Zonula Occludens-1 Alters Connexin43 Gap Junction Size and Organization by Influencing Channel Accretion

Abstract

Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens (ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1 (PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover. Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 association with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.

Full text

Molecular Biology of the Cell
Vol. 16, 5686–5698, December 2005
Zonula Occludens-1 Alters Connexin43 Gap Junction Size
and Organization by Influencing Channel Accretion
Andrew W. Hunter, Ralph J. Barker, Ching Zhu, and Robert G. Gourdie
Department of Cell Biology and Anatomy, Cardiovascular Developmental Biology Center, Medical University
of South Carolina, Charleston, SC 29425
Submitted August 9, 2005; Revised September 13, 2005; Accepted September 15, 2005
Monitoring Editor: Asma Nusrat
Regulation of gap junction (GJ) organization is critical for proper function of excitable tissues such as heart and brain, yet
mechanisms that govern the dynamic patterning of GJs remain poorly defined. Here, we show that zonula occludens
(ZO)-1 localizes preferentially to the periphery of connexin43 (Cx43) GJ plaques. Blockade of the PDS95/dlg/ZO-1
(PDZ)-mediated interaction between ZO-1 and Cx43, by genetic tagging of Cx43 or by a membrane-permeable peptide
inhibitor that contains the Cx43 PDZ-binding domain, led to a reduction of peripherally associated ZO-1 accompanied by
a significant increase in plaque size. Biochemical data indicate that the size increase was due to unregulated accumulation
of gap junctional channels from nonjunctional pools, rather than to increased protein expression or decreased turnover.
Coexpression of native Cx43 fully rescued the aberrant tagged-connexin phenotype, but only if channels were composed
predominately of untagged connexin. Confocal image analysis revealed that, subsequent to GJ nucleation, ZO-1 associ-
ation with Cx43 GJs is independent of plaque size. We propose that ZO-1 controls the rate of Cx43 channel accretion at
GJ peripheries, which, in conjunction with the rate of GJ turnover, regulates GJ size and distribution.
INTRODUCTION
The gap junction (GJ) is a plaque-like aggregate of intercel-
lular channels that facilitates cytoplasmic interchange of
ions, second messengers, and other molecules ⬍1 kDa be-
tween cells (Goodenough et al., 1996). Frequent and variably
sized GJ channel aggregates couple most cells in animal
tissues. In excitable organs such as the heart and brain, GJs
show distinctive organizational patterns that configure ex-
tended intercellular pathways for stable and long-term
propagation of action potential (Lo, 2000). The channels
comprising individual GJ plaques are composed of proteins
encoded by the connexin family of genes (Willecke et al.,
2002). Assembly of GJs from connexin monomers is thought
to proceed in a multistep process (Musil and Goodenough,
1991, 1993). First, six connexins oligomerize into a
hemichannel, called a connexon, followed by trafficking to
the plasma membrane. Subsequently, a connexon docks
with a second connexon from the apposed membrane of an
adjacent cell to form an intercellular channel. In a process
that occurs either simultaneous with or after this docking
step, channels aggregate to form the functional organelle of
cell-cell communication—the GJ plaque.
The gating of single channels within a GJ plaque is regu-
lated by various stimuli, including voltage, pH, and phos-
phorylation (Saez et al., 2003). Intercellular communication is
also regulated at the level of the plaque as a whole by factors
that affect the abundance, size, and cellular distribution of GJ
channel aggregates (Hall and Gourdie, 1995; Bukauskas et
al., 2000; Spach et al., 2000; Johnson et al., 2002; Lauf et al.,
2002). Irregularities in the extent and geometry of gap junc-
tional contacts have been implicated in cardiac and neural
electrophysiological pathologies in humans, including isch-
emic heart disease (Smith et al., 1991), hypertrophic cardio-
myopathy (Sepp et al., 1996), heart failure (Dupont et al.,
2001), and medial temporal lobe epilepsy (Fonseca et al.,
2002). At present, the mechanisms governing higher order
aspects of GJ organization and function in health and dis-
ease are poorly understood.
Nonetheless, there is a growing understanding that an
array of proteins interact with connexins, potentially medi-
ating linkage of GJs to other junction types, signal transduc-
tion molecules, and the cytoskeleton (Giepmans, 2004). One
connexin-interacting molecule that has received particular
attention is zonula occludens (ZO)-1. Originally discovered
in association with tight junctions (Stevenson et al., 1986),
ZO-1 is a member of the membrane-associated guanylate
kinase (MAGUK) family of proteins that function in protein
targeting, signal transduction, and determination of cell po-
larity (Anderson, 1996). MAGUKs, such as ZO-1, synaptic
protein PSD95, and the Drosophila tumor suppressor discs
large (dlg), are characterized by an N-terminal array of
protein-binding domains that includes one or more PDS95/
dlg/ZO-1 (PDZ) domains. Previous immunoprecipitation
and yeast two-hybrid studies showed that a short PDZ-
binding motif at the C terminus of connexin43 (Cx43) inter-
acts with the second of three PDZ domains in ZO-1 (Giep-
mans and Moolenaar, 1998; Toyofuku et al., 1998).
Subsequently, other connexins have been reported to inter-
act with ZO-1 via a consensus C-terminal PDZ-binding do-
main similar to that of Cx43 (Jin et al., 2000; Kausalya et al.,
2001; Laing et al., 2001; Nielsen et al., 2003; Li et al., 2004).
Initially, ZO-1 function at GJs was assumed to be analo-
gous to its presumed role at tight junctions. Namely, ZO-1
This article was published online ahead of print in MBC in Press
(http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–08–0737)
on September 29, 2005.
Address correspondence to: Andrew W. Hunter (huntera@
musc.edu) or Robert G. Gourdie (gourdier@musc.edu).
Abbreviations used: AJ, adherens junction; Cx43, connexin43; GJ,
gap junction; MOI, multiplicity of infection; PDZ, PDS95/dlg/ZO-1;
ZO, zonula occludens.
5686 © 2005 by The American Society for Cell Biology

was envisaged as a passive scaffolding molecule that stabi-
lizes GJs through cytoskeletal anchoring. However, subse-
quent reports have indicated that interactions between con-
nexins and ZO-1 may encompass other, more dynamic
functions. In particular, changes in Cx43–ZO-1 interaction
have been noted during remodeling of GJs in cardiomyo-
cytes and other cell types (Defamie et al., 2001; Barker et al.,
2002; Segretain et al., 2004). Moderation of Cx43–ZO-1 inter-
action by c-Src has lead others to hypothesize a role for ZO-1
in the regulation of GJ channel activity (Giepmans et al.,
2001a; Toyofuku et al., 2001; Duffy et al., 2004; Sorgen et al.,
2004). As such, definitive roles for ZO-1 interaction with
connexins remain to be determined. Here, we provide evi-
dence that ZO-1 regulates the cellular distribution of Cx43,
and consequently the size of GJ plaques, by controlling the
rate of channel accretion at plaque perimeters.
MATERIALS AND METHODS
Animals
Hearts were harvested from 2- to 3-d-old Holtzman Sprague Dawley rats.
Animal care was in accordance with institutional guidelines at the Medical
University of South Carolina, Charleston, SC (Animal welfare assurance
#A4328-01).
Antennapedia Peptides
Antennapedia peptides (Lindgren et al., 2000) were generated at Medical
University of South Carolina or by Research Genetics (Huntsville, AL). The
inhibitor peptide includes the Cx43 C-terminal amino acids 374–382 (RPRP-
DDLEI) that encompass the ZO-1-binding sequence. A control peptide was
generated by reversing the Cx43 amino acid sequence (IELDDPRPR). Both
peptides contain a 16-amino acid antennapedia internalization vector (RQP-
KIWFPNRRKPWKK) linked to the N terminus of the Cx43 (or reversed Cx43)
sequence; peptides were N-terminally biotinylated.
Cell Culture
Neonatal cardiomyocytes were isolated with a Neonatal Cardiomyocyte Iso-
lation System (LK003300; Worthington Biochemicals, Freehold, NJ) according
to manufacturer’s instructions. Cultures were generated from 8 to 12 hearts.
Dispersed cardiomyocytes were plated on coverglass or culture dishes coated
with 0.2% gelatin (Sigma-Aldrich, St. Louis, MO). Cytosine arabinoside (Sig-
ma-Aldrich) was used to inhibit fibroblast proliferation. Cardiomyocytes
were cultured as described previously (Barker et al., 2002) as monolayers for
72 h before peptide treatments.
HeLa cells stably expressing either native Cx43 or Cx43-EGFP (Jordan et al.,
1999) were cultured as described previously (Hunter et al., 2003) for 24–48 h
before peptide treatments or adenoviral infection. Newly confluent cultures
were used for all experiments.
Adenovirus expressing human Cx43 (Qbiogene, Carlsbad, CA) was added
to cells (at various multiplicities of infection [MOIs]) in Opti-MEM and
allowed to adsorb for several hours before addition of culture medium.
Adenoviral expression progressed for 24 h subsequent to further manipula-
tions.
Peptides were added to cells at 30
␮
M, which falls within the effective range
(1–100
␮
M) for antennapedia peptides (Lindgren et al., 2000). Media contain-
ing peptides were replenished every 24 h.
Determination of Connexin Expression Levels,
Stoichiometry, and Turnover
Cx43 expression was assessed in cardiomyocytes and HeLa Cx43 cells cul-
tured for 48 and 72 h, respectively, with or without peptides. Cells were
solubilized in hot 4% SDS sample buffer for 5 min, sheared, and boiled for 5
min. Samples were resolved by 10% SDS-PAGE and immunoblotted with
Cx43 (71-0700; Zymed Laboratories, South San Francisco, CA) and glyceral-
dehyde-3-phosphate dehydrogenase (GAPDH) (Research Diagnostics,
Flanders, NJ) antibodies. Sample loading was normalized to GAPDH signal.
To determine stoichiometry and turnover in connexin-expressing HeLa
cells, metabolic labeling with [
35
S]methionine (35–100
␮
Ci/ml; PerkinElmer
Life and Analytical Sciences, Boston, MA), pulse-chase analysis, and immu-
noprecipitations using Cx43 antibodies (C6219; Sigma-Aldrich) were per-
formed as described previously (VanSlyke and Musil, 2000). Immunoprecipi-
tated Cx43 and Cx43-GFP were resolved by 10% SDS-PAGE. Dried gels were
exposed to phosphor screens;
35
S-labeled connexins were detected using a
Storm PhosphorImager and quantified using ImageQuant (Molecular Dy-
namics, Sunnyvale, CA). Curve fitting of pulse-chase data were performed
with IGOR Pro (Wavemetrics, Lake Oswego, OR).
Pull-Down Assays
Glutathione S-transferase (GST)-fusion proteins composed of either the PDZ1
(amino acids 1–106) or PDZ2 (amino acids 173–261) domain of human ZO-1
were generated as described previously (Nielsen et al., 2002) and isolated from
isopropyl
␤
-d-thiogalactoside-induced DH5
␣
bacteria according to standard
procedures.
HeLa Cx43 cells were lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2
mM EGTA, 1% NP-40, 0.25% Na-deoxycholate, 100
␮
M phenylmethylsulfonyl
fluoride (PMSF), and 1⫻ Complete protease inhibitors (Roche Diagnostics,
Indianapolis, IN) for 30 min at 4°C and then centrifuged at 16,000 ⫻ g for 10
min at 4°C. Fusion proteins (2–3
␮
g) coupled to glutathione-Sepharose 4B
beads (GE healthcare, Little Chalfont, Buckinghamshire, United Kingdom)
were added to clarified lysates containing peptides (100
␮
M). Reactions were
incubated sequentially with GST-PDZ1 and GST-PDZ2 beads, each for1hat
4°C. Pelleted beads were washed three times with lysis buffer. Pelleted
material was resolved by 10% (Cx43) and 10 –20% (peptides) SDS-PAGE.
Western blot detection was performed with streptavidin-horseradish perox-
idase (HRP) (GE Healthcare) and Cx43 antibodies (C6219; Sigma-Aldrich).
Triton X-100 Fractionation Assay
Peptide-treated HeLa Cx43 cells were lysed in 1% Triton X-100 in phosphate-
buffered saline (PBS) plus 100
␮
M PMSF and 1⫻ Complete protease inhibitors
for1honicewith occasional vortexing and then centrifuged at 10,000 ⫻ g for
5 min at 4°C. Triton-soluble fractions (supernatants) were removed, and
Triton-insoluble fractions (pellets) were resuspended in an equal volume of
lysis buffer. Equal volumes of Triton-soluble and -insoluble fractions were
resolved by 10% SDS-PAGE and immunoblotted with Cx43 antibody (71-
0700; Zymed Laboratories). Signal was detected by alkaline phosphatase-
based chemiluminescence (CDP-Star; Tropix, Bedford, MA) exposed to Hy-
perfilm ECL (GE Healthcare). Blots were digitized using a UMAX PowerLook
scanner and VueScan (Hamrick Software, Phoenix, AZ). Quantitative densi-
tometry was performed with ImageJ (National Institutes of Health; http://
rsb.info.nih.gov/ij/); soluble/insoluble ratios were calculated from area-un-
derpeak measurements; insoluble Cx43 isoform ratios were calculated from
peak height measurements.
Fluorescence Labeling
Cells grown on coverglass were fixed in 2% paraformaldehyde in PBS for 10
min, blocked with 1% bovine serum albumin, 0.1% Triton X-100 in PBS, and
immunolabeled with Cx43 (610062; BD Transduction Laboratories, Lexington,
KY; MAB3067; Chemicon International, Temecula, CA) and ZO-1 (61-7300;
Zymed Laboratories) antibodies. Signal was detected directly by green fluo-
rescent protein (GFP) fluorescence or by secondary antibodies conjugated
with Alexa488, Alexa546 (Invitrogen, Carlsbad, CA), or Cy5 (Jackson Immu-
noResearch Laboratories, West Grove, PA). Biotinylated peptides were de-
tected using streptavidin-Cy5 (Jackson ImmunoResearch Laboratories). To
delineate plasma membrane, tetramethylrhodamine B isothiocyanate-wheat
germ agglutinin (TRITC-WGA) (Sigma-Aldrich) was added (5
␮
g/ml) to live
HeLa Cx43 cultures for 5 min before fixation. Samples were mounted in
SlowFade (Invitrogen).
Confocal Microscopy
Optical sections were captured with a TCS SP2 AOBS laser scanning confocal
microscope equipped with a 63⫻/1.4 numerical aperture oil immersion ob-
jective (Leica Microsystems, Deerfield, IL). Lasers used were Ar (488 nm), Kr
(568 nm), and HeNe (633 nm). Pinhole (⫽airy disk), gain, and black level
settings were held constant. Potential overlaps in emission spectra were
eliminated by sequential scanning and tuning of the AOBS. For quantitative
analyses of single optical sections, scan format and zoom were adjusted to
give x-y pixel sizes of 68 ⫻ 68 nm for myocyte images and 116 ⫻ 116 nm for
HeLa cell images. Maximum projections were generated from z-series with
x-y-z voxel dimensions of 68 ⫻ 68 ⫻ 400–730 nm for myocyte images and
124 ⫻ 124 ⫻ 124 nm for HeLa cell images.
Image Analysis
GJ lengths, GJ number, total Cx43 area, and individual plaque areas were
measured from single optical sections as described previously (Green et al.,
1993; Hunter et al., 2003). HeLa cell analysis was modified by limiting mea-
surements to Cx43 signal colocalized with TRITC-WGA signal at cell–cell
borders. Parameter means were calculated from image averages, except in the
case of GJ mean lengths and size distributions, which were determined from
the entire sampled populations. Quantification of ZO-1 colocalization with
individual Cx43 plaques was carried out according to Zhu et al. (2005). Linear
regression analysis of Cx43–ZO-1 colocalization was performed in IGOR Pro.
Statistics
Multiple data sets were compared using analysis of variance (p ⬎ 0.05
rejected as not significant) and unpaired, two-tailed Bonferroni t tests (p ⬎
0.05/3 ⬇ 0.017 rejected as not significant). Size distributions were compared
using nonparametric chi-square and Mann-Whitney rank sum tests. Where
ZO-1 Affects Cx43 Gap Junction Dynamics
Vol. 16, December 2005 5687

appropriate, asterisks (*) indicate significant differences between inhibitor and
no peptide treatments; hash marks (#) indicate significant differences between
inhibitor and reverse peptide treatments.
RESULTS
Native Cx43 Rescues the Abnormal Cx43-GFP Gap
Junction Phenotype in HeLa Cells
HeLa cells do not express significant levels of any known
connexin and thus are considered communication deficient.
Previously, we showed that HeLa cells stably expressing a
GFP-tagged Cx43 form sheetlike GJs of abnormally large
size (Hunter et al., 2003); by contrast, stable expression of
untagged native Cx43 resulted in small, discontinuous GJs
uniformly distributed at cell–cell interfaces (Hunter et al.,
2003). From these observations, we concluded that fusion of
GFP to the C terminus of Cx43 disrupts regulatory mecha-
nisms that control GJ size and cellular distribution.
The fact that Cx43-GFP assembles into smaller GJs when
expressed in communication-competent cells (Jordan et al.,
1999; our unpublished data) suggested to us that the pres-
ence of untagged native Cx43 is sufficient to rescue the
abnormal GJ dynamics of Cx43-GFP. Consistent with our
hypothesis, adenoviral expression of native Cx43 in HeLa
Cx43-GFP cells restored GJs to normal size and organiza-
tional patterns (Figure 1). We quantified this effect by sys-
tematically modulating expression of the two Cx43 species
using varying MOIs of Cx43 adenovirus and then measuring
GJ lengths after 24 h of coexpression. As native Cx43 expres-
sion levels were increased in HeLa Cx43-GFP cells, the fre-
quency of smaller GJs also increased. Complete rescue of the
aberrant Cx43-GFP phenotype occurred at MOI 10 (Figure
1A), as assessed by comparison with the GJ length distribu-
tions in HeLa cells stably expressing only native Cx43
(Hunter et al., 2003) or expressing only native Cx43 after
adenoviral infection at MOI 10 (Figure 1A, bottom).
A shift toward smaller Cx43-GFP GJs with increased lev-
els of native Cx43 is reflected in a plot of mean GJ lengths
versus Cx43 adenovirus MOI (Figure 1B). Relative to the
mean length of pure Cx43-GFP GJs, the mean length in HeLa
Cx43-GFP cells coexpressing native Cx43 at MOI 10 was
reduced approximately threefold and was not significantly
different from the mean length in HeLa cells expressing only
native Cx43 at the same MOI (Figure 1B). This convergence
indicates that GJ size variation, and in particular the abnor-
mally large size of Cx43-GFP junctions in the absence of
native Cx43, is not a simple function of increasing Cx43
expression.
GJ Size Control Is Critically Dependent on Availability of
Free Cx43 C Termini within the Connexon Pool
The partial reduction in mean length observed at MOI 5
(Figure 1) indicates that Cx43-GFP GJ size is normalized
gradually after introduction of native Cx43 and suggests
that the actual amount of native Cx43 sufficient to rescue the
Cx43-GFP phenotype likely falls between that expressed by
viral MOIs of 5 and 10. But exactly how much native Cx43 is
Figure 1. Coexpression of native Cx43 in HeLa Cx43-GFP cells
normalizes gap junction size and distribution. (A) Confocal optical
sections of GFP fluorescence in HeLa Cx43-GFP cells coexpressing
increasing levels of native Cx43 (i.e., increasing viral MOI) and
corresponding length distributions generated from measurements
of membrane-localized Cx43-GFP plaques. Bottom, length distribu-
tion of Cx43 GJs after viral expression of native Cx43 at MOI 10 in
wild-type HeLa cells. Bar, 20
␮
m. (B) Mean length of Cx43-GFP GJs
as a function of native Cx43 viral expression (mean ⫾ SE). Coex-
pression of native Cx43 at MOI 10 resulted in a ⬎3-fold reduction in
the mean length of mixed GJs relative to pure Cx43-GFP GJs (MOI
0). Asterisk (*) indicates that the GJ size distribution in HeLa Cx43-
GFP cells expressing native Cx43 at MOI 10 was significantly dif-
ferent (p ⬍ 0.001) from the GJ distribution in HeLa Cx43-GFP cells
expressing native Cx43 at MOI 0 or 5, based on comparisons of GJ
populations (n ⬎ 1000 for all conditions) using a Mann–Whitney
rank sum test. The difference between length distributions in HeLa
Cx43-GFP and wild-type HeLa at MOI 10 was not significant.
A. W. Hunter et al.
Molecular Biology of the Cell5688

produced at these MOIs? More precisely, how many func-
tional Cx43 C termini are sufficient to normalize GJ size and
organizational pattern in HeLa Cx43-GFP cells?
To quantify the amounts of virally expressed native Cx43
relative to stably expressed Cx43-GFP, HeLa Cx43-GFP cells
were metabolically labeled after infection with increasing
amounts of Cx43 adenovirus. After immunoprecipitation,
35
S-labeled connexins were resolved by SDS-PAGE, and the
relative amounts of the two connexin species were measured
by phosphorimaging. At MOI 10—the Cx43 viral expression
level sufficient for complete rescue of Cx43-GFP GJ size—we
found a native Cx43:Cx43-GFP stoichiometry of ⬃5:1 (Figure
2A). This ratio represents a bulk distribution in rescued cells
of ⬃80% native Cx43 versus ⬃20% Cx43-GFP (Figure 2B),
which is consistent with a population of connexons that on
average contain one or fewer Cx43-GFP molecules.
Three lines of evidence argue in favor of random mixing
of native Cx43 and Cx43-GFP into heteromeric connexons.
First, if tagged and untagged Cx43 segregated exclusively
into homomeric connexons, then we might expect to see
regions of Cx43 immunofluorescence devoid of GFP fluores-
cence; instead, Cx43 immunofluorescence and GFP fluores-
cence displayed nearly identical, overlapping patterns in
cells coexpressing native Cx43 and Cx43-GFP (Figure 2C;
Jordan et al., 1999). Second, motifs governing Cx43 oligomer-
ization are located outside of the cytoplasmic C-terminal
domain (Falk et al., 1997; Maza et al., 2005). And third,
C-terminally tagged Cx43 constructs have been shown to
co-oligomerize with each other and with untagged Cx43
(Lauf et al., 2001; Sarma et al., 2002). Based on knowledge of
the relative expression levels of the two Cx43 species (Figure
2, A and B), and assuming random codistribution of con-
nexins within connexons (Figure 2C), a probability equation
(Burt et al., 2001) was used to generate the expected fre-
quency distributions of Cx43:Cx43-GFP connexon stoichio-
metries (Figure 2D). The probabilistic connexon distribu-
tions suggest that when native Cx43 is expressed in HeLa
Cx43-GFP cells at levels both necessary and sufficient for
normalization of GJ organization (i.e., Cx43 viral expression
at MOIs 5–10; Figure 1), the majority of connexons (⬎65%)
must be composed predominately of native Cx43 (ⱖ4 per
connexon); furthermore, nearly all connexons (⬎99%) must
contain at least one native Cx43 (Figure 2D). Clearly, a
channel population comprised of connexons with a single
intact Cx43 C terminus is not sufficient to reestablish GJ size
control. A conservative interpretation is that connexons con-
taining even a single Cx43-GFP cannot contribute effectively
to regulation of GJ size.
C-terminal Tagging of Cx43 Does Not Interfere with Gap
Junction Turnover
Connexins turn over at faster rates (t
1/2
⫽ 1–5 h; Fallon and
Goodenough, 1981; Musil et al., 1990; Laird et al., 1991;
Beardslee et al., 1998) than most membrane proteins (t
1/2
⬎
24 h; Hare and Taylor, 1991). One explanation for the for-
mation of large Cx43-GFP GJs is that they are resistant to
degradation and therefore turnover more slowly than GJs
composed of native Cx43. We tested this possibility using
pulse-chase assays of HeLa cells expressing either native
Cx43 or Cx43-GFP, or both proteins (Figure 3). Short pulse
labeling (40 min) of connexin-expressing cells indicated that
native Cx43 and Cx43-GFP turnover at similar rates, with
half-lives (t
1/2
⫽ 2–3 h) in the range expected for connexins
(Figure 3, A and B).
Previous studies argue against the formation of a meta-
bolically stable pool of Cx43 in GJ plaques (Laird et al., 1995;
Musil et al., 2000). However, if GFP-tagged Cx43 were to
segregate into pools with vastly different turnover kinetics
(e.g., stable junctional versus labile nonjunctional Cx43-
GFP), then short-interval metabolic labeling would not pen-
etrate preexisting, long-lived GJs. We therefore performed
long pulse labeling (ⱖ15 h) to ensure more uniform
35
S-
labeling in the event that the turnover rate of plaque-incor-
porated Cx43-GFP was appreciably slower than that of non-
junctional Cx43-GFP. However, even after protracted
radiolabeling, the kinetics of Cx43-GFP turnover was not
notably different from those of native Cx43 (Figure, 3, C and
D). In fact, when expressed in HeLa cells, the half-life of
Cx43-GFP (t
1/2
⬇ 2 h) seemed to be consistently shorter than
that of native Cx43 (t
1/2
⬇ 3–5 h; Figure 3, A–D)—a result
completely at odds with the hypothesis that the abnormally
large Cx43-GFP GJs arise from resistance to degradation.
When native Cx43 was coexpressed in HeLa Cx43-GFP
cells, the turnover rate of both Cx43 species fell in the range
of rates measured when native Cx43 was expressed alone
(Figure 3E). Thus, the half-life of Cx43-GFP was increased
slightly in the presence of native Cx43 at levels sufficient to
normalize GJ size. The convergence of the half-life of Cx43-
GFP to that of native Cx43 in coexpressing cells provides
further evidence of random coassembly of native Cx43 and
Cx43-GFP into heteromeric connexons. More importantly,
the increase in Cx43-GFP half-life is opposite to what might
have been expected if coexpression of native Cx43 rescued
GJ size in HeLa Cx43-GFP cells by increasing the turnover
rate of Cx43-GFP. These observations confirm that C-termi-
nal tagging of Cx43 does not interfere with GJ turnover and
allow us to draw an important conclusion: the abnormal size
Figure 2. Reduction of Cx43-GFP plaque size by coexpression of
native Cx43 requires a majority of connexons be composed predom-
inately of native Cx43. (A) Representative gel showing changes in
expression levels of Cx43-GFP and native Cx43 in HeLa Cx43-GFP
cells after infection with increasing Cx43 adenovirus MOI. Connexin
stoichiometries (mean ⫾ SE; n ⫽ 3) at each MOI were determined by
measuring band intensities with a PhosphorImager. (B) Connexin
stoichiometries presented as bulk expression levels of Cx43-GFP
and native Cx43 relative to total connexin. (C) Representative sets of
confocal images showing Cx43-GFP and native Cx43 codistribute in
HeLa Cx43-GFP cells coexpressing Cx43 at viral MOI 10. Note the
lack of Cx43 domains devoid of GFP fluorescence. Bar, 10
␮
m. (D)
Distribution of connexon stoichiometries expected when native
Cx43 is coexpressed in HeLa Cx43-GFP at levels necessary (MOI 5)
and sufficient (MOI 10) to normalize GJ size and distribution.
ZO-1 Affects Cx43 Gap Junction Dynamics
Vol. 16, December 2005 5689

of Cx43-GFP GJs cannot be explained by the formation of
degradation-resistant plaques.
A Peptide Inhibitor of Cx43–ZO-1 Interaction Based on
the PDZ-binding Domain of Cx43
Our results with Cx43-GFP suggested that tagging of Cx43
interfered with a regulatory mechanism that controls GJ
size. C-terminal tagging of Cx43, including with GFP, has
been shown by immunoprecipitation to profoundly disrupt
binding of ZO-1 (Giepmans and Moolenaar, 1998; Giepmans
et al., 2001b), suggesting a role for ZO-1 in regulating the size
and organization of Cx43 GJs. However, manipulation of
Cx43 through genetic tagging might affect the binding of
proteins other than ZO-1. Therefore, to directly target ZO-1
activity, we synthesized a peptide comprising the PDZ-
binding domain of Cx43 linked to an antennapedia internal-
ization sequence (Figure 4A).
At concentrations between 10 and 100
␮
M, antennapedia
peptides were detected at relatively uniform levels in cells
up to 24 h after addition to culture media (Figures 4B and 5,
E–F and H–I). Once inside cells, we hypothesized that the
inhibitor peptide should compete for binding to ZO-1,
thereby reducing its availability for interaction with the
PDZ-binding domain of endogenous Cx43. As a control
peptide, we used the same antennapedia sequence but re-
versed the Cx43 PDZ-binding sequence; thus, the reverse
control peptide no longer has a free C-terminal isoleucine
(Figure 4A), a Cx43 residue necessary for interaction with
the second PDZ domain in ZO-1 (Giepmans and Moolenaar,
1998). In accord with our expectations, the inhibitor peptide
but not the reverse peptide was pulled down by a recombi-
nant polypeptide comprising the PDZ2 domain of ZO-1
(Figure 4C). The specificity of this interaction was corrobo-
rated by the observation that the PDZ1 domain of ZO-1 did
not pull down the inhibitor or reverse peptide (Figure 4C).
Having demonstrated specificity of targeting to the PDZ2
domain, we next tested whether the inhibitor peptide dis-
rupted ZO-1 interaction with Cx43. Inhibitor or control pep-
tides were added to lysates from HeLa Cx43 cells and then
either the PDZ1 or PDZ2 domains of ZO-1 were used to pull
down Cx43. Consistent with interference of Cx43–ZO-1
binding, the amount of Cx43 pulled down by PDZ2 in the
presence of inhibitor peptide was significantly reduced rel-
ative to Cx43 pulled down in the presence of control peptide
or no peptide (Figure 4D). As expected, no Cx43 was pulled
down by PDZ1 irrespective of the presence or absence of
peptides. Based on these data, we conclude that the inhibitor
peptide disrupts the interaction between Cx43 and ZO-1.
Disruption of Cx43–ZO-1 Interaction Increases the Size of
Cx43 GJs
To characterize the effects of the inhibitor peptide on GJs,
HeLa Cx43 cells were treated with the inhibitor, reverse, or
no peptide for 72 h. At the end of the culture period, living
cells were first exposed to TRITC-WGA to label cell mem-
branes and then fixed and immunolabeled for Cx43 (Figure
5, A–F). As shown qualitatively in Figure 5, A–C, exposure
to the inhibitor peptide resulted in increased levels of Cx43
at WGA-defined cell membranes relative to controls. In-
creased cell border-localized Cx43 also was observed in
primary cultures of neonatal cardiomyocytes treated with
inhibitor peptide (Figure 5I).
Quantitatively, treatment with inhibitor peptide increased
the average dimensions of individual Cx43 plaques in both
HeLa Cx43 cells and neonatal cardiomyocytes relative to
controls (Figure 6A). This increase in mean GJ size in re-
sponse to inhibitor peptide was highly significant with
larger GJs occurring at greater frequency (at the expense of
smaller GJs) in both cell types (Figure 6B). In addition to
increased GJ size, the total area of membrane-localized
plaques in HeLa Cx43 cells was significantly increased by
exposure to inhibitor peptide relative to no peptide controls
(Figure 6C); and consistent with increased plaque fusion (or
decreased fission) events, HeLa Cx43 cells showed a slight
reduction in the number of membrane-localized GJs in in-
hibitor-treated versus control cells (Figure 6D), although we
could not establish this as a definitive trend.
Figure 3. Native Cx43 and Cx43-GFP turnover at similar rates in
HeLa cells. HeLa cells expressing native Cx43 (A and C) or Cx43-
GFP (B and D) or both connexin species (E) were pulse labeled with
[
35
S]methionine for 40 min (A, B, and E) or ⱖ15 h (C and D),
followed by chase with unlabeled methionine. At the designated
times during the chase period,
35
S-labeled connexins were quanti
-
tatively immunoprecipitated from HeLa cells lysates and resolved
using SDS-PAGE. Band intensities were measured with a Phospho-
rImager. Plots show levels of
35
S-labeled connexins remaining, nor
-
malized to the amount of
35
S-labeled connexin at time zero, as a
function of chase time (mean ⫾ SD; n ⫽ 2). Curves are exponential
fits to the data (weighted by their uncertainties) which yielded the
half-life (t
1/2
)of
35
S-labeled connexins (mean ⫾ SE).
A. W. Hunter et al.
Molecular Biology of the Cell5690

We can rule out the possibility that the observed increases
in plaque size (Figure 6A) and overall levels of immunode-
tectable Cx43 GJs at cell–cell borders (Figure 6C) were due
simply to an increase in Cx43 expression, because the inhib-
itor peptide did not significantly affect the total amount of
Cx43 in HeLa cells or cardiomyocytes (Figure 6E). This
agrees with the recent observation that Cx43 abundance was
unaltered after disruption of Cx43–ZO-1 interaction in os-
teoblastic cells (Laing et al., 2005). Moreover, this accords
with our finding that the size of Cx43-GFP plaques was
reduced after increasing the expression of native Cx43 (Fig-
ures 1 and 2).
Collectively, the data presented in Figure 6 suggest that
the observed increase in GJ plaque size in the presence of the
inhibitor peptide is due mainly to unregulated accretion of
connexons from nonjunctional pools, with a possible contri-
bution from increased plaque fusions.
Peptide Inhibition of Cx43–ZO-1 Interaction Leads to
Redistribution of Cx43 from Nonjunctional Pools to GJ
Plaques
Unaltered levels of total Cx43 in peptide-treated cells (Figure
6E) suggested that, rather than accumulation of newly up-
regulated Cx43, the increased dimensions of individual
Cx43 plaques at cell borders might be due to a redistribution
of preexisting Cx43. We tested this possibility using a bio-
chemical fractionation assay that distinguishes junctional
from nonjunctional connexins. HeLa Cx43 cells were treated
with peptides for 72 h and then lysed in ice-cold buffer
containing 1% Triton X-100. Under these conditions non-
junctional connexins are solubilized, whereas GJ plaques
remain insoluble (Musil and Goodenough, 1991). After lysis,
the two populations of connexins were fractionated by cen-
trifugation and resolved using SDS-PAGE. The Triton-solu-
ble fraction resolves predominately as an indistinguishable
collection of faster-migrating isoforms (NP/P0) that repre-
sent nonjunctional Cx43, which is largely (although not ex-
clusively) unphosphorylated (Figure 7A; Musil and Good-
enough, 1991; Solan et al., 2003). In contrast, the Triton-
insoluble gap junctional fraction resolves as at least three
distinct Cx43 isoforms, NP/P0 and two phosphorylated spe-
cies, P1 and P2 (Figure 7A).
Generally, it is thought that unphosphorylated Cx43 as-
sembles into connexons en route to the plasma membrane
(Musil and Goodenough, 1991; Musil and Goodenough,
1993). Phosphorylation events that result in gel mobility
shifts occur subsequent to delivery to the plasma membrane
and are closely linked to accretion of connexons into Triton-
insoluble GJ plaques (Musil and Goodenough, 1991). The
banding patterns shown in Figure 7A follow the well-estab-
lished relationship between detergent solubility, gel mobil-
ity, and phosphorylation state with respect to the life cycle of
connexins (Musil and Goodenough, 1991). Band intensity
profiles reveal a shift in the relative amounts of the various
Cx43 isoforms after treatment with inhibitor peptide (Figure
7B). Quantitative densitometry showed that the inhibitor
peptide caused a significant reduction in the level of soluble
relative to insoluble Cx43 (Figure 7C), which suggests
that the amount of endogenous Cx43 pulled down from HeLa
lysates by GST-PDZ2 beads was significantly reduced by the pres-
ence of inhibitor peptide. Peptide inhibition of PDZ2-Cx43 interac-
tion was reproducible (n ⫽ 3). Comparison of nonspecific GST
fusion protein band intensities confirms equal loading across con-
ditions.
Figure 4. A peptide inhibitor that binds specifically to the second
PDZ domain of ZO-1 and blocks interaction with Cx43. (A) Domain
organization of full-length Cx43 and antennapedia peptides. (B)
Detection of biotinylated peptide (0–100
␮
M) in HeLa cells after 2 h.
Streptavidin-Cy5 fluorescence intensity shows a dose-dependent
increase with increasing peptide concentration. Cell nuclei are
stained with DAPI. Bar, 15
␮
m. (C) Western blots showing that
inhibitor and reverse peptides (⬃3.5 kDa) both were detected by
streptavidin-HRP (top blot), but only inhibitor peptide was detected
by an antibody that recognizes the C terminus of Cx43 (bottom blot).
Only inhibitor peptide was pulled down by purified ZO-1 GST-
PDZ2 fusion protein-coupled beads; neither peptide was pulled
down by GST-PDZ1 beads. (D) Representative Cx43 blot showing
ZO-1 Affects Cx43 Gap Junction Dynamics
Vol. 16, December 2005 5691

greater incorporation of connexons into GJ plaques. Further-
more, within the insoluble fraction, we detected a significant
increase in the amounts of the P1 and P2 phosphoisoforms
relative to controls (Figure 7D). The increases in Cx43-P1
and -P2 seemed to be linked because the ratio of P2/P1 was
not affected by peptide treatment (Figure 7D). The increase
in Triton-insoluble Cx43, in conjunction with the appearance
of elevated levels of phosphorylated Cx43 species known to
populate mature GJs, supports the idea that the inhibitor
peptide increases GJ size by promoting the accretion of
nonjunctional connexons into gap junctional plaques.
ZO-1 Localization at GJ Peripheries Depends on PDZ-
mediated Interaction with Cx43
In addition to increasing the extent of Cx43 GJs, both C-
terminal tagging of Cx43 and the inhibitor peptide had
Figure 5. Peptide inhibition of Cx43–ZO-1 interaction increases the extent of membrane-localized Cx43 GJ plaques in HeLa cells and
cardiomyocytes. Confocal optical sections of HeLa Cx43 cells (A–F) and neonatal cardiomyocytes (G–I) cultured for 72 or 24 h, respectively,
without peptide (A, D, and G), or with reverse (B, E, and H) or inhibitor peptide (C, F, and I). All cells were immunolabeled for Cx43 (A–I).
Peptides were detected with streptavidin-Cy5 (D–I). WGA-TRITC delineates HeLa cell membranes (A–C). Arrowheads (C and I) denote
accumulation of membrane-localized Cx43 plaques in inhibitor-treated cells. Bars, 20
␮
m (A–F); 80
␮
m (G–I).
A. W. Hunter et al.
Molecular Biology of the Cell5692

effects on the pattern of colocalization between ZO-1 and
Cx43 (Figure 8). Interestingly, ZO-1 was often seen associ-
ated at the peripheries of GJs in HeLa Cx43 cells (Figure 8A).
By contrast, ZO-1 localization in HeLa Cx43-GFP cells
seemed to be independent of GJs (Figure 8B). Although
ZO-1 staining was seen along the edges of some large Cx43-
GFP plaques (Figure 8B, insets), this localization seemed to
be coincidental, with no evidence of actual overlap. How-
ever, when native Cx43 was coexpressed in HeLa Cx43-GFP
cells, colocalization of ZO-1 at plaque peripheries was rees-
tablished (Figure 8C, insets). This suggests that the mecha-
nistic link between restoration of ZO-1 localization at GJ
peripheries and size reduction of aberrantly large Cx43-GFP
GJs is the formation—after introduction of native Cx43—of
heteromeric connexons that are competent to bind ZO-1.
Similar to the HeLa Cx43-GFP phenotype, isolated cardi-
omyocytes treated with the inhibitor peptide produced
larger GJs that seemed to have less colocalized ZO-1 than
GJs in control cells (Figure 8, D–F). Again, most of the ZO-1
that colocalized with Cx43 in cardiomyocytes seemed re-
Figure 6. Quantification of gap junction patterning in HeLa cells
and cardiomyocytes after peptide inhibition of Cx43–ZO-1 interac-
tion. (A) The area of individual Cx43 plaques increased significantly
(*
#
p ⱕ 0.001) after 72 h (HeLa Cx43) and 48 h (cardiomyocytes)
treatments with inhibitor peptide relative to controls (means ⫾ SE;
n ⱖ 30). (B) Size distributions of Cx43 plaques in cells treated with
inhibitor or no peptide. Inhibitor distributions are significantly dif-
ferent (chi-square test yielded *
#
p ⬍ 0.001; n ⱖ 780 for all conditions)
from control distributions (reverse peptide distributions omitted for
clarity). (C) Total area of membrane-localized Cx43 plaques was
increased significantly (*p ⫽ 0.013) by inhibitor peptide (means ⫾
SE; n ⱖ 30). (D) Cells treated with inhibitor peptide displayed a
slight reduction in GJ number (means ⫾ SE; n ⱖ 30). (E) Western
blots of HeLa and cardiomyocyte lysates show that peptide treat-
ment does not alter Cx43 protein expression levels. GAPDH blots
serve as loading controls.
Figure 7. The formation of larger GJ plaques after disruption of
Cx43–ZO-1 interaction results from a redistribution of Cx43. (A)
Representative gel of Triton fractionation assay. (B) Corresponding
plot of band intensities generated by densitometric analysis. Note
the change in peak profiles in the inhibitor condition. (C) Shifts in
subcellular Cx43 distribution are expressed as ratios of fractionated
Cx43 isoforms. Exposure to inhibitor peptide for 72 h significantly
reduced (*p ⫽ 0.0001;
#
p ⫽ 0.015) the level of Triton-soluble relative
to Triton-insoluble Cx43 in HeLa cells. (D) The shift of Cx43 from
soluble to insoluble pools induced by inhibitor peptide was accom-
panied by significant increases in Cx43-P1 (*p ⫽ 0.012;
#
p ⬍ 0.006)
and -P2 (*p ⱕ 0.003;
#
p ⬍ 0.005) phosphoisoforms (relative to
NP/P0) within the insoluble fraction.
ZO-1 Affects Cx43 Gap Junction Dynamics
Vol. 16, December 2005 5693

stricted to the periphery of GJ plaques rather than their
interior. In fact, some plaques displayed near continuous
runs of ZO-1 that circumscribed the perimeter of the GJ;
however, only a fraction of ZO-1 at plaque perimeters
seemed to overlap directly with Cx43 (Figure 8, D–F, arrow-
heads).
Quantitative analysis of confocal images confirmed that
the inhibitor peptide reduced ZO-1 colocalization with Cx43
Figure 8. Disruption of Cx43–ZO-1 interaction reduces the preferential association of ZO-1 with the perimeter of Cx43 GJ plaques. (A–C)
Maximum projection z-series of HeLa cells expressing native Cx43 (A), Cx43-GFP (B), or both GFP-tagged and native Cx43 (C). After 24 h,
cells were fixed and immunolabeled for Cx43 and ZO-1. Note that ZO-1 localizes to the periphery of small, discontinuous native Cx43 GJ
plaques (A, arrowhead) but is excluded from large, sheetlike Cx43-GFP plaques (B). Coexpression of native Cx43 (viral MOI 5) in HeLa
Cx43-GFP cells reduces plaque size and restores ZO-1 colocalization at plaques perimeters (C, arrowhead). (D–F) Maximum projection
z-series of neonatal cardiomyocytes treated for 48 h with the inhibitor (F), reverse (E), or no peptide (D). Note the striking pattern of ZO-1
colocalization with Cx43 plaque edges (D–F, arrowheads), which is reduced in inhibitor-treated cells. Asterisks (*) in top panels (A–F) denote
image areas enlarged in lower panels (the second set of bottom panels in D–F are enlargements from different images). Bars, 10
␮
m (A–C,
top); 5
␮
m (A–C, bottom); 5
␮
m(D–F, top); 2.5
␮
m (D–F, bottom).
A. W. Hunter et al.
Molecular Biology of the Cell5694

GJs. The average area of individual Cx43 plaques colocal-
ized with ZO-1 was decreased significantly in isolated car-
diomyocytes treated for 48 h with the inhibitor (17.5 ⫾ 1.3%)
compared with reverse (23.1 ⫾ 1.6%) and no peptide (25.5 ⫾
2.0%) controls (Figure 9A). Fitting a line to a plot of the area
of Cx43 colocalized with ZO-1 versus GJ size (Figure 9B)
revealed a positive correlation (r ⱖ 0.7 for each condition
when data were weighted by their uncertainties) between GJ
size and the area of colocalized ZO-1 that was independent
of peptide treatment. However, the differential effects of the
peptide treatments were manifest as a significant decrease in
the slope (equal to ZO-1 colocalization as a percentage GJ
area) of the line fitted to the inhibitor data (21.08 ⫾ 0.008%)
relative to the slopes derived from fits to the reverse (28.23 ⫾
0.009%) and no peptide (28.43 ⫾ 0.011%) control data (Fig-
ure 9B). Plaques ⬎3.44
␮
m
2
were excluded from the fitting
process due to a single nonlinear outlier in the no peptide
data at these GJ sizes. Nevertheless, the colocalization values
associated with GJs ⬎3.44
␮
m
2
in cells treated with either
the inhibitor or reverse peptide fell within the 95% confi-
dence interval for the linear fits shown in Figure 9B. Because
the data were weighted by their uncertainties for linear
regression analysis (Figure 9B), mean colocalization values
obtained from linear fits differ slightly from those calculated
by unweighted averaging (Figure 9A).
Linear extrapolation of the data for the different peptide
conditions did not produce significantly different intercepts;
interestingly, however, the intercepts for all three conditions
were significantly different from zero (Inh p ⬍ 0.002, Rev p
⬍0.005, and NP p ⬍ 0.001) and were consistently negative
values, which correspond to positive ordinal intercepts (Fig-
ure 9B). This suggests that, rather than being coassembled
with Cx43, ZO-1 is recruited to GJs after they reach a critical
size. Taking the average of the ordinal intercepts (assuming
that the intercepts from the three conditions are equivalent)
yielded a critical GJ size of 9300 ⫾ 400 nm
2
⬇ 100 channels,
assuming 90-Å center-to-center hexagonal packing.
Together, the similar disruptive effects of the inhibitor
peptide and C-terminal tagging of Cx43 show that localiza-
tion of ZO-1 to GJ plaques is likely dependent on a binding
interaction between the C terminus of Cx43 and the PDZ2
domain of ZO-1.
DISCUSSION
The shape, extent, and distribution of gap junctional plaques
are recognized as important variables in the regulation of
intercellular coupling (Evans and Martin, 2002) and cou-
pling-dependent processes such as action potential spread in
heart and brain (Severs et al., 2004). Here, we provide evi-
dence that reduction in ZO-1 interaction with Cx43 is asso-
ciated with increases in the size of GJ plaques. First, we
demonstrated that disruption of ZO-1 binding by genetic
tagging of the C terminus of Cx43 with GFP leads to the
formation of abnormally large GJs when the tagged con-
struct is expressed in connexin-deficient HeLa cells; the ab-
normal Cx43-GFP phenotype was rescued by coexpression
of significant levels of native Cx43 (⬎80% of the total con-
nexin). Second, we showed that targeted disruption of
Cx43–ZO-1 interaction using a peptide inhibitor based on
the PDZ-binding domain of Cx43 also caused a significant
increase in the mean size of Cx43 GJs in two cell types,
primary neonatal cardiomyocytes and a HeLa cell line ex-
pressing native Cx43. Third, we found that the increase in GJ
size arose from a redistribution of Cx43 from nonjunctional
pools to GJ plaques, with a possible contribution from in-
creased plaque fusion events. And finally, both GFP tagging
and the inhibitor peptide caused a significant reduction in
the amount of ZO-1 associated at the peripheries of GJ
plaques. Together, these data suggest a function for ZO-1 in
regulating the growth of Cx43 GJs by controlling the rate at
which connexons are incorporated into GJ plaques at their
peripheries.
Previously, we demonstrated that ZO-1 shows low-to-
moderate levels of colocalization with Cx43 GJs in adult rat
heart (Barker et al., 2002). The present study confirms and
extends this finding, demonstrating quantitatively in iso-
lated cardiomyocytes that colocalization between ZO-1 and
Cx43 in individual GJ plaques is limited to ⬃28% overlap
(Figure 9B). Likewise, Nielsen et al. (2003) found that the
overall extent of colocalization between ZO-1 and Cx46 or
Cx50 in the eye lens was limited. Duffy et al. (2004) have also
confirmed in cultured astrocytes that colocalization of ZO-1
with Cx43 at cell borders is confined to a partial overlap.
Figure 9. Quantitative image analysis of ZO-1 association with
Cx43 GJs after peptide inhibition. (A) The average level of ZO-1
colocalization within individual plaques Cx43 (means ⫾ SE) was
significantly reduced (*p ⫽ 0.001;
#
p ⫽ 0.006) in inhibitor-treated
cardiomyocytes. (B) Cx43 area colocalized with ZO-1 (means ⫾ SE)
as a function of GJ size in myocytes. GJ sizes are means derived
from binned data. Lines are linear regression fits weighted by
uncertainties in GJ area colocalized with ZO-1; black dotted line is
the fit to the reverse peptide data (omitted for clarity). Slopes
correspond to the fractional area of individual Cx43 plaques colo-
calized with ZO-1, which was significantly reduced (*
#
p ⬍ 0.001) in
inhibitor-treated cells.
ZO-1 Affects Cx43 Gap Junction Dynamics
Vol. 16, December 2005 5695

Overall, the data suggest that in astrocytes, lens and heart a
significant proportion of the connexin pool localized at cell
borders does not associate with ZO-1.
What then is the functional significance of the limited
interaction between ZO-1 and connexins? The high resolu-
tion confocal images presented here provide a detailed per-
spective of the spatial relationship between ZO-1 and Cx43
within GJ plaques. These images reveal that the majority of
ZO-1 does not overlap directly with plaque interiors; rather,
ZO-1 seems to associate preferentially with the edges of GJs.
EM data from Li et al. (2004) also support the idea that ZO-1
associates preferentially with the perimeter of GJ plaques.
That the perimeter is a specialized microdomain of GJ
plaques was established by studies that demonstrated that
addition of C-terminal-tagged Cx43 occurred at the periph-
ery of GJ plaques (Gaietta et al., 2002; Lauf et al., 2002).
Although there seems to be a general spatial correlation
between ZO-1 and sites of connexin recruitment into GJs, it
is unlikely that ZO-1 enhances GJ assembly because C-
terminal-truncated forms of Cx43 lacking the ZO-1 binding
domain are able to form functional GJs (Fishman et al., 1991;
Liu et al., 1993). More specifically, mutations targeting the
PDZ-binding domain of Cx43 and other connexins known to
interact with ZO-1 suggest that ZO-1 is dispensable for the
formation or ongoing aggregation of connexons into func-
tional GJs (Toyofuku et al., 2001; Nielsen et al., 2003; Li et al.,
2004). Likewise, fusion of epitope tags or GFP to the C
terminus of Cx43, which results in loss of ZO-1 binding
(Giepmans and Moolenaar, 1998; Giepmans et al., 2001b),
does not impede the formation of GJ plaques (Jordan et al.,
1999; Bukauskas et al., 2000; Gaietta et al., 2002; Lauf et al.,
2002; Hunter et al., 2003). Furthermore, as we show here,
reduction of Cx43–ZO-1 interaction increases rather than
decreases the size of Cx43 plaques. Collectively, these data
indicate that a role for ZO-1 in promoting the formation and
growth of GJ plaques, although not ruled out, is unlikely.
Based on increased Cx43–ZO-1 colocalization after enzy-
matically or chemically induced GJ internalization, we and
others have postulated that reduction of ZO-1 binding to
Cx43 inhibits GJ internalization via formation of so-called
annular GJs. (Barker et al., 2002; Segretain et al., 2004). How-
ever, our analysis of Cx43-GFP turnover in HeLa cells ar-
gues strongly against an explanation for the redistribution of
Cx43 and plaque expansion resulting from peptide inhibi-
tion of Cx43–ZO-1 interaction based solely on suppression
of GJ internalization. Cx43-GFP, a connexin incompetent to
interact with ZO-1, was found to turnover at a rate nearly
identical to that of native Cx43 (Figure 3). Thus, GJs com-
prised exclusively of Cx43-GFP, despite being abnormally
large, continue to turnover with a half-life (t
1/2
⬇ 2h)
consistent with turnover rates in vivo (Fallon and Good-
enough, 1981; Beardslee et al., 1998) and with other systems
expressing tagged Cx43 (Gaietta et al., 2002; Segretain and
Falk, 2004). Moreover, cells expressing Cx43-GFP generate
annular GJs (Laird et al., 2001; Segretain and Falk, 2004), a
finding we have confirmed using live imaging of HeLa
Cx43-GFP cells (our unpublished data). And although Duffy
et al. (2004) confirmed coassociation between Cx43 and ZO-1
during enzymatically induced Cx43 internalization in astro-
cytes, pH-induced internalization of Cx43 was preceded by
a loss of Cx43–ZO-1 interaction. Therefore, increased Cx43–
ZO-1 colocalization after certain membrane internalization
events remains an unresolved issue, but it seems to be
independent of normal Cx43 turnover. Finally, if ZO-1 were
to play a prominent role in GJ internalization we might
expect to see ZO-1 localize preferentially to the center of
GJs—the major site of plaque removal—rather than the GJ
periphery—the major site of plaque assembly. Together,
these findings provide strong evidence against the direct
involvement of ZO-1 in the machinery of GJ internalization
and/or degradation.
Disruption of Cx43–ZO-1 interaction does seem to be as-
sociated with unconstrained growth of Cx43 channel aggre-
gates. We propose that ZO-1 actively constrains GJ size by
regulating the rate of channel accretion. Qualitatively, the
preferential localization at the perimeter of GJ plaques
places ZO-1 in a prime location—the site of plaque assem-
bly—to play a role in control of connexon incorporation into
GJs. But the most compelling evidence for ZO-1 control of
connexon accretion comes from observations that the inhib-
itor peptide caused a quantitative reduction in the amount
of ZO-1 colocalized within individual plaques, concomitant
with quantitative increases in Triton-insoluble Cx43 (at the
expense of the Triton-soluble pool) and plaque size. We
think these events are linked causally, which makes sense.
Even a slight reduction in ZO-1 binding at plaque perime-
ters might allow connexon accretion to proceed at a faster
rate. If the turnover rate remained constant, then an increase
in the rate of channel aggregation would be expected over
time to result in larger GJ plaques. Likewise, if the rate of
connexon accretion increased but connexin expression re-
mained constant, then we would expect to see an increase in
junctional (i.e., Triton-insoluble) Cx43 relative to nonjunc-
tional (i.e., Triton-soluble) Cx43. Our combined data sets fit
both the conditions and the expectations of the above-men-
tioned suppositions. The appearance of increased levels of
Triton-insoluble Cx43-P1 and -P2 isoforms, both of which
are constituents of mature GJs (Musil and Goodenough,
1991), in inhibitor-treated cells lends further support to our
proposal that ZO-1 plays a role in connexon accretion.
Importantly, although we think ZO-1 controls the rate of
connexon accretion and therefore exerts a level of control
over plaque expansion, ZO-1 does not sense GJ size. We
infer this from the linear relationship between GJ size and
the level of ZO-1 associated with individual plaques: ZO-1
continues to associate with larger GJs at levels equivalent (in
terms of percentage of plaque coverage) to that of smaller
GJs (Figure 8H). This suggests that subsequent to plaque
nucleation the mechanism of ZO-1 recruitment to GJs oper-
ates irrespective of plaque size. Thus, ZO-1 does not seem to
cap GJ size in absolute terms. Instead, the average GJ size at
steady state is set by a combination of the rates of connexon
accretion and GJ turnover.
Our conclusion that ZO-1 constrains the growth of GJs is
seemingly at odds with the findings of Laing et al. (2005),
who reported reduced appositional Cx43 immunostaining
and decreased GJ-mediated dye transfer after overexpres-
sion of a dominant-negative ZO-1 mutant in osteoblastic
cells. It is noteworthy that the truncated mutant used by
Laing et al. (2005) included N-terminal regions of ZO-1 that
extend beyond the PDZ2 domain, including the entire PDZ1
domain. The N-terminal half of ZO-1 is known to be a
functional component in cadherin-based cell adhesion (Itoh
et al., 1997). In fact, a truncated ZO-1 mutant similar to that
of Laing et al. (2005) was shown to disrupt cadherin-based
adherens junctions (AJs; Ryeom et al., 2000). Because AJs are
prerequisite for the formation and maintenance of Cx43 GJs
(Meyer et al., 1992; Hertig et al., 1996; Wei et al., 2005), the
effects of the Laing et al. (2005) mutant could be interpreted
as secondary due to decreased GJ stability, rather than
caused directly by loss of ZO-1 binding to Cx43.
Left unanswered is the question of how ZO-1 arrives at
plaque perimeters. Much of the ZO-1 that surrounds GJs
does not colocalize with Cx43. Presumably, ZO-1 not asso-
A. W. Hunter et al.
Molecular Biology of the Cell5696

ciated directly with Cx43 at plaque edges is complexed with
another cell junction type, the obvious candidate being the
AJ. The recent discovery that Cx43 coassembles with N-
cadherin (Wei et al., 2005) raises the possibility that AJs
nucleate GJs, with ZO-1 recruited first to AJs then passed
along to Cx43 once the GJ achieves a critical density of
channels, as suggested by our colocalization analysis (Figure
9B). Thus, it is unsurprising that overexpression of ZO-1 can
lead to enhanced GJ-mediated dye transfer (Laing et al.,
2005) because ZO-1 might promote the formation of AJs,
which in turn serve as GJ nucleation sites. The important
point is that ZO-1 does not inhibit the formation of GJs, we
think it merely constrains the rate of GJ growth after they
form.
The recent finding that ZO-2 also binds to the C terminus
of Cx43 (Singh et al., 2005) presents the intriguing prospect
that ZO-1 and ZO-2 compete for influence over Cx43 GJ
patterning. Because both ZO proteins interact with the PDZ-
binding motif in Cx43, the effects of our genetic tagging and
peptide inhibition on GJ size control might be influenced by
alterations in the activity of ZO-2 as well as ZO-1. At
present, we cannot exclude this possibility, but given that
Cx43 interacts predominately with ZO-1 in quiescent cells
(Singh et al., 2005), the effects of peptide inhibition in post-
mitotic cardiomyocytes reported here are likely due to the
specific disruption of ZO-1 activity. Nevertheless, distin-
guishing the activities of ZO-1 and ZO-2 with respect to
Cx43 GJ dynamics poses a formidable challenge, especially
considering the potential for functional redundancy and
compensation (Umeda et al., 2004).
Finally, control of Cx43 channel accretion has important
consequences for the regulation of GJ-mediated cell–cell
communication versus cell communication with the extra-
cellular environment via hemichannels (Goodenough and
Paul, 2003). Channel clustering is thought to lead to activa-
tion of individual intercellular channels within GJ plaques
(Bukauskas et al., 2000). A natural assumption is that by
controlling the rate of channel aggregation, ZO-1 directly
influences the level of cell–cell communication. But by lim-
iting connexon incorporation into GJ plaques, ZO-1 might
also effectively maintain a pool of nonjunctional Cx43
hemichannels that remain free to communicate with the
extracellular space. Changes in the rate of connexon accre-
tion by modulating ZO-1 action at plaque perimeters might
then be expected to provide a control point for dynamic
switching between junctional and nonjunctional communi-
cation mediated by Cx43 channels. In conjunction with fast
turnover rates, this type of control offers the possibility of
shifting the balance from one type of communication to the
other on relatively rapid time scales, thus allowing cells to
respond quickly to changes in their environment.
ACKNOWLEDGMENTS
We thank Dr. Ben Giepmans for ZO-1 GST-PDZ fusion proteins, assistance
with turnover assays, and helpful discussion; Jane Jourdan and Dr. Yuhua
Zhang for technical assistance; Dr. Stewart Denslow for advice on statistics;
Dr. Klaus Willecke for HeLa Cx43 cells; and Dr. Dale Laird for the Cx43-GFP
construct. This work was supported by National Institutes of Health Grants
HL07260 (to A.W.H.) and HL56728, HL36059, and HD39946 (to R.G.G.).
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