ARTICLE |
Correspondence to: Hideaki Tamaki, Dept. of Anatomy, Kitasato Univ. School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan. E-mail: tamaki@kitasato-u.ac.jp
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Summary |
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We examined the effects of specific inhibitors, brefeldin A (BFA) and okadaic acid (OA), on the ultrastructural organization of the Golgi apparatus and distributions of amylase, Golgi-associated proteins, and cathepsin D in the rat parotid acinar cells. BFA induced a rapid regression of the Golgi stack into rudimentary Golgi clusters composed of tubulovesicules, in parallel with a redistribution of the Golgi-resident proteins and a coat protein (ß-COP) into the region of the rough endoplasmic reticulum (rER) or cytosol. The rapid disruption of the Golgi stack could also be induced by the effect of OA. However, redistribution of the Golgi proteins in rER or cytosol could not be observed and ß-COP was not dispersed but was retained on the rudimentary Golgi apparatus. These findings suggested that the mechanism of OA in inducing degeneration of the Golgi stack was markedly different from that of BFA. In addition, missorting of amylase, a Golgi protein, and cathepsin D into incorrect transport pathways is apparent in the course of the disruption of the Golgi stack by OA. These Golgi-disrupting effects are reversible and the reconstruction of the stacked structure of the Golgi apparatus started immediately after the removal of inhibitors. In the recovery processes, missorting was also observed until the integrated structure of the Golgi apparatus was completely reconstructed. This suggested that the integrated structure of the Golgi apparatus was quite necessary for the occurrence of normal secretory events, including proper sorting of molecules. (J Histochem Cytochem 50:16111623, 2002)
Key Words: Golgi apparatus, okadaic acid, brefeldin A, parotid gland, immunogold electron, microscopy
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Introduction |
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MOST MODERN KNOWLEDGE about the Golgi apparatus and intracellular traffic through the organelle has been accumulated by application of genetic and biochemical approaches and immunofluorescent cytochemistry to unravel the operation of this organelle in yeast, cultured cells, or cell-free systems (for review see
The fungal metabolite BFA has been known to induce rapid and reversible disassembly of the Golgi stack into tubules and vesicles, resulting in the redistribution of Golgi-resident enzymes into the endoplasmic reticulum in a reversible manner (
OA, a monocarboxylic polyether isolated from marine sponges, is a potent inhibitor of the protein phosphatases 1 and 2a and has been used to investigate the role of dephosphorylation in various regulated cell events (
In addition, the disassembly and reassembly processes in Golgi apparatus induced by BFA or OA were found to be a quite useful model to analyze not only the mechanism for the establishment and maintenance of the integrated stacked structure but also the relationship between the structure and function of the Golgi apparatus (
Detailed investigations at the ultrastructural level in highly specialized and parenchymal cells have been limited, mainly because of the difficulties in preservation of antigenicity and this organelle's complicated morphology. We conducted an examination on the importance of the stacked structure and functions of the Golgi apparatus in the rat parotid acinar cell, because this cell type has the great advantage of containing highly developed and organized secretory components that are easily defined morphologically (
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Materials and Methods |
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Materials
The monoclonal antibody (MAb) raised from the Golgi fraction of guinea pig pancreas, GF-1 (mouse IgM), has been described previously (-amylase antiserum, the rabbit anti-calf brain cathepsin D antiserum, anti-ß-COP MAb (mouse IgG1), and BFA were purchased from Sigma (St Louis, MO). Anti-mannosidase II MAb (mouse IgG2b) was obtained from Berkeley Antibody (Richmond, CA). OA was from Wako Pure Chemicals (Tokyo, Japan). BFA and OA were prepared as stock solutions in methanol and stored at -20C. Goat anti-mouse IgG1 labeled by FITC and goat anti-mouse IgG2b labeled by rhodamine were purchased from Southern Biotechnology Associates (Birmingham, AL). Biotinylated goat anti-mouse IgM and HRP-conjugated streptavidin were purchased from Vector Laboratories (Burlingame, CA). Goat anti-mouse IgM labeled by 15-nm colloidal gold, goat anti-mouse IgG1, and goat anti-rabbit IgG labeled by 10-nm gold particles were purchased from Zymed (San Francisco, CA) and EY Laboratories (San Mateo, CA). Dulbecco's modified Eagle's minimal essential medium (MEM) was a product of Nissui Pharmaceutical (Tokyo, Japan). DL-isoproterenolHCl was from Nakarai Tesque (Kyoto, Japan). Medium-grade LR White resin was obtained from London Resin (Basingstoke, Hampshire, UK). All other chemicals were analytical or electron microscopic grade.
Experimental Procedure
Male Wistar rats were injected IP with isoproterenol at a dose of 2 mg/100 g body weight to induce salivary secretion. One hour later, both lobes of parotid glands were excised immediately after exsanguination through the abdominal aorta under ether anesthesia and were dissected into small pieces of lobules. These tissue pieces were incubated in MEM containing 25 mM HEPES, 0.3 mg/ml sodium bicarbonate (pH 7.2), and one of the Golgi-disrupting drugs, either BFA or OA at 37C with constant oscillation in a water bath incubator. The working concentration of the drugs added to the medium and the incubation time were as follows: 0.5 µM OA, up to 30 min or 2.5 µg/ml BFA, up to 90 min. To examine the recovery phase from the effects of BFA or OA, the tissue pieces treated with the Golgi-disrupting drugs as described above were rinsed three times with fresh medium containing no drug and chase incubation was conducted. After these treatments, tissue pieces were processed for immunocytochemistry. At least three incubations from three animals were conducted for every treatment.
SDS-PAGE and Immunoblotting
To evaluate the dynamics of the GF-1 antigen molecule under the effect of OA, immunoblotting for GF-1 was conducted. Tissue pieces were incubated in MEM with or without 0.5 µM OA up to 30 min as above. Then the tissues and culture medium were separated. The medium was concentrated by ultrafiltration to prepare for immunoblotting. The tissue slices were washed with ice-cold PBS and homogenized in a glass homogenizer with a Teflon pestle. Soluble and membrane-bound proteins were separated into aqueous and detergent phase by phase separation with Triton X-114 solution (
Immunofluorescent Cytochemistry
Parotid tissue pieces were fixed with PLP solution at 4C for 2 hr. After cryoprotection with gum sucrose, cryostat sections about 6 µm thick were cut and nonspecific staining was blocked with 1% BSAPBS for 1 hr. The sections were incubated with a mixture of anti-ß-COP and anti-mannosidase II in 1% BSAPBS at RT for 1 hr. After washing in PBS, the sections were incubated with a mixture of FITC-conjugated anti-mouse IgG1 and rhodamine-conjugated anti-mouse IgG2b for 1 hr. Negative control staining was conducted by replacing the primary antibodies with 1% BSAPBS. Crossreaction of secondary antibodies against IgG subclass was also examined by swapping the combination with primary antibodies. They were then observed using a confocal scanning laser microscope (MRC-1000; Bio-Rad, Hercules, CA). Serial optical sections (collected at 1-µm intervals) in the z-axis were collected and overlaid for the final images shown.
Immunogold Electron Microscopy
Tissue pieces were fixed with 0.05% glutaraldehyde4% paraformaldehyde in 0.1 M cacodylate buffer (pH 7.2) for 1 hr. After cryoprotection with 2.3 M sucrose in PBS (
High-resolution immunoelectron microscopy using postfixation with ferocyanide-reduced osmium and embedding in LR white resin was conducted as previously described (
Quantitative Evaluation of Immunogold Labeling
To confirm missorting of GF-1 antigen and cathepsin D, the density of labeling expressed as number of gold particles per µm2 of the different cell compartments (secretory granules, Golgi apparatus, lysosomes, vesicles/ER/cytosol) were evaluated on at least five electron micrographs of the drug-treated cells and untreated cells. The outlines of each cell compartment were traced on a digitizing tablet with a stylus pen and were fed into the TRI image analyzer (Ratoc System Engineering; Tokyo, Japan), and then area measurement was performed. Mean labeling density (±SD) for each cell compartment was determined.
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Results |
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Dynamics of the Golgi-resident Enzyme and Coat Protein
Fig 1 illustrates the distributional change in a Golgi-resident enzyme, mannosidase II (Man II), and a COP-I coat protein (ß-COP), in rat parotid acinar cells under the effects of Golgi-disrupting agents. In untreated acinar cells, ß-COP was mainly associated with the region of the Golgi apparatus in a reticular form on which Man II was co-localized (Fig 1a1c). Rapid redistribution of Man II into the region of the endoplasmic reticulum (ER) was obvious in the cells 90 min after BFA treatment (Fig 1f). ß-COP also lost its association with the Golgi region and dispersed throughout the cytoplasm (Fig 1d and Fig 1e). OA also disrupts the reticular structure of the Golgi apparatus into many fragments but, in contrast to BFA, redistribution of Man II into the ER could not be observed. Man II was closely associated with the fragmented Golgi apparatus. ß-COP was markedly accumulated in the region of the disrupted Golgi on which Man II was retained (Fig 1g1i).
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Immunogold electron microscopy on ultrathin cryosections clarified the distributional change in the ß-COP-accompanied structural changes in the Golgi apparatus induced by BFA or OA. Fig 2a shows the immunogold reaction of anti-ß-COP on an ultrathin cryosection of normal rat parotid acinar cells. Gold labeling is abundant mostly on the Golgi stack. Fig 2b shows the rapid redistribution of ß-COP to the entire cytoplasm in BFA-treated cells. It is apparent that gold labeling is decreased on the degenerating Golgi apparatus and is dispersed throughout the cytoplasm. However, ß-COP is closely restricted on the degenerating Golgi apparatus without dispersion to the cytoplasm in the cells 30 min after OA treatment (Fig 2c). In the high-power view, the Golgi cluster was found to be composed of ß-COP-positive and -negative vesicles. Both vesicles show somewhat regional distributions in the cluster (Fig 2d).
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Distributional Changes of Exportable Proteins and GF-1 Antigenicity
Next, ultrastructural change was collated with the distributional dynamics of exportable proteins and GF-1 antigen, and the effect of BFA and OA was compared by the double-labeling method at the electron microscopic level.
Untreated Cells. Fig 3a shows an electron micrograph of an untreated rat parotid acinar cell doubly stained with anti-amylase and GF-1 immunocytochemistry. The cytoplasmic organelles are well developed for the exocrine function in this cell. The rough endoplasmic reticulum (rER) is tightly stacked in the basal region of the cell. The Golgi apparatus consists of four or five layers of flattened cisternae. Condensing vacuoles of various shapes and sizes are present at the concave face of the Golgi stack. A number of secretory granules are accumulated in the apical cytoplasm. The immunoreactivity of amylase, a major secretory enzyme in this cell, as detected by smaller gold particles (10 nm in diameter) is distributed through the rER, Golgi apparatus, condensing vacuoles, and secretory granules. It is apparent that amylase is gradually condensed in the manner of a regulatory secretion pathway, whereas larger (15 nm) gold particles labeling GF-1 antigenicity are localized on the membranes of the concave trans-region of the Golgi stack, the trans-most discontinuous membranes identified as the trans-Golgi network (TGN), and condensing vacuoles. However, secretory granules, rER, lysosomes, cytosol, and the cell surfaces are completely negative in the reaction. The GF-1 antigen was retained on the membranes of the trans-Golgi area and was never sorted into the post-Golgi transport pathway in normal acinar cells. Immunoreactivity for lysosomal enzyme, cathepsin D, was detectable only on the contents of lysosomes, and the secretory granules are completely negative (Fig 3b). Quantitative analysis of immunoelectron micrographs further confirmed these observations. GF-1 antigenicity was mostly detectable on the Golgi apparatus (Table 1, untreated), and cathepsin D was on lysosomes (Table 2, untreated).
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Isoproterenol-stimulated Cells. Isoproterenol induced complete exocrine release of secretory granules from rat parotid acinar cells, followed by the formation of new granules. Fig 4 shows the Golgi area of the acinar cell after 60 min of secretory stimulation. The apical cytoplasm contained no secretory granules, but newly formed granules were observed in the concaved trans-face of the Golgi apparatus. The area of the trans-Golgi component was markedly extended, and GF-1 reactivity on the trans-Golgi membranes was also apparently increased. These morphological findings strongly suggested that biosynthesis and intracellular transport were enhanced and accelerated by the secretory stimulation. However, GF-1 antigen was still retained on the trans-Golgi membranes and was never transported into newly formed secretory granules, as well as other post-Golgi traffic in such stimulated cells.
Effect of BFA on Stimulated Cells
Next we examined effect of BFA on the morphological and functional alterations of the Golgi apparatus during the reconstitution phase of secretory granules in the isoproterenol-treated cells. In parotid acinar cells at 90 min after treatment with BFA, the cisternae of the rER were markedly dilated and filled with electron-lucent material, and the Golgi apparatus completely lost its stacked structure and degenerated into Golgi clusters composed of various shapes and sizes of tubulovesicles. Because of the inhibition of protein transport, neither secretory granules nor condensing vacuoles were formed during the BFA treatment. Both GF-1 and anti-amylase immunoreactivities were decreased on the tubulovesicles of the disrupted Golgi cluster and were markedly redistributed into the dilated cisternae of the rER (Fig 5a).
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Such effects of BFA have been known to be fully reversible, and gradual reconstruction of the stacked structure of the Golgi apparatus could also be observed after withdrawal of the drug in the parotid acinar cells (
Effect of OA on Stimulated Cells
OA also completely disrupted the Golgi stack of acinar cells, within 30 min, into clusters of small vesicles about 70 nm in diameter. However, the manner of the disruption of the Golgi stack differed considerably from that by BFA. The tubular profile of the membrane was very rare and the vesicles in the Golgi clusters were more uniform in shape and size than those obtained by BFA. Neither dilation of the rER nor redistribution of the GF-1 antigen to the rER could be observed during OA treatment. In addition, the formation of the secretory granules was not stalled during the degeneration of the stack (Fig 6a). A small number of the granules, which were smaller than those in normal cells or the cells in the recovery phase from BFA, were observed in the cytoplasm. The granules contained not only amylase but also GF-1 antigenicity. The antigenicity of a lysosomal enzyme, cathepsin D was also abnormally detectable in these granules (Fig 6b; Table 2). In addition, amylase and GF-1 antigen were sorted onto the constitutive pathway. Both antigenicities were detectable on very small vesicle-like structures (Table 2) that apparently differed from secretory granules in morphology and were released into not only the luminal space (Fig 6c) but also into the basolateral space between acinar cells (Fig 6d) without secretory stimulation.
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Such missorting and constitutive secretion were confirmed by biochemical assay using immunoblotting for GF-1 antigen. GF-1 antigen is a Golgi membrane-bounded protein and is separated in the detergent phase in untreated cells. In OA-treated cells, however, the protein is transferred to the soluble aqueous phase and is released into the culture medium without secretory stimulation (Fig 7). As above, marked abnormality of sorting and retention mechanisms also took place in the degenerating Golgi apparatus by OA. The Golgi stack-disrupting effect of OA was reversible, similar to that of BFA. Fig 8a shows a cell at 1 hr after the removal of OA. In the clusters of small vesicles, some tubular profiles have started to appear on which GF-1 antigenicity is condensed. These tubular structures should be considered as a primitive form of the reforming Golgi stack. At 2 hr after removal of OA (Fig 8b), re-formation of the Golgi stack continued, from which condensing vacuoles arose. However, GF-1 antigenicity was still distributed through the re-forming stack rich in vesicular membranes and was partially separated from the limiting membranes, resulting in co-localization with amylase in the vacuole contents. At this stage, it was apparent that recovery of sorting machinery had not been completed. At 4 hr after removal (Fig 8c), the re-formation of the Golgi stack was nearly completed. GF-1 antigenicity was restricted to the trans-region of the Golgi apparatus. The condensing vacuole containing only amylase was formed from a well-defined trans-Golgi network. In OA treatment, the normal sorting or retention mechanism was also recovered on the completion of stack formation and establishment of the cis-trans direction of the Golgi apparatus.
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Discussion |
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Disruption of the Golgi Stack by BFA and OA
We demonstrated that the Golgi apparatus of parotid acinar cells shows complete and reversible disorganization of the stacked structure into rudimentary Golgi clusters composed of vesicles or tubules under the effects of both BFA and OA. BFA induced redistribution of Man II and ß-COP from the Golgi apparatus into the entire cytoplasm. BFA may inhibit attachment of ß-COP to membranes, resulting in the dispersion of ß-COP and disturbance of COP-I transport vesicle formation. The interruption of ß-COP-associated transport induced by BFA should cause a redistribution of a Golgi membrane protein into the ER, followed by disorganization of the stacked structure.
It is widely known that BFA rapidly and reversibly blocks intracellular transport by preventing the association of ß-COP with the donor membranes to cause a concomitant rapid redistribution of Golgi enzymes into the rER and vesiculation of the Golgi stack in many cell types. These phenomena were explained to be a discrete event and, once the transport from rER to the Golgi was inhibited by BFA, the microtubule-dependent tubular structure emanated from the Golgi stack and then the tubule fused to the rER (
OA also induced disorganization of the stack, but the mechanism seemed to be different from that of BFA. OA is a potent inhibitor of protein phosphatase, and has various effects on cellular events that related to intracellular traffic, such as (a) impairment of protein synthesis, (b) inhibition of fluid-phase endocytosis, (c) inhibition of regulated exocytosis, and (d) arrest of transport from the rER to the Golgi apparatus (
Missorting by a Disorganized Golgi Stack
The most interesting finding in this study was the abnormality of sorting and retention machinery in both the degenerating and regenerating Golgi apparatus. The GF-1 antigen molecule, which is normally attached to the membranes of the trans-Golgi and condensing vacuoles, became detached from membranes and was transported via regulative as well as constitutive pathways. Amylase and cathepsin D were also abnormally sorted into incorrect pathways.
Another possible explanation of the expression of the GF-1 antigen into the luminal and the basolateral space is the inhibition of the recycling pathway between the Golgi apparatus and the cell surface by increased phosphorylation induced by OA.
Granule Formation
The distributional changes in the GF-1 antigenicity on the Golgi clusters is another interesting finding in the present study. The Golgi cluster induced by BFA showed faint reactivity for GF-1 and could not produce new secretory granules. Granule formation was re-initiated as soon as the GF-1 antigen was condensed on the reconstructing Golgi stack, whereas the Golgi cluster induced by OA, on which strong GF-1 antigenicity could be detected, had the capability of producing small amounts of secretory granules. A high-resolution immunoelectron microscopic study during the treatments with BFA or OA and during the recovery phase revealed that both degenerating and regenerating Golgi stacks retained the capability of forming secretory granules if the GF-1-positive trans-Golgi elements were maintained but that the sorting machinery within an incomplete stack was considerably disturbed. Normal secretory function was concluded to be fully active only when recovery of the structural integrity of the Golgi apparatus was completed.
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Acknowledgments |
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Supported in part by a grant-in-aid for General Scientific Research (no. 11470008) from the Ministry of Education, Science and Culture of Japan and by the All Kitasato Project Study Grant from the Kitasato Gakuen Foundation.
We thank Mr Osamu Katsumata for technical assistance. The electron microscopy was performed at the Electron Microscopic Laboratory Center of the Kitasato University School of Medicine.
Received for publication December 19, 2001; accepted June 12, 2002.
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