Department of Medicine, Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.
Gap junction channels mediate intercellular communication by allowing the direct transfer of ions and
small aqueous molecules between neighboring
cells. Gap junction channel proteins, or connexins, have
been identified with sizes in the range of 26-56 kD (for reviews see 6, 7, 17, 21, 32). Channels composed of different connexins have distinct properties, and most tissues express more than one connexin. There is increasing evidence that multiple connexins can interact to form heteromeric gap junction channels (25, 52).
Regulation at the level of gene expression is clearly one
important way that gap junction composition can be regulated (5, 10, 12, 14, 31, 50, 58). Connexin transport and
assembly into gap junction channels are other possible
points where cells can regulate the formation of gap junction channels (17, 38). A number of studies have shown
that newly synthesized connexins are transported through
the normal secretory apparatus (18, 39, 40, 46). However,
in contrast with most multimeric membrane protein complexes that are formed in the ER (3, 13, 28), the identity of
the site of connexin oligomerization remains controversial (32). Both ER (33) and Golgi apparatus (41) have been
suggested as sites for connexin assembly into gap junction
channels or hemichannels.
In previous studies we have examined the expression of
connexins in human and rat osteoblastic cells (11, 30, 53).
All of the cells examined express Cx43 ( Cx46 expression has typically been associated with
plasma membrane gap junction channels in lens (14, 22,
25, 29, 43, 49, 57). We found that Cx46 was expressed by
primary rat osteoblastic cells and two osteosarcoma cell
lines, ROS-17/2.8 (ROS) and UMR 106-01 (UMR) cells.
In contrast with Cx43 and Cx45, Cx46 was largely retained
in an intracellular perinuclear compartment. Little, if any,
Cx46 accumulated on the cell surface, as determined by
immunofluorescence microscopy. Instead, Cx46 was retained in a trans-aspect of the Golgi apparatus, probably
the TGN. In contrast, we found that Hela cells stably
transfected with rat Cx46 (Hela/Cx46) formed functional
gap junction channels by Cx46 at the plasma membrane.
In addition to the differential transport of Cx43 and
Cx46 by ROS cells, these cells also showed differences in
connexin assembly. While Cx43 was assembled into hexameric gap junction hemichannels by ROS cells, the intracellular pool of Cx46 remained as unassembled monomers. Thus, transport and assembly of Cx43 and Cx46 are
differentially regulated by osteoblastic cells. These data
are discussed in the context of potential roles for connexin
oligomerization in the regulation of transport.
Cells
ROS 17/2.8 and UMR 106-01 cells were cultured in MEM (No. 11095-056;
GIBCO BRL, Gaithersberg, MD) containing 10% heat-inactivated bovine calf serum (BCS)1 (Hyclone, Logan, UT), 2 mM glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids (GIBCO BRL), 5 U/ml penicillin, and 5 µg/ml streptomycin (MEM + BCS). Primary osteoblastic cells
were isolated from fetal rat calvaria as previously described (51) and cultured in MEM + BCS for no more than six passages. Hela cells were
kindly provided by Jeanette Pingle and Matthew Thomas (Washington
University, St. Louis, MO).
RNA Blots
Total cellular RNA was isolated using guanidium isothiocyanate and
probed for rat Cx46 and human Antibodies
A number of immunological reagents were generous gifts from the following researchers. Tissue-culture supernatant from murine hybridoma cells
producing anti-Cx50 (MP70; Rabbit anti-Cx46 IgG was produced using a 6His-tagged Cx46 fusion
protein. A cDNA consisting of the COOH-terminal tail sequence of rat
Cx46 was produced by PCR amplification using a full-length rat Cx46
cDNA as a template (sense primer: 5 Polyclonal serum that recognized Cx46 was initially identified by immunoblot analysis of rat lens total protein preparations that showed reactive bands with the expected Mr at 53 and 68 kD, consistent with previous
reports (26, 29, 57). Preincubation of anti-Cx46 antiserum with antigen
eliminated binding to both bands in samples prepared from lens and ROS
cells (see Fig. 3, lanes 2 and 4). The perinuclear staining pattern obtained
for osteoblasts labeled with anti-Cx46 (e.g., see Fig. 2) was eliminated by
preincubation of the antiserum with Cx46-his protein (not shown). Also,
cells that do not express Cx46 did not show any labeling by immunofluorescence or immunoblotting (see Fig. 4).
Immunofluorescence Microscopy
Cells were initially cultured on glass coverslips for 1-3 d before examination. The cells were washed twice in PBS, and then fixed and permeabilized for 2 min at room temperature using methanol/acetone (1:1). The
cells were washed once with PBS containing 0.5% Triton X-100 and then
twice with PBS containing 0.5% Triton X-100 and 2% heat-inactivated
normal goat serum (PBS/TX/HIGS). The cells were then incubated with
primary antiserum diluted to optimal titer in PBS/TX/HIGS for 45-60 min
at room temperature. After washing three times with PBS/TX/HIGS, the
cells were further incubated with fluorescent anti-IgG diluted in PBS/TX/
HIGS for 45-60 min. The cells were then washed and viewed by fluorescence microscopy using appropriate optics. For Cx46 labeling, anti-Cx46
antiserum was diluted 1:1,000 into PBS/TX/HIGS, and the secondary antiserum was rhodamine-conjugated goat anti-rabbit IgG (Boehringer Mannheim Biochemicals, Indianapolis, IN) diluted 1:500 in PBS/TX/HIGS. For
double labeling experiments, Cx43 was labeled using monoclonal antiserum from Zymed Laboratories (South San Francisco, CA), and staining
with fluorescent lectins was substituted for labeling with antisera to visualize some organelles. In some instances, images were obtained with a BioRad MRC-1000 confocal fluorescence microscopy system (Hercules, CA);
all other images were obtained by epifluorescence microscopy using a
Zeiss Axioscope (Thornwood, NY) and GIPSSPC image processing system (Georgia Instruments, Roswell, GA).
Protein Preparation and Immunoblotting
Total cell protein samples were prepared as previously described (30) and
resolved by SDS-PAGE using standard methods and 10% polyacrylamide
gels. The proteins were then electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (transfer buffer: 50 mM Tris, 380 mM glycine, 0.025% (wt/vol) SDS, 20% MeOH), blocked with blotto (40 mM Tris, 5% (wt/vol) Carnation powdered milk, 0.1% (vol/vol) Tween20) for 1 h at room temperature, and then blocked overnight with antiserum diluted into blotto. The membranes were then washed, and specific
bands were detected using HRP-conjugated goat anti-rabbit IgG (Tago,
Burlingame, CA) and enhanced chemiluminescence (ECL; Amersham Intl.,
Little Chalfont, UK). Cx50 immunoblots were detected using peroxidaseconjugated goat anti-mouse IgG + IgM (Boehringer Mannheim Biochemicals). After a series of exposures, films showing unsaturated bands were digitized and quantitated using an image analysis system (Georgia Instruments).
Sucrose Gradient Velocity Centrifugation
This procedure was adapted from that of Musil and Goodenough (40).
Cells cultured for 1-3 d in 100-mm tissue-culture dishes were washed
twice with PBS, and then scraped into PBS at 4°C and centrifuged at 500 g
for 5 min. The cell pellet was resuspended in 2.75 ml incubation buffer
(0.14 M NaCl, 5.3 mM KCl, 0.35 mM Na2PO4, 0.35 mM KH2PO4, 0.8 mM
MgCl2, 2.7 mM Hepes, pH 7.4) containing protease (10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonylchloride, 2 µg/ml leupeptin, 1 µg/ml
pepstatin) and phosphatase inhibitors (1 mM NaVO4, 10 mM NaF) at 4°C
and homogenized with a ball-bearing cell homogenizer (1). The preparation was then centrifuged at 500 g for 5 min to obtain a postnuclear supernatant and brought to 1% Triton X-100. After a 30-min incubation at 4°C,
the sample was centrifuged at 100,000 g for 30 min to remove Triton-insoluble material, and then layered onto a 3.6-ml 5-20% sucrose gradient in
incubation buffer + 0.1% Triton X-100 on top of a 0.2-ml 25% sucrose
cushion. Samples from intact lens were treated in a similar manner, except
that they were minced with a razor blade and incubated in 1% Triton X-100
for 12 h. The gradient was centrifuged using an SW55 rotor in a model L7-55
ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA) for 16 h at
148,000 g. After centrifugation, samples were collected from the bottom of
the tube and diluted 1:1 with denaturing SDS sample buffer. Connexins
were detected by immunoblotting as described above.
Immunoprecipitation
ROS cells were incubated in methionine-deficient MEM containing 10%
dialyzed BCS for 1 h at 37°C, and then labeled for 5 h at 37°C with 300 µCi
per dish 35S-Trans label (ICN Pharmaceuticals, Costa Mesa, CA). The
cells were then harvested, and Triton X-100-soluble and -insoluble fractions were prepared as described above. The insoluble pool was resuspended, both fractions were diluted with PBS containing 0.25% BSA and 0.2% gelatin, and SDS was added to 0.5% final concentration. The samples were precleared with protein A-Sepharose and preimmune serum for
90 min at room temperature, and then incubated overnight with specific antiserum and protein A-Sepharose that was preblocked with BSA and gelatin. The beads were washed, resuspended in reducing SDS-PAGE sample
buffer, and heated to 68°C to release proteins associated with the beads.
Transfection
Rat Cx46 cDNA was excised with EcoRI and inserted into the expression
vector pSFFVneo (8). Cx46/pSFFVneo was transfected into Hela cells by
calcium phosphate precipitation. The cells were incubated overnight at
37°C, and then shocked by treatment with 15% glycerol for 2 min. The
cells were then washed and plated in selective medium containing 1 mg/ml
G-418. Clones of G-418-resistant cells expressing Cx46 were identified by
immunofluorescence and immunoblotting.
Cx46 Is Expressed by Osteoblasts
By Northern blot analysis, both ROS and UMR cells were
found to express Cx46 mRNA (Fig. 1). The intracellular
distribution of Cx43 and Cx46 was examined by indirect
immunofluorescence (Fig. 2). Consistent with our previous
work (30, 53), ROS and primary rat calvarial cells showed
high levels of Cx43 expression localized to areas where the
cells were in close contact, corresponding to gap junctions.
While UMR cells express relatively low levels of Cx43 as
compared with ROS cells (53), we were able to locate areas where Cx43 was immunolocalized to the cell surface
(Fig. 2 c). In contrast, all three cell types showed expression of Cx46. In each case, Cx46 was predominantly localized to an intracellular compartment in the perinuclear region of the cell.
Lens cells typically show the highest levels of Cx46 expression (14, 22, 25, 29, 43, 49, 57). When total lens protein
extracts were examined by Western blotting, two forms of
Cx46 were found with Mr of 68 and 53 kD (Fig. 3, lanes 1 and 5). This is consistent with previous reports, where the
68-kD band corresponds to a phosphorylated form of
Cx46 (26, 29) and may also have other posttranslational
modifications (29). In contrast, immunoblots of total proteins isolated from ROS, UMR, and primary osteoblastic
cells derived from rat calvaria showed that these cells expressed only the 53-kD form of Cx46. This finding suggests
that Cx46 expressed by osteoblastic cells is likely to be
nonphosphorylated, although low levels of Cx46 phosphorylation cannot be ruled out. Osteoblasts do not show a general deficiency in connexin phosphorylation, since multiple forms of Cx43 corresponding to phosphorylated species were observed in immunoblots of total protein samples from ROS cells (Fig. 3, lane 9) (38, 39). Also, ROS
cells showed qualitatively similar amounts of Cx43 and
Cx46 expression, as determined by immunoblotting (Fig.
3, lanes 9 and 10).
Elfgang et al. (16) found that Hela cells were highly deficient in connexin expression. We confirmed by Western
blotting and immunofluorescence that wild-type Hela cells
did not express Cx46 (Fig. 4), Cx43, or Cx50 (not shown).
Thus, Hela cells were stably transfected with rat Cx46
cDNA (Hela/Cx46) to examine the targeting of Cx46 in
nonosteoblastic cells.
In contrast with osteoblasts, Cx46 was present at the cell
surface of Hela/Cx46 cells, as well as in intracellular compartments (Fig. 4 c). While these cells were not extensively
coupled, Cx46 expressed by Hela/Cx46 cells formed functional gap junction channels, as determined by intercellular transfer of microinjected Lucifer yellow (Fig. 5). Texas
red ovalbumin did not transfer between Hela/Cx46 cells.
This indicates that retention of Cx46 in an intracellular compartment depends upon the cell type examined. Furthermore, since Cx46 was readily detected at the surface of
Hela/Cx46 cells, it is likely that accumulation of appreciable amounts of Cx46 on the osteoblast cell surface would
be visible by immunofluorescence microscopy. However,
this does not rule out the possibility that Cx46 is transported to the cell surface and rapidly cleared by reinternalization.
The expression of Cx46 by transfected Hela cells was
also examined by immunoblotting (Fig. 4 a). Hela/Cx46
cells showed both the 68- and 53-kD forms of Cx46 that
are found associated with lens. This contrasts with results
obtained with osteoblastic cells, where only the lower molecular mass form was present (Fig. 3). Thus, the ability to
produce the 68-kD form of Cx46 correlated with the ability to form gap junctions containing Cx46. Whether one
process is a prerequisite for the other is not known at
present.
Cx46 Is Retained in the TGN
The identity of the intracellular compartment containing
Cx46 was determined with a series of fluorescence colocalization experiments in ROS cells. To label endocytic
compartments, cells were incubated at 37°C for 1 h with
medium containing the fluorescent fluid phase marker, Lucifer yellow. ROS cells labeled in this manner were fixed
and immunostained for Cx46, and then examined by fluorescence microscopy. In these double-labeled cells, fluorescently labeled endocytic compartments and intracellular vesicles containing Cx46 showed distinct intracellular
distributions (Fig. 6, a and b).
ER in fixed and permeabilized cells was preferentially
labeled using a fluorescent lectin, fluorescein-conjugated
conconavalin A (FITC-ConA), which recognizes the high
mannose core oligosaccharide component of glycoproteins. Areas of the ER that are prominently labeled with
FITC-ConA, such as the nuclear envelope and lattices in
the periphery of the cell, showed little labeling for Cx46 (Fig. 6, c and d).
The Cx46-containing compartment in ROS cells colocalized with the Golgi apparatus by double-labeling immunofluorescence using a marker for the medial-Golgi stack
(anti-GCI) (9). As shown in Fig. 7, a and b, both the antiGCI and Cx46 showed prominent perinuclear localization.
However, these two proteins were in distinct compartments. This was revealed by treatment of the cells with
brefeldin A (BFA), which preferentially disassembles cis and medial aspects of the Golgi apparatus (54). When
ROS cells were preincubated for 5 min with BFA and then
fixed and examined by immunofluorescence, the GCI
marker redistributed to the periphery of the cell, while the
Cx46 staining pattern was relatively intact (Fig. 7, c and d).
This suggests that Cx46 was located in a secretory compartment beyond the medial Golgi, either the trans-Golgi
cistern or the TGN.
To further examine this possibility, we performed colocalization experiments using a monoclonal anti-rat TGN38
antibody (24). As shown in Fig. 7, e and f, both Cx46 and
TGN38 colocalize to the perinuclear region of the cell. The
distribution of these two proteins was not affected by shortterm treatment with BFA (not shown). However, in cells
incubated for 30 min in the presence of BFA, both Cx46
and TGN38 condensed into the same concentrated spot in
the nuclear region (Fig. 7, g and h). This is similar to the
pattern of TGN38 labeling in normal rat kidney (NRK)
cells treated for 30-60 min with BFA (34, 48). Taken together, these observations suggest that the intracellular
pool of Cx46 likely corresponds to an aspect of the TGN.
We attempted to stimulate the transport of Cx46 to the
plasma membrane by plating osteoblastic cells under a variety of culture conditions. Under the conditions examined, little, if any, Cx46 was found to be localized to the
cell surface. We also incubated ROS cells for short periods
(2-12 h) before immunofluorescence labeling with a number of agents known to affect bone cell metabolism (47),
such as Osteoblasts Retain Cx46 as a Monomer
To examine the assembly of Cx43 and Cx46, we used techniques previously described by Musil and Goodenough
(40) using 1% Triton X-100 at 4°C, conditions that enable
the solubilization of intact Cx43 hexamers in other cell
types. Cx43 and Cx46 were solubilized to different extents
(Fig. 8). Note that Cx43 already assembled into gap junctional plaques is resistant to solubilization under these
conditions (39). As determined by immunoprecipitation analysis, ~50% of the Cx43 in ROS cells was soluble in
1% Triton X-100 at 4°C, while virtually all of the Cx46 was
solubilized under the same conditions.
Detergent-extracted connexin preparations were resolved into monomers and multimers by sucrose gradient
velocity sedimentation (Fig. 9). Cx43 from ROS cells resolved into two fractions. The major fraction centered at
8% sucrose corresponds to Cx43 monomers, while the
peak at 15% sucrose corresponds to multimers. Multimeric Cx43 consisted of 21.4 ± 2.8% (n = 3) of the total Triton
X-100-soluble Cx43. The preponderance of Cx43 monomers over hexamers was expected, since the Triton-soluble fraction of Cx43 is likely to contain mostly newly synthesized Cx43 (39, 40). However, some monomeric Cx43
may be due to spontaneous disassembly of hexameric Cx43
during the course of the experiment.
Treatment of NRK cells with BFA inhibits the oligomerization of Cx43, suggesting that Cx43 is assembled into
oligomers in a late Golgi apparatus compartment (40). If
this is the case, then treatment of ROS cells with monensin, which inhibits cis to medial transport through the
Golgi apparatus, should also inhibit Cx43 oligomerization.
As shown by immunofluorescence microscopy in Fig. 10,
monensin-treated ROS cells showed Cx43 and Cx46 retained in a similar perinuclear region of the cell. This is
consistent with results obtained with monensin-treated
cardiac myocytes (45). When oligomerization of Cx43 was
examined in monensin-treated cells, only 8% (n = 2) of
the Triton-soluble Cx43 isolated from monensin-treated
cells was assembled into multimers, as compared with control cells that had two- to threefold more Cx43 in the hemichannel form (Fig. 11). Also, since we could detect
this difference in the amount of Cx43 assembled into
hemichannels, it is likely that nonspecific aggregation of
Cx43 did not occur during the solubilization and sucrose
gradient fractionation procedure.
In contrast with Cx43, Cx46 solubilized from ROS cells
migrated as a single peak centered at 8% sucrose (Fig. 9 b).
Since nearly all of the Cx46 was soluble in 1% Triton X-100,
this suggests that all of the Cx46 was being retained by
ROS cells in a monomeric form.
One possible concern in using Triton X-100-solubilized
preparations is that Cx46-containing oligomers may not be
stable under these conditions. To examine this possibility,
we prepared Triton X-100-soluble extracts from both
Hela/Cx46 cells and whole rat lens. While Hela/Cx46 cells
were solubilized using the same conditions used for ROS
cells, extensive incubation with 1% Triton X-100 at 4°C
was required to solubilize detectable amounts of material from rat lens. Both the 53- and 68-kD forms of Cx46 were
solubilized under these conditions (Figs. 12 and 13).
When Triton X-100-soluble extracts from lens were examined by sucrose gradient fractionation, the majority of
the Cx46 was found to be in oligomeric forms (Fig. 12 a).
We also examined this gradient for Cx50, another abundant lens connexin (14, 15). The profile for Cx50 was very
comparable to that observed for Cx46 in lens (Fig. 12 b).
Similar results were obtained by sucrose gradient analysis
of Cx46 solubilized from Hela/Cx46 cells, although the
peak at 14-18% was significantly reduced (Fig. 13 a). The
sucrose gradient profiles of Cx46 isolated from lens and
Hela/Cx46 cells are consistent with the notion that multimers containing Cx46 were largely stable in the presence
of Triton X-100. This is further supported by Fig. 13 b,
where Cx46 extracted from Hela/Cx46 cells and then dissociated by treatment with 0.2% SDS migrated on the gradient as a single peak at 8-9% sucrose.
In this paper we have examined the expression of Cx46 by
osteoblastic, lens, and transfected Hela cells. Osteoblastic
cells retained Cx46 as a monomer in an intracellular compartment that is likely to be an aspect of the TGN. In contrast, Cx46 was assembled into oligomers by lens and
Hela/Cx46 cells and transported to the cell surface. This
raises the possibility that connexin assembly and transport
are interlinked and regulated processes.
Connexins are assembled into a hemichannel in an intracellular compartment before transport to the plasma
membrane (33, 40). This hemichannel then pairs with another hemichannel on a neighboring cell to form a complete gap junction channel. There is evidence supporting
both ER and Golgi apparatus as sites for connexin oligomerization. Kumar et al. (33) have found by EM that BHK
cells transfected with Cx32 ( We used sucrose gradient fractionation to examine the
oligomerization state of Cx43 and Cx46 present in Triton
X-100 extracts. Treatment of ROS cells with monensin,
which inhibited cis- to medial-Golgi transport, also reduced Cx43 hemichannel formation by >62% (Fig. 10).
While this evidence is not conclusive, it is consistent with a
trans-Golgi compartment as the site of Cx43 oligomerization in this system. Since Cx43 assembly was not completely inhibited, it is possible that Cx43 hemichannels
may also be assembled in the ER. Alternatively, some of
the hemichannels that we detected in the presence of monensin may be due to Cx43 disassembled from gap junctions
that is en route to degradation.
We found that Cx46 oligomers were stable in Triton X-100
(Figs. 12 and 13). Sucrose gradients have also been used to
analyze connexin channels isolated from ovine lens (25).
Analogous to chick, ovine, and bovine lens (25, 29), it is
likely that most hemichannels isolated from rat lens contained Cx50 in addition to Cx46. Preliminary analysis of
ROS and UMR cells indicates that these cells do not express Cx50 (not shown). An absolute requirement for
Cx50 is not likely for Cx46 incorporation into gap junction channels, since we found that Hela/Cx46 cells were able to
form functional gap junctions in the absence of Cx50, albeit at relatively low levels. Formation of gap junctions
purely from Cx46 has also been described in other systems
(59, 60).
Cx46 was retained in a monomer, rather than a hemichannel form, by ROS cells. Based on colocalization experiments shown above, it seems likely that osteoblasts
transported Cx46 to compartments where connexin oligomerization occurs. This is consistent with a requirement
for connexin oligomerization as a prerequisite for transport from the TGN and/or retention at the cell surface.
The notion of quality control at the level of the Golgi apparatus is supported by the observation that some mutant
viral glycoproteins exit the ER without being properly assembled, but are retained in the Golgi apparatus (20, 42).
Immunofluorescence was used to determine whether
Cx46 was present at the plasma membrane of osteoblastic
cells. This technique does not completely rule out the possibility that Cx46 transport to the cell surface occurs, but
suggests that it is rapidly reinternalized after delivery to
the plasma membrane. TGN38 (48) and furin (37, 56) are
examples of proteins that continuously recycle at low levels between the TGN and plasma membrane, yet are not readily detectable at the cell surface by conventional indirect immunofluorescence. Furthermore, the communication-deficient cell lines L929 and S180, which do not show
accumulation of Cx43 at the plasma membrane by immunofluorescence (41), contain a pool of Cx43 that is accessible by surface biotinylation (39). Interestingly, S180 and
L929 cells show low levels of Cx43 phosphorylation (41), analogous to our observations with Cx46 in osteoblasts.
However, these situations are not completely comparable,
since both S180 and L929 cells assemble Cx43 into hemichannels (40). Cx43 hemichannels at the cell surface have
also been observed in other systems (35).
It is also possible that Cx46 is transported by osteoblasts
along a regulated secretory pathway that is distinct from
the transport pathway for Cx43. Rat uterine myometrial cells
provide a precedent for the stimulated transport of connexins to form gap junctions (2, 10, 19, 23, 44). Whether
the intracellular compartment containing Cx46 is a regulated secretory compartment is not known at present.
However, storage vesicles that accumulate other transmembrane proteins subject to stimulated transport to the
plasma membrane, such as GLUT4, accumulate in the
perinuclear region in other cell types (27, 36).
Since Cx43 and Cx46 had distinct intracellular distributions in osteoblastic cells, this indicates that these two connexins were not randomly intermixing in these cells. It is
not known whether Cx43 and Cx46 can form heteromeric
channels in other systems, and there is no a priori reason
that these two Since Cx43 and Cx46 have different fates in osteoblastic
cells, our data support the notion that connexin transport
is a specific process and not due to the bulk flow of connexins along a "default" secretory pathway. Also, oligomerization seems to be interconnected with connexin targeting and transport to the cell surface. Further work will
be required to find structural determinants that either target connexins to specific intracellular locations or control connexin transport at the level of gap junction channel assembly.
1) in junctional
plaques at the cell surface. In addition, some osteoblasts
also produce Cx45 (
6), which also shows plasma membrane localization (53). In this paper we have characterized a third endogenous connexin expressed by rat osteoblastic cells, Cx46 (
3).
Materials and Methods
-actin as previously described (30).
8) was from Drs. J. Kistler (University of
Auckland, New Zealand) and D. Goodenough (Harvard University, Boston, MA) (14, 15). Tissue-culture supernatant containing monoclonal murine anti-rat TGN38 was from Dr. G. Banting (University of Bristol, UK)
(24). Production of rabbit polyclonal antiserum that recognizes Cx43 was
previously described (4).
-CGAGA GCGAC ATATG CTAGA
GATTT ACCAC CT-3
/antisense primer: 5
-AGCGA GCGGA TCCTT TCTAC CTGTT GATTT GA-3
). The resulting PCR product was amplified and inserted into the pET-15b vector (Novagen, Madison, WI) to produce a bacterial expression vector containing cDNA encoding for a fusion
protein containing the Cx46 COOH terminus, a thrombin cleavage site,
and six histidine residues (Cx46-his). Bacterial expression of Cx46-his was
induced by treatment with isopropylthio-
-d-galactoside, cell extracts
were prepared, and Cx46-his was affinity purified using a nickel/His-Bind
chelation resin column (Novagen). This product was used to induce polyclonal antiserum in rabbits by established protocols.
Fig. 3.
Immunoblot for Cx46 in lens and osteoblasts. (Lanes 1-4)
Samples from rat lens (lanes 1 and 2) and ROS (lanes 3 and 4)
were resolved by SDS-PAGE, electrophoretically transferred to
PVDF membranes, and then immunoblotted for Cx46. For lanes
2 and 4, the antiserum was preincubated with Cx46-his before immunoblotting, which blocked the recognition of Cx46. (Lanes 5-8)
Samples from rat lens (lane 5), ROS (lane 6), UMR (lane 7), and
primary rat calvarial cells (lane 8) were normalized for total protein and immunoblotted for Cx46. All three osteoblastic cell
types expressed the 53-kD form of Cx46. (Lanes 9 and 10) Samples from ROS cells corresponding to total cell protein were immunoblotted for either Cx43 (lane 9) or Cx46 (lane 10). Roughly
10-fold more protein was loaded for these lanes than the corresponding sample in lane 6. Migration of molecular weight standards is indicated in the figure, and dots correspond to specific
protein bands.
[View Larger Version of this Image (60K GIF file)]
Fig. 2.
Intracellular distribution of Cx46 in osteoblasts. (a and
b) Colocalization. ROS cells on glass coverslips were fixed, permeabilized, and double immunolabeled with monoclonal anti-
Cx43 mouse IgG and polyclonal anti-Cx46 rabbit IgG. The cells
were then labeled with rhodamine-conjugated goat anti-mouse
IgG and FITC-conjugated goat anti-rabbit IgG to visualize Cx43
(a) and Cx46 (b) by confocal microscopy. (Arrowheads) Cx43 localized to the cell surface; (arrows) Cx46 in the perinuclear region of the cell. (c-f) Single label localization. UMR cells (c and
d) or rat calvarial cells (e and f) on glass coverslips were fixed,
permeabilized, and immunolabeled with either anti-Cx43 (c and
e) or anti-Cx46 (d and f) antiserum and rhodamine-conjugated
goat anti-rabbit IgG. c-f were obtained by epifluorescence microscopy. These cells also show the characteristic perinuclear accumulation of Cx46. Note that the immunofluorescence shown in
c represents an area showing very high levels of Cx43 expression
by UMR cells. Bar, 10 µm.
[View Larger Version of this Image (87K GIF file)]
Fig. 4.
Expression of Cx46 by transfected Hela cells. (a) Samples corresponding to Hela cells (lane 1) and Hela/Cx46 cells
(lane 2) were normalized for total protein, resolved by SDS/PAGE,
electrophoretically transferred to PVDF, and then immunoblotted for Cx46. Faint nonspecific bands present in both lanes are
also observed in Western blots using preimmune serum (not
shown). (b and c) Untransfected Hela cells (b) or Hela/Cx46 cells
(c) plated on glass coverslips were fixed, permeabilized, and then
immunolabeled with anti-Cx46 and rhodamine-conjugated goat
anti-rabbit IgG. Both images were obtained using the same exposure conditions. Hela/Cx46 cells showed Cx46 present both on
the cell surface (arrows) and in the perinuclear region of the cell.
[View Larger Version of this Image (86K GIF file)]
Results
Fig. 1.
Northern blot analysis
of Cx46 mRNA. Total RNA was
isolated from either ROS (lanes
1 and 3) and UMR (lanes 2 and
4) cells, subjected to agarose gel
electrophoresis, transferred to
membranes, and then hybridized
with a radiolabeled cDNA probe
for Cx46 (lanes 1 and 2). The
membranes were then stripped
and reprobed for actin as a control for mRNA loading (lanes 3 and 4). Dashes correspond to
28S and 18S rRNA.
[View Larger Version of this Image (47K GIF file)]
Fig. 5.
Dye transfer by Hela/Cx46 cells. Untransfected Hela
cells and Hela/Cx46 cells were cultured on glass coverslips. (a and
b) Phase-contrast (a) and fluorescence (b) images of one nontransfected Hela cell in a monolayer that was microinjected with
10 mg/ml Lucifer yellow (*). Hela cells showed no intercellular
transfer of Lucifer yellow. (c-f) Hela/Cx46 cells were microinjected with a mixture of 10 mg/ml Lucifer yellow and 5 mg/ml
Texas red ovalbumin. While Texas red was retained in the microinjected cell (c and e), Hela/Cx46 cells showed intercellular transfer of Lucifer yellow to some nearest neighbors (d and f).
[View Larger Version of this Image (94K GIF file)]
Fig. 6.
Cx46 is not localized to either endocytic or ER compartments. (a and b) ROS cells on glass coverslips were incubated
for 1 h at 37°C in medium containing 10 mg/ml Lucifer yellow to
label endocytic compartments (a). The cells were then fixed with
paraformaldehyde, permeabilized using 0.2% Triton X-100,
immunostained for Cx46 (b), and visualized by epifluorescence
microscopy. The intracellular distribution of Lucifer yellow was
distinct from the pattern observed for Cx46. (c and d) ROS cells
on glass coverslips were fixed and permeabilized using methanol/
acetone (50/50) and labeled with FITC-ConA (c). The cells were
then immunostained using anti-Cx46 antiserum and rhodamineconjugated goat anti-rabbit IgG (d). c and d were obtained by
confocal microscopy. Colocalization between Cx46 and FITCConA, which preferentially labeled elements of the ER, was limited. In particular, note that low levels of Cx46 labeling were
present in the nuclear envelope (arrowhead).
[View Larger Version of this Image (128K GIF file)]
Fig. 7.
Cx46 is in a transGolgi compartment. (a and
b) ROS cells on glass coverslips were fixed, permeabilized, and immunolabeled with mAbs that recognize a
medial-Golgi protein (GCI).
The cells were also immunostained for Cx46 using polyclonal antiserum, and then
labeled with rhodamine-conjugated goat anti-mouse IgG
(a) and FITC-conjugated goat
anti-rabbit IgG (b). (c and d)
ROS cells on glass coverslips
were preincubated in MEM
containing 10 µg/ml brefeldin A (BFA) for 5 min at
37°C. The cells were then
fixed, permeabilized, and immunolabeled for GCI (c) and
Cx46 (d). a-d were obtained
by confocal microscopy. While BFA disrupts the medial Golgi, Cx46 remains in the perinuclear region (arrow). (e and f) ROS cells on
glass coverslips were fixed, permeabilized, and immunolabeled with mAbs that recognize TGN38 (e) and Cx46 using polyclonal antiserum (f). (g and h) ROS cells on glass coverslips were preincubated in MEM containing 10 µg/ml BFA for 30 min at 37°C. The cells were
then fixed, permeabilized, and immunolabeled for TGN38 (g) and Cx46 (h). e-h were obtained by epifluorescence microscopy. Under
these conditions, both proteins condense into a dot in the center of the cell (arrowheads).
[View Larger Version of this Image (86K GIF file)]
-glycerophosphate (2.3-5.8 mM) + ascorbate
(0.1-0.25 mg/ml), dexamethasone (10-250 µM), PMA (0.1 µM) ± bromo-A23187 (0.1 µg/ml), parathyroid hormone
(0.125-1.0 µM),
-estradiol (0.5-50 µg/ml), 1,25-dihydroxy-vitamin D3 (5-25 ng/ml), and dibutyryl-cAMP (0.1 mM), as well as a variety of plating densities and mechanical stress. None of these treatments altered the intracellular distribution of Cx46 in ROS cells.
Fig. 8.
Triton solubility of Cx43 and Cx46 in ROS cells. ROS
cells were metabolically radiolabeled with 35S-Trans label, harvested, homogenized, and solubilized in 1% Triton X-100 at either room temperature (lane 1) or 4°C (lanes 2-5) for 30 min. Triton-soluble (lanes 1, 2, and 4) and -insoluble (lanes 3 and 5)
fractions were analyzed by immunoprecipitation using antisera
that recognizes either Cx43 (lanes 1-3) or Cx46 (lanes 4 and 5).
Note that the Cx46 band (lane 4) was somewhat distorted due to
the presence of high levels of IgG heavy chain required for immunoprecipitation. At 4°C, Cx46 showed complete Triton X-100 solubility, while Cx43 was only partially soluble (lane 2). Migration of molecular weight standards is indicated in the figure, and dots correspond to specific protein bands.
[View Larger Version of this Image (76K GIF file)]
Fig. 9.
Sucrose gradient analysis of Cx43 and Cx46 oligomerization. ROS cells were harvested and homogenized, and Triton
X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g
for 30 min, and the resulting Triton-soluble fraction was layered onto a 5-20% sucrose gradient containing 0.1% Triton X-100
(with a 25% sucrose cushion). The gradient was centrifuged for
16 h at 148,000 g. Fractions were collected from the bottom of the tube, diluted 1:1 with sample buffer, resolved by SDS-PAGE,
and electrophoretically transferred to PVDF membranes. Cx43 (a
and c) and Cx46 (b and d) were detected by immunoblotting (c
and d) and quantified by densitometry (a and b). Lanes in c and d
are labeled with the corresponding sample sucrose concentration,
which was determined by refractometry.
[View Larger Version of this Image (26K GIF file)]
Fig. 10.
Cx43 and Cx46 are retained in the same compartment
in the presence of monensin. ROS cells on glass coverslips were
preincubated in MEM containing 10 µM monensin for 4 h at 37°C.
The cells were then fixed, permeabilized, and immunolabeled for
Cx43 (a) and Cx46 (b) as described in Fig. 1.
[View Larger Version of this Image (130K GIF file)]
Fig. 11.
Monensin inhibits Cx43 oligomerization in ROS cells.
Cells were incubated in either the absence (a) or presence (b) of
10 µM monensin for 4 h at 37°C. The cells were then harvested
and homogenized, and Triton X-100 was added to a final concentration of 1%. After an incubation for 30 min at 4°C, the preparation was centrifuged at 50,000 g for 30 min, and the resulting supernatant was analyzed by sucrose gradient fractionation as
described above. Cx43 in control (a and c) and monensin-treated
(b and d) cells were detected by immunoblotting (c and d) and
quantified by densitometry (a and b). Monensin treatment, which
inhibits transport from the cis- to medial-Golgi compartments,
also inhibited Cx43 oligomerization.
[View Larger Version of this Image (26K GIF file)]
Fig. 12.
Connexin hemichannels from rat lens are stable in Triton X-100. Connexins solubilized from rat lenses were analyzed
by sucrose gradient fractionation as described above. Cx46 (a and
c) and Cx50 (b and d) were detected by immunoblotting and
quantified by densitometry (a and b). Symbols in a correspond to
measurements of total Cx46 () and the 53-kD form of Cx46 (
).
[View Larger Version of this Image (28K GIF file)]
Fig. 13.
Connexin hemichannels from Hela/Cx46 cells are stable in Triton X-100. Connexins solubilized from Hela/Cx46 cells
were further incubated at 4°C for 1 h in either the absence (a and
c) or presence (b and d) of 0.2% SDS, and then analyzed by sucrose gradient fractionation as described above. Symbols correspond to measurements of total Cx46 () and the 53-kD form of
Cx46 (
). In the presence of SDS, Cx46 oligomers dissociated
into monomers that migrated on the sucrose gradient as a single
peak centered at 8-9% sucrose.
[View Larger Version of this Image (27K GIF file)]
Discussion
1) form intracellular gap junctional structures in the ER as well as the plasma membrane. This indicates that Cx32 assembly into hemichannels also occurred at the ER and is consistent with "classical" models for transmembrane protein assembly (3, 13,
28). An alternative model has been proposed by Musil and
Goodenough (40). Using sucrose gradient fractionation to
analyze connexin assembly, they found that Cx43 is assembled in a late Golgi compartment of NRK and CHO cells, most likely the TGN. When cells were treated with BFA,
oligomerization was inhibited. Similar results were obtained using temperature-sensitive mutant cell lines deficient in ER to Golgi transport.
-connexins cannot intermix. In fact, Cx43
has been observed to colocalize near lens gap junctional
plaques that also contain Cx46 (14). Also, Cx43 and Cx46
expressed by Xenopus oocytes can form functional heterotypic channels (55, 59).
Received for publication 27 June 1996 and in revised form 2 April 1997.
1. Abbreviations used in this paper: BCS, bovine calf serum; BFA, brefeldin A; NRK, normal rat kidney; PVDF, polyvinylidene difluoride.We are grateful to Dr. S. Kornfeld and the anonymous reviewers for their very helpful comments. We also thank A. Robertson for technical assistance with the RNA blot and Dr. R. Civitelli for providing calvarial cells.