Research Article
3299
Cadherin point mutations alter cell sorting and
modulate GTPase signaling
Hamid Tabdili1,*, Adrienne K. Barry2,*, Matthew D. Langer1,*, Yuan-Hung Chien2,`, Quanming Shi1,§,
Keng Jin Lee1,", Shaoying Lu4 and Deborah E. Leckband1,2,3,**
1
Department
Department
Department
4
Department
2
3
of
of
of
of
Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, IL 61801, USA
Biochemistry, University of Illinois, Urbana-Champaign, IL 61801, USA
Chemistry, University of Illinois, Urbana-Champaign, IL 61801, USA
Bioengineering, University of Illinois, Urbana-Champaign, IL 61801, USA
*These authors contributed equally to this work
`
Present address: Salk Institute, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA
§
Present address: California Institute for Quantitative Biosciences, University of California at Berkeley, Berkeley, CA 94720, USA
"
Present address: Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA
**Author for correspondence (leckband@uiuc.edu)
Journal of Cell Science
Accepted 22 March 2012
Journal of Cell Science 125, 3299–3309
2012. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.087395
Summary
This study investigated the impact of cadherin binding differences on both cell sorting and GTPase activation. The use of N-terminal
domain point mutants of Xenopus C-cadherin enabled us to quantify binding differences and determine their effects on cadherindependent functions without any potential complications arising as a result of differences in cytodomain interactions. Dynamic cell-cell
binding measurements carried out with the micropipette manipulation technique quantified the impact of these mutations on the twodimensional binding affinities and dissociation rates of cadherins in the native context of the cell membrane. Pairwise binding affinities
were compared with in vitro cell-sorting specificity and ligation-dependent GTPase signaling. Two-dimensional affinity differences
greater than five-fold correlated with cadherin-dependent in vitro cell segregation, but smaller differences failed to induce cell sorting.
Comparison of the binding affinities with GTPase signaling amplitudes further demonstrated that differential binding also proportionally
modulates intracellular signaling. These results show that differential cadherin affinities have broader functional consequences than
merely controlling cell-cell cohesion.
Key words: Cadherin, Cell sorting, GTPase, Micropipette manipulation, Recognition
Introduction
The intercellular adhesion cadherin proteins are essential for
maintaining the structural integrity of tissues. During
morphogenesis, they are required for cell patterning, and, in
mature tissues, they regulate crucial barrier functions (Gumbiner,
2005; Takeichi, 1995). The classical cadherins are the most
extensively studied proteins in the cadherin superfamily. There are
.20 known subtypes, which exhibit the same overall fold, but differ
in their primary structure and tissue expression patterns. A central
question is whether subtype-dependent sequence differences alter
cadherin-mediated intercellular binding, and the implications of
those differences for cadherin-dependent cell functions.
Investigations of differences between cadherin subtypes mainly
focused on cadherin-dependent cell segregation. This is in part
due to important in vitro studies suggesting that cell sorting
depends on both the identities and surface densities of the
expressed cadherin subtypes (Nose et al., 1988; Steinberg, 1963;
Steinberg, 2007). Those findings suggested that that subtypedependent differences in intercellular adhesion energies direct
cell sorting in vitro and possibly in vivo (Steinberg, 1963). This
focused attention on cadherin affinities and their relationship
to adhesion energies or surface tension thought to influence
cell segregation (Foty and Steinberg, 2005; Steinberg, 1963;
Steinberg, 2007).
The N-terminal cadherin domain is the main locus of cadherin
binding differences that influence in vitro sorting. In structures of
complexes of the extracellular segment of Xenopus C-cadherin
(Boggon et al., 2002) and truncated fragments of N-cadherin (Shan
et al., 2000; Shapiro et al., 1995) or E-cadherin (Häussinger et al.,
2004; Pertz et al., 1999; Tomschy et al., 1996), the W2 on the first
extracellular domain (EC1) inserts into a hydrophobic pocket on
the EC1 domain of the adjacent cadherin. The high degree of
sequence similarity among EC1 domains of type I classical
cadherins begs the question of how this conserved binding motif
supports cell binding selectivity. Yet, mutations in the W2 binding
pocket alter cell-cell cohesion and sorting. Exchanging the Nterminal domain of E-cadherin with that of P-cadherin, or
substituting residues 78 and 83 on mouse E-cadherin with the
corresponding P-cadherin sequence altered the aggregation
specificity of cells expressing the E-cadherin mutants (Nose
et al., 1990). The A78M mutation abolished N-cadherin function
(Tamura et al., 1998). Despite these qualitative observations, links
between sequence differences, quantified affinities, and cadherindependent functions have not been established.
Solution binding affinities of recombinant, soluble fragments
indicated that affinities differing by at least 5 fold correlated with
in vitro cell sorting, assuming similar cadherin expression levels
(Katsamba et al., 2009). However, semi-quantitative estimates of
Journal of Cell Science
3300
Journal of Cell Science 125 (14)
relative cell adhesion (Niessen and Gumbiner, 2002), quantified,
protein-level adhesion energies (Prakasam et al., 2006b),
strengths of single cadherin bonds (Shi et al., 2008), or
cohesive energies of cell aggregates (Duguay et al., 2003) do
not always correlate with in vitro cell sorting outcomes.
In vivo, the role of cadherin binding differences in cell sorting
is less clear. Differential cadherin expression correlates with
retinal cell patterning in Drosophila, for example (Hilgenfeldt
et al., 2008). Yet, cortical tension, rather than cell cohesion,
appears to direct germ cell positioning in zebrafish embryos
(Krieg et al., 2008). A possibility is that differential adhesion is
unimportant in vivo, but correlations between mutations that
impair cadherin adhesion and gastric cancer (Becker et al., 1999;
Handschuh et al., 1999; Handschuh et al., 2001) suggest that
altered cadherin adhesion also modulates signaling. Differential
cadherin adhesion could more broadly influence cell behavior
through signaling. For example, affinity-dependent Rho GTPase
signal amplitudes (Braga, 2002; Drees et al., 2005; Noren et al.,
2003) could also modulate cortical tension, cell cycle
progression, and differentiation (Fournier et al., 2008;
Levenberg et al., 1999). The broader impact of such binding
differences on both sorting and signaling has not been
considered.
This study investigated the impact of cadherin binding site
mutations on two-dimensional affinities, in vitro cell sorting, and
GTPase signaling. Comparisons of ectodomain mutants rather
than cadherin subtypes enabled us to focus on affinity
differences, independent of differences in cytoplasmic domain
interactions. The use of cadherins that cause Chinese Hamster
Ovary (CHO) cells to segregate from each other (Shi et al., 2008),
also facilitated determinations of the impact of binding
differences on cell sorting. Selected Xenopus C-cadherin
mutants were based on sequence differences between amino
acids near docked W2 in the hydrophobic pocket of N-cadherin.
Micropipette measurements then quantified the affinities of fulllength C-cadherin mutants in the native context of the cell
membrane. These cadherin properties were compared with both
in vitro cell sorting outcomes and ligation-dependent GTPase
signaling (Becker et al., 1999; Handschuh et al., 1999;
Handschuh et al., 2001).
Results
Design and expression of C-cadherin mutants
CHO cells that express the same densities of Xenopus C-cadherin
(C-CHO) and chicken N-cadherin (N-CHO) sort out in both
hanging drops and in agitated cell suspensions (Shi et al., 2008).
Here we used these proteins as models to investigate the impact
of binding site mutations on affinities, in vitro cell sorting, and
GTPase signaling. On the basis of sequence and structural
comparisons of docked W2 at EC1-EC1 interfaces of Xenopus Ccadherin and mouse N-cadherin (Fig. 1A,B), three sites in the
EC1 domain of C-cadherin were mutated to the corresponding
amino acid in chicken N-cadherin (Fig. 1C). The EC1 domain of
mouse N-cadherin (Fig. 1B) is 98% identical to that of chicken
N-cadherin. The K8NS10P double mutant potentially alters the
docked W2 orientation (Pokutta and Weis, 2007). The other two
mutations S78A and M92I involve more polar residues lining the
W2 binding pocket that were postulated to play a greater role in
modulating the affinity (Patel et al., 2003). Two other mutants
Q23G and E83V did not express sufficiently well for these
biophysical studies.
Fig. 1. Crystal structure of the EC1-EC1 complex. (A) Xenopus Ccadherin (Protein Data Bank access code 1L3W). (B) Murine N-cadherin
(Protein Data Bank access code 1NCG). Both structures were generated with
Visual Molecule Dynamics (VMD) (Humphrey et al., 1996). The positions of
the amino acids mutated in this study are labeled blue, red and green.
(C) Sequence alignment between C-cadherin and N-cadherin EC1 domains.
Red letters indicate the loci of amino acid mutations. (D) Western blots of
CHO-K1 cells expressing C-cadherin mutants and WT C-cadherin. The
biotinylated cell surface cadherin was detected with an antibody against the
cytoplasmic domain of C-cadherin.
Clones that express the C-cadherin mutants were selected
according to expression level, by quantitative FACS and by
Western blots of cell surface proteins. Comparisons of in vitro
cell sorting and quantitative GTPase activation measurements
require cell populations that express similar cadherin surface
densities. The following clones (cadherin surface densities in
parentheses) were selected for these studies: WT C-cadherin (18/
mm2), K8NS10P (20/mm2), S78A (22/mm2), M92I (19/mm2), and
WT N-Cadherin (16/mm2). The expression levels of WT and
mutant C-cadherins were compared to each other and to an actin
loading control, by Western blots of the biotinylated surface
proteins (Fig. 1D). The Western blots agreed with FACS
measurements, which used antibodies against the ectodomains.
C-cadherin mutations alter in vitro cell sorting patterns
In vitro hanging-drop assays (Foty and Steinberg, 2004) showed
that C-CHO sorted out from N-CHO in this assay (Fig. 2A,D),
when both surface expression levels of cadherin and cell densities
(106 cells/ml) were similar (Shi et al., 2008). The K8NS10P
mutant did not affect cell sorting relative to the WT
C-cadherin: cells expressing K8NS10P sorted from N-CHO but
intermixed with C-CHO (Fig. 2B–D). By contrast, the S78A
mutation completely switched the sorting specificity, such that
cells expressing S78A sorted from WT C-CHO but intermixed
Cadherin mutations alter cell sorting
3301
Journal of Cell Science
Fig. 3. Schematic of the micropipette experiment. CHO cell and the RBC
held in opposing pipettes is illustrated at the top. The proteins on the cells
(bottom) include the full-length cadherin expressed on the CHO cell (bottom
left) and Fc-tagged extracellular domains captured by anti-Fc antibodies,
which are covalently bound to the RBC (bottom right).
Fig. 2. Hanging-drop cell-sorting assay. Cells expressing the different
C-cadherin mutants were intermixed with an equal number of CHO cells
expressing either WT C-cadherin (C-CHO) or WT N-cadherin (N-CHO). The
cells expressing the different cadherin mutants were labeled with DiI (red)
and the cells expressing C-CHO or N-CHO were labeled with DiO (green).
(A) Different combinations of C-CHO and N-CHO. (B) C-CHO mixed with
CHO cells expressing different C-cadherin mutants. (C) N-CHO mixed with
cells expressing different C-cadherin mutants. (D) Quantification of the
percentage of different cell types in aggregates. More than 50 aggregates of
three or more cells were scored as red, green, or red and green. (E) The Rac1
inhibitor NSC27366 did not affect the intermixing of C-CHO (top) or the
segregation of N-CHO and C-CHO (bottom). Experimental conditions are
given in the text. Scale bars: 100 mm.
with WT N-CHO (Fig. 2B–D). The M92I cells intermixed with both
C-CHO and N-CHO (Fig. 2B–D). The Rac1 inhibitor NSC 23766
did not affect the sorting outcomes (Fig. 2E). Although the
mechanisms and parameters determining these in vitro assays are
not completely defined, the outcomes identify cadherin differences.
Biophysical properties of homophilic and heterophilic
cadherin bonds
Micropipette measurements (Fig. 3) quantified the biophysical
differences of the cadherin bonds that correlate with the sorting
behavior. These measurements, which are not single molecule
force measurements, quantify the probability of cell-cell binding
as a function of contact time, cadherin surface densities, binding
affinities, and dissociation rates. The binding probability P is the
number of detected binding events nb divided by the total number
of cell-cell touches nb/N. This established approach has been used
to quantify the kinetics and two-dimensional affinities of selectin/
lectin, T-Cell-receptor/MHC, integrin/ligand, and C-cadherin
bonds (Chesla et al., 2000; Chien et al., 2008; Huang et al.,
2004; Huang et al., 2007; Huang et al., 2010). A significant
advantage of these measurements is the quantification of twodimensional protein affinities in the native environment of the
membrane.
The two-dimensional affinities and dissociation rates for
pairwise cadherin interactions were determined from the timedependent binding probabilities P(t) measured between CHO
cells expressing cadherins and red blood cells (RBCs) modified
with either WT Fc-tagged C-cadherin ectodomains (CEC1-5-Fc)
or WT Fc-tagged N-cadherin ectodomains (NEC1-5-Fc). We
previously showed that the initial (,40 seconds) cadherin
binding kinetics are independent of the cytoplasmic domain
(Chien et al., 2008). Because the two-dimensional affinity and
adhesion energy of the Xenopus CEC12-Fc fragment are lower
than both CEC1-5-Fc and the EC1245-Fc fragments (ChappuisFlament et al., 2001; Chien et al., 2008; Zhu et al., 2003), these
studies used the full extracellular segment immobilized on RBCs
to probe cadherin kinetics.
Fig. 4A shows the binding probability P(t) for homophilic Ncadherin adhesion, as a function of cell-cell contact time. As
reported for C-cadherin (Chien et al., 2008), the kinetic profiles
exhibits two distinct stages. The first step is a fast rise to a
limiting plateau at P1,0.4 – that is, 40% of cell-cell touches
resulted in a binding event (Fig. 4A). This is followed by a 2–5
second lag, and a subsequent rise to a second plateau at a binding
probability of P2,0.7 (Fig. 4A). The time-course for heterophilic
C-cadherin/N-cadherin binding is qualitatively similar (Fig. 4B).
The differences in the plateau amplitudes, e.g. P1 and P2 reflect
differences in the protein surface densities, the two-dimensional
affinities, and the dissociation rates.
The previous study mapped the fast, first step to EC1 (Chien
et al., 2008), which is the putative specificity-determining region
(Harrison et al., 2005; Klingelhöfer et al., 2000; Nose et al.,
1990). In this study, we therefore analyzed the affinities and
dissociation rates for this first binding step. Although the
3302
Journal of Cell Science 125 (14)
The limiting probability for the first step as t?? is P1 (cf.
Fig. 4A). By rearranging Eqn 2, it is easily shown that
2Ln(12P1) is a linear function of the product of the receptor
and ligand densities mL 6 mR:
Journal of Cell Science
{Ln(1{P)~mL |mR Ac Ka
Fig. 4. Binding probability P as a function of the intercellular contact
time for cadherin interactions. (A) N-CHO (15/mm2) and NEC1-5-Fc
(69/mm2) on the RBC. (B) N-CHO (15/mm2) and CEC1-5-Fc (19/mm2) on the
RBC. (C) CHO cells expressing S78A (46/mm2) and CEC1-5-Fc (33/mm2)coated RBCs. Binding occurs in two stages. P1 indicates the first binding
plateau, and P2 indicates the second binding plateau. The solid lines are the
best, nonlinear least-squares fits of the first binding stage (,10 seconds) to
Eqn 2, with the best-fit parameters given in Table 1. The dashed lines are the
95% confidence intervals for the fits. Each time point indicates the mean and
s.d. of ,150 measurements.
subsequent steps in the kinetic pathway require additional
cadherin domains (Chien et al., 2008), here we address the
impact of quantitative changes in EC1-dependent bonds on in
vitro cell sorting and signaling.
A simple binding model describes the first, EC1-dependent
binding step (Chesla et al., 1998; Zhang et al., 2005):
kf
RzL < RL;
kr
This is a convenient equation for analyzing data obtained with
cells that express a range of receptor densities. If Eqn 1 and the
kinetic rate Eqn 2 describe the data, then plots of 2Ln(12P1)
versus the product mL 6mR would be linear with a slope of AcKa.
Plots of 2Ln(12P1) versus mL 6 mR (Fig. 5) are linear for
both homophilic and heterophilic binding by WT N-cadherin and
WT C-cadherin for a range of different cadherin densities, e.g.
variable mL 6 mR. This confirms that the fast, first step is
described by the mechanism in Eqn 1. The slopes reflect the
different affinities of the indicated binding interactions (Eqn 3).
The linear plots also confirm that Eqn 2 mathematically describes
the first binding step. Consequently, EC1-EC1 binding affinities
for different pairwise cadherin interactions can be obtained either
from slopes of graphs as in Fig. 5 or from nonlinear least squares
fits of Eqn 2 to kinetic profiles (see Fig. 4), without the need for
measurements at several different cadherin surface densities.
The weighted, nonlinear least-squares fit of Eqn 2 to the binding
probability curves (Fig. 4A–C) gave values of 116261024 mm2
and 0.660.2 seconds21 for Ka and the dissociation rate,
respectively for homophilic C-cadherin bonds (Table 1). The
similarly determined values for the homophilic N-cadherin bond
are, respectively 1.960.361024 mm2 and 1.160.4 seconds21. The
lesser N-cadherin affinity relative to the homophilic C-cadherin
bond agrees with prior molecular-level adhesion measurements
(Prakasam et al., 2006b). The two-dimensional affinity of the
heterophilic bond between WT N-cadherin and CEC1-5-Fc is
3.260.761024 mm2, and the dissociation rate is 0.860.4
seconds21. These results are summarized in Table 1.
C-cadherin binding site mutations alter EC1-dependent
affinities and kinetic rates
All binding kinetics measured between different C-cadherin
mutants and either CEC1-5-Fc or NEC1-5-Fc on RBCs exhibited
ð1Þ
where kf and kr are the forward and reverse rates, respectively,
and the two-dimensional, equilibrium binding affinity is Ka 5
kf/kr. The kinetic rate equation describing the time evolution of
the binding probability P(t) for the above reaction, in terms of
the two-dimensional receptor-ligand affinity Ka (mm2), the
dissociation rate kr (seconds21), the cell-cell contact area Ac
(mm2), and the receptor and ligand surface densities mL and mR
(#/mm2) (Chesla et al., 1998) is:
P(t)~1{expf{mR mL AC Ka ð1{expð{kr tÞÞg:
ð3Þ
ð2Þ
In these measurements, Ac is controlled at ,3 mm2, and mL and
mR are quantified by FACS (Zhang et al., 2005).
Fig. 5. Plot of 2Ln(12P1) against the product of the cadherin surface
densities mL6mR for different pairwise C- and N-cadherin interactions.
The cadherins expressed on the CHO cell and the Fc-tagged ectodomain on
the RBC are indicated in the figure, with the black diamonds indicating
homophilic C-cadherin bonds, open squares indicating homophilic
N-cadherin bonds, and the black triangles indicating heterophilic
N-cadherin/C-cadherin bonds. The solid lines are linear least-squares fits to
the data, with the best-fit parameters given in Table 1.
Cadherin mutations alter cell sorting
3303
Table 1. Two-dimensional cadherin affinities and dissociation rates
Expressed cadherin
Journal of Cell Science
C-cadherin
N-cadherin
N-cadherin
C-cadherin
K8NS10P
K8NS10P
S78A
S78A
M92I
M92I
mR (mm22)
Cadherin-Fc on red blood cells
mL (mm22)
kr (second21)
Ka (61024 mm2)
R2
18
15
15
14
41
41
46
46
16
16
C-cadherin
N-cadherin
C-cadherin
N-cadherin
C-cadherin
N-cadherin
C-cadherin
N-cadherin
C-cadherin
N-cadherin
10
69
19
38
6
9
16
9
16
44
0.5960.21
1.1360.37
0.8260.37
1.3260.26
1.2060.28
1.5960.27
1.4460.60
1.7460.84
2.3060.72
1.9260.55
10.662.2
1.8560.26
3.260.7
3.4860.20
10.3060.79
2.560.1
1.6560.25
2.3560.37
4.2960.54
1.8860.23
0.89
0.99
0.97
0.90
0.87
0.8
0.94
0.74
0.95
0.93
two-stage kinetics (cf. Fig. 4C). The effects of the point
mutations on the C-cadherin affinities and dissociation rates
were then similarly quantified from fits of the first binding step
(t,10 seconds) to Eqn 2. Fig. 4C shows the kinetics and model
fit for binding between the S78A mutant and CEC1-5-Fc.
Relative to the WT C-cadherin, this mutation reduced the
two-dimensional affinity for CEC1-5-Fc more than six-fold to
1.6560.2561024 mm2. At 2.3560.3761024 mm2, the twodimensional affinity between S78A and N-Cadherin was
slightly less than the WT C-cadherin/NEC1-5-Fc bond (Table 1).
The affinity of M92I for CEC1-5-Fc was 2.5-fold lower than
WT C-cadherin, but the affinity for NEC1-5-Fc was similar to
WT C-cadherin, within experimental error. The two-dimensional
affinity measured between M92I and CEC1-5-Fc was
the dissociation
rate was
4.360.561024 mm2, and
2.360.7 seconds21 (Table 1). The fitted parameters for M92I
binding to NEC1-5-Fc are in Table 1. By contrast, the double
mutant K8NS10P, which is postulated to alter the W2 orientation
in the binding site, bound CEC1-5-Fc with a statistically similar
affinity as WT C-cadherin, at 10.360.861024 mm2, and the
dissociation rate of 1.260.3 seconds21 was slightly higher. The
affinity and dissociation rate for the K8NS10P/NEC1-5-Fc bond
are in Table 1.
summarized in supplementary material Table S1. K8NS10P
ligation to CEC1-5-Fc increased Rac1-GTP 762-fold over
initial Rac1-GTP levels (Fig. 6B; supplementary material
Ligation-dependent Rac1 activation
Quantitative measurements of Rac1-GTP levels in cells seeded
on either CEC1-5-Fc or NEC1-5-Fc substrata demonstrated the
correlation between binding affinities and signaling. In these
comparisons between signaling amplitudes, the immobilized
protein (ligand) densities, the cell type, the overall protein
scaffold, including the cytoplasmic domain, the cadherin
expression levels, and the measurement time are the same.
Therefore, the only known variable is the affinity between the
cadherins mediating cell attachment. Rac1-GTP in C-CHO cells
increased up to 45 minutes after attachment to CEC1-5-Fc
substrata (56103 cadherin/mm2) (Fig. 6A), similar to prior reports
(Noren et al., 2003). By contrast, N-CHO on either CEC1-5-Fc or
NEC1-5-Fc (56103 cadherin/mm2) did not exhibit any change in
Rac1-GTP levels. The difference between N-CHO and C-CHO
was not due to differences in cadherin expression (Table 1). Due
to the robust Rac1 activation in C-CHO at 45 minutes, we
compared Rac1-GTP levels 45 minutes after seeding different
cells onto either CEC1-5-Fc or NEC1-5-Fc substrata (Fig. 6B).
A key finding is that Rac1-GTP levels increase with the twodimensional cadherin affinity (Fig. 6C). In C-CHO attached to
CEC1-5-Fc, the Rac1-GTP increased by a factor of 6.360.3
relative to t50 minutes (Fig. 6B). The quantitative data are
Fig. 6. Rac1-GTP levels in cells cultured on either CEC1-5-Fc or NEC15-Fc substrata. (A) Western blot analysis of the Rac1-GTP levels in C-CHO
(21 cadherins/mm2) seeded on CEC1-5-Fc substrata (56104/mm2) at t50, 15
and 45 minutes. The top, middle, and bottom panels indicate the Rac1-GTP,
the total Rac1, and the actin loading control, respectively. (B) Normalized
Rac1-GTP 45 minutes after attaching cells to the different cadherin-coated
substrata. The cadherin subtype expressed on the CHO cell is indicated below
each bar. Gray bars indicate the use of CEC1-5-Fc substrata, and white bars
denote NEC1-5-Fc substrata. The cadherin surface densities are in Table 1.
(C) Plot of the two-dimensional cadherin binding affinity versus Rac1-GTP
levels 45 minutes after cell adhesion. Data are means 6 s.d.
Journal of Cell Science
3304
Journal of Cell Science 125 (14)
Table S1). The latter is statistically similar to WT C-cadherin
(P50.69). The M92I mutant, with an intermediate affinity for
WT CEC1-5-Fc between K8NS10P and S78A, triggered a 3.7fold increase in Rac1-GTP (Fig. 6B; supplementary material
Table S1). By contrast, in cells expressing S78A, the Rac1-GTP
was 0.660.2. Rac1-GTP levels were unchanged in N-CHO
seeded on CEC1-5-Fc. Conversely, on NEC1-5-Fc substrata,
the Rac1-GTP levels in WT C-CHO and N-CHO were 0.960.1
and 0.860.3, respectively (Fig. 6B; supplementary material
Table S1). The K8NS10P cell adhesion to NEC1-5-Fc similarly
did not activate Rac1-GTP. Fig. 6C shows the correlation
between C-cadherin affinities and Rac1-GTP activation.
To address possible differences in GTPase signaling at cell-cell
junctions, global Rac1 activation was quantified in confluent cell
monolayers, following a calcium switch. Although it was only
possible to analyze Rac1 activation at homophilic cell-cell
junctions, the results were qualitatively similar to signaling
triggered by adhesion to immobilized, recombinant ectodomains
(supplementary material Fig. S1).
Dynamic fluorescence imaging monitored Rac1-GTP
accumulation at nascent cell-cell junctions, by quantifying the
localization of the YFP-PBD-PAK reporter for active Rac1 (or
Cdc42) at junctions between WT C-CHO, following a calcium
switch (supplementary material Movie 1). Although it was not
feasible to test heterophilic interactions, the qualitative trends for
homophilic cadherin ligation are similar to the Rac1 assays
(Fig. 6B; supplementary material Fig. S1). The signal amplitude
was greater and persisted longer at C-CHO junctions compared to
N-CHO (Fig. 7A,B; supplementary material Fig. S2). Differences
in YFP-PBD-PAK accumulation at these junctions were
significant (P,0.05) at 40 minutes after calcium stimulation
(Fig. 7). In addition to diminished signal amplitudes, N-CHO
exhibited more extensive ruffling and increased motility
(supplementary material Movie 2). Treatment with NSC 23766,
which inhibits Rac1 without affecting Cdc42 or RhoA (Gao et al.,
2004), abolished YFP-PBD-PAK localization (supplementary
material Movie 3), confirming that YFP-PBD-PAK localization
in these experiments reflects Rac1 rather than Cdc42 activity
(Gao et al., 2004).
The K8NS10P mutant triggered greater YFP-PBD-PAK
accumulation at junctions than the S78A mutant or N-CHO. At
40 minutes, the intensity was less than C-CHO (Fig. 7;
supplementary material Fig. S2, Movie 4). By contrast, the
S78A mutant exhibited only transient increases in junctional
YFP-PBD-PAK (Fig. 7A; supplementary material Fig. S2, Movie
5). Rac1 activity extended throughout the intercellular junction,
but the PBD-PAK-YFP localization did not persist over the timelapse measurement. Signal amplitudes were significantly
diminished (P,0.05) in the S78A mutant compared with
C-CHO, 40 minutes after calcium stimulation (Fig. 7B). Both
N-CHO and the S78A mutant displayed less stable junctions than
C-CHO. Junction persistence was reduced or even abolished
during observations, as some cell pairs separated from each other.
Similarly, at 40 minutes, the Rac1 activity in the S78A mutant
was not statistically different than N-CHO.
These aggregate dynamic imaging studies (Fig. 7;
supplementary material Fig. S2) are qualitatively similar to
results obtained with the Rac1 immuno-capture assay (Fig. 6B).
The exception is the K8NS10P mutant, which is discussed below.
The dynamic imaging also confirmed that cadherin junctions are
loci of Rac1 activation.
Fig. 7. Rac1 accumulation at cell-cell junctions following a calcium switch.
(A) Time course of the mean fluorescence intensity (MFI) of YFP-PBD-PAK at
cadherin-expressing CHO cell junctions. Curves represent averages of
independent experiments (WT C-CHO or N-CHO, n53; K8NS10P, n57;
S78A, n55). The YFP intensity was quantified within intercellular junctions
and normalized to values before calcium stimulation at t50 minutes. The data
were averaged at 5 minute intervals using Matlab software. Error bars represent
the s.e.m. interpolated-value at each time point. (B) Histogram of the
normalized MFI of YFP-PBD-PAK at junctions 40 minutes after calcium
activation. Error bars are the s.e.m. See supplementary material Movies 1–5.
Ligation-dependent RhoA activation
Quantitative RhoA-GTP levels in confluent cell monolayers,
following a calcium switch, were compared with Rac1-GTP
activated under similar conditions (supplementary material Fig.
S3). Neither WT C-cadherin nor K8NS10P, which have similar
affinities for CEC1-5-Fc, activated significant levels RhoA-GTP
(supplementary material Fig. S3). By contrast, nascent N-CHO
junctions increased RhoA-GTP 2.5-fold, 90 minutes after
calcium addition. Interestingly, the S78A mutant activated
similar RhoA-GTP levels to N-CHO, but with slower kinetics.
This inverse correlation between Rac1 and RhoA agrees with
reports suggesting that RhoA and Rac1 have antagonistic effects
(Comunale et al., 2007; Wildenberg et al., 2006).
Cortical tension
Magnetic twisting cytometry (MTC) measurements tested
whether cadherin ligation might affect the global stiffness of
these cells. Probing N-CHO and C-CHO with fibronectinmodified beads interrogated global changes in cytoskeletal
tension through integrin bonds (Potard et al., 1997). The results
were mixed. The expressed cadherin subtypes altered cell rigidity
to different extents (supplementary material Fig. S4). The
substratum ligand also influenced cell stiffness, but not in a
way that compares simply to ligation-dependent GTPase activity.
This suggests a more complex relationship between cadherin
ligation and cell mechanics.
Journal of Cell Science
Cadherin mutations alter cell sorting
Discussion
The main findings of this study are (1) the identification of a
C-cadherin binding site mutation that substantially attenuates
the affinity and correspondingly switches cell aggregation
specificity, (2) the demonstration that, above an apparent
threshold difference in cadherin affinities, in vitro cell sorting
correlates with quantitative differences between two-dimensional
affinities of cell surface cadherins, and (3) cadherin affinity
differences modulate GTPase signaling.
Despite the highly conserved W2 binding site, these kinetic
measurements also demonstrate that small sequence differences
in the binding pocket can generate substantial affinity differences
between cadherin subtypes. The switch in cell sorting specificity
by the C-cadherin point mutation S78A correlates with a six-fold
decrease in the two-dimensional affinity for WT C-cadherin. By
contrast, the modest 2.5-fold decrease in the M92I affinity caused
cells to intermix with both WT C-CHO and WT N-CHO. These
results suggest that relatively large differences in twodimensional binding affinities for the same overall protein
scaffold, e.g. C-cadherin, correlate with CHO cell sorting in vitro.
The affinity appears to be particularly sensitive to sequence
variations at positions 78 and 83; namely, S78A substantially
reduced the C-cadherin affinity, A78M ablated N-cadherin
adhesive function (Tamura et al., 1998), and mutations at
positions 78 and 83 in human E-cadherin altered the in vitro
sorting patterns of L1 cells expressing the proteins (Nose et al.,
1990). The A78M mutation also allosterically altered epitope
accessibility on EC1 (Harrison et al., 2005). Distinct from earlier
reports, this study quantified the biophysical differences of the
membrane-bound proteins that altered cell adhesion and sorting.
The K8NS10P mutation did not significantly alter the
two-dimensional affinity for CEC1-5-Fc, Rac1 activation at
45 minutes, or the RhoA-GTP levels at 45 and 90 minutes.
However, there are differences between Rac1 activation
following CEC1-5-Fc ligation (Fig. 6B) versus junction
activation following a calcium switch (Fig. 7A; supplementary
material Figs S1,S2). This difference is attributed to the ligands
used in the two assays. In the adhesion assay (Fig. 6B), one
protein ligand, or half of the strand dimer, is wild-type
C-cadherin, but in the calcium switch assays, both proteins are
identical. Prior biophysical measurements of the W2A mutant
showed that adhesion between WT C-cadherin and the W2A
mutant is intermediate between homophilic WT C-cadherin and
homophilic W2A adhesion (Prakasam et al., 2006a). We would
thus expect the two-dimensional affinities between CEC1-5-Fc
and the mutants to be somewhat higher than between identical
mutants. These Rac1 assays are consistent with this trend, and
suggest that, although K8NS10P does not detectably alter the
affinity for CEC1-5-Fc, it may lower the affinity between
identical mutants with a corresponding change in Rac1 signaling.
Prior biophysical studies identified multiple bonds between
cadherin ectodomains that require different domains (Bayas et al.,
2006; Bibert et al., 2002; Chappuis-Flament et al., 2001; Chien
et al., 2008; Perret et al., 2004; Shi et al., 2008; Sivasankar et al.,
1999; Sivasankar et al., 2001; Tsukasaki et al., 2007; Zhu et al.,
2003). All five EC domains are needed to recapitulate the kinetic
signature of the intact protein (Chien et al., 2008). The fast first
step requires EC1, but the lag and second step require EC3
(Chien et al., 2008) and are modulated by N-glycosylation on
EC2 and EC3 (Langer et al., 2012). These studies therefore
focused on the first binding step between EC1 domains. Our
3305
results show that two-dimensional affinity differences associated
with EC1-dependent binding correlate with in vitro sorting and
signaling. Thus, only the initial, fast step was considered here.
The logarithm of these two-dimensional affinities are
proportional to the protein adhesion energies (Prakasam et al.,
2006b). However, in micropipette measurements (1) the affinities
and kinetic rates are not determined by mechanically breaking
cadherin bonds, and (2) micropipette measurements probe the
full-length cadherins in the native environment of the cell
membrane. The micropipette data demonstrate that cadherins
with similar two-dimensional affinities (, ,3-fold differences),
and correspondingly similar adhesion energies do not induce cell
segregation, at the expression levels considered. This agrees with
prior studies (Prakasam et al., 2006b; Shi et al., 2008), and could
explain the absence of correlations between adhesion and sorting
in some cases (Duguay et al., 2003; Niessen and Gumbiner, 2002;
Prakasam et al., 2006b).
Importantly, a 3-fold difference in the three-dimensional
(solution) affinity corresponds to a difference in bond energies
of ,1 kcal/mole at 37 ˚C that could be readily offset by cadherin
expression levels or cortical tension (Duguay et al., 2003;
Kalantarian et al., 2009; Krieg et al., 2008; Manning et al., 2010;
Steinberg and Takeichi, 1994; Winklbauer, 2009). The capacity
of cadherin affinities to regulate actin organization through
GTPase signaling introduces an additional mechanism that could
augment or offset differential cadherin adhesion.
The two-dimensional C-cadherin affinities correlate with
ligation-dependent Rac1 activation. Thus, binding differences
due to mutations or binding to heterophilic ligands would
similarly modulate signaling. Because we compared proteins
with the same overall backbone and cytoplasmic domain, this
correlation might not apply for general comparisons across
cadherin subtypes due to possible differences in their interactions
with GTPases (Anastasiadis et al., 2000; Boulter et al., 2010). Ccadherin and E-cadherin activate Rac1 (Noren et al., 2003; Yap
and Kovacs, 2003), but N-cadherin ligation triggers RhoA
activation (Charrasse et al., 2002; Comunale et al., 2007; Marrs
et al., 2009; Taulet et al., 2009). Whether the latter is due to low
N-cadherin affinity and Rac1/RhoA antagonism (Boulter et al.,
2010; Burridge and Doughman, 2006; Comunale et al., 2007;
Wildenberg et al., 2006), or to differences in GTPase interactions
with N-cadherin complexes (Anastasiadis et al., 2000) remains to
be determined.
The two-dimensional affinity measurements were determined
from data acquired within 45 seconds of cell-cell contact, while
the signaling and sorting assays range from 45 minutes to
48 hours, respectively. Importantly, the measured affinities
reflect intrinsic, equilibrium (time-independent) properties of
cadherin bonds. This is implicit in Eqn 2 where Ka is defined as
the two-dimensional equilibrium binding-constant. Solution
binding constants obtained from kinetic, e.g. surface plasmon
resonance measurements are similarly intrinsic properties, and
are similarly compared with cell sorting on timescales of 4 and
48 hours (Katsamba et al., 2009; Takeichi and Nakagawa, 2001).
The same physical chemical justification for such comparisons
applies here.
Given the role of cadherin-dependent GTPase activation
in cytoskeletal regulation (Takaishi et al., 1997) and cell
cycle control (Liu et al., 2006), this connection between
cadherin affinities and GTPase signaling is expected to have
broader consequences for cadherin-dependent cell functions.
3306
Journal of Cell Science 125 (14)
Compromised cadherin adhesion is associated with cancer. For
example, the E-cadherin exon 8 deletion is associated with
human gastric and breast cancers, and it reduces Rac1 signaling,
with a corresponding increase in RhoA activity (Deplazes et al.,
2009). Our findings indicate that differential cadherin binding
affinities play more diverse physiological and mechanical roles
than merely modulating cell cohesion.
Materials and Methods
Plasmids and cell lines
Journal of Cell Science
The cDNAs for the full length Xenopus C-cadherin and the C-cadherin W2A
mutant in pEE14 plasmids were gifts from B. Gumbiner (University of Virginia,
Charlottesville, VA). The cDNA encoding the full-length chicken N-cadherin in
the pEGFP-N1 plasmid was from Andre Sobel (Institut du Fer a Moulin, Gif-surYvette, France). These plasmids were transfected into Chinese Hamster Ovary
(CHO-K1) cells using Lipofectamine2000 (Invitrogen, Carlsbad, CA). CHO-K1
cells stably expressing the full length C-cadherin were selected as described
(Brieher et al., 1996). CHO-K1 cells expressing the full-length chicken N-cadherin
were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing
10 v/v% FBS and 400 mg/ml G418 (Sigma-Aldrich, St Louis, MO). For live-cell
imaging experiments, CHO cell lines were transfected with a plasmid encoding
YFP-PBD-PAK (from Y. Wang, University of Illinois, Urbana, IL) using Fugene 6
(Roche, IN). The YFP-PBD-PAK construct contains the YFP-tagged p21 binding
domain (PBD) of p21-activated kinase (PAK). At 12 hours after transfection, cells
were plated at low confluence on 35 mm glass bottom dishes (Cell E&G) coated
with 20 mg/ml fibronectin (Sigma).
Biotinylation and western blot
Cadherin surface expression was assessed with a standard biotinylation assay. Cells
were detached from substrates with 0.01 v/v% trypsin in HBSS containing 2 mM
CaCl2. The cells were washed, resuspended in PBS. Then 0.5 ml were treated with
0.5 ml of 1 mg/ml Sulfo-NHS-SS-biotin (Pierce) for 25 minutes at 4 ˚C, and then
washed with PBS. Prior to cell lysis, 50 ml of streptavidin-coated beads were washed
twice with ice-cold lysis buffer (50 mM Tris-HCl, 10 mM MgCl2, 200 mM NaCl, 1
w/v% Triton X-100, 5 v/v% glycerol, and Roche complete protease inhibitors at
pH 7.5) and pelleted by centrifugation at 12,000 g. The beads were stored on the ice
prior to adding 1 ml of lysate. The biotinylated cells were lysed, and the lysate was
clarified by centrifugation at 4 ˚C. Then 1 ml of supernatant was incubated with
streptavidin beads (Sigma) and overnight at 4 ˚C. The beads were then washed with
ice-cold lysis buffer, and centrifuged for 1 minute at 12,000 g and 4 ˚C. The collected
beads were boiled in SDS-PAGE buffer, and the proteins were separated by SDSPAGE. In western blots, the primary antibody used was a monoclonal, mouse anti-Ecadherin antibody against the conserved cytoplasmic domain (BD Transduction,
Clone 36/E-cadherin). The secondary antibody was horseradish peroxidase
conjugated, polyclonal anti mouse IgG (Sigma).
FACS quantification of cadherin surface expression levels
Cadherin surface expression levels were quantified by flow cytometry (Chesla et al.,
1998; Chien et al., 2008). Cells were labeled with protein-specific antibodies against
the ectodomains. C-cadherin expressing cells were labeled with anti-C-cadherin
antibody (C/EP/B-Cadherin (clone xC-12), Santa Cruz Biotechnology, Santa Cruz,
CA) followed by the secondary fluorescein isothiocyanate (FITC)-conjugated antigoat IgG (whole molecule; Sigma). Chicken N-cadherin was detected with
monoclonal mouse anti-N-cadherin (Clone GC-4, Sigma) and then fluoresceinisothiocyanate (FITC)-conjugated anti-mouse IgG (whole molecule; Sigma). The
antibody labeling was in phosphate buffered saline (PBS) containing 1 w/v% bovine
serum albumin (BSA) at pH 7.4. The fluorescence intensities of labeled cells were
measured with an LSR II flow cytometer (BD Biosciences) (Zhang et al., 2005). The
fluorescence intensity calibration curve was obtained with calibrated FITC-labeled
standard beads (Bangs Laboratories, Fishers, IN) (Zhang et al., 2005).
Hanging-drop sorting assay
Cell sorting measurements used the hanging-drop method (Foty and Steinberg,
2005). Briefly, cells with cadherin expression levels within 15% of each other were
labeled with DiI or DiO (Molecular Probes, Eugene, OR) by incubating 80%
confluent monolayers with growth medium supplemented with 5 ml/ml of the
appropriate dye for 60 minutes. After washing to remove excess dye, cells were
detached with 0.01% in trypsin in Hank’s Balanced Salt Solution (HBSS)
(Invitrogen, Carlsbad, CA) supplemented with 2 mM CaCl2 (Nose et al., 1988).
Cells were resuspended in HBSS with 2 mM CaCl2 and 5 v/v% FBS at 16106 cell/
ml. A 10 ml aliquot of each of the two cell suspensions was mixed on the lid of a 10
cm Petri dish, inverted over a dish containing 10 ml of PBS (Invitrogen, Carlsbad,
CA). After incubating the hanging drops in an incubator at 37 ˚C under 5% CO2,
cell aggregates were imaged after 24 and 48 hours under a 106 objective with a
Zeiss Axiovert 200 inverted fluorescence microscope equipped with a Zeiss
Axiocam MR camera. To quantify sorting, at least 50 aggregates of three or more
cells were scored as containing red cells, green cells, or both red and green cells
(Niessen and Gumbiner, 2002).
Surface modification of erythrocytes with oriented cadherin extracellular
domains
Erythrocytes were isolated from human whole blood collected from healthy
donors. The whole blood was stored in Vacutainers, and proper protocols were
followed for handling human-derived materials. The erythrocytes were isolated
with Histopaque 1119 (Sigma). To 12 ml of Histopaque 1119 in a 50 ml centrifuge
tube, a mixture of 7 ml whole blood and 7 ml of 0.9 w/v% NaCl was slowly
transferred to the tube containing Histopaque. The mixture was centrifuged at 800
g for 20 minutes at room temperature. The supernatant was discarded as biological
waste, and the remaining cells were resuspended in 7 ml of 0.9 w/v% NaCl prior to
the addition of 1.5 ml of 6 w/v% Dextran. The cells were incubated at 23 ˚C for
45 minutes, during which they settled to the bottom of the tube. After discarding
the supernatant, the red blood cells (RBC) were washed twice at room temperature
with 0.9 w/v% NaCl, and resuspended in 12 ml EAS45 (2.0 mM adenine, 110 mM
dextrose, 55 mM mannitol, 50 mM NaCl, 10 mM glutamine and 20 mM
Na2HPO4, at pH 8.0) (Dumaswala et al., 1996). The RBC suspension in EAS45
can be stored at 4 ˚C up to 3 weeks, after which the RBCs are treated with bleach
and discarded.
Antibodies were covalently coupled to the RBCs using the CrCl3 coupling
method (Gold and Fudenberg, 1967; Kofler and Wick, 1977). Approximately 106
RBCs were washed with 0.85 w/v% NaCl, and resuspended in 250 ml of 0.85%
NaCl with 1 mg of either goat polyclonal anti-human immunoglobulin G (IgG) Fc
or goat polyclonal anti-mouse IgG Fc antibodies (Sigma). The CrCl3 solution was
diluted to below 0.01 w/v% with 0.02 mM sodium acetate containing 0.85 w/v%
NaCl. A 250 ml aliquot of CrCl3 solution was mixed with 250 ml of the RBC/
antibody mixture and incubated at 23 ˚C for 5 minutes. The reaction was stopped
with 500 ml of PBS with 0.5 mM EDTA and 1% BSA. The cells were then washed
twice. The concentration of CrCl3 determined the density of antibodies
immobilized to the surface of the RBCs.
After the antibody immobilization, approximately 20,000 modified RBCs were
incubated with 3 ml of 1 mg/ml Cadherin-Fc fragments. The resulting cadherin
surface densities were quantified by flow cytometry (Chesla et al., 1998; Chien
et al., 2008).
Micropipette measurements of cell binding kinetics
The binding probability was determined as a function of contact time with the
micropipette manipulation technique (Chesla et al., 1998; Chien et al., 2008; Evans
et al., 2004; Huang et al., 2010; Zhang et al., 2005). The binding probability P(t) is
the ratio of the number of binding events nb to the total NT cell-cell touches, nb/NT.
A cadherin-expressing CHO cell and a RBC modified with Fc-tagged cadherin
were partially aspirated into opposing micropipettes (Fig. 3). The cells were
maintained in the chamber with L15 medium (Invitrogen, Carlsbad, CA)
supplemented with 1 w/v% BSA. Cells were visualized with a 1006 oilimmersion objective on a Zeiss Axiovert 200 microscope, and images were
recorded with a DAGE-MTI CCD100 CCD camera (DAGE-MTI, Michigan City,
IN). Automated piezo-electric controllers were programmed to cyclically bring the
two cells into contact for a defined period. The contact area was controlled at
,3 mm2 (,1 mm diameter). Adhesion events are identified from the surface
deformation of the RBC during separation and recoil at adhesive failure. Each cell
pair was tested for 50 cell-cell touches (NT550), and each contact time represents
measurements with at least three different cell pairs (N.150). The reported
probabilities P are the mean 6 s.d. from the mean.
Data fitting and parameter estimation
Both the two-dimensional, equilibrium affinity and the dissociation rate for the
initial rise in binding probability were determined from non-linear least squares fits
of the binding probability data to Eqn 2 (OriginLab, Northampton, MA). The data
range described by the simple binding mechanism (Eqn 1) was identified with a
non-linear lack-of-fit test (Neill, 1988). The test determines the ratio of the
deviation of the model from the mean to the variability in the data, normalized to
an F-distribution (Eqn 4)
n
P
F~
ni (Pmean {Ppred )2 =(n{2)
i
ni
n P
P
:
ð4Þ
(Pdata {Ppred )2 =(N {2)
i j~1
Here, ni is the number of measurement repetitions, n is the number of distinct time
points, Pmean is the mean probability at each data point, Pmodel is the predicted
probability, Pdata is each individual probability observed, and N is the total number
of observations. When the test statistic is larger than the critical value on an F
distribution with (N2n, n22) degrees of freedom, the model is rejected. To
Cadherin mutations alter cell sorting
calculate the parameters in Table 1, the maximum number of data points that did
not fail the lack-of-fit-test was used.
Journal of Cell Science
Rac1 activation assay
Rac1-GTP was determined with a published Rac1-GTP immuno pull-down assay
(Benard and Bokoch, 2002; Noren et al., 2003). Cells expressing cadherins at ,20/
mm2 were seeded onto substrata coated with cadherin ectodomains. CEC1-5-Fc
and NEC1-5-Fc surface densities, determined by isotope labeling (Yeung et al.,
1999), were 56103 molecules/mm2 and 46103 molecules/mm2, respectively. To
prepare these substrata, 10 cm, non tissue culture polystyrene plates (Fisher
Scientific, Pittsburgh, PA) were incubated with 5 ml of 30 mg/ml cadherin EC1-5Fc in HEPES buffer (20 mM HEPES, 150 mM NaCl, 5 mM CaCl2, 1 mM MgCl2,
pH 7.5) for 1 hour at 23 ˚C, and stored overnight at 4 ˚C before use. Controls used
plates coated with poly-L-lysine (PLL). At least 30 minutes prior to use, the coated
plates were washed with HEPES buffer, and equilibrated with serum-free, phenol
red-free DMEM at 37 ˚C.
Cells were maintained at confluence for 2 days before the experiment, detached
from tissue culture plates with 0.01% trypsin in 16 HBSS, supplemented with
1 mM CaCl2 (Nose, 1988), and then collected by centrifugation. Cells were then
re-suspended in serum-free DMEM prior to seeding at 3–46106 cells on the coated
dishes. At defined intervals, the plates were washed twice with ice-cold HEPES
buffer and lysed with 750 ml ice-cold lysis buffer per plate. Cells were removed
with a cell scraper, and the lysate was clarified by centrifugation at 14,000 g for
2 minutes at 4 ˚C. Then 20 ml of the clarified supernatant was analyzed for total
Rac1 by western blot, with anti-Rac1 antibody (Cytoskeleton, Denver, CO).
The remaining lysate was added to 30 ml of GST-PBD beads. Just prior to use,
the beads were washed with ice-cold lysis buffer (50 mM Tris-HCl, 10 mM
MgCl2, 200 mM NaCl, 1% v/v Nonidet P-40, 5% v/v glycerol, and Roche
complete protease inhibitors at pH 7.5) for 10 minutes with gentle shaking at 4 ˚C.
The beads were collected by centrifugation and stored on ice.
Each time point required 30 ml beads. After mixing the lysis buffer and beads,
the GST-PBD bead slurry was centrifuged and washed three times with ice-cold
lysis buffer. After the final wash, the beads were collected by centrifugation, and
boiled in SDS-PAGE buffer. Western blots with anti-Rac1 antibody (Cytoskeleton
Inc., Denver, CO) determined the amount of Rac1-GTP in the lysate. The Rac1GTP was normalized by the Rac1-GTP at t50 minutes, defined by cells in
suspension, and compared to the total Rac1 in the cells and to the actin loading
control. Rac1-GTP levels showed a robust increase at 45 minutes (Fig. 6), so
Rac1-GTP was determined 45 minutes after seeding cells on different substrata.
Rac1 activation at junctions following a calcium switch
Rac1-GTP activation at cell-cell junctions was determined with the Rac1-GTP
immuno pull-down assay described above. The cells were maintained at
confluence for 2 days. Then, 12 hours before the experiment, cells were
incubated in medium with 0.05 v/v% FBS and no calcium. After 12 hours, the
medium in one flask was switched to DMEM containing 0.05 v/v% FBS and
1.8 mM CaCl2, and incubated for 45 minutes at 37 ˚C. Cells in the second flask
(t50 minutes) were washed twice with ice-cold HEPES buffer, lysed with 1500 ml
ice-cold lysis buffer, and removed with a cell scraper. This step was repeated
45 minutes after the addition of calcium. The relative amounts of total Rac1 and
Rac1-GTP in the cell lysates were determined as described above. Actin was used
as a loading control.
3307
RhoA activation assay
Ligation-activated RhoA-GTP was quantified following cadherin activation with a
calcium switch with a commercial G-LISA kit (BK 124; Cytoskeleton, Denver,
CO). Confluent cells in 10 mm Petri dishes were serum starved for 24 hours,
before addition of 4 mM EGTA, which disrupts cell-cell junctions (Noren et al.,
2003). After a 1 hour incubation with EGTA, the medium was exchanged with
medium containing 1.8 mM calcium, but lacking serum. Duplicate samples were
analyzed at t50, 45 and 90 minutes. After specific time periods, plates were
immediately treated with ice-cold lysis buffer, as described for Rac1 assays. Cell
lysates were scraped into tubes and clarified by centrifugation, before snapfreezing in liquid nitrogen. Lysates at t50 minutes were prepared by resuspending cells in lysis buffer.
The protein concentration in the cell lysates was normalized to the t50 minute
sample, and equal amounts of total protein were incubated in duplicate in a 96-well
GLISA assay plate. RhoA-GTP levels were determined with a microplate
spectrophotometer (Molecular Devices SpectraMax M2). The change in RhoAGTP was normalized relative to the corresponding zero time-point samples.
Dynamic fluorescence imaging
Prior to imaging, cell-cell junctions were disrupted by incubation for 4–6 hours at
37 ˚C with calcium-free culture medium without phenol red (Braga, 2002; Noren
et al., 2003). Live cell imaging was performed at 37 ˚C with a Nikon Eclipse Ti
inverted microscope equipped with a cooled charge-coupled device camera
(QuantEM 512SC; Photometrics) using MetaFluor 6.2 software (Universal
Imaging). Time-lapse images were acquired every 1–2 minutes using a 406 or
1006 oil objective lens (Nikon). When switching to the calcium-containing
medium, imaging was paused to allow replacement of calcium-free medium with
standard medium containing 1.8 mM calcium without phenol red. To inhibit Rac1,
cells were incubated with 50 mM of sterile NSC 23766 (Tocris Bioscience,
Ellisville, MO) for 12 hours at 37 ˚C (Gao et al., 2004). The Rac1 inhibitor was
added to the calcium-containing medium used for the calcium switch imaging
experiments.
Image processing and the creation of time-lapse movies were done with
MetaMorph software (Universal Imaging). In order to quantify YFP-PBD-PAK at
cell-cell junctions (Deplazes et al., 2009), the background-subtracted mean
fluorescence intensity (MFI) of YFP within the maximal area of a single junction
was recorded using ImageJ64 software (National Institutes of Health, USA). Only
two cells in contact were examined so that a single cell-cell junction could be
clearly defined for analysis. In order to correct for differences in transfection
efficiency, each trace was normalized to the average basal fluorescence intensity
before calcium stimulation. The normalized YFP intensity at cell-cell junctions
was plotted against time using the spline interpolation feature, and curves were
smoothed by loess, provided in MatLab software (Mathworks). The standard error
of the interpolated mean value was calculated using the Excel function, and a twotailed Student’s t-test determined whether detected differences were statistically
significant.
Acknowledgements
We thank Saiko Rosenberger and Dr Sandy McMasters for technical
assistance; Shaoying Lu for assistance with image analysis; and
Yingxiao Wang for discussions, reagents, and microscope and
software usage.
Cortical tension measurement
Differences in cortical tension were determined by quantifying the global cell
stiffness with Magnetic Twisting Cytometry (MTC) and magnetic beads covalently
modified with fibronectin (Wang et al., 1993). Glass-bottom Petri dishes were
incubated overnight with an anti-immunoglobulin Fc antibody, followed by rinsing
and incubation with 0.5 mg of either CEC1-5-Fc or NEC1-5-Fc for 4 hours at 4 ˚C.
After rinsing with HEPES buffer, the surfaces were incubated with 1 w/v% BSA at
room temperature for 30 minutes. Ferromagnetic beads (4.9 mm; Spherotech),
chemically activated with ethyl-3-(dimethylaminopropyl)-carbodiimide and Nhydroxysuccinimide (Prakasam et al., 2006a; Prakasam et al., 2006b), were
covalently modified with fibronectin.
Cells stably expressing different cadherins were grown to confluence, detached
with PBS containing 3.5 mM EDTA and 1w/v% BSA, collected by centrifugation,
and seeded at low density on the cadherin-Fc coated substrata, in medium
supplemented with 0.05 v/v% FBS. These measurements focused on isolated cells
to eliminate interference from cell-cell adhesion. After 4 hours at 37 ˚C,
fibronectin-coated beads were incubated with the cells for 20 minutes. All MTC
measurements were performed on an inverted microscope (Leica) using a 206
objective and a cooled charge-coupled device camera (Orca2; Hamamatsu
Photonics). After initial bead magnetization, an oscillating magnetic field
perpendicular to the bead magnetic moment was applied for a defined period.
The bead magnetic moment constant of 0.12 Pa/Gauss was calibrated as described
(Wang et al., 1993). The bead displacements were measured and converted to the
complex modulus (Wang et al., 1993).
Funding
This work was supported by the National Science Foundation [grant
number CBET 0853705]; the National Institutes of Health [grant
numbers R21 HD059002 and HL098472]; and the American Heart
Association [grant number 10PRE3840004 to A.K.B.]. Deposited in
PMC for release after 12 months.
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.087395/-/DC1
References
Anastasiadis, P. Z., Moon, S. Y., Thoreson, M. A., Mariner, D. J., Crawford, H. C.,
Zheng, Y. and Reynolds, A. B. (2000). Inhibition of RhoA by p120 catenin. Nat. Cell
Biol. 2, 637-644.
Bayas, M. V., Leung, A., Evans, E. and Leckband, D. (2006). Lifetime measurements
reveal kinetic differences between homophilic cadherin bonds. Biophys. J. 90, 13851395.
Becker, K. F., Kremmer, E., Eulitz, M., Becker, I., Handschuh, G., Schuhmacher,
C., Müller, W., Gabbert, H. E., Ochiai, A., Hirohashi, S. et al. (1999). Analysis of
E-cadherin in diffuse-type gastric cancer using a mutation-specific monoclonal
antibody. Am. J. Pathol. 155, 1803-1809.
Journal of Cell Science
3308
Journal of Cell Science 125 (14)
Benard, V. and Bokoch, G. M. (2002). Assay of Cdc42, Rac, and Rho GTPase
activation by affinity methods. Methods Enzymol. 345, 349-359.
Bibert, S., Jaquinod, M., Concord, E., Ebel, C., Hewat, E., Vanbelle, C., Legrand,
P., Weidenhaupt, M., Vernet, T. and Gulino-Debrac, D. (2002). Synergy between
extracellular modules of vascular endothelial cadherin promotes homotypic
hexameric interactions. J. Biol. Chem. 277, 12790-12801.
Boggon, T. J., Murray, J., Chappuis-Flament, S., Wong, E., Gumbiner, B. M. and
Shapiro, L. (2002). C-cadherin ectodomain structure and implications for cell
adhesion mechanisms. Science 296, 1308-1313.
Boulter, E., Garcia-Mata, R., Guilluy, C., Dubash, A., Rossi, G., Brennwald, P. J.
and Burridge, K. (2010). Regulation of Rho GTPase crosstalk, degradation and
activity by RhoGDI1. Nat. Cell Biol. 12, 477-483.
Braga, V. M. (2002). Cell-cell adhesion and signalling. Curr. Opin. Cell Biol. 14, 546556.
Brieher, W. M., Yap, A. S. and Gumbiner, B. M. (1996). Lateral dimerization is
required for the homophilic binding activity of C-cadherin. J. Cell Biol. 135, 487-496.
Burridge, K. and Doughman, R. (2006). Front and back by Rho and Rac. Nat. Cell
Biol. 8, 781-782.
Chappuis-Flament, S., Wong, E., Hicks, L. D., Kay, C. M. and Gumbiner, B. M.
(2001). Multiple cadherin extracellular repeats mediate homophilic binding and
adhesion. J. Cell Biol. 154, 231-243.
Charrasse, S., Meriane, M., Comunale, F., Blangy, A. and Gauthier-Rouvière, C.
(2002). N-cadherin-dependent cell-cell contact regulates Rho GTPases and betacatenin localization in mouse C2C12 myoblasts. J. Cell Biol. 158, 953-965.
Chesla, S. E., Selvaraj, P. and Zhu, C. (1998). Measuring two-dimensional receptorligand binding kinetics by micropipette. Biophys. J. 75, 1553-1572.
Chesla, S. E., Li, P., Nagarajan, S., Selvaraj, P. and Zhu, C. (2000). The membrane
anchor influences ligand binding two-dimensional kinetic rates and three-dimensional
affinity of FcgammaRIII (CD16). J. Biol. Chem. 275, 10235-10246.
Chien, Y. H., Jiang, N., Li, F., Zhang, F., Zhu, C. and Leckband, D. (2008). Two
stage cadherin kinetics require multiple extracellular domains but not the cytoplasmic
region. J. Biol. Chem. 283, 1848-1856.
Comunale, F., Causeret, M., Favard, C., Cau, J., Taulet, N., Charrasse, S. and
Gauthier-Rouvière, C. (2007). Rac1 and RhoA GTPases have antagonistic functions
during N-cadherin-dependent cell-cell contact formation in C2C12 myoblasts. Biol.
Cell 99, 503-517.
Deplazes, J., Fuchs, M., Rauser, S., Genth, H., Lengyel, E., Busch, R. and Luber, B.
(2009). Rac1 and Rho contribute to the migratory and invasive phenotype associated
with somatic E-cadherin mutation. Hum. Mol. Genet. 18, 3632-3644.
Drees, F., Pokutta, S., Yamada, S., Nelson, W. J. and Weis, W. I. (2005). Alphacatenin is a molecular switch that binds E-cadherin-beta-catenin and regulates actinfilament assembly. Cell 123, 903-915.
Duguay, D., Foty, R. A. and Steinberg, M. S. (2003). Cadherin-mediated cell adhesion
and tissue segregation: qualitative and quantitative determinants. Dev. Biol. 253, 309323.
Dumaswala, U. J., Wilson, M. J., José, T. and Daleke, D. L. (1996). Glutamine- and
phosphate-containing hypotonic storage media better maintain erythrocyte membrane
physical properties. Blood 88, 697-704.
Evans, E., Leung, A., Heinrich, V. and Zhu, C. (2004). Mechanical switching and
coupling between two dissociation pathways in a P-selectin adhesion bond. Proc.
Natl. Acad. Sci. USA 101, 11281-11286.
Foty, R. A. and Steinberg, M. S. (2004). Cadherin-mediated cell-cell adhesion and
tissue segregation in relation to malignancy. Int. J. Dev. Biol. 48, 397-409.
Foty, R. A. and Steinberg, M. S. (2005). The differential adhesion hypothesis: a direct
evaluation. Dev. Biol. 278, 255-263.
Fournier, A. K., Campbell, L. E., Castagnino, P., Liu, W. F., Chung, B. M., Weaver,
V. M., Chen, C. S. and Assoian, R. K. (2008). Rac-dependent cyclin D1 gene
expression regulated by cadherin- and integrin-mediated adhesion. J. Cell Sci. 121,
226-233.
Gao, Y., Dickerson, J. B., Guo, F., Zheng, J. and Zheng, Y. (2004). Rational design
and characterization of a Rac GTPase-specific small molecule inhibitor. Proc. Natl.
Acad. Sci. USA 101, 7618-7623.
Gold, E. R. and Fudenberg, H. H. (1967). Chromic chloride: a coupling reagent for
passive hemagglutination reactions. J. Immunol. 99, 859-866.
Gumbiner, B. M. (2005). Regulation of cadherin-mediated adhesion in morphogenesis.
Nat. Rev. Mol. Cell Biol. 6, 622-634.
Handschuh, G., Candidus, S., Luber, B., Reich, U., Schott, C., Oswald, S., Becke, H.,
Hutzler, P., Birchmeier, W., Höfler, H. et al. (1999). Tumour-associated E-cadherin
mutations alter cellular morphology, decrease cellular adhesion and increase cellular
motility. Oncogene 18, 4301-4312.
Handschuh, G., Luber, B., Hutzler, P., Höfler, H. and Becker, K. F. (2001). Single
amino acid substitutions in conserved extracellular domains of E-cadherin differ in
their functional consequences. J. Mol. Biol. 314, 445-454.
Harrison, O. J., Corps, E. M., Berge, T. and Kilshaw, P. J. (2005). The mechanism of
cell adhesion by classical cadherins: the role of domain 1. J. Cell Sci. 118, 711-721.
Häussinger, D., Ahrens, T., Aberle, T., Engel, J., Stetefeld, J. and Grzesiek, S.
(2004). Proteolytic E-cadherin activation followed by solution NMR and X-ray
crystallography. EMBO J. 23, 1699-1708.
Hilgenfeldt, S., Erisken, S. and Carthew, R. W. (2008). Physical modeling of cell
geometric order in an epithelial tissue. Proc. Natl. Acad. Sci. USA 105, 907-911.
Huang, J., Chen, J., Chesla, S. E., Yago, T., Mehta, P., McEver, R. P., Zhu, C. and
Long, M. (2004). Quantifying the effects of molecular orientation and length on twodimensional receptor-ligand binding kinetics. J. Biol. Chem. 279, 44915-44923.
Huang, J., Edwards, L. J., Evavold, B. D. and Zhu, C. (2007). Kinetics of MHC-CD8
interaction at the T cell membrane. J. Immunol. 179, 7653-7662.
Huang, J., Zarnitsyna, V. I., Liu, B., Edwards, L. J., Jiang, N., Evavold, B. D. and
Zhu, C. (2010). The kinetics of two-dimensional TCR and pMHC interactions
determine T-cell responsiveness. Nature 464, 932-936.
Humphrey, W., Dalke, A. and Schulten, K. (1996). VMD: visual molecular dynamics.
J. Mol. Graph. 14, 33-38, 27-38.
Kalantarian, A., Ninomiya, H., Saad, S. M., David, R., Winklbauer, R. and
Neumann, A. W. (2009). Axisymmetric drop shape analysis for estimating the
surface tension of cell aggregates by centrifugation. Biophys. J. 96, 1606-1616.
Katsamba, P., Carroll, K., Ahlsen, G., Bahna, F., Vendome, J., Posy, S.,
Rajebhosale, M., Price, S., Jessell, T. M., Ben-Shaul, A. et al. (2009). Linking
molecular affinity and cellular specificity in cadherin-mediated adhesion. Proc. Natl.
Acad. Sci. USA 106, 11594-11599.
Klingelhöfer, J., Troyanovsky, R. B., Laur, O. Y. and Troyanovsky, S. (2000).
Amino-terminal domain of classic cadherins determines the specificity of the
adhesive interactions. J. Cell Sci. 113, 2829-2836.
Kofler, R. and Wick, G. (1977). Some methodologic aspects of the chromium chloride
method for coupling antigen to erythrocytes. J. Immunol. Methods 16, 201-209.
Krieg, M., Arboleda-Estudillo, Y., Puech, P. H., Käfer, J., Graner, F., Müller, D. J.
and Heisenberg, C. P. (2008). Tensile forces govern germ-layer organization in
zebrafish. Nat. Cell Biol. 10, 429-436.
Langer, M. D., Guo, H. B., Shashikanth, N., Pierce, M. and Leckband, D. E (2012).
N-glycosylation alters cadherin-mediated intercellular binding kinetics. J. Cell Sci.
125, 2478-2485.
Levenberg, S., Yarden, A., Kam, Z. and Geiger, B. (1999). p27 is involved in Ncadherin-mediated contact inhibition of cell growth and S-phase entry. Oncogene 18,
869-876.
Liu, W. F., Nelson, C. M., Pirone, D. M. and Chen, C. S. (2006). E-cadherin
engagement stimulates proliferation via Rac1. J. Cell Biol. 173, 431-441.
Manning, M. L., Foty, R. A., Steinberg, M. S. and Schoetz, E. M. (2010). Coaction of
intercellular adhesion and cortical tension specifies tissue surface tension. Proc. Natl.
Acad. Sci. USA 107, 12517-12522.
Marrs, G. S., Theisen, C. S. and Brusés, J. L. (2009). N-cadherin modulates voltage
activated calcium influx via RhoA, p120-catenin, and myosin-actin interaction. Mol.
Cell. Neurosci. 40, 390-400.
Neill, J. W. (1988). Testing for Lack of fit in Nonlinear Regression. Ann. Stat. 16, 733740.
Niessen, C. M. and Gumbiner, B. M. (2002). Cadherin-mediated cell sorting not
determined by binding or adhesion specificity. J. Cell Biol. 156, 389-400.
Noren, N. K., Arthur, W. T. and Burridge, K. (2003). Cadherin engagement inhibits
RhoA via p190RhoGAP. J. Biol. Chem. 278, 13615-13618.
Nose, A., Nagafuchi, A. and Takeichi, M. (1988). Expressed recombinant cadherins
mediate cell sorting in model systems. Cell 54, 993-1001.
Nose, A., Tsuji, K. and Takeichi, M. (1990). Localization of specificity determining
sites in cadherin cell adhesion molecules. Cell 61, 147-155.
Patel, S. D., Chen, C. P., Bahna, F., Honig, B. and Shapiro, L. (2003). Cadherinmediated cell-cell adhesion: sticking together as a family. Curr. Opin. Struct. Biol. 13,
690-698.
Perret, E., Leung, A., Feracci, H. and Evans, E. (2004). Trans-bonded pairs of Ecadherin exhibit a remarkable hierarchy of mechanical strengths. Proc. Natl. Acad.
Sci. USA 101, 16472-16477.
Pertz, O., Bozic, D., Koch, A. W., Fauser, C., Brancaccio, A. and Engel, J. (1999). A
new crystal structure, Ca2+ dependence and mutational analysis reveal molecular
details of E-cadherin homoassociation. EMBO J. 18, 1738-1747.
Pokutta, S. and Weis, W. I. (2007). Structure and mechanism of cadherins and catenins
in cell-cell contacts. Annu. Rev. Cell Dev. Biol. 23, 237-261.
Potard, U. S., Butler, J. P. and Wang, N. (1997). Cytoskeletal mechanics in confluent
epithelial cells probed through integrins and E-cadherins. Am. J. Physiol. 272, C1654C1663.
Prakasam, A., Chien, Y. H., Maruthamuthu, V. and Leckband, D. E. (2006a).
Calcium site mutations in cadherin: impact on adhesion and evidence of
cooperativity. Biochemistry 45, 6930-6939.
Prakasam, A. K., Maruthamuthu, V. and Leckband, D. E. (2006b). Similarities
between heterophilic and homophilic cadherin adhesion. Proc. Natl. Acad. Sci. USA
103, 15434-15439.
Shan, W. S., Tanaka, H., Phillips, G. R., Arndt, K., Yoshida, M., Colman, D. R. and
Shapiro, L. (2000). Functional cis-heterodimers of N- and R-cadherins. J. Cell Biol.
148, 579-590.
Shapiro, L., Fannon, A. M., Kwong, P. D., Thompson, A., Lehmann, M. S., Grübel,
G., Legrand, J. F., Als-Nielsen, J., Colman, D. R. and Hendrickson, W. A. (1995).
Structural basis of cell-cell adhesion by cadherins. Nature 374, 327-337.
Shi, Q., Chien, Y. H. and Leckband, D. (2008). Biophysical properties of cadherin
bonds do not predict cell sorting. J. Biol. Chem. 283, 28454-28463.
Sivasankar, S., Brieher, W., Lavrik, N., Gumbiner, B. and Leckband, D. (1999).
Direct molecular force measurements of multiple adhesive interactions between
cadherin ectodomains. Proc. Natl. Acad. Sci. USA 96, 11820-11824.
Sivasankar, S., Gumbiner, B. and Leckband, D. (2001). Direct measurements of
multiple adhesive alignments and unbinding trajectories between cadherin extracellular
domains. Biophys. J. 80, 1758-1768.
Steinberg, M. S. (1963). Reconstruction of tissues by dissociated cells. Some
morphogenetic tissue movements and the sorting out of embryonic cells may have
a common explanation. Science 141, 401-408.
Cadherin mutations alter cell sorting
Journal of Cell Science
Steinberg, M. S. (2007). Differential adhesion in morphogenesis: a modern view. Curr.
Opin. Genet. Dev. 17, 281-286.
Steinberg, M. S. and Takeichi, M. (1994). Experimental specification of cell sorting,
tissue spreading, and specific spatial patterning by quantitative differences in cadherin
expression. Proc. Natl. Acad. Sci. USA 91, 206-209.
Takaishi, K., Sasaki, T., Kotani, H., Nishioka, H. and Takai, Y. (1997). Regulation of
cell-cell adhesion by rac and rho small G proteins in MDCK cells. J. Cell Biol. 139,
1047-1059.
Takeichi, M. (1995). Morphogenetic roles of classic cadherins. Curr. Opin. Cell Biol. 7,
619-627.
Takeichi, M. and Nakagawa, S. (2001). Cadherin-dependent cell-cell adhesion. In
Current Protocols in Cell Biology, pp 9.3.1-9.3.15. New York: Wiley.
Tamura, K., Shan, W. S., Hendrickson, W. A., Colman, D. R. and Shapiro, L.
(1998). Structure-function analysis of cell adhesion by neural (N-) cadherin. Neuron
20, 1153-1163.
Taulet, N., Comunale, F., Favard, C., Charrasse, S., Bodin, S. and GauthierRouvière, C. (2009). N-cadherin/p120 catenin association at cell-cell contacts occurs
in cholesterol-rich membrane domains and is required for RhoA activation and
myogenesis. J. Biol. Chem. 284, 23137-23145.
Tomschy, A., Fauser, C., Landwehr, R. and Engel, J. (1996). Homophilic adhesion of
E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.
EMBO J. 15, 3507-3514.
3309
Tsukasaki, Y., Kitamura, K., Shimizu, K., Iwane, A. H., Takai, Y. and Yanagida, T.
(2007). Role of multiple bonds between the single cell adhesion molecules, nectin and
cadherin, revealed by high sensitive force measurements. J. Mol. Biol. 367, 996-1006.
Wang, N., Butler, J. P. and Ingber, D. E. (1993). Mechanotransduction across the cell
surface and through the cytoskeleton. Science 260, 1124-1127.
Wildenberg, G. A., Dohn, M. R., Carnahan, R. H., Davis, M. A., Lobdell, N. A.,
Settleman, J. and Reynolds, A. B. (2006). p120-catenin and p190RhoGAP regulate
cell-cell adhesion by coordinating antagonism between Rac and Rho. Cell 127, 10271039.
Winklbauer, R. (2009). Cell adhesion in amphibian gastrulation. Int. Rev. Cell Mol.
Biol. 278, 215-275.
Yap, A. S. and Kovacs, E. M. (2003). Direct cadherin-activated cell signaling: a view
from the plasma membrane. J. Cell Biol. 160, 11-16.
Yeung, C., Purves, T., Kloss, A. A., Kuhl, T. L., Sligar, S. and Leckband, D. (1999).
Cytochrome c Recognition of Immobilized, Orientational Variants of Cytochrome
b5: Direct Force and Equilibrium Binding Measurements. Langmuir 15, 6829-6836.
Zhang, F., Marcus, W. D., Goyal, N. H., Selvaraj, P., Springer, T. A. and Zhu, C.
(2005). Two-dimensional kinetics regulation of alphaLbeta2-ICAM-1 interaction by
conformational changes of the alphaL-inserted domain. J. Biol. Chem. 280, 4220742218.
Zhu, B., Chappuis-Flament, S., Wong, E., Jensen, I. E., Gumbiner, B. M. and
Leckband, D. (2003). Functional analysis of the structural basis of homophilic
cadherin adhesion. Biophys. J. 84, 4033-4042.