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Integrin nanoclusters can bridge thin matrix fibres to form cell–matrix adhesions

Abstract

Integrin-mediated cell–matrix adhesions are key to sensing the geometry and rigidity of extracellular environments and influence vital cellular processes. In vivo, the extracellular matrix is composed of fibrous arrays. To understand the fibre geometries that are required for adhesion formation, we patterned nanolines of various line widths and arrangements in single, crossing or paired arrays with the integrin-binding peptide Arg-Gly-Asp. Single thin lines (width ≤30 nm) did not support cell spreading or formation of focal adhesions, despite the presence of a high density of Arg-Gly-Asp, but wide lines (>40 nm) did. Using super-resolution microscopy, we observed stable, dense integrin clusters formed on parallel (within 110 nm) or crossing thin lines (mimicking a matrix mesh) similar to those on continuous substrates. These dense clusters bridged the line pairs by recruiting activated but unliganded integrins, as verified by integrin mutants unable to bind ligands that coclustered with ligand-bound integrins when present in an active extended conformation. Thus, in a fibrous extracellular matrix mesh, stable integrin nanoclusters bridge between thin (≤30 nm) matrix fibres and bring about downstream consequences of cell motility and growth.

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Fig. 1: Cell–matrix adhesions form across closely spaced nanofibre mimetic substrates and trigger cell spreading.
Fig. 2: The protein FAK is phosphorylated to a greater extent on 2D than on 1D patterns.
Fig. 3: Single-fibre mimetics do not support robust integrin cluster formation.
Fig. 4: Line pairs support more stable adhesion nanoclusters than single lines.
Fig. 5: Activation is sufficient and ligand binding is not necessary.
Fig. 6: Proposed model for assembly of adhesion nanoclusters.

Data availability

Data supporting the findings of this study are available within the article (and its Supplementary Information files), and from the corresponding author upon reasonable request.

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Acknowledgements

We thank G. Giannone, Neurosciences Bordeaux, France and the Michael W. Davidson group, The Florida State University, Tallahassee, FL, USA for DNA constructs. We thank H. Wolfenson for his help with initial experiments, P. Kathirvel for cloning the double-mutant β3 construct and M. Lee for help with illustrations. This work was supported by intramural funds from the Mechanobiology Institute. R.C. is supported by Singapore National Research Foundation’s CRP grant (No. NRF2012NRF-CRP001-084), and M.P.S. received National Institutes of Health (NIH) grant support related to this project (no. RO1-GM113022). S.J.W. and H.C. were supported by the National Science Foundation under award no. CMMI-1300590 and NIH Common Fund Nanomedicine program grant no. PN2 EY016586. The Columbia Nano Initiative provided cleanroom and processing facilities. This work was performed in part at the Center for Nanoscale Materials, a US Department of Energy Office of Science User Facility, and was supported by the US Department of Energy, Office of Science under contract no. DE-AC02-06CH11357.

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R.C. and M.P.S conceived and designed the experiments. H.C. and S.J.W. designed and prepared the AuPd and Ti nanopatterned substrates. R.C. and H.C. standardized the functionalization of AuPd substrates with RGD. R.C. standardized the functionalization of Ti substrates and performed the experiments, analysed the data and wrote the manuscript. R.C., M.P.S., S.J.W. and H.C., prepared the manuscript.

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Correspondence to Rishita Changede or Michael P. Sheetz.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–9, Supplementary Video Legends 1–3 and Supplementary Table 1

Reporting Summary

Supplementary Video 1

MEFs do not spread well on single-line 1D geometry

Supplementary Video 2

MEFs spread well and form large adhesions on line pair 2D geometry

Supplementary Video 3

Dynamics of b3GFP on single lines and line pairs

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Changede, R., Cai, H., Wind, S.J. et al. Integrin nanoclusters can bridge thin matrix fibres to form cell–matrix adhesions. Nat. Mater. 18, 1366–1375 (2019). https://doi.org/10.1038/s41563-019-0460-y

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