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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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Synthesis and characterization of fluorescently labeled Cl−-sensing ligands. (A, left) TMR-labeled Tf and α2M were conjugated to BAC-dextran by a disulfide linker to generate dual wavelength Cl− indicators (see Materials and methods). (right) TMR-labeled CTb was conjugated to BAC-dextran by a thioether linker. (B) Elution profiles of BAC-dextran-Tf-TMR (left) and BAC-dextran-α2M-TMR (right) for gel filtration chromatography on Sephacryl 300HR. Profiles of BAC and TMR fluorescence shown for original purified conjugates (top) and after isolation from cells (bottom) in which endosomes were labeled, chased at 37°C for 15 min (Tf) or 45 min (α2M), and lysed by sonication and freeze/thaw. Arrow indicates void volume.
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fig1: Synthesis and characterization of fluorescently labeled Cl−-sensing ligands. (A, left) TMR-labeled Tf and α2M were conjugated to BAC-dextran by a disulfide linker to generate dual wavelength Cl− indicators (see Materials and methods). (right) TMR-labeled CTb was conjugated to BAC-dextran by a thioether linker. (B) Elution profiles of BAC-dextran-Tf-TMR (left) and BAC-dextran-α2M-TMR (right) for gel filtration chromatography on Sephacryl 300HR. Profiles of BAC and TMR fluorescence shown for original purified conjugates (top) and after isolation from cells (bottom) in which endosomes were labeled, chased at 37°C for 15 min (Tf) or 45 min (α2M), and lysed by sonication and freeze/thaw. Arrow indicates void volume.

Mentions: The goal of this paper is to develop ratioable Cl−-sensitive fluorescent ligands for receptor-mediated internalization in order to do the following: (a) measure [Cl−] in early/recycling and late endosomes; (b) define quantitatively the role of Cl− conductance in vacuolar acidification; (c) investigate the mechanisms responsible for the low [Cl−] early after endosome formation; and (d) measure [Cl−] in Golgi compartments. Transferrin (Tf) and α2-macroglobulin (α2M) were chosen as endosomal markers on the basis of a substantial body of evidence that these ligands are targeted by receptor-mediated endocytosis to early/recycling and late endosomes, respectively (Yamashiro et al., 1984; Dautry-Varsat, 1986; Yamashiro et al., 1989; Mukherjee et al., 1997). The cholera toxin B-subunit (CTb) was chosen as a Golgi compartment marker on the basis of data showing efficient Golgi compartment labeling in Vero cells after retrograde transport (Schapiro et al., 1998). The requirements of the indicators included the following: uptake by a receptor-mediated mechanism; bright, long wavelength fluorescence; Cl− sensitivity in the range 0–100 mM; pH insensitivity; and stability in cells. As in our previous work (Sonawane et al., 2002), BAC was used as the Cl−-sensitive chromophore; however, BAC could not be conjugated to the protein ligands directly because BAC fluorescence was quenched by >90% after conjugation to proteins, even when long spacers were introduced. Our strategy was to synthesize 1:1 covalent conjugates of TMR-labeled ligands with BAC-labeled dextran (BAC-dextran; see Fig. 1 A). Cell labeling with the fluorescent ligands at nanomolar concentrations in the labeling solution gave remarkably brighter endosomes than labeling with high concentrations of the fluorescent dextran, and permitted a pulse-internalization protocol in which perfusate temperature was increased rapidly after cell surface labeling at low temperature. Ratioable, pH-sensitive Tf, α2M, and CTb ligands were also synthesized to compare endosomal/Golgi compartment [Cl−] and pH. Our data establish new classes of targetable Cl− indicators, define the principle determinants of [Cl−] in early/recycling and late endosomes, and provide the first data on [Cl−] in the Golgi compartment.


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

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

Synthesis and characterization of fluorescently labeled Cl−-sensing ligands. (A, left) TMR-labeled Tf and α2M were conjugated to BAC-dextran by a disulfide linker to generate dual wavelength Cl− indicators (see Materials and methods). (right) TMR-labeled CTb was conjugated to BAC-dextran by a thioether linker. (B) Elution profiles of BAC-dextran-Tf-TMR (left) and BAC-dextran-α2M-TMR (right) for gel filtration chromatography on Sephacryl 300HR. Profiles of BAC and TMR fluorescence shown for original purified conjugates (top) and after isolation from cells (bottom) in which endosomes were labeled, chased at 37°C for 15 min (Tf) or 45 min (α2M), and lysed by sonication and freeze/thaw. Arrow indicates void volume.
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Related In: Results  -  Collection

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

fig1: Synthesis and characterization of fluorescently labeled Cl−-sensing ligands. (A, left) TMR-labeled Tf and α2M were conjugated to BAC-dextran by a disulfide linker to generate dual wavelength Cl− indicators (see Materials and methods). (right) TMR-labeled CTb was conjugated to BAC-dextran by a thioether linker. (B) Elution profiles of BAC-dextran-Tf-TMR (left) and BAC-dextran-α2M-TMR (right) for gel filtration chromatography on Sephacryl 300HR. Profiles of BAC and TMR fluorescence shown for original purified conjugates (top) and after isolation from cells (bottom) in which endosomes were labeled, chased at 37°C for 15 min (Tf) or 45 min (α2M), and lysed by sonication and freeze/thaw. Arrow indicates void volume.
Mentions: The goal of this paper is to develop ratioable Cl−-sensitive fluorescent ligands for receptor-mediated internalization in order to do the following: (a) measure [Cl−] in early/recycling and late endosomes; (b) define quantitatively the role of Cl− conductance in vacuolar acidification; (c) investigate the mechanisms responsible for the low [Cl−] early after endosome formation; and (d) measure [Cl−] in Golgi compartments. Transferrin (Tf) and α2-macroglobulin (α2M) were chosen as endosomal markers on the basis of a substantial body of evidence that these ligands are targeted by receptor-mediated endocytosis to early/recycling and late endosomes, respectively (Yamashiro et al., 1984; Dautry-Varsat, 1986; Yamashiro et al., 1989; Mukherjee et al., 1997). The cholera toxin B-subunit (CTb) was chosen as a Golgi compartment marker on the basis of data showing efficient Golgi compartment labeling in Vero cells after retrograde transport (Schapiro et al., 1998). The requirements of the indicators included the following: uptake by a receptor-mediated mechanism; bright, long wavelength fluorescence; Cl− sensitivity in the range 0–100 mM; pH insensitivity; and stability in cells. As in our previous work (Sonawane et al., 2002), BAC was used as the Cl−-sensitive chromophore; however, BAC could not be conjugated to the protein ligands directly because BAC fluorescence was quenched by >90% after conjugation to proteins, even when long spacers were introduced. Our strategy was to synthesize 1:1 covalent conjugates of TMR-labeled ligands with BAC-labeled dextran (BAC-dextran; see Fig. 1 A). Cell labeling with the fluorescent ligands at nanomolar concentrations in the labeling solution gave remarkably brighter endosomes than labeling with high concentrations of the fluorescent dextran, and permitted a pulse-internalization protocol in which perfusate temperature was increased rapidly after cell surface labeling at low temperature. Ratioable, pH-sensitive Tf, α2M, and CTb ligands were also synthesized to compare endosomal/Golgi compartment [Cl−] and pH. Our data establish new classes of targetable Cl− indicators, define the principle determinants of [Cl−] in early/recycling and late endosomes, and provide the first data on [Cl−] in the Golgi compartment.

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