Distinct biophysical mechanisms of focal adhesion kinase mechanoactivation by different extracellular matrix proteins

Abstract

Matrix mechanics controls cell fate by modulating the bonds between integrins and extracellular matrix (ECM) proteins. However, it remains unclear how fibronectin (FN), type 1 collagen, and their receptor integrin subtypes distinctly control force transmission to regulate focal adhesion kinase (FAK) activity, a crucial molecular signal governing cell adhesion/migration. Here we showed, using a genetically encoded FAK biosensor based on fluorescence resonance energy transfer, that FN-mediated FAK activation is dependent on the mechanical tension, which may expose its otherwise hidden FN synergy site to integrin α5. In sharp contrast, the ligation between the constitutively exposed binding motif of type 1 collagen and its receptor integrin α2 was surprisingly tension-independent to induce sufficient FAK activation. Although integrin α subunit determines mechanosensitivity, the ligation between α subunit and the ECM proteins converges at the integrin β1 activation to induce FAK activation. We further discovered that the interaction of the N-terminal protein 4.1/ezrin/redixin/moesin basic patch with phosphatidylinositol 4,5-biphosphate is crucial during cell adhesion to maintain the FAK activation from the inhibitory effect of nearby protein 4.1/ezrin/redixin/moesin acidic sites. Therefore, different ECM proteins either can transmit or can shield from mechanical forces to regulate cellular functions, with the accessibility of ECM binding motifs by their specific integrin α subunits determining the biophysical mechanisms of FAK activation during mechanotransduction.FRET biosensor | intracellular tension | substrate rigidity C ells can sense and respond to the mechanical microenvironment by converting forces into biochemical signals inside the cells; that is, mechanotransduction (1). Focal adhesions (FAs) are the major sites of interaction between a cell and its extracellular matrix (ECM) microenvironment, and thus, outside mechanical signals can be sensed at FAs through transmembrane receptor integrins. In particular, it has been shown that matrix elasticity can control the cell fate (2) by modulating the interactions between ECM proteins and their receptor integrins (3, 4). The modifications in ECM-integrin bonds can be further translated into biochemical signals through FA proteins (5). For example, mechanical stretching of Crk-associated substrate (p130 CAS ) promotes its phosphorylation by Src family kinases (6). Unfolding purified talin rod domain by mechanical stretching allows the recruitment of vinculin, potentially reinforcing the connection between integrin and actin (7). Myosin II-dependent endogenous tension has also been shown to directly unfold vinculin in living cells (8). However, it remains unclear how focal adhesion kinase (FAK), a major downstream molecule of integrin signaling, is regulated by mechanical signals.Various ECM proteins including fibronectin (FN) and type 1 collagen (Col I) are specifically recognized by integrin subtypes with different combinations of α and β subunits, allowing diver...

The integrin–collagen connection – a glue for tissue repair?

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E-cadherin-mediated force transduction signals regulate global cell mechanics

“…Dynamic changes in Src kinase activity at cadherin adhesions were visualized with a membrane-targeted FRET-based KRas-Src reporter (Yingxiao Wang, UCSD, San Diego CA) (Seong et al, 2013;Wang et al, 2005). Transiently transfected MCF7 cells were cultured on collagen-I-coated glass-bottomed dishes (Glass #1.5, Cell E&G, Houston TX) in Phenol-Red-free DMEM supplemented with 0.5% FBS, 1 mM sodium pyruvate, and 1% (v/v) penicillin-streptomycin (Corning Cell Grow, Manassas, VA), for 6 h prior to the measurements, in order to reduce serum-induced Src kinase activity Wang et al, 2005).…”

Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization

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