell traction force. Cell. Mol. Bioeng. 6, 449–459 (2013)

    PubMed  Google Scholar 

  23. Bangasser, B. L., Rosenfeld, S. S. & Odde, D. J. Determinants of maximal force transmission in a motor-clutch model of cell traction in a compliant microenvironment. Biophys. J. 105, 581–592 (2013)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  24. Chan, C. E. & Odde, D. J. Traction dynamics of filopodia on compliant substrates. Science 322, 1687–1691 (2008)

    ADS  CAS  PubMed  Google Scholar 

  25. Peterson, L. J. et al. Simultaneous stretching and contraction of stress fibers in vivo. Mol. Biol. Cell 15, 3497–3508 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Humphries, J. D. et al. Vinculin controls focal adhesion formation by direct interactions with talin and actin. J. Cell Biol. 179, 1043–1057 (2007)

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Kanchanawong, P. et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Choi, C. K. et al. Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 10, 1039–1050 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Prager-Khoutorsky, M. et al. Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing. Nat. Cell Biol. 13, 1457–1465 (2011)

    CAS  PubMed  Google Scholar 

  30. Oakes, P. W., Beckham, Y., Stricker, J. & Gardel, M. L. Tension is required but not sufficient for focal adhesion maturation without a stress fiber template. J. Cell Biol. 196, 363–374 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Engler, A. et al. Substrate compliance versus ligand density in cell on gel responses. Biophys. J. 86, 617–628 (2004)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  32. Pallarola, D. et al. Interface immobilization chemistry of cRGD-based peptides regulates integrin mediated cell adhesion. Adv. Funct. Mater. 24, 943–956 (2014)

    CAS  PubMed  Google Scholar 

  33. Liu, Y. et al. Nanoparticle tension probes patterned at the nanoscale: impact of integrin clustering on force transmission. Nano Lett. 14, 5539–5546 (2014)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Allen, M. D. et al. Altered microenvironment promotes progression of preinvasive breast cancer: myoepithelial expression of αvβ6 integrin in DCIS identifies high-risk patients and predicts recurrence. Clin. Cancer Res. 20, 344–357 (2014)

    CAS  PubMed  Google Scholar 

  35. Roca-Cusachs, P. et al. Integrin-dependent force transmission to the extracellular matrix by alpha-actinin triggers adhesion maturation. Proc. Natl Acad. Sci. USA 110, E1361–E1370 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Butler, J. P., Tolic-Norrelykke, I. M., Fabry, B. & Fredberg, J. J. Traction fields, moments, and strain energy that cells exert on their surroundings. Am. J. Physiol. Cell Physiol. 282, C595–C605 (2002)

    CAS  PubMed  Google Scholar 

  37. Alcaraz, J. et al. Microrheology of human lung epithelial cells measured by atomic force microscopy. Biophys. J. 84, 2071–2079 (2003)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  38. Hutter, J. L. & Bechhoefer, J. Calibration of atomic-force microscope tips. Rev. Scient. Instr. 64, 1868–1873 (1993)

    ADS  CAS  Google Scholar 

  39. Sengupta, P., Jovanovic-Talisman, T. & Lippincott-Schwartz, J. Quantifying spatial organization in point-localization superresolution images using pair correlation analysis. Nat. Protocols 8, 345–354 (2013)

    CAS  PubMed  Google Scholar 

  40. Veatch, S. L. et al. Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting. PLoS One 7, e31457 (2012)

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kong, F., García, A. J., Mould, A. P., Humphries, M. J. & Zhu, C. Demonstration of catch bonds between an integrin and its ligand. J. Cell Biol. 185, 1275–1284 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Ghibaudo, M. et al. Traction forces and rigidity sensing regulate cell functions. Soft Matter 4, 1836–1843 (2008)

    ADS  CAS  Google Scholar 

  43. Molloy, J. E., Burns, J. E., Kendrick-Jones, J., Tregear, R. T. & White, D. C. Movement and force produced by a single myosin head. Nature 378, 209–212 (1995)

    ADS  CAS  PubMed  Google Scholar 

  44. Litvinov, R. I. et al. Resolving two-dimensional kinetics of the integrin alphaIIbbeta3-fibrinogen interactions using binding-unbinding correlation spectroscopy. J. Biol. Chem. 287, 35275–35285 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Roca-Cusachs, P., Iskratsch, T. & Sheetz, M. P. Finding the weakest link—exploring integrin-mediated mechanical molecular pathways. J. Cell Sci. 125, 3025–3038 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Spanish Ministry of Economy and Competitiveness (grants BFU2016-79916-P and BFU2014-52586-REDT to P.R.-C.; BFU2015-65074-P to X.T.; DPI2015-64221-C2-1-R to J.M.G.-A.; PI14/00280 to D.N.; SAF2016-75241-R (MINECO-FEDER) to L.A.), the European Commission (grant agreement SEP-210342844 to X.T. and P.R.-C.), the Generalitat de Catalunya (grant 2014-SGR-927), the European Research Council (CoG-616480 to X.T. and StG 306571 to J.M.G.-A.), Obra Social ‘La Caixa’, Fundació la Marató de TV3 (project 20133330 to P.R.-C.), the German Science Foundation (DFG SFB1129 P15 to E.A.C.-A.), and the EMBO Young Investigator Programme. A.E.-A., R.O., and L.A. were supported respectively by a Juan de la Cierva Fellowship (Spanish Ministry of Economy and Competitiveness, fellowship number IJCI-2014-19156), an FI fellowship (Generalitat de Catalunya), and a Ramon y Cajal Fellowship (Spanish Ministry of Economy and Competitiveness). The support of the Max Planck Society and the Alexander von Humboldt foundation (to I.P.) is acknowledged. We thank P. Oakes, J. Spatz, J. L. Jones, M. D. Allen and the members of the P.R.-C. and X.T. laboratories for technical assistance and discussions.

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Contributions

R.O. and P.R.-C. conceived the study; R.O., L.A., D.N., X.T., E.A.C.-A. and P.R.-C. designed the experiments; R.O., T.W., A.E.-A., J.J.U., I.P. and P.D. performed the experiments; J.E., C.M.-P., J.M.G.-A. and P.R.-C. carried out the theoretical modelling; and R.O. and P.R-C. wrote the manuscript. All authors commented on the manuscript and contributed to it.

Corresponding authors

Correspondence to Elisabetta Ada Cavalcanti-Adam or Pere Roca-Cusachs.

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Reviewer Information Nature thanks M. Mrksich and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Nanopattern swelling on gels.

a, Scanning electron micrograph of a quasi-hexagonal 100-nm ordered pattern on a glass surface (from one of two independent experiments). b, Scanning electron micrograph of a quasi-hexagonal 100-nm ordered pattern on a polyacrylamide gel. c, Corresponding histograms showing the distribution of distances between nanodots and their first-order neighbours on glass and polyacrylamide substrates of rigidity 30 kPa (300 particles; two independent experiments). d, Corresponding quantification of mean distance between nanodots on polyacrylamide gels as a function of rigidity (n = 300 particles per condition; two independent experiments). Scale bar, 200 nm.

Extended Data Figure 2 Cell binding to nanopatterned substrates is specific to α5β1 integrins, cRGD, and nanodots.

a, Images showing breast myoepithelial cells plated on 30 kPa substrates with ligand spacing of 50 nm, under conditions that either allow integrin-mediated cell binding (cRGD + nanodots; top left) or do not (the remaining three images). RGE is a peptide with low affinity for α5β1 integrin; abα5β1 is an antibody that blocks α5β1 integrin. b, Corresponding quantification of the percentage spread of cells (n = 30/30/30/22 fields of view; three independent experiments). Scale bar, 100 μm; ***, P < 0.001. Error bars, mean ± s.e.m.

Extended Data Figure 3 Further characterization of cell response to rigidity and nanodot spacing.

a, Quantification of the fluorescence intensity of staining for phosphorylated paxillin (phospho-paxillin) at the cell edge (two different regions per cell; n = 10/10/11, 10/10/11, 10/11/11, 10/10/11, 10/10/11, 10/10/11, 10/10/11, 10/10/11 cells on 50/100/200-nm-spaced substrates and increasing rigidity; two independent experiments). The effect of both ligand spacing and rigidity was significant (P < 0.05; two-way ANOVA). Rather than measuring focal adhesions, this complementary measurement integrates phospho-paxillin recruitment in both adhesions and surrounding areas. The same trends were observed in Fig. 1e. b, Cell-spreading area (n = 13/13/11, 11/16/11, 11/19/11, 16/13/11, 13/16/11, 13/14/11, 13/13/11 cells on 50/100/200-nm-spaced substrates as rigidity increases; two independent experiments). Although nanodot spacing did affect cell spreading, we note that, on the 50-nm- and 100-nm-spaced substrates, the rigidities inducing adhesion formation and collapse are not associated with changes in cell spreading. c, Examples of cells transfected with GFP−paxillin, seeded on 30 kPa and 150 kPa substrates, with nanodots spaced 50 nm and 100 nm apart. The right-hand images of each pair correspond to rectangles marked in red in the left-hand image. d, Corresponding quantification of focal-adhesion length (ten adhesions per cell; n = 11/11, 10/10 cells for 50/100-nm-spaced substrates as rigidity increases; two independent experiments) **, P < 0.005; ***, P < 0.001, two-way ANOVA. Scale bar, 20 μm. Error bars, mean ± s.e.m.

Extended Data Figure 4 Effect of rigidity and nanodot spacing on different cell types and ligands.

a, Staining of phospho-paxillin-containing adhesions in myoepithelial cells, seeded on polyacrylamide substrates of different rigidities or on glass, with either 50-nm- or 100-nm-spaced nanodots coated with the collagen-mimicking GFOGER peptide. Right-hand images are magnifications of the red rectangular regions in the left-hand images. b, Corresponding quantification of focal-adhesion length (mean of at least three adhesions per cell for n = 15 cells per condition; two independent experiments). ch, As for panels a and b, but for different cell types (HUVECs, MEFs or MCF 10As) seeded on cRGD-coated nanodots. At least three focal adhesions were analysed per cell. For 50/100-nm-spaced substrates and increasing rigidity, n = 16/16, 16/15, 15/16, 15/15 cells (HUVECs), n = 15/15, 16/15, 15/15, 14/14 cells (MEFs), and n = 15/15, 15/15, 15/15, 10/10 cells (MCF 10As); two independent experiments. Scale bars, 20 μm. Error bars, mean ± s.e.m.

Extended Data Figure 5 Adhesion collapse is not associated with changes in nanodot spacing between paxillin clusters.

a, STORM super-resolution images of phospho-paxillin stainings in cells seeded on 100-nm-spaced patterns on 30 kPa or 150 kPa gels. Left, overview images of different focal adhesions; scale bar, 5 μm. Right, magnified images; scale bar, 300 nm. White circles show examples of phospho-paxillin clusters. Two independent experiments. b, Pair-correlation functions (g(r)) of phospho-paxillin clusters as a function of distance in different adhesions (marked with different colours). In all cases, a first peak is observed at around 100 nm, indicating the periodicity of the cluster pattern. c, Histogram showing the distances between neighbouring phospho-paxillin clusters (n = 409 and 197 clusters for 30 kPa and 150 kPa substrates respectively; two independent experiments). No significant differences were observed.

Extended Data Figure 6 Spatial distribution of ordered and disordered nanopatterns.

a, b, Scanning electron micrographs of ordered and disordered nanopatterns on glass for average nanodot spacings of 50 nm (a) and 100 nm (b). Two independent experiments. c, d, Histograms showing the distribution of interparticle distances for ordered and disordered patterns with spacings of 50 nm (c) and 100 nm (d) (n = 300 particles for all the conditions measured in two independent experiments). Scale bar, 100 nm.

Extended Data Table 1 Model parameters
Extended Data Table 2 Preparation details on micellar nanolithography
Extended Data Table 3 Polyacrylamide gel rigidities measured by atomic force microscopy

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Oria, R., Wiegand, T., Escribano, J. et al. Force loading explains spatial sensing of ligands by cells. Nature 552, 219–224 (2017). https://doi.org/10.1038/nature24662

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