| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Regular Articles |
From the Department of Medicine, Gastroenterology Division, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
6ß4,
6ß1, and 3ß1) to wounding. To model this event
in vitro, monolayers of T84 cells,
well-differentiated colon carcinoma cells, were damaged by
aspiration and the ensuing response was analyzed by a combination of
time-lapse video microscopy, fluorescence confocal microscopy
and antibody inhibition assays. We show that wound healing begins with
retraction of the monolayer. 6ß4 integrin is localized on the
basal surface in structures referred to as type II hemidesmosomes that
persist throughout this early stage. We hypothesize that these
structures adhere to the substrate and function to retard retraction.
Once retraction ceases, the wound is contracted initially by
actin purse strings and then lamellae. Purse strings and lamellae
produce a pulling force on surrounding cells, inducing them to
flatten into the wound. In the case of lamellae, we detected
actin suspension cables that appear to transduce this pulling force. As
marginal cells produce lamellae, their basal type II
hemidesmosomes disappear and the 6 integrins appear evenly
distributed over lamellae surfaces. Antibodies directed against the
6 subunit inhibit lamellae formation, indicating that
redistribution of the 6 integrins may contribute to the protrusion
of these structures. Antibodies directed against the 3ß1 integrin
also reduce the size and number of lamellae. This integrin’s
contribution to lamellae extension is most likely related to its
localization at the leading edge of emerging protrusions. In
summary, wounds in epithelial sheets initially retract,
and then are contracted by first an actin purse string and then
lamellae, both of which serve to pull the surrounding cells
into the denuded area. The 6 integrins, particularly
6ß4, help contain retraction and both the 6 integrins
and 3ß1 integrin contribute to lamellae formation.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Epithelial sheets respond to injury by mobilizing their actin cytoskeleton. Two different types of responses have been noted.5,6 One involves lamellae formation, a key feature of restitution both in vivo and in vitro.4,7 Lamellae are large, flat, cytoplasmic protrusions that are extended by the marginal cells into the denuded area. Much of what is known about lamellae is derived from the study of solitary migrating cells such as fibroblasts.8 During fibroblast locomotion the actin cytoskeleton within the lamella associates with integrins on the surface. The traction to pull the cell forward is provided by the adhesion between the integrins and their specific extracellular matrix ligands. Eventually the lamella contracts, detaching the rearward part of the cell and allowing it to translocate. Although this process seems relevant to epithelial wound healing, many normal epithelia reseal defects without loss of cell contact.9,10 In the case of restitution, marginal cells surrounding gut injuries are thought to use lamellae to migrate into the wound, but cells are not observed to detach from their neighbors and translocate into the injury.1,2,4,7,11 This observation raises the question of how lamellae participate in the healing process. Other epithelial wounds heal by a process called purse string contraction in which cells marginal to the damage arrange their actin in a belt that tightens to close the injury.5,6 It is thought that small wounds (<0.008 mm2) heal by purse string contraction, whereas larger wounds use lamellae.6 However, recent studies of corneal abrasions suggest that the two processes may not be mutually exclusive.12 It is unclear from previous findings whether actin purse strings form during gut epithelial wound healing or whether lamellae that form during restitution preclude these structures.
We were interested in understanding the underlying molecular processes
that drive restitution. To model the processes in vitro we
used T84 cells, well-differentiated human intestinal carcinoma cells
that are polarized along their apical to basal axis and express
well-developed intercellular junctions.13
Previously we
found that the laminin family of extracellular matrix proteins and the
integrins that bind these proteins are instrumental in
restitution.14
Our findings targeted the laminin-binding
integrins, 6ß1, 6ß4, and 3ß1, as part of the molecular
machinery responsible for epithelial wound healing, but the point at
which these integrins participate in the healing process is unknown. It
would be informative to correlate the changes in cell morphology that
occur on wounding with changes in the function and distribution of
laminin-binding integrins.
Here, we examine the mechanism of epithelial cell movement during wound
closure by following the mobilization of the actin cytoskeleton and
laminin-binding integrins in response to injury of T84 monolayers. We
show that lamellae formation is a later step in a progression of
cytoskeletal rearrangements that begins with actin purse strings. The
6 integrins and 3ß1 integrin, which we found to be instrumental
in lamellae formation, redistribute to distinctive locales on the
lamellae during wound resealing. We conclude that the purpose of both
the actin purse string and lamellae is to allow the usually columnar
T84 cell to flatten greatly, providing as much cytoplasmic coverage of
the denuded area as possible.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
T84 colon carcinoma cells were cultured as described previously.13,14 Briefly, cells were grown in DME-low glucose/Ham’s F12 (GIBCO, Grand Island, NY) supplemented with 15 mmol/L Hepes, 6% normal calf serum, 2 mmol/L L-glutamine, 50 µg/ml streptomycin, and 50 U/ml penicillin. Cells were grown for 2 to 3 days after reaching confluency before being used in assays.
Antibodies
The following monoclonal antibodies specific for integrin subunits
were used in the present study: mouse antibody UM-A9 (integrin ß4
subunit), provided by Dr. Thomas Carey (University of Michigan, Ann
Arbor, MI); rat antibody GoH3 (integrin 6 subunit) and mouse
antibody against CD29 (integrin ß1 subunit), purchased from
Immunotech (Marseille, France); mouse antibody PIB5 (integrin 3
subunit), purchased from Becton Dickinson (San Jose, CA). Mouse and rat
IgG was purchased from Sigma (St. Louis, MO). Fluorescein-conjugated
goat anti-rat and goat anti-mouse IgG were purchased from Jackson
ImmunoResearch Laboratories, Inc. (West Grove, PA).
Immunofluorescence Microscopy
T84 cells were dissociated with trypsin-EDTA and, except where mentioned in the figure legends, plated onto glass coverslips. The cells were grown in a 37°C, 5% CO2 atmosphere for 10 to 14 days. Subsequently, the confluent monolayers were wounded by aspiration through a micropipet tip. These were made by pulling a heated glass Pasteur pipet so that a very narrow bore resulted.
At various times after wounding, cells were fixed for 10 minutes with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) with 7% sucrose followed by 0.4% Triton X-100 in Tris-buffered saline (TBS, pH 7.4) for 2 minutes. In some cases, noted in the figure legend, cells were permeabilized before fixation for 30 seconds on ice in F1 buffer (10 mmol/L Pipes, pH 6.8, 0.5% Triton-X 100, 300 mmol/L sucrose, 100 mmol/L potassium chloride, 3 mmol/L magnesium chloride, 10 mmol/L ethyleneglycoltetraacetic acid, and 2 mmol/L phenylmethylsulfonyl fluoride). After fixation, the coverslips were incubated in a blocking solution (3% bovine serum albumin and 1% normal donkey serum in TBS) for 1 hour at room temperature. After incubation with either primary antibody or nonspecific IgG, the coverslips were washed in TBS (3 times, 10 minutes each) and incubated for 1 hour at room temperature in the appropriate fluorescein-conjugated secondary antibody.1:100 In most experiments the secondary antibody was accompanied by rhodamine-phalloidin (1 µg/ml, Sigma) or rhodamine-phalloidin and Hoechst stain (0.5 µg/ml, Sigma). All antibodies and fluorescent compounds were diluted in blocking solution. After staining, the coverslips were washed in TBS and mounted in a mixture (8:2) of glycerol and PBS, pH 8.5, containing 1% propylgallate. Slides were examined by confocal imaging using a Zeiss LSM410 Laserscan Microscope equipped with a peripheral Argon-UV laser for exciting Hoechst fluorescence (Carl Zeiss, Thornwood, NY).
Video Microscopy and Function Blocking Experiments
T84 cells were grown for 10 to 14 days on 35-mm tissue culture dishes. Circular wounds were made using micropipet tips. The healing process was examined by time-lapse video-microscopy using a Nikon Diaphot 300 inverted microscope with phase contrast optics, equipped with a stage warmer. Images were captured using a CCD camera (Dage-MTI, Michigan City, IN), a frame-grabber (Scion) and a 7600 Power Macintosh computer. Films of wounds healing at 37°C, 5% CO2 were made by capturing images every 2 minutes for up to 12 hours. Some wounds were filmed for various amounts of time and then fixed and stained for immunofluorescence. For function-blocking experiments, T84 cell monolayers were wounded, fresh medium containing function-blocking integrin antibodies was added, and then images of the wounds were captured so that their initial diameters could be measured. After incubation for 2 hours in a 37°C, 5% CO2 incubator, images were captured again for a final wound diameter measurement. Alternatively, images of wounds were captured for measurements of initial diameters and then wound healing was allowed to progress until the first appearance of lamellae. At this point, function-blocking antibodies were added. After two hours, images were captured again for measurement of final wound diameters. Wound diameters were measured using IP lab spectrum software (Scanalytics, Fairfax, VA). The initial and final diameters for each wound were used to determine effects of the monoclonal antibodies on wound closure.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To identify the sequence of events involved in the closure of T84
wounds (in this case, 0.018 mm2, approximately
400 cells), we used time-lapse video microscopy (Figure 1)
. Initially after wounding, the wound
edge actually retracted for 10 minutes and the surface area of the
wound increased by ~14%. At this time, a refractile ring that
surrounded the wound edge was evident. This ring was similar in
appearance to actin purse strings that have been shown to contract
smaller wounds.5,6
Subsequent to the appearance of the
refractile ring, the cells surrounding the wound edge elongated and
increased their surface area approximately twofold, a process that
resulted in the initiation of wound closure. Although the surface area
of the wound was reduced by 30% at 40 minutes relative to its largest
size at 10 minutes, surface protrusions such as lamellae were not
evident in cells at the wound edge. In fact, distinct lamellae
extending from a few cells abutting the damage were not seen until 60
minutes after wounding. Lamellae protrusion was not synchronous, but
gradually most of the marginal cells produced these extensions, while
the refractile ring became less evident. At 120 minutes after wounding,
the denuded area was covered entirely by lamellae. Based on this video
analysis, we conclude that the closure of T84 wounds involves a
sequence of morphologically-defined events that involve an initial
retraction of the wound edge, subsequent refractile ring formation
accompanied by cell elongation, and, finally, lamellae extension.
|
. Indeed, an intense band of F-actin,
characteristic of an actin purse string, was seen around the
circumference of such wounds (Figure 2A)
. In fact, even larger wounds
exhibited actin purse string formation within 30 minutes after wounding
(Figure 2B)
. Interestingly, cells that were damaged as a result of
wounding were excluded and appeared in the denuded area outside the
actin purse string. Cross-sectional images revealed that these damaged
cells were detached from the substrate (Figure 2C)
.
|
.
Moreover, rhodamine-phalloidin staining revealed that cells surrounding
the wound rearranged their stress fibers orthogonally to the wound edge
such that these stress fibers stretched toward the actin purse string
(Figure 3A)
. Cross-sectional images
highlighted the impression of an intact monolayer tapered down to the
actin purse string (Figure 3B)
. Collectively, these images demonstrate
that cells distant from the wound contribute to closure by elongating,
and that actin purse strings, which generate a contractive force, are
associated with this elongation.
|
. However, we observed regions in the
monolayer adjacent to the lamellae that contained no detectable F-actin
(Figure 4A)
. Cross-sectional analysis revealed that these
F-actin-deficient regions corresponded to the bodies of the marginal
cells that produced the lamellae and actually contained fine F-actin
cables that were suspended above the basal plane (Figure 4B)
. For this
reason, images taken at this basal plane gave the appearance of F-actin
holes in the monolayer. These F-actin suspension cables extended from
the marginal cell’s rearward apicolateral cell surface to the dense
actin network at the base of the lamella. This unique arrangement of
actin suggested that the cables were under tension (Figure 4B)
. A
cross-section taken in a neighboring region revealed shorter actin
suspension cables (Figure 4C)
. These cross-sectional views imply that
the cable’s suspension from the rearward apicolateral cell surface
down to the lamella base is related to the extent to which the cell
containing it elongates. Together, the images presented in Figure 4
demonstrate that lamellae serve to stretch the marginal cells that
produce them and result in the formation of actin suspension cables
that bridge the rearward, lateral cell surface with the lamella base.
|
6 Integrins and
3ß1 Integrin during Wound Healing with Changes in Cell Morphology
Induced by Wounding
Previously we demonstrated that 3ß1 and the 6 integrins
were involved in wound resealing of T84 cell monolayers. In the current
study, we determined at which stage (retraction, actin purse string
formation or lamellae extension) these integrins contributed to the
healing process. Wounds were allowed to reseal for 2 hours in the
presence of nonspecific antibody or antibodies against the 3 or 6
integrin subunit (Figure 5a)
. All of the
wounds, regardless of treatment, exhibited refractile actin purse
strings, suggesting that neither the 3ß1 integrin nor the 6
integrins were involved in the formation of this structure. Wounds
treated with nonspecific antibody closed 35%, whereas those treated
with 6 subunit antibody were 11% larger compared with their initial
sizes. These data demonstrate that cells treated with the 6 subunit
antibody were unable to halt retraction within 2 hours of wounding,
suggesting that the 6 integrins promote the cessation of retraction.
In contrast, wounds treated with 3 subunit antibody were 12%
smaller. Thus, injured monolayers treated with 3 subunit antibody
were able to halt retraction within the 2-hour time span, but resealed
more slowly than those treated with nonspecific antibody.
|
3ß1 integrin
and the 6 integrins to this process, inhibitory antibodies were
added just as lamellae were beginning to emerge, rather than
immediately after wounding (Figure 5b)
. This strategy allowed wound
resealing to proceed past the retraction and purse string stages before
the inhibitory antibodies were added. Wounds were analyzed 2 hours
later for the contribution of lamellae to wound closure. In the
presence of an 3-specific antibody, lamellae contributed only 10%
of the surface area to wound healing as compared to 25% in the
presence of nonspecific antibody. When antibodies against the 6
integrin subunit were added at this later time point, lamellae
formation was also reduced by 50% relative to that seen with
nonspecific antibody. These findings demonstrate that both the 6
integrins and 3ß1 integrin participate in lamellae formation.
We examined the localization of the 3, 6, and ß4 subunits in
cells at distinct stages of wound closure to obtain additional insights
into their contribution to wound healing. We focused on the 6ß4
integrin because a significant fraction of the 6 signal colocalizes
with the ß4 subunit on the basal surface of intact T84 monolayers in
plaque-like structures.14
The basal surface was examined
by immunofluorescence microscopy soon after wounding, as retraction
ceased and purse strings formed (Figure 6A)
. Superimposing a relatively apical
section containing the forming actin purse string (in blue) on a basal
section containing actin stress fibers (in red) and 6ß4 integrin
(in green) demonstrated that intact plaques remained on the basal
surface of marginal cells during retraction. Higher magnification
revealed that basal plaques occupied almost the entire basal surface of
T84 cells and contained striations through which actin stress fibers
ran (Figure 6B)
. These actin-striated plaques are most likely Type II
hemidesmosomes, described in several mammary and colon carcinoma cell
lines.15,16
Besides resembling type II hemidesmosomes in
appearance, they contained the protein HD-1, also known as
plectin, but lacked the proteins BP-1 and BP-2 (data not shown).
These findings indicate that 6ß4 integrin is found in type II
hemidesmosomes, basal structures that survive injury and thus are
present during retraction.
|
6 integrins on lamellae to
determine how they participate in the formation of these processes.
Examination of marginal cells revealed that 6ß4 integrin does not
reside in plaques on those producing lamellae. Rather, 6ß4
integrin was dispersed on the marginal cell surface and was not found
in the characteristic plaque pattern, in which it alternates with
actin. This was seen in a wound fully encircled by lamellae and stained
for nuclei (Figure 6C
, blue), actin (red), and 6ß4 integrin
(green). Marginal cells exhibited a uniform distribution of the ß4
subunit, although cells located rearward to the marginal cells
displayed this integrin in basal plaques stretched toward the injury
(Figure 6D)
. Close inspection of a lamella revealed the ß4 subunit
distributed between the fine actin filaments radiating out from the
lamella base (Figure 6E)
. To detect 6ß1 integrin on lamellae,
double-staining experiments were performed with antibodies to the 6
and ß1 integrin subunit. Points of colocalization, indicating the
presence of 6ß1 integrin, were dispersed over these extensions
(Figure 6F)
. These images demonstrate that the 6 integrins respond
to injury by distributing themselves over lamellae.
The localization of 3ß1 integrin during lamellae formation was
also analyzed. Before the appearance of large, adherent lamellae, small
cytoplasmic protrusions were often found emerging into the denuded
region in areas where the actin purse string was no longer intact
(Figure 7)
. A distinctive feature of
these small protrusions was that they were observed when wounds were
stained for the 3 integrin subunit, but not when they were stained
for the 6 or ß4 integrin subunit. They also stained weakly for
actin. Approximately 1 out of 5 of these emerging lamellae exhibited
more intense 3 integrin subunit staining at their leading edge than
on their bodies. Optical cross-sectioning revealed that this intense
band corresponded to a concentration of 3 integrin subunit at the
outermost edge into which thin actin filaments ran (Figure 7D)
. The
3 subunit on more mature lamellae was evenly distributed across
these larger extensions and was not found as concentrated at the edge
of larger lamellae as it was on small emerging ones (Figure 7E)
. The
localization of 3ß1 integrin on the leading edge of small
protrusions and the antibody inhibition data suggest that this integrin
plays a distinct role in lamellae formation.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
6ß1, 6ß4,
and 3ß1 because they have been implicated in epithelial sheet
healing.14
We found that after an initial retraction,
wound closure progressed by a series of actin rearrangements that
generated a contractive or pulling force on the cells surrounding the
defect, inducing them to flatten into the denuded area, thereby sealing
it. The laminin-binding integrins participated at different points
during the healing process. The 6 integrins, particularly 6ß4,
helped to contain retraction, and both the 6 integrins and 3ß1
integrin contributed to lamellae formation. Actin purse strings were first described in studies that demonstrated the ability of small epithelial wounds to heal without any contribution from lamellae.5,6 These studies show that actin purse strings mechanically pull on marginal cells, drawing them together. When purse string formation is inhibited by the inactivation of endogenous Rho, small, embryonic skin wounds fail to heal.17 In this study we show that purse strings also participate in the resealing of larger wounds that later produce lamellae. Our results demonstrate that closure begins after purse strings form and before lamellae appear. In the case of larger wounds, the force generated by the purse string appears to be transduced to more remote cells, probably via cell-to-cell contacts. The result is that remote cells stretch toward the injury, increasing their surface area on the average twofold as their basal stress fibers become aligned at right angles to the purse string. These findings suggest that actin purse strings contribute to the resealing of larger wounds by providing a pulling force that induces surrounding cells to flatten, thus increasing coverage of the denuded area.
Lamellae, a key feature of restitution, appear after the purse string
completely encircles the wound. We suggest that the wounds made in this
study (with surface area of >50 cells), cannot be closed solely by
actin purse contraction but require further efforts provided by
lamellae. The signal to extend lamellae may be related to the
down-regulation of adherens junctions that link the filamentous actin
components together to form the purse string.12
It has
been inferred from studies of migrating, isolated cells that the
primary function of lamellae during epithelial wound healing is that of
a motile apparatus. In contrast, the images presented here indicate
that the primary function of lamellae is to generate a pulling force on
distal cells, much like purse strings. In fibroblasts, the pulling
force generated by lamellae results in the detachment of the cell tail
and translocation of the cell.18,19
However, in intestinal
epithelium, the marginal cells do not detach from their rearward
neighbors.2,4,7,11
Rather, the force generated by lamellae
is transferred rearward, inducing cells that are distant from the wound
to flatten as they stretch toward the injury. This force can be
detected by the fact that 6ß4-containing basal plaques become
aligned orthogonally to the wound edge in cells that are rearward to
those abutting the damage. Cell flattening serves to cover the denuded
area in addition to surface area provided by the lamellae themselves.
A novel finding in our study is that marginal cells rearrange their actin cytoskeleton to produce an actin suspension cable. There is no equivalent of this structure in solitary migrating cells, and to our knowledge it has not been described previously. We propose that this actin suspension cable is used to pull the rearward cells toward the lamellae at the wound margin. Actin cables course down from the rearward, apicolateral surface of the marginal cell into the dense actin array at the base of the lamellae. This array in the lamellae of solitary migrating fish keratocytes consists of actin and myosin arranged to produce the contractive force required for detachment of the trailing edge.20 Our data suggest that this contractive force in T84 cell monolayers and other epithelial sheets is transduced along actin suspension cables to cell-cell contacts at the surface distal from the lamellae. In this way the contractive force is communicated to surrounding cells, inducing them to flatten.
Past work by our group pertaining to extracellular matrix interactions
during wound healing demonstrated that laminins secreted by T84 cells,
particularly laminin-5, and the laminin-binding integrins 6ß1 and
6ß4 contribute to closure.14
However, these studies
did not pinpoint the stages during resealing at which these integrins
act. Here we show that the 6 integrins act during retraction and
lamellae formation. We hypothesize that 6ß4 integrin within type
II hemidesmosome-basal plaques is responsible for generating substratum
adhesion that helps contain retraction. This is suggested by the
observation that Type II hemidesmosomes share important features with
classical hemidesmosomes, complex structures that mediate very tight
adhesion of the epidermis to the underlying basal lamina of the
skin.21-26
Both types of hemidesmosome link the keratin
cytoskeleton to the cell substratum via 6ß4
integrin.16,27
Our hypothesis that the 6ß4 integrin
is responsible for containing retraction is supported by the sharp
distinction seen during retraction between nonadherent cells lying in
the damaged area and the adherent cells that express basal plaques. It
is likely that 6ß4 integrin within the type II hemidesmosome
attaches to laminin-5, one of its ligands that is secreted as a basal
sheet by T84 cells.14,28
Our previous finding that
antibodies to laminin-5 added at the time of injury, like those against
6 integrin subunit, allow the monolayer to retract unimpeded lend
credence to the idea that 6ß4 integrin within the basal plaques
mediates attachment to laminin-5, helping to halt
retraction.14
Antibody inhibition data also demonstrated that the 6
subunit-containing integrins participate in lamellae protrusion during
T84 wound resealing. Localization studies revealed that on extending
lamellae, marginal cells no longer exhibit alternating actin and
6ß4 integrin characteristic of type II hemidesmosomes, nor the
lateral cell surface expression of 6ß1 integrin seen in intact
monolayers.14
Instead, 6ß4 and 6ß1 integrin are
found dispersed over the lamellae, indicating that both integrins
redistribute as lamellae extend. A similar observation has been made
during corneal wound healing. In this case, 6ß4 integrin relocates
over the cell surface and classical hemidesmosomes disassemble on
corneal epithelial cells bordering the injury.10,24,29
We
suggest that redistribution of 6ß4 and 6ß1 integrin is
important for lamellae formation and that the basis for 6-subunit
antibody inhibition is interference with the rearrangement of either
one or both of these integrins. In agreement with this conclusion,
6ß4 integrin has been shown to promote the formation and
stabilization of lamellae in carcinoma cells by stimulating cyclic
AMP-related and phosphoinositide 3-OH kinase
pathways.30-32
It is intriguing to speculate that the
surface relocation and release of 6ß4 integrin from
hemidesmosome-like structures coincides with its signaling to these
pathways during epithelial wound healing.
Antibody inhibition assays also demonstrate that 3ß1 integrin
participates in lamellae formation. Images presented here suggest that
3ß1 integrin plays a prominent role in lamellae formation because
they show this integrin concentrated at the leading edge of small
protrusions where actin filaments terminate. Similarly, 3ß1
integrin associates with actin in keratinocytes and in doing so
participates in cell spreading as opposed to cell
anchoring.23
Its role in lamellae formation may also help
explain the finding that 3ß1 integrin is important for pancreatic
carcinoma cell migration.33
The observed concentration of
3ß1 integrin at the leading edge of small lamellae suggests that
it moves over the cell surface during T84 wound resealing similarly to
ß1 integrins during fibroblast migration.34
In
fibroblast migration, integrins are preferentially transported to the
cell’s leading edge where they bind to their extracellular matrix
ligand and then associate with the actin cytoskeleton. The actin
cytoskeleton exerts a rearward force on the adherent integrin resulting
in the traction required to move the cell forward. Our data indicate
that 3ß1 integrin on T84 cells preferentially moves toward the
leading edge of emerging lamellae. We hypothesize that it then binds to
laminin-5, one of its ligands, helping to generate the traction or a
pulling force required for lamella growth.35
Eventually,
this force is transmitted rearward and serves to pull on surrounding
cells.
Our findings on T84 epithelial sheet wound healing can be summarized in
the following model. On injury, the monolayer first retracts.
Retraction ceases, at least in part, due to the adhesion mediated by
6ß4 integrin in type II hemidesmosomes on the basal surface of the
cells. Once retraction ceases, the actin purse string exerts a pulling
force on the monolayer. The result is that cells flatten into the
denuded area and the wound begins to contract. Type II hemidesmosomes
remain intact during this process even though the cells stretch to
cover 2x more surface area. Some wounds do not close entirely by the
efforts of actin purse strings and, in these cases, lamellae are
extended by marginal cells. During lamellae extension, type II
hemidesmosomes disassemble and actin suspension cables are formed.
Disassembly of type II hemidesmosomes is part of lamellae formation and
is accompanied by redistribution of the 6 integrins. 3ß1
integrin contributes to the initial phase of lamellae protrusion by
helping to create the adhesive traction required to form actin
suspension cables. The actin suspension cables pull on the rearward
cells and flatten them, similarly to the actin purse string, creating
more cytoplasmic coverage of the denuded area.
|
Footnotes |
|---|
M. M. L. was supported by a Crohn’s and Colitis Foundation of America Career Development Award. I. R. and A. M. were supported by National Institutes of Health grant CA44704.
Accepted for publication November 29, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
6ß4 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci USA 1990, 87:8970-89746ß4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol 1991, 113:907-9173ß1 in focal adhesions and 6ß4/bullous pemphigoid antigen in a new stable anchoring contact (SAC) of keratinocytes: relation to hemidesmosomes. J Cell Biol 1990, 111:3141-31546ß4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing. J Cell Biol 1991, 115:1737-17506ß4 integrin is a receptor for both laminin and kalinin. Exp Cell Res 1994, 211:360-367[Medline]
6ß4 integrin and bullous pemphigoid antigens during epithelial wound healing. Exp Cell Res 1993, 207:86-98[Medline]
6ß4 integrin promotes carcinoma invasion. Cell 1997, 91:949-960[Medline]
6ß4 functions in carcinoma cell migration on laminin-1 by mediating the formation and stabilization of actin-containing motility structures. J Cell Biol 1997, 139:1873-11846ß4 integrin stimulates lamellae formation and chemotactic migration of invasive carcinoma cells. J Cell Biol 1998, 143:1749-17603ß1 in epithelial basement membranes. Cell 1991, 65:599-610[Medline]
This article has been cited by other articles:
 |
|
 |
|
  L. S. Chaturvedi, C. P. Gayer, H. M. Marsh, and M. D. Basson Repetitive deformation activates Src-independent FAK-dependent ERK motogenic signals in human Caco-2 intestinal epithelial cells Am J Physiol Cell Physiol, June 1, 2008; 294(6): C1350 - C1361. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Grasso, J. A. Hernandez, and S. Chifflet Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture Am J Physiol Cell Physiol, October 1, 2007; 293(4): C1327 - C1337. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Voltan, I. Castagliuolo, M. Elli, S. Longo, P. Brun, R. D'Inca, A. Porzionato, V. Macchi, G. Palu, G. C. Sturniolo, et al. Aggregating Phenotype in Lactobacillus crispatus Determines Intestinal Colonization and TLR2 and TLR4 Modulation in Murine Colonic Mucosa Clin. Vaccine Immunol., September 1, 2007; 14(9): 1138 - 1148. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gunzel, P. Florian, J. F. Richter, H. Troeger, J. D. Schulzke, M. Fromm, and A. H. Gitter Restitution of single-cell defects in the mouse colon epithelium differs from that of cultured cells Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2006; 290(6): R1496 - R1507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Rashid, J. N. H. Stern, and A. R. Ahmed Identification of an Epitope within Human Integrin {alpha}6 Subunit for the Binding of Autoantibody and Its Role in Basement Membrane Separation in Oral Pemphigoid J. Immunol., February 1, 2006; 176(3): 1968 - 1977. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Sanders and M. D. Basson p130cas but Not Paxillin Is Essential for Caco-2 Intestinal Epithelial Cell Spreading and Migration on Collagen IV J. Biol. Chem., June 24, 2005; 280(25): 23516 - 23522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chifflet, J. A. Hernandez, and S. Grasso A possible role for membrane depolarization in epithelial wound healing Am J Physiol Cell Physiol, June 1, 2005; 288(6): C1420 - C1430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Brun, C. Mastrotto, E. Beggiao, A. Stefani, L. Barzon, G. C. Sturniolo, G. Palu, and I. Castagliuolo Neuropeptide neurotensin stimulates intestinal wound healing following chronic intestinal inflammation Am J Physiol Gastrointest Liver Physiol, April 1, 2005; 288(4): G621 - G629. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. C. Kurten, P. Chowdhury, R. C. Sanders Jr., L. M. Pittman, L. W. Sessions, T. C. Chambers, C. S. Lyle, B. J. Schnackenberg, and S. M. Jones Coordinating epidermal growth factor-induced motility promotes efficient wound closure Am J Physiol Cell Physiol, January 1, 2005; 288(1): C109 - C121. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. A. Sanders and M. D. Basson Collagen IV regulates Caco-2 migration and ERK activation via {alpha}1{beta}1- and {alpha}2{beta}1-integrin-dependent Src kinase activation Am J Physiol Gastrointest Liver Physiol, April 1, 2004; 286(4): G547 - G557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. M. Waters, P. H. S. Sporn, M. Liu, and J. J. Fredberg Cellular biomechanics in the lung Am J Physiol Lung Cell Mol Physiol, September 1, 2002; 283(3): L503 - L509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Van Aelst and M. Symons Role of Rho family GTPases in epithelial morphogenesis Genes & Dev., May 1, 2002; 16(9): 1032 - 1054. [Full Text] [PDF]
|
|
|
|
 |
|
 T. L. Davis, F. Buerger, and A. E. Cress Differential Regulation of a Novel Variant of the {alpha}6 Integrin, {alpha}6p Cell Growth Differ., March 1, 2002; 13(3): 107 - 113. [Abstract] [Full Text] [PDF]
|
|
