Figure 2. Vectorial actin polymerization at focal adhesions halts upon increased contractility and formation of ventral stress fibers.

(A) Representative images of U2OS cells grown on 26 kPa polyacrylamide dishes with fluorescent nanobeads together with the corresponding force maps. Adhesions located at the ends of ventral stress fibers (orange arrowheads) apply stronger forces to their substrate compared to adhesions located at the ends of dorsal stress fibers (red arrowheads). Bar, 10 um. (B) Quantification of traction forces at adhesions located in the ends of dorsal and ventral stress fiber adhesions. Mean +/- SEM, n = 20 cells, 4–8 adhesions per cell. (C) Recovery of GFP-actin signal was measured next to dorsal stress fiber adhesions (dsf FA, red box) and ventral stress fiber adhesions (vsf FA, yellow box). Bar, 5 um. (D) Representative example of a fluorescence-recovery-after-photobleaching (FRAP) experiment performed on a GFP-actin expressing cell at a ventral stress fiber (vsf) region close to a focal adhesion. Yellow box indicates the photobleached region and the orange arrowhead the distal end of the ventral stress fiber. Scale, 3 um. (E) Quantification of the recovery speed (μm/min) for GFP-actin from focal adhesions located at the tips of dorsal stress fibers (dsf) or ventral stress fibers (vsf). Means +/- SEM, n (dorsal stress fibers) = 11; n (ventral stress fibers) = 17. (F) Activation of PA-GFP-actin in focal adhesions is followed by centripetal flow of photoactivated actin along the dorsal stress fiber. In contrast, PA-GFP-actin activated at a focal adhesion located at the tip of ventral stress fiber does not distribute from the adhesion to the stress fiber. Activated PA-GFP-actin is in green, focal adhesion marker mCherry-zyxin in red and the yellow dashed lines show the borders of the photoactivated region. Bar, 2,5 μm.

DOI: http://dx.doi.org/10.7554/eLife.06126.005

Figure 2—figure supplement 1. Alignment of focal adhesions linked to the ends of transverse arcs.

(A) Thick actomyosin bundles derived through arc fusion apply tension to focal adhesions leading to enlargement and alignment of those focal adhesions that are linked to the ends of the actomyosin bundle through dorsal stress fibers. Eventually, this results in a formation of a straight ventral stress fiber, where the focal adhesions are aligned along the direction of the actomyosin bundle. Focal adhesions and stress fibers were visualized by expression of GFP-paxillin and mCherry-actin, respectively. The two ‘terminal’ focal adhesions, whose alignments during the process are shown in the insets, are indicated by red arrowheads in the larger cell frames. Bars, 10 μm. (B) Fusion of transverse arcs into thicker actomyosin structures correlates with the alignment of the ‘terminal’ focal adhesions along the direction of the arc bundle. Stress fibers were visualized by expression of GFP-CaP3. Bar, 5 μm.

DOI: http://dx.doi.org/10.7554/eLife.06126.006

Figure 2—figure supplement 2. Actin dynamics in focal adhesions located at the tips of dorsal and ventral stress fibers.

The dynamics of GFP-actin in focal adhesions of U2OS cells were examined by fluorescence recovery after photobleaching (FRAP). Recovery curves with mean +/- SD are shown. n (dorsal stress fiber adhesions) = 11; n (ventral stress fiber adhesion) = 12.

DOI: http://dx.doi.org/10.7554/eLife.06126.007

Figure 2—figure supplement 3. Inhibition of myosin light chain phosphorylation results in formation of abnormally long dorsal stress fibers.

(A) Examples of a control U2OS cell and a cell incubated for 2 h with ROCK kinase inhibitor, Y27632. This inhibitor results in an abnormal morphology of transverse arcs or their total disapparance. Y27632 –treated cells also contain fewer dorsal stress fibers, which appear abnormally long and thin. Red arrowheads indicate dorsal stress fibers. Bar, 10 μm. (B) Quantification of the lengths of dorsal stress fibers in control and Y27632 -treated cells. Mean lengths (+/- SD) of dorsal stress fibers from 20 cells is shown. (C) Inhibition of myosin light chain kinase (MLCK) by ML-7 leads to a loss of transverse arcs and concomitant defects in formation of ventral stress fibers. Bar, 10 μm. (D) Dorsal stress fibers are abnormally long in ML-7 treated cells. Mean length (+/- SD) of 55 dorsal stress fibers measured from control and ML-7 treated cells.

DOI: http://dx.doi.org/10.7554/eLife.06126.008

Figure 2—figure supplement 4. Expression of dominant inactive Rif leads to abnormal elongation of dorsal stress fibers.

(A) Video frames from a U2OS cells expressing GFP-actin alone (right panel) or GFP-actin and Rif-TN. In control cells, ventral stress fibers (yellow arrowhead) are generated through fusion of relatively short dorsal stress fibers (red arrows) and transverse arcs. Rif-TN expressing cells lack transverse arcs and concomitant formation of ventral stress fibers. Importantly, dorsal stress fibers in Rif-TN expressing cells are abnormally long and continue elongating through the observation period. Bar, 10 μm. (B) Images of phalloidin stained control and Rif-TN expressing cells. Dorsal stress fibers are indicated by red arrowheads. (C) Quantification of the lengths of dorsal stress fibers length from 20 control and Rif-TN expressing cells. Mean lengths of the dorsal stress fibers (+/- SEM) are shown.

DOI: http://dx.doi.org/10.7554/eLife.06126.009