\u03b1V-class integrins exert dual roles on \u03b15\u03b21 integrins to strengthen adhesion to fibronectin

Mitasha Bharadwaj,Niko Beerenwinkel,Jonne Helenius,Georgina P Colo,Reinhard Fässler,Nico Strohmeyer,Daniel J Müller,Herbert B Schiller

doi:10.1038/ncomms14348

Abstract

Upon binding to the extracellular matrix protein, fibronectin, αV-class and α5β1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Both integrin classes have been functionally specified, however their specific roles in immediate phases of cell attachment remain uncharacterized. Here, we quantify the adhesion of αV-class and/or α5β1 integrins expressing fibroblasts initiating attachment to fibronectin (≤120 s) by single-cell force spectroscopy. Our data reveals that αV-class integrins outcompete α5β1 integrins. Once engaged, αV-class integrins signal to α5β1 integrins to establish additional adhesion sites to fibronectin, away from those formed by αV-class integrins. This crosstalk, which strengthens cell adhesion, induces α5β1 integrin clustering by RhoA/ROCK/myosin-II and Arp2/3-mediated signalling, whereas overall cell adhesion depends on formins. The dual role of both fibronectin-binding integrin classes commencing with an initial competition followed by a cooperative crosstalk appears to be a basic cellular mechanism in assembling focal adhesions to the extracellular matrix.

Highlights

Upon binding to the extracellular matrix protein, fibronectin, aV-class and a5b1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion
To determine how a5b1 and aV-class integrins contribute to the initiation of cell adhesion, we quantified the adhesion forces of a5b1 and/or aV-class integrin-expressing mouse kidney fibroblasts to FN by single-cell force spectroscopy (SCFS) (Fig. 1a)
For SCFS, a single fibroblast was attached to concanavalin A (ConA)-functionalized atomic force microscopy (AFM) cantilever and incubated for 7–10 min to ensure firm adhesion of the fibroblast to the cantilever

Summary

Introduction

Upon binding to the extracellular matrix protein, fibronectin, aV-class and a5b1 integrins trigger the recruitment of large protein assemblies and strengthen cell adhesion. Cell adhesion mediated by FN-binding integrins leads to the formation of nascent adhesions that eventually mature into large focal adhesions and convert into central or fibrillar adhesions[5,6] While both integrin classes bind the tripeptide sequence Arg-Gly-Asp (RGD) in the 10th type III module of FN (FNIII10)[7,8], a5b1 integrins require the Pro-His-Ser-Arg-Asn (PHSRN) synergy site in the FNIII9 module, which is in close proximity to the RGD motif, to establish cell adhesion[9]. It is not clear, whether a5b1 and aV-class integrins function individually and/or cooperate with each other during the first few seconds and minutes of adhesion initiation. Specific perturbation experiments identified signalling pathways involved in the early crosstalk between both FN-binding integrin classes

Methods

Results

Conclusion