* Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan; The translational movement of E-cadherin, a
calcium-dependent cell-cell adhesion molecule in the
plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle
tracking (SPT) and optical tweezers (OT). The wild
type (Wild) and three types of artificial cytoplasmic
mutants of E-cadherin were expressed in L-cells, and
their movements were compared. Two mutants were
E-cadherins that had deletions in the COOH terminus
and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild);
these are called Catenin-minus and Short-tailed in this
paper, respectively. The third mutant, called Fusion, is
a fusion protein between E-cadherin without the catenin-binding site and E-CADHERIN is a calcium-dependent cell-to-cell recognition/adhesion molecule in epithelial tissues,
and a transmembrane protein that spans the plasma
membrane once (Takeichi, 1988 Some cytoplasmic proteins, including The association of newly synthesized Recently, we have demonstrated the existence of two
major types of interactions between membrane-spanning
proteins and the membrane-associated portion of the cytoskeleton (membrane skeleton) (Sako and Kusumi, 1994 Department of Medical Chemistry,
Abstract
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm
colloidal gold or 210-nm
latex particles via a monoclonal
antibody to the extracellular domain of E-cadherin for
SPT or OT experiments, respectively. E-cadherin on
the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed
could be dragged an average of 1.1 and 1.8 µm by OT
(trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 × 10
10
and 2.1 × 10
10 cm2/s, respectively. Approximately 40%
of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 µm2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by
OT. Average Dmicro for Fusion measured by SPT was
small (0.2 × 10
10 cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of
two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin
in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through
catenin(s) (like Fusion) and (b) a corralling effect of
the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane
skeleton that contribute to the tethering and corralling
effects as measured by the dragging experiments were
30 and 5 pN/µm, respectively, indicating a difference in
the skeletal structures that produce these two effects.
Introduction
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
, 1991
). E-cadherin is localized in cell-to-cell adherens junctions and is also found in dilute homogeneous distributions over the free surface
of cells (Bacallao et al., 1989
). Cadherin molecules on the
free cell surface may be surveying new physical contacts
with other cells or may be on their way to the assembly of
adherens junctions.
- and
-catenins
and p120, are bound to the cytoplasmic domain of E-cadherin (Ozawa et al., 1989
; McCrea et al., 1991
; Reynolds
and McCrea, 1994
).
-Catenin is an F-actin binding protein (Rimm et al., 1995
). Binding of E-cadherin to actin
through
-catenin is essential for cadherin-mediated cell
adhesion (Hirano et al., 1992
; Nagafuchi et al., 1994
; Watabe et al., 1994
). p120 is a
-catenin-, plakoglobin-related protein which makes a complex with E-cadherin and
-catenin
(Peifer et al., 1994
; Jou et al., 1995
; Shibamoto et al., 1995
).
-catenin with the
cadherin-catenin complex takes place at the plasma membrane (Hinck et al., 1994
). However, it is not known if all
cadherin molecules are bound to the actin cytoskeleton, in
addition, the stage of their assembly into adherens junctions at which they start being associated with actin filaments is also unclear. Furthermore, little knowledge is
available regarding the mechanical properties of the actin
filaments that are associated with cadherin molecules, although such information is necessary for understanding
the mechanical basis of cadherin-based cell-cell adhesion.
,
1995
). The first type of interaction is binding to the membrane skeleton (Fig. 1 A, Tether model). The second type
of interaction is that where membrane proteins are confined in compartments bounded by the network of the
membrane skeleton (Fig. 1 B, Fence model). In this model,
membrane proteins are not tethered to the membrane
skeleton and are free to undergo Brownian diffusion, but
are corralled in the membrane skeleton meshes because of
the steric hindrance of the cytoplasmic domain of membrane proteins and the membrane skeleton. The movements of membrane proteins, including transferrin receptor and
2-macroglobulin receptor in a fibroblast (Sako
and Kusumi, 1994
, 1995
), E-cadherin, transferrin receptor,
and EGF receptor in a keratinocyte (Kusumi et al., 1993
),
and band 3 in erythrocyte (Sheetz et al., 1980
; Tsuji and
Ohnishi, 1986
; Tsuji et al., 1988
), can be explained using
these two models.
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Fig. 1.
A tether model (A) and a fence model (B) proposed as
mechanisms for the regulation of lateral movements of E-cadherin (Sako and Kusumi, 1995 ). These models are shown together with the optical tweezers experiments which may make it
possible to differentiate and characterize these two mechanisms.
(A) E-cadherin tethered to a cytoskeletal filament can be
dragged only the length the filament is stretched, with a force of
1 pN or less. (B) An E-cadherin molecule free from tethering
may be temporarily trapped within a compartment enclosed by
the membrane skeleton fence. The particle-protein complex can
pass across the fence if the dragging force by OT is high enough.
For transferrin receptor in the plasma membrane of NRK cells,
the force needed to pass across the fence is 0.05-0.1 pN (Sako
and Kusumi, 1995
).
These two models are based on findings obtained using
single particle tracking (SPT)1 (De Brabander et al., 1985;
Gelles et al., 1988
; Kucik et al., 1989
) and optical tweezers
(OT) (Ashkin et al., 1986
, 1987
). For example, in the
plasma membrane of normal rat kidney (NRK) fibroblast cells, movements of 80-90% of the particles bound to the
transferrin receptor were temporarily confined within
compartments with an average diagonal length of 600 nm.
Lateral diffusion over the cell surface takes place as a result of consecutive hops from one compartment to an adjacent compartment, which occurs an average of once every
20-30 s (intercompartmental hop diffusion). This population exhibited microscopic diffusion coefficients within compartments (Dmicro) >1.5 × 10
10 cm2/s, suggesting free
diffusion within a compartment. Such molecules could be
dragged freely by OT until they hit the membrane skeleton fence. With a trapping force of 0.1 pN, half of this population escaped from OT at the boundaries of the membrane compartment. The remaining transferrin receptors
(10-20%) exhibited Dmicro <1.5 × 10
10 cm2/s and could
not be dragged much even under a trapping force of 0.8 pN. It was concluded that these molecules are tethered to the membrane undercoat structures or the membrane
skeleton (Sako and Kusumi, 1994
, 1995
).
In the present study, we again used SPT and OT and studied the mechanisms of the regulation of the movements of the wild type and three artificial cytoplasmic mutants of E-cadherin. These molecules were artificially expressed in mouse L cells, in which the expression of intrinsic cadherin is not detectable.
The structures of the E-cadherins studied in this work
are shown in Fig. 2. The wild-type E-cadherin (EL 1a,
Nose et al., 1988
, called "Wild" in this paper) has a cytoplasmic domain of 152 amino acids (aa) at the COOH terminus. It includes a binding domain for
- and
-catenins
in the region 7-72 aa from the COOH terminus (Nagafuchi and Takeichi, 1989
; Ozawa et al., 1990
). Two mutants
(EL
21 and EL
24; Nagafuchi and Takeichi, 1988
) have deletions of 36 and 131 aa at the COOH terminus, leaving
116 and 21 aa in the cytoplasmic domain, respectively.
These molecules lack a catenin-binding site(s), and are
called "Catenin-minus" and "Short-tailed" in this paper,
respectively. They cannot mediate cell-cell adhesion. One
mutant (nE
CL1, called "Fusion" in this paper (Nagafuchi et al., 1994
) is a fusion molecule of the COOH-terminal half (aa 508-906) of
-catenin and E-cadherin that lost
72 aa in the COOH terminus. Fusion does not bind to either
- or
-catenin. However, fusion can mediate cell-cell
adhesion, probably because of the presence within the
molecule of a part of
-catenin that is capable of binding
to actin filaments (Nagafuchi et al., 1994
). The structures
of these cadherin molecules differ only in the cytoplasmic
domain. The ectoplasmic and transmembrane domains are
the same. The cytoplasmic domain is expected to affect the mobility of these molecules through (a) binding (ability or
inability) to catenins and (b) the size (steric effect) of the
cytoplasmic domain. Binding to catenin(s) would lead to
tethering of cadherins to actin filaments near the cytoplasmic surface of the plasma membrane (see Fig. 1 A). The
size of the cytoplasmic domain would affect the probability for the molecules to hop over the fence of the membrane skeleton network into an adjacent compartment (see Fig. 1 B).
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In addition to observing the movements, E-cadherin on
the free cell surface was dragged laterally along the plasma
membrane by OT. By observing the response of E-cadherin to this dragging force, E-cadherin molecules that are
either bound to or corralled in the membrane skeleton can
be distinguished, and the mechanical properties of the membrane skeleton that regulate the movements of E-cadherin
can be analyzed. We found that the mobility of Fusion was
restricted by tethering to actin filaments through its -catenin portion, whereas the movements of Catenin-minus and
Short-tailed were mainly regulated by the corralling effect
of the membrane skeleton network. Half of Wild were
tethered to, and the other half were confined by, the membrane skeleton. Both tethering and corralling structures
were found to be elastic.
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Materials and Methods |
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Cells
Mouse L-cells expressing Wild and mutant molecules after transfection and cloning were grown in MEM supplemented with 10% FCS. Cells cultured on a cover slip for 2 d after plating were used for the experiments. Cells transfected with cDNAs of Wild and Fusion showed cell-cell adhesion activity and, under optimal conditions, exhibited a cobblestone morphology typical of epithelial cells, whereas cells transfected with cDNAs of Catenin-minus and Short-tailed did not.
Preparation of Colloidal Gold and Latex Particles Coated with Anti-E-Cadherin mAb
Colloidal gold particles of 40 nm in diameter coated with anti-E-cadherin
mAb (ECCD-2; Shirayoshi et al., 1986) were prepared as described previously (G40; Kusumi et al., 1993
). Gold particles were incubated with
ECCD-2 IgG at a ratio of 500 IgG molecules/particle. This is the minimal
protecting amount (the lowest concentration of a protein concentration in
a solution that is used to pretreat the gold particles necessary to stabilize
the gold particles in suspension and avoid aggregation and sedimentation
of gold particles) of IgG. To test the effect of multiple binding of E-cadherins to gold particles, gold particles coated with smaller amounts of
E-cadherin specific Fab were prepared. In this case, gold particles were incubated with ECCD-2 Fab at a ratio of 100 Fab molecules/particle. This is
about 1/10 the molar amount of the minimal protecting amount of Fab.
Latex particles coated with ECCD-2 were prepared in the following way. ECCD-2 (300 µg) in 840 µl PBS (150 mM NaCl, 10 mM sodium phosphate, pH 7.2) was centrifuged at 12,000 g for 10 min. The supernatant was mixed with 60 µl of a suspension (2% solid) of 210-nm-diam latex particles (Polysciences Inc., Warrington, PA), vortexed for 10 s, and then incubated for 3 h at room temperature. BSA (10% in water, pH 7.0) was added as a stabilizer to a final concentration of 1%. After incubating for 1 h at room temperature, 5 ml PBS was added and the mixture was centrifuged at 12,000 g for 30 min. The precipitate was resuspended in 6 ml PBS by brief sonication and washed by two additional runs of centrifugation. After the final centrifugation, the precipitate was resuspended in 1 ml of HBSS buffered with Pipes, pH 7.2, containing 1% BSA (HBSS-BSA) by sonication, filtered through a 0.45-µm filter (Millipore Corp., Bedford, MA), and then stored at 4°C (L210).
Optical Trapping and Single Particle Tracking
Cells on a cover slip were incubated with 80 µl of G40 or L210 suspension for 30 min at room temperature, washed three times with HBSS-BSA, and then mounted in MEM (less NaHCO3) containing 10% FCS buffered with 5 mM Pipes, pH 7.2, on a slide glass with spacers of 0.2-mm-thick adhesive tape. The particles to be dragged or observed were selected randomly from over the entire cell surface, except for regions of cell-cell contact.
The optical trapping apparatus was the same as that used by Sako and
Kusumi (1995). Complexes of L210 and E-cadherin were captured with
the focused beam of an Nd/YAG laser (
= 1,064 nm) and dragged laterally along the plasma membrane by moving the laser beam.
The maximal trapping force was 0.8 pN, and the dragging velocity was
0.6 µm/s. SPT was carried out as described previously (Kusumi et al.,
1993; Sako and Kusumi, 1994
, 1995
) using video-enhanced Nomarski microscopy. All experiments were performed at 37°C.
Movements of G40 and L210 particles on the cell surface were recorded on a laser disk video recorder (TQ3100-F; Panasonic, Osaka, Japan). Video sequences were digitized frame by frame with an image processor (DVS-3000; Hamamatsu Photonics, Hamamatsu, Japan) and (x, y)
coordinates of particles in each video frame were calculated by a personal
computer using the method described by Gelles et al. (1988). Usually, movements during 16.7 s (500 video frames) were recorded for SPT with G40.
Data Analysis
Data analysis was basically the same as described previously (Kusumi et al.,
1993; Sako and Kusumi, 1994
, 1995
). The mean square displacement
(MSD) that is averaged over a trajectory at each time interval (
t) was
calculated from the trajectory of a particle. Dmicro was calculated as the
slope of the MSD-
t plot for 67-133 ms (2-4 video frames, the displacement between time 0 and 67 ms was not included to avoid high frequency
noise) by least-square fitting.
To determine the motional mode for each trajectory, MSD between 0 and 5 s (MSD5) was used (Kusumi et al., 1993). The method is briefly described below. Consider particles undergoing simple Brownian diffusion
at an average rate of Dmicro. MSD5 for simple Brownian particles after ensemble averaging over all of the particles will be 4 × Dmicro × 5 s. If MSD5
for a test particle is significantly greater than or less than 4 × Dmicro × 5 s,
the probability that the particle is not undergoing simple diffusion increases; it may be undergoing directed movement or confined diffusion,
respectively. Therefore, we introduce a convenient parameter to characterize a trajectory of a test particle in terms of its deviation in MSD from the
ensemble averaged MSD expected for simple Brownian particles possessing the same Dmicro as the test particle, i.e., RD (for relative deviation) = MSD5/(4 × Dmicro × 5 s) (Kusumi et al., 1993
). Since diffusion is a stochastic process, we generated 2,000 simple Brownian trajectories in a computer and obtained the distribution of the ratio MSD5/(4 × Dmicro × 5 s)
(= RDsim). For each experimental trajectory, we calculated MSD5/(4 × Dmicro × 5 s) (= RDexp), and determined whether this value was within
2.5% from either end of the distribution of RDsim. When RDexp was within
the middle 95% of the distribution of RDsim, the trajectory was classified as simple Brownian diffusion. When RDexp was within 2.5% from the high
(low) end of the distribution of RDsim, the trajectory was classified as directed (confined) diffusion.
The size of the confinement area and the drift velocity of directed
movement were estimated by fitting the MSD-t plot from
t = 0-5 s using equations we derived previously (Kusumi et al., 1993
).
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Results |
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Specificity and Multiplicity of Binding
E-cadherin molecules on the living cell surface were labeled with 40-nm colloidal gold particles or 210-nm
latex particles coated with anti-E-cadherin monoclonal antibody (G40 or L210, respectively). An average of 20 G40 or
L210 particles per cell were bound to the cell surface. The
number of particles bound to the cells vary from one cell
to another (10-40 particles per cell), and its variations for
different cell types were smaller than cell-to-cell variations. Difference in the type of particles, i.e., gold and latex particles, made no difference in terms of the number of
particles bound to the cell surface for all cell types used in
the present work (similar ranges and averages as above).
Particles coated only with BSA, without antibody IgG or Fab, were not bound to cells (only at a level of a few particles per cell for all types of cells used in the present work).
These results suggest high binding specificities of these
probes. Previously, we have shown that incubation of keratinocytes expressing higher levels of E-cadherin with the
particles in the presence of a 100-fold excess amount of
free ECCD-2 IgG (which was premixed with the particles
before addition to cells) reduced the binding of these particles to only several particles/cell. This result again indicates that the binding is specific for E-cadherin.
This result also suggests that the avidity effect, i.e., the
increases in the effective binding rate and/or the effective binding constant of ECCD-2 IgG because of multivalency
of the particles, was small. In the present investigation,
G40 coated with antibody Fab at molar concentrations
5-10 times less than those for antibody IgG (G40-Fab and
G40-IgG, respectively) were bound to the cell surface
equally well as particles coated with antibody IgG at higher
concentrations. As described above, both gold and latex
particles were bound to the cell surface at a similar level.
Taken together, these results suggest that these particles were bound to a single or a small group of E-cadherin molecules. Difference in motional characteristics of G40-IgG
and G40-Fab (and also L210-IgG) is described later in the
present report (virtually no difference). Mecham et al.
(1991) also suggested that the gold particles behave as individual ligand molecules and can be used to predict both
the location and binding properties when they studied the
elastin/laminin binding protein using single particle tracking.
The level of cross-linking by the particle probes would also depend on the concentration of cadherin on the cell surface (expression levels) and local concentration or intrinsic aggregation of cadherin. Another possibility is that induction of small aggregation by gold particles causes formation of large aggregates around the particle-induced aggregates. To examine such possibilities, E-cadherins were stained by indirect immunofluorescence, and the stained cells were observed by confocal laser scanning fluorescence microscopy (Fig. 3, the focus is set at about the height of the free cell surface near peripheries of the cell). Amounts of various cadherins expressed in cells look similar to each other (Fig. 3, A-D). Small aggregates outside the region of cell-cell contact may exist, but the amounts of aggregates are similar for different cadherins (larger punctates seen in wild-type cells are localized intracellularly, perhaps representing intracellular pools). Addition of G40 did not change the staining patterns (Fig. 3, E-H).
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The tendency that Fusion becomes aggregated after addition of the particle probes on the cell surface is not more than the tendencies of others. In addition, the extracellular domain is the same for all types of E-cadherin molecules. Therefore, it is not very likely that one type of cadherin mutants used in this work stuck to the particles more than others.
Lateral Dragging of Particle-E-Cadherin Complexes
A single-beam gradient optical trap (Ashkin et al., 1986,
1987
) was used to capture and drag L210-E-cadherin complexes laterally along the plasma membrane. The maximal
trapping force was 0.8 pN and the optical trap was moved
at a velocity of 0.6 µm/s (Sako and Kusumi, 1995
) for up to
5 µm. The direction of dragging was chosen randomly.
Since we are interested in the mechanisms that regulate
the movements and assembly process of E-cadherin, particles outside the region of cell-cell contact were selected
for the dragging experiments. Particles bound to E-cadherin at sites of cell-cell contact exhibited small movements
(Kusumi et al., 1993
). Complexes of 40-nm
colloidal gold
particles with anti-E-cadherin IgG or Fab could not be
captured by OT for unknown reasons. We have noted that
gold particles cannot be captured when coated with some
mAbs. This is a major reason why we used L210 for the dragging experiments. An added benefit of using L210 was
that the maximal trapping force with L210 was greater
than that with G40 by a factor of ~3.
In our previous experiments of dragging receptors for
transferrin and 2-macroglobulin, the results obtained by
these two types of probes were basically the same (Sako
and Kusumi, 1995
). We also found that characteristics of
diffusion for G40 and L210 attached to either transferrin
receptor or
2-macroglobulin receptor were the same, except that the diffusion rate of G40-labeled receptors is
20% greater than that of L210-labeled receptors. In the
present investigation, characteristics of diffusion of Wild
labeled with G40-Fab or L210-IgG were found to be similar to each other. For all types of cadherin molecules, diffusion properties were the same for G40-IgG and G40-Fab
as shown later (see below; and see Table II b). Therefore,
the results of the dragging experiment would have been
similar for G40-Fab and L210-IgG if G40 could have been
used for dragging experiments. However, due caution in
terms of the effect of cross-linking of cadherin molecules is
required in interpreting the present data.
Transferrin receptor molecules tethered to the membrane skeleton/cytoskeleton network could be clearly distinguished from free transferrin receptor molecules by
dragging them with OT (Sako and Kusumi, 1995). Membrane protein molecules tethered to the cytoskeleton or
the membrane skeleton may be dragged only short distances (Fig. 1 A, right), and after escaping from OT, they may return to their initial positions before being dragged.
On the other hand, membrane proteins that are free from
the tether of the cytoskeleton may be dragged much further. Even if they encounter the membrane skeleton fence
in the dragging path, they can pass through the fence if the
trapping force is sufficiently strong (Fig. 1 B, right). In the
case of transferrin receptor in the plasma membrane of
NRK cells, half of the particles passed across the membrane skeleton fence at a trapping force of 0.05-0.1 pN. If
the trapping force was insufficient, the molecules tended to escape from the OT at the fence (Fig. 1 B, center).
Fig. 4 shows typical trajectories of E-cadherin during dragging and after escape from the OT. The distance from the initial trap point to the farthest point reached by the particle in OT dragging was measured, and is called "escape distance (desc)" in this report.
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The time course of a typical dragging experiment is
shown in Fig. 5 A. The displacements of a particle-cadherin complex and the center of the OT are plotted against
time after the start of dragging. The OT was moved along
the sample plane at a constant rate (0.6 µm/s). If the only
force that is exerted on the complex besides that from the
OT is hydrodynamic drag in the lipid bilayer, the force
from the plasma membrane is small and the complex follows the OT. In the case shown in Fig. 5 A, the complex
more or less followed for OT for up to 1.4 s from the start
of dragging, or up to 0.78 µm. After 1.4 s, the complex
started to lag behind OT, indicating that some additional
force from the cell started to act on the complex. This additional force is likely to be due to the membrane skeleton/cytoskeleton. In this report, the distance the complex
followed the OT with little lag is called the "freely dragged
distance (dfd)." As described below, the freely dragged distance is different from the "barrier-free path" (BFP, the
distance from the start point to the farthest point reached
by the particle with dragging) defined by Edidin et al.
(1991).
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The lag of the complex behind the center of the OT increased up to 2.8 s, at which point the complex escaped
from the OT. At the escape point, the force exerted by the
OT becomes the maximum value of 0.8 pN. Since the distance from the start point to the escape point is the escape
distance, the escape distance in this report is the same as
the BFP in Edidin et al. (1991), but at the maximum dragging force of 0.8 pN. In the experiment shown in Fig. 5 A,
the escape distance is 1.32 µm. In principle, the freely
dragged distance is not dependent on the trapping force, whereas the escape distance is.
Escape Distance and Freely Dragged Distance
Histograms of the escape distance are shown in Fig. 6. Fusion exhibited very short escape distances (Figs. 3 D and 5 D; and Table I). In contrast, E-cadherin mutants that lacked the catenin-binding domain (Catenin-minus and Short-tailed) could be dragged an average of >1 µm (mean; Figs. 6, B and C; and Table I). In particular, Short-tailed could be dragged farthest.
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The distributions of the freely dragged distance for cadherins are shown in Fig. 7. E-cadherin molecules that are not bound to the membrane skeleton/cytoskeleton are expected to be dragged freely until they encounter the compartment boundaries (Fig. 5 B, left). On the other hand, cadherin molecules that are bound to the cytoskeleton must start to lag behind the OT immediately after the initiation of dragging (Fig. 5 B, right).
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The freely dragged distance for Fusion was only 20 nm (median), whereas Catenin-minus and Short-tailed showed much greater freely dragged distances (Fig. 7, note the logarithmic scale for the abscissa). In many cases (80%), Fusion started to lag behind the OT immediately after the start of dragging (dfd <50 nm). Some particles showed freely dragged distances <10 nm as seen in Fig. 6. Since our time resolution is limited to 33 ms, which reduces spatial resolution of a moving particle, the extremely short dragged distances simply indicate that the dragged distance was very small, or the backward movement induced by the force from the cell was initiated very early during dragging, and superimposed in the first several video frames. (Instrumental spatial precision is 1.5 nm.)
On the other hand, 65 and 82% of Catenin-minus and Short-tailed showed freely dragged distances of >50 nm, respectively. Freely dragged distances for Short-tailed were generally much greater than those for Catenin-minus. The cytoplasmic domain of Short-tailed (21-aa long) is substantially smaller than that of Catenin-minus (116-aa long). Therefore, the data on freely dragged distances are consistent with the membrane skeleton fence model, since Short-tailed should collide with the membrane skeleton fences less often than Catenin-minus.
These results are consistent with previous observations
suggesting that the COOH-terminal region of -catenin is
responsible for linking E-cadherin to actin filaments (Nagafuchi et al., 1994
; Rimm et al., 1995
). The binding affinity between
-catenin and F-actin must be high because almost all of the Fusion molecules exhibited the characteristics
of a tethered molecule.
The escape distances for Wild showed a broad distribution (Fig. 6 A). About half (58%) of Wild could be dragged <400 nm, whereas many (34%) Wild molecules could be dragged >1 µm. These results suggest that there are two populations of Wild; one is tethered to the cytoskeleton and the other is not.
The distribution of the freely dragged distance for Wild (Fig. 7 A) also suggests the presence of two populations. About half (45%) of Wild showed freely dragged distances <50 nm, whereas ~1/3 of Wild could be freely dragged >500 nm. This result again suggests that about half of Wild is tethered to the cytoskeleton, while the other half is free and only confined by the presence of membrane skeleton corrals.
Elasticity of the Membrane Skeleton/Cytoskeleton Network that Interacts with E-Cadherins
In the dragging experiments, many E-cadherin molecules showed rebound motion toward their initial positions after they escaped from the OT (Fig. 4), which indicates that the barriers for lateral dragging of E-cadherin are elastic. The elasticity of the membrane skeleton/cytoskeleton with which E-cadherins interact was estimated based on the response to the dragging by OT.
First, the distance from the point of initial encounter of
E-cadherin with the membrane skeleton fence to the point
at which E-cadherin escaped from the trap was measured.
This distance is the same as the extension (strain) of the
membrane skeleton, and called msk in the present paper,
i.e.,
msk = desc
dfd (Fig. 5 B). For example,
msk is 0.54 µm in the case of Fig. 5 A.
The maximum trapping force of the OT used in the
dragging experiments was 0.8 pN. Since this force equals
the force from the membrane skeleton, at the escape
point, 0.8 (pN) = kmsk × msk, where kmsk is the effective
spring constant of the membrane skeleton/cytoskeleton with which the particle-cadherin complex was interacting.
In this expression, the elasticity of the membrane skeleton
is approximated by a simple spring. This assumption is
good for small extension of the cytoskeleton, and has been
found to be true in the case of red blood cells for the extent of deformation seen in the present experiment
(Kusumi et al., 1997
). The same Hookean expression can
be used for E-cadherin molecules attached to the skeleton and those corralled by the skeleton. In the latter case, the
origin is simply shifted to the point of initial encounter of
the E-cadherin molecule to the membrane skeleton fence,
whereas in the former case, the origin is the point where
dragging was initiated. kmsk is 1.5 pN/µm in the case shown
in Fig. 5 A.
It is important to realize that, in the present method,
kmsk is estimated only when the particle escaped from the
OT. In addition, since it is possible that more than one
membrane skeleton fence is encountered during dragging
of the distance msk, the estimated value of kmsk should be
understood as the maximal estimate for the kmsk of the
membrane skeleton fence.
Histograms of kmsk's are shown in Fig. 8. Fusion exhibited kmsk's greater than those for Catenin-minus and Short-tailed. Again, the distribution of Wild falls between these two extreme distributions.
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It is likely that the force exerted on Short-tailed molecules from the membrane skeleton as they encounter during dragging is smaller than that on other E-cadherin. In the present investigation, we did not intend to measure such force. We only measured kmsk in the case where a particle escaped from the optical trap (in which case the escape force was 0.8 pN). To measure the dragging force required to move E-cadherin over the fence, particularly for E-cadherin with smaller cytoplasmic domains, much more refined method and instrumentation are required because the method has to be sensitive to all encounters of E-cadherin with the membrane skeleton fence, including those that involve very small force.
In Fig. 9, kmsk is plotted as a function of the freely dragged distance for each particle. kmsk of the tether for Fusion is broadly distributed (1-100 pN/µm). However, kmsk for Fusion that exhibited freely dragged distances of <50 nm tended to be greater than that for Fusion that exhibited freely dragged distances of >50 nm. kmsk for Short-tailed was distributed in the range of 1-10 pN/µm, and there seemed to be no evident relationship between kmsk and the freely dragged distance for Short-tailed. The plot for Wild looks like a mixture of those for Short-tailed and Fusion. The distribution of kmsk for Catenin-minus is similar to that for Short-tailed, although more points were in the region of a short, freely dragged distance, and some points in this region showed somewhat greater values for kmsk.
|
These results indicate that the cytoskeleton to which cadherins bind has greater effective spring constants than the membrane skeleton that is involved in corralling of cadherin molecules. kmsk for Fusion is likely to reflect the spring constant of the cytoskeleton to which cadherin is bound, and is estimated to be ~30 pN/µm (Fig. 8 D). The maximal values of kmsk of the membrane skeleton, which acts like a picket fence for dragging of cadherin can be estimated from those found in the dragging of Short-tailed and Catenin-minus, and was found to be 3.5-7.0 pN/µm (Fig. 8, B and C). This result suggests that kmsk of the cytoskeleton that tethers E-cadherin is greater than that of the general membrane skeleton by a factor of more than six (comparison in terms of median values).
Diffusion of E-Cadherins Observed by SPT
Movements of E-cadherins were followed by SPT. E-cadherin molecules on the cell surface were labeled with 40-nm
gold particles through monoclonal anti-E-cadherin antibody (ECCD-2), and movements of individual gold particles were observed by video-enhanced differential interference contrast microscopy (Kusumi et al., 1993).
Diffusion theories predict that simple increase in mass of
the Brownian particles does not affect the diffusion rate
(Berg, 1993). As long as the size of the particles are within
the range of Brownian particles, the diffusion rate is determined by the balance between the hydrodynamic dragging
force, which is dependent on the surface area (or radius, or
their equivalents), and thermal agitation (kT) (Berg, 1993;
Saffman and Delbrück, 1975
). In addition, since the viscosity of lipid bilayers is greater than that of water by a factor
of about 100, the diffusion rate (more specifically, hydrodynamic dragging force) is mainly determined by the
membrane-spanning domain unless there are other specific interactions such as those between the membrane
protein and the membrane skeleton. Therefore, simple increase of the size of the extracellular domain by addition of G40 or L210 would not greatly affect the diffusion rate.
In fact, in the present experiment, diffusion properties of
Wild were the same for both G40 and L210 particles. Previously, we found that diffusion rates of transferrin receptor and
2-macroglobulin receptor decreased only 20%
when the probe was changed from G40 to L210 (Sako and
Kusumi, 1994
). Diffusion rates of lipids in various cells and
liposomes measured with G40 were practically the same as
those measured by fluorescence redistribution after photobleaching (Lee et al., 1991
; Kusumi, A., unpublished
observations). In the present study, since all cadherin molecules used have the same extracellular and membrane-spanning domains, the difference in diffusion characteristics (and in responses to the dragging force exerted by
laser tweezers) for different cadherins could be ascribed to
changes induced by different cytoplasmic domains of these
cadherins.
Typical trajectories observed by SPT for 16.7 s (500 video frames) are shown in Fig. 10. These trajectories are those that showed median MSD values at 5 s for each type of molecule (see the Materials and Methods). The area the movements covered was largest for Short-tailed, and smallest for Fusion.
|
The microscopic diffusion coefficient Dmicro for each particle was calculated from MSD during 67-167 ms (see Materials and Methods), which represents the diffusion rate in this time window and a space scale of <500 nm. Fig. 11 shows the distributions of Dmicro, and their mean and median values are listed in Table II.
|
|
Dmicro for Fusion shows a peak of ~2 × 1012 cm2/s (Fig.
11 D), which may represent local and/or fluctuating movements of the cytoskeleton to which Fusion is bound. Aggregation of Fusion molecules may occur through a putative self-assembly domain of
-catenin (Nagafuchi et al.,
1991
), or through self-association of the extracellular domain of E-cadherin (Shapiro et al., 1995
), but the dependence of the diffusion coefficients of membrane proteins
on their size (i.e., aggregation) is slight (because the diffusion rate in a two-dimensional plane only depends on the
logarithm of the radius of a diffusing unit; Saffman and
Delbrück, 1975
). If the difference in Dmicro by a factor of
100 on average between Fusion and Short-tail were to be
explained solely by aggregation of Fusion, the aggregate size of Fusion would have to be like >10,000 monomers.
Such large aggregates of Fusion molecules were not detected by indirect immunofluorescence microscopy (Figs.
3, D and H). These results suggest that the small Dmicro
(and short dfd) for Fusion is mainly because of tethering to
the cytoskeleton.
Dmicro values for Catenin-minus and Short-tailed exhibited peaks at ~0.9 × 1010 and ~3 × 10
10 cm2/s, respectively, which are greater than that for Fusion by a factor of
~100. These results are consistent with the dragging data
that suggest that Fusion was tethered to the cytoskeleton, whereas Catenin-minus and Short-tailed were not bound
to the cytoskeleton. Only small subpopulations of Short-tailed and Catenin-minus showed Dmicro values indicative
of a bound component (Fig. 11, B and C). This may be due
to binding to the cytoskeleton mediated by another membrane protein that is associated with the cytoskeleton.
The distribution of Dmicro for Wild is broad, and covers
the distributions for both Fusion and Catenin-minus (or
Short-tailed). Wild molecules that exhibited Dmicro values
in the same range as Fusion molecules (<1.5 × 1011 cm2/s)
may be bound to the cytoskeleton, whereas those with
Dmicro values >1.5 × 10
11 cm2/s are probably free from
the cytoskeleton.
In conclusion, the distribution of Dmicro for each cadherin represents a superposition of two populations. One
may consist of molecules bound to the cytoskeleton and
the other may consist of molecules free from the cytoskeleton. The bound and unbound fractions typically have
Dmicro values smaller or greater than ~1.5 × 1011 cm2/s,
respectively.
Since gold particles can cross-link E-cadherin, and since
the degree of cross-linking may vary from one type of cadherin to another because of different aggregation levels on
the cell surface (although the aggregate size is small as
shown in Fig. 3), SPT was performed with gold particles
coated with anti-cadherin Fab with 1/5 molar amounts of
IgG. The results of SPT are summarized in Table II b,
which shows that the diffusion characteristics of gold-Fab
particles are very similar to those of gold-IgG particles in
all types of cells used in this work. In addition, diffusion characteristics of L210 bound to Wild are similar to those
of G40-Fab and G40-IgG (data not shown). These results
in turn suggest that these probes did not induce formation
of large aggregates, and that the different diffusion characteristics observed for different cadherins were not created
by cross-linking by the particle probes but were due to different cytoplasmic domains of these cadherins. As discussed previously, most of the IgG and Fab molecules on
the surface of the gold or latex particles are likely to be denatured, which reduces the effective valency of these particle probes (Sako and Kusumi, 1994; Kusumi et al., 1997
).
In some trajectories, they show behaviors that suggest interconversion of bound and unbound states. However, since diffusion is a stochastic process, it is very difficult to prove that the particular changes observed in a trajectory is statistically meaningful. At this stage of the investigation, we would like to refrain from making specific comments on these. However, we would like to point out the possibility that the spread in Dmicro for Wild seen in Fig. 11 could partially be due to interconversion between the free and the bound states. Another possible reason for the spread in Dmicro could be because of coexistence of particles that are bound to different number of E-cadherin molecules.
Movements of transferrin receptor in transfectants expressing Wild (EL 1a) and Short-tailed (EL
24) were
observed by SPT (Tables II and III). Dmicro and the confinement area (see below) for transferrin receptor were
similar in both clones, indicating that the differences observed among various E-cadherins depend on the structural differences in their cytoplasmic domains, rather than
on differences in cellular structures.
|
Although most of the Catenin-minus and Short-tailed
molecules appear to be free from tethering to the cytoskeleton, Dmicro for these molecules (1-2 × 1010 cm2/s) was
smaller than that expected for membrane proteins diffusing freely in the plasma membrane (1-4 × 10
9 cm2/s; Jacobson et al., 1987
), i.e., Dmicro of E-cadherin is reduced by
mechanisms other than long-term binding to the cytoskeleton. Dmicro for transferrin receptor was 10-fold smaller in
L cells (~10
10 cm2/s; Table II) than in NRK cells (~10
9
cm2/s; Sako and Kusumi, 1994
). The mechanism that reduces Dmicro of these proteins in the plasma membrane of
L cells is not known. This reduction may be because of a
crowding effect by other membrane proteins, particularly
in their extracellular domains (Sheetz, 1993
), interaction
with the extracellular matrix (Lee et al., 1993
), and/or a
percolation effect of immobile or slowly diffusing obstacles (Saxton, 1987
). The association of a cadherin-catenin complex with other proteins has been reported (Balsamo
and Lilien 1990
; Nelson, et al., 1990; Itoh et al., 1993;
Hinck et al., 1994
; Hoschuetzky et al., 1994
).
Directed Movements of E-Cadherins Bound to the Cytoskeleton
Movements of E-cadherin observed by SPT were statistically classified into three types of motion, i.e., simple diffusion, confined diffusion, and directed movement (Kusumi
et al., 1993), within a time window of 5 s. The results are
shown in Fig. 12 with further classification; values for Dmicro
greater or smaller than 1.5 × 10
11 cm2/s correspond to
mostly unbound and bound cadherin molecules, respectively (as discussed in the previous section).
|
Considerable fractions of Wild and Fusion undergo directed movement (8 and 20%, respectively; Table III). On
the other hand, Catenin-minus and Short-tailed did not
show directed movement. (Because of the statistical nature of this classification method, a percentage of <2.5 for
directed movement is insignificant, see Materials and
Methods.) Representative trajectories of particles classified as directed movement are shown in Fig. 13, A (Wild) and B (Fusion). Such movements of Wild and Fusion reflected the superposition of rather linear and uniform motion and random diffusion, in which Dmicro was smaller
than the overall average by a factor of 6-13 (Table II and
III). The average drift velocity was ~20-30 nm/s (Table
III), which is comparable to the velocity of the tread milling of actin filament in the cytoplasm (Wang, 1985; Sheetz et al., 1989
). These properties suggest that directed movement is related to the movement of cytoskeletal filaments
rather than to membrane flow.
|
Confined Diffusion of Unbound E-Cadherins
A large fraction (30-40%) of Wild, Catenin-minus, and
Short-tailed molecules exhibited confined diffusion, as
shown in Fig. 12. This mode is more clearly seen in the
population that showed Dmicro values >1.5 × 1011 cm2/s
(50%).
We previously proposed that the compartmentalized
structure caused by the membrane skeleton fence is a basic feature of the plasma membrane (Kusumi and Sako,
1996). According to this model, particles that are classified
as exhibiting simple diffusion are undergoing "apparently
simple diffusion." They may be located in larger compartments and/or have smaller Dmicro values. For example, in
the time scale used for classifying the mode in this report
(5 s), particles exhibiting Dmicro values <0.75 × 10
10 cm2/s
will not feel the boundaries of compartments that are
>0.15 µm2, which is the average compartment size for
Wild and Catenin-minus (Table III). This may explain why
few particles with Dmicro values <1.5 × 10
11 cm2/s are
classified into the confined diffusion mode (Fig. 12).
Therefore, for the movements of cadherins that are not
bound to the cytoskeleton, compartmentalization of the
plasma membrane, perhaps because of membrane skeletal
barriers, plays a major role in determining their mobility.
The confinement area was the same for Wild, Catenin-minus, and transferrin receptor (0.15 µm2 on average), but was greater (0.5 µm2) for Short-tailed, which has a very small cytoplasmic domain (21 aa) (Table III). These results support the notion that the membrane skeleton fence effect is a mechanism for regulating the diffusion of cadherins that are not bound to the cytoskeleton.
Effect of Cytochalasin D on E-Cadherin Diffusion
SPT measurements were carried out in the presence of 1 µM
cytochalasin D to examine the effect of partial destruction
of actin filaments. The effect was dramatic and almost all
particles started undergoing slow simple Brownian diffusion after the drug treatment. Dmicro decreased by a factor
of 10-50 except for Fusion, which showed a decrease by a
factor of 2.5 (Table II c). Although these results do not directly support the fence or the tether models, they strongly
suggest the involvement of actin filaments in regulation of
the movement of cadherin. Such reduction of Dmicro and
increase in the simple Brownian mode in the cells treated
with cytochalasin D were previously observed for receptors for transferrin and 2-macroglobulin in NRK cells
(Sako and Kusumi, 1994
). It is possible that membrane proteins were trapped in the membrane-bound aggregates
of actin filaments formed by the cytochalasin treatment.
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Discussion |
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---|
Cytoplasmic Regulation of the Movements of E-Cadherin in the Plasma Membrane by the Membrane Skeleton/Cytoskeleton Network
Based on motion analysis by SPT and lateral dragging by
OT of transferrin receptor, 2-macroglobulin receptor,
EGF receptor, and E-cadherin, we have proposed two
mechanisms for the cytoplasmic regulation of movements
of membrane proteins in the plasma membrane, i.e., tethering to the cytoskeleton and temporal confinement
within the membrane skeleton mesh (Fig. 1; Kusumi and
Sako, 1996
). The results of other groups have also suggested the cytoplasmic regulation of the movement of
plasma membrane proteins by the membrane skeleton/cytoskeleton network (Sheetz et al., 1980
, 1989
; Kucik et al.,
1989
; De Brabander et al., 1991
; Edidin and Stroynowski,
1991
; Schmidt et al., 1993
; Wang et al., 1994
). In the
present study, we examined the cytoplasmic regulation of
E-cadherin using the wild type and three cytoplasmic mutants.
Fig. 14 shows a model which we propose to explain the results obtained in this research. Tethering to the cytoskeleton, as shown in Fig. 14 A, was typical of Fusion. The small Dmicro for Fusion may reflect the local movement and conformational fluctuation of the cytoskeleton to which Fusion is bound. In addition, some of the tethered molecules are transported directly. Catenin-minus and Short-tailed do not exhibit tethering to the cytoskeleton, but are confined within submicrometer scale membrane compartments (Fig. 14, C and D, respectively). A decrease in the size of the cytoplasmic domain of E-cadherin from 116 aa (Catenin-minus) to 21 aa (Short-tailed) produces an increase in the compartment size by a factor of four (Table III). The possibility of colliding with the fence of the membrane skeleton network should be greater for a molecule with a greater cytoplasmic domain.
|
The results for Wild suggest the presence of two populations of Wild: one that is tethered to the membrane skeleton/cytoskeleton and another that is unbound but confined by the membrane skeleton (Fig. 14, A-C). About
half of Wild showed an escape distance (Fig. 6) and a
freely dragged distance (Fig. 7) as short as those for Fusion, whereas the other half showed values similar to those for Catenin-minus and Short-tailed. In addition, the distribution of Dmicro for Wild appeared to be a superposition of
those for Fusion (1012-1.5 × 10
11 cm2/s) and Short-tailed
(1.5 × 10
11-10
9 cm2/s) (Fig. 11).
Similar SPT and OT experiments were carried out with
other mutants of E-cadherin that have a deletion inside
(aa 814-849; EL 32) or outside (aa 774-813, EL
33; and
aa 751-773, EL
34) of the catenin-binding domain (Nagafuchi and Takeichi, 1989
). The results are summarized
in Table IV. Dmicro is expected to be smaller for mutants
with actin-binding capability (i.e., with catenin- binding
sites), which was observed. Dmicro for EL
32 is close to
catenin-minus, whereas those for EL
33 and 34 are similar to Wild. Confinement areas for the particles undergoing confined diffusion are similar to Wild and Catenin-
minus, which is also consistent with the above model. Escape distance was only measured with EL
33, and found
to be ~200 nm, which is close to Wild. This again is consistent with the above model.
|
We found a submicron scale meshwork on the cytoplasmic surface of the dorsal part of the plasma membrane,
much of which was consisted of actin filaments (Kawasaki
et al., 1995). Actin filaments may be involved in the membrane skeleton fence structure that restricts the movement
of E-cadherin within compartments, consistent with the effect of cytochalasin D. The cytoskeleton to which E-cadherin is tethered is likely composed of actin filaments
(Hirano et al., 1987
; Nagafuchi and Takeichi, 1989
; Ozawa
et al., 1990
).
-Catenin has been proposed to mediate E-cadherin-
actin linkage (Nagafuchi and Takeichi, 1989
; Ozawa et al.,
1989
, 1990
). The COOH-terminal half of
-catenin possesses regions that are homologous to the actin-binding region of vinculin (Nagafuchi et al., 1994
; Johnson and Craig,
1995
). Recently, the COOH-terminal part of
-catenin consisting of 447 aa was reported to bind directly to F-actin
(Rimm et al., 1995
). On the other hand, a yeast two-hybrid assay and an in-vitro binding assay between recombinant
E-cadherin and catenins have suggested that
-catenin
mediates the association between E-cadherin and
-catenin
(Jou et al., 1995
).
Activities of Cell-Cell Adhesion and of Actin Binding of E-Cadherin Are Highly Correlated
In the cell aggregation assay, it was shown that Fusion has
higher activity in inducing aggregation of cells. The cells
expressing Fusion were found to be flat even in the
metaphase of cytokinesis (Nagafuchi et al., 1994). These
observations indicate that Fusion has greater cell adhesion
activity than Wild.
The present results indicate that half of Wild molecules
are free from tethering, whereas almost all of Fusion molecules are bound to the cytoskeleton. Since tethering to the
cytoskeleton mediated by -catenin is necessary for E-cadherin to exhibit cell adhesion activity (Watabe et al., 1994
),
greater activity of Fusion can be explained by its binding
to the actin skeleton.
Related to this correlation is the present finding that the spring constant of the membrane skeleton involved in tethering is greater than that involved in corralling by a factor of six. Actin bundles may be involved in tethering of E-cadherin, which may help strengthen the cell adhesion activities.
Resistance to Detergent Extraction and Tethering to the Membrane Skeleton/Cytoskeleton
Previous studies have assumed that most of the Wild and
Fusion molecules located inside cell-cell contact regions
could not be extracted by a nonionic detergent, 2.5% NP-40
(Nagafuchi and Takeichi, 1989; Nagafuchi et al., 1994
) or a
mixture of 1% NP-40 and 1% Triton X-100 (Ozawa et
al., 1989
, 1990
). Although considerable amounts of E-cadherin molecules are diffusely distributed over the cell surface, it has been difficult to find out whether or not they
are bound to the cytoskeleton. Such population of cadherin is important because these cadherins provide the
ready pool for new cell-cell association, and perhaps they
may be surveying new physical contacts with other cells.
However, detergent extraction was incapable of distinguishing bound and unbound components of E-cadherin.
Wild and Fusion molecules were largely extracted from the free cell surface with nonionic detergents as examined
by immunofluorescence microscopy (Nagafuchi and Takeichi, 1989
; Nagafuchi et al., 1994
).
The SPT and OT experiments in the present study showed that half of Wild and most of Fusion are linked to the cytoskeleton, even outside cell-cell contact sites. This clearly shows that even membrane proteins that are bound to the cytoskeleton can be extracted by nonionic detergents, i.e., extractability with nonionic detergents is no guarantee for unbinding of the membrane protein from the cytoskeleton. Mechanical assays, such as SPT and OT, are more direct and reliable than detergent extraction methods.
Mechanical Properties of the Interaction between Membrane Proteins and the Membrane Skeleton/Cytoskeleton Network
Both the tether and fence structures interacting with E-cadherin could be deformed using OT at a dragging force of
0.8 pN, and rebound motion of E-cadherin after its escape
from OT indicates that the tether and fence are elastic
(Fig. 4). The median values for spring constants of the
tether and fence estimated from the dragging experiments
were 30 and 3.5 pN/µm, respectively (Fig. 8; 8 and 3 pN/µm
on average). Previously, we found that the effective spring
constants of the membrane skeleton involved in the tether and fence were ~12 and 3 pN/µm, respectively, in a dragging experiment of transferrin receptor in NRK cells
(Sako and Kusumi, 1995).
In both L (expressing E-cadherins) and NRK (transferrin receptor) cells, the effective spring constant for tethers is greater than that for fences, suggesting that the structure of the membrane skeleton and the cytoskeleton involved in these interactions are different. In the case of E-cadherin, tethers may be bundles of actin filaments, whereas fences may be thinner bundles or single actin filaments. The average fluctuation of the elastic filaments for tethers (kmsk = 0.03 pN/nm) and fences (kmsk = 0.0035 pN/nm) can be estimated from the elastic constants, and was found to be 10 and 30 nm, respectively [since (1/2)kmsk < x2 > = (1/2) kBT = 2 pN·nm, < x2 >1/2 = < 4/kmsk >1/2 nm].
One way to estimate fluctuation of the cytoskeleton is to examine the trajectories of directed movements, and evaluate the mean fluctuation perpendicular to the direction of drift movement (because the cytoskeleton is responsible for such directed movements). This was done using MSD150 in the perpendicular direction to the directed movement, and was found to be 21 ± 2 and 40 ± 20 nm for Wild and Fusion, respectively, in general agreement with those estimated from the spring constant. This agreement suggests again that the cytoskeleton is responsible for forming tethers and the fences for E-cadherin.
Calcium-induced Assembly of E-Cadherin during the Formation of Adherens Junctions
In the course of the calcium-induced formation of cell-to-cell
adherens junctions in epithelial cells in vitro, E-cadherin on the dorsal cell surface assembles into cell-cell contact
sites. This assembly occurs via the movement of E-cadherin in the plasma membrane (Kusumi et al., 1988).
Therefore, the mechanism that regulates the movement of
E-cadherin on the cell surface is important for E-cadherin
assembly and the formation of adherens junctions (McNeill et al., 1993
).
E-cadherin in adherens junctions is linked to actin filaments (Hirano et al., 1987; Nagafuchi et al., 1994
), whereas
data by SPT and OT indicate that only part of the E-cadherin on the free cell surface (outside the cell-cell contact
region) is associated with the cytoskeleton in both cultured
mouse keratinocyte (Kusumi et al., 1993
) and L cells. Regulation mechanisms, such as the binding of E-cadherin to
the cytoskeleton, cytoskeleton-dependent transport of bound
and corralled E-cadherin, and changes in the association
states of E-cadherin, which would greatly affect binding and corralling of E-cadherin by the membrane skeleton,
may play important roles in the assembly of E-cadherin at
cell-cell contact sites.
![]() |
Footnotes |
---|
Received for publication 29 July 1997 and in revised form 8 January 1998.
Address all correspondence to Akihiro Kusumi, Department of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. Tel.: 011-81-52-789-2969. Fax: 011-81-52-789-2968. E-mail: akusumi{at}bio.nagoya-u.ac.jp ![]() |
Abbreviations used in this paper |
---|
MSD, square displacement averaged over a single particle's trajectory (running average over a single trajectory) ; OT, optical tweezers; SPT, single particle tracking.
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