Limits...
Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands.

Sonawane ND, Verkman AS - J. Cell Biol. (2003)

Bottom Line: In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05.The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20.Cl- accumulation was prevented by bafilomycin but restored by valinomycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.

Show MeSH

Related in: MedlinePlus

Confocal microscopy of J774 cells after staining with fluorescently labeled Cl−sensing ligands. Micrographs show BAC (green) and TMR (red) fluo-rescence ligands of Tf (A) and a2M (B). Cells were labeled with 300 nM BAC-dextran-Tf-TMR or 100 nM BAC-dextran-α2M-TMR for 15 min at 4°C. Micrographs taken before (0 min) and at indicated times after perfusion with 37°C buffer. “Excess Tf” and “excess α2M” indicate inclusion of 10 μM of the respective unlabeled ligand at the time of incubation with the fluorescent ligand. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172765&req=5

fig2: Confocal microscopy of J774 cells after staining with fluorescently labeled Cl−sensing ligands. Micrographs show BAC (green) and TMR (red) fluo-rescence ligands of Tf (A) and a2M (B). Cells were labeled with 300 nM BAC-dextran-Tf-TMR or 100 nM BAC-dextran-α2M-TMR for 15 min at 4°C. Micrographs taken before (0 min) and at indicated times after perfusion with 37°C buffer. “Excess Tf” and “excess α2M” indicate inclusion of 10 μM of the respective unlabeled ligand at the time of incubation with the fluorescent ligand. Bar, 10 μm.

Mentions: Fig. 2 shows a series of confocal micrographs of J774 cells after labeling with BAC-dextran-Tf-TMR (A) and BAC-dextran-α2M-TMR (B). The cell surface was stained after incubation with nanomolar concentrations of fluorescent ligands for 15 min at 4°C (Fig. 2, A and B, 0 min). The green BAC fluorescence was relatively dim because of the high extracellular [Cl−]. Removal of the internalization solution, cell washing, and warming to 37°C resulted in prompt internalization. The staining pattern of BAC-dextran-Tf-TMR was characteristic of early/recycling endosomes, as also seen for Tf-labeled with FITC and TMR (FITC-Tf-TMR; not depicted). At longer times, there was decreased staining because of ligand recycling to the cell surface. Inclusion of excess unlabeled Tf in the internalization solution blocked BAC-dextran-Tf-TMR binding and uptake (Fig. 2 A, left). In dual-labeling studies, BAC-dextran-Tf-TMR and FITC-TMR-Tf colocalized throughout the chase period (not depicted). For BAC-dextran-α2M-TMR, there was again a surface pattern initially (Fig. 2 B). Endosomes became brighter and larger over time (as also seen for FITC-TMR-α2M; not depicted), which is characteristic of ligand transport from early to late endosomes. Excess unlabeled α2M blocked labeling. In dual-labeling studies, BAC-dextran-α2M-TMR and FITC-TMR-α2M colocalized throughout the chase period (not depicted).


Determinants of [Cl-] in recycling and late endosomes and Golgi complex measured using fluorescent ligands.

Sonawane ND, Verkman AS - J. Cell Biol. (2003)

Confocal microscopy of J774 cells after staining with fluorescently labeled Cl−sensing ligands. Micrographs show BAC (green) and TMR (red) fluo-rescence ligands of Tf (A) and a2M (B). Cells were labeled with 300 nM BAC-dextran-Tf-TMR or 100 nM BAC-dextran-α2M-TMR for 15 min at 4°C. Micrographs taken before (0 min) and at indicated times after perfusion with 37°C buffer. “Excess Tf” and “excess α2M” indicate inclusion of 10 μM of the respective unlabeled ligand at the time of incubation with the fluorescent ligand. Bar, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172765&req=5

fig2: Confocal microscopy of J774 cells after staining with fluorescently labeled Cl−sensing ligands. Micrographs show BAC (green) and TMR (red) fluo-rescence ligands of Tf (A) and a2M (B). Cells were labeled with 300 nM BAC-dextran-Tf-TMR or 100 nM BAC-dextran-α2M-TMR for 15 min at 4°C. Micrographs taken before (0 min) and at indicated times after perfusion with 37°C buffer. “Excess Tf” and “excess α2M” indicate inclusion of 10 μM of the respective unlabeled ligand at the time of incubation with the fluorescent ligand. Bar, 10 μm.
Mentions: Fig. 2 shows a series of confocal micrographs of J774 cells after labeling with BAC-dextran-Tf-TMR (A) and BAC-dextran-α2M-TMR (B). The cell surface was stained after incubation with nanomolar concentrations of fluorescent ligands for 15 min at 4°C (Fig. 2, A and B, 0 min). The green BAC fluorescence was relatively dim because of the high extracellular [Cl−]. Removal of the internalization solution, cell washing, and warming to 37°C resulted in prompt internalization. The staining pattern of BAC-dextran-Tf-TMR was characteristic of early/recycling endosomes, as also seen for Tf-labeled with FITC and TMR (FITC-Tf-TMR; not depicted). At longer times, there was decreased staining because of ligand recycling to the cell surface. Inclusion of excess unlabeled Tf in the internalization solution blocked BAC-dextran-Tf-TMR binding and uptake (Fig. 2 A, left). In dual-labeling studies, BAC-dextran-Tf-TMR and FITC-TMR-Tf colocalized throughout the chase period (not depicted). For BAC-dextran-α2M-TMR, there was again a surface pattern initially (Fig. 2 B). Endosomes became brighter and larger over time (as also seen for FITC-TMR-α2M; not depicted), which is characteristic of ligand transport from early to late endosomes. Excess unlabeled α2M blocked labeling. In dual-labeling studies, BAC-dextran-α2M-TMR and FITC-TMR-α2M colocalized throughout the chase period (not depicted).

Bottom Line: In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05.The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20.Cl- accumulation was prevented by bafilomycin but restored by valinomycin.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
Chloride concentration ([Cl-]) was measured in defined organellar compartments using fluorescently labeled transferrin, alpha2-macroglobulin, and cholera toxin B-subunit conjugated with Cl--sensitive and -insensitive dyes. In pulse-chase experiments, [Cl-] in Tf-labeled early/recycling endosomes in J774 cells was 20 mM just after internalization, increasing to 41 mM over approximately 10 min in parallel to a drop in pH from 6.91 to 6.05. The low [Cl-] just after internalization (compared with 137 mM solution [Cl-]) was prevented by reducing the interior-negative Donnan potential. [Cl-] in alpha2-macroglobulin-labeled endosomes, which enter a late compartment, increased from 28 to 58 mM at 1-45 min after internalization, whereas pH decreased from 6.85 to 5.20. Cl- accumulation was prevented by bafilomycin but restored by valinomycin. A Cl- channel inhibitor slowed endosomal acidification and Cl- accumulation by approximately 2.5-fold. [Cl-] was 49 mM and pH was 6.42 in cholera toxin B subunit-labeled Golgi complex in Vero cells; Golgi compartment Cl- accumulation and acidification were reversed by bafilomycin. Our experiments provide evidence that Cl- is the principal counter ion accompanying endosomal and Golgi compartment acidification, and that an interior-negative Donnan potential is responsible for low endosomal [Cl-] early after internalization. We propose that reduced [Cl-] and volume in early endosomes permits endosomal acidification and [Cl-] accumulation without lysis.

Show MeSH
Related in: MedlinePlus