Vinculin tension distributions of individual stress fibers within cell-matrix adhesions

Abstract

Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell-ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force.

Keywords: Cell mechanics; Fluorescence resonance energy transfer; Focal adhesions; Laser ablation; Mechanotransduction; Stress fibers.

Fig. 1.

Mapping tension distributions of single stress fibers. The panels depict representative mCherry-Lifeact (top row), vinculin tension sensor (VinTS) donor (second row), VinTS FRET (third row), and FRET ratio (bottom row) images immediately before (0 min) and after (2 min and 4 min) laser ablation. The SF ablation site is indicated by the arrow in the mCherry image at 0 min. In the FRET ratio maps, all vinculin signal outside of the FAs depicted was masked out to facilitate analysis (see the Materials and Methods). The hotspots in the FRET ratio maps depict regions of increased FRET ratios following SF ablation, reflecting tension reduction. The contrast and brightness of all fluorescence images were optimized for clarity of presentation (see the Materials and Methods). Scale bar: 50 µm.

Fig. 2.

SF incision reduces total tension in cellular FAs. (A) Overall mean normalized FRET ratio versus time for ‘4-Image Tracking’, i.e. including only FAs that were clearly visualized throughout all four time points of the experiment. The FRET ratio values were normalized to the corresponding FRET ratio at time 0 min. The statistics are based on 546 focal adhesions from 17 cells for peripheral SF (PSF) ablation, 376 FAs from 10 cells for central SF (CSF) ablation, and 164 FAs from 8 cells for non-irradiated control cells. The increase in the control FRET ratio is presumably due to fluorophore photobleaching that was not fully corrected. [See supplementary material Fig. S3 for vinculin tail-less sensor (Grashoff et al., 2010) results]. (B) Changes in overall mean normalized FRET ratios across adjacent time points for the set of FAs considered in A (see the text). The FRET ratio changes were normalized to the corresponding FRET ratio at the first time point of each two-minute interval. (C) Changes in overall mean normalized FRET ratios for FAs that could be tracked across two temporally consecutive images (‘2-Image Tracking’). As in B, FRET ratio changes were normalized to the corresponding FRET ratio at the first time point of each two-minute interval. The statistics are based on 681–716 FAs from 17 cells for peripheral SF ablation, 458–485 FAs from 10 cells for central SF ablation, and 226–232 FAs from 8 cells for controls. *P<0.05 compared with corresponding control; ⁺P<0.05 for CSF vs PSF; ^#P<0.05 compared with the corresponding ‘0 to 2 min’ group. In all cases, the data are means±s.e.m. and statistical comparisons were performed using two-tailed Student's t-tests.

Fig. 3.

Coordinate system for measuring relative orientations of SFs and FAs. (A) Schematic of coordinate system. We defined FA angles as the angles between the long axis of each FA and the axial orientations of the cell (green solid arrow) or the severed SF (yellow solid arrow). The cell-referenced angle system is also shown (dotted arrow and labels). For clarity, only the image of the SF network (mCherry-Lifeact) is shown. (B,C) Examples of FA orientations showing their long and short axes. (D) Cell with FA orientations (arrows) overlaid upon a FRET intensity image, with region enclosed by white square zoomed and shown in (E). The angle of a selected FA is illustrated as θ with the severed SF axis as the reference. The colors in (B–E) reflect arbitrary units of VinTS FRET channel fluorescence intensity. White scale bars: 50 µm; yellow scale bars: 1 µm.

Fig. 4.

SFs distribute tension to FAs in an angle-dependent manner. (A–C) Distributions of normalized FRET ratio changes in individual FAs following SF ablation. The FRET ratio changes were calculated from 2-image FA tracking (Fig. 2C) over the indicated time intervals, and then plotted as a function of the angle between FA long axis and the reference axis (SF axis for ablated cells and cell long axis for non-ablated controls, as shown in Fig. 3A). The corresponding central and peripheral SF-ablation subpopulations are shown in supplementary material Fig. S8. The FRET ratio changes were normalized to the corresponding FRET ratio at the first time point of each two-minute interval. The horizontal box lines indicate 25th, 50th and 75th percentiles, the whisker ends indicate 5th and 95th percentiles, and the solid dots indicate mean values. Each data point represents one FA. (D–F). Time-dependent histograms of angles between FA and SF orientations for (D) all FAs, (E) tension-reduced (TR) FAs, and (F) tension-enhanced (TE) FAs. The curves are the corresponding Gaussian curve fits. The histograms for central and peripheral SF-ablation subpopulations are shown in supplementary material Fig. S9. (G–I). Breadth of tension distributions as a function of time and SF location. (G) Gaussian widths for all FAs, with the red, green, and blue curves (all, tension-reduced, and tension-enhanced) corresponding to the Gaussian fits shown in D–F, respectively. (H,I) Gaussian widths for (H) peripheral SF (PSF) and (I) central SF (CSF) subpopulations.

Fig. 5.

FAs undergo dynamic tension transitions following SF ablation. Tension state transition plots for (A) all SFs, (B) peripheral SF (PSF), and (C) central SF (CSF) subpopulations. FAs were analyzed from 4-image tracking data depicted in Fig. 2A,B. In these diagrams, each of the nine tiles represents the category into which a given FA falls [tension-reduced (TR), tension-enhanced (TE), non-tension-reduced/tension-enhanced] for a given time interval, with the color representing the number of FAs (see scale on right-hand side). The lines between temporally adjacent tiles reflect FAs transitioning from one state to another, with the thickness of each line indicating the number of FAs undergoing that particular transition (which can include remaining in the same state). Scale bar thickness: 50 FAs (note variation of scale between panels).

Fig. 6.

Structural model of SF tension distribution across cellular FAs. (A–C) Confocal fluorescence images depicting actin cytoskeleton (phalloidin). Red boxes highlight regions where lateral interconnections between central SFs are clearly visible. Arrows indicate peripheral SFs, which exhibit these lateral connections to a much lesser extent. (D) Live-cell image illustrating actomyosin cytoskeletal structure (mCherry-Lifeact, red) and FAs [overlaid VinTS donor (blue) and FRET (green) channels]. White dotted arrows indicate FAs that are associated with the transverse actomyosin structures. Scale bar: 20 µm. (E–G) A simplified model for tension re-distribution after (F) central and (G) peripheral SF photo-disruption, following disruption of the mechanical equilibrium depicted in (E). FAs are represented by green ovals, with a darker green color indicating higher tension experienced by that FA. Blue arrows represent the involved force vectors along SFs, with stronger force reflected by thicker arrows. The red solid or dashed lines depict the SFs. The ablation site is indicated by an ‘X’. In this model, the interconnected central SFs re-direct tension to a broader array of FAs with a diversity of cellular locations and orientations, which preserves cell shape after SF ablation. Conversely, peripheral SFs, which are not as structurally coupled into the total SF network, distribute dissipated tension to a narrower segment of cellular FAs with similar orientations. Thus, peripheral SF disruption is more likely to trigger FA rupture and cellular contraction. TE, tension-enhanced FA; TR, tension-reduced FA.