(549b) Nanoscale Architecture of Tension Generation within Focal Adhesions

Mechanical
interactions between cells and the extracellular matrix (ECM) exert a profound
influence on cell migration, proliferation, and stem-cell differentiation. However, the mechanisms by which
cells generate and detect mechanical force remains poorly understood, in part
due to a lack of methods that visualize molecular-scale forces in living cells.
We used a Förster resonance energy transfer (FRET)-based molecular tension
sensor (MTS) to directly visualize cellular forces at the molecular level. The greatly enhanced
spatial resolution of this technique allowed us, for the first time, to observe
patterns of force generation within integrin-based adhesions, termed focal
adhesions (FAs). We
observe strikingly complex distributions of tensions within individual FAs. We
find that α_Vβ₃
integrin localizes to high force regions, whereas α₅β₁
integrin is broadly distributed along
the cell?s basal surface. Canonical FA proteins (paxillin, talin, vinculin, α-actinin,
and actin) show distinct patterns of colocalization with respect to tension
generation. Paxillin is most closely associated with
regions of low MTS FRET, while the recruitment of talin
and vinculin, which have been previously proposed to act as cellular mechanosensors, show moderate spatial correlation with
local tension generation. α-Actinin shows a complex localization
relative to tension, which may reflect its distinct roles in FA nucleation and
in stabilizing actin bundles in mature FAs. We applied Bayesian localization microscopy (S.
Cox et al., Nature Methods 2012) to
obtain super-resolution images of both paxillin and MTS FRET. Preliminary
analysis of these data suggests that FAs may be comprised of distinct,
submicron-sized structural assemblies with possible roles in coordinated force
transmission and mechanical signal transduction.

Figure 1. FRET-based MTSs measure tension at integrin adhesions. (a) MTSs are site-specifically labeled
with a FRET donor and FRET acceptor, and present the RGD sequence from fibronectin
to promote cell adhesion. The (GPGGA)₈
sequence acts as an entropic spring that is stretched upon the application of
force. Sensors are covalently attached to a glass coverslip; the PEG brush
prevents nonspecific cell and sensor attachment. Integrin heterodimers attach
to the RGD domain and apply load generated by the cell cytoskeleton. (b) GFP-labeled myosin
regulatory light chain (green) and inverted FRET signal (red). Bright red
indicates areas of high tension (low MTS FRET), which cluster around the cell
periphery. (c) Correlation
between FA protein recruitment (paxillin, vinculin, talin, and α-actinin) and tension within individual
FAs. (d) Left: raw fluorescence images of (i)
GFP-Paxillin and (ii) the FRET donor channel (white indicates high tension). Center: corresponding Bayesian localization
microscopy super-resolution images. Right:
insets show submicron regions of tension generation and a correspondence between
paxillin localization (green) and tension (red).