Fig. 6: DAFS variant VcnTSs experience reduced, but not negligible load.
From: Molecular basis and cellular functions of vinculin-actin directional catch bonding
a Schematic of TSMod and VcnTS constructs in loaded and unloaded states. b Schematic of VcnTS in the FA, loaded by actomyosin contractility. c–g FA masked acceptor intensity images are shown for single Vcn−/− MEFs expressing WT VcnTS, DAFS variants of VcnTS and a previously validated variant with reduced actin affinity, VcnTS I997A. h Representative cytoplasm acceptor intensity shown for a single Vcn−/− MEF expressing unloaded FRET control, TSMod. i–m, Corresponding FA masked FRET efficiency shown for (c–g). n Corresponding cytoplasm FRET efficiency shown in h. o Box-whisker plots are shown for cell-averaged FRET efficiency of VcnTS and VcnTS DAFS variants compared to expected unloaded FRET efficiency (dotted line) controls (n = 232, 85, 79, 97, 58, and 69) biologically independent cells respectively, examined from over N = 3 independent experimental days. Box depicts the median as the center value, the 25th percentile as the lower bound, and the 75th percentile as the upper bound. Whiskers extend 1.5 times the IQR from the bottom and top of box, or to the minimum and maximum of the data if the data does not extend to the whiskers. Values outside the whiskers are plotted as individual points. One-way ANOVA paired with a Steel-Dwass test was used for statistical analysis. Differing letters denote significant difference at p < 0.05. See Supplementary Table 8 for a detailed listing of exact p-values. Source data are provided as a Source Data file. Scale bars are 10 μm in all images, and insets are 10 μm in length and width.
