Medical Research Council Group in Periodontal Physiology, Faculty of Dentistry, University of Toronto, Toronto, Ontario, Canada M5S 3E2
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ABSTRACT |
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Despite their significance in
wound healing, little is known about the molecular determinants of
cell-to-cell adhesion and gap junctional communication in fibroblasts.
We characterized intercellular adherens junctions and gap junctions in
human gingival fibroblasts (HGFs) using a novel model. Calcein-labeled
donor cells in suspension were added onto an established, Texas red dextran (10 kDa)-labeled acceptor cell monolayer. Cell-to-cell adhesion
required Ca2+ and was >30-fold stronger than
cell-to-fibronectin adhesion at 15 min. Electron micrographs showed
rapid formation of adherens junction-like structures at ~15 min that
matured by ~2-3 h; distinct gap junctional complexes were
evident by ~3 h. Immunoblotting showed that HGF expressed -catenin
and that cadherins and connexin43 were recruited to the
Triton-insoluble cytoskeletal fraction in confluent cultures. Confocal
microscopy localized the same molecules to intercellular contacts of
acceptor and donor cells. There was extensive calcein dye transfer in a
cohort of Texas red dextran-labeled cells, but this was almost
completely abolished by the gap junction inhibitor
-glycyrrhetinic
acid and the connexin43 mimetic peptide GAP 27. This
donor-acceptor cell model allows large numbers (>105) of
cells to form synchronous cell-to-cell contacts, thereby enabling the
simultaneous functional and molecular studies of adherens junctions and
gap junctions.
adherens junctions; fluorescence dye; cell adhesion
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INTRODUCTION |
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INTERCELLULAR ADHESION AND COMMUNICATION are essential components of tissue differentiation and remodeling. The intercellular adherens junctions mediated by cadherins are essential for tissue morphogenesis (17) and are crucial for the maintenance of solid tissues as well as cell recognition and cell sorting during development (34). Of equal importance, intercellular communication through gap junction channels plays a crucial role not only in coordinating electrical signals in excitable cells but also in facilitating intercellular signaling in nonexcitable cells during development, growth regulation, and cell differentiation (20). Gap junctional communication is also responsible for cellular organization in oocytes (23) and the regulation of epidermal wound healing (14, 21).
Both adherens junctions and gap junctions are thought to be important in cellular signaling in connective tissue cells. For example, cadherin-mediated intercellular adhesion is required for human osteoblast differentiation (6), and gap junctional communication can modulate gene expression in osteoblastic cells (22). Despite their significance in tissue remodeling and wound healing, there are few studies on adherens junctions and gap junctions in human fibroblasts. Periodontal connective tissues provide a good model for study of intercellular adhesion and communication in vivo because the fibroblasts from these tissues form extensive adherens junctions and gap junctions (3, 32). However, neither the molecular components of adherens junctions (e.g., cadherins, catenins, and actin) (17) and gap junctions (4) nor the kinetics of formation have been well characterized in fibroblasts.
Previous studies of intercellular contacts in fibroblasts involved observation of colliding lamella of two adjacent cells in the x-y plane (13, 29, 30). Studies based on this and similar model systems present several limitations in that only a few cells can be examined at any time; because cell processes are moving over a substrate, observation of intercellular contacts is affected by cell-to-substrate interactions. Cognizant of these limitations, we developed and characterized a simple intercellular adhesion model that allows the study of key events in the early stages of intercellular adhesion that is independent of cell-to-substrate interactions. Because a large number of synchronized intercellular contacts were established with this model, biochemical studies of intercellular adhesive and gap junctional proteins and their regulation are possible. Our results indicate that the double-label synchronized cohort model provides a simple and effective approach to studying functional interactions in intercellular adhesion and communication.
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MATERIALS AND METHODS |
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Reagents.
Primary antibodies against human antigens including mouse monoclonal
anti-connexin43 (Clone 2) and anti--catenin (Clone 14) were
purchased from Transduction Laboratories (Lexington, NY); mouse
monoclonal anti-connexin26 antibody (Clone CX-12H10) and anti-connexin32 antibody (Clone CX-2C2) were purchased from Zymed Laboratories (San Francisco, CA). Pan-cadherin (Clone CH-19) and FITC-goat anti-mouse antibodies, tetramethylrhodamine isothiocyanate (TRITC)-phalloidin, and
-glycyrrhetinic acid (BGA) were purchased from Sigma Chemical (St. Louis, MO). Calcein-AM, Texas red dextran (10 kDa), and FITC-dextran (70 kDa) were purchased from Molecular Probes
(Eugene, OR). Connexin43 mimetic peptides including GAP 20 (amino acid
sequence EIKKFKYGC), GAP 27 (amino acid sequence SRPTEKTIFII), and
modified GAP 27 (amino acid sequence SRPTEKTIF) were synthesized by the
Alberta Peptide Institute (Edmonton, Alberta, Canada).
Cell culture.
Human gingival fibroblasts (HGFs) were derived from primary explant
cultures as described (27). Cells from passages
6-15 were grown as monolayers in T-75 flasks. Full growth
medium consisted of -MEM, antibiotics [0.017% penicillin G (Ayerst
Lab, Montreal, PQ), 0.01% gentamycin sulfate (Life Technologies, Grand
Island, NY) in
-MEM], and 10% (vol/vol) heat-inactivated fetal
bovine serum (FBS; ICN Biomedicals, Costa Mesa, CA). Two days
before each experiment, cells were harvested with 0.01% trypsin, and ~100,000 cells were plated into 35-mm-diameter culture dishes (Falcon, Becton Dickinson, Mississauga, ON). The cells were grown to
confluence before all experiments except when sparse cultures were used
as indicated.
Intercellular adhesion assay.
To characterize the functional properties and the intercellular
adhesion molecules in our simplified model, HGF were grown overnight to
a confluent monolayer in -MEM (supplemented with 10% FBS and
antibiotics). Another set of HGF (donor cells) were fluorescence-labeled with calcein-AM (5 µg/ml) and DiI-CM (10 µg/ml) for 1 h at 37°C, followed by three washes with
-MEM.
These cells were then plated onto the established cell monolayer
(acceptor cells). Attachment and spreading of the plated cells were
monitored and recorded at specific time points (0-180 min) with a
fluorescent microscope coupled with a charge-coupled device
(CCD) camera (Princeton Instruments, NJ). Quantification of
relative adhesivity under different experimental conditions was done by
counting the number of donor cells per high-power microscope field that
remained attached after three washes with PBS. The acceptor cell
monolayer was grown overnight to ensure that the acceptor cells were
highly adherent to the tissue culture plate and were not detached
during jet washing.
Immunocytochemistry.
To identify and localize specific molecules involved in cell-to-cell
adhesion, immunocytochemistry was performed for cadherins (using
pan-cadherin antibody), -catenin, and the gap junction protein
connexin43. Cells grown on coverslips were fixed with methanol at
20°C for 10 min, blocked with 1:1,000 mouse serum in PBS for 10 min, incubated with primary antibody (1:100 dilution) for 1 h at
room temperature, washed 3 times with PBS containing 0.2% BSA, and
incubated with FITC-conjugated goat anti-mouse (1:100). Nonspecific
control staining was performed on a separate coverslip using secondary
antibody only. Coverslips were washed with PBS and mounted with an
antifade mounting medium (ICN Biomedicals). For visualization of actin
filaments, cells were stained with TRITC-phalloidin and examined using
a ×40, 1.3 numerical aperture (NA) oil-immersion objective under
epifluorescence optics and confocal imaging (Leica Confocal
Laser-Scanning Microscopy, Heidelberg, Germany).
Confocal microscopy. Laser-scanning confocal microscopy was used to locate and identify adhesive and gap junctional proteins at the intercellular interface between donor and acceptor cells. For FITC-labeled probes, excitation was set at 488 nm and emission was collected with a 530/20-nm barrier filter. For TRITC, excitation was set at 530 nm and emission was collected at 620/40 nm. Cells were imaged with a ×63 oil-immersion lens (NA = 1.4), and transverse optical sections were obtained from the level of cell attachment at the substratum of the acceptor cell to the dorsal surface of the donor cell (as verified by phase-contrast microscopy). The cell-to-cell interface was estimated to be located at about the middle optical section between the cells and further verified by visual assessment of the position of the nuclei of the top and bottom cells (4',6-diamidino-2-phenylindole staining).
Immunoblotting.
Cells were washed once with PBS and lysed directly with 2% SDS Laemmli
sample buffer for production of whole cell lysates or with 1% Triton
X-100 in PBS for production of cytoskeletal fractions. The cytoskeletal
buffer also contained 5 mM EDTA, 50 µM VO4, 10 mM NaF,
and protease inhibitors (2 mM phenylmethylsufonyl fluoride, 10 µg/ml
aprotinin, and 1 µg/ml leupeptin). The Triton X-100 insoluble
fraction (i.e., cytoskeletal pellet) was solubilized with 2% SDS
sample buffer. Proteins were separated by SDS-PAGE (10% acrylamide)
and transferred to nitrocellulose membranes. Cadherins, -catenin,
connexin43, connexin26, and connexin32 were detected using
anti-pan-cadherin monoclonal antibody (Clone CH-19), anti-
-catenin
monoclonal antibody (Clone 14), anti-connexin43 monoclonal antibody
(Clone 2), anti-connexin26 monoclonal antibody (Clone CX-12H10), and
anti-connexin32 antibody (Clone CX-2C2). Blots were blocked for 1 h with 5% skim milk in Tris-buffered saline (TBS) and incubated with
the indicated antibody in 0.1% Tween-TBS. Blots were washed
with 0.5% Tween-TBS for 30 min. Primary antibody was detected using
affinity-purified, peroxidase-conjugated goat antibody (Chemicon
International, Temecula, CA) for 1 h at room temperature, washed 5 times in TBS-Tween, and developed by chemiluminescence (Amersham,
Oakville, ON).
Flow cytometry and quantification of dye coupling.
HGF monolayers (acceptor cell population) were grown on 35-mm dishes in
the presence of Texas red dextran (10 kDa; 1 mg/ml) overnight for
intracellular loading through endocytosis (31). Texas red
dextran in acceptor cells was not able to pass through the gap
junctions because of its size. Single cell suspensions of donor HGF
were prepared, labeled with 0.05 µg/ml calcein-AM, incubated at
37°C for 45 min, and followed by a 2× PBS wash. Cell counts were
obtained with an electronic cell counter (Coulter Electronics, Hialeah,
FL) and included separate estimates of acceptor cells (number of cells
per 35-mm dish) and donor cells (number of cells per milliliter of
growth medium). An aliquot (1 ml) of donor cells was added to each
35-mm dish of acceptor cells so that the ratio of donor to acceptor
cells was 1:4. Cocultures were incubated in -MEM with 10% FBS at
37°C for various time periods (30, 60, 120, 240, and 360 min) to
allow formation of cell-to-cell adhesions and gap junctions. At the
indicated time points, cocultures were gently washed once with PBS to
remove unbound cells.
Electron microscopy. Microspheres (2 µm; Polysciences, Warrington, PA) that were phagocytosed by fibroblasts after overnight incubation were used to discriminate donor cells from acceptor cells. Permeabilization of cells was obtained with 10% PHEM (0.6 M PIPES, 0.25 M HEPES, 0.1 M EGTA, 20 mM MgCl2, and 0.75% Triton X-100). Fixation was done with 1% glutaraldehyde. After 30 min, samples were embedded in Lowicryl-K4M, and thin sections were placed on nickel grids. The grids were stained with uranyl acetate and lead citrate and observed under an electron microscope (Hitachi-60).
Statistical analysis. For continuous variable data, means and SE of the mean were computed, and when appropriate, comparisons between two groups were made with unpaired Student's t-tests with statistical significance set at P < 0.05.
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RESULTS |
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Intercellular adhesion.
We studied cell-to-cell adhesion and gap junctional communication in
fibroblasts with a model in which two-color fluorescence labeling
of donor and acceptor cell populations were used for discrimination and tracking (Fig.
1A). Calcein-labeled donor
cells were added onto the Texas red dextran-labeled acceptor cell
monolayer as a substrate for attachment. Cell morphology and calcein
dye transfer from donor to acceptor cells were studied at various time
points after the coincubation was started. The donor cells rapidly
formed attachments with the acceptor cells; membrane ruffles on the
acceptor cell dorsal surface were observed within 15 min after
coincubation at 37°C (Fig. 1B). With increased time, the donor cells continued to spread on the acceptor cells, and by 60 min,
there was evidence of calcein dye transfer from the donor to the
acceptor cells, indicating the formation of functional gap junctions.
Within 180 min, the donor cells became well spread, and by
phase-contrast microscopy, blended in with the acceptor cells. More
extensive dye coupling was also evident by 180 min. These data
indicated that fibroblasts can form adhesive cell-to-cell junctions
(<15 min) and gap junctions (<60 min) shortly after they are in
contact with each other.
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Morphology of intercellular contacts formation.
We initially characterized cell-to-cell junctions in HGF monolayers
(i.e., no donor cells) by transmission electron microscopy. At sites of
contacting cell processes, HGFs formed structures with the morphology
of gap junctions and adherens junctions (Fig. 2, A and B). Unlike
epithelial cells that form abundant and well-defined intercellular
junctions (2), we found that cell-to-cell contacts in
fibroblasts often involved overlapping cell processes that complicate
the study of cell-to-cell contacts in the x-y plane. Therefore, to investigate the ultrastructure of cell-to-cell junctions during their formation, donor cells labeled with phagocytosed microspheres were used to distinguish donor from acceptor cells. Cells
were prepared for electron microscopy at various times after the
addition of donor cells. Adherens junction-like structures were formed
within 15 min of cell-to-cell contact (Fig. 2, C and D) that were coincident with membrane ruffling seen by
phase-contrast microscopy (Fig. 1B). Adherens junctions were
frequently adjacent to vesicles in the donor cells (Fig.
2E). Abundant close contacts between donor and acceptor
cells were formed within 60 min of coincubation (Fig. 2F).
Close contacts continued to develop over ~2 h, and well-defined
adherens junctions were evident after ~3 h of attachment (Fig.
2G). Distinct structures that resembled gap junctions were
evident also after ~3 h (Fig. 2H). The structure of the
adherens and gap junctions in HGFs appeared to "mature" over the
course of 3 h after the initial cell-to-cell contact. Thus, within
3 h, close contacts between the plasma membranes of donor and
acceptor cells developed into well-defined structures resembling that
of adherens junctions and gap junctions in 3-day-old HGF monolayer
cultures (Fig. 2, A and B).
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Cadherin, -catenin, and connexin43 expression and
localization.
Because electron micrography showed that adjacent human
fibroblasts were linked by structures that resembled adherens junctions and gap junctions, we used immunoblotting to study the expression of
proteins responsible for intercellular adhesion. High levels of
cadherins (using pan-cadherin antibody) and
-catenin (Fig. 3A) indicated that these two
proteins may play a role in intercellular adhesion in fibroblasts. A
relatively low level of P-cadherin and cadherin-5 was also detected in
HGF (data not shown). HGFs strongly expressed both the phosphorylated
(46 kDa) and the unphosphorylated isoforms (43 kDa) of connexin43 but
not connexin26 or connexin32 (Fig. 3A).
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Kinetics of intercellular adhesive contacts.
We compared the rate of formation of cell-to-cell and cell-to-substrate
adhesive contacts. Donor cells were added to highly adherent acceptor
cell monolayer and allowed to attach for various lengths of time at
37°C (15, 30, 60, and 180 min) before vigorous washing with PBS. We
optimized previously plating conditions by overnight attachment so that
the acceptor cells were not detached by jet washing. The
DiI-chloromethylbenzamido (CM)-labeled donor cells remaining attached
to the HGF acceptor monolayer (cell-to-cell) or those that were freshly
plated on the fibronectin-coated surface (cell-to-substrate) were
counted in a fluorescence microscope and used to estimate the rate of
adhesive contact formation. Based on counts of DiI-CM-labeled cells,
cell-to-cell adhesive contacts formed at a faster rate than
cell-to-substrate contacts (Fig. 5). The
difference in adhesion rate was most significant during the time of
early contact formation (>30-fold at 15 min; P < 0.001), suggesting that cell-to-cell adhesive contacts may be stronger than cell-to-substrate contacts at their early stages of formation. Cell-to-cell adhesion was Ca2+ dependent because when the
same adhesion assay was repeated in Ca2+-free medium
containing 2 mM EGTA, no donor cells remained attached on the acceptor
cell monolayer after washing.
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Quantification of gap junctional communication.
We used flow cytometry to measure the amount of dye transfer between
donor-acceptor couples in a 3-h time course. Gap junctional communication was quantified by measuring the mean calcein fluorescence of the acceptor population (Gate R2; Figs.
6 and 7).
The consistency in calcein fluorescence among samples at each time
point (Fig. 8) and the general trend of
calcein increase in the acceptor cells over time indicate that our
model generates a synchronous cohort of dye-coupled cells, findings
that are consistent with previous studies of single cells forming
junctions in the x-y plane (9). When dye
coupling was measured over time, there was a dramatic increase after
~120 min of cell-to-cell contact at 37°C (Fig. 8). This finding was
consistent with the electron microscopy data that showed progressive
formation of gap junctions between the donor and the acceptor cells
over time and in which distinct gap junctional structures appeared
after ~120-180 min of cell-to-cell contact. The increase in
cell-to-cell adhesion (Fig. 5) and the increase in gap junctional
communication (Fig. 8) exhibited different kinetics. The rate of
increase in cell-to-cell adhesion was highest in the first 60 min,
whereas the rate of increase of calcein dye transfer was dramatically
increased only after ~120 min. This apparent delay in gap junctional
communication is consistent with the generally accepted theory that gap
junctions are formed after cell-to-cell adherens junctions are formed
(10).
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Inhibition of gap junction communication. We investigated whether the formation of functional gap junctions is temperature dependent by conducting cell-to-cell adhesion assays at 4°C for 3 h. Whereas cell-to-cell adhesion was only slightly reduced at 4°C compared with control cells when incubated at 37°C (<20% reduction), dye transfer was significantly inhibited to <5% of control (P < 0.001; Fig. 6), indicating that the formation of functional gap junctions is indeed temperature dependent.
We perturbed donor-acceptor cell couples with different inhibitors of gap junctional communication, including BGA (8, 33) and several different connexin43 mimetic peptides (5, 9). BGA, a saponin that causes gap junction disassembly and connexin43 dephosphorylation (16), significantly reduced dye transfer in a dose-dependent manner (to <10% of control at 20 µM, P < 0.001; Figs. 6 and 7) without affecting cell-to-cell adhesion (105% of control at 20 µM, P > 0.2). Specific blockade of gap junctional communication was achieved by incubation with the connexin43 mimetic peptide GAP 27 (amino acid sequence SRPTEKTIFII) that has the same amino acid sequence as a part of the extracellular loop of connexin43. This peptide likely acts by perturbing connexin-connexin interactions that probably maintain channel integrity (5, 9). Accordingly, significant inhibition by GAP 27 of dye transfer was observed at 500 µM (<10% of control, P < 0.001; Figs. 6 and 7). On the other hand, a truncated version of GAP 27 (amino acid sequence SRPTEKTIF) that lacks the two isoleucine residues required for membrane insertion did not inhibit dye transfer (Figs. 6 and 7). Further, a connexin-mimetic peptide that resembles part of the intracellular loop of connexin43 (GAP 20; amino acid sequence EIKKFKYGC) (5, 9) was included as a negative control and also did not inhibit dye transfer (Figs. 6 and 7). ![]() |
DISCUSSION |
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Double-label synchronized cohort model. Studies of epithelial cells (29) and developing tissues (1) have provided much of our current understanding of intercellular adhesive structures and gap junctional communication. However, there has been only limited characterization and less understanding of intercellular adhesion and gap junctions in fibroblasts despite the apparent importance of these structures in wound healing (14, 21) and remodeling of connective tissues. To facilitate the study of intercellular adhesion and gap junctional communications, we have developed and characterized a simple model using human fibroblasts derived from the periodontium. In these tissues, gap junctions and adherens junctions are prominent structures of adjacent fibroblasts in vivo (3, 32).
Most previous studies of intercellular adhesions and gap junctions in cultured fibroblasts have been limited to morphological examinations in the x-y plane (13, 30, 35) of only a few cells attached on a substrate. We considered that the ability to study the formation of intercellular contacts without undue interference from cell-to-substrate interactions is important for resolving the dynamics of cell-to-cell interactions. In addition, many studies of cell-to-cell contacts use transformed cells (28, 30) or epidermal cells (12), and there are few reports on normal human fibroblasts. The new model presents several useful features. First, it creates conditions in which a large number of intercellular contacts form in a relatively short time period, thereby enabling studies of synchronized intercellular adhesive events. With this approach, we have characterized their morphology at discrete stages of their development by transmission electron microscopy, confocal microscopy, and optical sectioning. These data showed that the donor-acceptor model not only features types of intercellular junctions similar to those that formed in monolayer cultures in the x-y plane but that they are much more prominent and more easily recognized. Because large numbers of cells can be analyzed, the model also enables biochemical analysis of intercellular junctions that are associated with the cytoskeleton during the formation of intercellular adhesions (e.g., immunoblotting). Furthermore, functional assays of intercellular adhesion and gap junctional communication by dye transfer measurements with flow cytometry can be performed. A second useful feature relates to the design of the model. Because donor cells are added onto a confluent acceptor cell monolayer, the attachment and subsequent spreading of donor cells depend largely on cell-to-cell interactions between the donor and acceptor cells and only minimally on cell-to-substrate interactions between acceptor cells and their substratum. Third, because the donor and the acceptor cells are separately labeled with different fluorescence dyes, any donor, acceptor, or dye-coupled acceptor populations can be preferentially selected by fluorescence-activated cell sorting for later analysis, including biochemical analysis. The model could therefore be used to test the permeability of gap junctions to specific molecules between donor and acceptor cells. Molecules to be tested could be loaded into the cytoplasm of donor cells by electroporation (11), scrape loading, or endocytosis. For example, we have shown that endocytosed FITC or Texas red dextran (>10 kDa) does not transfer between cells after a 6-h coincubation. In general, gap junctions are formed by connexin subunits that are a family of proteins including connexins43, 26, 32, and 45 (15). Another advantage of using human gingival fibroblasts is that of simplification: only connexin43 was detected by immunoblotting. Notably, the degree of dye transfer inhibition in HGFs was significantly more than previous reports in which the GAP 27 peptide was used to inhibit gap junctional communication in other cell types (5, 9). With the availability of inhibitory peptides, our model provides a sensitive, specific, and simplified assay for study of gap junctions.Intercellular adhesion.
We found that intercellular adhesive contacts formed rapidly (<15 min)
between donor and acceptor fibroblasts and were typical of the
appearance of adherens junctions (3, 32). The
intercellular adhesion was Ca2+ dependent. Our
immunofluorescence and confocal images showed clustering of cadherins,
-catenin, and connexin43 at intercellular contacts. We also found an
enrichment of these proteins in the Triton-insoluble cytoskeletal
fraction in confluent cell cultures. These observations are consistent
with the notion that intercellular adherens junctions are
cadherin-mediated complexes comprised of cytoplasmic plaque proteins
such as catenins and are connected to the actin cytoskeleton
(17).
Interdependence between intercellular adhesion and gap junctions. Previous reports have shown a dependence of gap junction formation on cadherin-mediated intercellular adhesion in epidermal cells (19) as well as an interdependence between these two types of junctions (24). The pattern of spatial association between gap junctions and cell adhesion junctions is likely an important factor in maturation of mammalian cardiac tissues (1). In thyroid cells, neoplastic alterations of the complex cellular network established by adhesion receptors and gap junctions can lead to an imbalance of cell-to-cell communication: this imbalance allows transformed cells to escape from the tissue to generate metastases (28). Therefore, the ability to study simultaneously both adherens and gap junctions is of considerable biological importance and can be realized with this model system. We have shown that structures resembling adherens junctions appeared well before gap junctional plaques. Kinetic studies showed that adhesive cell-to-cell contacts increased most rapidly during the initial 60 min of coincubation that was followed by a dramatic increase in dye transfer starting at ~120 min. This temporal relationship suggests that intercellular adhesion is a prerequisite for gap junction formation. Presumably, the close apposition of adjacent cell membranes mediated by adherens junctions facilitates cell-to-cell interactions. These interactions include connexon-to-connexon couples that lead in turn to the formation of gap junctions.
The ability to measure intercellular adhesion and gap junctional communication simultaneously by flow cytometry enhances the analytical power of our model. We showed that although the gap junctional inhibitor BGA had no effect on cell-to-cell adhesion, GAP 27 peptide significantly reduced adherence of donor cells by >90%. Because BGA dephosphorylates connexins (16, 18) while GAP 27 probably inhibits the formation of gap junctions by perturbing connexin-connexin interactions (5, 9), we interpret these data as indicating that connexon assembly has a more important effect on intercellular adhesion than does connexin phosphorylation. We conclude that the synchronized cell cohort model reported here provides information on the structure and kinetics of the formation of intercellular adherens and gap junctions in human fibroblasts. The data suggest that it is a sensitive, specific, and quantitative model for investigating the dynamics of intercellular adherens junctions and gap junctions. The model should facilitate studies of intercellular signaling in fibroblasts and other stromal cells that are involved in wound healing and tissue remodeling. ![]() |
ACKNOWLEDGEMENTS |
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We thank Cheung Lo for assistance with cell cultures, Lowell Langille and Greg Downey for advice on immunostaining and confocal microscopy, and Sela Cheifetz for helpful comments on the manuscript.
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FOOTNOTES |
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This project was supported by a Medical Research Council (MRC) of Canada Group grant and Maintenance grant as well as a Heart and Stroke Foundation grant (to C. McCulloch) and MRC Fellowship (to K. Ko).
Address for reprint requests and other correspondence: K. Ko, Rm. 244, Fitzgerald Bldg., Univ. of Toronto, 150 College St., Toronto, Ontario M5S 3E2, Canada (E-mail: kevin_ko{at}hotmail.com).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 8 December 1999; accepted in final form 26 January 2000.
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