Metavinculin modulates force transduction in cell adhesion sites

Figures

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  Fig. 1. Metavinculin is immobilized in focal adhesions (FAs) and displays enhanced binding to talin. a Representative images of vinculin-deficient (vinc(−/−)) cells expressing vinculin–venus (V-V) or metavinculin–venus (M-V). Co-staining with a paxillin antibody indicates the independent localization of each vinculin isoform to FAs. b V-V and M-V both rescue the spreading defect of vinc(−/−) cells, which display reduced cellular eccentricity 2 h after cell adhesion when compared to the parental cell line (vinc(f/f)). (n = 23, 32, 27, 30 cells). Two-sided Kolmogorov–Smirnov test: ***p < 0.001. The bar chart shows the mean values ± SD. c V-V and metavinculin–mCherry (M–C) co-localize in FAs when co-expressed in vinc(−/−) cells. d Talin turnover rates are comparable in V-V- or M-V-expressing vinc(−/−) cells, as revealed by FRAP analysis of cells with co-expressed talin-1-TagBFP-HA (T1-B-HA). (n = 12, 12 cells). e Metavinculin resides in FA longer than vinculin as shown by FRAP analysis of vinc(−/−) cells expressing V-V or M-V. (n = 7, 5 cells). f Co-immunoprecipitation (IP) experiments using the venus-tag as bait demonstrate an increased association of talin with metavinculin (IB:…

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  Fig. 2. Force transduction in FAs is vinculin isoform-dependent. a Representative images of vinculin-deficient (vinc(−/−)) cells expressing vinculin tension sensor (V-TS), metavinculin tension sensor (M-TS), and the no-force control (Con-TS) 4 h after spreading on FN-coated glass coverslips show localization of all constructs to FAs (YPet), which are visualized by paxillin staining. Scale bar: 20 µm, in zoom: 5 µm. b Expression of V-TS or M-TS rescues the spreading defect of vinc(−/−) cells; data of the parental (vinc(f/f)) and vinc(−/−) cells are the same as in Fig. 1b. (n = 23, 32, 21, 14 cells). The bar chart shows the mean values ± SD. c Live-cell FLIM measurements of vinc(−/−) cells expressing FL-based tension sensors demonstrate FRET efficiency differences between V-TS and M-TS. Impairing actin binding by inserting the I997A mutation into vinculin (V-TS-I997A) and I1065A into metavinculin (M-TS-I1065A) strongly reduces tension and eliminates vinculin isoform-specific differences. (n = 73, 73, 74, 72, 73 cells). d Live-cell FLIM measurements of FL-based Con-TS, V-TS, and M-TS expressed in talin-deficient cells (tln1−/−tln2−/−), seeded on pLL-coated dishes and treated with Y-27632,…

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  Fig. 3. Vinculin isoform-specific differences in force transduction are observed in FAs and adherens junctions (AJs) of muscle cells. a Representative images of HL-1 cells expressing FL-based vinculin tension sensor (V-TS), metavinculin tension sensor (M-TS), and the no-force control (Con-TS) show localization of all fusion proteins to FAs (YPet), which are co-stained with a paxillin antibody. Nuclei are visualized with DAPI (blue). b FLIM measurements of FAs in HL-1 cells expressing FL-based sensors demonstrate vinculin isoform-specific force transduction also in muscle cells. (n = 81, 92, 84 cells). c Representative images of HL-1 cells expressing V-TS, M-TS, and Con-TS show localization of all tension sensors to AJs (YPet). The signal of co-stained α-catenin (α-cat) is used to outline AJs. d FLIM measurements of AJs in HL-1 cells reveal vinculin isoform-specific force transduction in cell–cell contacts. (n = 65, 53, 48 cells). Boxplots show median, 25th, and 75th percentile with whiskers reaching the last data point within 1.5× interquartile range. Two-sided Kolmogorov–Smirnov test: ***p < 0.001, **p < 0.01, *p < 0.05. Scale bar: 20 µm, in zoom: 5 µm. Source data and exact p values…

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  Fig. 4. Metavinculin knockout mice display a normal hypertrophic response upon transverse aortic constriction (TAC). a Western blot analysis of tissue lysates from 6-month-old mice shows complete loss of metavinculin in knockout M(−/−) and ~50% reduction of expression in heterozygous M(+/−) animals, compared to wild-type M(+/+) littermates. (GA: gastrocnemius muscle, V: vinculin, M: metavinculin, Tub: tubulin). b Histological analysis of M(+/+) and M(−/−) heart tissue of 6- and 13-month-old mice indicates normal morphology of the cardiac muscle. Scale bar: 40 µm. c, d Representative immunohistochemistry of ventricular tissue from 13-month-old M(+/+) and M(−/−) mice, respectively, reveals unchanged intercalated disk (ICD) structures as indicated by β-catenin (β-cat) and (meta)vinculin (V/M) staining. Nuclei are visualized with DAPI (blue). Scale bar: 20 µm, in zoom: 10 µm. e Heart weight analysis of sham- or TAC-operated mice, expressed as the ventricular weight (VW) normalized to tibia length (TL) shows no difference between M(+/+) and M(−/−) mice (n = 10, 10, 9, 9 mice). The bar chart shows the mean values ± SD. f Echocardiography assessment shows the expected decrease in fractional…