Transient frictional slip between integrin and the ECM in focal adhesions under myosin II tension

Abstract

Background: The spatiotemporal regulation of adhesion to the extracellular matrix is important in metazoan cell migration and mechanosensation. Although adhesion assembly depends on intracellular and extracellular tension, the biophysical regulation of force transmission between the actin cytoskeleton and extracellular matrix during this process remains largely unknown.

Results: To elucidate the nature of force transmission as myosin II tension is applied to focal adhesions, we correlated the dynamics of focal adhesion proteins and the actin cytoskeleton to local traction stresses. Under low extracellular tension, newly formed adhesions near the cell periphery underwent a transient retrograde displacement preceding elongation. We found that myosin II-generated tension drives this mobility, and we determine the interface of differential motion, or "slip," to be between integrin and the ECM. The magnitude and duration of both adhesion slip and associated F-actin dynamics is strongly modulated by ECM compliance. Traction forces are generated throughout the slip period, and adhesion immobilization occurs at a constant tension.

Conclusions: We have identified a tension-dependent, extracellular "clutch" between integrins and the extracellular matrix; this clutch stabilizes adhesions under myosin II driven tension. The current work elucidates a mechanism by which force transmission is modulated during focal adhesion maturation.

FIGURE 1. FA maturation on rigid and compliant substrata

(Upper panel) On glass, GFP-paxillin puncta appears near the cell edge, remains immobilized and elongates within minutes. (Lower panel) On a compliant substrate (2.8kPa gel), GFP-paxillin puncta appears near the cell edge and undergoes retrograde displacement prior to immobilization and elongation. Dashed lines highlight initial location of distal edge of FA puncta. Arrowheads indicate proximal end of FA. Solid black line indicates cell boundary. Color combined image of FA at 0:00 (red) and 27:45 (greed). Time stamp in min:sec. Scale bar = 1 µm.

FIGURE 2. Cytoskeletal remodeling and traction stress recovery upon myosin reactivation by blebbistatin removal

Images of GFP-actin (Top), mApple-paxillin (Middle) and traction stress magnitude at times prior to blebbistatin treatment (Control) and at times indicated after blebbistatin removal. Time is indicated in min:sec. Gray line indicates cell boundary. Scale bar = 5µm.

FIGURE 3. F-actin and FAs move retrogradely as myosin-II is reactivated

(A) (Top) Color combined images of mApple-paxillin at successive times post blebbistatin removal, early (red) and late (green) times. Insets show magnification of outlined regions. Scale bar = 5 µm. (Middle) GFP-actin images overlaid with actin flow vectors (red arrows). Time indicates the initial time point used in velocity calculation. Red arrow indicates trajectory scale = 4.5 µm/min. Dashed white line indicates typical linescan across cell front used in analysis with location of adhesion and actin patche (yellow square with black outline) and lamellar actin (light blue square). (Bottom) Heat-scale map of traction stress with scale bar indicated at right (from blue to red). Times are indicated in seconds. (B) Traction stress (violet), F-actin flow (green), paxillin speed (red), and substrate speed (black) over time post blebbistatin removal. F-actin flow was tracked at actin patches associated with FA. Data reflect mean plus standard error for n_FA=26 and n_cell=5. (C) Displacement of actin patches colocalized with adhesions (yellow square, black outline), and at 1.2 µm proximal to adhesion puncta within the lamella (blue square). Approximate locations of these data points indicated in A, middle panel. Horizontal dashed line is a line of zero slope.

FIGURE 4. FA displacement occurs at the integrin-ECM interface

(A) Color combined images in time of FA proteins upon blebbistatin inactivation, (top) 0 sec (red) and 40 sec (green) and (bottom) 20 sec (red) and 40 sec (green). Scale bar = 1µm. Arrowheads indicate adhesion puncta. (B) Speeds of FA proteins and beads in substrate over time after blebbistatin inactivation. (C) Total displacement of adhesion proteins and substrate prior to adhesion immobilization. No statistical significant difference between FA proteins; substrate displacement is statistically significantly different from FAs (p<0.0001). Data reflect the mean and standard error for all samples, with sample sizes: α₅ (n_FA=8; n_cell=6), α_v (n_FA=9; n_cell=4), talin (n_FA=6; n_cell=3), paxillin (n_FA=26; n_cell=16), and vinculin (n_FA=12; n_cell=3).

FIGURE 5. Minimal Exchange of Integrin within FA during frictional slip

(A) Vertical montage of images of GFP-tagged α_v, α₅, talin, and paxillin. Prebleached adhesions shown at t=0 sec (top, white solid arrows), photobleaching occurred between 0 and 5 seconds. Post-bleach images for α_v, α₅, talin and paxillin at times indicated at left. Times indicated in seconds. Reappearance of adhesions post-bleach in α₅, talin and paxillin images are indicated by outlined arrows. (B) Recovery profiles for FA proteins in (A) over time. Dashed line demarcates termination of FA slip (t~15 sec). Data shown for 5 FA per protein. (C) Intensity profiles of GFP-α_v from line scans subtending an individual adhesion at t=0 sec (red), t=5 sec (green) and t=15 sec (blue). Color-combined image of GFP-α_v at times indicated in plot. Dashed lines indicate FA width. Scale bar = 0.5 µm. LP indicates lamellipodium, while CB marks the cell body. (D) Traction stress and integrin fluorescence were normalized to data at t=0 sec and plotted over time. Data reflect average values taken from 18 FAs from 5 cells. (E) Ratios of relative increase in traction stress to relative change in integrin intensity at adhesion immobilization.

FIGURE 6. FA slip modulated by ECM stiffness

(A) Adhesion immobilization time (open squares, left) and total displacement during adhesion slip (solid circles, right) plotted against substrate stiffness. (B) Lamellar F-actin retrograde flow at the onset of blebbistatin removal plotted against substrate stiffness. (C) Total displacement during adhesion slip plotted for F-actin, adhesions, and substrate on 2.8kPa and 0.6kPa gels. (D) Relative (normalized to the 2.8 kPa case) changes in substrate strain (deformation) and traction stress (force) for different substrate stiffnesses. Sample size distribution: Glass (n_FA=8, n_cell=2), 2.8kPa (n_FA=36, n_cell=5), 1.5kPa (n_FA= 8, n_cell=3), 0.6 kPa (n_FA=8, n_cell=3). P-values for data compared to 0.6kPa case; * p<0.01, ** p<0.001.

FIGURE 7. Force dependent ‘clutch’ between the integrin and ECM

(A) Schematic illustrating mobility of cytoskeletal components at different levels of force. Adhesion assembly: integrins are weakly coupled to the ECM. Frictional slip: Myosin-II drives fast retrograde velocity of F-actin and associated integrins (v, open arrow); traction force magnitude is low (small F, solid arrow). Adhesion Immobilization: At a critical tension, integrin-ECM immobilization occurs, traction force increases, myosin-dependent actin flow slows and FA elongation commences. (B) Schematic showing that interfacial slip between the integrin/ECM (dashed line) dominates in early adhesions under low force while slip at the actin/FA interface (solid line) dominates and persists in large adhesions under high force. (C) Semi-log plot of frictional drag coefficient versus traction stress, determined by calculation based on interpolated data points shown in Figure S7C. Inset: a semi-log plot of integrin/ECM slip rate versus time after blebbistatin removal, calculated from interpolated data from Figure S7C.