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Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
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.

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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.
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Figure 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.

Mentions: 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).


Connexin46 is retained as monomers in a trans-Golgi compartment of osteoblastic cells.

Koval M, Harley JE, Hick E, Steinberg TH - J. Cell Biol. (1997)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139843&req=5

Figure 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.
Mentions: 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).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110, USA. koval@id.wustl.edu

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus