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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.

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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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Contributions

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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