Figure 1.
Focal Adhesion Maturation in Wild Type Cells.
(a) Time-lapse images of a U2OS cell expressing GFP-actin and mApple-paxillin, with images of traction stress magnitude and overlaid traction stress vectors. Time is in min:sec. (b) Plot of focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion (FA) indicated in (a) as a function of time. (c) Region of a U2OS cell with overlaid actin retrograde flow vectors. (d) Histogram of FA lifetimes in U2OS cells. (e) Histogram of FA elongation rate, measured by the rate of change in the length during FA elongation. (f) Histogram of focal adhesion loading rate, measured by the rate of change of the traction stress during FA elongation.
Figure 2.
Focal Adhesion Morphology and Traction Forces under Treatment with ROCK inhibitor Y-27632.
(a) Images of F-actin visualized by fluorescent phalloidin (top row) and paxillin immunofluorescence (middle row) in U2OS cells treated with the indicated concentrations of the rho-kinase inhibitor, Y-27632. (b) Box plots of FA lengths in U2OS cells as a function of Y-27632 concentration (n = 101 (WT), 78 (1 µM), 134 (2 µM), 117 (5 µM), 124 (10 µM) FAs). (c) Representative GFP-actin images (top row) and traction stress maps (bottom row) and for U2OS cells treated with 2, 5 and 10 µM Y-27632. (d) Plot of the total traction force exerted by U2OS cells as a function of Y-27632 concentration (n = 23 (WT), 13 (1 µM), 8 (2 µM), 14 (5 µM), 6 (10 µM) cells).
Figure 3.
Focal Adhesion Maturation under Treatment with ROCK inhibitor Y-27632.
(a) Time-lapse images of GFP-actin and mApple-paxillin in U2OS cell treated with 2 µM Y-27632, with images of traction stress magnitude. Time is in min:sec. (b) Focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion indicated in (a) as a function of time. Focal adhesion assembly characteristics in varied Y-27632 concentrations: (c) FA elongation rate, (d) FA loading rate, (e) FA disassembly rate, (f) FA lifetime under indicated conditions (n = 10 FA for each condition for FA elongation, loading rate, disassembly rate, and lifetime). (g) Lamellar actin retrograde flow speeds in varied conditions (n = 2 cells per condition, 5 to 7 regions per cell). (h) Box plot of peak traction stress reached during focal adhesion lifetimes in cells treated with Y-27632. “Stable FA” indicates FA that had lifetime greater than 30 minutes, while “Unstable FA” indicates FA with lifetime less than 30 minutes.
Figure 4.
Focal Adhesion Stability is Rescued at Low Tension by Over-expression of α-actinin and Dia1.
(a) Representative GFP-paxillin image and traction stress map of a U2OS cell treated with 5 µM Y-27632 and over-expressing mApple-α-actinin. (b) Total traction force generated by cells treated with 5 µM Y-27632 and over-expressing either mApple-α-actinin or mCherry-Dia1 (n = 9 cells per condition). (c) Time-lapse GFP-paxillin and traction stress map images of a U2OS cell treated with 5 µM Y-27632 and over-expressing mApple-α-actinin. Time is in min:sec. (d) Focal adhesion length (black squares) and traction stress (blue circles) at the focal adhesion as a function of time for red oval indicated in (c). Focal adhesion assembly characteristics for cells treated with 5 µM Y-27532 and over-expressing either mApple-α-actinin or mCherry-CA-Dia1: (e) FA elongation rate, (f) FA loading rate, (g) FA disassembly rate, (h) FA lifetime and (i) Maximum FA stress attained by FAs (n = 12 FA for each condition for FA elongation, loading rate, disassembly rate, and lifetime).