Article |
Address correspondence to Alan S. Verkman, Cardiovascular Research Institute, 1246 Health Sciences East Tower, Box 0521, University of California, San Francisco, San Francisco, CA 94143-0521. Tel.: (415) 476-8530. Fax: (415) 665-3847. E-mail: verkman{at}itsa.ucsf.edu
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Abstract |
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Key Words: endocytosis; transferrin; ratio imaging; chloride channel; cholera toxin
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Introduction |
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We recently developed a ratioable Cl--sensitive fluorescent indicator for direct measurement of Cl- concentration ([Cl-])* in endosomes labeled by fluid-phase endocytosis (Sonawane et al., 2002). A green-fluorescing Cl--sensitive chromophore 10,10'-bis[3-carboxypropyl]-9,9'-biacridinium dinitrate (BAC) was conjugated to amino dextran (40 kD) together with the red-fluorescing Cl--insensitive chromophore 5- (and 6) carboxytetramethylrhodamine (TMR). The principle finding was that endosomal [Cl-] in two different cell lines increased from 25 to 60 mM in parallel to a decrease in endosomal pH from 6.95 to 5.30. The low endosomal [Cl-] of
20 mM measured at the earliest time point of 1 min was an intriguing observation because the extracellular solution contained 137 mM Cl-. Although quantitative measurements of endosomal [Cl-] could be made, the relatively dim BAC fluorescence (even with maximal BAC-dextran labeling and concentration in the internalization solution, 18 mg/ml) and the fluid-phase dye uptake mechanism precluded specific targeting to defined endosomal or secretory compartments, or analysis of the early kinetics of endosomal [Cl-].
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 2-macroglobulin (
2M) 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 0100 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,
2M, 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.
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Results |
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Receptor-mediated endocytosis of Tf and 2-M conjugates
Fig. 2 shows a series of confocal micrographs of J774 cells after labeling with BAC-dextran-Tf-TMR (A) and BAC-dextran-2M-TMR (B). The cell surface was stained after incubation with nanomolar concentrations of fluorescent ligands for 15 min at 4°C (Fig. 2, A and B, 0 min). The green BAC fluorescence was relatively dim because of the high extracellular [Cl-]. Removal of the internalization solution, cell washing, and warming to 37°C resulted in prompt internalization. The staining pattern of BAC-dextran-Tf-TMR was characteristic of early/recycling endosomes, as also seen for Tf-labeled with FITC and TMR (FITC-Tf-TMR; not depicted). At longer times, there was decreased staining because of ligand recycling to the cell surface. Inclusion of excess unlabeled Tf in the internalization solution blocked BAC-dextran-Tf-TMR binding and uptake (Fig. 2 A, left). In dual-labeling studies, BAC-dextran-Tf-TMR and FITC-TMR-Tf colocalized throughout the chase period (not depicted). For BAC-dextran-
2M-TMR, there was again a surface pattern initially (Fig. 2 B). Endosomes became brighter and larger over time (as also seen for FITC-TMR-
2M; not depicted), which is characteristic of ligand transport from early to late endosomes. Excess unlabeled
2M blocked labeling. In dual-labeling studies, BAC-dextran-
2M-TMR and FITC-TMR-
2M colocalized throughout the chase period (not depicted).
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Similar kinetic experiments were done for receptor-mediated endocytosis of the fluorescent 2M-conjugated Cl- and pH indicators. Fig. 4 A (left) shows that R/G versus [Cl-] for BAC-dextran-
2M-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-
2M-TMR fluorescence ratios. The increase in [Cl-] in BAC-dextran-
2M-TMRlabeled endosomes was greater than that seen for BAC-dextran-Tf-TMRlabeled 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
2M labeled with FITC and TMR (FITC-
2M-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-
2M-TMRlabeled endosomes, which can enter a late endosomal compartment, than for FITC-Tf-TMRlabeled endosomes (Fig. 3 B, right) that remain in an early/recycling compartment. Endosomal acidification was reversed by bafilomycin.
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As a further test of the conclusion that endosomal Cl- conductance is substantially greater than K+ conductance under the conditions of our experiments, molar H+ entry was related quantitatively to Cl- entry using endosomal buffer capacity (ß) to convert pH change (pH) to molar H+ entry. Using the NH4Cl pulse method, the average ß was 28 mM/pH unit for Tf-labeled endosomes in the pH range 6.056.95 and 22 mM/pH unit for
2M-labeled endosomes in the pH range of 5.206.85 (Fig. 6 D). Thus, the molar quantity of H+ accumulation in endosomes during a drop in pH from 6.95 to 6.04 for Tf (ß ·
pH = 25 mM) agreed well with the molar quantity of Cl- accumulation (22 mM; Fig. 3 A). For
2M, the molar quantity of H+ accumulation (ß ·
pH = 36 mM) also agreed with the molar quantity of Cl- accumulation (31 mM; Fig. 4 A, right). If significant endosomal K+ exit occurred during acidification, the molar ratio of Cl-/H+ transport across the endosomal membrane should be much less than one.
Rapid reduction in endosomal [Cl-] after endosome formation
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 05, 20, and 60 s after internalization at 37°C in presence of PBS (left three panels). Surface labeling was seen at 05 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 4560 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.
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Discussion |
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Fluorescent pH-sensing Tf and 2M ligands have been used to define the acidification kinetics of endosomes targeted to early/recycling and late endosomal compartments. Tf ligands are targeted to early and recycling endosomes that are relatively alkaline (pH 6.06.8) compared with late endosomes and lysosomes (Yamashiro et al., 1984; Yamashiro and Maxfield, 1987; Mellman, 1996). In general, early endosomes accumulate at the cell periphery a few minutes after internalization and later fuse to form recycling endosomes that are distributed near the nucleus. In contrast,
2M ligands do not recycle to the cell surface but progress rapidly through the endosomal pathway to late, relatively acidic endosomes (pH
5; Yamashiro et al., 1989; Zen et al., 1992). We found here that the kinetics of Cl- accumulation in early/recycling and late endosomes approximately paralleled endosomal acidification.
Several lines of functional evidence supported the conclusion that endosomal Cl- conductance provides the major route for counter ion movement to permit endosomal acidification by electrogenic H+ pumping. Endosomal Cl- accumulation was blocked >80% by the vacuolar H+ pump inhibitor bafilomycin but was restored by the K+ ionophore valinomycin, providing evidence that intrinsic endosomal K+ conductance is very low. The molar coupling ratio of H+ to Cl- entry was approximately unity, indicating that Cl- entry rather than K+ exit accounted for the majority of counter ion movement. Endosomal acidification was remarkably impaired by replacement of Cl- by gluconate, and endosomal acidification and Cl- accumulation were inhibited in parallel by a Cl- channel blocker. In these studies, we made no attempt to define the molecular identity of endosomal Cl- channel(s), and thus cannot extrapolate our results obtained (mostly J774 and CHO cells) to other cell types where the expression pattern of endosomal ion channels may differ. However, the methods developed here can be readily applied to other systems, such as testing the roles of Cl- channels ClC-5 in kidney proximal tubule cells or ClC-3 in hippocampal neurons.
[Cl-] in Golgi compartment was measured by internalization of the ratioable fluorescent conjugate BAC-dextran-CTb-TMR and retrograde transport via the secretory pathway. As found previously for the pH-sensitive fluorescent indicator CF-CTb (Schapiro et al., 1998), the Cl--sensitive conjugate localized to the Golgi compartment of Vero cells after a 30-min chase time at 37°C. Golgi compartment [Cl-] was 49 mM and pH was 6.42. This pH is in the range of 5.96.7 reported in Golgi compartment of different cell types using a variety of labeling methods (Seksek et al., 1995; Kim et al., 1996; Farinas and Verkman, 1999; Wu et al., 2000; Chandy et al., 2001; Demaurex et al., 1998). Golgi compartment [Cl-] was insensitive to the cAMP agonist forskolin but decreased substantially after H+ pump inhibition by bafilomycin and resultant Golgi compartment alkalinization. Inhibition of Golgi compartment H+ leak by ZnCl2 decreased the rates of Cl- exit and alkalinization following bafilomycin. The molar coupling ratio of H+ to Cl- exit following bafilomycin was approximately unity, suggesting that Cl- is the principal counter ion involved in Golgi compartment acidification in Vero cells. This conclusion is somewhat different from that of Demaurex et al. (1998) in a different cell type (CHO cells), where both Cl- and K+ contributed to overall Golgi compartment counter ion permeability. Whether cell type or methodological differences account for the different interpretations is unclear. Last, we note that the data here do not address whether Cl- is an important regulator of steady-state Golgi compartment pH. Indeed, there is good evidence that H+ leak is high in Golgi compartment and probably a more important determinant of steady-state Golgi pH than Golgi membrane potential (Kim et al., 1996; Farinas and Verkman, 1999; Wu et al., 2000).
The low endosomal [Cl-] just after internalization was an intriguing observation because the aqueous lumen of a nascent endosome is exposed to a high extracellular [Cl-] at the time of internalization. The kinetic data here show that the [Cl-] sensed by the fluorescently labeled transferrin ligand was initially >120 mM before internalization but decreased rapidly to 20 mM (t1/2
30 s) after initiation of internalization. The reduction in [Cl-] was not inhibited by NPPB, suggesting that Cl- transport across the endosomal membrane is not involved in this process. We tested the hypothesis that the low [Cl-] is related to the interior-negative Donnan potential produced by negative charges on membrane proteins that face the outside solution when at the plasma membrane and the lumen after endosome formation. Partial neutralization of surface charge by inclusion of polylysine after ligand binding resulted in increased endosomal [Cl-]. Conversion of the surface charges from negative to positive by exposure to pH 4.0 buffer at the time of endosome formation prevented the low [Cl-] observed under control conditions, without changing the rate of dye internalization. These results support the hypothesis that the initial low [Cl-] is produced by an interior-negative Donnan potential.
The low [Cl-] and aqueous volume in endosomes early after internalization provides a simple and elegant mechanism to permit endosomal acidification without lysis. We propose that endosomes form as flattened structures with very high surface/volume ratio and hence a substantial interior-negative Donnan potential. [Cl-] in nascent endosomes is low compared with that in the external solution just as endosomes form. Active H+ entry by the vacuolar proton pump is accompanied by secondary active Cl- entry to maintain electroneutrality. The chemical gradient of Cl- from cytoplasm (generally 3070 mM) to early endosomes (20 mM) provides a favorable electrochemical driving force for Cl- entry in response to H+ pumping. The Donnan potential decreases as endosomes acidify (titrating fixed negative charges) and accumulate ions by transport and fusion events. To achieve full acidification in late endosomes, the H+ entry is accompanied by a substantial increase in [Cl-]. The low [Cl-] after internalization, thus, permits the acidification-driven increment in [Cl-] without development of a large opposing (endosome to cytoplasm) Cl- gradient. Furthermore, the low internal aqueous volume of the nascent endosome permits the accumulation of osmoles and, thus, water without reaching a critical volume where endosome lysis occurs. Validation of our hypothesis will require measurements of endosomal membrane potential, volume, and K+ concentration in order to define the electrochemical forces driving protons, ions, and water across the endosomal membrane.
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Materials and methods |
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Cell culture
J774.1 macrophages (ATCC No. TIB-67) were obtained from American Type Culture Collection and grown in DME-H21. CHO-K1 cells (ATCC No. CCL-61) were grown in Ham's F-12K medium, and Vero cells (ATCC No. CCL-81) were grown in MEM containing 2 mM glutamine. All media were supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Cells were cultured on 18-mm-diam round coverslips at 37°C in 95% air/5% CO2, and used just before confluence.
Synthesis of fluorescently labeled ligands for [Cl-] measurements
Synthesis of TMR-Tf-SH and TMR-2M-SH (Fig. 1 A, left). A mixture of diferric Tf (14 nM in 1 ml PBS) or
2M (7.1 nM in 2 ml PBS) and TMR-succinimidyl ester (40 nM, from DMSO stock solution) were stirred slowly at room temperature for 1 h. Unreacted dye was removed by gel filtration (Sephadex G25, PBS). Using molar absorbance data, dye/protein ratios were 2.1:1 (TMR-Tf) and 2.4:1 (TMR-
2M). Equal volumes of diferric TMR-Tf or TMR-
2M (10 µM in degassed PBS) and iminothiolane (13 µM PBS containing 5 mM EDTA, pH 8) were mixed and incubated for 1 h at room temperature in the dark under N2. Unreacted iminothiolane was removed by gel filtration (Sephadex G25). Sulfhydryl group/ligand molar labeling ratios were 0.91:1 (TMR-Tf-SH) and 0.96:1 (TMR-
2M-SH) as measured using Ellman's reagent (extinction coefficient 13,600 M-1cm-1, 412 nm). No ligand polymerization was found by SDS-PAGE.
Synthesis of BAC-dextran-S-S-2Py.
5 µM amino dextran (40 kD) was stirred with 7 µM SPDP in 20 ml aqueous NaHCO3 (0.1 M, pH 8) at room temperature for 1 h. The dextran conjugate was dialyzed (25-kD cut-off) for 24 h against 0.1 M NaHCO3, and then against 10 mM PBS for 36 h at 4°C. The pyridyl/dextran molar labeling ratio was 0.95 as measured by liberation of pyridine-2-thion after reduction (extinction coefficient, 8,100 M-1cm-1; 343 nm). 5 µM purified amino dextran-S-S-2Py was reacted with 50 mM BACsuccinimidyl ester (prepared as per Sonawane et al., 2002) in 50 ml aqueous NaHCO3 (0.1 M, pH 8) at room temperature for 3 h. The reaction mixture was dialyzed and concentrated for conjugation with TMR-Tf-SH or TMR-2M-SH.
Synthesis of BAC-dextran-Tf-TMR and BAC-dextran-2M-TMR.
7 nM TMR-Tf-SH or 7 nM TMR-2M-SH was mixed with BAC-dextran-S-S-2Py (8.8 nM in 2 ml PBS containing 5 mM EDTA, pH 8) and incubated for 18 h at room temperature under N2. Unreacted TMR-Tf-SH (or TMR-
2M-SH) and BAC-dextran-S-S-2Py were removed by gel filtration (Sephadex G-100). BAC-dextran-Tf-TMR and BAC-dextran-
2M-TMR were lyophilized and stored at -20°C in a dessicator in the dark.
Synthesis of BAC-dextran-CTb-TMR (Fig. 1 A, right).
TMR-CTb was synthesized as described above for TMR-Tf (TMR/CTb molar ratio, 2.2:1). Equal volumes of TMR-CTb (10 µM, PBS) and MBS (13 µM in PBS, pH 7) were incubated for 1 h at room temperature in the dark under N2. Unreacted MBS was removed by gel filtration (Sephadex G25). The maleimide benzoyl group/ligand molar labeling ratio was 0.96:1. BAC-dextran-S-S-2Py (synthesized above) was reduced with mercaptoethanol (1:3 molar excess) for 1 h to give BAC-dextran-SH which was purified by dialysis (25-kD cut-off) against 10 mM PBS containing 5 mM EDTA for 20 h at 4°C. TMR-CTb-MB (77 nM, 0.5 ml PBS) was mixed with BAC-dextran-SH (88 nM in 2 ml PBS containing 5 mM EDTA, pH 7) and incubated for 10 h at room temperature under N2. Unreacted TMR-CTb-SH and BAC-dextran-SH were removed by gel filtration (Sephadex G-100). BAC-dextran-CTb-TMR was lyophilized and stored at -20°C.
Synthesis of fluorescently labeled ligands for pH measurements
TMR-Tf or TMR-2M (5 nM) as prepared above were mixed with FITC (50 nM from DMSO stock) in 1 ml aqueous NaHCO3, pH 8, and gently stirred for 1 h. The reaction products FITC-Tf-TMR and FITC-
2M-TMR were purified by gel filtration chromatography (Sephadex G-25). Thin layer chromatography showed no free dye contamination of the fluorescently labeled ligands. Molar labeling ratios were 5.1:1:2.1 (FITC/Tf/TMR) and 5.8:1:2.4 (FITC/
2M/TMR). CF-CTb-TMR was synthesized and purified identically, except that carboxyfluorescein (CF) succinimidyl ester was used in place of fluorescein isothiocynate. Molar labeling ratio was 4.9:1:2.2 (CF/CTb/TMR).
Characterization of the ratioable fluorescent Cl- ligands
Molecular masses of the conjugates (before and after cell internalization) were determined by column chromatography (Sephacryl 300HR). Eluted fractions were assayed for BAC and TMR fluorescence. Fluorescence spectra, molar extinction coefficients, and quantum yields were measured using a fluorimeter (Fluoromax-3). In fluorescence quenching studies, microliter aliquots of NaCl (1-M stock) were added to 3 ml of fluorescent ligand (10 µM in 5 mM Na2HPO4-NaH2PO4, pH 7.4). Stern-Volmer constant (Ksv) was calculated from the slope of F0/F - 1 versus [Cl-] plots (F0/F - 1 = Ksv [Cl-]), where F0 is BAC fluorescence in the absence and F in the presence of Cl-.
Endosome labeling and kinetics of endosomal [Cl-] and pH
Cells were incubated in serum-free medium for 15 min at 37°C before experiments. For receptor-mediated endocytosis, the cell surface was labeled by incubation with the Cl- or pH indicators (300 nM for Tf, 100 nM for 2M) for 15 min in PBS (containing 1 mM CaCl2 and MgCl2) at 4°C. Coverslips were washed twice with ice-cold PBS and transferred to a precooled perfusion chamber (0°C) containing ice-cold perfusate. Sets of BAC and TMR images (for Cl-) or FITC and TMR images (for pH) were acquired at specified times after rapid warming by perfusion at 37°C in a microincubator (PDMI-2; Harvard Apparatus). In some experiments, the perfusate contained 200 nM bafilomycin A1, 530 mM NH4Cl, 10 µM valinomycin, 100 µM NPPB, or 5 mM acetate or pthalate buffer, pH 4. In Cl--free experiments, perfusate Cl- was replaced by gluconate and cells were incubated with the Cl--free perfusate for 2 h before measurements. In some experiments, cells were incubated in polylysine solution (0.1% in PBS) at 4°C for 5 min before experiments. In some experiments, the 37°C perfusate was titrated to pH 4.0 to alter surface protein charge. In some experiments, cells were pretreated with 200 µM ouabain in PBS for 20 min.
Golgi compartment [Cl-] and pH
Cells were incubated in serum-free medium for 30 min at 37°C, washed three times with ice-cold PBS, and incubated with Cl- or pH indicators (200 nM) for 30 min in PBS at 4°C. Coverslips were washed three times with ice-cold PBS, warmed, and maintained at 37°C for 45 min, which is when a typical Golgi compartment pattern was observed. Sets of BAC and TMR images (for Cl-) or CF and TMR images (for pH) were acquired. In some experiments, the perfusate contained 200 nM bafilomycin, 520 mM NH4Cl, or 200 µM ZnCl2.
Calibration protocols
For in vivo Cl- calibrations (BAC/TMR fluorescence ratio vs. [Cl-]), perfusate and endosomal [Cl-] were equalized by incubating cells for 1520 min at 37°C in 120 mM KCl/KNO3, 20 mM NaCl/NaNO3, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, pH 7.4, with [Cl-] from 0 to 120 mM (NO3- replacing Cl-). Solutions contained the ionophores nigericin (10 µM), valinomycin (10 µM), CCCP (5 µM) and monensin (10 µM), and the H+ pump inhibitor bafilomycin (200 nM). For in vivo pH calibrations (TMR/FITC fluorescence ratio vs. pH), cells were incubated with high K+ solutions containing nigericin, valinomycin, and bafilomycin, pH adjusted to 48. For calibration of Golgi compartment Cl- and pH indicators, Vero cells were incubated with calibration buffers at 45 min after internalization.
Endosomal and Golgi compartment buffer capacity
Buffer capacity (ß) was determined from the rapid increase in endosomal/Golgi compartment pH in response to addition of 530 mM NH4Cl to the perfusate. ß was computed from the equation:
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Fluorescence microscopy
Experiments were performed using a Leitz upright fluorescence microscope equipped with a coaxial-confocal attachment and 14-bit cooled (-30°C) charge-coupled device camera as described previously (Zen et al., 1992). Cells were initially identified and focused using dim red light to avoid photobleaching. Fluorescence was collected using a 100x oil immersion objective (numerical aperture, 1.4; Plan-Apo [Nikon]). Images (500-ms acquisition time) were obtained using a custom filter set for BAC (excitation 470 ± 5 nm, dichroic 505 nm, emission 535 ± 20 nm), and standard TMR and FITC filter sets. Serial image acquisitions indicated <3% BAC photobleaching per image acquisition, and <1% photobleaching for TMR and FITC. In time course studies, endosomes in different cells were imaged for different time points except where indicated.
Ratio image analysis
Custom software was written in Labview to compute area-integrated background-subtracted pixel intensities as described previously (Sonawane et al., 2002). Four regions of cells containing well-demarcated endosomes were identified by rectangular boxes in each TMR (red) image; for each region, four nearby regions outside of the endosome/cell were identified for background determination. The same regions were identified automatically in the BAC or FITC (green) image. After background subtraction, R/G intensity ratios were computed from area-integrated intensities. Three pairs of images were analyzed for each time point. Similarly, background-subtracted intensities of BAC (or CF) and TMR were determined for a labeled perinuclear region of well-defined Golgi compartments for computation of R/G. For studies of the early kinetics of endosomal [Cl-], R/G was determined in individual endosomes. Area-integrated intensities of individual endosomes were calculated using a rectangular annular region just outside of the endosome-containing rectangle as a background (Zen et al., 1992).
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Acknowledgments |
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Submitted: 21 November 2002
Revised: 11 February 2003
Accepted: 12 February 2003
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References |
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