375363 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 αVβ3 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).