Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling

Abstract

Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the Tension Gauge Tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of ~40 pN to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.Cells sense and respond to the mechanical properties of the surrounding extracellular matrix (ECM) and neighboring cells. Reciprocally, cells also apply force on the ECM and transmit mechanical signals to neighboring cells. These mechanical interactions activate intracellular signaling pathways and regulate such diverse processes as cell adhesion, polarization, migration, proliferation and differentiation (1, 2). As a result, by tuning bulk mechanical properties like stiffness, texture, and geometry of the substrate, researchers have gained insight into processes such as stem cell differentiation (3) and tumor metastasis (4). Singlemolecule force spectroscopy has been used to study various mechano-sensitive membrane receptors including integrin, cadherin and Notch (5-8). However, these approaches cannot reveal the single molecule forces required for physiological functions because they either measure collective forces exerted through many molecules or probe molecular unbinding or unfolding forces only. More recently, FRET-based force sensors were developed and inserted to target sites to monitor cellular forces (9, 10), but great efforts must be taken to prepare the sensors and to track and interpret the fluorescence signal. Here, we describe a platform termed Tension Gauge Tether (TGT) that allows us to determine the single molecule forces required for mechanical signaling in cells.In TGT, a ligand is covalently conjugated to a tether that ruptures at a critical force, which we term `tension tolerance' or T tol , and is immobilized on a solid surface through the tether (Fig. 1). Cells are plated on the surface and membrane receptors engage with and apply tension to the ligands. If signal activation through the receptor requires a molecular tension larger than T tol , the tether will rupture, abolishing signal activation. In contrast, if the required tension is smaller than T tol , the tether will endure, activating the receptor-mediated signaling. By engineering a series of tethers with different T tol values, the tension required for signal activation can be determined by observing receptor-regulated cell activities which * To whom correspondence should be addressed. tjha@il...

Talin determines the nanoscale architecture of focal adhesions

“…For example, if talin is nearly colinear with the actin stress fiber, that is, θ Talin ∼ θ SF , the majority of the actomyosin contraction is expected to be channeled through talin. In particular, this may correspond to early cell spreading when large integrin tensions >50 pN have been implicated (52). Such high tension would also lead to talin becoming significantly extended, consistent with the observation of talin molecules spanning >200 nm in cells (39).…”

“…In other words, talin may exert a pulling force on both the integrin and the surrounding plasma membrane. Consistent with this, a number of studies have documented how membrane tension may influence integrin-ECM interaction (51,52). An interesting possibility is that such in-plane tensions may influence other FAs in the vicinity, thus providing a degree of local mechanical coordination.…”

“…Several recent studies have focused on single integrin tension measurements at FAs, reporting a wide range of force magnitude, from a few piconewtons to >50 pN (52)(53)(54)(55)(56). Although our experiments do not directly measure tension, the range of various forces can be estimated based on the measured geometry and in vitro talin force/extension data (38).…”

Rapid disorganization of mechanically interacting systems of mammary acini

“…Cells use multiple approaches for integrating mechanical cues with biochemical cues provided by soluble factors; for example, there is extensive multilayered cross-talk between integrin and TGF-β signaling (16,17). It is now also clear that key developmental regulators, such as Notch, can be directly activated by mechanical force (18,19).…”

“…Cells use multiple approaches for integrating mechanical cues with biochemical cues provided by soluble factors; for example, there is extensive multilayered cross-talk between integrin and TGF-β signaling (16,17). It is now also clear that key developmental regulators, such as Notch, can be directly activated by mechanical force (18,19).Less is known about the interactions of organized multicellular structures with the ECM and how those interactions affect tissue architecture, composition, and stability, as well as the molecular pathways by which these effects are mediated. In certain situations, contractile multicellular structures are able to mechanically reorganize biopolymer networks over long distances and in a highly directional manner, generating what are variously referred to as fibers, tracts, cables, straps, or lines.…”