Two Distinct Regimes of Integrin Molecular Forces

Abstract

Forces transmitted by integrins activate focal adhesion kinase (FAK) and regulate cell adhesion, spreading and migration. However, the magnitudes and sources of the molecular forces involved with these mechanical regulations remain largely unknown. Here we discovered two distinct regimes of integrin molecular forces generated by cell membrane and actomyosin, respectively, and that they govern different cellular functions. Using integrin ligands linked with tension gauge tether (TGT), we showed that TGT with 54 pN tension tolerance (Ttol) is specifically ruptured at focal adhesions (FA) in CHO-K1 cells or central regions of cell-substratum contact area in neutrophil-like dHL-60 cells, indicating that remarkably strong forces can be applied to a single integrin. This force is abolished by blebbistatin treatment which inhibits myosin II, suggesting that this >54 pN integrin force is generated by actomyosin. However, blebbistatin treatment does not abolish the rupture of TGT with Ttol of 33 pN and lower, consistent with the previously reported ∼40 pN integrin force generated by cell membrane during cell adhesion. FAK activation and cell spreading beyond initial adhesion also require ∼ 40 pN integrin force. dHL-60 cells were poorly polarized and migration rate was significantly reduced on 54 pN TGT but not on unrupturable TGT or fibronectin coated surfaces. Collectively, our results demonstrated that cell membrane generates ∼40 pN molecular force on integrins and this force activates FAK and mediates cell initial adhesion and spreading, while actomyosin generates a >54 pN force to single integrins and this force mediates neutrophil-like cell polarization and migration. The discovery and decoupling of these two regimes of integrin forces was enabled by TGT which measures molecular forces and also regulates cellular functions by restricting integrin molecular forces under a designed level, providing a global molecular force control for cell mechanics study.