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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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Early kinetics of endosomal [Cl−]: investigation of the role of Donnan potential. (A) Fluorescence micrographs (BAC and TMR fluorescence) of cells at indicated times after heating to 37°C. Where indicated (low pH), the 37°C perfusate was at pH 4.0 during internalization. (B) Time course of endosomal [Cl−] measured using BAC-dextran-Tf-TMR. Where indicated, cells were incubated with polylysine (0.1%, 10,000 kD) at 4°C for 5 min before internalization (polylysine), or a pH 4.0 perfusate was used as in A (low pH), or cells were incubated with high K+ buffer containing ionophores for 20 min before internalization (n = 4). [Cl−] was measured in individual endosomes/endosome-like structures by ratio image analysis. (C) Rate of endosomal internalization of BAC-dextran-TF-TMR measured from total fluorescence of cell homogenates (n = 4). Internalization was done for 1 min as in A, and cells were washed five times at 4°C before homogenization. Blank indicates zero internalization time (heating to 37°C omitted).
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fig7: Early kinetics of endosomal [Cl−]: investigation of the role of Donnan potential. (A) Fluorescence micrographs (BAC and TMR fluorescence) of cells at indicated times after heating to 37°C. Where indicated (low pH), the 37°C perfusate was at pH 4.0 during internalization. (B) Time course of endosomal [Cl−] measured using BAC-dextran-Tf-TMR. Where indicated, cells were incubated with polylysine (0.1%, 10,000 kD) at 4°C for 5 min before internalization (polylysine), or a pH 4.0 perfusate was used as in A (low pH), or cells were incubated with high K+ buffer containing ionophores for 20 min before internalization (n = 4). [Cl−] was measured in individual endosomes/endosome-like structures by ratio image analysis. (C) Rate of endosomal internalization of BAC-dextran-TF-TMR measured from total fluorescence of cell homogenates (n = 4). Internalization was done for 1 min as in A, and cells were washed five times at 4°C before homogenization. Blank indicates zero internalization time (heating to 37°C omitted).

Mentions: As mentioned in the Introduction, an unanticipated finding in our previous measurements of endosomal [Cl−] using a fluid-phase indicator was the low endosomal [Cl−] of ∼25 mM at the first time point that could be measured, much lower than the [Cl−] of 137 mM in the extracellular solution. Using fluorescently labeled transferrin, we investigated this phenomenon by first defining the early kinetics of [Cl−] sensed by BAC-dextran-Tf-TMR after membrane surface labeling and rapidly induced receptor internalization. Fig. 7 A shows BAC and TMR fluorescence micrographs of J774 cells taken at 0–5, 20, and 60 s after internalization at 37°C in presence of PBS (left three panels). Surface labeling was seen at 0–5 s with dim BAC fluorescence because of exposure of BAC-dextran-Tf-TMR to the external solution. Few well-demarcated endosomes were observed by 20 s, though many hazy nascent endosomes were seen having dim BAC fluorescence. Many well-demarcated endosomes were seen by 45–60 s with brighter BAC fluorescence indicating low [Cl−]. In contrast, endosome BAC fluorescence was low (indicating high [Cl−]) at 60 s when internalization was done in low pH buffer (see below) (Fig. 7 A, right panels). Fig. 7 B shows the kinetics of average endosomal [Cl−] determined by ratio image analysis of individual endosome structures at indicated times after solution heating to 37°C. [Cl−] was ∼120 mM early after internalization, where ratio imaging was done in blurred endosome-like structures that may or may not represent sealed vesicles. [Cl−] decreased rapidly to ∼20 mM over 60 s. One possible mechanism for the rapid drop in [Cl−] is a strong interior-negative Donnan potential created by proteins at the inner surface of the endosome-limiting membrane. Other mechanisms include rapid fusion with endosomes containing low [Cl−] and rapid volume expansion without entry of Cl− from the cytoplasm. If a negative Donnan potential is responsible, then we reasoned that collapse of the Donnan potential should reduce or prevent the initial drop in [Cl−]. Two approaches were used to diminish the Donnan potential: inclusion of polylysine after surface labeling to neutralize cell-surface negative charges, and internalization during exposure to a low pH external buffer to protonate anionic proteins at the cell surface.


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

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

Early kinetics of endosomal [Cl−]: investigation of the role of Donnan potential. (A) Fluorescence micrographs (BAC and TMR fluorescence) of cells at indicated times after heating to 37°C. Where indicated (low pH), the 37°C perfusate was at pH 4.0 during internalization. (B) Time course of endosomal [Cl−] measured using BAC-dextran-Tf-TMR. Where indicated, cells were incubated with polylysine (0.1%, 10,000 kD) at 4°C for 5 min before internalization (polylysine), or a pH 4.0 perfusate was used as in A (low pH), or cells were incubated with high K+ buffer containing ionophores for 20 min before internalization (n = 4). [Cl−] was measured in individual endosomes/endosome-like structures by ratio image analysis. (C) Rate of endosomal internalization of BAC-dextran-TF-TMR measured from total fluorescence of cell homogenates (n = 4). Internalization was done for 1 min as in A, and cells were washed five times at 4°C before homogenization. Blank indicates zero internalization time (heating to 37°C omitted).
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fig7: Early kinetics of endosomal [Cl−]: investigation of the role of Donnan potential. (A) Fluorescence micrographs (BAC and TMR fluorescence) of cells at indicated times after heating to 37°C. Where indicated (low pH), the 37°C perfusate was at pH 4.0 during internalization. (B) Time course of endosomal [Cl−] measured using BAC-dextran-Tf-TMR. Where indicated, cells were incubated with polylysine (0.1%, 10,000 kD) at 4°C for 5 min before internalization (polylysine), or a pH 4.0 perfusate was used as in A (low pH), or cells were incubated with high K+ buffer containing ionophores for 20 min before internalization (n = 4). [Cl−] was measured in individual endosomes/endosome-like structures by ratio image analysis. (C) Rate of endosomal internalization of BAC-dextran-TF-TMR measured from total fluorescence of cell homogenates (n = 4). Internalization was done for 1 min as in A, and cells were washed five times at 4°C before homogenization. Blank indicates zero internalization time (heating to 37°C omitted).
Mentions: As mentioned in the Introduction, an unanticipated finding in our previous measurements of endosomal [Cl−] using a fluid-phase indicator was the low endosomal [Cl−] of ∼25 mM at the first time point that could be measured, much lower than the [Cl−] of 137 mM in the extracellular solution. Using fluorescently labeled transferrin, we investigated this phenomenon by first defining the early kinetics of [Cl−] sensed by BAC-dextran-Tf-TMR after membrane surface labeling and rapidly induced receptor internalization. Fig. 7 A shows BAC and TMR fluorescence micrographs of J774 cells taken at 0–5, 20, and 60 s after internalization at 37°C in presence of PBS (left three panels). Surface labeling was seen at 0–5 s with dim BAC fluorescence because of exposure of BAC-dextran-Tf-TMR to the external solution. Few well-demarcated endosomes were observed by 20 s, though many hazy nascent endosomes were seen having dim BAC fluorescence. Many well-demarcated endosomes were seen by 45–60 s with brighter BAC fluorescence indicating low [Cl−]. In contrast, endosome BAC fluorescence was low (indicating high [Cl−]) at 60 s when internalization was done in low pH buffer (see below) (Fig. 7 A, right panels). Fig. 7 B shows the kinetics of average endosomal [Cl−] determined by ratio image analysis of individual endosome structures at indicated times after solution heating to 37°C. [Cl−] was ∼120 mM early after internalization, where ratio imaging was done in blurred endosome-like structures that may or may not represent sealed vesicles. [Cl−] decreased rapidly to ∼20 mM over 60 s. One possible mechanism for the rapid drop in [Cl−] is a strong interior-negative Donnan potential created by proteins at the inner surface of the endosome-limiting membrane. Other mechanisms include rapid fusion with endosomes containing low [Cl−] and rapid volume expansion without entry of Cl− from the cytoplasm. If a negative Donnan potential is responsible, then we reasoned that collapse of the Donnan potential should reduce or prevent the initial drop in [Cl−]. Two approaches were used to diminish the Donnan potential: inclusion of polylysine after surface labeling to neutralize cell-surface negative charges, and internalization during exposure to a low pH external buffer to protonate anionic proteins at the cell surface.

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