469577 Single Integrin Force Measurements Reveal the Distribution of Forces Exerted By Living Cells
Integrins are heterodimeric transmembrane proteins that mediate cell attachment to the extracellular matrix (ECM) and play key roles in interpreting mechanical cues from a cells environment. In particular, mechanical forces sensed and transmitted through integrin-ligand interactions are a critical aspect of integrin-based signaling that affects migration, proliferation, and differentiation. However, the forces that individual integrins experience in living cells have yet to be fully characterized. Here, we use Förster resonance energy transfer (FRET)-based molecular tension sensors (MTSs) to measure the distribution of mechanical forces exerted by individual integrin heterodimers in living cells at the single-molecule level. We engineered two MTS variants: MTSGPGGA, to probe a low force range (0-8 pN), and MTSHPst, for examining intermediate forces (8-12 pN). We find that a large fraction of integrins exert modest forces of <12 pN, even within defined integrin adhesion complexes. This finding is intriguing given that the load-bearing capacity of integrins has been reported to be on the order of tens of piconewtons. For integrins specifically within adhesion complexes, we see that the measured force per integrin falls into distinct subpopulations. These subpopulations may correspond to specific adhesion structures or modes of attachment, and may be crucial to understanding how mechanical forces activate downstream signaling. In ongoing work, we take advantage of the unique capabilities of MTSs to determine how subpopulations of load-bearing integrins are altered in response to external mechanical perturbations, and how (and whether) distinct integrin subtypes bear differing levels of mechanical load.
Figure 1. (a) Molecular tension sensors (MTSs) present an RGD-based cell-binding domain and use different force-sensitive linkers flanked by organic fluorophores. MTSRGD measures forces in the 0 to 8 pN range, while MTSHPst measures forces in the 8 to 12 pN range. (b) MTSs are attached to a functionalized PEG coverslip. Integrin heterodimers bind and exert force on the cell-binding domain, stretching the force-sensitive domain and decreasing the FRET efficiency. (c) Relative fraction of molecules measured under adhesions in live cells as measured from MTSRGD (Blue) and MTSHPst (Red).