Vol. 279, Issue 1, C147-C157, July 2000
Biochemical and functional characterization of intercellular
adhesion and gap junctions in fibroblasts
Kevin
Ko,
Pamela
Arora,
Wilson
Lee, and
Christopher
McCulloch
Medical Research Council Group in Periodontal Physiology, Faculty
of Dentistry, University of Toronto, Toronto, Ontario, Canada M5S
3E2
 |
ABSTRACT |
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
| |
INTRODUCTION |
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.
| |
MATERIALS AND METHODS |
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.
For qualitative observation of dye coupling, cocultures were monitored
and images were recorded at specific time points (0, 15, 30, 60, and
180 min) with a fluorescence microscope coupled to a CCD camera.
Quantification of dye coupling between the donor and acceptor cells was
assessed by dual-excitation flow cytometry. Single cell suspensions
were prepared from the coculture monolayer by trypsinization (0.01%
vol/vol trypsin in PBS for 10 min). The suspension was neutralized with
growth medium, pelleted, and resuspended in PBS for flow cytometry
analysis. Three samples were analyzed for each experimental group, and
>105 cells were analyzed in each sample. Samples were
analyzed with a FACStar Plus flow cytometer (Becton Dickinson FACS
Systems, Mountain View, CA) at a sheath pressure of 11 lb/in.2 with an Innova 70 argon laser (Coherent Laser
Products, Palo Alto, CA) at a light regulation mode setting of 250 mW
and a wavelength of 488 nm. Emitted fluorescence was divided between
two detectors by beam splitters and band-pass filters for green
fluorescence (515-545 nm) and red fluorescence (606-644 nm).
Photomultiplier tube voltage settings were determined for each
experiment on the basis of thresholds established from unlabeled cell
samples and from a mixture of calcein-loaded and Texas red
dextran-loaded cells. Evidence of dye coupling was indicated by the
appearance of cells that exhibited suprathreshold staining for both
calcein and Texas red. Negative controls included 1)
incubation of FITC-dextran (70 kDa)-labeled donor cells with Texas red
dextran (10 kDa)-labeled acceptor monolayers, and 2) use of
a porous filter (Falcon membrane, 0.4 µm) to separate donor
and acceptor cells. In both cases, no cells with both red and green
fluorescence were detected for up to 6 h of incubation.
To further characterize the model system, two different inhibitors of
gap junction communication were used to assess whether these agents
inhibit dye coupling in the above assay. The inhibitors included BGA
(20-100 µM) (8, 33) and GAP 27 peptide
[synthetic connexin-mimetic peptide; GAP 20 and truncated GAP
27 peptides were used as controls (5, 8); 500 µM]. Cells were pretreated with the above inhibitors for 3 h
before experiments.
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.
| |
RESULTS |
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.
View larger version (62K):
[in this window]
[in a new window]
|
Fig. 1.
A: schematic diagram showing the model system
used in the study of intercellular adhesion and gap junctional
communication in human gingival fibroblasts (HGFs). Fluorescence dyes
are used to distinguish the two cell populations. Calcein-labeled
(green) donor cells are added onto Texas red dextran-labeled (red)
acceptor cell monolayers as substrates for attachment. At the early
stage (<15 min) of cell-to-cell contact, the donor cell is rounded and
adherens junctions are starting to form between the donor and acceptor
cells. At later stages (60-180 min), the donor cell spreads on the
acceptor cell and forms membrane ruffles. More adherens junctions and
gap junctions form as indicated by transfer of calcein from donor to
acceptor cells. B: phase-contrast (top) and
epifluorescence micrographs (bottom) of donor and acceptor
cells at indicated times of cell-to-cell contact. Calcein-labeled donor
cells were added at time 0 to the acceptor cell
monolayer and incubated at 37°C. Note that donor cells were rounded
at time 0, started to form membrane ruffles and filopodia
within 15 min, spread well within 60 min, and were very well spread and
formed dye couples to acceptor cells underneath by 180 min.
|
|
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).
View larger version (126K):
[in this window]
[in a new window]
|
Fig. 2.
A and B: electron micrographs
showing intercellular adherens junctions (large arrows) and gap
junctions (arrow heads) in gingival fibroblast monolayers
(A, ×30,000) and intercellular adherens junctions between
overlapping filopodia from 2 adjacent fibroblasts (B,
×20,000) from a 3-day-old culture. C-H: electron
micrographs showing adherens junctions (large arrows) and gap junctions
(arrow heads) between donor (Dn) and acceptor (Ac) cells in the
intercellular adhesion model at 15 min (C, ×4,000;
D, ×15,000; E, ×35,000), 60 min (F,
×20,000), and 180 min (G, ×30,000; H, ×30,000)
of cell-cell contact. Donor cells with intracellular microspheres
(b) as marker were added to acceptor cell monolayer, and
cells were fixed at different times after 1 wash. Note the formation of
filopodia and intercellular adherens junctions within 15 min of
cell-cell attachment (C, inset; ×10,000). In
E, note the abundance of exocytic vesicles (v) adjacent to
early (~15 min) intercellular contacts. Also, note abundant rough
endoplasmic reticulum (C, F, G) in the
donor cells. Increased contact area between donor and acceptor cells
was evident by 60 min (F). At 180 min, well-formed,
electron-dense adherens junctions (G) and distinct gap
junctional plaques (H) appeared between donor and acceptor
cells.
|
|
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).
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 3.
Immunoblots showing expression of cadherins and connexins
in human gingival fibroblasts (A) and the association of
these proteins with the cytoskeleton (B) in sparse
(lanes 1 and 3) and confluent cultures
(lanes 2 and 4). Immunoblots were
probed with pan-cadherin (for cadherins), -catenin, connexin43,
connexin26, and connexin32 antibodies. Whole cell lysates of 3-day-old
confluent HGF culture showed presence of cadherins, -catenin, and
connexin43 but not connexin32 and connexin26 (A). The band
at 46 kDa corresponds to the phosphorylated form of connexin43
(23). B: lane 1: Triton-insoluble
fraction of sparse HGF culture; lane 2: Triton-insoluble
fraction of confluent HGF culture; lane 3: whole cell lysate
of sparse HGF culture; lane 4: whole cell lysate of
confluent HGF culture. Sparse cell cultures with minimal cell-cell
contacts expressed low levels of cadherins and connexin43. Cadherins,
-catenin, and connexin43 were recruited to the Triton-insoluble
fraction of the cell when cells were grown to confluence. Similar
results were obtained in 3 independent experiments. Note the increased
amount of phosphorylated species of connexin43 (46 kDa) in confluent
cells (B, lanes 2 and 4) further
enriched in the Triton-insoluble fraction.
|
|
Because cadherins, -catenin, and connexins are normally concentrated
at the cell-to-cell contacts (17) but not in
cell-to-substrate contacts (e.g., focal adhesion complexes), we
investigated the distribution of these proteins and their relationship
with the cytoskeleton in monolayer cultures. Triton-insoluble cell
lysates were prepared from sparse (no cell-to-cell contacts) and
confluent cell cultures (abundant cell-to-cell contacts). Western blot
analysis showed that in sparse cultures, cadherins, although present in whole cell lysates (Fig. 3B, lane 3), were not
recruited to the Triton-insoluble cytoskeletal fraction (Fig.
3B, lane 1). In contrast, -catenin was
associated with the cytoskeletal fraction in the absence or presence of
intercellular contacts (Fig. 3B, lanes 1 and 2). Connexin43 was minimal in the cytoskeletal
fraction of sparse cultures (Fig. 3B, lane 1) but was
abundant in confluent cultures (Fig. 3B, lane 2).
Because Triton extraction was enriched for the slower migrating,
presumably phosphorylated species of connexin43 (46 kDa) that are
incorporated into junctional plaques (26,
35), the abundance of phosphorylated connexin43 in
confluent HGF monolayers (Fig. 3B, lane 2)
suggests that they may be able to form gap junctions with cytoskeletal
associations under these conditions.
Immunofluorescence microscopy was used to determine the localization of
intercellular attachment proteins at cell-to-cell contacts in monolayer
cell cultures. In confluent monolayers, cadherins, -catenin, and
connexin43 were concentrated at cell-to-cell contact points (Fig.
4A) where filopodia meet and
overlap. Both cadherins and -catenin were concentrated in discrete
linear structures perpendicular to the peripheral margin of the cell.
The cell monolayer that we studied (Fig. 4A) exhibited two
types of contacts: 1) cell-to-substrate contacts with the
tissue culture plate (e.g., focal adhesion complexes), and
2) intercellular contacts between adjacent cells (i.e.,
adherens junctions and gap junctions). The appearance and distribution
of cadherins and -catenin were very different from those found in
typical focal adhesion complexes, consistent with the fact that these
cadherins and -catenin are intercellular adherens junctional
proteins. Although cytoplasmic staining for connexin43 was evident,
very strong connexin43 staining appeared as punctate junctional plaques
at intercellular contacts.
View larger version (39K):
[in this window]
[in a new window]
|
Fig. 4.
A: gingival fibroblasts stained for
connexin43, cadherins, and -catenin with anti-connexin43 antibody,
pan-cadherins antibody, -catenin antibody, and FITC-conjugated goat
anti-mouse antibody. Note the localization of connexin43 at the
intercellular junctional plaques and the staggered pattern of cadherins
and -catenin at the cell-cell junctions. B: confocal
images show clustering of connexin43, cadherins, and -catenin at the
cell-to-cell interface by a series of optical sections from the bottom
of the acceptor cell to the top of the donor cell. Only the
section immediately (1 µm) above or below the cell-cell interface is
shown (B, top diagram). Note the slight difference in
spatial distribution between connexin43 and cadherins or -catenin.
|
|
Immunocytochemistry and confocal microscopy were used to localize
connexin43, -catenin, and cadherins at intercellular contacts in
z-axis scans of donor-acceptor cell preparations. Optical
sections (2 µm apart; perpendicular to the z-axis of the
culture dish; Fig. 4, top diagram) midway between
the nuclei of the donor (top) cell and the acceptor cell
(bottom) showed clustering of cadherins, -catenin, and
connexin43 at the level of cell-to-cell contact in both donor and
acceptor cells (Fig. 4B). Staining for cadherins, -catenin, and connexin43 was not observed at acceptor cell-substrate sites. Notably, during early cell-to-cell contact formation, cadherins and -catenin were concentrated more toward the periphery of donor cells, a finding that is consistent with electron microscopy showing cell-to-cell contacts at the tip of the filopodia of donor cells (Fig.
2C). Unlike adherens junctions, staining for gap junctions with connexin43 exhibited a more dispersed distribution at the cell-to-cell interface.
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.
View larger version (14K):
[in this window]
[in a new window]
|
Fig. 5.
Line plot comparing the strength of adhesion between
cell-to-cell and cell-to-substrate adhesion. DiI-chloromethylbenzamido
(CM)-labeled HGFs were added to HGF monolayer (cell-to-cell adhesion)
or 10 µg/ml fibronectin-coated glass surface (cell-to-substrate
adhesion) and incubated at 37°C before vigorous jet washing and
fixation at 15, 30, 60, and 180 min. Cells were counted from each of 4 randomly chosen microscope fields (×40). For each sample,
mean ± SE of mean number of cells is shown. The data shown are
representative of 3 independent experiments. Note that cell-to-cell
adhesion was significantly stronger than cell-substrate adhesion during
the early stages of adhesion (t < 30 min), but not in
the later stages (t > 60 min.)
|
|
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).
View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
Flow cytographs showing the effects of various
inhibitors of gap junctional communication. Dye transfer was indicated
by increased calcein fluorescence of the acceptor cell population
(upper left quadrant). Significant dye transfer occurred
within 180 min of incubation at 37°C in the presence (ii) or absence
(iii) of serum when compared with time 0 (i). Note the
absence of calcein-labeled donor cells in (i) but their presence in the
bottom right quadrant at later times (ii-ix,
t = 180 min). Low temperature significantly
inhibited dye transfer (iv). The gap junctional communication blocker
-glycyrrhetinic acid (BGA) significantly reduced calcein dye
transfer at 20 µM (v), and further reduction was evident at 100 µM
(vi). The connexin43 mimetic peptide GAP 27 significantly reduced the
extent of dye transfer (viii), whereas its truncated version GAP 27-II
(viii) and GAP 20 (ix) had relatively no effect. These results are
representative of 3 parallel samples (n = 3) in 2 independent experiments.
|
|
View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Histograms showing the effects of various gap junctional
communication blockers. Each data point is an average of the mean
calcein fluorescence of the acceptor cell population after 180-min
incubation with calcein-labeled donor cells from 3 parallel samples.
Note the marked inhibition by BGA and the connexin43 mimetic peptide
GAP 27. Note also the lack of significant inhibition by GAP 27-II and
GAP 20 peptides, 2 other connexin43 mimetic peptides used here as
negative controls.
|
|
View larger version (11K):
[in this window]
[in a new window]
|
Fig. 8.
Line plot showing the increase in mean calcein
fluorescence (mean ± SE) of acceptor cell population over time
after coincubation with donor cells. Donor cells (calcein-labeled) were
added to the acceptor cell monolayer (Texas red dextran-labeled) for
various lengths of time before washing and trypsinization for flow
cytometric analysis. Each point represents the average of 3 parallel
samples. Similar results were obtained in 5 independent experiments.
Note the dramatic increase in calcein dye transfer from the donor to
the acceptor cells after the initial 120 min.
|
|
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 |
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).
For functional analysis, we employed a jet-washing assay to study
cell-to-cell adhesive strength. This method has been used previously to
estimate relative adhesive strength of collagen-coated beads to
fibroblasts (7). The increased number of DiI-CM-labeled donor cells remaining attached to acceptor cells after jet washing provides an estimate of the increase in intercellular adhesive strength
over time. Presumably, the progressive maturation of the adherens
junctions strengthens intercellular adhesion by expansion of
cell-to-cell contact sites into larger junctional plaques as shown by
our electronmicrographic data and by forming more connections to the
actin cytoskeleton. The adherens junctions that formed evidently
engaged the cytoskeleton because cadherins, -catenin, and connexin43
were recruited into the cytoskeletal network when there were abundant
cell-to-cell contacts.
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 |
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.
| |
FOOTNOTES |
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.
| |
REFERENCES |
1.
Angst, BD,
Khan LU,
Severs NJ,
Whitely K,
Rothery S,
Thompson RP,
Magee AI,
and
Gourdie RG.
Dissociated spatial patterning of gap junctions and cell adhesion junctions during postnatal differentiation of ventricular myocardium.
Circ Res
80:
88-94,
1997[Abstract/Free Full Text].
2.
Balda, MS,
and
Matter K.
Tight junctions.
J Cell Sci
111:
541-547,
1998[Abstract].
3.
Beertsen, W,
and
Everts V.
Junctions between fibroblasts in mouse periodontal ligament.
J Periodontal Res
16:
524-541,
1980.
4.
Beyer, EC,
Paul DL,
and
Goodenough DA.
Connexin family of the gap junction proteins.
J Membr Biol
116:
187-194,
1990[ISI][Medline].
5.
Chaytor, AT,
Evans WH,
and
Griffith TM.
Peptides homologous to extracellular loop motifs of connexin 43 reversibly abolish rhythmic contractile activity in rabbit arteries.
J Physiol (Lond)
503:
109-119,
1997.
6.
Cheng, SL,
Lecanda F,
Davidson MK,
Warlow PM,
Zhang SF,
Zhang L,
Suzuki S,
St. John T,
and
Civitelli R.
Human osteoblasts express a repertoire of cadherins, which are critical for BMP-2-induced osteogenic differentiation.
J Bone Miner Res
13:
633-644,
1998[ISI][Medline].
7.
Chou, DH,
Lee W,
and
McCulloch CAG
TNF- inactivation of collagen receptors: implications for fibroblast function and fibrosis.
J Immunol
156:
4354-4362,
1996[Abstract].
8.
Davidson, JS,
Baumgarten IM,
and
Harley EH.
Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid.
Biochem Biophys Res Commun
134:
29-36,
1986[ISI][Medline].
9.
Dora, KA,
Martin PEM,
Chaytor AT,
Evans WH,
Garland CJ,
and
Griffith TM.
Role of heterocellular gap junctional communication in endothelium-dependent smooth muscle hyperpolarization: inhibition by a connexin-mimetic peptide.
Biochem Biophys Res Commun
254:
27-31,
1999[ISI][Medline].
10.
Fujimoto, K,
Nagafuchi A,
Tsukita S,
Kuraoka A,
Ohokuma A,
and
Shibata Y.
Dynamics of connexins, E-cadherin and -catenin on cell membranes during gap junction formation.
J Cell Sci
110:
311-322,
1997[Abstract].
11.
Glogauer, M,
and
McCulloch CAG
Introductions of large molecules into viable fibroblasts by electroporation: optimization of loading and identification of labeled cellular components.
Exp Cell Res
200:
227-234,
1992[ISI][Medline].
12.
Gloushankova, NA,
Alieva NO,
Krendel MF,
Bonder EM,
Feder HH,
Vasiliev JM,
and
Gelfand IM.
Cell-cell contact changes the dynamics of lamellar activity in nontransformed epitheliocytes but not in their ras-transformed descendants.
Proc Natl Acad Sci USA
94:
879-883,
1997[Abstract/Free Full Text].
13.
Gloushankova, NA,
Krendel MF,
Alieva NO,
Bonder EM,
Feder HH,
Vasiliev JM,
and
Gelfand IM.
Dynamics of contacts between lamellae of fibroblasts: essential role of the actin cytoskeleton.
Proc Natl Acad Sci USA
95:
4362-4367,
1998[Abstract/Free Full Text].
14.
Goliger, JA,
and
Paul DL.
Wounding alters epidermal connexin expression and gap junction-mediated intercellular communication.
Mol Biol Cell
6:
1491-1501,
1995[Abstract].
15.
Goodenough, DA,
Goliger JA,
and
Paul DL.
Connexins, connexons, and intercellular communication.
Annu Rev Biochem
65:
475-502,
1996[ISI][Medline].
16.
Guan, X,
Wilson S,
Schlender KK,
and
Ruch RJ.
Gap-junction disassembly and connexin 43 dephosphorylation induced by 18 -glycyrrhetinic acid.
Mol Carcinog
16:
157-164,
1996[ISI][Medline].
17.
Gumbiner, BM.
Cell adhesion: the molecular basis of tissue architecture and morphogenesis.
Cell
84:
345-357,
1996[ISI][Medline].
18.
Guo, Y,
Martinez-Williams C,
Gilbert KA,
and
Rannels DE.
Inhibition of gap junction communication in alveolar epithelial cells by 18-glycyrrhetinic acid.
Am J Physiol Lung Cell Mol Physiol
276:
L1018-L1026,
1999[Abstract/Free Full Text].
19.
Jongen, WMF,
Fitzgerald DJ,
Asamoto M,
Piccoli C,
Slaga TJ,
Gros D,
Takeichi M,
and
Yamasaki H.
Regulation of connexin 43-mediated gap junctional intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin.
J Cell Biol
114:
545-555,
1991[Abstract/Free Full Text].
20.
Kumar, NM,
and
Gilula NB.
The gap junction communication channel.
Cell
84:
381-388,
1996[ISI][Medline].
21.
Lampe, PD,
Nguyen BP,
Gil S,
Usui M,
Olerud J,
Takada Y,
and
Carter WG.
Cellular interaction of integrin 31 with laminin 5 promotes gap junctional communication.
J Cell Biol
143:
1735-1747,
1998[Abstract/Free Full Text].
22.
Lecanda, F,
Towler DA,
Ziambaras K,
Cheng SL,
Koval M,
Steinberg TH,
and
Civitelli R.
Gap junctional communication modulates gene expression in osteoblastic cells.
Mol Biol Cell
9:
2249-2258,
1998[Abstract/Free Full Text].
23.
Li, R,
and
Mather JP.
Lindane, an inhibitor of gap junction formation, abolishes oocyte directed follicle organizing activity in vitro.
Endocrinology
138:
4477-4480,
1998[Abstract/Free Full Text].
24.
Meyer, RA,
Laird DW,
Revel J-P,
and
Johnson RG.
Inhibition of gap junction and adherens junction assembly by connexin and A-CAM antibodies.
J Cell Biol
119:
179-189,
1992[Abstract/Free Full Text].
25.
Musil, LS,
Cunningham BA,
Edelman GM,
and
Goodenough DA.
Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines.
J Cell Biol
111:
2077-2088,
1990[Abstract/Free Full Text].
26.
Musil, LS,
and
Goodenough DA.
Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques.
J Cell Biol
115:
1357-1374,
1991[Abstract/Free Full Text].
27.
Pender, N,
and
McCulloch CAG
Quantitation of actin polymerization in two human fibroblast sub-types responding to mechanical stretching.
J Cell Sci
100:
187-193,
1991[Abstract/Free Full Text].
28.
Potter, E,
Schonermark M,
Bock O,
Hoang-Vu C,
Munari-Silem Y,
Rousset B,
and
Brabant G.
Cell adhesion receptors and gap junctions in normal and neoplastic transformed thyrocytes.
Exp Clin Endocrinol Diabetes
104, Suppl 4:
24-28,
1996.
29.
Prowse, DM,
Cadwallader GP,
and
Pitts JD.
E-cadherin expression can alter the specificity of gap junction formation.
Cell Biol Int
21:
833-843,
1997[ISI][Medline].
30.
Ragsdale, GK,
Phelps J,
and
Luby-Phelps K.
Viscoelastic response of fibroblasts to tension transmitted through adherens junctions.
Biophys J
73:
2798-2808,
1997[Abstract/Free Full Text].
31.
Roederer, M,
Bowser R,
and
Murphy RF.
Kinetics and temperature dependence of exposure of endocytosed material to proteolytic enzymes and low pH: evidence for a maturation model for the formation of lysosomes.
J Cell Physiol
131:
200-209,
1987[ISI][Medline].
32.
Shore, RC,
Berkovitz BKB,
and
Moxham B.
Intercellular contacts between fibroblasts in the periodontal connective tissues of the rat.
J Anat
133:
67-76,
1981[ISI][Medline].
33.
Spanakis, SG,
Petridou S,
and
Masur SK.
Functional gap junctions in corneal fibroblasts and myofibroblasts.
Invest Ophthalmol Vis Sci
39:
1320-1328,
1998[Abstract/Free Full Text].
34.
Takeichi, M.
Cadherin cell adhesion receptors as a morphogenetic regulator.
Science
251:
1451-1455,
1991[Abstract/Free Full Text].
35.
Yonemura, S,
Itoh M,
Nagafuchi A,
and
Tsukita S.
Cell-to-cell adherens junction formation and actin filament organization: similarities and differences between nonpolarized fibroblasts and polarized epithelial cells.
J Cell Sci
108:
127-142,
1995[Abstract].
Am J Physiol Cell Physiol 279(1):C147-C157
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society
TOP
ABSTRACT
INTRODUCTION
