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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 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).
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Figure 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).

Mentions: 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.


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 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).
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Related In: Results  -  Collection

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Figure 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).
Mentions: 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.

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