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 micro
Trang 1connexin43–eYFP in Madin–Darby canine kidney cells
can be disrupted by the oculodentodigital dysplasia
mutation L90V
Jana Chtchetinin1,2, Wes D Gifford1,2, Sichen Li1,2, William A Paznekas3, Ethylin Wang Jabs3,4 and Albert Lai1,2
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 the basolateral surface of Madin–Darby canine kidney (MDCK) cells in 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 basolateral targeting of Cx43–eYFP Additionally, substitution of a sequence containing the transferrin receptor internalization signal, LSYTRF, for PGYKLV(284–289) also disrupted basolateral targeting Taken together, these results indicate that Y286 in its native amino acid sequence is necessary for targeting Cx43–eYFP to the basolateral 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 the L90V mutation disrupted 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–Darby canine kidney; ODDD, oculodentodigital dysplasia; DPBS, dulbecco’s phosphate buffered saline.
Trang 2functions [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 of the 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–Darby canine kidney (MDCK) cells has
previously been examined, but not in the 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 in basolateral targeting of Cx43
At least 60 mutations in Cx43 have been discovered
that cause oculodentodigital dysplasia (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 of the relationship
between genotype and phenotype, although functional
evaluations of most of the 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 the basolateral membrane domain
of MDCK cells We also found that Y286 in the sequence PGYKLV(284–289) is necessary for basolat-eral targeting of Cx43–eYFP In addition, we found that the L90V ODDD mutation disrupts the selective delivery of Cx43–eYFP to the basolateral 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 of human 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 of the four mutaloca-tions 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 in the total cell lysate of cells expressing only eGFP No GFP expression was detected after surface biotinylation of cells 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 of the constructs Confocal images in the 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)
Trang 3WT 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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Fig 1 Schematic diagram of the 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 in the cyto-plasmic tail This figure has been modified from one that has been published previously [43].
eGFP Wild-type Y286A LSYTRF F52dup L90V
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Surface
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Cx43-eYFP
eGFP
Cx43-eYFP
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LSYTRF
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Uninfected
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eGFP Wild-type Y286A LSYTRF F52dup L90V
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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 by the 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.
Trang 4individual 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) Localilocali-zation of plaques in the XZ
plane is shown in Fig 7 (see below)
To confirm these results, we performed selective
sur-face biotinylation on the apical and basolateral sursur-faces
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 the basolateral 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 of the distribution between the two
domains using a fluorescence plate reader revealed that
86% of the 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 of the Cx43–eYFP
constructs We confirmed that sodium butyrate treat-ment did not affect results by performing confocal microscopy in the 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 in the 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 degradainternaliza-tion of Cx43 [47] To determine whether Y286 is involved in the basolateral targeting
of WT Cx43–eYFP, we substituted an alanine for Y286 of the Cx43–eYFP sequence and examined the surface distribution of the 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
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Fig 3 WT Cx43–eYFP is selectively targeted to the basolateral 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 in the top panels Lines drawn through the XY plane
in the bottom panels indicate the location of the Z sections WT Cx43–eYFP was expressed on the basolateral 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.
Trang 5(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 of the surface protein using a
fluorescence plate reader indicated that, similar to
Y286A, 74% of the 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 of basolateral 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 the basolateral targeting of 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 the L90V mutant because it appeared to have an increased amount of plaques in the glial cell processes compared to WT (unpublished observation) Confocal Z sections of cells expressing the F52dup mutant construct showed predominantly basolateral expression, indicating that this mutation does not alter polarized targeting of 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 of the L90V 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 of the L90V mutant construct is not restricted to the basolateral surface, with 57% of the 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 of cells expressing WT and apically distributed mutant constructs
With the exception of the F52dup mutant, all mutant constructs formed plaques with the same frequency
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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.
Trang 610 µm
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Fig 5 L90V but not the F52dup ODDD
mutation disrupts basolateral targeting 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 the L90V mutant construct
showing that basolateral targeting of
Cx43–eYFP was disrupted (two independent
clones) Scale bar = 10 lm.
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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).
Trang 7and 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 in cells 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 of the 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 in the 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 of the 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-dependent basolateral expression in MDCK cells based on the presence of YKLV(286– 289) within the amino acid sequence of the cyto-plasmic tail Using confocal microscopy and selective surface biotinylation, we have shown that WT Cx43–eYFP is targeted to the basolateral membrane domain of MDCK cells (Figs 3A and 6A) The selec-tive expression of Cx43–eYFP on the basolateral
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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 by the WT construct are shown, and are representative of plaques seen in all mutants Plaques were found to span the entire membrane (A) or part of the 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 by the Y286A mutant construct Arrows indicate plaques Scale bar = 10 lm.
Trang 8domain of MDCK cells is consistent with other
stud-ies showing that Cx43 and other connexins are
typi-cally distributed on the basolateral surface of
polarized cells [5,11–13] As all experiments were
per-formed in the presence of sodium butyrate to increase
expression of the 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 of the 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 of the filter-grown monolayer
We demonstrated that Y286 in the cytoplasmic
domain of Cx43 is necessary for basolateral sorting of
Cx43–eYFP in MDCK cells, as evidenced by the
absence of selective basolateral targeting of the Y286A mutant construct (Fig 4A,B) Therefore, Y286 of the tetrapeptide sequence YKLV(286–289) represents a crit-ical tyrosine residue of the common basolateral sorting motif YXXø [18] To further characterize this signal, we then substituted PGYKLV(284–289) by the 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 by the 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 the basolateral surface From our data, it remains unclear by what mechanism gap junctional plaques are retained at the basolateral 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 of the L90V mutant Cx43–eYFP construct may indicate the presence of another basolat-eral sorting determinant located in the 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 of the 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 the L90V and not the F52dup
Time (h)
WT Y286A
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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.
Trang 9mutation [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 of the other mutants We
expected to see a greater difference between WT and
Y286A given that assay of the 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 of the 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 targeting of 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 the basolateral 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 the basolateral domain of
endothe-lial cells, based on findings that other basolateral
sort-ing signals active in MDCK cells are recognized for
basolateral targeting in endothelial cells [53] By
simi-lar mechanisms, Cx43-dependent neuronal migration
along glial fibers via gap junctional adhesion during
development may require polarized targeting of 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 targeting of 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)1penicillin and
100 lgÆmL)1streptomycin (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%
CO2humidified atmosphere
Generation of wild-type and mutant Cx43–eYFP fusion constructs
Generation of the WT, L90V and F52dup Cx43–eYFP fusion constructs in the 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¢-GATCATATCGATACAGCAGCGGAG TTT-3¢ (forward) and 5¢-GATCATATCGATGCCGCT TTACTTGTA-3¢ (reverse) PCR products were digested with
Trang 10ClaI 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 in the 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 of the 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 CaCl2
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 of the 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 255
8G syringe three times (Becton-Dickinson, Franklin Lakes, NJ, USA) Lysates were centrifuged for 10 min at high speed at 4C, then 750 lL of the total lysate was combined with 75 lL streptavidin–agarose beads (Novagen, Gibbstown, NJ, USA) and incubated overnight on a rotating shaker at 4C 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 Victor2 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· 106
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⁄ Pi NHS-LC-biotin (2 mL) was applied to each plate for 4 h
on a shaker at 4C All rinses were performed as described above in ‘Isolation of surface protein by biotinylation on tissue culture plates’ using NaCl⁄ Pior 100 mm glycine dis-solved in NaCl⁄ Piinstead 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