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Volume 270, Number 10, Issue of March 10, 1995 pp. 4967-4970
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Direct Measurement of trans-Golgi pH in Living Cells and Regulation by Second Messengers (*)

(Received for publication, November 4, 1994; and in revised form, January 10, 1995)

Olivier Seksek (§),  ,  Joachim Biwersi ,  A. S. Verkman (¶)

From the Departments of Medicine and Physiology, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0521

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the endocytic compartment, an acidic pH plays a key role in receptor and ligand sorting, vesicular transport, and protein degradation. In the secretory compartment, indirect estimates of trans-Golgi pH based on partitioning of weak bases and following viral infection suggest a mildly acidic pH of >6.0. We developed a liposome microinjection method to introduce fluorescent indicators into the aqueous compartment of trans-Golgi in living cells. In the presence of ATP and at 37 °C, 70-nm diameter liposomes delivered their fluid-phase contents selectively into the trans-Golgi compartment as assessed by colocalization with the trans-Golgi stain N- {6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-sphingosine (C(6)-NBD-ceramide). Liposome fusion was ATP- and temperature-dependent and blocked by N-ethylmaleimide but not by guanosine 5`-O-(3-thiotriphosphate) (GTPS). trans-Golgi pH in skin fibroblasts was 6.17 ± 0.02 (S.E., n = 174) as measured by ratio imaging confocal microscopy using fluorescein and rhodamine-based indicators and an in vivo calibration procedure. trans-Golgi pH increased to 6.8 ± 0.1 by cAMP agonists and to 6.5 ± 0.1 by protein kinase C activation. These results provide the first direct measurement of trans-Golgi pH in living cells and demonstrate pH regulation by second messengers.

INTRODUCTION

It is well documented that a vacuolar-type proton pump maintains a relatively acidic pH in some organelles of the endosomal and secretory compartments(1, 2, 3) . An acidic pH relative to cytosol was demonstrated in trans-Golgi by accumulation of the lysosomotropic agent 3-(2,4-dinitroanilino)-3`-amino-N-methyl-dipropylamine(4, 5) . Two other lines of evidence suggest that trans-Golgi pH is >6.0, including analysis of pH-dependent fusion of viral spike proteins resulting in viral infectivity (6) and pH-dependent ligand binding to mannose 6-phosphate receptors(7) . A mildly acidic pH in trans-Golgi may play an important role in the transport of secretory proteins into secretory granules and in the post-translational processing of newly synthesized proteins(3, 8) .

Cytosolic pH has been studied extensively by fluorescence methods over the past decade with the development of a variety of pH-sensitive dyes and cell-trapable acetoxymethylester derivatives(9, 10) . New developments in fluorescence ratio imaging microscopy have enabled the mapping of pH in single cells (11) and in individual vesicles of the endosomal pathway(12, 13) . However, because the secretory compartments remain relatively inaccessible to fluid-phase fluorescent markers, there has been no method to selectively label in vivo the lumen of endoplasmic reticulum, Golgi, and secretory vesicles for direct measurement of pH.

We report here a novel method to deliver aqueous-phase fluorescent indicators into the lumen of the trans-Golgi compartment in living cells. Our strategy was inspired by a vesicle fusion method applied previously in semi-intact cells(14) , where liposomes were shown to fuse selectively with the trans-Golgi. Here, trans-Golgi in living human skin fibroblasts was labeled by cytoplasmic microinjection of 70-nm diameter liposomes containing membrane-impermeable fluorophores. A pH-sensitive fluorophore (fluorescein sulfonate) and a pH-insensitive fluorophore (sulforhodamine 101) were introduced into the trans-Golgi for direct measurement of pH by ratio imaging confocal microscopy. Average trans-Golgi pH was found to be 6.17 ± 0.02, and an unexpected regulatory effect of second messengers was demonstrated.

MATERIALS AND METHODS

Cells and Reagents

Normal human skin fibroblasts (American Type Culture Collection CCD 187 Sk) were grown at 37 °C in DME-H21 medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin and used between passages 10 and 20. Fluorescein sulfonate (FS), (^1)sulforhodamine 101 (SR), N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-sphingosine (C(6)-NBD-ceramide), and N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TMR-PE) were purchased from Molecular Probes (Eugene, OR). Defatted bovine serum albumin (BSA), monensin, CCCP, bafilomycin A(1), 8-(4-chlorophenylthio)-adenosine 3`,5`-cyclic monophosphate (CPT-cAMP), N-ethylmaleimide, and GTPS were obtained from Sigma, and dioleoylphosphatidylcholine was from Avanti Polar (Alabaster, AL). Platelet-derived growth factor B/B was from Boehringer Mannheim.

Preparation of Liposomes

Multilamellar vesicles were prepared by dispersing dry dioleoylphosphatidylcholine in 25 mM HEPES, 115 mM KCl, 2.5 mM MgCl(2), (SR (5 mM) + FS (0 or 30 mM)) or TMR-PE (1 mol %), pH 7.2. Multilamellar vesicles were then frozen in liquid nitrogen and thawed at 40 °C for five cycles. Small uniform sized liposomes were obtained by extrusion through a series of polycarbonate filters (Nuclepore, Pleasanton, CA) of decreasing pore size (450 220 100 50 nm). External SR and FS were removed by Sephadex G-50 size-exclusion gel chromatography. Liposome diameter was measured to be 70 ± 1 nm (S.D.) by quasi-elastic dynamic light scattering (Coulter model N4, Hialeah, FL).

Microinjection Procedure

Cell microinjection was performed using glass needles prepared from thin-walled filament capillaries (FHC, Brunswick, ME) drawn to a fine tip (0.5-µm hole diameter) with a vertical needle puller (Kopf, Tujunga, CA) and back-filled with injection solutions. Filled needles were mounted in the holder of an Eppendorf micromanipulator (model 5170), and cells were injected using an automatic Eppendorf microinjector (model 5242) over 0.5 s. Cells used for microinjection were 2-day-old cultures (80% confluent) grown on 18-mm diameter glass coverslips.

C(6)-NBD-ceramide Labeling

To stain the trans-Golgi membrane(15) , 50 nmol of C(6)-NBD-ceramide was dissolved in 200 µl of ethanol and injected into 10 ml of 10 mM HEPES-buffered minimal essential medium containing 0.34 mg of BSA. The solution was dialyzed overnight at 4 °C against HEPES-buffered minimal essential medium. Cells were incubated with the C(6)-NBD-ceramide-BSA complex for 5 min at 37 °C, washed with PBS, and mounted in the perfusion chamber for observation.

Ratio Imaging Confocal Microscopy

Fluorescence microscopy was carried out on a Leitz epifluorescence microscope equipped with a Nipkow wheel coaxial-confocal attachment (Technical Instruments, San Francisco, CA). Cells were mounted in a perfusion chamber and viewed with a Nikon Plan-Apo times 100 oil immersion objective (numerical aperture, 1.4). Confocal fluorescence images were detected by a cooled CCD camera (AT200; Photometrics, Tucson, AZ) with a back-thinned 14-bit detector (TK512CB; Tektronix). (C(6)-NBD-ceramide or FS) and (SR or TMR-PE) were visualized with standard fluorescein and rhodamine filter sets, respectively. Image pairs (SR and FS) were acquired (exposure time, 500 ms) for the same field containing one or more cells. Dark current and shading corrections were applied. Analysis was performed using PMIS software (Photometrics) on each image pair; the trans-Golgi contour on the SR image was drawn, and the integrated pixel intensity (I) was calculated over the delimited area. The same area on the FS image was used for determination of integrated intensity, I. For calculation of the FS-to-SR signal ratio, (I - B)/(I - B), the background signals, B and B, were determined over a region of cytosol near the trans-Golgi contour.

pH Calibration in Situ

After microinjection with liposomes containing FS and SR, cells were incubated for 30 min at 37 °C and then mounted in the perfusion chamber. Prior to calibration, cells were perfused for 20 min with 10 nM bafilomycin A(1) in PBS at 23 °C to inhibit the vacuolar proton pump. Cells were then incubated for 10 min with calibration solutions containing 10 nM bafilomycin A(1), 10 µM monensin, 1 µM CCCP in 125 mM KCl, 20 mM NaCl, 25 mM HEPES, 0.5 mM MgSO(4), 0.5 mM CaCl(2) titrated to specified pH values. FS-to-SR signal ratio was determined as a function of pH.

RESULTS AND DISCUSSION

Normal human skin fibroblasts were microinjected with uniform-sized liposomes of 70 ± 1 nm diameter containing selected fluid-phase fluorescent probe(s). Liposomes containing 5 mM SR (a water-soluble fluorescent probe) fused with trans-Golgi and delivered their aqueous phase contents at 37 °C with a half-time of 15 min. Maximum trans-Golgi fluorescence was observed at 30 min. Selective labeling of trans-Golgi was demonstrated with confocal microscopy by colocalization of SR (red) with the specific trans-Golgi lipid-phase stain C(6)-NBD-ceramide (green) (15) (Fig. 1A).

Figure 1: Selective labeling of the lumen of the trans-Golgi compartment in normal human skin fibroblasts. A, left: gallery of cells labeled with the specific lipid phase trans-Golgi marker, C(6)-NBD-ceramide (5 µM C(6)-NBD-ceramide-BSA complex, 5 min, 37 °C); right: same cells microinjected with a suspension of 70-nm diameter liposomes containing 5 mM sulforhodamine 101 in 2.5 mM ATP, 25 mM HEPES, 125 mM sucrose, 70 mM KCl, 2.5 mM MgCl(2) (pH 7.2) and incubated for 30 min at 37 °C. B, C(6)-NBD-ceramide image (left) and sulforhodamine 101 image (right) of a cell obtained after 0, 10, and 20 min of incubation at 37 °C. C, representative cell microinjected with the same liposome suspension but incubated at 23 °C. Scalebar, 2 µm; n, nucleus; arrow, non-microinjected cell.


After a 30-min cell incubation at 37 °C, it was estimated that 30-50% of microinjected liposomes fused with trans-Golgi (based on the ratio of trans-Golgi specific to whole cell fluorescence using liposomes containing the lipid phase marker TMR-PE). Additional incubation at 37 °C resulted in a progressive decline in trans-Golgi fluorescence (Fig. 1B) due to a combination of downstream and secretory traffic, and dye leakage. At 23 °C, however, labeling was stable for >60 min. Liposome fusion was not detected when cells were incubated at 23 °C instead of at 37 °C after microinjection (Fig. 1C), and fusion was inefficient (relative efficiency 0.2) when ATP was not included in the microinjection buffer. The sensitivity of fusion efficiency to added ATP may result from increased cytoplasmic ATP concentration and/or from replacement of ATP loss associated with microinjection. Fusion was completely blocked by addition to the microinjection buffer of 5 mMN-ethylmaleimide but not by up to 1 mM GTPS, suggesting that liposome fusion does not involve a GTP-dependent coating process(16) . Larger (200-nm diameter) and smaller (<40-nm diameter, prepared by probe sonication) liposomes did not fuse efficiently. Although we do not know the precise mechanism by which trans-Golgi is labeled selectively, the results above suggest that the liposomes introduced by microinjection may be misrecognized by the trans-Golgi as 70-nm diameter transport vesicles arising from an earlier compartment(16) .

For measurement of pH, the combination of SR (pH insensitive, red fluorescence) and FS (pK(a) 6.3, green fluorescence) was chosen based on their bright fluorescence, self-quenching at high concentrations(17) , non-overlapping fluorescence spectra, optimal pK(a), and low membrane permeability(10, 18) . After microinjection of liposomes containing SR and FS and a 30-min incubation at 37 °C, pairs of confocal images were recorded by a cooled CCD camera (Fig. 2A). trans-Golgi pH was calculated by quantitative image analysis from the FS-to-SR signal ratio after background subtraction. Absolute pH determination required in situ calibration of trans-Golgi FS-to-SR signal ratio versus pH. trans-Golgi pH was set equal to extracellular pH using bafilomycin A(1) (proton pump inhibitor, 10 nM) and the ionophore pair monensin (Na-H exchanger, 10 µM) + CCCP (protonophore, 1 µM) (Fig. 2B). The FS-to-SR signal ratio did not change when the concentrations of bafilomycin A(1), monensin, and/or CCCP were increased by 3-fold or when incubation with ionophores was extended to 30 min. Monensin in the presence of bafilomycin A(1) did not affect Golgi structure. Nigericin (K-H exchanger, 1 µM) could not be used for in situ calibration because it disrupted the Golgi structure, similar to results obtained using the Golgi-disrupting agents brefeldin A (5 µg/ml) and nocodazole (20 µg/ml). The apparent pK(a) of 6.3 measured in situ (Fig. 2C) was identical to that in cell-free aqueous solution. Time course studies indicated <5% indicator photobleaching or leakage occurred in 30 min under the conditions of our experiment.

Figure 2: trans-Golgi pH in normal human skin fibroblasts measured at 23 °C. Cells were microinjected with liposomes containing 5 mM SR and 30 mM FS and incubated at 37 °C for 30 min. A, colocalization of FS (left, green) and SR (middle, red); right, pseudocolored ratio image of FS/SR after background subtraction. B, FS, SR, and FS/SR images of a cell after perfusion for 10 min with calibration buffer at pH 6.2. C, in situ calibration curve of FS/SR signal ratio versus pH.


The FS-to-SR signal ratio was 0.48 ± 0.02 (S.E.) in 174 skin fibroblasts, corresponding to a trans-Golgi pH of 6.17 ± 0.02 (Fig. 2C). There was little pH variation in the lumen of the trans-Golgi compartment as shown by the representative pseudocolored ratio image in Fig. 2, right. Analysis of pH distributions obtained from separate cells indicated that mean trans-Golgi pH in 75% of cells was in the range 6.0-6.3. It is noted that the total intraliposomal volume microinjected into each cell (4 times 10 cm^3) was much smaller than cell volume (5.2 times 10 cm^3) or estimated Golgi volume (4 times 10 cm^3(19) ). Taken together with the incomplete fusion efficiency (30-50%) and the low intraliposomal buffer capacity (6 mM/pH unit at pH 7.0) compared with that in Golgi (50 mM/pH unit, measured by NH(4)Cl pulse technique, see Fig. 3), it is unlikely that the liposome fusion process affects trans-Golgi pH.

Figure 3: Effect of regulatory factors on trans-Golgi pH. Measurements were performed in microinjected cells after 30 min at 37 °C and an additional 30 min in PBS containing indicated compounds at 23 °C. Concentrations: 30 mM NH(4)Cl, 10 nM bafilomycin A(1), 10 nM platelet-derived growth factor B/B (PDGF), 1 µM phorbol 12-myristate 13-acetate (PMA), 0.5 mM CPT-cAMP. Cl-free buffer indicates replacement of Cl by the membrane-impermeant anion isethionate.


The influence of putative regulators of trans-Golgi pH was investigated (Fig. 3). As anticipated, inhibition of the vacuolar proton pump by bafilomycin A(1) or addition of a weak base (NH(4)Cl) caused trans-Golgi alkalinization. trans-Golgi pH was mildly increased by protein kinase C activation by phorbol 12-myristate 13-acetate, and platelet-derived growth factor, whereas protein kinase A activation by forskolin or a cell-permeable cAMP analog (CPT-cAMP) remarkably elevated trans-Golgi pH to 6.8 ± 0.1. Interestingly, a smaller but significant cAMP-induced alkalinization was reported in early endosomes from Swiss 3T3 fibroblasts labeled with a fluorescent transferrin(12) . To determine whether intracellular cAMP-stimulated Cl channels (20, 21, 22) were responsible for the alkalinization, experiments were performed in which Cl was replaced by the impermeant anion isethionate. Cytosolic Cl activity decreased from 55 to <5 mM by this maneuver as measured by SPQ fluorescence(23) . Cl removal itself caused a small trans-Golgi alkalinization but did not abolish the large cAMP-dependent alkalinization.

Our results establish an effective method to label the aqueous phase of trans-Golgi in living cells and provide the first direct measurement of trans-Golgi pH. The acidic lumenal pH is consistent with the identification of multiple pH-dependent events in the secretory pathway, including the sorting and storage of numerous secretory proteins(3, 24, 25, 26) , aggregation of pancreatic secretory proteins(27) , protein post-translational modifications by sialyltransferases(26) , binding of KDEL to its receptor(28) , and activation of virus fusion protein(7) . In addition, recent experiments on permeabilized cells suggest that pH between 6 and 6.2 in the trans-Golgi is optimal for enzymatic cleavage of prosomatostatin(29) .

The introduction by liposome fusion of fluorescent indicators of calcium, monovalent ions, and membrane potential should enable measurements of trans-Golgi ion activities and mechanisms of protein processing and secretion. The strong alkalinization of trans-Golgi by cAMP agonists is an unexpected finding that may provide an explanation, when taken together with recent data on effects of lumenal pH on vesicular transport(30) , for the cAMP-dependent inhibition of vesicular transport in some cell types (31) . Last, the ability to quantify trans-Golgi pH should facilitate direct examination of the ``defective organelle acidification hypothesis'' proposed to be the cellular basis of cystic fibrosis(32) .

FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL42368 and DK43840 and Grant R613 from the National Cystic Fibrosis Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by a fellowship from the American Lung Association.

To whom correspondence should be addressed: 1246 Health Sciences East Tower, University of California, San Francisco, CA 94143-0521. Tel.: 415-476-8530; Fax: 415-665-3847; verkman{at}itsa.ucsf.edu.

(^1)
The abbreviations used are: FS, fluorescein sulfonate; SR, sulforhodamine 101; C(6)-NBD-ceramide, N-{6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]caproyl}-sphingosine; TMR-PE, N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; BSA, defatted bovine serum albumin; CCCP, carbonyl cyaninide m-chlorophenylhydrazone; CPT-cAMP, 8-(4-chlorophenylthio)-adenosine 3`,5`-cyclic monophosphate; GTPS, guanosine 5`-O-(3-thiotriphosphate); PBS, phosphate-buffered saline.

ACKNOWLEDGEMENTS

We thank Dr. H. Pin Kao for writing the ratio imaging software.

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