(776e) 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 cell’s
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: MTS_GPGGA, to
probe a low force range (0-8 pN), and MTS_HPst, 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. MTS_RGD measures forces in the 0 to 8 pN
range, while MTS_HPst 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 MTS_RGD (Blue) and MTS_HPst
(Red).