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. 2015 Nov 27;5:17225. doi: 10.1038/srep17225

Figure 4. Release of tension removes VASP, zyxin and TES from junctions.

Figure 4

(A) HUVECs treated with Y-27632 for 10 minutes (and subsequently fixed and IF for vinculin, VE-cadherin and actin) show an absence of radial stress fibres and FAJs (no vinculin in cell-cell junctions), indicating a lack tension on the adherens junctions. (B–D) HUVECs were virally transduced with expression vectors for α-catenin (tagged by mCherry) as a constitutive junction marker and the indicated tension-regulated proteins (tagged by eGFP) and live, wide-field imaging was performed to capture the result of releasing tension by addition of the Rock inhibitor Y-27632. Still images from these time-lapse series, just prior to Y-27632 treatment and 10 minutes after treatment are shown. Also see Supplemental movies 4-7. (E) Pixel intensity of either p120, vinculin, VASP, zyxin or TES was measured over time, in several cell-cell junctions (defined by α-catenin as junctional marker; n ≥ 6) over several time lapse experiments. Pixel intensity before addition of Y-27632 (at 3.5 minutes) was normalized to 1. Vinculin, VASP, zyxin and TES show clear protein decay over time after Y-27632 addition, compared to p120 as control. (F) After normalizing the minimum protein intensity to 0, the protein decay of vinculin, VASP, zyxin and TES starts immediately after Y-27632 addition. Furthermore, the protein dynamics of vinculin, VASP, zyxin and TES are similar after Y-27632 addition.