Abstract
Focal adhesions (FAs) link the extracellular matrix to the actin cytoskeleton to mediate cell adhesion, migration, mechanosensing and signalling. FAs have conserved nanoscale protein organization, suggesting that the position of proteins within FAs regulates their activity and function. Vinculin binds different FA proteins to mediate distinct cellular functions, but how vinculin’s interactions are spatiotemporally organized within FAs is unknown. Using interferometric photoactivation localization super-resolution microscopy to assay vinculin nanoscale localization and a FRET biosensor to assay vinculin conformation, we found that upward repositioning within the FA during FA maturation facilitates vinculin activation and mechanical reinforcement of FAs. Inactive vinculin localizes to the lower integrin signalling layer in FAs by binding to phospho-paxillin. Talin binding activates vinculin and targets active vinculin higher in FAs where vinculin can engage retrograde actin flow. Thus, specific protein interactions are spatially segregated within FAs at the nanoscale to regulate vinculin activation and function.
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Acknowledgements
The authors thank S. Craig (Johns Hopkins University) for the cDNA encoding the vinculin FRET activation biosensor, G. Danuser (UT Southwestern) for the FRET biosensor image analysis package, W. Shin for maintenance of the Waterman Lab microscopes, D. Honemond and S. Thacker for administrative assistance, T. Kanchanawong (National University of Singapore) for sharing and discussing iPALM protocols, and members of the Waterman Lab, G. Alushin (NHLBI), H. Elliot (Harvard Medical School) and J. Taraska (NHLBI) for helpful discussions. Financial support: Division of Intramural Research, NHLBI (L.B.C. and C.M.W.); Howard Hughes Medical Institute (G.S. and H.F.H.); GM081764 (S.L.C.), GM080568 (S.L.C.).
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L.B.C. and C.M.W. conceived the study and wrote the manuscript with input from all authors. L.B.C., C.M.W. and S.L.C. designed experiments. L.B.C. performed and analysed most experiments. L.B.C. and G.S. performed iPALM imaging. M.A.B. and M.W.D. designed new cDNA constructs and performed cloning. G.S. and H.F.H. conceived of, built and maintained iPALM instrumentation and developed iPALM processing tools. L.B.C., M.A.B., M.W.D. and C.M.W. designed the summary cartoon.
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Integrated supplementary information
Supplementary Figure 4 Vinculin WT iPALM measurements do not significantly vary between experiments.
(a) Mean of Z-median measurements from individual FAs in cells expressing WT vinculin-N-tdEos and imaged in three independent experiments. (b) Averaged Z-position frequency histograms of molecules within FAs. Solid line, mean frequency; Shaded region, bootstrapped 95% confidence about the mean. Significance tested with two-sample KS test. (c) Mean fraction of molecules localized to each of the three FA layers in FAs. Colouring in b,c used to highlight the three FA layers as in Fig. 1. Graphs in a–c represent measurements of n = 21 FAs for 2 cells (Day1), n = 60 FAs from 3 cells (Day2), and n = 35 FAs from 3 cells (Day3). Data in all bar graphs are represented as mean ± 95% bootstrapped confidence intervals. Significance tested with one way ANOVA followed by post-hoc Tukey test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ∗∗∗∗∗p < 0.00001, ns: not significant.
Supplementary Figure 5 Talin nano-scale position is rescued by re-expression of WT Vinculin.
(a) Western blots of vinculin (top) and tubulin (loading control, bottom) protein in lysates of WT HFFs versus HFFs expressing vinculin siRNA for 72 h (KD). (b) Representative iPALM renderings from wild-type (WT) HFF cells expressing Talin-C-tdEos. (c,d) Representative iPALM rendering from HFF cells treated with siRNAs targeting vinculin (Vcl KD) and additionally expressing Talin-C-tdEos (c) or Talin-C-tdEos with Vinculin-mCerulean (c). (a,c data duplicated from Fig. 2 for comparison purposes). In b–d the colourscale represents Z-position (nm), FAs oriented with the distal tip facing up, and scale bar = 1 micron. Histograms of the Z-position of the molecules within individual FAs (white boxes in b–d) displayed next to the colourscale. (e) Mean of Z-median measurements of the position of molecules from individual FAs in cells expressing Talin-C-tdEos (TlnC) in WT HFFs (WT), TlnC in vinculin KD HFFs (Vcl KD) or in cells expressing TlnC and vinculin-mCerulean in vinculin KD HFFs (Res). (f,g) Averaged Z-position frequency histograms of molecules within FAs. Solid line, mean frequency; Shaded region, bootstrapped 95% confidence about the mean. Significance tested with two-sample KS test. (h,i) Mean fraction of molecules localized to each of the three FA layers in FAs from cells in e. Colouring in f–i used to highlight the three FA layers as in Fig. 1. Graphs in e–i represent measurements of n = 95 FAs from 5 TlnC WT cells, n = 66 FAs from 5 TlnC KD cells, and n = 79 FAs from 5 TlnC rescue cells. Data in all bar graphs are represented as mean ± 95% bootstrapped confidence intervals with significance tested with one way ANOVA followed by post-hoc Tukey test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ∗∗∗∗∗p < 0.00001, ns: not significant.
Supplementary Figure 6 Active vinculin is oriented with the tail above the head.
(a,b) Representative iPALM rendering from an HFF cell expressing N-terminally tagged wild-type constitutively active (CA, N773/E775A) vinculin-N-tdEos (a, data duplicated from Fig. 3 for comparison purposes), or C-terminally tagged CA-vinculin-C-tdEos (b). In a,b the colourscale represents Z-position (nm), FAs oriented with the distal tip facing up, and scale bar = 1 micron. Histograms of the Z-position of the molecules within individual FAs (white boxes in a,b) displayed next to the colourscale. (c) Mean of Z-median measurements from individual FAs. (d) Averaged Z-position frequency histograms of molecules within FAs. Solid line, mean frequency; Shaded region, bootstrapped 95% confidence about the mean. Significance tested with two-sample KS test. (e) Mean fraction of molecules localized to each of the three FA layers. Colouring in d,e used to highlight the three FA layers as in Fig. 1. Graphs in c–e represent measurements of n = 82 FAs from 8 CA-vinculin-N-tdEos cells and n = 148 FAs from 6 CA-vinculin-C-tdEos expressing cells. Data in all bar graphs are represented as mean ± 95% bootstrapped confidence intervals with significance tested with one way ANOVA. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, ∗∗∗∗∗p < 0.00001, ns: not significant.
Supplementary Figure 7 Characterization of and controls for the vinculin activation FRET biosensor.
(a) mTurquoise (mTurq, top row) NeonGreen (NeonGr, middle row) and processed FRET ratio image (bottom row) of HFF cells either expressing mTurqouise fused to NeonGreen by a 10 amino acid linker (Cytosolic control probe), expressing the first 400 amino acids of vinculin (Vcl) fused to mTurquoise and NeonGreen (FA-targeted control probe), co-expressing mTurquoise and NeonGreen, or co-expressing vinculin-mTurquoise and vinculin-NeonGreen. Cartoon schematics of the different FRET probes are displayed beneath the images. Scale bar = 5 micron. In a,d, The FA mask (grey lines) was created from the mTurq images and superimposed onto FRET ratio images. (b) Quantification of the mean FRET ratio value for the constructs described in a in regions confined within FAs (FA) or in non-FA cytosolic regions (cyto). N = number of cells. (c) Scatterplot of the cytoplasmic mTurquoise Intensity versus Cytoplasmic FRET ratio measured in cells expressing FA-targeted control probe, WT-Vinculin FRET probe, co-expressing mTurquoise and NeonGreen, or co-expressing vinculin-mTurquoise and vinculin-NeonGreen. Each point represents measurements from a single cell. (d) mTurquoise (mTurq, top row) and processed FRET ratio image (middle row) of HFF cells expressing wild type (WT) vinculin FRET biosensor (left) or constitutively active (CA, N773/E775A) vinculin FRET biosensor (right). Scale bar = 5 micron. Bottom row: cartoon schematic of the WT vinculin FRET biosensor, numbers refer to amino acid positions in full-length vinculin. (d) Quantification of the mean FRET ratio value inside FAs (FA) and outside FAs (Cyto) from n = 18 WT-vinculin FRET expressing cells and n = 16 CA-vinculin FRET expressing cells. Data in all bar graphs are represented as mean ± 95% confidence intervals with significance tested with ANOVA test (∗∗∗∗∗p < 0.00001) followed by Tukey test post-hoc analysis. (∗ difference is significant at p < 0.05 cutoff, ns: not significant).
Supplementary Figure 8 Validation of paxillin knockdown and immunoprecipitation experiments, and the effects of paxillin overexpression on vinculin nanoscale localization.
(a) Western blots of Paxillin (top), Hic-5 (middle) or tubulin (loading control, bottom) protein in lysates of WT HFFs (WT) versus HFFs coexpressing paxillin and Hic-5 siRNA for 48 h (KD). (b) Western blots from Fig. 5d with molecular size markers labelled. (c) Independent co-immunoprecipitation displayed as a replicate experiment of Fig. 5d and Supplementary Fig. 5b. In b,c samples are from HFFs expressing vinculin siRNA for 72 hrs and additionally expressing GPF (1), WT-Vinculin-GFP (2), CA(N773/E775A)-Vinculin-GFP (3), A50I-Vinculin-GFP (4), or CA-A50I-Vinculin-GFP (5). (d,e) Representative iPALM renderings from HFF cells expressing WT-vinculin-tdEos (Vcl) and WT-paxillin-mCerulean (d) or WT-vinculin-tdEos (Vcl) and WT-paxillin-mCerulean in a paxillin/hic5 siRNA (Pxn KD) background (e). In d,e the colourscale represents Z-position (nm), FAs oriented with the distal tip facing up, scale bar = 1 micron. Histograms of the Z-position of the molecules within individual FAs (white boxes in d,e) are displayed next to the colourscale. (f) Mean of Z-median measurements of molecules in individual FAs. (g) Averaged Z-position frequency histograms of molecules within FAs. Solid line, mean frequency; Shaded region, bootstrapped 95% confidence about the mean. Significance tested with two-sample KS test. (h) Mean fraction of molecules localized to each of the three FA layers. Colouring in g,h used to highlight the three FA layers as in Fig. 1. Graphs in f–h represent measurements of n = 110 FA from 5 WT cells coexpressing WT-vinculin-N-tdEos and WT-paxillin-mCerulean and n = 111 FA from 5 PxnKD cells coexpressing WT-vinculin-N-tdEos and WT-paxillin-mCerulean. Data in all bar graphs represented as mean ± 95% bootstrapped confidence intervals with significance tested by one-way ANOVA.
Supplementary Figure 9 Spatial characterization of FRET biosensors and temporal characterization of paxillin phosphorylation during myosin-II dependent FA maturation.
(a,d) mTurquoise (mTurq, top) and processed mTurquoise/NeonGreen FRET ratio images (FRET, bottom) of a protruding region of an HFF cell expressing the CA(N773/E775A)-vinculin FRET biosensor (a) or the first 400 amino acids of vinculin (Vcl) fused to mTurquoise and NeonGreen (FA-targeted control probe, d). The FA mask (grey lines) was created from the mTurq image and superimposed onto FRET ratio image. Scale bar = 5 microns. (b,e) Values for mTurquoise intensity (blue line) and FRET ratio (red line) along the 5 μm long yellow line in a,d. Dark grey shaded areas are in the cytosolic (cyto) regions adjacent to the FA (FA, light grey shaded area) (c,f) Quantification of the mean FRET ratio of the distal (Dist) 1/3 and proximal (Prox) 1/3 of FA linescans. Data represented as mean ± standard error of measurements of n = 19 FA from 5 CA-vinculin FRET cells (c) or n = 18 FA from 7 control probe cells (f). Significance tested with one way ANOVA. In c,f, ‘Dist’ refers to the end of the FA facing the leading edge, and ‘Prox’ refers to the end of the FA facing the cell centre. (c) Uncropped western blots from Fig. 8r with molecular size markers labelled.
Supplementary Figure 10 Model of vinculin activation and nanoscale positioning during FA formation and maturation.
Speculative model for the role of vinculin-protein interactions in vinculin nanoscale localization and function during focal adhesion maturation. See text for details.
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Case, L., Baird, M., Shtengel, G. et al. Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nat Cell Biol 17, 880–892 (2015). https://doi.org/10.1038/ncb3180
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DOI: https://doi.org/10.1038/ncb3180
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