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. 2013 Nov 26;110(48):19372-7.
doi: 10.1073/pnas.1307405110. Epub 2013 Nov 12.

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

Jihye Seong  1 , Arash Tajik, Jie Sun, Jun-Lin Guan, Martin J Humphries, Susan E Craig, Asha Shekaran, Andrés J García, Shaoying Lu, Michael Z Lin, Ning Wang, Yingxiao Wang
Affiliations

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

Jihye Seong et al. Proc Natl Acad Sci U S A. .

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.

Keywords: FRET biosensor; intracellular tension; substrate rigidity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
FAK activation is dependent on substrate rigidity coupled with FN, but not Col I. (A and B) The representative images (A) and the average ECFP/YPet ratio values (B) of FAK biosensor in HT1080 cells cultured on the FN- or Col I-coated PA gels with different stiffness, as indicated (n = 18–31). The color bar on the left in A shows the ECFP/YPet ratio values, with cold and hot colors representing low and high ratio values, as indicated. FN and Col I group are displayed by black and white circles, respectively, in B. (C) The traction in HT1080 cells on the FN- or Col I-coated PA gels of 0.6 kPa (black) and 40 kPa (white) (n = 3). (D) The average ECFP/YPet ratios of FAK biosensor in HT1080 cells after 2 h of adhesion on the FN-coated PA gels of 0.6 kPa and 40 kPa, with (white, 10 µM; gray, 15 µM) or without (black) the pretreatment of ML7 (n = 24–34). (E) The average ECFP/YPet ratio values of FAK biosensor in HT1080 cells adhered on the Col I-coated 0.6 kPa gel for 2 h with or without 10 µM of ML7 or Y-27632 (n = 30–33). (F and G) The average ECFP/YPet ratio values of FAK biosensor in HT1080 cells with or without FAK mutant FRNK, on PA gels of 0.6 kPa (black) and 40 kPa (white) coupled with (F) FN (n = 18–21) or (G) Col I (n = 11–21). Graphs show mean ± SEM. *Significant difference (P < 0.05). (Scale bar, 10 µm.)
Fig. 2.
Fig. 2.
Soluble collagen can induce FAK activation via integrin α2β1 in low-tensional states. (A and B) The representative images (A) and the average ECFP/YPet ratio values (B) of FAK biosensor in HT1080 cells suspended for 1 h and subsequently treated with or without 40 µg/mL of Col I or FN for another 1 h (n = 45–50). The “Ad on Col I” group represents the FAK activity in the cell adhered on the Col I-coated glass surface. The ECFP/YPet ratio of each group was normalized by the value of suspension group without any treatment. (C) The average ECFP/YPet ratios of FAK biosensor in the suspended HT1080 cells treated with 1% DMSO as a control, or with 40 µg/mL Col I together with 1% DMSO, 10 µM ML7, or 1 µM PF228 (n = 14–19). The ECFP/YPet ratio of each group was normalized by the value of the suspension DMSO group. (D) The average ECFP/YPet ratios of FAK biosensor by the incubation of 40 µg/mL of Col I in the suspended HT1080 cells expressing FRNK or treated by 10 µg/mL inhibitory antibody of integrin α2β1 (n = 20–37). The ECFP/YPet ratio of each group was normalized by the value of the control suspension group. (E) The average ECFP/YPet ratio values of FAK biosensor near the attachment area of beads coated with 75 µg/mg bead of PDL or 25, 75, or 150 µg/mg bead of GFOGER peptide, in cells adhered on PDL-coated surface (n = 12–30). (F) The representative ECFP/YPet ratio images of FAK biosensor in cells with beads coated with PDL or GFOGER peptide (75 µg/mg bead). (G) The representative images and the average ECFP/YPet ratio values of FAK biosensor near the attachment area of GFOGER bead before (Control) and after the treatment with 10 µM of ML7 (n = 14–26). Graphs show mean ± SEM. *Significant difference (P < 0.05). (Scale bar, 10 µm.)
Fig. 3.
Fig. 3.
Activation of FAK in suspended and adhesion cells. (A) The representative ECFP/YPet ratio images of FAK biosensor in the suspended HT1080 cells treated with 40 µg/mL of RGD only or together with SNAKA51 (α5 activating antibody, 10 µg/mL), mAb11 (control antibody, 10 µg/mL), PHSRN (synergy peptide, 240 µg/mL), or HPRNS (control peptide, 240 µg/mL). (B) The average ECFP/YPet ratio values of FAK biosensor in the suspended HT1080 cells with the incubation of SNAKA51 (10 µg/mL), RGD peptide (40 µg/mL), RGD and SNAKA51 with or without 10 µM ML7, or RGD and mAb11 (10 µg/mL) (n = 23–38). The ECFP/YPet ratio of each group was normalized by the value of the control suspension group. (C) The average ECFP/YPet ratio values of FAK biosensor in the suspended HT1080 cells with the incubation of RGD peptide (40 µg/mL) and PHSRN (240 µg/mL), without or together with SNAKA51 (10 µg/mL) or 10 µM ML7, or the incubation of RGD and HPRNS (240 µg/mL) (n = 17–35). The ECFP/YPet ratio of each group was normalized by the value of the control suspension group. (D) The average ECFP/YPet ratios of FAK biosensor in the suspended HT1080 cells treated with Col I (40 µg/mL), 12G10 (β1 activating antibody, 10 µg/mL), or K20 (control antibody for 12G10, 10 µg/mL) (n = 14–27). The ECFP/YPet ratio of each group was normalized by the value of the control suspension group. (E) The average ECFP/YPet ratio values of FAK biosensor in the suspended HT1080 cells after the treatment of Col I, with or without the pretreatment of inhibitory antibody for integrin β1 (MAB1965) (n = 25–44). The ECFP/YPet ratio of each group was normalized by the value of control suspension group. (F) The average ECFP/YPet ratio values of FAK biosensor in cells seeded on the 40 kPa gel coated with FN (n = 19). Graphs show mean ± SEM. *Significant difference (P < 0.05). (Scale bar, 10 µm.)
Fig. 4.
Fig. 4.
FERM basic patch is required to protect the FAK activation during cell adhesion from the inhibitory effect of nearby acidic sites. (A and B) The representative images (A) and the average ECFP/YPet ratio values (B) of FAK biosensor in HT1080 cells expressing the control (black) or KAKTLRK mutant (white) during cell adhesion on the FN-coated glass surface. (C) The average ECFP/YPet ratio values of FAK biosensor in cells expressing KAKTLRK mutant (white) or KAK-EDQ mutant (gray). (D) The average ECFP/YPet ratio values of FAK biosensor in cells with (gray) or without WT-EDQ mutant (black) (n = 10–15). Graphs show mean ± SD. (E and F) The average ECFP/YPet ratios of FAK biosensor in cells expressing different FAK mutants after adhesion on (E) FN (n = 29–44) or (F) Col I (n = 27–65). Graphs show mean ± SEM. *Significant difference (P < 0.05). (Scale bar, 10 µm.)
Fig. 5.
Fig. 5.
Proposed model of FAK mechanoactivation mechanisms via different ECM and integrin subtypes during cell adhesion process. (A) Integrin α5β1 can be fully activated in the tensioned state where both RGD peptide (yellow circle) and synergy site (red circle) bind to α5 and β1 subunits, respectively. Because FN synergy site is exposed only in the high-tensional state, the FAK activation via integrin α5β1 is dependent on the mechanical environment. In contrast, integrin α2β1 can directly bind to the constitutively exposed GFOGER motif (orange circle) in Col I, thus causing the activation of integrin α2β1 and FAK independent of mechanical tension. (B) Integrin activation can recruit and induce the transphosphorylation of FAK. This leads to the FAK activation, which is maintained by the interaction between the FERM basic patch (blue oval) and PIP2 to prevent the inhibitory interaction of myosin II with FERM acidic sites (red oval).
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References

    1. Wang N, Tytell JD, Ingber DE. Mechanotransduction at a distance: Mechanically coupling the extracellular matrix with the nucleus. Nat Rev Mol Cell Biol. 2009;10(1):75–82. - PubMed
    1. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell. 2006;126(4):677–689. - PubMed
    1. Trappmann B, et al. Extracellular-matrix tethering regulates stem-cell fate. Nat Mater. 2012;11(7):642–649. - PubMed
    1. Huebsch N, et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat Mater. 2010;9(6):518–526. - PMC - PubMed
    1. Bershadsky AD, Balaban NQ, Geiger B. Adhesion-dependent cell mechanosensitivity. Annu Rev Cell Dev Biol. 2003;19:677–695. - PubMed

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