Báo cáo khoa học: Tyrosine-dependent basolateral targeting of human connexin43–eYFP in Madin–Darby canine kidney cells can be disrupted by the oculodentodigital dysplasia mutation L90V ppt
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Tyrosine-dependentbasolateraltargetingof human
connexin43–eYFP inMadin–Darbycaninekidney cells
can bedisruptedbytheoculodentodigital dysplasia
mutation L90V
Jana Chtchetinin
1,2
, Wes D. Gifford
1,2
, Sichen Li
1,2
, William A. Paznekas
3
, Ethylin Wang Jabs
3,4
and Albert Lai
1,2
1 Department of Neurology, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
2 Henry E. Singleton Brain Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
3 Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA
4 Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY, USA
Introduction
Connexin 43 (Cx43) is a ubiquitously expressed gap
junctional subunit that mediates intercellular commu-
nication via the formation of gap junctions and hemi-
channels [1]. In addition, Cx43 may promote normal
cellular migration and development by enabling inter-
cellular adhesion [2]. Cx43 is expressed in many polar-
ized cell types, such as brain endothelial cells, thyroid
epithelial cells, and cholangiocytes [3–6]. Cx43 is also
highly expressed in astrocytes, a cell type that exhibits
a polarized phenotype and participates in polarized
Keywords
basolateral; connexin43; oculodentodigital
dysplasia; tyrosine
Correspondence
A. Lai, 710 Westwood Plaza, Suite 1-230,
Reed Neurological Research Center,
Los Angeles, CA 90095, USA
Fax: +1 310 825 0644
Tel: +1 310 825 5321
E-mail: albertlai@mednet.ucla.edu
(Received 10 February 2009, revised 16
September 2009, accepted 25 September
2009)
doi:10.1111/j.1742-4658.2009.07407.x
Polarized membrane sorting of connexin 43 (Cx43) has not been well-char-
acterized. Based on the presence of a putative sorting signal, YKLV(286–
289), within its C-terminal cytoplasmic domain, we hypothesized that Cx43
is selectively expressed on thebasolateral surface ofMadin–Darby canine
kidney (MDCK) cellsin a tyrosine-dependent manner. We generated stable
MDCK cell lines expressing human wild-type and mutant Cx43–eYFP, and
analyzed the membrane localization of Cx43–eYFP within polarized mono-
layers using confocal microscopy and selective surface biotinylation. We
found that wild-type Cx43–eYFP was selectively targeted to the basolateral
membrane domain of MDCK cells. Substitution of alanine for Y286 dis-
rupted basolateraltargetingof Cx43–eYFP. Additionally, substitution of a
sequence containing the transferrin receptor internalization signal,
LSYTRF, for PGYKLV(284–289) also disruptedbasolateral targeting.
Taken together, these results indicate that Y286 in its native amino acid
sequence is necessary for targeting Cx43–eYFP to thebasolateral mem-
brane domain of MDCK cells. To determine whether the F52dup or L90V
oculodentodigital dysplasia -associated mutations could affect polarized
sorting of Cx43–eYFP, we analyzed the expression of these Cx43–eYFP
mutant constructs and found that theL90Vmutationdisrupted basolateral
expression. These findings raise the possibility that some oculodentodigitial
dysplasia-associated mutations contribute to disease by altering polarized
targeting of Cx43.
Abbreviations
Cx43, connexin 43; eYFP, enhanced yellow fluorescent protein; MDCK, Madin–Darbycanine kidney; ODDD, oculodentodigital dysplasia;
DPBS, dulbecco’s phosphate buffered saline.
6992 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS
functions [7–10]. So far, there have been limited studies
examining the expression of Cx43 in polarized cells,
and there is little information regarding characteriza-
tion ofthe involved sorting signals. Previous studies
have demonstrated basolateral expression of Cx43 and
other connexins in various polarized cell types [5,11–
13]. The trafficking of a Cx43–GFP chimera expressed
in Madin–Darbycaninekidney (MDCK) cells has
previously been examined, but not inthe context of
polarized monolayers [14].
MDCK cells are a well-characterized model system
for the study of polarized trafficking to distinct apical
and basolateral domains that are separated by tight
junctions [15–17]. In MDCK cells, basolaterally
expressed membrane proteins often depend on a tyro-
sine or dileucine-based sorting signal located in the
cytoplasmic tail. A common tyrosine-based consensus
sorting motif is YXXø (where Y is tyrosine, X is any
amino acid, and Ø is an amino acid with a bulky hydro-
phobic side chain) [18]. These polarized sorting signals
are recognized in other cell types as well [19–21]. For
example, the vesicular stomatitis virus glycoprotein
(VSV-G) protein, which is targeted to the basolateral
membrane domain in MDCK cells, has a tyrosine-based
signal directing it to the somatodendrite versus the axon
in neurons and the myelin sheath versus the soma in
oligodendrocytes [22,23]. The existence of such a signal
in the C-terminus of Cx43 led us to hypothesize that
Y286 is involved inbasolateraltargetingof Cx43.
At least 60 mutations in Cx43 have been discovered
that cause oculodentodigitaldysplasia (ODDD), a rare
human developmental disorder characterized by defects
in the craniofacial bones, loss of tooth enamel, and
abnormal soft tissue separation of two or three digits
[24,25]. Depending on the particular Cx43 mutation, a
wide range of other abnormalities, including neurologi-
cal and cardiac abnormalities, are observed [26–37]. As
yet, there is no clear understanding ofthe relationship
between genotype and phenotype, although functional
evaluations of most ofthe mutations that have been
studied have demonstrated reduced gap junction activ-
ity [38–43]. Several ODDD mutations have been found
to cause altered trafficking of Cx43. For example, the
C260fsX306 mutation (leading to truncation of most
of the Cx43 cytoplasmic tail) and the G60S mutation
have been found to cause impaired cell membrane
expression of Cx43 and hence impaired intercellular
communication [44,45]. Interestingly, the G138R and
G143S ODDD mutations have been found to cause
enhanced hemichannel function with absent gap junc-
tional signaling in HeLa cells, and these mutations
were associated with decreased Cx43 degradation [46].
These studies provide evidence that abnormal mem-
brane protein trafficking may be responsible for the
altered function of Cx43 associated with disease.
In this study, we determined that a fusion between
Cx43 and enhanced yellow fluorescent protein (Cx43–
eYFP) is targeted to thebasolateral membrane domain
of MDCK cells. We also found that Y286 in the
sequence PGYKLV(284–289) is necessary for basolat-
eral targetingof Cx43–eYFP. In addition, we found
that theL90V ODDD mutation disrupts the selective
delivery of Cx43–eYFP to thebasolateral membrane
domain. These results imply that aberrant polarized
sorting of Cx43 may be associated with particular
ODDD phenotypes.
Results
Stable expression of wild-type and mutant
Cx43–eYFP constructs in MDCK cells
To examine the localization ofhuman Cx43–eYFP
constructs in MDCK cells, we stably expressed eYFP-
tagged wild-type (WT) and mutant Cx43 cDNA fusion
constructs in MDCK (strain II) cells. As indicated in
Fig. 1, eYFP-tagged Cx43 constructs comprise the Cx43
sequence joined at its C-terminus to eYFP by an eight-
amino-acid ‘linker’ segment (SRDPPVAT). The loca-
tions ofthe four mutations analyzed (Y286A, LSYTRF,
L90V and F52dup) are also indicated. We confirmed by
western blot analysis that Cx43–eYFP constructs were
properly translated in MDCK cells (Fig. 2A). Using a
Cx43 antibody, a prominent band migrating at approxi-
mately 70 kDa, the predicted molecular weight of full-
length Cx43–eYFP, was detected in total and surface
lysates for WT and each mutant cell line, but not in
uninfected control cells. Similarly, using the polyclonal
GFP antibody, a prominent band migrating at approxi-
mately 70 kDa was detected in total and surface lysates
for WT and each mutant cell line, but not in uninfected
control cells. For reference, a band at approximately
29 kDa was detected inthe total cell lysate of cells
expressing only eGFP. No GFP expression was detected
after surface biotinylation ofcells expressing only
eGFP, demonstrating the selectivity of surface biotiny-
lation for cell surface proteins. As the amounts of pro-
tein loaded for surface and total Cx43–eYFP western
blots were not normalized to each other, no conclusions
can be drawn from these experiments regarding the
relative level of surface expression ofthe constructs.
Confocal images inthe XY plane show that all Cx43–
eYFP constructs are predominantly expressed at the cell
membrane. Gap junction aggregates, or plaques, can
also be seen at areas of cell–cell contact (arrow) for all
constructs except the F52dup mutant (Fig. 2B).
J. Chtchetinin et al. Basolateral sorting of Cx43 in MDCK cells
FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 6993
WT Cx43–eYFP is selectively expressed on the
basolateral domain of MDCK cells
To determine the steady-state polarized membrane
distribution of WT Cx43–eYFP in MDCK cells,
polarized monolayers expressing WT Cx43–eYFP were
cultured on filter inserts and examined using confocal
microscopy. Reconstructed Z sections (XZ plane) show
that WT Cx43–eYFP is selectively expressed on the
basolateral surface of MDCK cells (Fig. 3A–C; three
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SRDPPVAT
eYFP
NH
2
L
Extracellular
L90V
F52dup
Y286A
Lipid bilayer
Cytoplasm
LSYTRF (284–289)
Linker
Fig. 1. Schematic diagram ofthe Cx43–
eYFP amino acid sequence indicating the
predicted topology of Cx43 and the position
of eYFP and its fusion to the C-terminus via
an eight amino acid linker (SRDPPVAT).
Cx43 is predicted to span the plasma
membrane four times, and has cytoplasmi-
cally located N- and C-termini. The locations
of the amino acid mutations examined in
this study are indicated. F52dup is located
in the extracellular domain, L90V is located
in the second transmembrane domain, and
Y286A and LSYTRF are located inthe cyto-
plasmic tail. This figure has been modified
from one that has been published previously
[43].
Uninfected
eGFP
Wild-type
Y286A
LSYTRF
F52dup
L90V
72 -
56 -
34 -
26 -
Total
Surface
Anti-Cx43 Anti-GFP
43 -
Cx43-
eYFP
eGFP
Cx43-
eYFP
10 µm
Wild-type
Y286A
LSYTRF
10 µm
10 µm
10 µm
10 µm
F52dup
L90V
Uninfected
10 µm
Uninfected
eGFP
Wild-type
Y286A
LSYTRF
F52dup
L90V
72 -
56 -
34 -
26 -
43 -
A
B
Fig. 2. Expression of Cx43–eYFP constructs
in MDCK cells. (A) Surface and total protein
were isolated by biotinylation and analyzed
by western blot. Using Cx43 and GFP
antibodies to confirm proper translation of
Cx43–eYFP constructs, bands of approxi-
mately 70 kDa representing the full-length
fusion proteins were obtained from cells
expressing WT and mutant Cx43–eYFP.
(B) Confocal XY images showing the XY
plane from above the apical surface of cell
monolayers. Uninfected control MDCK cells
showed minimal background fluorescence.
WT and mutant Cx43–eYFP constructs were
expressed on the cell membrane. Gap
junction plaques (indicated bythe arrow in
the WT image) formed at points of cell–cell
contact in all mutants with the same
frequency and morphology as WT except
for F52dup, which formed plaques much
less frequently. Scale bar = 10 lm.
Basolateral sorting of Cx43 in MDCK cells J. Chtchetinin et al.
6994 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS
individual cell lines are shown). For comparison, lim-
ited signal was detected in uninfected control cells
(Fig. 3D), and cells infected with eGFP only showed a
diffuse cytoplasmic signal but no specific surface locali-
zation (Fig. 3E). Localization of plaques inthe XZ
plane is shown in Fig. 7 (see below).
To confirm these results, we performed selective sur-
face biotinylation on the apical and basolateral surfaces
and western blot analysis using the Cx43 antibody in
order to visualize the apical ⁄ basolateral distribution of
Cx43–eYFP. This analysis confirmed that the majority
of the signal is found on thebasolateral surface of cell
lines expressing WT Cx43–eYFP, although there
appeared to be a small amount of apically expressed
WT Cx43–eYFP (Fig. 6A). Five independent WT
clones were tested, all yielding similar results. The
results for three are shown. No signal could be detected
for the apical or basolateral surfaces of uninfected con-
trol cells at either 43 or approximately 70 kDa, and,
similarly, no signal could be detected at approximately
29 kDa for the apical or basolateral surface of cells
expressing only eGFP (Fig. 6B). Additionally, quantita-
tive analysis ofthe distribution between the two
domains using a fluorescence plate reader revealed that
86% ofthe surface WT Cx43–eYFP was located on the
basolateral surface (Fig. 6G).
As described in Experimental procedures, all experi-
ments were performed after sodium butyrate pre-incu-
bation to increase expression ofthe Cx43–eYFP
constructs. We confirmed that sodium butyrate treat-
ment did not affect results by performing confocal
microscopy inthe absence of sodium butyrate, dye
transfer experiments with and without butyrate, and
transepithelial resistance measurements with and
without butyrate on a selected cell line (Fig. S1).
Y286 inthe context of its native sequence
PGYKLV(284–289) is necessary for selective
basolateral expression of Cx43–eYFP in MDCK
cells
Y286 is contained within a putative tyrosine-based
sorting signal, YKLV(286–289), and within a PPXY
motif, PPGY(283–286), which is a ubiquitin ligase
(NEDD4) binding site that is involved in internaliza-
tion and degradation of Cx43 [47]. To determine
whether Y286 is involved inthebasolateral targeting
of WT Cx43–eYFP, we substituted an alanine for
Y286 ofthe Cx43–eYFP sequence and examined the
surface distribution ofthe resulting Y286A mutant
construct in polarized MDCK cell monolayers. Confo-
cal analysis of XZ sections revealed that the Y286A
mutation causes Cx43–eYFP to be expressed predomi-
nantly on the apical surface, indicating that Y286 is
necessary for the proper basolateral distribution of
Cx43–eYFP in MDCK cells (Fig. 4A,B). Western blot
analysis of apical/basolateral surface biotinylation frac-
tions confirmed this predominantly apical distribution
10 µm
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A C
B
DE
Fig. 3. WT Cx43–eYFP is selectively targeted to thebasolateral domain of MDCK cells. (A–C) Three independent MDCK clones expressing
WT Cx43–eYFP were cultured on filter inserts. Z sections of cell monolayers are shown inthe top panels. Lines drawn through the XY plane
in the bottom panels indicate the location ofthe Z sections. WT Cx43–eYFP was expressed on thebasolateral membrane domain of MDCK
cells. (D) Z section of a monolayer of uninfected MDCK cells showing background fluorescence. (E) Z section of a monolayer of MDCK cells
expressing only eGFP showing a diffuse cytoplasmic pattern. Scale bar = 10 lm.
J. Chtchetinin et al. Basolateral sorting of Cx43 in MDCK cells
FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 6995
(Fig. 6C), and quantitative analysis showed that 78%
of the surface signal for this mutant construct was
located on the apical surface (Fig. 6G). Data from two
individual cell lines are shown.
To determine the effect of substitution of a
sequence containing the internalization signal of the
transferrin receptor in place of PGYKLV(284–289),
we expressed a mutant construct in which
PGYKLV(284–289) was replaced by LSYTRF [48].
Confocal Z sections of MDCK cells expressing the
LSYTRF mutation showed that the Cx43–eYFP sig-
nal was apparently equally distributed on the apical
and basolateral surfaces (Fig. 4C,D). Western blot
analysis of selective surface biotinylation fractions
confirmed that cells expressing the LSYTRF mutant
construct express Cx43–eYFP at similar levels on the
apical and basolateral surfaces (Fig. 6E). Interest-
ingly, quantification ofthe surface protein using a
fluorescence plate reader indicated that, similar to
Y286A, 74% ofthe LSYTRF surface signal resides
on the apical surface (Fig. 6G). As Y286 is intact in
this mutant construct but its surrounding sequence
is altered, these findings strongly suggest that the
context in which Y286 exists is important for main-
tenance ofbasolateral targeting.
The L90V but not the F52dup ODDD mutation
disrupts basolateral sorting of Cx43–eYFP in
MDCK cells
To determine whether ODDD-associated mutations
affect thebasolateraltargetingof Cx43–eYFP, we
examined the localization of F52dup and L90V
mutant Cx43–eYFP constructs in polarized MDCK
cell monolayers. These mutants were selected for
analysis based on our previous study of ODDD
mutants in C6 rat glioma cells [43]. We chose the
F52dup mutant because it failed to form gap junction
plaques and theL90V mutant because it appeared to
have an increased amount of plaques inthe glial cell
processes compared to WT (unpublished observation).
Confocal Z sections ofcells expressing the F52dup
mutant construct showed predominantly basolateral
expression, indicating that this mutation does not
alter polarized targetingof Cx43–eYFP (Fig. 5A,B).
These results were confirmed by western blot analysis
of the selective surface biotinylation fractions, with
nearly all signal (97%) found on the basolateral
surface (Fig. 6D,G). In contrast, confocal analysis of
the distribution oftheL90V mutant construct in
polarized monolayers revealed that this mutation
causes Cx43–eYFP to be distributed on both the api-
cal and basolateral surfaces (Fig. 5C,D). Western blot
analysis of selective surface biotinylation fractions
confirmed that surface expression oftheL90V mutant
construct is not restricted to thebasolateral surface,
with 57% ofthe signal being found on the apical sur-
face (Fig. 6F,G). The L90V-1 clone appeared to be
exclusively expressed on the apical surface (Fig. 6F).
The results for two independent clones for each
mutant construct are shown.
Gap junction plaques reside predominantly on
the lateral surface ofcells expressing WT and
apically distributed mutant constructs
With the exception ofthe F52dup mutant, all mutant
constructs formed plaques with the same frequency
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CD
Fig. 4. Y286 in its native context is neces-
sary for selective basolateral expression of
Cx43–eYFP in MDCK cells. (A,B) Z sections
of MDCK monolayers expressing the Y286A
mutant construct showing predominantly
apical distribution of Cx43–eYFP (two inde-
pendent clones). (C,D) Z sections of
MDCK monolayers expressing the LSYTRF
mutant construct showing signal on both
the apical and basolateral membranes (two
independent clones). Scale bar = 10 lm.
Basolateral sorting of Cx43 in MDCK cells J. Chtchetinin et al.
6996 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS
10 µm
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CD
Fig. 5. L90V but not the F52dup ODDD
mutation disrupts basolateraltargeting of
Cx43–eYFP in MDCK cells. (A,B) Z sections
of MDCK monolayers expressing the
F52dup mutant construct showing that
this mutation does not affect basolateral
targeting of Cx43–eYFP (two independent
clones). (C,D) Z sections of MDCK monolay-
ers expressing theL90V mutant construct
showing that basolateraltargeting of
Cx43–eYFP was disrupted (two independent
clones). Scale bar = 10 lm.
A
WT-1 WT-2 WT-3
UninfectedUninfected
eGFP
Y286A-1 Y286A-2
LSYTRF-1 LSYTRF-2
F52dup-1 F52dup-2
72 -72 -
72 -
72 -
72 -
72 - 29 -43 -
L90V-2L90V-1
abab
B
C
abab ab ab
ab
ab ab
ab
D
ab ab
ab ab
F
E
G
WT Y286A LSYTRF F52dup L90V
Apical
14%
Basolateral 86%
78%
74%
26%
97%
57%
43%
3%
22%
20%
40%
60%
80%
100%
WT Y286A LSYTRF F52dup L90V
Surface expression (%)
Cell line
Apical
Basolateral
*
*
*
0%
Fig. 6. Apical ⁄ basolateral cell surface
distribution of Cx43–eYFP. Selective surface
biotinylation followed by western blot
analysis using the Cx43 antibody showed
that WT and the F52dup mutant construct
were expressed predominantly on the
basolateral surface (A,D), but the Y286A,
LSYTRF and L90V mutant constructs were
not exclusively distributed on the basolateral
surface (C,E,F). (B) Uninfected MDCK cells
showed no bands at 43 or 70 kDa. The
absence of bands at approximately 29 kDa
in control cells expressing only eGFP
indicates that non-specific biotinylation of
cytoplasmic protein did not occur. Two or
three individual cell lines are shown for
each construct. (G) Surface expression as
quantified by a fluorescence plate reader
following selective surface biotinylation.
The percentage of signal found on the
basolateral surface of Y286A, LSYTRF and
L90V was significantly different from that of
WT (*). Error bars indicate SEM. n = 3–6
(varies between cell lines).
J. Chtchetinin et al. Basolateral sorting of Cx43 in MDCK cells
FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 6997
and morphology as WT (Fig. 2B). The F52Dup
mutant formed large gap junction plaques far less fre-
quently, consistent with our previous results when the
F52dup mutant was expressed in C6 rat glioma cells
[43]. The various plaques formed incells expressing
the WT construct were representative of plaques seen
in all mutant cell lines (Fig. 7A–E). Plaques formed
most frequently on the lateral surface ofthe cell
membrane and spanned either the entire area of cell–
cell contact (Fig. 7A) or a smaller area closer to the
basal (Fig. 7B) or apical (Fig. 7C) membrane. Plaques
were also sometimes seen apparently unbound to the
cell membrane near the basal surface (Fig. 7D) or
near the apical membrane (Fig. 7E); however, these
examples occurred less frequently. Mutants with api-
cal expression formed plaques predominantly on the
lateral surface; a representative plaque formed by the
Y286A mutant is shown (Fig. 7F). We found no dif-
ference inthe relative frequencies of these types of
plaques between WT and the various mutants (data
not shown).
Degradation from the cell membrane is impaired
for Y286A Cx43–eYFP
The tyrosine-based signal containing Y286 also con-
forms to a putative lysosomal degradation signal [47].
To determine whether any ofthe mutations inhibit
degradation of surface Cx43–eYFP, we performed
pulse–chase surface biotinylation experiments on all of
the cell lines (grown as monolayers on 60 mm cell
culture dishes). The amount of Cx43–eYFP present
after 2, 4 and 8 h chase intervals was measured by a
fluorescence plate reader and normalized to the
amount present at time 0. We found that surface WT
Cx43–eYFP was rapidly degraded, with a half-life of
under 2 h. We performed western blots probed with
the GFP and Cx43 antibodies to confirm that we were
properly measuring the disappearance of intact Cx43–
eYFP without tracking degradation products contain-
ing eYFP (data not shown). The Y286A mutation
resulted in a slightly longer half-life of surface Cx43–
eYFP compared to WT, indicating that Y286A has a
role in mediating degradation from the cell surface
(Fig. 8). The L90V, LSYTRF and F52dup mutations
had minimal effect on degradation of Cx43–eYFP
from the surface compared to WT (Fig. S2).
Discussion
We sought to determine the localization of surface
expression of Cx43–eYFP in polarized MDCK cells
and whether two ODDD mutations could alter this
distribution. We hypothesized that Cx43–eYFP would
have tyrosine-dependentbasolateral expression in
MDCK cells based on the presence of YKLV(286–
289) within the amino acid sequence ofthe cyto-
plasmic tail. Using confocal microscopy and selective
surface biotinylation, we have shown that WT
Cx43–eYFP is targeted to thebasolateral membrane
domain of MDCK cells (Figs 3A and 6A). The selec-
tive expression of Cx43–eYFP on the basolateral
10 µm
X
Y
X
Z
10 µm
X
Y
10 µm
X
Y
10 µm
X
Y
10 µm
X
Y
X
Z
X
Z
X
Z
X
Z
10 µm
X
Y
X
Z
ABC
DEF
Fig. 7. Gap junction plaques are located predominantly on the lateral surface of cell monolayers expressing WT and mutant constructs. Z
sections generated from areas containing the various types of plaques expressed bythe WT construct are shown, and are representative of
plaques seen in all mutants. Plaques were found to span the entire membrane (A) or part ofthe membrane (B,C). Less frequently, plaques
were found to be apparently suspended near the basal membrane (D) or the apical membrane (E), rather than at cell–cell junctions. (F) Z
section of a representative plaque formed bythe Y286A mutant construct. Arrows indicate plaques. Scale bar = 10 lm.
Basolateral sorting of Cx43 in MDCK cells J. Chtchetinin et al.
6998 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS
domain of MDCK cells is consistent with other stud-
ies showing that Cx43 and other connexins are typi-
cally distributed on thebasolateral surface of
polarized cells [5,11–13]. As all experiments were per-
formed inthe presence of sodium butyrate to increase
expression ofthe Cx43–eYFP constructs, as previ-
ously shown using this model system [49], we used a
variety of approaches to confirm that sodium butyrate
incubation did not alter the distribution of Cx43–
eYFP or disrupt the integrity ofthe monolayer
(Fig. S1). First, we performed confocal experiments in
the absence of sodium butyrate pre-incubation and
found similar distributions between the apical and ba-
solateral surface for all constructs. We then performed
dye transfer assays on monolayers expressing each
construct and found no consistently significant
changes in dye transfer after sodium butyrate pre-
incubation compared to untreated monolayers. Lastly,
we performed transepithelial resistance measurements
on a representative cell line and found that the
sodium butyrate did not alter the transepithelial resis-
tance ofthe filter-grown monolayer.
We demonstrated that Y286 inthe cytoplasmic
domain of Cx43 is necessary for basolateral sorting of
Cx43–eYFP in MDCK cells, as evidenced by the
absence of selective basolateraltargetingofthe Y286A
mutant construct (Fig. 4A,B). Therefore, Y286 of the
tetrapeptide sequence YKLV(286–289) represents a crit-
ical tyrosine residue ofthe common basolateral sorting
motif YXXø [18]. To further characterize this signal, we
then substituted PGYKLV(284–289) bythe LSYTRF
sequence containing the transferrin receptor internaliza-
tion signal (YTRF), and found that selective basolateral
targeting of Cx43–eYFP was not preserved (Fig. 4C,D).
This finding was not unexpected given that the transfer-
rin receptor internalization signal does not contain
basolateral targeting information [48]. Interestingly, gap
junction plaques were found predominantly on the lat-
eral membrane domain, even in cell lines expressing con-
structs that have apical expression (Fig. 7). This raises
the possibility that the biotinylation assay does not cap-
ture this population completely, possibly due to poor
accessibility of fully assembled gap junctions to sulfo-
NHS-LC-biotin (see methods). Alternatively, this pla-
que population may be a component of small basolat-
eral fraction of Cx43-eYFP detected for Y286A and the
other apically expressed mutant Cx43–eYFP constructs.
Despite our inability to determine which assembly states
are efficiently captured bythe biotinylation assay, the
combination of confocal microscopy with the biotinyla-
tion data strongly suggest that trafficking of Cx43, pre-
sumably as undocked Cx43 connexons (hemichannels),
is directed to thebasolateral surface. From our data, it
remains unclear by what mechanism gap junctional
plaques are retained at thebasolateral surface.
We found that the ODDD-associated L90V mutant
disrupts basolateral expression of Cx43–eYFP without
affecting the rate of surface degradation, whereas
F52dup does not affect either basolateral expression or
degradation (Figs 5 and 6). The finding of altered
basolateral expression oftheL90V mutant Cx43–eYFP
construct may indicate the presence of another basolat-
eral sorting determinant located inthe second trans-
membrane domain of Cx43. Although not as common
as cytoplasmic sorting signals, transmembrane sorting
signals have been identified. For example, the gastric
H,K-ATPase has an apical sorting signal in its 4th
transmembrane domain, although the exact amino
acids responsible have not been identified [50]. Studies
have shown that Cx43 oligomerizes into connexons in
the ER or Golgi prior to delivery to the cell membrane
[1]. Therefore, an alternative hypothesis is that the
L90V mutation may affect oligomerization of Cx43
subunits, which impairs recognition ofthe Y286-based
sorting signal. Overall, these findings may provide an
explanation for the additional phenotypic features of
neurodegeneration and hearing loss observed in
ODDD patients with theL90V and not the F52dup
Time (h)
Fluorescence remaining (%)
0 246
8
WT
Y286A
20
10
70
40
30
50
60
80
90
100
*
*
*
Fig. 8. The Y286A mutant construct shows impaired degradation
compared to WT. Surface protein degradation assays on tissue cul-
ture dishes were performed by labeling surface protein with mem-
brane-impermeable sulfo-NHS-LC-biotin and lysing cells at 0, 2, 4
and 8 h. eYFP fluorescence was quantified using a fluorescence
plate reader. Fluorescence remaining (%) was calculated by normal-
izing to the reading at time 0 for each cell line. The Y286A mutation
slightly impaired degradation of Cx43–eYFP from the surface
compared to WT. For each cell type, at least three independent
experiments were performed on two clones. Values shown are
means ± SEM.
J. Chtchetinin et al. Basolateral sorting of Cx43 in MDCK cells
FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 6999
mutation [25]. Characterization of polarized trafficking
of other ODDD-associated mutants in MDCK cells
will be necessary to correlate aberrant polarized traf-
ficking with a particular phenotype.
Consistent with other studies, we found that Cx43–
eYFP is rapidly degraded from the surface, with a
half-life of about 2 h (Fig. 7) [47,51]. We detected a
slight but significant decrease in surface protein degra-
dation between WT and the Y286A mutant but not
between WT and any ofthe other mutants. We
expected to see a greater difference between WT and
Y286A given that assay ofthe Y286A mutant in
SKHep1 cells demonstrated that the mutation
increased the half-life of total cellular Cx43 from 2 to
6 h [51]. The disparity between these results may be
due to the difference in cell lines used or to the fact
that our assay examined degradation of surface protein
as opposed to total protein. However, the lack of
an appreciable effect on degradation ofthe construct
containing the substituted transferrin internalization
signal suggests that another signal may be involved
in the degradation of Cx43 other than the
PGYKLV(284–289) sequence.
Our findings imply that targetingof Cx43 to specific
domains of polarized cells may be crucial for its func-
tional regulation, by concentrating or restricting inter-
cellular interactions to a specific plasma membrane
domain. For example, astrocytes have a polarized mor-
phology with formation of specialized endfeet that
make contacts with endothelial cells [9,10]. Cx43 has
been found to be abundantly expressed at the connec-
tion of blood vessels and astrocytic endfeet [52].
Although there are no known studies correlating tar-
geting to thebasolateral domain of MDCK cells with
targeting to astrocytic endfeet, we predict that Cx43 is
selectively targeted to the astrocytic endfeet, based on
the finding that the VSV-G protein is targeted to the
processes that form the myelin sheath in oligodendro-
cytes, another glial cell type [22]. We also predict that
Cx43 is targeted to thebasolateral domain of endothe-
lial cells, based on findings that other basolateral sort-
ing signals active in MDCK cells are recognized for
basolateral targetingin endothelial cells [53]. By simi-
lar mechanisms, Cx43-dependent neuronal migration
along glial fibers via gap junctional adhesion during
development may require polarized targetingof Cx43
[17]. Alteration of polarized expression may explain
the central nervous system developmental abnormali-
ties found in ODDD. Lastly, processes such as glioma
migration along white matter or endothelial basement
membrane paths may also utilize Cx43-dependent
mechanisms that rely on proper targetingof Cx43 in
polarized cells [54,55].
Experimental procedures
Cell culture
MDCK (strain II) cells expressing the RSV(A) receptor
[obtained from Dr G. Odorizzi, Department of Molecular,
Cellular, and Developmental Biology (MCDB), University
of Colorado, Boulder, CO, USA] and DF-1 cells (purchased
from the American Type Culture Collection, Manassas, VA,
USA) were maintained in DMEM ⁄ F12 (Mediatech, Hern-
don, VA, USA) supplemented with 10% fetal bovine serum
(Lonza, Walkersville, MD, USA), 100 UÆmL
)1
penicillin and
100 lgÆmL
)1
streptomycin (Lonza, Walkersville, MD, USA).
293T cells (obtained from Dr P. Mischel, Department of
Pathology, University of California, Los Angeles, CA, USA)
were maintained in Iscove’s Modified Dulbecco’s Medium
(IMDM) (Hyclone, Logan, UT, USA) supplemented with
10% fetal bovine serum, 100 UÆmL
)1
penicillin and
100 lgÆmL
)1
streptomycin. All cells were grown in a 5%
CO
2
humidified atmosphere.
Generation of wild-type and mutant Cx43–eYFP
fusion constructs
Generation ofthe WT, L90V and F52dup Cx43–eYFP
fusion constructs inthe pEYFP-N1 vector has been
described previously [43]. To introduce the Y286A and
LSYTRF mutations into the Cx43 sequence, two-stage
mutagenesis was performed using the WT plasmid as the
template. An upstream forward primer and a mutagenic
reverse primer were used to amplify a 5¢ product carrying
the mutation, and an overlapping 3¢ product was amplified
using a forward mutagenic primer (complement of the
mutagenic reverse primer) and a downstream reverse pri-
mer. The 5¢ and 3¢ Cx43 amplification products were com-
bined and amplified using HindIII forward and XmaI
reverse adapter primers, and the resultant altered Cx43
sequences were cloned into pEYFP-N1. The following
mutagenic forward primers were used: Y286A, 5¢-GATCA
TGAATTGTTTCTGTCGCCAGTAACCAGCTTGGCCC
CAGGAGGAGACATAGGCG-3¢; LSYTRF, 5¢-GCAAG
AAGAATTGTTTCTGTCGCCAGTGAACCGGGTATAT
GACAAAGGAGACATAGGCGAGAGGGGAGC-3¢. The
complementary sequences were used as reverse primers.
Subcloning of Cx43–eYFP constructs into
BH-RCAS and pLPCX retroviral expression
vectors
Mutant and WT Cx43–eYFP fusion constructs were ampli-
fied using the following adapter primers containing ClaI sites
(underlined): 5¢-GATCAT
ATCGATACAGCAGCGGAG
TTT-3¢ (forward) and 5¢-GATCAT
ATCGATGCCGCT
TTACTTGTA-3¢ (reverse). PCR products were digested with
Basolateral sorting of Cx43 in MDCK cells J. Chtchetinin et al.
7000 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS
ClaI and ligated into ClaI-linearized BH-RCAS, a repli-
cation-competent retroviral vector derived from the Rous
sarcoma virus [56]. In addition, the insert encoding Y286A–
eYFP was excised from the BH-RCAS vector using ClaI and
inserted into the ClaI-linearized pLPCX vector. Therefore,
one Y286A cell line was made using pLPCX and one was
made using BH-RCAS. Cx43-coding sequences were verified
at the UCLA Sequencing Core Facility.
Retroviral expression of Cx43–eYFP constructs in
MDCK cells
These procedures have been described previously [43,56,57].
Briefly, transfection of DF-1 cells with BH-RCAS constructs
encoding wild-type and mutant Cx43–eYFP was performed
using Superfect (Qiagen, Valencia, CA, USA) in 60 mm
tissue culture plates according to the manufacturer’s instruc-
tions. Prior to infection, the RSV(A) receptor had been
expressed inthe MDCK cells, rendering them susceptible to
infection. MDCK cells containing the RSV(A) receptor were
selected using 0.5 mgÆmL
)1
G418 (Sigma, St Louis, MO,
USA). For transfection with pLPCX vectors, 293T cells were
co-transfected with 10 lg ofthe designated pPLCX con-
struct, 5 lg Hit-60 (a plasmid expressing MLV gag-pol) and
5 lg VSV-G using Hepes-buffered saline and 150 mm CaCl
2
.
For infection of MDCK cells, conditioned medium was
collected from transfected DF-1 or 293T cells (containing
recombinant virus particles), filtered using a 0.45 lm filter
(Whatman, Florham Park, NJ, USA), supplemented with
5 lgÆmL
)1
polybrene (Sigma) and added to target MDCK
cells. For each construct, multiple clonal cell lines were
derived from the selected populations by limited dilution.
Isolation of surface protein by biotinylation on
tissue culture plates
To extract protein for western blot analysis (Fig. 2A),
MDCK cells expressing wild-type and mutant Cx43–eYFP
constructs were cultured on 60 mm tissue culture plates.
Sodium butyrate (10 mm; Alfa Aesar, Ward Hill, MA, USA)
dissolved in complete cell culture medium was added to cells
24 h prior to the experiment to boost protein expression.
Confocal microscopy, paracellular dye flux assays and trans-
epithelial resistance measurements were used to confirm that
addition of 10 mm sodium butyrate does not alter the polar-
ized membrane properties of MDCK cells (Fig. S1). Cells
were kept on ice for the duration ofthe experiment. Cells
were rinsed (all washes were brief – about 1 minute) three
times with cold Dulbecco’s Phosphate Buffered Saline
(DPBS). Membrane impermeable sulfo-NHS-LC-biotin
(2 mL; Pierce, Rockford, IL, USA) dissolved in DPBS (at a
concentration of 0.5 mgÆmL
)1
) was applied to each plate for
30 min. The labeling reaction was quenched by three rinses
with 100 mm glycine (Fisher, Fair Lawn, NJ, USA) dissolved
in DPBS, and cells were washed once more with DPBS. Cells
were lysed using lysing buffer containing 0.5% SDS (Tekno-
va, Hollister, CA, USA), 1% nonidet P-40 (United States
Biological, Swampscott, MA, USA) and 0.25% sodium
deoxycholate (Sigma), supplemented with Complete Mini
protease inhibitors (Roche Diagnostics, Indianapolis, IN,
USA), 1 lm sodium vanadate (Fisher), 1 lm sodium fluoride
(Fisher) and 1 lm phenylmethanesulfonyl fluoride (Sigma),
for 30 min. Lysates were passed through a 25
5
8
G syringe
three times (Becton-Dickinson, Franklin Lakes, NJ, USA).
Lysates were centrifuged for 10 min at high speed at 4 °C,
then 750 lL ofthe total lysate was combined with 75 lL
streptavidin–agarose beads (Novagen, Gibbstown, NJ, USA)
and incubated overnight on a rotating shaker at 4 °C. On the
following day, beads were rinsed four times with DPBS.
After the fourth rinse, the beads (which remained suspended
in approximately 175 lL of DPBS) were transferred to a
96-well plate, and fluorescence was quantified using a Wallac
Victor
2
plate reader (Perkin-Elmer, Waltham, MA, USA)
with 485 nm excitation and 535 nm emission filters. These
beads were prepared for western blot as indicated below.
Selective isolation of surface protein from the
apical and basolateral domains by biotinylation
For polarized protein distribution studies, cells were seeded
at a high density (0.5–1 · 10
6
cells per filter, depending on
cell line), and cultured on Corning PET Transwell perme-
able filter supports for 5 days (Corning Incorporated, Corn-
ing, NY, USA). Experiments were performed as described
above with a few adjustments. Membrane impermeable
sulfo-NHS-LC-biotin was added to either the apical or
basolateral side, and DPBS was added to the side not
receiving sulfo-NHS-LC-biotin. Prior to lysing, filters were
cut out, and placed into new six-well plates.
Isolation of total protein by biotinylation on
tissue culture plates
This procedure was performed as described above with a
few changes. Membrane permeable NHS-LC-biotin (Pierce,
Rockford, IL, USA) was used instead of sulfo-NHS-LC-
biotin. A 40 mm solution of NHS-LC-biotin was prepared
in dimethylsulfoxide, and then diluted 10-fold in NaCl ⁄ P
i
.
NHS-LC-biotin (2 mL) was applied to each plate for 4 h
on a shaker at 4 °C. All rinses were performed as described
above in ‘Isolation of surface protein by biotinylation on
tissue culture plates’ using NaCl ⁄ P
i
or 100 mm glycine dis-
solved in NaCl ⁄ P
i
instead of DPBS.
Paracellular permeability of MDCK monolayers
MDCK cells expressing WT and mutant Cx43–eYFP were
plated at high density on filter inserts and cultured for
J. Chtchetinin et al. Basolateral sorting of Cx43 in MDCK cells
FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 7001
[...]... mL of DPBS were added to thebasolateral side Because sodium fluorescein is membrane-impermeable, it can only cross the monolayer via the paracellular pathway, thus the accumulation of dye on thebasolateral side represents paracellular flux Accumulation ofthe dye was determined by taking 25 lL aliquots every 30 mins for 3 h from thebasolateral chamber, dissolving them in 1.5 mL DPBS, and quantifying... (1997) Structural requirements for basolateral sorting ofthehuman transferrin receptor inthe biosynthetic and endocytic pathways ofMadin–Darbycaninekidneycells J Cell Biol 137, 1255–1264 49 Odorizzi G, Pearse A, Domingo D, Trowbridge IS & Hopkins CR (1996) Apical and basolateral endosomes of MDCK cells are interconnected and contain a polarized sorting mechanism J Cell Biol 135, 139–152 50 Dunbar... FL, USA) to the membrane and measuring the induced voltage drop Resistance was calculated using Ohm’s law (V = IR) The current was delivered using silver chloride electrodes in both the apical and basolateral compartments ofthe culture dishes The MDCK cell monolayer provides a barrier between the apical and basolateral compartments The voltage drop over the membrane was measured using an instrumentation... centrifuged for 10 min at high speed at 4 °C All ofthe supernatant (containing the protein) was then loaded into pre-cast Tris ⁄ Hepes ⁄ SDS gels (Pierce) Protein was transferred to nitrocellulose paper using a Trans-Blot SD semi-dry transfer cell apparatus (Bio-Rad, Hercules, CA, USA) Immunoblotting was performed by incubation in primary antibodies diluted in 1% milk in Tris-buffered saline using a 1 : 500... cognate basolateral trafficking in myelin biogenesis J Neurosci Res 86, 1007–1016 FEBS Journal 276 (2009) 6992–7005 ª 2009 The Authors Journal compilation ª 2009 FEBS 7003 Basolateral sorting of Cx43 in MDCK cells J Chtchetinin et al 23 Winckler B & Mellman I (1999) Neuronal polarity: controlling the sorting and diffusion of membrane components Neuron 23, 637–640 24 Laird DW (2008) Closing the gap on... made in DPBS, and, as a control, in NaCl ⁄ Pi, which creates calcium-free conditions that disrupt tight junctions in MDCK cells and therefore cause transepithelial resistance to be drastically decreased Western blot analysis of Cx43–eYFP fusion proteins Streptavidin–agarose beads were boiled in sample buffer containing b-mercaptoethanol and dithiothreitol for 10 min 7002 to strip off protein Then the. .. varies between cell lines) The results are normalized to time 0 for each cell line Values given are means ± SEM Significance was determined using Student’s t-test P values £ 0.05 were considered to be significant Acknowledgements This work was made possible through funding from the American Brain Tumor Association ⁄ Michael Reiss Fellowship (A.L.), Art ofthe Brain (A.L.), unrestricted funds from the Cancer... Center of Santa Barbara (A.L.), and National Institutes of Health grant R01 DE13849 (E.W.J.) We thank Seema Tiwari-Woodruff PhD (Department of Neurology, UCLA School of Medicine) for the use of her microscope, Guido Faas PhD (Department of Neurology, UCLA School of Medicine) for performing all ofthe transepithelial resistance measurements, and Olga Vagin PhD (Department of Physiology, UCLA School of Medicine)... not affect the polarized distribution of Cx43–eYFP constructs or the integrity of MDCK cell monolayers Fig S2 LSYTRF, F52dup and L90V mutant constructs show the same rate of degradation as wild-type This supplementary material canbe found inthe online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied bythe authors... C, Kiessling F & Kubler W (1998) Polarized expression of heterologous membrane proteins transfected in a human endothelial-derived cell line Eur J Cell Biol 75, 353–361 54 Oliveira R, Christov C, Guillamo JS, de Bouard S, Palfi S, Venance L, Tardy M & Peschanski M (2005) Contribution of gap junctional communication between tumor cells and astroglia to the invasion ofthe brain parenchyma byhuman glioblastomas . Tyrosine-dependent basolateral targeting of human
connexin43–eYFP in Madin–Darby canine kidney cells
can be disrupted by the oculodentodigital dysplasia
mutation. require-
ments for basolateral sorting of the human transferrin
receptor in the biosynthetic and endocytic pathways of
Madin–Darby canine kidney cells. J Cell