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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 Tf-labeled early/recycling endosomes. (A, left) In situ calibration of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles). For solution measurements, Cl− was replaced by NO3−. For cell measurements, J774 cells were labeled with BAC-dextran-Tf-TMR for 15 min at 4°C, and incubated for 25 min at 37°C with buffers containing specified [Cl−] and ionophores. Data are mean ± SEM for measurements on n = 4 separate sets of experiments (10–12 cells from each culture at each time point). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-Tf-TMR and perfusion at 37°C (n = 4). Where indicated, 200 nM bafilomycin or 200 μM ouabain was present in the incubation solution and perfusate. (B, left) Calibration of FITC-Tf-TMR red to green fluorescence ratio (R/G) as a function of pH (n = 4). (right) Time course of endosomal acidification after labeling with FITC-Tf-TMR (n = 4).
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fig3: Kinetics of endosomal [Cl−] and pH in Tf-labeled early/recycling endosomes. (A, left) In situ calibration of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles). For solution measurements, Cl− was replaced by NO3−. For cell measurements, J774 cells were labeled with BAC-dextran-Tf-TMR for 15 min at 4°C, and incubated for 25 min at 37°C with buffers containing specified [Cl−] and ionophores. Data are mean ± SEM for measurements on n = 4 separate sets of experiments (10–12 cells from each culture at each time point). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-Tf-TMR and perfusion at 37°C (n = 4). Where indicated, 200 nM bafilomycin or 200 μM ouabain was present in the incubation solution and perfusate. (B, left) Calibration of FITC-Tf-TMR red to green fluorescence ratio (R/G) as a function of pH (n = 4). (right) Time course of endosomal acidification after labeling with FITC-Tf-TMR (n = 4).

Mentions: Calibration of R/G (TMR/BAC fluorescence ratio) versus [Cl−] was done by incubating dye-loaded cells with high K+ buffer containing a mixture of ionophores and bafilomycin. Fig. 3 A (left) shows that the dependence of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) on [Cl−] was similar in aqueous solution (closed circles) and in cells (open circles). The kinetics of endosomal [Cl−] was measured after labeling the cell surface at 4°C followed by rapid warming to 37°C (defined as time 0). Fig. 3 A (right) shows that endosomal [Cl−] in J774 cells increased from 18 to 40 mM during the 15-min chase period. A similar time course of increasing endosomal [Cl−] from 24 to 46 mM was found for CHO cells (not depicted). Inclusion of bafilomycin in the extracellular solution reduced endosomal Cl− accumulation by >50%.


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 Tf-labeled early/recycling endosomes. (A, left) In situ calibration of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles). For solution measurements, Cl− was replaced by NO3−. For cell measurements, J774 cells were labeled with BAC-dextran-Tf-TMR for 15 min at 4°C, and incubated for 25 min at 37°C with buffers containing specified [Cl−] and ionophores. Data are mean ± SEM for measurements on n = 4 separate sets of experiments (10–12 cells from each culture at each time point). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-Tf-TMR and perfusion at 37°C (n = 4). Where indicated, 200 nM bafilomycin or 200 μM ouabain was present in the incubation solution and perfusate. (B, left) Calibration of FITC-Tf-TMR red to green fluorescence ratio (R/G) as a function of pH (n = 4). (right) Time course of endosomal acidification after labeling with FITC-Tf-TMR (n = 4).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172765&req=5

fig3: Kinetics of endosomal [Cl−] and pH in Tf-labeled early/recycling endosomes. (A, left) In situ calibration of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) as a function of [Cl−] in solution (closed circles) and living cells (open circles). For solution measurements, Cl− was replaced by NO3−. For cell measurements, J774 cells were labeled with BAC-dextran-Tf-TMR for 15 min at 4°C, and incubated for 25 min at 37°C with buffers containing specified [Cl−] and ionophores. Data are mean ± SEM for measurements on n = 4 separate sets of experiments (10–12 cells from each culture at each time point). (right) Time course of endosomal [Cl−] after labeling at 4°C with BAC-dextran-Tf-TMR and perfusion at 37°C (n = 4). Where indicated, 200 nM bafilomycin or 200 μM ouabain was present in the incubation solution and perfusate. (B, left) Calibration of FITC-Tf-TMR red to green fluorescence ratio (R/G) as a function of pH (n = 4). (right) Time course of endosomal acidification after labeling with FITC-Tf-TMR (n = 4).
Mentions: Calibration of R/G (TMR/BAC fluorescence ratio) versus [Cl−] was done by incubating dye-loaded cells with high K+ buffer containing a mixture of ionophores and bafilomycin. Fig. 3 A (left) shows that the dependence of BAC-dextran-Tf-TMR red to green fluorescence ratio (R/G) on [Cl−] was similar in aqueous solution (closed circles) and in cells (open circles). The kinetics of endosomal [Cl−] was measured after labeling the cell surface at 4°C followed by rapid warming to 37°C (defined as time 0). Fig. 3 A (right) shows that endosomal [Cl−] in J774 cells increased from 18 to 40 mM during the 15-min chase period. A similar time course of increasing endosomal [Cl−] from 24 to 46 mM was found for CHO cells (not depicted). Inclusion of bafilomycin in the extracellular solution reduced endosomal Cl− accumulation by >50%.

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