Post on 28-Feb-2022
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1. MATERIALS AND METHODS
Sample Preparation
The skin was removed from newborn mice and was placed in phosphate buffered saline
supplemented with 0.5 mM MgCl2 and 0.9 mM CaCl2. Pieces were cut ~2 mm in diameter and
immediately transferred into specimen carriers for high pressure freezing. Excess buffer was
removed with a paper point and the specimen chamber was filled with either 1-hexadecene, yeast
paste or 10% ficoll was added to eliminate air pockets; 1-hexadecene produced marginally better
results. Specimens were then frozen within 1 min in a high pressure freezer - either the Balzers
HPM 010 (Bal-Tec Products, Middlebury, CT) or the Leica EM PACT (Leica Microsystems Inc.,
Bannockburn, Ill.). In the latter case the operating pressure and rate of temperature change were
measured at ~2050 bar and ~17000°C/second, respectively. For freeze substitution, these
samples were placed in a solution of acetone containing 1% OsO4 and 0.1% uranyl acetate at -
99ºC for 6-8 hr, followed by -90ºC for 24-48 hr, -60ºC for 24 hr, and -30ºC for 18 hr;
temperature was maintained in a Balzers FSU 010 and, during transitions, was increased slowly:
2 hr for the first transition, 6 hr for the others. After slowly warming to 0ºC, the sample was
washed three times in pure acetone and warmed to room temperature. Tissue was carefully
separated from the sample carrier and transferred to a glass vial for infiltration with LX112 resin
(1:1 weight ratio of NMA and DDSA, Ladd Research Industries, Williston VT). Samples were
placed in flat molds and polymerization was done at 45ºC for 18-24 hr followed by 60ºC for 24-
48 hr. 30-70 nm sections were placed on 200 mesh fine bar hexagonal grids (Ted Pella, Inc.,
Redding CA) that were coated with formvar. These sections were stained with 3% uranyl acetate
in 70% methanol for 3 min, washed with water, stained with SATO lead stain for 2 min and again
rinsed with water. For tomographic data collection, sections were treated with 0.02% polylysine
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for 7-10 min, rinsed with water, and incubated 1-2 min with a solution of 10 nm colloidal gold
(Ted Pella, Inc., Redding CA) prior to a final water rinse. The colloidal gold was dispersed with
a bath sonicator for 10-15 min prior to application and its concentration was empirically adjusted
to provide a density of particles suitable for fiducial alignment. Finally, the section was coated
on both sides with 5-10 nm of evaporated carbon.
Electron Microscopy
Images were recorded with CM200FEG electron microscope (FEI Corp., Eindhoven,
Netherlands) equipped with a 1k x 1k Multiscan 794 CCD camera (Gatan Corp., Pleasanton CA)
using either the standard single-tilt specimen holder or a model 670 ultra high tilt holder (Gatan
Corp.). An automated system was used to collect the data consisting of EMACT (1) and
EMSCOPE (2). Samples were pre-irradiated with ~105 electrons/nm2 prior to collecting images
at 1º intervals either between +/-60º or +/-75º, depending of the holder. Dual axis tilt series were
obtained by removing the sample, rotating the grid ~90º, reinserting the sample and collecting a
second tilt series. Microscope magnification was 38 - 66kx, providing 0.55-0.93 nm/pixel after
binning the CCD images to 512x512. Defocus was 0.4-0.5 µm, which placed the first zero in the
contrast transfer function beyond the resolution limit of the images. The cumulative electron
dose during imaging was ~105 electrons/nm2.
Image analysis and 3D reconstruction
After assembling images into a single file with PRIISM (3), alignment was done with IMOD
(4) using 10-20 colloidal gold particles as fiducial markers. For dual-axis tilt series, each single-
tilt series was reconstructed individually with IMOD, aligned by cross-correlation and averaged
in real space. Although anisotropy in resolution is often a problem in tilt reconstructions by
electron microscopy, the use of dual-axis tilt series with full coverage up to +/-75 degrees made
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the corresponding artifacts negligible. Indeed, the definition of features is virtually
indistinguishable in X-Y and Y-Z sections. Fig S2 illustrates the effect of tilt range and of adding
the second tilt axis on anisotropy in the resolution. Effects of anisotropic resolution include the
presence of a halo, the elongated shapes of gold particles, and conical streaking in Y-Z sections;
all are quite evident in single-tilt axis reconstructions with a range of +/-60°. Increasing the tilt
range to +/-75°, or adding a second axis both produce significant improvements. The best results
are obtained by using dual-axis tomography with a range of +/-75º.
Segmentation and modeling
Overall, image tilt series were collected from 14 different desmosomes, 8 of which were
selected for tomographic reconstruction and 5 of which were used for delineation of the
molecular components. The reconstruction was segmented with AMIRA (TGS Inc., San Diego,
CA) in an iterative process that involved delineating molecular densities and fitting the x-ray
structure of C-cadherin 1L3W (5). Three videos are included as part of these supporting online
materials: the first two show intercellular densities as one scans through the sections composing
the 3D map of desmosomes "R" and "P", and the third shows the verification of the atomic fit
relative to the densities within the tomogram for desmosome "P". Even casual inspection of the
tomograms reveals distinct densities crossing the intercellular space, which are consistent with
the size of cadherin molecules (videos 1 and 2). Initially, this region of the tomogram was
displayed with a rather high density threshold to reveal numerous curved densities running
between the membrane surface and the midline. Depending on the threshold, a portion of these
densities were discontinuous, making the fit within a group of tangled molecules ambiguous.
These discontinuous densities were used for initial fitting of C-cadherin x-ray structures, starting
with well separated densities near the membrane surface and attempting to follow the relevant
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density into the midline. Fitting only involved rigid-body docking of the entire x-ray structure
without any changes of domain geometries or secondary structure. After fitting several
molecules within a local region, lower density thresholds were considered in refining the
molecular envelopes and the fitting was reevaluated to minimize molecular clashes and to
resolve ambiguities about density assignments within the midline. These assessments involved
scrolling back and forth through the original map to ensure that the original stain distribution
accurately represented the location of the molecules (video 3). This fitting accounted for the vast
majority of densities in the map, though some density remained near the membrane surface. It is
possible that this extra density corresponds to the E48 antigen, which is a GPI anchored protein
of 128 residues that is expressed in keratinocytes and squamous tumor cells (6) and that co-
purifies with desmosomes from bovine muzzle (7). This protein belongs to the Ly-6 family and
has been shown to function in selectin signaling pathway (8, 9). Although early immunolabeling
studies suggested its presence in the midline, its small size and GPI anchor are more likely to
place it next to the surface of the membrane.
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2. SUPPORTING TEXT
Comparisons of Cadherin Sequence
The N-terminal domain (EC1) has the highest sequence homology across the cadherin
family with N-cadherin, for example, showing 55% identity to C-cadherin and 37% identity to
desmosomal cadherins; identities for the entire extracellular region (EC1-EC5) are 44% between
N- and C-cadherin and 32-37% between these and the desmosomal cadherins (Table S1). This
high level of conservation is consistent with a common structure for classical and desmosomal
cadherins (10), especially considering that sequence identities amongst individual EC domains of
classical cadherins are considerably lower (e.g., 5-24% for C-cadherin, Table S2 ) yet have been
shown to adopt a consistent fold (5). This fold is likely stabilized by a conserved pattern of Pro,
Gly and hydrophobic core residues, which also extends to the more distantly related proteins in
the immunoglobulin family (10, 11). A lower conservation for the fifth extracellular domain
(EC5) reflects its unique features, which include a series of conserved cysteines and a substantial
truncation and genetic polymorphism in desmoglein (12, 13).
Flexibility between EC Domains
Local variations in the cadherin densities within our tomographic map suggest molecular
flexibility that is consistent with variability in the angle between individual EC domains in x-ray
structures. There is good evidence in the literature to support flexibility in the angles between
individual EC domains. In particular, the angle between these domains is variable, as shown
both by comparison of two-domain structures from N-and E-cadherins (14) and by pairwise
comparison of domains composing the EC1-EC5 structure of C-cadherin. The latter shows
interdomain angles from 106-137º with an ~180º disparity in the skew angle of EC3/EC4 relative
to the others (5). The linker regions between successive EC domains represent hinges for this
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flexibility and calcium binding by these linkers appears to modulate the magnitude of this
flexibility. As a result, calcium has documented roles in progressively straightening of the
molecule (15) and in reducing proteolytic susceptibility (16). On an atomic level, these roles
reflect the recruitment of side chains from the adjacent domains to these calcium binding sites,
the geometry of which enforces an obtuse angle between domains and structures the linker
peptide.
Flexibility in the Intermolecular EC1 Domain Interface
A different kind of structural flexibility is evident in the intermolecular EC1 interactions that
we have identified from our desmosome maps. For molecules involved in W-, S- and λ-shapes,
there is considerable variability in the angle between interacting molecules (Fig. S5). Similar
variability was seen in the W-shape in x-ray structures from various crystal forms displaying
intermolecular angles ranging between 54 and 88º (5). This observation is consistent with the
innate flexibility in the N-terminus of EC1, which is further manifested by its variable
disposition in the x-ray structures. Specifically, although the short β-strand at the N-terminus
was involved in stabilizing the first strand dimer seen in EC1 constructs of N-cadherin (17), the
latest C-cadherin structure shows no such N-terminal strand involved in the strand-dimer
exchange (5). In fact, the first 6 residues (DWVIPP) do not form hydrogen bonds to any other
piece of secondary structure. Furthermore, the main chain of these N-terminal residues is also
unconstrained in structures of EC1/EC2 constructs from both E- and N-cadherins, where the
critical Trp is either disordered or inserted into its own hydrophobic pocket, respectively (15,
18). Thus, the primary stabilizing element of the EC1 intermolecular interface would appear to
be the Trp side chain within the hydrophobic pocket.
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3. SUPPORTING FIGURES
Fig. S1. Projection image of an ultra-thin (~35 nm) section from skin prepared with high
pressure freezing and freeze sustitution. Although this section is too thin for the tomographic
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analysis of cadherin packing, the intermediate filaments and extracellular domains of cadherins
are more clearly seen in the projection image due to minimal superposition. In many cases,
individual cadherin molecules can be seen crossing from the membrane to the midline. The
white bar corresponds to 500 nm.
Fig. S2. Determinants of isotropic resolution. (A-B) X-Y slices from samples K and R,
respectively (Table 1). Sample K has a tilt range of +/-60° and white rings are visible around the
gold particles, reflecting artifacts from missing data (i.e., tilt angles between 60-90°). Sample R
has a tilt range of +/-75° and as a result these artefacts are much diminished. Images were
recorded at 38kx and 66kx respectively, accounting for the apparent difference in gold particle
size. (C-D) Y-Z sections from single-axis tilt series with tilt range +/- 60° show significant
artifacts from the missing wedge of data, namely a white zone around the gold particles at the
top of the section and conical tails coming down at 30° angles from each gold particle. (E-F) Y-Z
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sections from dual-axis tilt series with tilt range +/- 60° showing a reduction of the above-
mentioned artifacts. (G-H) Y-Z sections from single axis tilt series with tilt range +/- 75 degrees.
Main artifacts are a white zone around the gold particles and a slightly elongated shape of these
spherical particles, which reflects anisotropic resolution. (I-J) Y-Z sections from dual-axis tilt
series with tilt range +/-75°. Artifacts are minimal with round gold particles and minimal white
zone. The resolution of features within the section also appears to be isotropic, which allows for
the most accurate delineation and fitting of individual molecules.
Fig. S3. Fitting of molecules to desmosome P. (A) An alternative configuration for a 4-way
molecular networks that consists of a λ-shape plus one additional molecule in trans. (B-C)
Groups of molecules at the midline consist of two or more networks that provide the potential for
a variety of alternative, uncharacterized intermolecular interactions.
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Fig. S4. C-cadherin molecules fitted to desmosome P. (A) α-carbon trace of 136 molecules after
fitting to the densities shown in Fig. 2A. (B-C) EC5 domains on apposing extracellular
membrane surfaces show a tendency to form pairs (some of which are marked with red circles).
Molecules along the upper, left border of this surface are missing because this region was not
fitted with the x-ray structure.
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Fig. S5. Intermolecular interactions between EC1 domains have a variable geometry. (A)
Archetypal W-, S- and λ-shapes with molecules 0 and 1 representing a W-shape with
symmetrical Trp2 strand exchange. Molecules 2 and 3 form alternative S-shapes with molecule
0: 2 acts as a donor of Trp2, whereas 3 acts as an acceptor of Trp2. Similarly, molecules 4 and 5
form alternative λ-shapes with molecule 0, with 4 acting as Trp2 donor and 5 as Trp2 acceptor.
(B) Range of orientations of molecules assigned to the W-shape with the red pair corresponding
closely to that seen in the C-cadherin x-ray structure (5). For the most part, these molecules are
related by a rotation within the plane of the image. (C-D) Range of orientations for molecules
acting as Trp2 donors for the S- and λ-shapes with the magenta and green molecules
corresponding to those in (A). These molecules are roughly related by rotations about a vertical
axis running through the molecular interface.
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4. SUPPORTING TABLES
Table S1. Sequence identities between cadherins and their EC1 domains.
Numbers below the diagonal correspond to % sequence identity for the entire extracellular
portion of the molecule, whereas numbers below the diagonal refer to only the EC1 domain.
Alignments and identity determination were peformed by CLUSTALW. Sequences were all
from mouse, except C-cadherin which was from Xenopus. Accession numbers were N-cadherin:
AAH22107, C-cadherin: IJXLCP, Desmocollin 1: NP_038532 and Desmoglein 2: NP_031909.
N-cadherin C-cadherin Desmocollin Desmoglein
N-cadherin - 55 37 37
C-cadherin 44 - 37 34
Desmocollin 34 37 - 35
Desmoglein 32 32 31 -
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Table S2. Sequence identities amongst individual EC domains of N- and C-cadherin.
Numbers above the diagonal correspond to % sequence identity for EC domains of C-cadherin,
whereas those below the diagonal refer to N-cadherin. Sequence information and alignment
methods are described in Table II.
EC1 EC2 EC3 EC4 EC5
EC1 - 24 10 20 13
EC2 30 - 7 20 5
EC3 20 14 - 15 11
EC4 21 15 10 - 5
EC5 6 11 2 11 -
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5. SUPPORTING REFERENCES AND NOTES
1. J. C. Fung et al., J. Struc. Biol. 116, 181 (1996).
2. N. Kisseberth, M. Whittaker, D. Weber, C. S. Potter, B. Carragher, J. Struc. Biol. 120, 309
(1997).
3. H. Chen, D. D. Hughes, T. A. Chan, J. W. Sedat, D. A. Agard, J. Struc. Biol. 116, 56 (1996).
4. J. R. Kremer, D. N. Mastronarde, J. R. McIntosh, J. Struc. Biol. 116, 71 (1996).
5. T. J. Boggon et al., Science 296, 1308 (2002).
6. R. H. Brakenhoff et al., J. Cell Biol. 129, 1677 (1995).
7. A. H. Schrijvers et al., Exp. Cell Res. 196, 264 (1991).
8. R. Eshel et al., J. Biol. Chem. 275, 12833 (2000).
9. R. Eshel et al., Int. J. Cancer 98, 803 (2002).
10. L. Shapiro, P. D. Kwong, A. M. Fannon, D. R. Colman, W. A. Hendrickson, Proc. Natl.
Acad. Sci. U.S.A. 92, 6793 (1995).
11. M. Overduin et al., Science 267, 386 (1995).
12. F. Nollet, P. Kools, F. van Roy, J. Mol. Biol. 299, 551 (2000).
13. S. Puttagunta, M. Mathur, P. Cowin, J. Biol. Chem. 269, 1949 (1994).
14. K. Tamura, W. S. Shan, W. A. Hendrickson, D. R. Colman, L. Shapiro, Neuron 20, 1153
(1998).
15. O. Pertz et al., EMBO J. 18, 1738 (1999).
16. M. Takeichi, J. Cell. Biol. 75, 464 (1977).
17. L. Shapiro et al., Nature 374, 327 (1995).
18. B. Nagar, M. Overduin, M. Ikura, J. M. Rini, Nature 380, 360 (1996).
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6. CAPTIONS TO MOVIES
Video 1. Scanning through the sections composing the tomographic map of desmosome
"R". This same map was used for Fig. 1e,f. These sections have been cut parallel to the untilted
sample, same as Fig. 1e and the movie therefore starts and ends with the upper and lower faces
of the section. Densities have been segmented with colored lines both in the intercellular space
and in the cytoplasmic plaque; colors used are blue for cadherins, red for the membrane, orange
and light green for the two zones composing the cytoplasmic plaque, and dark green for the
intermediate filaments. Discrete densities are clearly seen crossing the intercellular space that
correspond to cadherin molecules. The large black circles at one end of the section correspond to
the colloidal gold particles that were deposited on one surface of the section.
Video 2. Scanning through the sections composing the tomographic map of desmosome "P".
This map was used for extensive molecular delineation and fitting depicted in Fig. 2. These
sections have been cut parallel to the untilted sample as in Fig. 1e and in video 1. Segmentation
is shown in the forward half of the movie for the membrane (cyan) and a significant number of
individual cadherin molecules (various colors). The colloidal gold particles are visible at one
surface of the section and their larger size reflects the higher magnification used for desmosome
"P" relative to "R" (see Table 1).
Video 3. This video illustrates the use of Amira to compare segmented densities as well as
fitted coordinates with the original densities in the original tomogram. Sections through the
tomogram appear as black and white and are scanned backward and forward to intersect with the
3D shapes of segmented densities and fitted coordinates. Although the movie starts with the
majority of desmosome P, it focuses on several molecules toward one end of the desmosome,
which includes some of the same molecules shown in Fig. 2b. As expected, high densities are
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present in the map wherever the section intersects the 3D shapes. This is shown for both X-Y
and X-Z sections.