Figure 3. Local relaxation of ventral stress fibers induces vectorial actin polymerization at focal adhesion.

(A) The effect of tension on actin polymerization at focal adhesions was monitored by fluorescence recovery after photobleaching (FRAP) in laser-ablated (indicated by red box/arrowhead) and intact ventral stress fibers (indicated by yellow box/arrowhead). FRAP experiment was initiated 10 seconds after ablation. Symbol (¤) indicates the ablation site. (B) Kymographs recorded along the center of ablated and non-ablated ventral stress fiber regions (shown in the red and yellow boxes to the left from the kymograps) reveal that non-ablated and ablated fibers display differences in actin dynamics. In contractile control fibers, the rate of vectorial actin polymerization is slow (0,023 μm/min +/- 0,007 μm/min; SEM; n = 17), whereas relaxation induces vectorial actin polymerization from the adhesion located at the end of ablated ventral stress fiber (0,257 μm/min +/- 0,035 μm/min; SEM; n = 12). See also Figure 7B for a graphical representation of the data. (C) Photoactivation of GFP-PA-actin in contractile (yellow box) and relaxed (red box) ventral stress fibers. (D) Kymograph analysis performed along the center of indicated ventral stress fiber regions (shown in yellow and red boxes to the left from the kymographs) demonstrate induction of vectorial actin polymerization at the focal adhesion located in the end of an ablated ventral stress fiber. However, no detectable vectorial actin polymerization occurred in the non-ablated ventral stress fiber. Photoactivation was performed 10 s after ablation of the contractile stress fiber. Bars, 10 μm.

DOI: http://dx.doi.org/10.7554/eLife.06126.014

Figure 3—figure supplement 1. Method for laser ablation of ventral stress fibers.

Ablation of contractile stress fibers was achieved by using full laser power of 405 laser line (3I Marianas microscope) for 3 x 200 ms pulses. Symbol (¤) indicates ablation sites. Breakage of the fibers was confirmed by following retraction of mCherry-actin bundle with 561 channel for 10 seconds (A) or by immediate recruitment of a repair protein zyxin to the ablation site (B). Bar, 10 um. (C) Ablation of a ventral stress fiber leads to an immediate decrease in cell-mediated forces at focal adhesions located at the tips of the stress fiber. A representative example is shown in the figure, and quantification of the data from six adhesions at the ends of ablated stress fibers from three different cells revealed 27,3 +/- 7,0 (SD)% decrease in tension at the area around focal adhesions. Traction forces were measured both before, and 0,5 seconds after ablation. Bar, 10 um.

DOI: http://dx.doi.org/10.7554/eLife.06126.015