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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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Kinetics of endosomal [Cl−] and pH in α2M-labeled endosomes. (A, left) In situ calibration of BAC-α2M-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles; n = 4 sets of experiments). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-α2M-TMR and perfusion at 37°C. After the initial chase period of 45 min, 200 nM bafilomycin was added to the perfusate (n = 4). (B, left) Calibration of FITC-α2M-TMR red to green fluorescence ratio (R/G) as a function of pH. (right) Time course of endosomal pH after labeling with FITC-α2M-TMR. Where indicated 200 nM bafilomycin was added to the perfusate (n = 4).
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fig4: Kinetics of endosomal [Cl−] and pH in α2M-labeled endosomes. (A, left) In situ calibration of BAC-α2M-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles; n = 4 sets of experiments). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-α2M-TMR and perfusion at 37°C. After the initial chase period of 45 min, 200 nM bafilomycin was added to the perfusate (n = 4). (B, left) Calibration of FITC-α2M-TMR red to green fluorescence ratio (R/G) as a function of pH. (right) Time course of endosomal pH after labeling with FITC-α2M-TMR. Where indicated 200 nM bafilomycin was added to the perfusate (n = 4).

Mentions: Similar kinetic experiments were done for receptor-mediated endocytosis of the fluorescent α2M-conjugated Cl− and pH indicators. Fig. 4 A (left) shows that R/G versus [Cl−] for BAC-dextran-α2M-TMR in cells was similar to that in solution. Absolute R/G ratios differed from those in the BAC-dextran-Tf-TMR calibration because of differences in chromophore labeling ratios. Fig. 4 A (right) shows the kinetics of endosomal [Cl−] in J774 cells measured from BAC-dextran-α2M-TMR fluorescence ratios. The increase in [Cl−] in BAC-dextran-α2M-TMR–labeled endosomes was greater than that seen for BAC-dextran-Tf-TMR–labeled endosomes in Fig. 3 A (right). The endosomal [Cl−] accumulation was reversed by addition of bafilomycin at 45 min after endocytosis. Interestingly, endosomal [Cl−] after bafilomycin became lower (∼30 mM) than that in the cytoplasm in these cells (∼45 mM). This observation is consistent with coupled H+/Cl− exit, though it is difficult to make quantitative predictions because of unknown electrochemical driving forces (membrane potential, [K+], and [Na+]). For pH measurements, calibrations of endosomal and solution R/G (TMR/FITC fluorescence ratio) for α2M labeled with FITC and TMR (FITC-α2M-TMR; Fig. 4 B, left) were similar to those in Fig. 3 B (left) for FITC-Tf-TMR. Fig. 4 B (right) shows substantially greater endosomal acidification for FITC-α2M-TMR–labeled endosomes, which can enter a late endosomal compartment, than for FITC-Tf-TMR–labeled endosomes (Fig. 3 B, right) that remain in an early/recycling compartment. Endosomal acidification was reversed by bafilomycin.


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

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

Kinetics of endosomal [Cl−] and pH in α2M-labeled endosomes. (A, left) In situ calibration of BAC-α2M-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles; n = 4 sets of experiments). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-α2M-TMR and perfusion at 37°C. After the initial chase period of 45 min, 200 nM bafilomycin was added to the perfusate (n = 4). (B, left) Calibration of FITC-α2M-TMR red to green fluorescence ratio (R/G) as a function of pH. (right) Time course of endosomal pH after labeling with FITC-α2M-TMR. Where indicated 200 nM bafilomycin was added to the perfusate (n = 4).
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fig4: Kinetics of endosomal [Cl−] and pH in α2M-labeled endosomes. (A, left) In situ calibration of BAC-α2M-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles; n = 4 sets of experiments). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-α2M-TMR and perfusion at 37°C. After the initial chase period of 45 min, 200 nM bafilomycin was added to the perfusate (n = 4). (B, left) Calibration of FITC-α2M-TMR red to green fluorescence ratio (R/G) as a function of pH. (right) Time course of endosomal pH after labeling with FITC-α2M-TMR. Where indicated 200 nM bafilomycin was added to the perfusate (n = 4).
Mentions: Similar kinetic experiments were done for receptor-mediated endocytosis of the fluorescent α2M-conjugated Cl− and pH indicators. Fig. 4 A (left) shows that R/G versus [Cl−] for BAC-dextran-α2M-TMR in cells was similar to that in solution. Absolute R/G ratios differed from those in the BAC-dextran-Tf-TMR calibration because of differences in chromophore labeling ratios. Fig. 4 A (right) shows the kinetics of endosomal [Cl−] in J774 cells measured from BAC-dextran-α2M-TMR fluorescence ratios. The increase in [Cl−] in BAC-dextran-α2M-TMR–labeled endosomes was greater than that seen for BAC-dextran-Tf-TMR–labeled endosomes in Fig. 3 A (right). The endosomal [Cl−] accumulation was reversed by addition of bafilomycin at 45 min after endocytosis. Interestingly, endosomal [Cl−] after bafilomycin became lower (∼30 mM) than that in the cytoplasm in these cells (∼45 mM). This observation is consistent with coupled H+/Cl− exit, though it is difficult to make quantitative predictions because of unknown electrochemical driving forces (membrane potential, [K+], and [Na+]). For pH measurements, calibrations of endosomal and solution R/G (TMR/FITC fluorescence ratio) for α2M labeled with FITC and TMR (FITC-α2M-TMR; Fig. 4 B, left) were similar to those in Fig. 3 B (left) for FITC-Tf-TMR. Fig. 4 B (right) shows substantially greater endosomal acidification for FITC-α2M-TMR–labeled endosomes, which can enter a late endosomal compartment, than for FITC-Tf-TMR–labeled endosomes (Fig. 3 B, right) that remain in an early/recycling compartment. Endosomal acidification was reversed by bafilomycin.

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