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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.

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[Cl−] and pH in Golgi compartment of Vero cells. (A) Cells were labeled with 200 nM BAC-dextran-CTb-TMR for 30 min at 4°C. Micrographs showing BAC (green) and TMR (red) fluo-rescence taken before (0 min) and at 45 min after perfusion at 37°C. (White arrows) Fluorescently stained Golgi complex. Bar, 10 μm. (B) In situ calibration of BAC-dextran-CTb-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and cells (open circles; n = 4). (C) Golgi compartments [Cl−]. Where indicated, 200 μM ZnCl2 and/or 200 nM bafilomycin or 10 μM forskolin were present. (D) Time course of Golgi compartment pH in the presence of 200 μM ZnCl2 and/or 200 nM bafilomycin (n = 4). Time 0 corresponds to the time of surface labeling.
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fig5: [Cl−] and pH in Golgi compartment of Vero cells. (A) Cells were labeled with 200 nM BAC-dextran-CTb-TMR for 30 min at 4°C. Micrographs showing BAC (green) and TMR (red) fluo-rescence taken before (0 min) and at 45 min after perfusion at 37°C. (White arrows) Fluorescently stained Golgi complex. Bar, 10 μm. (B) In situ calibration of BAC-dextran-CTb-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and cells (open circles; n = 4). (C) Golgi compartments [Cl−]. Where indicated, 200 μM ZnCl2 and/or 200 nM bafilomycin or 10 μM forskolin were present. (D) Time course of Golgi compartment pH in the presence of 200 μM ZnCl2 and/or 200 nM bafilomycin (n = 4). Time 0 corresponds to the time of surface labeling.

Mentions: Fig. 5 A shows labeling of Vero cells by BAC-dextran-CTb-TMR. At time 0 (before warming), a membrane labeling pattern was seen in which the green BAC fluorescence was relatively dim because of the high extracellular [Cl−]. Incubation at 37°C resulted in prompt internalization, giving a Golgi compartment staining pattern by 30 min (Fig. 5 A, 45 min). The staining pattern was similar to that reported for internalization of FITC-CTb (Schapiro et al., 1998) and TMR-Verotoxin-B (Schapiro and Grinstein, 2000). The Golgi compartment staining pattern did not change for >4 h. In dual-labeling studies, BAC-dextran-CTb-TMR and CF-CTb-TMR colocalized in Golgi compartment (not depicted). [Cl−] and pH were calibrated in situ as done for the endosome studies (Fig. 5 B, Cl− calibration). Golgi compartment [Cl−] was 49 mM and stable for >120 min. Bafilomycin reduced Golgi compartment [Cl−] to 26 mM over 30 min (Fig. 5 C), during which time Golgi compartment pH increased from 6.42 to 7.04 (Fig. 5 D). Golgi compartment buffer capacity, as determined from the magnitude of pH increase in response to an NH4Cl pulse, was 43 mM/pH unit in the pH range of 6.4–7.1. Thus, bafilomycin caused the parallel exit of 26 mM H+ and 23 mM Cl−. Inhibition of proton leak by ZnCl2 decreased the rate of Cl− exit and alkalinization of Golgi compartments after bafilomycin (Fig. 5, C and D). These experiments support the conclusion that the Golgi compartment membrane is H+ permeable, and that H+ and Cl− transport are quantitatively coupled.


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

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

[Cl−] and pH in Golgi compartment of Vero cells. (A) Cells were labeled with 200 nM BAC-dextran-CTb-TMR for 30 min at 4°C. Micrographs showing BAC (green) and TMR (red) fluo-rescence taken before (0 min) and at 45 min after perfusion at 37°C. (White arrows) Fluorescently stained Golgi complex. Bar, 10 μm. (B) In situ calibration of BAC-dextran-CTb-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and cells (open circles; n = 4). (C) Golgi compartments [Cl−]. Where indicated, 200 μM ZnCl2 and/or 200 nM bafilomycin or 10 μM forskolin were present. (D) Time course of Golgi compartment pH in the presence of 200 μM ZnCl2 and/or 200 nM bafilomycin (n = 4). Time 0 corresponds to the time of surface labeling.
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fig5: [Cl−] and pH in Golgi compartment of Vero cells. (A) Cells were labeled with 200 nM BAC-dextran-CTb-TMR for 30 min at 4°C. Micrographs showing BAC (green) and TMR (red) fluo-rescence taken before (0 min) and at 45 min after perfusion at 37°C. (White arrows) Fluorescently stained Golgi complex. Bar, 10 μm. (B) In situ calibration of BAC-dextran-CTb-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and cells (open circles; n = 4). (C) Golgi compartments [Cl−]. Where indicated, 200 μM ZnCl2 and/or 200 nM bafilomycin or 10 μM forskolin were present. (D) Time course of Golgi compartment pH in the presence of 200 μM ZnCl2 and/or 200 nM bafilomycin (n = 4). Time 0 corresponds to the time of surface labeling.
Mentions: Fig. 5 A shows labeling of Vero cells by BAC-dextran-CTb-TMR. At time 0 (before warming), a membrane labeling pattern was seen in which the green BAC fluorescence was relatively dim because of the high extracellular [Cl−]. Incubation at 37°C resulted in prompt internalization, giving a Golgi compartment staining pattern by 30 min (Fig. 5 A, 45 min). The staining pattern was similar to that reported for internalization of FITC-CTb (Schapiro et al., 1998) and TMR-Verotoxin-B (Schapiro and Grinstein, 2000). The Golgi compartment staining pattern did not change for >4 h. In dual-labeling studies, BAC-dextran-CTb-TMR and CF-CTb-TMR colocalized in Golgi compartment (not depicted). [Cl−] and pH were calibrated in situ as done for the endosome studies (Fig. 5 B, Cl− calibration). Golgi compartment [Cl−] was 49 mM and stable for >120 min. Bafilomycin reduced Golgi compartment [Cl−] to 26 mM over 30 min (Fig. 5 C), during which time Golgi compartment pH increased from 6.42 to 7.04 (Fig. 5 D). Golgi compartment buffer capacity, as determined from the magnitude of pH increase in response to an NH4Cl pulse, was 43 mM/pH unit in the pH range of 6.4–7.1. Thus, bafilomycin caused the parallel exit of 26 mM H+ and 23 mM Cl−. Inhibition of proton leak by ZnCl2 decreased the rate of Cl− exit and alkalinization of Golgi compartments after bafilomycin (Fig. 5, C and D). These experiments support the conclusion that the Golgi compartment membrane is H+ permeable, and that H+ and Cl− transport are quantitatively coupled.

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