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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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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).
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Figure 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).

Mentions: 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).


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)

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).
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Related In: Results  -  Collection

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Figure 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).
Mentions: 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).

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