Nanoscale architecture of cadherin-based cell adhesions
Cristina Bertocchi
1
, Yilin Wang
1,8
, Andrea Ravasio
1
, Yusuke Hara
1
, Yao Wu
1
, Talgat
Sailov
1,9
, Michelle A. Baird
2,10
, Michael W. Davidson
2,3,#
, Ronen Zaidel-Bar
1,4
, Yusuke
Toyama
1,5,6
, Benoit Ladoux
1,7
, Rene-Marc Mege
7
, and Pakorn Kanchanawong
1,4,*
1
Mechanobiology Institute, Singapore, Republic of Singapore, 117411
2
National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL, USA,
32310
3
Department of Biological Science, The Florida State University, Tallahassee, FL, USA, 32306
4
Department of Biomedical Engineering, National University of Singapore, Republic of Singapore,
117583
5
Department of Biological Sciences, National University of Singapore, Singapore, Republic of
Singapore, 117543
6
Temasek Life Sciences Laboratory, National University of Singapore, Singapore, Republic of
Singapore, 117604
7
Institut Jacques Monod, Université Paris Diderot and CNRS UMR 7592, Paris, France
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 superresolution 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 upon 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
*
Correspondence: biekp@nus.edu.sg.
8
Current addresses: Department of Biology, South University of Science and Technology, Shenzhen, China
9
Singapore Centre on Environmental Life Sciences Engineering, Nanyang Technological University, Singapore, Republic of
Singapore
10
National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA
#
deceased
Author Contributions C.B. and Y.Wang performed the superresolution 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 3-D superresolution microscopy.
C.B., T.S., M.B., M.W.D., B.L., and R.M.M. designed and generated fusion constructs, 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.
Competing Financial Interests
The authors declare no competing financial interests.
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Published in final edited form as:
Nat Cell Biol
. 2017 January ; 19(1): 28–37. doi:10.1038/ncb3456.
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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.
Introduction
The question of how animal cells self-organize into complex and patterned structures at the
tissue and organism levels are intrinsically multiscale, depending on an intricate interplay of
local and long-range forces within tissues and cells, as well as exquisite coordination of sub-
cellular programs ranging from genetic and signaling pathways to cell morphodynamic
behaviors1,2. While recent advances in the understanding of these processes have
prominently focused at the length scale of tissues and cells3,4, much has remained
unexplored at the level of the very molecular machines that enable intercellular adhesions,
cytomechanical adaptation, and mechanotransduction processes underlying these
morphogenetic events. Cell-cell junctions mediated by the cadherin transmembrane
receptors are among the most important molecular machinery that interlink and coordinate
neighboring cells, participating in important cellular pathways including transcriptional
control, cell polarization, cytoskeletal regulation, and cellular mechanotransduction5–10.
Adhesions of cadherin recruit numerous proteins, collectively known as ‘cadhesome’11, to
form supramolecular complexes closely associated with the actin cytoskeleton. However, the
nanoscale dimension and the compositional complexity of the cadherin adhesions have long
defied available structure-determination or imaging techniques, and thus the structural
framework for understanding how such complex multiprotein assembly is physically
organized to perform biological functions has not been available.
Previously, astigmatism-based 3-D superresolution microscopy12 has been applied to
resolve nanocluster organization of cadherins in adherens junctions (AJs) where neighboring
epithelial cells form contact sites13,14. However, the spatial resolution thus attained,
>20-100 nm, poses a challenge for quantifying protein organization at the sub-20 nm
molecular length scale. Likewise, it has been difficult to decipher molecular organization of
cadhesome proteins from electron microscopy (EM) images15,16. Therefore, to provide a
structural framework for understanding cadherin-based cell adhesions, we adopted a
planarized biomimetic platform based on oriented cadherin-F
c
arrayed on IgG-coated
substrates17. This format confers a greater optical accessibility amenable to high-precision
(sub-20 nm) superresolution fluorescence microscopy techniques18–22, allowing molecular
scale interrogation with current fluorescent protein (FP) technologies.
In this study, we mapped protein organization within planar cadherin-based adhesions,
observing a compartmentalized nanoscale architecture, whereby the plasma membrane-
proximal cadherin-catenin compartment is physically segregated by ˜30 nm from the
uppermost compartment containing actin and actin regulatory proteins, bridged by an
interface compartment containing vinculin. We showed that the nanoscale positioning of
vinculin is determined by α-catenin. Upon conformational activation, vinculin extends ˜30
nm to bridge the cadherin-catenin and actin compartments, while also modulating the
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nanoscale positions of the actin polymerization regulators zyxin and VASP. The extended
conformation of vinculin requires both tension and tyrosine phosphorylation at residue Y822
by Abl kinase, while we also identified PTP1B as the tyrosine phosphatase that
dephosphorylates vinculin. The observed multi-layer nanoscale architecture of cadherin-
based adhesions appears to centrally position vinculin to act as an integrator of mechanical
and biochemical signals, suggesting how the cadherin-based adhesions could selectively
engage the actin cytoskeleton in response to regulatory input signals, effectively as a
‘molecular clutch’, to mediate intercellular interactions.
Results
Mapping protein position in planar cadherin-based adhesions by superresolution
microscopy
The multi-micron vertical (z) depth of the AJs in epithelial monolayer limited our ability to
map molecular scale organization by astigmatism-based superresolution microscopy14
(Supplementary Fig. 1a). We noted that the planar cadherin-coated substrate format have
been employed in earlier studies17,23–25 to obtain key molecular insights into interactions
between cadherin and associated proteins. On such platform cells formed cadherin-based
adhesions that recruited cadhesome proteins but not integrin-associated proteins
(Supplementary Fig. 1b-c), suggesting that salient protein-protein interactions are likely
recapitulated. To demarcate the plasma membrane position in this format, we first applied 3-
D Interferometric PhotoActivated Localization Microscopy19 (Supplementary Fig. 2a-b) to
image MDCK (Madin-Darby Canine Kidney) epithelial cells cultured on E-cadherin-coated
substrate, using DiD membrane-targeting fluorophores26. This clearly resolved dorsal and
ventral plasma membranes, with the z-position of the latter at ˜30-40 nm above the substrate
(Fig. 1a-c). We then imaged filamentous (F)-actin using AlexaFluor 647-phalloidin,
observing that F-actin bundles reside at a higher z-position, centering around ˜70-80 nm,
(Fig. 1d-e). The spatial separation of ˜30 nm between the ventral plasma membrane and the
actin cytoskeleton thereby minimizes direct cadherin-actin interaction. The F-actin angle of
approach is nearly parallel to the adhesion plane (Supplementary Fig. 3a-d), geometrically
comparable to the F-actin orientation around AJs4. Altogether these data are suggestive of
the nanoscale similarity between planar cadherin adhesions and native cell-cell contacts.
Nanoscale compartmentalization of E-cadherin-based adhesions
We next applied a surface-generated structured illumination technique20,21 (Supplementary
Fig. 2c-g) to characterize nanoscale organization of FP-conjugated cadhesome proteins
(Supplementary Fig. 4a). The fluorophore z-position relative to the substrate surface (z= 0
nm) was analyzed pixel-wise, with the median value, z
centre
, for adhesion region-of-interests
(ROI) used as the representative protein z-position27, while the z-position histograms denote
the spatial distribution of proteins (Fig. 3, Supplementary Note 1.3, Supplementary Fig.
2h,i). We observed that E-cadherin (cytoplasmic domain GFP fusion) is positioned at z
centre
= 46.6 nm, consistent with the dimensions of cadherin and other substrate components (Fig.
2b, 3a).
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Subsequently, we surveyed the nanoscale organization of key cadhesome proteins, observing
a surprising degree of compartmentalization along the z-dimension that effectively spans
between the plasma membrane and the actin cytoskeleton. Proteins observed in close
proximity to cadherin include p120-catenin (44.6 nm), β-catenin (57.5 nm, N-terminus; 50.4
nm, C-terminus), and α-catenin (40.2 nm, N-terminus). Their z-positions are consistent with
their close association with the E-cadherin cytodomain6, thus defining the cadherin-catenin
compartment. In contrast, actin-binding proteins were observed at significantly elevated z-
positions, largely coinciding with the actin cytoskeleton. Eplins were found at 93.6 nm (α-
isoform) and 87.2 nm (β-isoform), palladin at 94.2 nm, and α-actinin at 112.2 nm. A
number of proteins were observed at intermediate z-positions, including vinculin (54.1 nm,
N-terminus), zyxin (65.5 nm), VASP (vasodilator-stimulated protein; 66.5 nm), and vinexin
(64.5 nm, N-terminus; 63.1 nm, C-terminus) (Fig. 2a-b, 3a, Supplementary Figure 5,
Supplementary Tables 1-2). Our measurements suggest that these centrally-positioned
proteins likely play an important role as an interface compartment that mediates structural
connection and mechanical coupling between the cadherin-catenin and the actomyosin
compartments.
The conformation and nanoscale organization of
α-catenin
Since α-catenin and vinculin have been implicated as mechanotransducers28–31, we next
investigated their configurations and spatial organization within the cadherin adhesions.
Using a monoclonal antibody (α18) against the activated conformation of α-catenin32, we
observed prominent staining (Fig. 4b, Supplementary Fig. 4d-e) consistent with
measurements by fluorescence resonance energy transfer (FRET) conformation probe9 (Fig.
4c, Supplementary Fig. 6d). Furthermore, the high precision of our technique enables
inference of protein orientation and/or conformation via the use of the N- and C-terminal FP
fusion constructs (Supplementary Note 2, Supplementary Fig. 5e-h). We thus determined the
C-terminal z-position of α-catenin, obtaining z
centre
= 53.3 nm, compared to z
centre
= 40.2
nm for the N-terminus, indicative of an oriented and activated configuration (Fig. 3c-e, Fig.
4d, Supplementary Note 3). We next probed the z-position of the α-catenin vinculin binding
domain (VBD) by imaging vinculin head domain (Vd1, residue 1-258) N-terminal-tagged
with GFP, observing the z-position of 57.1 nm closely overlapping with the α-catenin C-
terminus, consistent with vinculin association to α-catenin (Fig. 4d). To further explore the
role of α-catenin in vinculin positioning, we imaged vinculin-FP expressed in MDCK cells
with stable α-catenin shRNA expression33 (Supplementary Fig. 4b). We found that with α-
catenin depleted, vinculin localizes to a higher z-position within the actomyosin
compartment, probably via the association with actin34 or actin-regulatory proteins such as
VASP or α-actinin. On the other hand, upon re-expression of α-catenin-FP, the intermediate
z-positioning of vinculin is restored (Supplementary Fig. 6a, Supplementary Note 3).
Activated vinculin spans between the cadherin-catenin and actin cytoskeletal
compartments
We next characterized how vinculin is organized within the cadherin adhesions. In MDCK,
wild-type (wt) vinculin C-terminus was observed at z
centre
= 59.6 nm, compared to 54.1 nm
for the N-terminus (Fig. 2b). Since vinculin conformation is able to switch between the
compact and the extended, uninhibited forms, the small N-C z-positional differences
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observed may correspond to either a relatively compact conformation, or an extended
conformation that is oriented nearly parallel to the plasma membrane (<10°). To differentiate
these, we first probed vinculin configuration by FRET. Using vinculin tension biosensor
(vinculin-TS)29 or vinculin conformation biosensor35, we observed comparatively high
FRET efficiencies, indicative of low vinculin tension and a relatively compact conformation,
respectively (Fig. 4f-g, Supplementary Fig. 6b-c,e-h). Additionally, the z-position of the
mTFP1 fluorophore within vinculin-TS (Fig . 4a) was mapped to report the z-position of the
vinculin linker region, obtaining z
centre
of 67.6 nm, further supporting a compact
conformation of vinculin, with the linker region arching upward (Fig. 4d-e, Supplementary
Fig. 5e-h, Supplementary Note 2). In contrast, when we probed the orientation of the
constitutively active vinculin-T12 mutant36, we observed a drastic upshift of the C-terminal
z-position (85.8 nm) relative to the N-terminus (53.7 nm) (Fig. 4d-e). Thus, activated
vinculin effectively spans ˜30 nm or greater, a distance comparable to the fully extended
length of vinculin37. Taken together, these results illustrate how α-catenin emplaces
vinculin in the interface zone, a central position that enables activated vinculin to robustly
couple the cadherin-catenin with the actomyosin compartments (Supplementary Note 3.4).
We also note that a more subtle interplay between α-catenin, β-catenin, and vinculin
configuration may also be present (Supplementary Fig. 6 a, Supplementary Note 3.2-3.3).
Our approach may thus be of further use in unraveling how the cadherin:β-catenin:α-catenin
module is structurally and mechanically integrated with the cortical actin
cytoskeleton15,23,28, an important long-standing question, but that which is beyond the
scope of the current study.
Vinculin conformation modulates nanoscale position of zyxin and VASP
We next investigated how vinculin conformational states may regulate the spatial
organization of other cadhesome components. We performed 2-color nanoscale z-mapping
experiments by imaging FP fusion of vinculin partners such as zyxin or VASP, together with
vinculin-T12. As shown in Fig. 4h-j, zyxin and VASP, in the presence of endogenous
vinculin (wt), were observed at z
centre
= 65.5 nm and z
centre
= 66.5 nm, respectively. In
contrast, upon co-expression with vinculin-T12, we observed significant upshifts in their z-
positions with z
centre
= 75.8 nm for zyxin and z
centre
= 82.9 nm for VASP. These results
suggest that vinculin conformation may modulate the positioning of proteins such as zyxin
and VASP that bind to its proline-rich linker region, which may in turn help promote actin
polymerization in cadherin adhesions5 (Supplementary Note 3.4).
Vinculin conformational switch is regulated by Abl kinase and PTP1B phosphatase
To probe how vinculin conformational transition is regulated, we mapped the C-terminal z-
position of vinculin (wt) under various biochemical or pharmacological perturbations. We
observed that vinculin remains in the compact state with the overexpression of contractility
effectors such as myosin IIA, myosin IIB, activated Src, or constitutively-active RhoA.
Interestingly, the overexpression of constitutively-active Rac1 or cdc42 led to even lower C-
terminal z-positions of vinculin of ˜ 46 nm, coinciding with the membrane-proximal
cadherin-catenin layer (Fig. 5a, Supplementary Table 3). This probably results from the
vinculin-tail (V
T
) phospholipid interaction31, and will be investigated in a more thorough
manner separately. Intriguingly, we found that the inhibition of tyrosine phosphatases by
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