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Multimeric connexin interactions prior to the trans-Golgi network

¹ University of Pennsylvania School of Medicine, Institute for Environmental Medicine and Department of Physiology and
² Department of Biology, University of Pennsylvania, Philadelphia, PA 19104, USA and
³ Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA

View larger version (42K): [in a new window]   Fig. 1. Cx43/ß-gal expression increased endogenous Cx43 expression. (A) Membrane-enriched fractions prepared from cells stably transfected with either ß-gal (con, lanes 1, 4) or Cx43/ß-gal (43ß1, lanes 2, 5; 43ß2, lane 3) were resolved by SDS-PAGE, transferred to PVDF and then immunoblotted using rabbit antisera to either Cx43 (lanes 1-3) or to ß-gal (4-5) and detected with enhanced chemiluminescence. The arrowhead indicates the band representing the Cx43/ß-gal fusion protein, while the arrow indicates ß-gal. The lower molecular mass band appearing in some samples of Cx43/ß-gal is a truncation product, as previously described (Sullivan and Lo, 1995). The 44 and 46 kDa Cx43 isoforms correspond to phosphorylated Cx43. Note the increase in amount of the faster migrating 42 kDa Cx43 isoform in cells expressing Cx43/ß-gal. The positions of molecular markers (kDa) are shown. (B) Immunoblots from 43ß1 and 43ß2 cells were quantified by densitometry to obtain the amount of Cx43/ß-gal expression relative to endogenous Cx43. Cx43/ß-gal expression by 43ß2 cells was five- to sixfold higher than for 43ß1 cells. (C) Immunoblots from control, 43ß1 and 43ß2 cells were quantified for the amount of the 42 kDa Cx43 isoform, normalized to the total level of Cx43 expressed by control cells. Values are means ± s.e.m. of triplicate preparations. *Statistically significant from control (P<0.05).

View larger version (120K): [in a new window]   Fig. 2. Cx43/ß-gal expression alters the intracellular distribution of endogenous Cx43. Control (A-C), 43ß1 (D-F) and 43ß2 (G-I) cells were fixed, permeabilized and then double-label immunostained using mouse anti-ß-gal (A,D,G) and rabbit anti-Cx43 (B,E,H), detected using Texas Red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies. With increasing Cx43/ß-gal expression, there was increased perinuclear localization of both the anti-ß-gal and anti-Cx43 signals (arrowheads). Also, while control cells had numerous areas showing labeling by Cx43 alone in areas where cells are in close apposition (arrows), this was less apparent for cells expressing high levels of Cx43/ß-gal. Note the plasma membrane regions of 43ß1 cells, which showed labeling by both anti-ß-gal and anti-Cx43 (open arrows). Bar, 10 µm.

View larger version (33K): [in a new window]   Fig. 3. Triton X-100 extraction of Cx43/ß-gal and Cx43. Membrane preparations obtained from control (con) or Cx43/ß-gal transfected cells (43ß1, 43ß2) were incubated in Triton X-100 at 4°C for 30 minutes and then centrifuged at 100,000 g for 30 minutes to separate Triton X-100 soluble (sol) and insoluble (ins) fractions. The soluble and insoluble fractions were then resolved by electrophoresis, and detected by immunoblot for Cx43/ß-gal (A) and Cx43 (B). Cx43 samples that had the best resolution of the three Cx43 isoforms are shown. The amount of Triton X-100 soluble Cx43 (C) and Cx43/ß-gal (D) was determined by densitometry. The amount of Cx43 in the Triton X-100 soluble fraction was significantly higher for 43ß2 cells, consistent with decreased incorporation into gap junction plaques. *Statistically significant from control (P<0.05).

View larger version (86K): [in a new window]   Fig. 4. Intracellular Cx43/ß-gal is Triton X-100 soluble. 43ß2 cells (A-D) or control cells (E,F) were incubated with PBS alone (A,C,E) or PBS containing 1% Triton X-100 (B,D,F) for 30 minutes at 15°C, then washed, fixed and immunostained using mouse anti-ß-gal antisera (A,B) and rabbit anti-Cx43 (C,D) or rabbit anti-Cx43 alone (E,F). The cells were then stained with FITC-conjugated goat anti-rabbit IgG and Texas Red-conjugated goat anti-mouse IgG. ß-gal images were obtained at 350 millisecond exposure with gain settings of 15 for unextracted cells (A) and 100 for Triton X-100 extracted cells (B), while Cx43 images were obtained at 1.5 second exposure with gain settings of 70 (C,E) and 200 (D,F). Triton X-100 extracted nearly all intracellular Cx43/ß-gal (arrowheads), revealing the Triton X-100 resistant pool of Cx43/ß-gal at the plasma membrane (arrows). Note the relatively higher Cx43 immunofluorescence signal from Triton X-100 extracted control cells (F), as compared to 43ß2 cells (D). Bar, 30 µm. (G-I) Control (G), 43ß1 (H) and 43ß2 (I) cells were processed for EM immunogold labeling as described in Materials and Methods and labeled using mouse anti-Cx43/15 nm gold-conjugated goat anti mouse IgG (arrows) and rabbit anti-ß-gal/5 nm gold-conjugated goat anti-rabbit IgG (arrowheads). Labeling for both Cx43 and Cx43/ß-gal was present in 43ß1 and 43ß2 cells (H,I), but not control cells (G). Bar, 100 nm.

View larger version (87K): [in a new window]   Fig. 5. Intracellular Cx43/ß-gal is in an early secretory compartment. 43ß2 cells were incubated in either the absence (A-D) or presence (E-H) of 5 µg/ml brefeldin A (BFA) for either 5 minutes (E,F) or 30 minutes (G,H) to collapse the cis and medial aspects of the Golgi apparatus into the ER. (A,B,E,F) The cells were then fixed, permeabilized and double-label immunostained using mouse anti-MG160 (A,E) and rabbit anti-ß-gal (B,F), which were detected using Texas Red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies, respectively. Arrowheads indicate perinuclear regions where there was good colocalization between MG160 and Cx43/ß-gal (A,B). Note that both MG160 and Cx43/ß-gal were translocated to the ER by BFA treatment, as revealed by an increase in nuclear membrane (E,F, arrows) and peripheral labeling. (C,G) 43ß2 cells (C) treated to preferentially label the TGN with C6-NBD-Cer (as described in Materials and Methods) showed perinuclear labeling, while 43ß2 cells pretreated with BFA for 30 minutes (G) showed a characteristic TGN labeling pattern (arrowheads). (D,H) In a parallel set of cells, treatment for 30 minutes with BFA (H) did not cause Cx43/ß-gal to accumulate in a condensed TGN structure. Bar, 20 µm.

View larger version (24K): [in a new window]   Fig. 6. Direct interaction between endogenous Cx43 with Cx43/ß-gal. (A) Membrane preparations from control (lanes 1, 3), 43ß1 (lanes 2, 4) and 43ß2 cells (lane 5) were solubilized in 1% Triton X-100 at 4°C for 30 minutes and then centrifuged at 100,000 g for 30 minutes to remove insoluble material. The supernatant was then incubated with mouse anti-ß-gal antiserum and goat anti-mouse IgG-conjugated magnetic beads, then magnetically isolated and solubilized in SDS-PAGE sample buffer. The resulting samples were resolved by electrophoresis, immunoblotted using rabbit antisera which recognizes either ß-gal (lanes 1, 2) or Cx43 (lanes 3-5), then detected by chemiluminescence. The arrowhead indicates bands corresponding to Cx43/ß-gal, while the dot indicates endogenous Cx43. (B,C) Membrane-enriched fractions from control cells (B) or 43ß2 cells (C) were solubilized in Triton X-100 and then analyzed by sucrose gradient fractionation as described in Materials and Methods. The solid line indicates fractions containing unmodified, endogenous Cx43 while the broken line in C corresponds to Cx43/ß-gal. Region 1 of the gradient (5%-11% sucrose) corresponds to Cx43 monomers, while Cx43 hexamers sediment at region 3 of the gradient (approx. 15%-18% sucrose), peaks that correspond to 5S (HRP) and 9S (catalase) standards, respectively. Expression of Cx43/ß-gal caused an increase in Cx43 which cosedimented in region 2 of the gradient (11%-15% sucrose). Region 4 of the gradient (18%-20% sucrose) from 43ß2 cells contained some Cx43/ß-gal complexes, but showed little, if any native Cx43.

View larger version (111K): [in a new window]   Fig. 7. Cx32 does not colocalize with Cx43/ß-gal. Control (con; A-C), 43ß1 (D-F) and 43ß2 (G-I) cells were transiently transfected with Cx32 and allowed to recover for 48 hours. The cells were fixed, permeabilized and then double-label immunostained using mouse anti-ß-gal (A,D,G) and rabbit anti-Cx32 (B,E,H) antisera, which were detected using Texas Red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies, respectively. The intracellular distribution of Cx32 was not affected by Cx43/ß-gal expression (arrows). Bar, 10 µm.

View larger version (101K): [in a new window]   Fig. 8. Cx46 transport is inhibited by Cx43/ß-gal. Control (con; A-C), 43ß1 (D-F) and 43ß2 (G-I) cells were transiently transfected with Cx46 and allowed to recover for 48 hours. The cells were fixed, permeabilized and then double-label immunostained using mouse anti-ß-gal (A,D,G) and rabbit anti-Cx46 (B,E,H) antisera, which were detected using Texas Red-conjugated goat anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies, respectively. Cells showed colocalization of Cx43/ß-gal with both Cx46 at the cell surface (arrows) and Cx46 retained in the perinuclear region of the cell (arrowheads). Note the increased retention of Cx46 by 43ß2 cells, as compared to 43ß1 cells. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 9. Cx43 and Cx43/ß-gal specifically interact with Cx46. (A) Membrane preparations from either non-transfected cells (lanes 1, 2, 7, 8) or cells transiently transfected with either Cx32 (lanes 3-6) or Cx46 cDNA (lanes 9-12) were Triton X-100 solubilized and immunopurified using mouse anti-ß-gal as described in Fig. 6. The resulting samples were resolved by electrophoresis, immunoblotted using rabbit antisera that recognizes either Cx32 (lanes 1-6) or Cx46 (lanes 7-12), then detected by chemiluminescence. Arrowheads denote bands corresponding to Cx32 (approx. 28 kDa), phosphorylated Cx46 (approx. 68 kDa) and nonphosphorylated Cx46 (approx. 53 kDa). Occasionally, the anti-Cx46 antibody recognized a non-specific band of apparent molecular mass approx. 75 kDa. Cx46, but not Cx32, was immunopurified using anti-ß-gal antiserum (lane 12), suggesting that Cx43/ß-gal and Cx46 formed a specific oligomeric complex. (B) To confirm the ability of unmodified Cx43 to coimmunoprecipitate with Cx46, wild-type Hela cells (‘H’, lanes 13, 15, 17) or Hela cells stably transfected with Cx43 (‘43’; lanes 14, 16, 18) were transiently transfected with Cx46 cDNA, then analyzed as described above for total Cx46 expression (lanes 13, 14), immunopurified Cx43 (lanes 15, 16) and coimmunopurified Cx46 (lanes 17, 18), except that anti-Cx43 was used for coimmunopurification. Consistent with the ability of Cx43 and Cx46 to form mixed oligomers, Cx46 was specifically coimmunopurified using Cx43 antibodies from Hela cells expressing both connexins (lane 16). (C-I) ROS cells (‘R’, lanes 19, 21) and type I-like alveolar epithelial cells, AEC (‘A’, lanes 20, 22) expressing endogenous Cx43 and Cx46 were also examined for the ability of Cx46 to coimmunopurify with anti-Cx43 IgG. By immunofluorescence, both cell types had Cx43 present at the plasma membrane (D,G); however, Cx46 showed differential localization to either TGN (E, arrows) or the plasma membrane (H, arrowheads), depending on cell phenotype. Bar, 10 µm. While comparable levels of Cx43 were immunopurified from both cell types using anti-Cx43 IgG (lanes 19, 20), Cx46 was only coimmunopurified with anti-Cx43 IgG from samples prepared from alveolar epithelial cells (lane 22).