76
Actin dynamics and cell–cell adhesion in epithelia
Valeri Vasioukhin and Elaine Fuchs*
Recent advances in the field of intercellular adhesion highlight
the importance of adherens junction association with the
underlying actin cytoskeleton. In skin epithelial cells a dynamic
feature of adherens junction formation involves filopodia, which
physically project into the membrane of adjacent cells,
catalyzing the clustering of adherens junction protein complexes
at their tips. In turn, actin polymerization is stimulated at the
cytoplasmic interface of these complexes. Although the
mechanism remains unclear, the VASP/Mena family of proteins
seems to be involved in organizing actin polymerization at these
sites. In vivo, adherens junction formation appears to rely upon
filopodia in processes where epithelial sheets must be
physically moved closer to form stable intercellular connections,
for example, in ventral closure in embryonic development or
wound healing in the postnatal animal.
Addresses
Howard Hughes Medical Institute, Department of Molecular Genetics
and Cell Biology, The University of Chicago, 5841 South Maryland
Avenue, Room N314, Chicago, Illinois 60637, USA
*e-mail: nliptak@midway.uchicago.edu
normally involved in adherens junction formation through
its ability to bind to β-catenin and link cadherins to the
actin cytoskeleton [1,4]. However, β-catenin leads a dual
life in that it can also act as a transcriptional cofactor when
stimulated by the Wnt signal transduction pathway [5].
α-catenin: more than just a bridge between
adherens junctions and the actin cytoskeleton
α-catenin was initially discovered as a member of the E-cadherin–catenin complex [6,7]. It is related to vinculin, an
actin-binding protein that is found at integrin-based focal
contacts ([8,9]; Figure 2). The amino-terminal domain of
α-catenin is involved in α-catenin/plakoglobin binding and
is also important for dimerization [10–14]. Its central segment can bind to α-actinin [11] and to vinculin [15], and it
partially encompasses the region of the protein necessary for
cell adhesion (which is the adhesion-modulation domain;
amino acids 509–643 [16••]). The carboxy-terminal domain
of both vinculin and α-catenin is involved in filamentousactin (f-actin) binding [17], and for α-catenin, this domain is
also involved in binding to ZO1 [16••].
Current Opinion in Cell Biology 2001, 13:76–84
0955-0674/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Cell–cell adhesion is required for tissue morphogenesis and
homeostasis. In these processes, epithelial cells utilize many
types of intercellular adhesion structures. This review concentrates on the adherens junction and places specific
emphasis on the role of the actin cytoskeleton in the formation and maintenance of these structures. Located at
cell–cell borders, adherens junctions are electron dense
transmembrane structures that associate with the actin
cytoskeleton [1]. In their absence, the formation of other
cell–cell adhesion structures is dramatically reduced.
The transmembrane core of adherens junctions consists of
cadherins, of which E-cadherin is the epithelial prototype.
Its extracellular domain is responsible for homotypic, calcium-dependent, adhesive interactions with E-cadherins
on the surface of opposing cells. Its cytoplasmic domain is
important for associations with other intracellular proteins
involved in the clustering of surface cadherins to form a
junctional structure (Figure 1). Through a site near its
transmembrane domain, cadherins bind directly to the
catenin p120ctn, and through a more central site within the
cytoplasmic domain, cadherins bind preferentially to
β-catenin, but can also bind plakoglobin, a close relative of
β-catenin. The mechanism of action of p120ctn is still
poorly understood, although it has been implicated in both
intercellular adhesion and cell migration [2]. Recent studies suggest that p120ctn promotes cell migration through
recruiting and activating small GTPases [3]. β-catenin is
α-catenin is the only catenin that can directly bind to actin
filaments [17], and E-cadherin–catenin complexes do not
associate with the actin cytoskeleton after α-catenin is
removed by extraction with detergent [18]. Cancer cell
lines lacking α-catenin still express E-cadherin and
β-catenin, but do not show proper cell–cell adhesion [19]
unless the wild-type gene is reintroduced into the cancer
cell [13,20,21]. This provides strong evidence that clustering of the E-cadherin–catenin complex and cell–cell
adhesion requires the presence of α-catenin.
Although intercellular adhesion is dependent upon association of the E-cadherin–β-catenin protein complex with
α-catenin and the actin cytoskeleton, it is unclear whether
α-catenin’s role goes beyond linking the two structures.
Fusion of a nonfunctional tailless E-cadherin (E C71) with
α-catenin resulted in a chimeric protein able to confer
cell–cell adhesion on mouse fibroblasts in vitro [22], and
generation of additional chimeric proteins enabled delineation of the region of α-catenin that is important for cell
aggregation [16••,23]. Not surprisingly, the essential domain
of α-catenin was its carboxy-terminal domain (~amino acids
510–906), containing the actin-binding site, which encompasses residues 630–906 of this domain [16••,23]. The
actin-binding domain is certainly a key component of
α-catenin, as evidenced by the fact that the remaining part
of α-catenin expressed as a β-geo fusion protein was unable
to compensate for α-catenin in the trophectoderm of developing mouse blastocysts [24]. Interestingly, however, the
carboxy-terminal domain of vinculin, which is similar to
α-catenin and also contains an actin-binding site, was not
able to substitute for α-catenin in in vitro transfection assays,
Actin dynamics and cell–cell adhesion in epithelia Vasioukhin and Fuchs
77
Figure 1
Schematic model of an idealized epithelial
adherens junction. The extracellular domain of
the transmembrane E-cadherin dimerizes and
interacts in a calcium-dependent manner with
similar molecules on neighboring cells. The
intracellular juxtamembrane part of E-cadherin
binds to p120ctn, an armadillo repeat protein
capable of modulating E-cadherin clustering.
The distal segment of E-cadherin’s
cytoplasmic domain can interact with
β-catenin or plakoglobin, armadillo repeat
proteins which in turn bind to α-catenin. The
carboxyl end of α-catenin binds directlys to
f-actin, and, through a direct mechanism,
α-catenin can link the membrane-bound
cadherin–catenin complex to the actin
cytoskeleton. Additionally, α-catenin can bind
to either vinculin or ZO1, and it is required for
junctional localization of zyxin. Vinculin and
zyxin can recruit VASP (and related family
members), which in turn can associate with
the actin cytoskeleton, providing the indirect
mechanism to link the actin cytoskeleton to
adherens junctions. ZO1 is also a member of
tight junctions family, providing a means to link
these junctions with adherens junctions.
f-actin
Plasma membranes
E-cadherin
Extracellular
- f-actin
- Vinculin
- p120ctn
- α-catenin
- α-actinin
- β-catenin
- ZO1
- VASP
- E-cadherin
Current Opinion in Cell Biology
nor was the actin-binding domain of α-catenin. Thus,
although the binding of α-catenin to the actin cytoskeleton
is required for cell–cell adhesion, α-catenin appears to have
some additional function(s) that go beyond its ability to link
E-cadherin–β-catenin complexes to actin filaments. The
domain encompassing residues 509–643 of α-catenin has
been referred to as an adhesion-modulation domain to
reflect this added, and as yet unidentified, function [16••].
Although simple connection of the E-cadherin–catenin
complex to the actin cytoskeleton does not appear to be sufficient to support adhesion we do not understand exactly
Figure 2
Schematic representation of α-E-catenin and
its functionally important regions. VH1, VH2
and VH3 are three regions sharing homology
to vinculin. The percentage amino acid identity
is indicated below, and the numbers
correspond to the amino acid residues of the
α-catenin polypeptide.
22
224
355
595 697
849 906
-COOH
VH3 34%
NH2VH1 27%
VH2 31%
α-E-catenin
Binding regions:
54
82
β-catenin/
plakoglobin
148
279
Dimerization
697
509
402
325
394
697
f-actin
Adhesion
modulation
domain
643
631
327
906
906
ZO1
906
Vinculin
α-actinin
Current Opinion in Cell Biology
78
Cytoskeleton
Figure 3
Low calcium 0.07 mM
High calcium 1.5 mM
Cytochalasin D
Passive adhesion
Normal
conditions
Active adhesion
Current Opinion in Cell Biology
Schematic representations of models for active and passive cell–cell
adhesion. Upon a switch from low to high calcium, cadherin-mediated
intercellular adhesion is activated in primary keratinocyte cultures.
Passive adhesion: in cells whose actin cytoskeleton has been largely
disrupted by cytochalasin D, cadherin–catenin complexes occur only
at sites where membranes of neighboring cells directly contact each
other. Active adhesion: neighboring cells with functional actin
cytoskeletons can draw their membranes together, forming a
continuous epithelial sheet. Cadherin–catenin complexes of proteins
are indicated in green, the actin cytoskeleton is depicted in red.
how α-catenin performs its function(s). Besides its association with β-catenin and f-actin, α-catenin binds to a
number of additional proteins, some of which are actinbinding proteins themselves. For example, α-actinin
coimmunoprecipitates with α-catenin, and the two interact
in a yeast two-hybrid system [11,25]. Additionally, the
localization of vinculin to cell–cell borders is dependent
upon the presence of α-catenin [15]. α-catenin can also
bind to the MAGUK (membrane-associated guanylate
kinase) family members ZO1 and ZO2 [26,16••]. Proteins
involved in regulating actin polymerization, such as VASP
(vasodilator-stimulated phosphoprotein) and Mena, also
localize to E-cadherin–catenin complexes, and this localization was impaired in primary keratinocytes deficient for
α-catenin [27••]. Taken together, these findings suggest
that the role for α-catenin might not simply be to link
E-cadherin–catenin complexes to the actin cytoskeleton
but rather to organize a multiprotein complex with multiple
actin-binding, bundling and polymerization activities.
Adherens junction formation in cells with a
disrupted actin cytoskeleton: active versus
passive intercellular adhesion
The decisive requirement for α-catenin’s actin-binding
domain in adherens junction formation underscores the
importance of the actin cytoskeleton in intercellular adhesion. Thus, it is perhaps not surprising that the majority of
f-actin in epithelial cells localizes to cell–cell junctions
[27••]. When epidermal cells are incubated in vitro in culture media with calcium concentrations below 0.08 mM
they are unable to form adherens junctions. However,
when the calcium concentrations are raised to the levels
naturally occurring in skin (1.5–1.8 mM), intercellular
adhesion is initiated. This switch in part promotes a calcium-dependent conformational change in the extracellular
domain of E-cadherin that is necessary for homotypic
interactions to take place [1]. In primary keratinocytes, disruption of the actin cytoskeleton by treatment with
cytochalasin D abolishes cell–cell junction formation in
this assay [27••]. Despite this impairment, some E-cadherin–catenin complexes are still deposited at sites where
the membranes of neighboring cells are in contact with
each other. Similar studies with the MDCK kidney epithelial cell line reveal that in the presence of cytochalasin D
cells are unable to form new cell–cell contacts, and the
young contacts (less than one hour old) disassemble [28].
Interestingly, the mature contacts (more than one hour old)
are not disrupted. These data suggest a role for the actin
cytoskeleton in facilitating the process that brings opposing membranes together and stabilizing them once
junction formation has been initiated. In this regard, the
formation of cell–cell adhesion can be divided into two categories: active adhesion, a process that utilizes the actin
cytoskeleton to generate the force necessary to bring
opposing membranes together, and passive adhesion, a
process which may not require actin if the membranes are
already closely juxtaposed and stabilized by the deposition
of cadherin–catenin complexes (Figure 3).
Dynamics of cadherin–catenin complexes and
actin during junction formation
In vitro studies
James Nelson and colleagues [28] have used retrospective
and real time immunofluorescence microscopy to monitor
E-cadherin and actin cytoskeletal dynamics during epithelial sheet formation in MDCK cells [28,29]. Upon initial
membrane contact, E-cadherin forms punctate aggregates
or puncta along regions where opposing membranes are in
contact with one another (Figure 4a). Each of these puncta
is contacted by a bundle of actin filaments that branch off
from the cortical belt of actin filaments underlying the cell
membrane. At later stages in the process, those segments of
the circumferential actin cables that reside along the zone
of cell–cell contacts disappear, and the resulting semi-circles of cortical actin align to form a seemingly single
circumferential cable around the perimeter of the two cells.
At the edges of the zone of cell–cell contact, plaques of
E-cadherin–catenin complexes connect the cortical belt of
actin to the line of adhesion (opposing arrows in Figure 4).
At the center of the developing zone of adhesion, E-cadherin puncta associate with small bundles of actin filaments
oriented perpendicular to the zone. Propagation of the seal
between the cells can be visualized as a line of E-cadherin
Actin dynamics and cell–cell adhesion in epithelia Vasioukhin and Fuchs
79
Figure 4
A model for actin and E-cadherin dynamics
during epithelial sheet formation in MDCK
cells and in primary mouse keratinocytes.
(a) In MDCK cells, a circumferential actin
cable (thick red line) surrounds cells. Multiple
E-cadherin-containing puncta (green dots)
form along the developing contact and
rapidly associate with small bundles of actin
filaments (thin red lines). As the contact
between cells lengthens, puncta continue to
develop at a constant average density, with
new puncta at the edges of the contact. The
segment of the circumferential actin cable
that underlies the developing contact
gradually ‘dissolves’, and merges into a large
cable, encompassing both cells. This is made
possible through cable-mediated connections
to the E-cadherin plaques at the edges of the
contact. As contact propagates, E-cadherin
is deposited along the junction as a
continuous line of staining. The actin
cytoskeleton reorganizes and is now oriented
along the cell–cell contact [28]. (b) In
primary keratinocytes, two neighboring cells
send out filopodia, which, upon contact, slide
along each other and project into the
opposing cell’s membrane. Filopodia are rich
in f-actin (thin red lines). Embedded tips of
filopodia are stabilized by puncta (green),
which are transmembrane clusters of
adherens junction proteins. This process
(a) MDCK cells
(b) Primary keratinocytes
- Filopodia
- E-cadherin, α-and β-catenins
- Zyxin, vinculin, VASP, Mena
- Actin filaments
- Actin monomer
Current Opinion in Cell Biology
draws regions of the two cell surfaces
together, which are then clamped by
desmosomes. Radial actin fibers (thick red
lines) reorganize at filopodia tips in a zyxin-,
vinculin-, VASP-, and Mena-dependent
fashion (yellow crescents). Actin
polymerization is initiated at stabilized puncta
(green crescents and dots), creating the
directed reverse force needed to push and
merge puncta into a single line as new
immunofluorescence along the sites of membrane contact.
At this point, actin filaments organize parallel to and just
underneath this line. Similar actin dynamics have been
observed in the rat hepatocyte line IAR-2 [30].
Primary mouse keratinocytes behave somewhat differently
(Figure 4b) [27••]. In contrast to MDCK and IAR-2 cells,
which primarily form lamellipodia, primary keratinocytes
grown on extracellular matrix extend numerous filopodia,
which are packed with actin cytoskeleton. When filopodia
from opposing cells make contact they slide along each
other and physically embed into the neighboring cell.
E-cadherin–catenin complexes cluster at the tips of
embedded filopodia. By immunofluorescence microscopy
these complexes can be visualized as a double row of perfectly aligned puncta, indicating that the degree to which
filopodia embed is a relatively fixed process, perhaps determined by the barrier of cortical actin cytoskeleton. At the
ultrastructural level, a prominent bundle of actin filaments
emanates from the developing adherens junction in the
host cell. Pulse labeling experiments with rhodamine–actin
reveal actin polymerization at these sites. At later stages of
cell–cell adhesion, the zipper of cadherin–catenin-labeled
puncta closes into a single row toward the center of the two
cells, whereas additional puncta are added at both ends of
the zipper. As an epithelial sheet forms, the actin filaments
reorganize from the perpendicular to a lateral orientation
parallel to the sealed membranes [27••].
puncta form at the edges. The actin-based
movement physically brings remaining
regions of opposing membranes together and
seals them into epithelial sheets. As filopodia
contain actin rather than keratin intermediate
filaments, they become natural zones of
adherens junctions, whereas the cell surface
flanking filopodia becomes fertile ground for
desmosome formation, alternating adherens
junctions and desmosomes.
Cadherin-mediated cell junction formation can be mimicked by exposing cells to glass beads coated with a dimeric
form of N-cadherin, which retains the structural and functional properties of cadherins [31]. These beads induce the
recruitment and clustering of N-cadherin–catenin complexes, accompanied by recruitment and redistribution of
actin filaments and local membrane remodeling involving
the extension of long filopodia (2–3 µm long). Some of
these stimulated cells extend large lamellipodia that envelop
and ultimately internalize the beads. Taken together, these
findings provide further evidence that actin dynamics
operating immediately beneath the cell membrane can
generate the necessary force to push cell membranes forward
and promote intercellular junction formation.
In vivo studies
How universal is this active process of intercellular adhesion and is it of importance in biological systems?
Although it is too early to predict the answer to this
question, there is some evidence to suggest that a filopodia-mediated mechanism for intercellular adhesion may
play a broad role in epithelial sheet formation in vitro and
in vivo. In vivo, a filopodia-based mechanism of intercellular junction formation has recently been described as a
natural process that occurs during embryonic development of Caenorhabditis elegans [32••,33]. This mechanism
involves cadherin–catenin complexes and appears to
function when free edges of sheets of epithelia must be
80
Cytoskeleton
Figure 5
(a)
(b)
Wound
Contraction of a tissue
to fill in a wound site
Propagation of adhesion
at the edges of contact
Current Opinion in Cell Biology
joined to create continuous tissues that seal the interior
of the organism from the outside environment. Similarly,
in Drosophila tracheal morphogenesis, a similar mechanism of filopodia extension is used when the cells at the
end of each developing branch fuse together [34]. E-cadherin accumulates at the sites where these filopodia
contact their target cells. The sealing of imaginal discs
during dorsal closure of the thorax may be another example of a filopodia-dependent mechanism that operates
during Drosophila development [35••], and lamellipodia
and filopodia also appear to participate in cell–cell adhesion involving DdCAD-1, a cadherin-like protein in
slime mold [36]. Interestingly, although DdCAD-1
seems to play a role in the formation of initial contacts, it
does not appear to be necessary for maintaining stable
contacts in this organism.
A filopodia-based mechanism for actively bringing cells
together may be particularly important within the epidermis and other stratified squamous epithelia that undergo
constant self renewal. The inner most layer of mammalian
epidermis harbors mitotically active cells that display
numerous filopodia-like interdigitations at sites of cell–cell
adhesion (C Bauer, E Fuchs, unpublished data), and as
these cells exit the basal layer, they create vacancies that
must be filled, perhaps by this active filopodia-based
mechanism of epithelial sheet formation. Such a mechanism might also be important in wound healing. Although
the process is not as uniform as the one described for primary keratinocytes, the mouse mammary tumor cell line
MTD1-A responds to a cultured ‘wound site’ by forming
thin cellular processes that protrude into neighboring cells
and assemble E-cadherin–catenin complexes at their tips
[37,38]. Taken together these findings suggest compelling
reasons for stratified tissues to utilize this type of mechanism. It is also interesting that the cell–cell contacts within
stratified epithelial tissues exhibit alternating desmosomes
and adherens junctions, a pattern which naturally results
from the type of intercellular adhesion described here
[27••]. Such patterns are not prominent in simple epithelial
Possible roles of myosin in cell–cell adhesion.
(a) A hypothetical ‘purse string’ model for
myosin-driven epithelial sheet closure at a
large circular wound site in the cornea of an
adult mouse. At the edge of wound site
epithelial cables of actin (red) appear to
extend from cell to cell, forming a ring around
the wound circumference. Contraction of
actin cables (arrows) driven by myosin can
lead to wound closure [68]. (b) Inside out
‘purse string’ model for contact propagation
(compaction) in MDCK cells. During contact
formation in MDCK cells, circumferential actin
cables contact cadherin–catenin plaques at
the edges of the contact (green triangles).
Contraction of actin cables driven by myosin
can lead to the contact expansion.
tissues, suggesting that there could be tissue-specific
variation in the extent to which this active mechanism of
intercellular adhesion is utilized.
A role for Rho, VASP and myosin families of
proteins in adherens junction formation
Roles for Rhos
What regulates the actin dynamics that are important for
cell–cell adhesion? The answer to this remains uncertain,
but the small GTPases of the Rho family seem to be likely
candidates, given that Rho, Rac1 and Cdc42 promote stress
fiber, lamellipodia and filopodia formation, respectively
[39]. Additionally, evidence has been accumulating that
implicates these factors in intercellular adhesion involving
cadherin–catenin complexes in epithelial cells. In vivo
mutagenesis studies in Drosophila reveal a role for Rac1 and
Rho in dorsal closure and/or in head involution, processes
that involve complex and well orchestrated rearrangements
of cells [40,41]. In contrast, Cdc42 appears to be involved in
regulating polarized cell shape changes [40]. In vitro, keratinocytes microinjected with dominant negative Rac1 or
with C3 toxin, a specific inhibitor of Rho, are unable to
form cadherin-based cell–cell contacts [42]. Similarly, overexpression of a constitutively active form of Rac1 or Cdc42
in MDCK cells increases junctional localization of E-cadherin–catenin complexes, whereas the dominant negative
forms of Rac1 and Cdc42, or C3 microinjection, have the
opposite effect [43,44]. Consistent with these data is the
finding that Tiam1, a guanine nucleotide exchange factor
for Rac1, increases E-cadherin mediated cell–cell adhesion,
inhibits hepatocyte growth-factor-induced cell scattering
and reverses the loss of adhesion in Ras-transformed cells
[45]. Together, these findings provide compelling evidence
that activation of the Rho family of small GTPases plays a
key role in the actin dynamics that are necessary for
adherens junction formation.
A priori, the Rho GTPases can mediate their effect on
adherens junction formation by changing actin cytoskeletal dynamics or by some other means. In cells expressing a
Actin dynamics and cell–cell adhesion in epithelia Vasioukhin and Fuchs
dominant negative Rac1 or Cdc42, cell–cell adhesion
involving a chimeric E-cadherin fused to α-catenin is considerably less affected than that involving a wild-type
E-cadherin [46]. This suggests that these small GTPases
influence adherens junction formation through promotion
of the assembly of stable E-cadherin–β-catenin–α-catenin
complexes. Activated Cdc42 and Rac1stabilize these complexes by inhibiting IQGAP1, a protein which interacts
with β-catenin and dissociates α-catenin from E-cadherin–β-catenin [47]. When the actin cytoskeleton is
unable to associate with E-cadherin–β-catenin complexes,
reduced cell–cell adhesion is observed. This explains how
Rac and Cdc42 promote cell–cell adhesion without directly
affecting the actin cytoskeleton.
The value of VASPs
We found that E-cadherin–catenin-enriched puncta, which
assemble during the first stages of epithelial sheet formation, are sites of de novo actin polymerization [27••]. This
led us to postulate that actin polymerization might provide
the force that is subsequently necessary to merge the double role of puncta into a single row and ultimately into an
epithelial sheet. Knowledge of how actin polymerization
might generate movement comes largely from studies of
the mechanism by which the pathogen Listeria monocytogenes pirates actin polymerization and utilizes it for
intracellular propulsion [48]. For this endeavor, these bacteria recruit two types of cellular components, the VASP
family of proteins and the Arp2/3 complex. The Arp2/3
protein complex is required for de novo nucleation of actin
filament polymerization, whereas VASP appears to accelerate
bacterial movement by about 10 fold [49••].
We found that antibodies against VASP and its close cousin
Mena localize to puncta [27••]. Moreover, expression of a
VASP dominant negative mutant in cultured keratinocytes
interfered with the formation of adherens junctions, and
when it was expressed in the basal layer of epidermis of
transgenic mice, this mutant perturbed cell–cell adhesion
and produced small blisters within the skin [27••]. Although
such approaches are always associated with the potential
caveat that the dominant negative protein may have
acquired a novel, unexpected function, the results suggest
that the association of VASP/Mena proteins at developing
adherens junctions may play a role in the reorganization
and/or polymerization of actin filaments that is needed to
seal epithelial sheets. Exploring the precise roles of these
proteins in more detail may be difficult in mice where at
least three different family members (VASP, Mena and Evl)
are coexpressed and may be functionally redundant.
In addition to its well established role in promoting Listeria
movement [49••,50,51], VASP is not only capable of binding to f-actin, it is also capable of stimulating actin
nucleation and polymerization in vitro [51–55]. For example, expression of the neural isoform of Mena in fibroblasts
induces the formation of actin-rich neurite-like extensions
[56], and axons in mice deficient for Mena fail to project
81
across the midline during development [57]. VASP family
members have also been implicated in the actin reorganization that takes place upon T-cell receptor (TCR)
activation [58•]. Although most studies have revealed
positive roles for VASP and its cousins in actin reorganization/polymerization, recent experiments have shown that
in certain instances these proteins act negatively in directing cell movement [59••,60••]. A further complication is
the finding that VASP family proteins can be phosphorylated, thereby inhibiting their actin nucleation and f-actin
binding ability [54,55].
Although a role for VASP family members in epithelial
sheet sealing seems attractive, another role for VASP may
be in the actin polymerization necessary for filopodia
extensions. In this regard, VASP family proteins localize to
the tips of filopodia during neural growth [57] and in
calcium-stimulated keratinocytes [27••]. VASP family proteins in this process might provide directionality to the
process of actin polymerization, reshaping f-actin into parallel bundles to produce and extend filopodia-like
structures from branched lamellipodial networks [61].
Interestingly, VASP is a profilin-binding protein and in
mice it genetically interferes with profilin [57].
Conversely, a mutant profilin defective in actin-binding
suppresses actin polymerization in the Cdc42–N-WASPinduced formation of microspikes [62]. Although
additional studies will be necessary to clarify the precise
function of VASP family members in actin dynamics, these
findings suggest a possible functional intersection
between VASP and Rho GTPases in coordinating the actin
dynamics necessary for intercellular adhesion.
The might of myosins
Although actin polymerization seems to be important in
generating the cellular movement necessary for intercellular adhesion, this does not rule out the possibility that the
myosin family of actin motor proteins may also play a role.
It is known, for instance, that cells can use myosin–actin
contractile forces to alter cell shape, and myosin II is a
ubiquitously expressed protein involved in such diverse
processes as cell spreading, cytokinesis, cell migration,
generation of tension within actin stress fiber networks and
retrograde flow of actin filaments at the leading edge of
moving cells [63–67]. Interestingly, mouse corneal cells at
a wound edge assemble cables of actin filaments anchored
to E-cadherin–catenin complexes. The cells surrounding
the wound site display myosin-II-associated actin filaments that are aligned in a structure resembling a purse
string (Figure 5a; [68]). It has been postulated that closure
of the wound may be achieved through myosin-directed
contraction of the actin filaments, in a mechanism similar
to that of pulling on a purse string. A similar, but insideout, mechanism may operate in the propagation of puncta
assembly at the edges of epithelial sheet sealing, as proposed by Adams and Nelson ([28]; Figure 5b). In this
model, circumferential actin cables engulf two cells as they
make initial contact, and subsequent contraction of actin
82
Cytoskeleton
cables propagates and expands the region of membrane
contact. Finally, there is some evidence that membraneassociated myosin I molecules are also involved in
adherens junction formation. Thus, the myosin I protein
myr3 localizes to adherens junctions in epithelial tissues
and in HeLa cells, and it is enriched at junctions induced
by overexpression of Cdc42 [69].
Overall, through guilt by association, myosins have been
implicated in cell–cell adhesion and in adherens junction
formation and although the models proposed are attractive
[70], direct experimental evidence is still lacking. BDM
(2,3-butanedione monoxime), a general inhibitor of
myosin function, had no obvious effect on intercellular
junction formation in our keratinocyte adhesion assays
(V Vasioukhin, E Fuchs, unpublished data). However, the
role of myosins clearly deserves a more detailed investigation, and this awaits the development of new and
improved inhibitors and activators of myosin action.
Conclusions
Significant progress has been made in the past few years in
elucidating the role of actin cytoskeletal dynamics in promoting epithelial cell adhesion. It is now clear that
α-catenin is the central protein linking the actin cytoskeleton to cadherin–catenin complexes at the cell membrane,
and the functional significance of this association has been
extensively studied using conditional gene targeting and
cultured cell reconstitution experiments. An active process
of epithelial sheet formation has been discovered that
involves the extension, protrusion, embedding and anchoring of filopodia into neighboring cell membranes. This
process stabilizes contacts between two membranes and
catalyzes adherens junction formation, which in turn promotes the sealing of epithelial cells into sheets. This
dynamic process necessitates a major role for actin polymerization and reorganization in cell–cell adhesion. It is
likely that this mechanism is both operative and important
during development, particularly where epithelial sheets
must be drawn together. Examples of such situations
include ventral closure, the sealing of imaginal discs,
wound healing, and the natural flux of cells through selfrenewing epithelial tissues such as the epidermis. Analysis
of cell–cell adhesion in keratinocytes reveals potential
roles for the VASP family of proteins and for de novo actin
polymerization at E-cadherin–catenin enriched puncta,
which represent the prelude to the mature adherens junction. Actin polymerization has emerged as a force that can
push membranes of neighboring cells together, the alignment
of which is necessary for intercellular adhesion.
Although a clearer image of actin’s role in orchestrating
cell–cell adhesion is developing, the exact mechanism is
still beyond our grasp. A major clue was the discovery that
α-catenin does not merely function as a bridge between
actin and cadherin–catenin complexes at the membrane
but in addition actively participates to recruit molecules
involved in actin dynamics. But several questions remain:
what is the function of α-catenin’s adhesion-modulation
domain, and how does α-catenin recruit and organize so
many proteins to the intracellular side of the junction?
What is the nature of this macromolecular structure and
how is it regulated? What are the precise roles of the Rho
family of GTPases, and the VASP/WASP family members
in regulating cadherin-mediated intercellular junction formation? Finally, how do the initial stages of adherens
junction formation impact on subsequent steps in actin
reorganization and in epithelial formation? A long and
interesting road of scientific investigation still lies ahead
for this exciting field of cell biology.
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