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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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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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.
The authors declare no competing interests.
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Supplementary Figs. 1–9, Supplementary Video Legends 1–3 and Supplementary Table 1
MEFs do not spread well on single-line 1D geometry
MEFs spread well and form large adhesions on line pair 2D geometry
Dynamics of b3GFP on single lines and line pairs