Nanoscale architecture of cadherin-based cell adhesions

Abstract

Multicellularity in animals requires dynamic maintenance of cell–cell contacts. Intercellularly ligated cadherins recruit numerous proteins to form supramolecular complexes that connect with the actin cytoskeleton and support force transmission. However, the molecular organization within such structures remains unknown. Here we mapped protein organization in cadherin-based adhesions by super-resolution microscopy, revealing a multi-compartment nanoscale architecture, with the plasma-membrane-proximal cadherin–catenin compartment segregated from the actin cytoskeletal compartment, bridged by an interface zone containing vinculin. Vinculin position is determined by α-catenin, and following activation, vinculin can extend 30 nm to bridge the cadherin–catenin and actin compartments, while modulating the nanoscale positions of the actin regulators zyxin and VASP. Vinculin conformational activation requires tension and tyrosine phosphorylation, regulated by Abl kinase and PTP1B phosphatase. Such modular architecture provides a structural framework for mechanical and biochemical signal integration by vinculin, which may differentially engage cadherin–catenin complexes with the actomyosin machinery to regulate cell adhesions.

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Figure 1: Interferometric photoactivated localization microscopy imaging of plasma membrane marker and F-actin.
Figure 2: Protein stratifications in MDCK and C2C12 cadherin-based adhesions.
Figure 3: z-dimension profiles of cadhesome proteins.
Figure 4: Nanoscale positions and conformations of vinculin and α-catenin in cadherin-based adhesions.
Figure 5: Vinculin conformation is modulated by tyrosine phosphorylation and tension.
Figure 6: Integration of mechanical and biochemical signals by vinculin regulates mechanical properties of cell–cell contacts.

References

  1. 1

    Heisenberg, C.-P. & Bellaïche, Y. Forces in tissue morphogenesis and patterning. Cell 153, 948–962 (2013).

    CAS  Article  Google Scholar 

  2. 2

    Guillot, C. & Lecuit, T. Mechanics of epithelial tissue homeostasis and morphogenesis. Science 340, 1185–1189 (2013).

    CAS  Article  Google Scholar 

  3. 3

    Lecuit, T. & Yap, A. S. E-cadherin junctions as active mechanical integrators in tissue dynamics. Nat. Cell Biol. 17, 533–539 (2015).

    CAS  Article  Google Scholar 

  4. 4

    Takeichi, M. Dynamic contacts: rearranging adherens junctions to drive epithelial remodelling. Nat. Rev. Mol. Cell Biol. 15, 397–410 (2014).

    CAS  Article  Google Scholar 

  5. 5

    Leerberg, J. M. et al. Tension-sensitive actin assembly supports contractility at the epithelial zonula adherens. Curr. Biol. 24, 1689–1699 (2014).

    CAS  Article  Google Scholar 

  6. 6

    Leckband, D. & de Rooij, J. Cadherin adhesion and mechanotransduction. Annu. Rev. Cell Dev. Biol. 30, 291–315 (2014).

    CAS  Article  Google Scholar 

  7. 7

    Niessen, C. M., Leckband, D. & Yap, A. S. Tissue organization by cadherin adhesion molecules: dynamic molecular and cellular mechanisms of morphogenetic regulation. Physiol. Rev. 91, 691–731 (2011).

    CAS  Article  Google Scholar 

  8. 8

    Borghi, N. et al. E-cadherin is under constitutive actomyosin-generated tension that is increased at cell–cell contacts upon externally applied stretch. Proc. Natl Acad. Sci. USA 109, 12568–12573 (2012).

    CAS  Article  Google Scholar 

  9. 9

    Kim, T. J. et al. Dynamic visualization of α-catenin reveals rapid, reversible conformation switching between tension states. Curr. Biol. 25, 218–224 (2015).

    CAS  Article  Google Scholar 

  10. 10

    Bertocchi, C., Vaman Rao, M. & Zaidel-Bar, R. Regulation of adherens junction dynamics by phosphorylation switches. J. Signal Transduct. 2012, 125295 (2012).

    Article  Google Scholar 

  11. 11

    Zaidel-Bar, R. Cadherin adhesome at a glance. J. Cell Sci. 126, 373–378 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Huang, B., Wang, W., Bates, M. & Zhuang, X. Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810–813 (2008).

    CAS  Article  Google Scholar 

  13. 13

    Truong Quang, B. A., Mani, M., Markova, O., Lecuit, T. & Lenne, P. F. Principles of E-cadherin supramolecular organization in vivo. Curr. Biol. 23, 2197–2207 (2013).

    CAS  Article  Google Scholar 

  14. 14

    Wu, Y., Kanchanawong, P. & Zaidel-Bar, R. Actin-delimited adhesion-independent clustering of e-cadherin forms the nanoscale building blocks of adherens junctions. Dev. Cell 32, 139–154 (2015).

    CAS  Article  Google Scholar 

  15. 15

    Buckley, C. D. et al. Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force. Science 346, 1254211 (2014).

    Article  Google Scholar 

  16. 16

    Yonemura, S., Itoh, M., Nagafuchi, A. & Tsukita, S. Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between non-polarized fibroblasts and polarized epithelial cells. J. Cell Sci. 108, 127–142 (1995).

    CAS  PubMed  Google Scholar 

  17. 17

    Gavard, J. et al. Lamellipodium extension and cadherin adhesion: two cell responses to cadherin activation relying on distinct signalling pathways. J. Cell Sci. 117, 257–270 (2004).

    CAS  Article  Google Scholar 

  18. 18

    Kanchanawong, P. et al. Nanoscale architecture of integrin-based cell adhesions. Nature 468, 580–584 (2010).

    CAS  Article  Google Scholar 

  19. 19

    Shtengel, G. et al. Interferometric fluorescent super-resolution microscopy resolves 3D cellular ultrastructure. Proc. Natl Acad. Sci. USA 106, 3125–3130 (2009).

    CAS  Article  Google Scholar 

  20. 20

    Ajo-Franklin, C. M., Ganesan, P. V. & Boxer, S. G. Variable incidence angle fluorescence interference contrast microscopy for z-imaging single objects. Biophys. J. 89, 2759–2769 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Paszek, M. J. et al. Scanning angle interference microscopy reveals cell dynamics at the nanoscale. Nat. Methods 9, 825–827 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Bertocchi, C., Goh, W. I., Zhang, Z. & Kanchanawong, P. Nanoscale imaging by superresolution fluorescence microscopy and its emerging applications in biomedical research. Crit. Rev. Biomed. Eng. 41, 281–308 (2013).

    Article  Google Scholar 

  23. 23

    Yamada, S., Pokutta, S., Drees, F., Weis, W. I. & Nelson, W. J. Deconstructing the cadherin-catenin-actin complex. Cell 123, 889–901 (2005).

    CAS  Article  Google Scholar 

  24. 24

    Kovacs, E. M., Goodwin, M., Ali, R. G., Paterson, A. D. & Yap, A. S. Cadherin-directed actin assembly: E-cadherin physically associates with the Arp2/3 complex to direct actin assembly in nascent adhesive contacts. Curr. Biol. 12, 379–382 (2002).

    CAS  Article  Google Scholar 

  25. 25

    Strale, P.-O. et al. The formation of ordered nanoclusters controls cadherin anchoring to actin and cell–cell contact fluidity. J. Cell Biol. 210, 333–346 (2015).

    CAS  Article  Google Scholar 

  26. 26

    Shim, S. H. et al. Super-resolution fluorescence imaging of organelles in live cells with photoswitchable membrane probes. Proc. Natl Acad. Sci. USA 109, 13978–13983 (2012).

    CAS  Article  Google Scholar 

  27. 27

    Liu, J. et al. Talin determines the nanoscale architecture of focal adhesions. Proc. Natl Acad. Sci. USA 112, E4864–E4873 (2015).

    CAS  Article  Google Scholar 

  28. 28

    Yonemura, S., Wada, Y., Watanabe, T., Nagafuchi, A. & Shibata, M. α-Catenin as a tension transducer that induces adherens junction development. Nat. Cell Biol. 12, 533–542 (2010).

    CAS  Article  Google Scholar 

  29. 29

    Grashoff, C. et al. Measuring mechanical tension across vinculin reveals regulation of focal adhesion dynamics. Nature 466, 263–266 (2010).

    CAS  Article  Google Scholar 

  30. 30

    Yao, M. et al. Force-dependent conformational switch of α-catenin controls vinculin binding. Nat. Commun. 5, 4525 (2014).

    CAS  Article  Google Scholar 

  31. 31

    Peng, X., Nelson, E. S., Maiers, J. L. & DeMali, K. A. New insights into vinculin function and regulation. Int. Rev. Cell Mol. Biol. 287, 191–231 (2011).

    CAS  Article  Google Scholar 

  32. 32

    Nagafuchi, A. & Tsukita, S. The loss of the expression of α catenin, the 102 kD cadherin associated protein, in central nervous tissues during development. Dev. Growth Differ. 36, 59–71 (1994).

    CAS  Article  Google Scholar 

  33. 33

    Benjamin, J. M. et al. αE-catenin regulates actin dynamics independently of cadherin-mediated cell–cell adhesion. J. Cell Biol. 189, 339–352 (2010).

    CAS  Article  Google Scholar 

  34. 34

    Sumida, G. M., Tomita, T. M., Shih, W. & Yamada, S. Myosin II activity dependent and independent vinculin recruitment to the sites of E-cadherin-mediated cell–cell adhesion. BMC Cell Biol. 12, 48 (2011).

    CAS  Article  Google Scholar 

  35. 35

    Chen, H., Cohen, D. M., Choudhury, D. M., Kioka, N. & Craig, S. W. Spatial distribution and functional significance of activated vinculin in living cells. J. Cell Biol. 169, 459–470 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Cohen, D. M., Chen, H., Johnson, R. P., Choudhury, B. & Craig, S. W. Two distinct head-tail interfaces cooperate to suppress activation of vinculin by talin. J. Biol. Chem. 280, 17109–17117 (2005).

    CAS  Article  Google Scholar 

  37. 37

    Milam, L. M. Electron microscopy of rotary shadowed vinculin and vinculin complexes. J. Mol. Biol. 184, 543–545 (1985).

    CAS  Article  Google Scholar 

  38. 38

    Bays, J. L. et al. Vinculin phosphorylation differentially regulates mechanotransduction at cell–cell and cell–matrix adhesions. J. Cell Biol. 205, 251–263 (2014).

    CAS  Article  Google Scholar 

  39. 39

    Nagar, B. et al. Crystal structures of the kinase domain of c-Abl in complex with the small molecule inhibitors PD173955 and imatinib (STI-571). Cancer Res. 62, 4236–4243 (2002).

    CAS  PubMed  Google Scholar 

  40. 40

    Ukropec, J. A., Hollinger, M. K., Salva, S. M. & Woolkalis, M. J. SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin. J. Biol. Chem. 275, 5983–5986 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Xu, G., Arregui, C., Lilien, J. & Balsamo, J. PTP1B modulates the association of β-catenin with N-cadherin through binding to an adjacent and partially overlapping target site. J. Biol. Chem. 277, 49989–49997 (2002).

    CAS  Article  Google Scholar 

  42. 42

    Chang, Y.-C., Nalbant, P., Birkenfeld, J., Chang, Z.-F. & Bokoch, G. M. GEF-H1 couples nocodazole-induced microtubule disassembly to cell contractility via RhoA. Mol. Biol. Cell 19, 2147–2153 (2008).

    CAS  Article  Google Scholar 

  43. 43

    Hara, Y., Shagirov, M. & Toyama, Y. Cell boundary elongation by non-autonomous contractility in cell oscillation. Curr. Biol. 26, 2388–2396 (2016).

    CAS  Article  Google Scholar 

  44. 44

    Tsai, T. Y. et al. Robust, tunable biological oscillations from interlinked positive and negative feedback loops. Science 321, 126–129 (2008).

    CAS  Article  Google Scholar 

  45. 45

    Peng, X., Maiers, J. L., Choudhury, D., Craig, S. W. & DeMali, K. A. α-Catenin uses a novel mechanism to activate vinculin. J. Biol. Chem. 287, 7728–7737 (2012).

    CAS  Article  Google Scholar 

  46. 46

    Peng, X., Cuff, L. E., Lawton, C. D. & DeMali, K. A. Vinculin regulates cell-surface E-cadherin expression by binding to β-catenin. J. Cell Sci. 123, 567–577 (2010).

    CAS  Article  Google Scholar 

  47. 47

    Critchley, D. R. Biochemical and structural properties of the integrin-associated cytoskeletal protein talin. Annu. Rev. Biophys. 38, 235–254 (2009).

    CAS  Article  Google Scholar 

  48. 48

    Abe, K. & Takeichi, M. EPLIN mediates linkage of the cadherin catenin complex to F-actin and stabilizes the circumferential actin belt. Proc. Natl Acad. Sci. USA 105, 13–19 (2008).

    CAS  Article  Google Scholar 

  49. 49

    Gingras, A. R. et al. Mapping and consensus sequence identification for multiple vinculin binding sites within the talin rod. J. Biol. Chem. 280, 37217–37224 (2005).

    CAS  Article  Google Scholar 

  50. 50

    Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    CAS  Article  Google Scholar 

  51. 51

    Shtengel, G. et al. Imaging cellular ultrastructure by PALM, iPALM, and correlative iPALM-EM. Methods Cell Biol. 123, 273–294 (2014).

    Article  Google Scholar 

  52. 52

    Thompson, R. E., Larson, D. R. & Webb, W. W. Precise nanometer localization analysis for individual fluorescent probes. Biophys. J. 82, 2775–2783 (2002).

    CAS  Article  Google Scholar 

  53. 53

    Brown, T. A. et al. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol. Cell. Biol. 31, 4994–5010 (2011).

    CAS  Article  Google Scholar 

  54. 54

    Betzig, E. et al. Imaging intracellular fluorescent proteins at nanometer resolution. Science 313, 1642–1645 (2006).

    CAS  Article  Google Scholar 

  55. 55

    Thomas, W. A. et al. α-Catenin and vinculin cooperate to promote high E-cadherin-based adhesion strength. J. Biol. Chem. 288, 4957–4969 (2013).

    CAS  Article  Google Scholar 

  56. 56

    Giannini, A. L., Vivanco, M. M. & Kypta, R. M. Analysis of β-catenin aggregation and localization using GFP fusion proteins: nuclear import of α-catenin by the β-catenin/Tcf complex. Exp. Cell Res. 255, 207–220 (2000).

    CAS  Article  Google Scholar 

  57. 57

    Roczniak-Ferguson, A. & Reynolds, A. B. Regulation of p120-catenin nucleocytoplasmic shuttling activity. J. Cell Sci. 116, 4201–4212 (2003).

    CAS  Article  Google Scholar 

  58. 58

    Ravasio, A. et al. Regulation of epithelial cell organization by tuning cell-substrate adhesion. Integr. Biol. 7, 1228–1241 (2015).

    CAS  Article  Google Scholar 

  59. 59

    Hodgson, L., Nalbant, P., Shen, F. & Hahn, K. Imaging and photobleach correction of Mero-CBD, sensor of endogenous Cdc42 activation. Methods Enzymol 406, 140–156 (2006).

    CAS  Article  Google Scholar 

  60. 60

    Jobin, C. M. et al. Receptor-regulated dynamic interaction between endothelial nitric oxide synthase and calmodulin revealed by fluorescence resonance energy transfer in living cells. Biochemistry 42, 11716–11725 (2003).

    CAS  Article  Google Scholar 

  61. 61

    Hutson, M. S. et al. Forces for morphogenesis investigated with laser microsurgery and quantitative modeling. Science 300, 145–149 (2003).

    CAS  Article  Google Scholar 

  62. 62

    Meijering, E., Dzyubachyk, O. & Smal, I. Methods for cell and particle tracking. Methods Enzymol. 504, 183–200 (2012).

    Article  Google Scholar 

  63. 63

    Ravasio, A. et al. Gap geometry dictates epithelial closure efficiency. Nat. Commun. 6, 7683 (2015).

    Article  Google Scholar 

Download references

Acknowledgements

We thank C. Ajo-Franklin (Lawrence Berkeley National Laboratory), G. Shtengel and H. Hess (Howard Hughes Medical Institute, Janelia Research Campus) for generous help with super-resolution microscopy instrumentation and analysis. We thank H. Chen (Protein Expression Facility, Mechanobiology Institute, Singapore) for generation of mutant constructs, H. T. Ong and A. Sathe (Mechanobiology Institute) for assistance with FRET analysis, C. Zhang (Mechanobiology Institute) for assistance with 3D graphical model, and J. Yan (National University of Singapore) for critical reading of the manuscript. P.K., C.B., Y.Wang, A.R., T.S. and R.Z.-B. are supported by Singapore National Research Foundation Fellowship awarded to P.K. (NRF-NRFF-2011-04), R.Z.-B. (NRF-RF2009-RF001-074), and Competitive Research Programme (NRF2012NRF-CRP001-084) to P.K. and R.Z.-B. Y.T. is supported by Mechanobiology Institute and National University of Singapore Startup Grants, and a Singapore Ministry of Education Tier 2 grant (MOE2015-T2-1-116). B.L. is supported by the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007–2013/ERC grant agreement no. 617233), the Mechanobiology Institute, and the Institut Universitaire de France, and USPC-NUS funding. B.L. and R.-M.M. are also supported by CNRS, the Human Frontier Science Program (grant RGP0040/2012) and Agence Nationale de la Recherche (ANR 2010 Blan1515).

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C.B. and Y.Wang performed the super-resolution imaging experiments and conducted data analysis. C.B. and A.R. performed and analysed FRET experiments. C.B., A.R., Y.H. and Y.T. designed and C.B. performed and analysed laser ablation experiments. Y.Wu and R.Z.-B. performed imaging of Eph4 cell–cell junctions by astigmatism-based 3D super-resolution microscopy. C.B., T.S., M.B., M.W.D., B.L. and R.-M.M. designed and generated fusion constructs, and provided new reagents and analytical tools. C.B. and P.K. designed the study and wrote the manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to Pakorn Kanchanawong.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Planarized biomimetic cadherin substrate.

(a) Superresolution microscopy of Adherens Junctions (AJs) in epithelial monolayer. Astigmatism-based 3D superresolution microscopy of Eph4 cell monolayer, showing the organization of E-cadherin (Top left and second to left), zoomed area corresponds to top left inset, and other key proteins (all other panels, cell–cell contacts aligned approximately vertically). E-cadherin, β-catenin, α-catenin, vinculin, and myosin IIA were probed using primary and Alexa Fluor 647-labeled secondary antibodies. F-actin is probed by Alexa Fluor 647-phalloidin. Color bar: z-position from-375 to 375 nm. Scale bars: 5 μm (top left), 1 μm (all other panels). (b) Model of planarized biomimetic (N- or E-) cadherin-Fc substrate. Silanized glass coverslips or silicon wafers were coated by anti-Fc IgG, followed by purified cadherin-Fc chimeric protein. Cells were seeded onto the substrate in absence of serum to avoid extracellular matrix deposition so that adhesions were formed primarily via cellular cadherin engagement to the substrate-bound cadherin-Fc. (c) E-cadherin-based adhesions recruited canonical cell–cell junction proteins. MDCK cells expressing EGFP fusions of E-cadherin, p120 catenin, β-catenin, and α-catenin, spreading on E-cadherin-coated substrate, fixed and imaged after 3 h. Cadherin and associated proteins localize to clusters at cell edge and lamellipodia. Scale bar, 5 μm.

Supplementary Figure 2 Nanoscale fluorescence imaging methods.

(a) Schematics for interferometric PhotoActivated Localization Microscopy (iPALM). For details see Methods section. (b) iPALM calibration curve, measured by piezo-based translation along z-axis at 8-nm step. Amplitudes of a fluorescent fiducial observed in EMCCD 1-3 exhibit oscillation with mutual 120° phase differences. (c) Principles of surface-generated structured illumination, (VIAFLIC, Variable Incidence Angle Fluorescence Interference Contrast; or SAIM, Scanning Angle Interference Microscopy)20,21. (d) Intensity of the fluorescence excitation field as a function of incidence angle (θinc) and fluorophore z-position (nm). (e) Montage showing the variation of fluorescence intensity with θinc (degree). (f) Topographic z-position map of fluorophores (E-cadherin-EGFP expressed in MDCK cell). Z-position is calculated pixel-by-pixel by least-square fitting of the measured angle-dependence curve (white) to theoretical model (green), as show in g. (h) Notched box plots and histograms for z-position calculated from the median of each adhesion ROI: first and third quartiles, median and confidence intervals; whiskers, 5th and 95th percentiles. Histogram bin size, 1 nm. The median of this distribution is the representative protein z-position, zcentre. n = 352 adhesions (nROI) pooled from 20 cells (ncell). (i) Profile of E-cadherin distribution along the z-axis. Normalized histogram (black, 1-nm bin) of pixel z-position. Also shown are the decomposition into 3 Gaussian functions along with the Gaussian centers (zI, zII, zIII), widths (σzI, σzII, σzIII), and relative amplitude (AI, AII, AIII). Peak z-position of the distribution (zpeak) and number of pixels analyzed (npixel) indicated.

Supplementary Figure 3 iPALM superresolution images of F-Actin.

(a) F-actin organization at the edge of a MDCK cell cultured on planar cadherin substrate. (bd) Side view iPALM images of regions 1–3 in a. Yellow lines approximate the envelope of the actin bundles, angles indicated. Horizontal scale indicated by the scale bars; vertical (z) scale indicated by colour bars. e, Zoomed-out view of the cell shown in a. Red box, area shown in a. White box, area of cell–cell contacts shown in f. (g) Histogram of actin density (number of single-molecule localizations per 5-nm bin) for white box area in f. Red arrow in f indicates horizontal axis direction in g. Peaks at 300 and 450 nm bracket the intercellular space seen as low density gap in e,f. Bright dots surrounded by dark halos in a,e are due to substrate-embedded fluorescent nanoparticles used as fiducials for calibration and alignment. Color bars, z-position (0–150 nm) relative to substrate surface, shown in b-d. Scale bars: 1 μm (a,f), 250 nm (bd), 5 μm (e).

Supplementary Figure 4 Localization and expression of cadhesome proteins.

(a) Fluorescence micrographs of MDCK epithelial monolayer expressing fusion constructs. Protein names indicate the FP used and tag position (for example, E-cadherin-EGFP: C-terminal EGFP tag): E-cadherin-EGFP; EGFP-p120 catenin; EGFP-β-catenin;α-catenin-EGFP; vinexin-mEmerald; EGFP-vinculin; zyxin-mEos2; mEos2-VASP; palladin-mEmerald; α-eplin-eGFP;β-eplin-EGFP. Scale bar: 10 μm. (b) Western blots of cell lysates from MDCK (control), and MDCK with stable expression of vinculin shRNA (Vcl KD) and α-catenin shRNA (α-cat KD), probed for vinculin, α-catenin. Loading control, α-tubulin. (c) Abl1 kinase and PTP1B are expressed in both MDCK and C2C12 cells. Western blot analysis of lysates from MDCK and C2C12, probed for Abl1 and PTP1B, with α-tubulin as loading control. (d) Epifluorescence micrographs of MDCK cells seeded on E-cadherin-Fc substrate, stained with α18 antibody for activated α-catenin, β-catenin, and vinculin. (Top row) Inverted contrast, single channel images. (Bottom row) Merged 2-channel images of α-catenin (green) and β-catenin (red), and α-catenin (green) and vinculin (magenta). (Bottom right) Zoom-in view for area in centre panel, highlighting relative distribution of α-catenin and vinculin at cell edges. Scale bar, 10 μm (5 μm for bottom right panel). (e) Epifluorescence micrographs of C2C12 cells seeded on N-cadherin-Fc substrate, stained with α18 antibody for activated α-catenin and β-catenin. Single channel (inverted contrast) and merged 2-channel images of α-catenin (green) and β-catenin (red). Scale bar, 10 μm.

Supplementary Figure 5 Measurements of protein positions and orientation in cadherin-based adhesions.

(a) and (c) Notched box plots and histograms for z-position of indicated proteins in MDCK (a) and C2C12 (c) cells: first and third quartiles, median and confidence intervals; whiskers, 5th and 95th percentiles.n values (number of adhesions) are described in Supplementary Tables 1 and 2. Histogram bin size, 1 nm. (b) and (d) Topographic map of z-positions (nm) for proteins not displayed in Fig. 2 in cadherin-based adhesions of MDCK (b) and C2C12 (c) cells. Colour bar indicates z-position relative to substrate surface. Scale bar, 10 μm. (ef) Inference of protein orientation from z-position of fluorophores. Difference in Z-position of N-terminus (blue) and C-terminus (red) z-position, ΔZNC, is a z-projection of the N-C distance in the molecular frame, rNC. For oblique protein orientation (θ < 90°), ΔZNC < rNC, while for perpendicular orientation (θ = 90°), ΔZNC = rNC. (g) Inference of protein conformation from 3-point z-position measurement. The z-position of the N-terminus, ZN, C-terminus, ZC, and mTFP1 in vinculin-tension sensor, ZTS, serve as constraints that can be satisfied only by the compact vinculin conformation. Illustrations approximate FP positions in a coarse-grained model of vinculin. (h) Elongated conformation of vinculin-T12. Since rNC ≥ ΔZNC, the large (30 nm) observed for ΔZNC indicates an elongated conformation, and a large separation between VH and VT domain of vinculin, indicating a relief of autoinhibition.

Supplementary Figure 6 Spatial organization and configurations of vinculin and α-catenin in cadherin-based adhesions.

(a) Notched box plots for zcenter of indicated proteins in MDCK α-catenin KD cells: first and third quartiles, median and confidence intervals; whiskers, 5th and 95th percentiles. n values (number of adhesions) are indicated in Supplementary Table 3. Absence of α − catenin results in an upshifted of vinculin z-position to the actin compartment (blue and red column on left, for N- and C-terminal vinculin, respectively), rescued upon α-catenin re-expression (orange). Positioning of vinculin T12 in MDCK α-catenin KD cells could be due to β-catenin interaction with open vinculin. (b,c) Tension (b) or conformation (c) of vinculin as reported by FRET sensors for cells on soft (15 kPa) and stiff (glass, 2 GPa) planar cadherin substrate. Box plots: median, first and third quartiles. Representative frames from FRET measurement by acceptor photobleaching for, f, α-cat-conf or α-cat-conf ΔABD, expressed in MDCK or C2C12 cells; g,h Vinculin tension-sensor (Vcl-TS) and tailless tension-sensor control (Vcl-TLTS), i,j, Vinculin tail-probe conformation sensor (Vcl-conf) expressed in MDCK or C2C12 cells plated on stiff (glass, 2 GPa) and soft substrate (15 kPa), respectively. (Left columns), acceptor channel images (YPet, venus, YFP) and donor channel images (ECFP, mTFP1, CFP) for pre-bleach and post-bleach. (Right columns), plots of relative intensity changes for acceptor (red) and donor (blue), measured from the ROI (white boxes on left) for three consecutive frames after acceptor photobleaching. Data are represented as mean ± s.e.m.; n values (number of measurements) are indicated in blue in b and c. , P < 0.05, , P < 5 × 10−4. Welch’s t-test. Scale bar, 5 μm.

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Bertocchi, C., Wang, Y., Ravasio, A. et al. Nanoscale architecture of cadherin-based cell adhesions. Nat Cell Biol 19, 28–37 (2017). https://doi.org/10.1038/ncb3456

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