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. 1999 Jan;80(1):51–57. doi: 10.1046/j.1365-2613.1999.00097.x

Low cytoplasmic pH causes fragmentation and dispersal of the Golgi apparatus in human hepatoma cells

Toshimichi Yoshida 1, Tomoko Kamiya 1, Kyoko Imanaka-Yoshida 1, Teruyo Sakakura 1
PMCID: PMC2517749  PMID: 10365087

Abstract

The centrosomal localization of the Golgi apparatus in interphase cells is thought to be maintained by retrograde microtubule-based motility. It is well established that, when intracellular pH is lowered, lysosomes and endosomes, also showing pericentrosomal localization, translocate towards the plus ends of microtubules within 15 min. In this study, we found that prolonged incubation in low pH medium (pH 6.6) with 20 mm Na acetate induced the fragmentation and dispersal of the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. The fraction of Golgi-dispersed cells increased in a time-dependent manner, and reached over 60% after the 16-h incubation. The cytoplasmic pH was dropped to approximately 7.10. Replacement with normal pH medium restored the structure and localization of the apparatus within 30 min. In the low pH condition, the microtubular network and endoplasmic reticulum appeared normal, and cytoplasmic dynein was still bound to the fragmented Golgi membranes. These findings suggest that low cytoplasmic pH suppresses the retrograde movement of the Golgi apparatus as well as that of lysosomes and endosomes.

Keywords: Golgi apparatus, intracellular pH, microtubule-based motility

The Golgi apparatus is a membranous organelle showing a distinctive structure. It is usually located in the centrosomal region of interphase cells as a central organelle for membrane traffic and sorting (Thyberg & Moskalewski 1985; Ho et al. 1989; Turner & Tartakoff 1993). During mitosis, the Golgi apparatus is rapidly disassembled into small fragments and dispersed throughout the cytoplasm (Zeligs & Wollman 1979; Lucocq et al. 1989). Treatment with okadaic acid causes fragmentation of the apparatus, in association with microtubule disorganization and arrested intracellular transport (Lucocq et al. 1991; Thyberg & Moskalewski 1992). Brefeldin A inhibits the membrane traffic into the Golgi apparatus, followed by Golgi disassembly (reviewed by Klausner et al. 1992; Lippincott-Schwartz 1993). Microtubule-disrupting agents such as nocodazole also induce Golgi dispersal (Thyberg & Moskalewski 1985; Ho et al. 1989; Turner & Tartakoff 1993; Cole et al. 1996). Other studies have shown an association of retrograde microtubule motors such as cytoplasmic dynein with the Golgi membranes, suggesting the involvement of microtubule-based motility in the centrosomal positioning (Corthésy-Theulaz et al. 1992; Vaisberg et al. 1996). These findings indicate that localization and structure of the Golgi apparatus is maintained by dynamic intracellular movements.

Lysosomes and late endosomes are also organelles located in the pericentrosomal region. The localization of these organelles is also maintained by microtubule-based motility (Matteoni & Kreis 1987; Lin & Collins 1992; Aniento et al. 1993). Several studies have demonstrated the redistribution of lysozomes and late endosomes by changes in cytoplasmic pH (Heuser 1989; Parton et al. 1991; Lin & Collins 1992). The lowering of intracellular pH (pHi) causes the translocation of these organelles towards the cell periphery, while alkalization produces a shift in distribution towards the centrosome. The redistribution of these organelles occurs quickly (15 min) after the change of extracellular pH (pHo). This short-term acidification has no affect on the microtubular network or the distribution of other organelles, including the Golgi apparatus (Parton et al. 1991).

In the present study, we examined the effects of prolonged acidification on the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. Prolonged low pHo (6.6) treatment induced the fragmentation and dispersal of the Golgi apparatus without changes in the microtubular network or endoplasmic reticulum (ER). We discuss the mechanisms of the Golgi fragmentation by the acidification of pHi.

Methods

Cell culture

The human hepatoma cell line PLC/PRF/5 was supplied by the Japanese Cancer Research Resources Bank, Tokyo, Japan. The cells were grown in Dulbecco's modified Eagle medium (DMEM, GIBCO, Grand Island, NY, USA) supplemented with 10% foetal calf serum (FCS, GIBCO). The cells were plated onto coverslips in 35-mm Petri dishes. Three days after plating, the dishes were washed three times with 2 ml of DMEM/10% FCS with 25 mm MOPS-NaOH and 5 mm NaHCO3 (MOPS-buffered DMEM), pH 7.4 or 6.6, or MOPS-buffered DMEM with 20 mm Na acetate. The dishes were then covered with Parafilm and incubated at 37°C for 0–24 h.

Measurement of intracellular pH (pHi)

The pHi was measured by a method described previously (Yoshida et al. 1993). In brief, the cells were loaded with 2 μm of a pH indicator, 2′,7′-bis-(2-carboxyethyl)-5(and-6) carbosyfluorescein, acetoxymetyl ester (BCECF-AM, Molecular Probes, Eugene, OR, USA) by incubation in 20 mm MOPS-NaOH-buffered loading medium (pH 7.4 or 6.6, 115 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl2, 13.8 mm glucose, 1.4 mm CaCl2, 5 mm NaHCO3, with or without 20 mm Na acetate) for 30 min at room temperature. The cells were washed twice and further incubated in 2 ml of the loading medium for at least 30 min to allow deesterification of the indicator. The coverslips were mounted on slide glasses with thin Teflon spacers. The measurement was performed at 30 °C using a fluorescence microphotometer (Nikon, Tokyo, Japan) with a 40x objective lens. The pH levels were estimated by measuring the ratios of fluorescence intensities of the cytoplasmic area at 440 and 480 nm excitation. The emission filter was 535 nm. The fluorescence ratios were compared with a calibration curve obtained by incubating BCECF-loaded cells in various pH media containing 10 μg/ml nigericin (Sigma, St. Louis, MO, USA) and with a high concentration of KCl substituted for NaCl in the loading medium.

Fluorescent staining of Golgi apparatus

The Golgi apparatus, particularly the trans- Golgi stacks, is labelled with NBD-ceramide (Pagano 1989). The cells grown on coverslips were fixed with 4% paraformaldehyde in 0.1 m PIPES buffer, pH 6.9, containing 2 mm EGTA and 2 mm MgCl2 for 15 min. After three washes in 10 mm HEPES-buffered saline (pH 7.4), the cells were incubated in a 10-fold diluted solution of C6 NBD-ceramide (Molecular Probes)/bovine serum albumin complex for 1 h at room temperature. The cells were washed twice in 10-fold diluted FCS solution, and then incubated in the solution for 1 h. After treatment with 2 mg/ml p-phenylenediamine, they were mounted onto slide glasses. The cells were observed under an Olympus fluorescence microscope (Olympus, Tokyo, Japan).

Immunofluorescence

The cells were incubated in the microtubule-stabilizing buffer (0.1 m PIPES-KOH pH 6.9, 5 mm MgSO4, 10 mm EGTA, 4% polyethylene glycol) containing 0.02% saponin for 2 min at 37 °C, and then fixed in the buffer containing 3.7% formaldehyde and 0.1% Triton X-100. The cells were washed several times in phosphate-buffered saline (PBS), treated with normal goat serum in PBS for 30 min, and incubated in affinity-purified rabbit anticytoplasmic dynein antibody (10 μg/ml) or affinity-purified rabbit anticalreticulin antibody (10 μg/ml) for 2 h at room temperature. The characterization of antibodies specific to bovine brain cytoplasmic dynein (Yoshida et al. 1992) and to bovine liver calreticulin (Ioshii et al. 1995) was reported previously. After several washes with PBS, the cells were treated with FITC-conjugated goat antirabbit IgG (100 × diluted; MBL, Nagoya, Japan) for 1 h. Following several washes, they were incubated in mouse monoclonal antiα-tubulin antibody (100-fold diluted; Cederlane, Ontario, Canada) for 1 h, and then treated with rhodamine-conjugated goat antimouse IgG antibody (100× diluted; Tago, Burlingame, CA, USA). The stained cells were observed using a fluorescent microscope installed with appropriate filters for double immunofluorescence. To label Golgi 58 kD protein and β-COP, the cells were fixed in 0.1 m phosphate buffer (pH 7.4) containing 3.7% formaldehyde and 0.2% Triton X-100. They were incubated with anti58 kD protein or antiβ-COP (50× diluted; Sigma, St. Louis, MO, U.S.A) overnight, and then with FITC-conjugated goat antimouse IgG (MBL) for 1 h.

Results

Golgi fragmentation and dispersal by low pHi

After 16-h incubation in the low pH (pH 6.6) medium with 20 mm Na acetate, the most cells showed dispersed Golgi fragments throughout the cytoplasm (Figure 1B), while the apparatus appeared to have a normal structure and localization in the physiological pH (7.4) medium (Figure 1A). Golgi 58 kD protein and β-COP were also labelled on the dispersed fragments in the low pH (Figure 1C,D, respectively). We counted cells showing the complete dispersion of the Golgi apparatus. The percentages of the cells and pHi under various conditions for 16 h are summarized in Table 1. The pHi of cells incubated in low pHo with 20 mm Na acetate was approximately 7.10. The frequency of Golgi dispersal was approximately 60%. Next, changes in the Golgi apparatus in a variety of incubation periods were examined. In a low-pH medium with Na acetate, the ratio of cells with Golgi dispersal increased in a time-dependent manner (Figure 2). The extent of the dispersal also became greater. When the low-pH medium was replaced with the normal medium at 16 h, the cells showing Golgi dispersal almost disappeared within 30 min (Figure 2). The fragmented Golgi membranes were quickly reorganized into the stacks at the centrosomal region. We examined how rapidly the pHi alters after the change of the pHo. The pHi of BCECF-loaded cells incubated in the low-pH buffer for 30 min was 7.0. After 1-, 4-, and 8-h culture in low pHo, the pHi was approximately 7.1, whereas the cells in normal pHo maintained around 7.4 of pHi (Figure 3). The pHi of the cells cultured for 16 h in low pHo and then incubated in normal pHo for 30 min was approximately 7.4. These findings indicate that prolonged low pHi causes the Golgi fragmentation and dispersal, and that normal pHi quickly restores the structure and localization.

Figure 1.

Figure 1

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The Golgi apparatus of hepatoma cells stained with NBD-ceramide after a 16-h incubation in normal (A) and low (B) pH media. The Golgi apparatus shows centrosomal positioning in most interphase cells incubated in normal pH medium (A), but fragmentation and dispersal was seen in the cells after the 16-h incubation in low pH medium (B). Golgi 58 kD protein and β-COP were also labelled on the dispersed fragments by the low pH (C and D, respectively). Bar; 50 μm for A and B; 30 μm for C and D.

Table 1.

Cytoplasmic pH and percentages of cells with dispersed Golgi fragments after a 16-h incubation in various media

graphic file with name iep0080-0051-t1.jpg

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* Total number of the measured cells in each group was 60 from three experiments. Values are mean ± SD of the experiments. ** More than 600 cells from three experiments were assessed. Values are mean ± SD of triplicate experiments.

Figure 2.

Figure 2

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Ratios of Golgi-dispersed cells in normal and low-pH media in a variety of incubation times. The ratio of Golgi-dispersed cells increased with the incubation time in the pH 6.6 medium (•), while the ratio was low in the pH 7.4 medium (▪). When the low-pH medium was replaced with normal pH medium at 16 h (○), the structure and localization of the Golgi apparatus was quickly restored.

Figure 3.

Figure 3

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Intracellular pH in normal and low-pH media in a variety of incubation times. The pHi of BCECF-loaded cells incubated in the low-pH buffer for 30 min was 7.0. After 1, 4 and 8 hours incubation in low pHo (•), the pHi was approximately 7.1, whereas the cells in normal pHo maintained around 7.4 (▪). The values are mean ± SD from the 20 cells.

The distribution of MTs, ER and cytoplasmic dynein in low pHi

We investigated whether prolonged low pHo would affect the MT network and membranous organelles other than the Golgi apparatus and lysosomes. After 16-h treatment, no changes in MTs were observed (Figure 4A). Immunofluorescence for calreticulin, localized in the ER (Ioshii et al. 1995), also showed a normal appearance of the ER with a polygonal tubular network throughout the cytoplasm after 16 h-acidification (Figure 4B). Thus, Golgi fragmentation is not a consequence of either disorganization of MT networks or cellular damage. It was reported that serum depletion and low extracellular Ca2+ caused the dissociation of cytoplasmic dynein from lysosomes, followed by lysosomal dispersion (Lin & Collins 1993). We therefore checked for any association between cytoplasmic dynein and the Golgi membrane. Although an immunofluorescence of cytoplasmic dynein by the usual fixation showed a diffuse cytoplasmic staining (Yoshida et al. 1990), a treatment by saponin allowed the preferential labelling of the Golgi membrane (Figure 4C). The Golgi fragments induced by acidification were still labelled (Figure 4D).

Figure 4.

Figure 4

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Effects of low cytoplasmic pH on microtubular network, endoplasmic reticulum, and binding of cytoplasmic dynein onto Golgi membranes. Cells were incubated in normal pH medium (C) or in low pH (A, B, D) for 16 h. Low pH for 16 h did not affect the microtubular network (A) or endoplasmic reticulum (B). Anti-cytoplasmic dynein antibody showed labelling of Golgi apparatus (C). Low pH induced Golgi dispersal, but the fragmented Golgi membranes retained the dynein staining (D). Doubule immunofluorescence for microtubules (A) and the dynein (D) is shown. Bar; 50 μm.

Discussion

Previous studies have demonstrated that the rapid translocation of lysosomes and endosomes towards the plus ends of microtubules, but not that of the Golgi apparatus, is caused by the lowering of the pHi. In this study, we demonstrated that the prolonged (16 h) incubation of hepatoma cells in acidic pHo causes the fragmentation and dispersal of the Golgi apparatus, being associated with a low pHi value. The treatment of microtubule depolymerizing drugs disrupts the Golgi apparatus (Thyberg & Moskalewski 1985; Ho et al. 1989; Turner & Tartakoff 1993; Cole et al. 1996). Okadaic acid, a phosphatase inhibitor, also induces Golgi disruption, accompanied by a loss of the microtubular network (Thyberg & Moskalewski 1992). The Golgi disruption observed in the present study can not be explained by the mechanisms, since low pHi does not affect the microtubular network. Brefeldin A, another drug inducing Golgi disassembly, prevents the assembly of coatomers on the membrane, followed by blocking of the ER-to-Golgi transport. In combination with the preserved Golgi-to-ER transport, Golgi membranes are redistributed into the ER (reviewed by Klausner et al. 1992; Lippincott-Schwartz 1993). This Golgi disassembly is completed much faster (within 30 min) than that induced by low pHi.

An association of MT-based motors, cytoplasmic dynein and kinesin, with the Golgi membrane has been demonstrated (Corthésy-Theulaz et al. 1992; Marks et al. 1994; Vaisberg et al. 1996). To account for the translocation of lysosomes and endosomes in low pHi, a model has been proposed that kinesin activity would be favoured over dynein in low pHi conditions and the organelles could move towards the plus ends of the MTs (Heuser 1989; Parton et al. 1991). This model is also acceptable as a mechanism of Golgi dispersal. However, there is an apparent difference of time-courses between Golgi dispersal, for 16 h, and the translocation of the latter organelles, within 15 min. In contrast, after replacement of the low pH medium with the normal pH medium, Golgi apparatus rearranges within 30 min as quickly as lysosomes and endosomes. A recent study has reported two novel dynein heavy chain-like proteins (DHCs 2 and 3) that are distinct from the conventional heavy chain of cytoplasmic dynein, and demonstrated the localization of DHC2 on the Golgi membranes and of DHC3 on lysosomes and endosomes (Vaisberg et al. 1996). Dynein family molecules bound to the Golgi membranes may be more resistant to the low pHi condition than those bound to lysosomes and endosomes. The difference in the time-courses of these events suggests different molecular machinery to maintain the positioning of the organelles. Recent studies also demonstrate the presence of a spectrin-based matrix associated with Golgi membranes (Devarajan et al. 1996; Beck et al. 1997; Lippincott-Schwartz 1998). The spectrin meshwork may provide structural integrity and stability to the membrane.

Thus, low pHi induced the fragmentation and dispersal of the Golgi apparatus as well as the translocation of lysosomes and endosomes. Various pathological conditions in vivo, such as ischemia and metabolic disorders, are known to cause low pHi, following low pHo with an increase in concentration of organic acids (Johnson et al. 1995; LaManna et al. 1995). A disorganization of the Golgi apparatus, possibly accompanied by the inactivation of membrane traffic, could account for the disturbed cellular functions in these conditions. Further attentions on changes of Golgi apparatus in the pathological tissues in vivo are necessary.

Acknowledgments

This research was supported in part by Grants-In-Aid from the Ministry of Education, Science, Culture and Sports, Japan, and by a Narishige Zoological Science Award.

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