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(Received for publication, April 20, 1995; and in revised form, July 21, 1995) From the
We transfected the cDNA for the cell-to-cell channel protein
connexin-43 (Cx43) into Morris hepatoma H5123 cells, which express
little Cx43 and lack gap junctional communication (open cell-to-cell
channels). We found that cells overexpressing Cx43 nonetheless lacked
open cell-to-cell channels, but that inhibition of glycosylation by
tunicamycin induced open channels in these cells. Tunicamycin also
induced biochemical changes in Cx43 protein; the level increased, and a
considerable fraction became phosphorylated and Triton X-100 insoluble,
in contrast to untreated cells where Cx43 was non-phosphorylated and
Triton X-100 soluble. Although tunicamycin caused the formation of open
channels, channels were not found aggregated into gap junctional
plaques, as they are when they have been induced by elevation of
intracellular cAMP. The results suggest that although Cx43 itself is
not glycosylated, other glycosylated proteins influence Cx43
posttranslational modification and the formation of Cx43 cell-to-cell
channels.
Cell-to-cell channels mediate intercellular communication by
providing a direct pathway for the exchange of molecules up to
1-2 kDa (Schwarzmann et al., 1981). The molecules
transferred include signaling molecules, which may play important roles
in tissue homeostasis (Loewenstein, 1981), cell growth control
(Loewenstein and Rose, 1992), and embryonic development (Warner, 1992).
The channels are known to cluster into often quite large aggregates,
forming the so-called gap junctions. In electron microscopic images of
freeze-fractured gap junctions, the channels show up as particles of
uniform size that span the two adjoining membranes (Kreutziger, 1968;
Goodenough and Revel, 1970). The channels are formed from membrane
proteins called connexins (Beyer et al., 1990; Kumar and
Gilula, 1992). More than a dozen different connexins have been
identified in vertebrates, all of which share similar topology and
amino acid sequence (Kumar and Gilula, 1992). One well studied and
widely expressed connexin is connexin-43 (Cx43), ( Progress has been made in understanding how
cell-to-cell channels are formed. For example, it is now known that
after synthesis, Cx43 (and probably the other connexins, too, see
Rahman et al.(1993)) is first assembled into multimeres called
connexons or hemichannels in the Golgi (Musil and Goodenough, 1993);
the hemichannels are then transported to the plasma membrane where they
find counterparts on the adjoining cell's membrane to form the
cell-to-cell channels proper. Yet, little is known about how the
cell-to-cell channels are concentrated into the gap junction plaques.
In some cells, a phosphorylated and Triton X-100 insoluble form of
Cx43, but not its non-phosphorylated and Triton X-100 soluble form, is
localized in the gap junctional plaques (Musil and Goodenough, 1991).
It is not clear whether Cx43 becomes Triton X-100 insoluble as a
consequence of hemichannel interlocking, i.e. channel
formation, or as a consequence of the clustering into gap junction
plaques. Neither is it clear what role phosphorylation has in Cx43
channel or gap junction formation. We have found previously that
inhibition of glycosylation by tunicamycin (Tm) greatly increases the
formation of open channels in a variety of Cx43-expressing cells (Wang
and Mehta, 1995). In the present study, we investigate whether any
biochemical changes in Cx43 are associated with Tm-induced channel
formation. For this, we constitutively expressed Cx43 in a cell line
that lacks open cell-to-cell channels and studied the effects of
tunicamycin on cell communication via Cx43 channels and on
phosphorylation, Triton X-100 solubility, and cellular localization of
Cx43.
The expression of Cx43 protein in MHD1 cells and in one Cx43
overexpressing clone, MHD1-43A, is shown in Fig. 1A. The MHD1-43A cells express Cx43
abundantly, manyfold higher than the parental MHD1 cells. And like in
the parental cells, Cx43 in the MHD1-43A cells is mainly in the
non-phosphorylated form, appearing as one major band of 42 kDa on
SDS-PAGE (Fig. 1A, lane 2; see also Fig. 1C, lane 1).
Figure 1:
Western blot analysis of Cx43 in MHD1
and MHD1-43A cells. A, overexpression of Cx43 in Morris
hepatoma cells. Lane 1, MHD1; lane 2, MHD1-43A. B, effect of Tm on Cx43 in MHD1 cells. Lane 1,
control (0.4% Me
Figure 2:
Tunicamycin induces gap junctional
communication in Cx43 overexpressor cells. A, cell-cell
transfer of Lucifer Yellow in MHD1-43A cells. panels a and b, control (0.4% Me
Figure 3:
Reduction of lectin binding after
tunicamycin treatment. Cell surface binding of rhodamine-labeled wheat
germ agglutinin (a and b) and D. biflorus agglutinin (c and d) in control (a and c) and tunicamycin-treated (b and d)
MHD1-43A cells.
Tunicamycin has been reported to inhibit
protein synthesis (Elbein, 1987), and inhibition of general protein
synthesis may somehow increase communication (Azarnia et al.,
1981). To test whether tunicamycin's effect on communication
could be due to general inhibition of protein synthesis, we treated
MHD1-43A cells with the protein synthesis inhibitor,
cycloheximide. As seen in Fig. 2B, communication
changed little, showing that the effect of tunicamycin cannot be
explained by general inhibition of protein synthesis.
Under control conditions, Cx43 in the
non-communicating MHD1 cells was predominantly Triton X-100 soluble (Fig. 4, lanes 1-3); Triton X-100 insoluble Cx43
was barely detectable, and this was so also after Tm treatment (Fig. 4, lanes 4-6). In contrast, forskolin
treatment, which increases Cx43 expression and induces communication as
well as Cx43 phosphorylation in MHD1 cells (Wang and Mehta, 1995),
induced Triton X-100 insoluble Cx43 (Fig. 4, lanes
7-9). This Triton-insoluble fraction included both
phosphorylated and non-phosphorylated Cx43, while the soluble fraction
consisted of non-phosphorylated Cx43.
Figure 4:
Western blot analysis of Triton X-100
solubility of Cx43 in MHD1 cells. Total (T), soluble (S), and insoluble (I) Cx43 of control (lanes
1-3), tunicamycin (lanes 4-6; 4 µg/ml, 8
h), and forskolin (lanes 7-9; 20 µM forskolin plus 50 µM phosphodiesterase inhibitor,
Ro-20-1724, 8 h) treated cells. The positions of molecular mass
markers are indicated on the left in
kDa.
The Cx43 in untreated,
basically non-communicating MHD1-43A and MHD1-43B cells was
also mainly Triton X-100 soluble (Fig. 5, lanes 1-3 and 7-9). But after tunicamycin treatment, with the
appearance of open channels and of phosphorylated Cx43 (the two upper
bands in lanes 4, 6, 10, and 12 of Fig. 5), part of the Cx43 became Triton X-100 insoluble. As in
the forskolin-treated MHD1 cells, this insoluble Cx43 consisted of both
non-phosphorylated and phosphorylated forms of Cx43 (Fig. 5, lanes 6 and 12), while the soluble Cx43 was mainly
non-phosphorylated (Fig. 5, lanes 5 and 11).
Figure 5:
Tunicamycin induces Triton-insoluble Cx43
in Cx43 overexpressor cells. Western blot analysis (same procedure as
in Fig. 4) of total, Triton-soluble, and Triton-insoluble Cx43
in MHD1-43A and MHD1-43B cells with or without tunicamycin
treatment is shown. Lanes 1-3, MHD1-43A control
cells; lanes 4-6, tunicamycin-treated MHD1-43A
cells; lanes 7-9: MHD1-43B control cells; lanes 10-12, tunicamycin-treated MHD1-43B cells.
The positions of molecular mass markers are indicated on the left in kDa. Note: do not compare band intensities of Fig. 4and Fig. 5; relative intensities of bands should only be compared
within one and the same blot.
Figure 6:
Immunostaining of Cx43 in MHD1 (a-c), MHD1-43A (d-f), and
MHD1-43B cells (g-i). Control (a, d, g), tunicamycin (b, e, h; 4 µg/ml, 8 h), and forskolin (c, f, i; 20 µM forskolin plus 50 µM
Ro-20-1724, 8 h) treated cells are
shown.
The lack of punctate staining is not due to an
intrinsic incapability of the cells to cluster cell-to-cell channels
into gap junction plaques; bright Cx43 plaque staining appeared in both
MHD1 and overexpressor cells after an 8-h forskolin treatment (Fig. 6, c, f, and i). This staining
is much stronger and more abundant in overexpressor cells than in
parental MHD1 cells, consistent with the much higher level of Cx43
protein (Fig. 1A) and of forskolin-induced
communication (data not shown) in the overexpressor cells. In the present study, we transfected cx43 cDNA into
cells that lack open cell-to-cell channels and express little Cx43 and
found that cells which overexpressed Cx43 nonetheless had few open
channels. The lack of open channels in the Cx43 overexpressors seems
not due to any null mutation in the cx43 cDNA. The ability of
cDNA-derived Cx43 to form open cell-to-cell channels is clearly evident
from the difference in communication between the parental MHD1 and the
overexpressor cells after tunicamycin treatment; extensive cell-to-cell
transfer of tracer was induced in Cx43 overexpressing cells (Fig. 2) but not in parental MHD1 cells (Wang and Mehta, 1995).
Instead, the failure of the exogenous Cx43 to make open channels points
to some cellular condition non-permissive for Cx43 to make open
channels, and inhibition of glycosylation remedies this condition,
allowing channel formation. Due to the lack of potential
glycosylation sites in their extracellular loops, connexins are
unlikely to be glycosylated. Connexin-32 is known not to be
glycosylated (Hertzberg and Gilula, 1979; Rahman et al.,
1993), and our result of a lack of decrease in apparent molecular mass
after tunicamycin treatment confirmed that Cx43 is not glycosylated
either. Therefore, a reduction of carbohydrates from cell surface
proteins other than Cx43 is a more likely cause for the observed
increase in communication. From a priori considerations,
glycoproteins on the cell surface can be expected to impose an
inhibitory effect on the formation of cell-to-cell channels and gap
junctions (Peracchia, 1985; Abney et al., 1987). It was shown
previously that lectins induced or fostered intercellular communication
in Aplysia neurons (Lin and Levitan, 1987) or in Xenopus oocytes (Levine et al., 1991), presumably by removing
bulky glycoprotein from the plasma membrane, and we have shown that
inhibition of glycosylation increased intercellular communication in a
variety of mammalian cells (Wang and Mehta, 1995). Carbohydrates may
interfere with any one of the steps occurring on the membrane during
the formation of open cell-to-cell channels and thereby result in
decreased communication. The extracellular domain of connexins is no
larger than 8-10 Å, smaller than that of many membrane
glycoproteins. One possibility therefore is that large membrane
glycoproteins interfere with hemichannel interlocking by hindering two
adjoining plasma membranes to come close enough to allow hemichannel
interaction. Little is known about how the hemichannels get to the
cell-cell contact sites and how channels become concentrated in the
junctional plaques. It is possible that hemichannels are transported
onto the plasma membrane at random sites and then laterally diffuse to
the cell-cell contacts; or, they could be directly inserted into these
sites. Bulky membrane glycoproteins may impede the lateral movement of
hemichannels on the plasma membrane, or the insertion of hemichannels
into the plasma membrane at random or specific sites may involve some
glycoprotein(s). Yet another possibility is that even when channels are
formed, bulky surface carbohydrates in the immediate vicinity of
channels produce some condition unfavorable for the channels to be in
the open state. Although tunicamycin treatment elevated the total
Cx43 level in Cx43 overexpressors, it is unlikely that this caused the
dramatic rise in communication. Instead, the greater total Cx43 protein
may reflect a higher stability of Cx43 protein in channels than in
hemichannels. We therefore interpret the increase in Cx43 to be the
consequence of hemichannel interlocking rather than the cause of it. In
agreement with this interpretation is that in the parental MHD1 cells,
where tunicamycin did not induce channel formation, the Cx43 protein
level did not rise; in fact, it was slightly diminished. This is
consistent with the reported inhibition of protein synthesis by
tunicamycin in other cells (Elbein, 1987). One unexpected result of
this study is that tunicamycin treatment induced Cx43 phosphorylation
in the Cx43 overexpressor cells, raising the possibility that
tunicamycin activates a kinase. Activation of protein kinase A
up-regulates junctional communication and induces Cx43 phosphorylation
in a variety of cells, including MHD1 cells (Wang and Mehta, 1995). But
because protein kinase A activation has other effects not seen with
tunicamycin treatment, e.g. stimulation of Cx43 transcription
(Wang and Mehta, 1995), it is unlikely that tunicamycin activates
protein kinase A. It is even less likely that tunicamycin activates
protein kinase C or a tyrosine kinase because, although these kinases
cause Cx43 phosphorylation, they inhibit communication (Crow et
al., 1990; Filson et al., 1990; Brisette et al.,
1991; Berthoud et al., 1992; Kanemitsu and Lau, 1993), whereas
tunicamycin increases communication. It is therefore unclear how Cx43
gets phosphorylated after tunicamycin treatment. Cx43 hemichannels are
probably not phosphorylated (Musil and Goodenough, 1993) and are in a
closed conformation. When they interlock, they must undergo a
conformation change, which enables them to switch to an open state. It
is possible that after the conformation change, Cx43 becomes a
substrate of an unidentified, constitutively active kinase and thus
gets phosphorylated. The function of this Cx43 phosphorylation is not
clear. There is no evidence that phosphorylation is a prerequisite for
channels to open. Musil and Goodenough(1991) showed in several cell
lines that a certain form of phosphorylated and Triton X-100 insoluble
Cx43 is correlated with its localization at the junctional plaques. We
found this to be true also in MHD1 cells, where forskolin induced the
appearance of junctional plaques concurrently with Cx43 phosphorylation
and Triton X-100 insolubility. However, we noticed differences. In the
cells used by Musil and Goodenough(1991), the Triton X-100 insoluble
fraction contained primarily phosphorylated Cx43, while in MHD1 and
Cx43 overexpressors, the Triton-insoluble fraction contained
additionally a substantial amount of non-phosphorylated Cx43. Another
difference is that in the overexpressor cells, after tunicamycin had
induced functional channels, phosphorylated and Triton X-100 insoluble
Cx43 was found, but junctional plaques were not seen. The latter result
would imply that Cx43 phosphorylation and Triton X-100 insolubility
correlate better with hemichannel interlocking or cell-to-cell channel
formation than with channel clustering into gap junction plaques, at
least not with plaques large enough to be resolved by immunostaining.
The clustering of channels into plaques that are detectable by
immunostaining seems to require elevation of cAMP, both in the MHD1 and
the Cx43 overexpressor cells.
Volume 270,
Number 44,
Issue of November 3, 1995 pp. 26581-26585
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)found in
the heart (Beyer et al., 1987) and many other tissues (Beyer et al., 1987; Micevych and Abelson, 1991) as well as in a
number of established cell lines (Musil et al., 1990; Mehta et al., 1992).
Materials
All culture media were from
Life Technologies, Inc., fetal bovine serum was from Hyclone
Laboratories (Logan, UT), Lucifer Yellow CH was from Molecular Probes,
and forskolin was from Calbiochem. Phosphodiesterase inhibitor
Ro-20-1724 was a gift from Dr. P. Sorter (Hoffman-LaRoche). Rhodamine-
or fluorescein isothiocyanate-labeled goat antirabbit IgG and alkaline
phosphatase were from Boehringer Mannheim; rhodamine-labeled lectins,
wheat germ agglutinin, and Dolichos biflorus agglutinin were
from EY laboratory (San Mateo, CA); all other reagents (molecular
biology grade or highest purity) were from Sigma.Cell Culture
A subclone (MHD1) of Morris
hepatoma H5123 cells (Borek et al., 1969) was isolated based
on its communication-enhancement response to elevation of cAMP. Cells
were grown as described previously (Wang and Mehta, 1995).Transfection of Cells
The expression
construct (pSGRcx43A) was made by inserting cx43 cDNA from rat
heart (Beyer et al., 1987) into the BamHI site of
plasmid pSG5 (Stratagene) as described in Mehta et al. (1991).
In pSGRcx43A, cx43 expression is driven by the SV40 promoter.
Subconfluent cells were harvested and cotransfected by electroporation
with pSGRcx43A and the expression vector for the geneticin resistance
gene, pMC1neo-poly(A) (Stratagene). Cells were then cultured in medium
containing 400 µg/ml geneticin (G418), and G418-resistant colonies
were isolated, propagated in 200 µg/ml G418, and screened by
Western blots for the expression of Cx43 protein.Treatments
Cells were seeded at 2 
10
/35-mm dish. 2 days later, when near confluent, they were
treated for experiments by replacing their medium with fresh medium
containing the relevant drug at the desired concentration. Forskolin,
Ro-20-1724, and tunicamycin were added from stock solutions in
dimethyl sulfoxide (Me
SO), with final MeSO not
exceeding 0.4%, a concentration which did not affect any of the
parameters we measured. For controls, cultures received fresh medium
containing 0.4% MeSO.Cell-Cell Transfer of Lucifer
Yellow
Micro-injection of fluorescent dye Lucifer Yellow
was performed as described (Wang and Mehta, 1995). The number of
fluorescent cells (excluding the injected one) was noted 5 min after
injection.Immunostaining and Lectin Binding to Cell
Surface
Immunostaining with affinity-purified anti-Cx43
antibody and surface binding of rhodamine-labeled lectins were
performed as described (Wang and Mehta, 1995). Stained cells were
viewed on a Nikon Diaphot fluorescence microscope with a 100 oil
immersion objective (for Cx43 immunostaining) or 40 objective
(for lectin binding). Images were photographed or captured on an
optical disk (Panasonic model TQ-2026F) with an SIT66 (DAGE MTI) video
camera and reproduced on a video printer (Hitachi).Western Blot
Lysis of cells, protein
separation by SDS-PAGE, and Western blot analysis for Cx43 were
performed as described (Wang and Mehta, 1995). The protein
concentration was determined with the Pierce BCA protein assay.Dephosphorylation of Cx43 by Alkaline
Phosphatase
After appropriate treatment, cells were lysed
in alkaline phosphatase buffer (100 mM Tris, pH 8.0, 100
mM NaCl, 5 mM MgCl) plus 2 mM PMSF and 0.6% SDS. The lysates were boiled, sheared with 27-gauge
needles, and diluted with 4 volumes of alkaline phosphatase buffer.
Half of the sample was treated with alkaline phosphatase (200 units/ml
sample) at 37 °C for 4 h, and the other half was incubated
untreated. The reaction was terminated by adding Laemmli sample buffer
and boiling for 5 min. Phosphatase-treated and untreated samples were
separated by SDS-PAGE and analyzed by Western blot.Separation of Triton X-100 Soluble and Insoluble
Fractions
The separation of Triton X-100 soluble and
insoluble material was done essentially according to the method of
Musil and Goodenough(1991). After appropriate treatment, cells from
6-cm dishes were scraped into 4 ml of phosphate-buffered saline
containing 2 mM PMSF, 10 mM NaF, 10 mM NEM.
Cells were then spun down, resuspended in 1 ml lysis buffer (5 mM Tris base, 5 mM EGTA, 5 mM EDTA plus 2 mM PMSF, 10 mM NaF, 10 mM NEM), incubated at 4
°C for 10 min, and disrupted by passing through a 25-gauge needle
25-30 times. The resulting cell lysates were brought to
isotonicity by addition of 100 µl of 10 phosphate-buffered
saline. Then 10% Triton X-100 was added to a final concentration of 1%
(v/v). The lysates were incubated at 4 °C for 30 min. One-third of
the samples (400 µl) was saved as total cell lysate, and the rest
(800 µl) was centrifuged at 100,000 g for 50 min
at 4 °C. After centrifugation, the supernatant (Triton X-100
soluble fraction) was carefully removed. 4 Laemmli buffer was
added to it and the total cell lysate to a final 1
concentration. The pellet (Triton X-100 insoluble fraction) was
resuspended with 1067 µl of lysis buffer containing 2 mM PMSF, 1 phosphate-buffered saline, 1% Triton X-100, and 1
Laemmli buffer. Equal volumes of total, Triton X-100 soluble,
and insoluble proteins were separated by SDS-PAGE, and Cx43 was
analyzed by Western blot.
Overexpression of Cx43
We used Morris
hepatoma H5123 cells (Borek et al., 1969), which express a low
level of Cx43 mRNA but neither Cx26 nor Cx32 mRNA (Mehta et
al., 1992). We transfected a subclone of Morris hepatoma cells,
MHD1 (see ``Experimental Procedures''), with cx43 cDNA from rat heart (Beyer et al., 1987). Several
overexpressing clones were obtained, and three of them (MHD1-43A,
-B, and -C) were used in this study. They all gave the same results.
SO, 8 h); lane 2, Tm-treated cells
(4 µg/ml, 8 h). C, effect of Tm on Cx43 in MHD1-43A
cells. Lane 1, control; lane 2, Tm. D,
dephosphorylation of Cx43 from Tm-treated MHD1-43A cells by
alkaline phosphatase. Lysates treated with (lane 1) and
without (lane 2) alkaline phosphatase. The positions of
molecular mass markers (in kDa) are indicated on the left for
Western blots A and B and on the right for
Western blots C and D (30 µg total
protein/lane).
Connexin-43 Overexpression Induces Few Open
Channels
Morris hepatoma cells lack gap junctional
communication (open cell-to-cell channels) (Borek et al.,
1969) and so do the cells of the subclone MHD1 (Wang and Mehta, 1995).
We tested junctional communication in three clones of Cx43
overexpressing cells by micro-injecting the channel-permeable
fluorescent tracer, Lucifer Yellow. Despite their high level of Cx43
protein, the overexpressors have few open channels; in most cases, the
tracer remained confined to the injected cells. A typical example for
MHD1-43A cells is shown in Fig. 2A (see also Fig. 2B).
SO, 8 h). Panels c and d, tunicamycin (4 µg/ml, 8 h). Phase contrast (a, c) and fluorescent (b, d)
images are shown. Photographs were taken 5 min after injection of
Lucifer Yellow into the asterisk-marked cells. B,
time course of the effect of tunicamycin and cycloheximide on
communication. Each data point represents the mean ±
S.E. from 30-50 injections in three experiments. Tm, 4 µg/ml;
cycloheximide (CHX), 60 µg/ml.
Inhibition of Glycosylation by Tunicamycin Reduces
Surface Carbohydrates and Induces the Formation of Open
Channels
Surface carbohydrates may have an inhibitory
effect on channel formation (Lin and Levitan, 1987; Levine et
al., 1991), and a reduction of surface carbohydrates by inhibition
of glycosylation has been found to correlate with increased channel
formation in several cell types (Wang and Mehta, 1995). We examined the
effect of a glycosylation inhibitor, tunicamycin (Tm) on the abundance
of surface carbohydrates and on communication in the Cx43
overexpressors. As in parental MHD1 cells (Wang and Mehta, 1995), Tm
greatly reduced surface carbohydrates as detected by lectin binding to
the cell surface (Fig. 3). Concomitant with this decrease, there
was a dramatic increase in communication; after an 8-h tunicamycin
treatment, Lucifer Yellow consistently transferred to more than 20
neighboring cells (Fig. 2A, panel d, and
2B). The time course of the communication increase in clones
MHD1-43A, -B, and -C is shown in Fig. 2B. After a
2-h delay, communication rose steadily to a maximum at 8-10 h.
This result is different from that obtained with the parental MHD1
cells where tunicamycin per se failed to induce communication
(Wang and Mehta, 1995).
Tunicamycin Treatment Induces Cx43 Phosphorylation
and an Increase in Total Cx43
To find out whether the
Tm-induced formation of open channels is associated with any
biochemical changes in Cx43, we compared the protein from treated and
untreated MHD1-43A cells in Western blots. One dramatic change
was the appearance of Cx43 of higher molecular mass in Tm-treated cells (Fig. 1C), representing phosphorylated Cx43; it
disappeared when cell lysates were treated with alkaline phosphatase (Fig. 1D). A second prominent change was an increase in
total Cx43 level (Fig. 1C). In contrast, no such
changes were seen in Cx43 from MHD1 cells after tunicamycin treatment (Fig. 1B).Tunicamycin Treatment Induces Triton X-100 Insoluble
Cx43
Musil and Goodenough(1991) have shown that in some
communication-incompetent cells, Cx43 is mainly non-phosphorylated and
Triton X-100 soluble. When these cells were made
communication-competent by expression of cell-cell adhesion molecules,
a form of Cx43 appeared that was phosphorylated and Triton X-100
insoluble. Since tunicamycin treatment induced communication and
phosphorylation of Cx43 in Cx43-overexpressing cells but not in the
parental MHD1 cells, we examined Cx43 Triton X-100 solubility in both
cells before and after tunicamycin treatment to see whether
communication correlated with Cx43 phosphorylation and Triton X-100
insoluble Cx43.
Tunicamycin Treatment Does Not Induce Channel
Clustering into Gap Junctional Plaques
In immunostaining of
communication-competent cells for cell-to-cell channel proteins,
punctate staining is seen at cell-cell contacts, representing the
aggregated channels in gap junction plaques (Dermietzel et
al., 1987; Beyer et al., 1990; Musil and Goodenough,
1991). No such punctate Cx43 staining was detected in untreated MHD1 or
Cx43 overexpressing cells (Fig. 6, a, d, and g). Tunicamycin did not induce punctate Cx43 plaque staining
in MHD1 cells (Fig. 6b) nor to a significant extent in
MHD1-43A or MHD1-43B cells; the Cx43 was still diffusely
distributed except that very fine dots were occasionally seen,
including on top of the cells where no cell-cell contacts could be
detected under the light microscope (Fig. 6, e and h).
)
We thank Dr. Werner R. Loewenstein for encouragement,
Tomas Lopez for technical help, and Dr. Dongyan Zhao (Dept. of
Microbiology and Immunology, University of Miami) for helpful
suggestions, especially in regard to transfection.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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