- Email: [email protected]
Swelling response of golgi apparatus cisternae in cells treated with monensin is reduced by cell injury
Cell Biology lnternational Reports, Vol. 16, No. 3, 1992
21 7
SWELLING RESPONSE OF GOLGI APPARATUS CISTERNAE IN CELLS TREATED WITH MONENSIN IS R E D U C E D BY CELL INJURY
Hilton H. Mollenhauer*, D. James Morr6t, And Nita Minnifieldt *Food Animal Protection Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, College Station, TX 77845; tDepartment of Medicinal Chemistry and Pharmacognosy and Department of Biological Sciences, Purdue University, West Lafayette, IN 47907
ABSTRACT The effect of mechanical stress on Golgi apparatus was examined in thin slices of rat liver. The findings should be of relevance both to electron microscopists who routinely mince tissue, and to biochemists who homogenize tissues to isolate membranous components. The swelling response of Golgi apparatus to monensin was used as an assay because the swelling response is distinct and is thought to result from a well-characterized metabolic process, namely the acidification of vesicles. The results showed that the swelling response was compromised by monensin as far away as 6-7 cells from a cut surface even though other aspects of cell ultrastructure were not altered from normal. The monensin-induced swelling response was also evaluated in isolated Golgi apparatus and found to be similar to that with tissue. Thus, mechanical stress such as commonly used to mince tissue or isolate tissue components, appears to markedly alter Golgi apparatus function compared to the situation in vivo. In this example, the altered response of Golgi apparatus to monensin indicated that some aspects associated with the ATP-dependant proton-pumping machinery of the transmost cisternae and trans Golgi network were compromised. INTRODUCTION Golgi apparatus of cells treated with the monovalent ionophore monensin respond by swelling of trans Golgi apparatus cisternae to form large vacuoles (Tartakoff and Vassalli, 1978; Ledger et al., 1980; Mollenhauer et al., 1982, 1987). The basis for the swelling is not well understood but is generally thought to be related to the well-known ability of monensin to disrupt Golgi apparatus function. The swelling seems localized to those Golgi apparatus regions with acidified interiors (Andersotl and Pathek, 1985). In this report, we show that isolated Golgi apparatus, or Golgi apparatus of injured cells, swell little, if at all, with monensin, and that whatever swelling that may occur, is restricted to the trans-most cisternae of a stack.
0309-1651/92/030217-4/$03.00/0
© 1992 Academic Press Ltd
21 8
Cell Biology International Reports, Vol. 16, No. 3, 1992
MATERIALS AND METHODS Liver slices (300-800 ~m thick) were excised from weanling outbred rats (SpragueDawley) 8-9 days old after the rats had been anesthetized with Rompun and Ketamine. Each liver slice contained an outer (uncut) surface of liver nodule. The slices were placed in a 400 ml beaker containing 5 ml of PBS or PBS and one of the following: 5/~! of 10-3 M monensin (Calbiochem) dissolved in ethyl alcohol, 50/xl of 10-4 M aqueous monensin, 5/.d ethyl alcohol, or 50/zl of water. The slices were then incubated for 30 min at 37* with gentle shaking. After incubation, the liver slices were prefixed in 3% glutaraldehyde + 0.05 M sucrose + 10% saturated picric acid, in 0.05 M PIPES, pH 7.4, for about 120 min. The slices were post-fixed for 90 min in 1% OsO 4 + 0.05 M sucrose + 0.05 M potassium ferricyanide in PIPES as above. Fixatives were at ice bath temperature except for the first 30-60 min which were at room temperature. The Golgi apparatus fractions were from livers of adult male Holtzman or SpragueDawley rats (ca. 200g) prepared as described (Morr6, 1971). All tissues and fractions were dehydrated in a graded series of ethyl alcohol and/or acetone, and embedded in either Epon 812 or a modified Spurr mixture (Mollenhauer, 1986). RESULTS The Golgi apparatus of the control liver slices (Fig. l) were essentially indistinguishable from those of comparably-fixed livers of whole animals where no swollen or vacuolated cisternae were observed. When the liver slices were treated with monensin and then tLxed in glutaraldehyde followed by osmium tetroxide, the cisternae of Golgi apparatus near the natural edges of the liver slices appeared swollen, and the amount of swelling increased progressively from cis to trans poles (Fig. 2). However, in Goigi apparatus adjacent to a cut edge of a liver slice, swelling was either absent or limited to the single trans-most cisterna (Fig. 3). Other ultrastructural aspects of cell architecture were relatively normal. These monensin effects were observed to a depth of about 6-7 cells into the liver slice apparently reflecting the limit of monensin penetration (Morr6 et al., 1987). In isolated Golgi apparatus stacks, only the trans-most cisterna (and often none) responded to monensin by swelling (Fig. 4; compare with the control stack of Fig. 5). In the monensin-treated preparations, swollen cisternae were present in only about 30% of the Golgi apparatus stacks. The results were the same with tissues from either the Holtzman or Sprague-Dawley strains of rats. DISCUSSION Monensin apparently causes swelling of Golgi apparatus cisternae in plant cells (Mollenhauer et al., 1982, 1988) and the amount and pattern of swelling is indicated by the usual glutaraldehyde and osmium tetroxide fixation. This conclusion is based on the fact that freeze substitution (which stabilizes tissue in milliseconds compared to minutes for chemical fixation; Bendayan, 1984) gives essentially the Same image as does fixation in glutaraldehyde followed by osmium tetroxide. Similar observations of freeze-substituted animal cells in culture have been reported by Yarowsky et al. (1986). Thus, it seems that swelling of cisternae is a primary physical response of both plant and animal Golgi apparatus to monensin. Cellular injury, such as excision of tissue slices, clearly alters the pattern of Golgi apparatus swelling (i.e., diminishes the number of swollen cisternae) implying that the trans cisternae (or at least some function of the trans cisternae) are diminished or,
Cell Biology International Reports, VoL 16, No. 3, 1992
21 9
Fig. 1. Control cell from a ca 400/.tin thick slice of liver excised from a 8-9 dy old rat and incubated for 30 min with constant agitation in growth medium. Golgi apparatus stacks (arrows) were typical of those in cells that were within 2-6 cells of the natural (i.e., uncut) edge of the liver lobe. Fig. 2. Cell equivalent to that of Fig. 1 except treated with 10/xM monensin. Swollen cisternae and vesicles were numerous. Swelling was progressive from cis to trans poles of the Golgi apparatus stacks (arrows). Fig. 3. Treated cell equivalent to that of Fig. 2 except that the Golgi apparatus (arrow) was from a cell adjacent to a cut edge of the tissue slice. Typically, in these Golgi apparatus, only the most trans cisterna (or no cisterna) was swollen. Note similarity of this response to that of the isolated rat liver Golgi apparatus of Fig. 4. Fig. 4. Isolated Golgi apparatus stack (arrows) from rat liver treated with 1 /zM monensin in the presence of 1 mM ATP, 1 mM MgCI 2 and 50 mM NaC1, pH 6.0 containing 0.05 M sucrose for 30 minutes at room temperature. A single swollen cisterna, presumably trans, was observed in 30% of the Golgi apparatus stacks. Other stacks showed no swollen cisternae. This contrasted with the situation in situ where virtually all trans cisternae were swollen when exposed to monensin (see Fig. 2). Fig. 5. As in Fig. 4 except incubated in medium without monensin. Swollen cisternae were rare.
220
Cell Biology International Reports, VoL 16, No. 3, 1992
sometimes, even eliminated. A similar response to injury (in this case dissolution of the cell wall) has been reported in plant protoplasts where little or no monensin-induced swelling response could be elicited prior to regeneration of a cell wall (Brightman et al., 1985). These data suggest that isolated Golgi apparatus should, also, exhibit diminished monensin-induced swelling response as was observed in this study (Figs. 4, 5). Monensin-induced swelling of Golgi apparatus cisternae is due, presumably, to the uptake of osmotically-active cations accompanied by a concomitant efflux of H + and entry of water (Geisow and Burgoyne, 1982). Maintenance of a pH gradient requires both sealed vesicles and the presence of a functional ATP-driven proton pump (Boss et al., 1984). Lack of swelling would imply an inability to achieve or maintain such a gradient. With isolated Golgi apparatus and Golgi apparatus of injured cells, only those cisternae already acidified appear to swell with monensin as if further Golgi apparatus swelling were dependent upon the continued formation of new Golgi apparatus cisternae. The formation of new cisternae apparently ceases when Goigi apparatus are isolated from the cell, and may be compromised upon cell injury as well. REFERENCES Anderson, R.G.W. and Pathek, R.K. (1985) Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell 40:635-643. Bendayan, M. (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications and limitations. Journal of Electron Microscopy Technique 1:243-270. Boss, W.F., Morr6, D.J. and Moilenhauer, H.H. (1984) Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient. European Journal of Cell Biology 34:1-8. Brightman, A.O., Boss, W.F. and Morr6, D.J. (1985) An atypical response of some Golgi apparatus of carrot protoplasts to the sodium-selective ionophore monensin. Plant Physiology 77:1 (Supplement). Geisow, M.J. and Burgoyne, R.D. (1982) Effect of monensin on chromaffin cells and the mechanism of organelle swelling. Cell Biology International Reports 6:933-939. Mollenhauer, H.H. (1986) Surfactants as resin modifiers and their effect on sectioning. Journal of Electron Microscopy Technique 3:217-222. Mollenhauer, H.H., Morr6, D.J. and Norman, J.O. (1982) Ultrastructural observations of maize root tips following exposure to monensin. Protoplasm a 112:117-126. Mollenhauer, H.H., Morr6, D.J. and Minnifield, N. (1987) Effect of fixation on Golgi apparatus swelling following treatment with monensin. Journal of Cell Biology 105:75a. Mollenhauer, H.H., Morr6, D.J. and Bracker, C.E. (1988) Swelling of Golgi apparatus cisternae of monensin-treated tissues is modulated by f'Lxation conditions. Protoplasma 145:66-69. Morr6, D.J. (1971) Isolation of Golgi apparatus. Methods in Enzymology 22:130-148. Morr6, D.J., Morr6, D.M., Mollenhauer, H.H. and Reutter, W. (1987) Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices. European Journal of Cell Biology 43:232-235. Tartakoff, A. and Vassalli, P. (1978) Comparative studies of intercellular transport of secretory proteins. Journal of Cell Biology 79:694-707. Yarowsky, P., Boyne, A.F., Wierwille, R. and Brooks, N. (1986) Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry. Journal of Neuroscience 6:859-866. Paper received 20.11.92. Paper accepted 13.01.92.
21 7
SWELLING RESPONSE OF GOLGI APPARATUS CISTERNAE IN CELLS TREATED WITH MONENSIN IS R E D U C E D BY CELL INJURY
Hilton H. Mollenhauer*, D. James Morr6t, And Nita Minnifieldt *Food Animal Protection Research Laboratory, U.S. Department of Agriculture, Agricultural Research Service, College Station, TX 77845; tDepartment of Medicinal Chemistry and Pharmacognosy and Department of Biological Sciences, Purdue University, West Lafayette, IN 47907
ABSTRACT The effect of mechanical stress on Golgi apparatus was examined in thin slices of rat liver. The findings should be of relevance both to electron microscopists who routinely mince tissue, and to biochemists who homogenize tissues to isolate membranous components. The swelling response of Golgi apparatus to monensin was used as an assay because the swelling response is distinct and is thought to result from a well-characterized metabolic process, namely the acidification of vesicles. The results showed that the swelling response was compromised by monensin as far away as 6-7 cells from a cut surface even though other aspects of cell ultrastructure were not altered from normal. The monensin-induced swelling response was also evaluated in isolated Golgi apparatus and found to be similar to that with tissue. Thus, mechanical stress such as commonly used to mince tissue or isolate tissue components, appears to markedly alter Golgi apparatus function compared to the situation in vivo. In this example, the altered response of Golgi apparatus to monensin indicated that some aspects associated with the ATP-dependant proton-pumping machinery of the transmost cisternae and trans Golgi network were compromised. INTRODUCTION Golgi apparatus of cells treated with the monovalent ionophore monensin respond by swelling of trans Golgi apparatus cisternae to form large vacuoles (Tartakoff and Vassalli, 1978; Ledger et al., 1980; Mollenhauer et al., 1982, 1987). The basis for the swelling is not well understood but is generally thought to be related to the well-known ability of monensin to disrupt Golgi apparatus function. The swelling seems localized to those Golgi apparatus regions with acidified interiors (Andersotl and Pathek, 1985). In this report, we show that isolated Golgi apparatus, or Golgi apparatus of injured cells, swell little, if at all, with monensin, and that whatever swelling that may occur, is restricted to the trans-most cisternae of a stack.
0309-1651/92/030217-4/$03.00/0
© 1992 Academic Press Ltd
21 8
Cell Biology International Reports, Vol. 16, No. 3, 1992
MATERIALS AND METHODS Liver slices (300-800 ~m thick) were excised from weanling outbred rats (SpragueDawley) 8-9 days old after the rats had been anesthetized with Rompun and Ketamine. Each liver slice contained an outer (uncut) surface of liver nodule. The slices were placed in a 400 ml beaker containing 5 ml of PBS or PBS and one of the following: 5/~! of 10-3 M monensin (Calbiochem) dissolved in ethyl alcohol, 50/xl of 10-4 M aqueous monensin, 5/.d ethyl alcohol, or 50/zl of water. The slices were then incubated for 30 min at 37* with gentle shaking. After incubation, the liver slices were prefixed in 3% glutaraldehyde + 0.05 M sucrose + 10% saturated picric acid, in 0.05 M PIPES, pH 7.4, for about 120 min. The slices were post-fixed for 90 min in 1% OsO 4 + 0.05 M sucrose + 0.05 M potassium ferricyanide in PIPES as above. Fixatives were at ice bath temperature except for the first 30-60 min which were at room temperature. The Golgi apparatus fractions were from livers of adult male Holtzman or SpragueDawley rats (ca. 200g) prepared as described (Morr6, 1971). All tissues and fractions were dehydrated in a graded series of ethyl alcohol and/or acetone, and embedded in either Epon 812 or a modified Spurr mixture (Mollenhauer, 1986). RESULTS The Golgi apparatus of the control liver slices (Fig. l) were essentially indistinguishable from those of comparably-fixed livers of whole animals where no swollen or vacuolated cisternae were observed. When the liver slices were treated with monensin and then tLxed in glutaraldehyde followed by osmium tetroxide, the cisternae of Golgi apparatus near the natural edges of the liver slices appeared swollen, and the amount of swelling increased progressively from cis to trans poles (Fig. 2). However, in Goigi apparatus adjacent to a cut edge of a liver slice, swelling was either absent or limited to the single trans-most cisterna (Fig. 3). Other ultrastructural aspects of cell architecture were relatively normal. These monensin effects were observed to a depth of about 6-7 cells into the liver slice apparently reflecting the limit of monensin penetration (Morr6 et al., 1987). In isolated Golgi apparatus stacks, only the trans-most cisterna (and often none) responded to monensin by swelling (Fig. 4; compare with the control stack of Fig. 5). In the monensin-treated preparations, swollen cisternae were present in only about 30% of the Golgi apparatus stacks. The results were the same with tissues from either the Holtzman or Sprague-Dawley strains of rats. DISCUSSION Monensin apparently causes swelling of Golgi apparatus cisternae in plant cells (Mollenhauer et al., 1982, 1988) and the amount and pattern of swelling is indicated by the usual glutaraldehyde and osmium tetroxide fixation. This conclusion is based on the fact that freeze substitution (which stabilizes tissue in milliseconds compared to minutes for chemical fixation; Bendayan, 1984) gives essentially the Same image as does fixation in glutaraldehyde followed by osmium tetroxide. Similar observations of freeze-substituted animal cells in culture have been reported by Yarowsky et al. (1986). Thus, it seems that swelling of cisternae is a primary physical response of both plant and animal Golgi apparatus to monensin. Cellular injury, such as excision of tissue slices, clearly alters the pattern of Golgi apparatus swelling (i.e., diminishes the number of swollen cisternae) implying that the trans cisternae (or at least some function of the trans cisternae) are diminished or,
Cell Biology International Reports, VoL 16, No. 3, 1992
21 9
Fig. 1. Control cell from a ca 400/.tin thick slice of liver excised from a 8-9 dy old rat and incubated for 30 min with constant agitation in growth medium. Golgi apparatus stacks (arrows) were typical of those in cells that were within 2-6 cells of the natural (i.e., uncut) edge of the liver lobe. Fig. 2. Cell equivalent to that of Fig. 1 except treated with 10/xM monensin. Swollen cisternae and vesicles were numerous. Swelling was progressive from cis to trans poles of the Golgi apparatus stacks (arrows). Fig. 3. Treated cell equivalent to that of Fig. 2 except that the Golgi apparatus (arrow) was from a cell adjacent to a cut edge of the tissue slice. Typically, in these Golgi apparatus, only the most trans cisterna (or no cisterna) was swollen. Note similarity of this response to that of the isolated rat liver Golgi apparatus of Fig. 4. Fig. 4. Isolated Golgi apparatus stack (arrows) from rat liver treated with 1 /zM monensin in the presence of 1 mM ATP, 1 mM MgCI 2 and 50 mM NaC1, pH 6.0 containing 0.05 M sucrose for 30 minutes at room temperature. A single swollen cisterna, presumably trans, was observed in 30% of the Golgi apparatus stacks. Other stacks showed no swollen cisternae. This contrasted with the situation in situ where virtually all trans cisternae were swollen when exposed to monensin (see Fig. 2). Fig. 5. As in Fig. 4 except incubated in medium without monensin. Swollen cisternae were rare.
220
Cell Biology International Reports, VoL 16, No. 3, 1992
sometimes, even eliminated. A similar response to injury (in this case dissolution of the cell wall) has been reported in plant protoplasts where little or no monensin-induced swelling response could be elicited prior to regeneration of a cell wall (Brightman et al., 1985). These data suggest that isolated Golgi apparatus should, also, exhibit diminished monensin-induced swelling response as was observed in this study (Figs. 4, 5). Monensin-induced swelling of Golgi apparatus cisternae is due, presumably, to the uptake of osmotically-active cations accompanied by a concomitant efflux of H + and entry of water (Geisow and Burgoyne, 1982). Maintenance of a pH gradient requires both sealed vesicles and the presence of a functional ATP-driven proton pump (Boss et al., 1984). Lack of swelling would imply an inability to achieve or maintain such a gradient. With isolated Golgi apparatus and Golgi apparatus of injured cells, only those cisternae already acidified appear to swell with monensin as if further Golgi apparatus swelling were dependent upon the continued formation of new Golgi apparatus cisternae. The formation of new cisternae apparently ceases when Goigi apparatus are isolated from the cell, and may be compromised upon cell injury as well. REFERENCES Anderson, R.G.W. and Pathek, R.K. (1985) Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments. Cell 40:635-643. Bendayan, M. (1984) Protein A-gold electron microscopic immunocytochemistry: methods, applications and limitations. Journal of Electron Microscopy Technique 1:243-270. Boss, W.F., Morr6, D.J. and Moilenhauer, H.H. (1984) Monensin-induced swelling of Golgi apparatus cisternae mediated by a proton gradient. European Journal of Cell Biology 34:1-8. Brightman, A.O., Boss, W.F. and Morr6, D.J. (1985) An atypical response of some Golgi apparatus of carrot protoplasts to the sodium-selective ionophore monensin. Plant Physiology 77:1 (Supplement). Geisow, M.J. and Burgoyne, R.D. (1982) Effect of monensin on chromaffin cells and the mechanism of organelle swelling. Cell Biology International Reports 6:933-939. Mollenhauer, H.H. (1986) Surfactants as resin modifiers and their effect on sectioning. Journal of Electron Microscopy Technique 3:217-222. Mollenhauer, H.H., Morr6, D.J. and Norman, J.O. (1982) Ultrastructural observations of maize root tips following exposure to monensin. Protoplasm a 112:117-126. Mollenhauer, H.H., Morr6, D.J. and Minnifield, N. (1987) Effect of fixation on Golgi apparatus swelling following treatment with monensin. Journal of Cell Biology 105:75a. Mollenhauer, H.H., Morr6, D.J. and Bracker, C.E. (1988) Swelling of Golgi apparatus cisternae of monensin-treated tissues is modulated by f'Lxation conditions. Protoplasma 145:66-69. Morr6, D.J. (1971) Isolation of Golgi apparatus. Methods in Enzymology 22:130-148. Morr6, D.J., Morr6, D.M., Mollenhauer, H.H. and Reutter, W. (1987) Golgi apparatus cisternae of monensin-treated cells accumulate in the cytoplasm of liver slices. European Journal of Cell Biology 43:232-235. Tartakoff, A. and Vassalli, P. (1978) Comparative studies of intercellular transport of secretory proteins. Journal of Cell Biology 79:694-707. Yarowsky, P., Boyne, A.F., Wierwille, R. and Brooks, N. (1986) Effect of monensin on deoxyglucose uptake in cultured astrocytes: energy metabolism is coupled to sodium entry. Journal of Neuroscience 6:859-866. Paper received 20.11.92. Paper accepted 13.01.92.