Traction stress in focal adhesions correlates biphasically with actin retrograde flow speed

Abstract

How focal adhesions (FAs) convert retrograde filamentous actin (F-actin) flow into traction stress on the extracellular matrix to drive cell migration is unknown. Using combined traction force and fluorescent speckle microscopy, we observed a robust biphasic relationship between F-actin speed and traction force. F-actin speed is inversely related to traction stress near the cell edge where FAs are formed and F-actin motion is rapid. In contrast, larger FAs where the F-actin speed is low are marked by a direct relationship between F-actin speed and traction stress. We found that the biphasic switch is determined by a threshold F-actin speed of 8-10 nm/s, independent of changes in FA protein density, age, stress magnitude, assembly/disassembly status, or subcellular position induced by pleiotropic perturbations to Rho family guanosine triphosphatase signaling and myosin II activity. Thus, F-actin speed is a fundamental regulator of traction force at FAs during cell migration.

Figure 1.

Traction stresses across the cell front. (A) Immunofluorescence of paxillin (red), serine-19–phosphorylated myosin II light chain marking activated myosin II (blue), and phalloiden staining of F-actin (green). Locations of lamellipodium, lamellipodium base, and lamella are indicated; distal and proximal directions are defined. (B) GFP-paxillin (inverted contrast) with traction stress vectors superimposed (Video 2, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). (C) Heat-scale plot of traction stress magnitude; segmented FAs indicated by black outlines (Video 2). White lines delineate boundaries between lamellipodium (LP), FAs, and cell body (CB). (D) Box plots of traction stresses in the lamellipodium, FAs, cell body, and background (BG) measured directly outside the cell. Box and whisker plots in all figures indicate the 25% (lower bound), median (middle line), and 75% (upper bound) nearest observations within 1.5 times the interquartile range (whiskers), 95% confidence interval of the median (notches) and near (+) and far (0) outliers. (E) FSM image of x-rhodamine F-actin with F-actin velocity vectors superimposed (Video 1). (F) Box plots of F-actin speed in the lamellipodium (LP), FAs, and cell body (CB). (G) Histograms of the F-actin speed across the entire cell front (left) and in areas of highest (>95%) traction stresses (right). (H) Heat scale plot of directional coupling, cosine of angle θ, between F-actin velocity and traction stress vectors; segmented FAs indicated by black outlines. Bars: (A) 10 μm; (B, C, and E) 3 μm; (H) 5 μm.

Figure 2.

Inhibition of myosin II activity constrains traction to the lamellipodium. (A–C) Cells were treated with 50 μM blebbistatin (+BLEB). (A) Heat scale plot of traction stress magnitude. (B) Mean traction stress (green) and F-actin speed (blue) as a function of distance from leading cell edge. (C) Immunofluorescence image of paxillin (red) and F-actin (green). Bars, 5 μm.

Figure 3.

Traction stress is biphasically correlated with F-actin speed in FAs. (A) Traction stress versus F-actin speed for all points throughout the cell front over 25 frames of a time-lapse movie. (B) Subset of traction stress versus F-actin speed from A for data located within segmented FAs. Data were grouped by F-actin speed (greater or less than 10 nm/s) and three values of traction stress (<20, 20–50, and >50 Pa) to obtain six “stress-speed” groups identified by different colors/symbols. (C) Inverted GFP-paxillin image with spatial location of stress-speed data groups plotted in B (Video 4, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). Bar, 3 μm. (D, top) Montage of GFP-paxillin images of a single FA over 15 min (Video 5). For each time point, the integrated area (second row), GFP-paxillin intensity (third row), traction stress (fourth row), and local F-actin speed (bottom) were determined. In the fourth row, the dashed line indicates 75% of maximum stress; the arrow indicates time (t_s) when traction stress exceeds this threshold. (E) F-actin speed at t_s (v(t_s)) as a function of t_s. Mean of v(t_s) = 12.7 ± 3 nm/s. Mean of t_s = 3 ± 2 min. (F) v(t_s) as a function of traction stress at t_s, σ(t_s). Mean of σ(t_s) = 65 ± 28 Pa. Data in E and F are from 26 FAs in four cells.

Figure 4.

The switch from an inverse to a direct correlation between F-actin speed and traction stress does not require FA disassembly and occurs at a specific F-actin speed. (A) Traction stress versus F-actin speed for data within FAs for 15 frames of a time-lapse video of a cell expressing CA-Rac. Data are grouped as in Fig. 3 B. (B) Inverted GFP-paxillin image with spatial location of stress/speed data points plotted in A. Bar, 3 μm. See Video 7, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1. (C) Mean traction stress as a function of distance from cell edge (left) and F-actin speed (right). Characteristic data are shown for a control cell, treatment with 50 μM blebbistatin (BLEB), expression of CA-Rac, and expression of CA-Rho. (D) The mean (squares) and standard deviation (error bars) of the distance away from the cell edge (left) and F-actin speed (right) associated with peak (>95%) traction stresses. Data reflects the mean of >100 data points from three cells.

Figure 5.

Inverse and direct correlations between traction stress and F-actin speed occur over similar ranges of F-actin speed despite perturbations to myosin II and Rho GTPase signaling. (A–D) Data ranges indicating the strongest inverse (blue) or direct (red) correlation between traction stress and F-actin speed (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200810060/DC1). Large blue and red arrows mark v_s and v_w, the F-actin speed delineating the upper and lower bounds, respectively, of the speed ranges. Gray symbols represent data outside the ranges; lines with slopes m_s and m_w show linear fits to data within the ranges. Small blue and red arrows mark σ_s and σ_w, the traction stress at v_s and v_w. Characteristic data are shown for control (A), blebbistatin-treated cells (B), and overexpression of CA-Rac (C) and CA-Rho (D). Mean slopes m_s (E, blue) and m_w (E, red), traction stresses σ_s (F, blue) and σ_w (F, red), and velocities v_s (G, blue) and v_w (G, red) for different conditions. In E–G, data reflects a mean of >1,000 data points for n = 3 cells. (H) Model for how F-actin dynamics are variably coupled to traction stress by initiation and assembly of FAs across the cell front; differently shaded regions reflect changes in the magnitude of traction stress measured at similar F-actin speeds for different conditions. Position within the cell front shown below; black bars represent FAs and crosshatching represents F-actin.