Pre-complexation of talin and vinculin without tension is required for efficient nascent adhesion maturation

Abstract

Talin and vinculin are mechanosensitive proteins that are recruited early to integrin-based nascent adhesions (NAs). In two epithelial cell systems with well-delineated NA formation, we find these molecules concurrently recruited to the subclass of NAs maturing to focal adhesions. After the initial recruitment under minimal load, vinculin accumulates in maturing NAs at a ~ fivefold higher rate than in non-maturing NAs, and is accompanied by a faster traction force increase. We identify the R8 domain in talin, which exposes a vinculin-binding-site (VBS) in the absence of load, as required for NA maturation. Disruption of R8 domain function reduces load-free vinculin binding to talin, and reduces the rate of additional vinculin recruitment. Taken together, these data show that the concurrent recruitment of talin and vinculin prior to mechanical engagement with integrins is essential for the traction-mediated unfolding of talin, exposure of additional VBSs, further recruitment of vinculin, and ultimately, NA maturation.

Particle Retracking Algorithm Capable of Quantifying Large, Local Matrix Deformation for Traction Force Microscopy

“…To assess the reconstructed traction elds' reasonability, we transfected the U2OS cells with paxillin-GFP, imaged its uorescence under TIRF, and segmented and detected focal adhesions (FAs), focal complexes (FCs) and nascent adhesions (NAs) as done previously (Fig. 6E) (2,17). The traction maps reconstructed from the displacement elds showed generally similar characteristics as in the displacement elds (Fig.…”

Nascent adhesions differentially regulate lamellipodium velocity and persistence

“…To experimentally test the finding that adhesion lifetime promotes lamellipodia protrusion velocity but limits persistence, we labeled adhesions in COS7 epithelial cells using transient expression of Paxillin-mApple and imaged adhesion and edge dynamics during 5 min of steady-state migration. We segmented the adhesions using focal adhesion analysis software for quantification of the adhesions' lifetime [51] and used morphodynamics software to track the edge motion [52] (Figure 6A and B). We noted that protrusions exhibited adhesions with heterogeneous lifetimes, in which clusters of short-living adhesions co-resided with a few longer-lifetime adhesions.…”

“…Nascent adhesions were detected and segmented using point source detection as previously described in [51,74]. Briefly, fluorescence images were filtered using the Laplacian of Gaussian filter and then local maxima were detected.…”

Real time observation of chaperone-modulated talin mechanics with single molecule resolution

“…[2][3][4][5] Talin rod segment (R1-R13) exhibits conformational changes and hierarchical unfolding of each rod domains under increasing mechanical tension. [6][7][8] Among them, R3 is the most unstable domain that not only unfolds at ~5pN but also exhibits equilibrium folding-unfolding transitions on a sub-second timescale by a single actomyosin contraction. 2,8,9 This force-dependent folding dynamics allow them to tune their interactome profile: such as, Rap1-interacting adapter molecule (RIAM) binds to folded R3 domain and protects from mechanical unfolding, whereas force above 5 pN results in stepwise unfolding of the domain with subsequent vinculin binding.…”