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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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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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Authors

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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The authors declare no competing financial interests.

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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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