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. 2023 Sep 28;25(10):1453–1464. doi: 10.1038/s41556-023-01238-1

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© The Author(s) 2023

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 5 — a, On flat areas, actin stress fibres (F-actin labelled by LifeAct-RFP) are anchored to ITGβ5-marked focal adhesion patches. Scale: full-size, 10 µm; insets, 5 µm. b, On nanopillar areas, both ITGβ5 and F-actin accumulate at nanopillars. However, nanopillars with high β5 accumulations usually do not have high levels of F-actin. A previous study shows that the membrane curvature around gelatin-coated nanopillars induces actin accumulation, but the accumulation is highly dynamic with a lifetime of 1-2 minutes³¹. As curved adhesions are stable and do not form on gelatin-coated nanopillars, we hypothesize that curved adhesions involve a different population of F-actin. Scale: full-size, 10 µm; insets, 5 µm. c, Dynamic correlation between ITGβ5-GFP and LifeAct-RFP in cells cultured on vitronectin-coated nanopillars. There are two distinct populations of the F-actin that accumulate at nanopillars: a stable population of F-actin at high-β5 nanopillars, and a dynamic population F-actin at low-β5 nanopillars. The F-actin intensity of the dynamic population is usually brighter than that of the stable population. Scale: 10 µm. d, On gelatin-coated nanopillars that do not induce β5 accumulation, only the highly dynamic population of F-actin was observed at nanopillars. Scale: 10 µm. e, Quantifications of time-dependent fluctuation confirm two F-actin populations: a stable population on high-β5 nanopillars and a dynamic population at low-β5 nanopillars. Nanopillars with the top 25% and bottom 25% ITGβ5 intensities were grouped as high-β5 and low-β5 nanopillars, respectively. The dynamic population of F-actin on vitronectin-coated nanopillars is similar to the dynamic F-actin on gelatin-coated nanopillars. Therefore, curved adhesions involve a stable subpopulation of F-actin at nanopillars. n = 103/103/153 nanopillars, from two independent cells. Medians (lines) and quartiles (dotted lines) are shown. P values calculated using Kruskal-Wallis test with Dunn’s multiple-comparison. Source numerical data are available in source data.

Source data