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Visualization of calcium-binding sites at plasma membrane of shock-frozen Acanthamoeba cells
Act a histochem. 68, 125 - 129 (1981)
M. N encki Instit ute of Experimental Biology, W arsaw, Poland
Visualization of calcium-binding sites at plasma membrane of shock-frozen Acanthamoeba cells By A"NDRZEJ SOBOTA and ALEKSANDRA PRZELEOKA With 5 figures (Recei ve d June 2, 1980)
Summary Elect ron·dense deposits appeal' at the protoplasmic side of plasma membrane in Acanthamoeba log· phase cells when fixed either with glutaraldehyde or formaldehyde supplemented with Ca 2 +. Similar deposits appear when the cells are preloaded wit h Ca2+ and thereafter shock· frozen and prepared for electron microscopic examina tion b y free ze·substitution t echnique. This suggests that their form ation reflects the presen('e of a physiologically active system involved in capturing excess of inflowing ('alcium .
Introduction Electron-dense calcium-dependent deposits appear at the protoplasmic side of the plasma membrane of some cells when they are fi xed in glutaraldehyde supplemented with calcium ions. The deposits are observed independently of whether the cells are subsequently postfixed with an osmium tetroxide-CaCl2 mixture, or not. They were found at the plasma membrane of cells active in food absorption, like insect intestinal epithelium (OSOHMAN et al. 1972), insect oocytes, but only at the stage of vitellogenesis (PRZELEOKA et al. 1976), and in amoebae (SOBOTA et al. 1977, STEOKEM et al. 1979), all highly active in pinocytosis. Moreover, the deposits were found in nerve cells the physiological functioning of which is virtually dependent on the calcium level. The presence of calcium in t he deposits was shown by use of the electron microprobe X-ray microanalysis (OSOHMA"N et a!. 1974, SOBOTA et al. 1978). It has been suggested that the appearence of the Ca-deposits reflects the presence of calcium-binding sites involved in regulating the intracellular calcium level. However, as the deposits are formed in the presence of glutaraldehyde, the question arises whether they might be a kind of a rtifacts induced by chemical fixation . To solve this question experiments were done in which Acanthamoeba cells were loaded with calcium under physiological conditions and prepared thereafter for electron microscope examination by shock-freezing and freeze-substitution technique.
Material and Methods Acantha11loebu castellanii cells (Neff strain) were grown a xenically, without aeration , at 28°C in darkness, as described previously (SOBOTA et al. 1977). Cells fro m log· phase cultures (5 to
126
A. SOBOTA and A. PRZELECKA
7 days) were collected by centrifugation at 600 g for 2 min. In order to load the cells with ~al('iulll they were resuspended in 100 mM HEPES buffer, pH = 7.4 containing either 10 or 50 m"~1 C"CI 2 • Cells at a final concentration of about 4 J 0 5 cells/ml were incubated in this medium for 120 min at 5 to 8°C. Under these conditions, it was shown by radio assay that the Acanthamoeba cells a('('uIllulate about 15 nIlloles Ca2+ per 1 mg cell dry weight per h. After incubation the cells were harvested by eentrifugation in tubE'S the bottoms of which were coverE'd with thin aluminium foil. Subsequently, the aluminium eups ('ontaining the cell pellet, removed from the tubes, were rapidly dried with filter paper and quenched eithE'!' in isopE'ntane or in propane both previously cooled to - H;O °C by liquid nitrogen. Freeze substitution of isoppntane quenehed samples was carried out in dry hexane containing 0.1 % osmium tetr'oxide (w/v), at about - ti5 °C for three weeks. The temperature was maintained by a solid carbon dioxide-ethanol bath. DUl'ing substitution the hexane-Os0 4 solution was continuously dried by means of a molecular sieve type 4 A. After fulfilling the substitution the tubes containing the samples were slowly warmed gradually up to 0 DC. Then the samples were transferred through a propylene oxide-Epon 812 mixture and embedded in Epon 812 (NEUMAXN 1973). The propane-gu(,Il(·hed samples were transferred into another portion of propane, cooled with liquid nitrogen as well and dried eontinuously by means of a molecular sieve, type 4A. In this freeze-substitution media the "Pll samples were maintained fol' 5 days at about -150°C. Ther'eatter, the samples were transferred into a eopper ehamber, also cooled with liquid nitrogen. Subsequently, the {'hamber was placed in vacuum and slowly, over 24 h, warmed up to room temperature. During this procedure propane evaporated slowly and the cells, devoided of this substitution fluid, were transferred dire<:tly into low-viseosity Spurr resin used as embedding media (Be-ROVINA, personal (·ommunieation). For comparison, some Acanthamoeba cells were fixed either in 2.5% glutaraldehyde or in 4% formaldehyde, both supplemented with 10 mM CaCl2 , and postfixed in 1 % OsO. containing 10 mM CaCI 2 , ac('ording to the routine procedure (OSCRMAN et al. 1972). Ultrathin cell sections were cut with an LKB II Ultratome using glass knives and examined in a JEM 100 B electron microscope after contrasting them with uranyl and lead salts, or without any additional contrast. The control samples were prepared for electron microseope examination in a similar mamwr as the experimental ones only without any exposure of the cells to the excess of calcium ions, either before, or during the fixation.
Results and Discussion In Acanthamoeba cells which have been loaded with calcium and prepared by the freeze-substitution technique, without any further exposure to calcium ions, numerous electron-dense deposits are observed at the protoplasmic side of their plasma membrane. The deposits form thin plaques adhering to the plasma membrane. They are well visible in uncontrasted sections. They appear both after using propane or hexaneosmium mixture as substitution media (Figs. 1 to 3). The presence of osmium tetroxide in the substitution media enhances only the electron contrast of the sample, without influencing the appearance of the deposits. A similar effect was obtained when osmium tetroxide was applied as postfixing agent for visualization of the calcium-dependent deposits by the conventional OSCIIMAN and WALL procedure (OSCIIMAN et al. 1972). It should be added that the poor preservation of the cell interior seen on the presented electron micrographs (Figs. 1 to 3) could be forseen. We consciously avoided soaking the cells before freezing in a cryoprotective fluid, as it is done in routinc practice. The rational was to prevent any chemical disturbances which could impair formation of the deposits. Glycerol which is most fre-
127
VislH,liz" l ion of calcium-binding sites
0.1.
0.1pm
I
i.1 ./
0.1 IJ m
11l1m
O.~m
2
3
Fig.!. Cr yofixed, he xane-osmium freeze-~ubstitute d Acanthamoeba cell. Before eryo[ixation the cells wt're in cubated in medium containing 50 111M C"CI2 fmd ]00 mM HE PES buffer, pH = 7.4 at 5 to il DC. Insert: Fragment of the cell sho wn in Fig. 1. - 0.1., i.l. - oute r and inner plasma membrane It'aflet. Uneontl'aste
quent.Jy applied as a cryoprotecting agent in Acanthamoeba cells was found to prevent formation of the calcium-related deposits, most probably owing to the extraction of the low molecular weight phosphat.ase responsible for their formation (SOBOTA et al. 1977, 1978). Another cryoprotector, namely DMSO (dimethylsulphoxide) apparently affects protein-lipid association in the cell membrane and this may also prevent formation of the deposits, as has been shown previously by treating the cells with Triton X 100, acting similarly in this respect (SOBOTA et al. 1977). The lack of any electron-dense structures in control unloaded cells examined without additional staining, independently of the Stl bstitution medium used for their preparation, confirmed the calcium relation of the deposits found in the cells loaded with calcium.
128
A.
SOBOTA
a nd A.
FRZELECKA
-
0.1 JJm
4
Fi g. 4. Acanthamopba cell fix ed in formaldehyde contain in g 10 mM CaCl z and postfixed in oSl1lium·CaCl 2 solution; d ectron·df'nse calcium-related deposits form tiny plaques adherin g the prot,oplasmic surface of the pl"811»\ membrane. Fig . 5. Acantharnoeba eell fix ed in glutaraldehyde containing 10 mM CaCl 2 and post{ixed in osmium-CaCl z solution; ,,1f'(,t ron-denRf' ealcium-rdated deposit'" fo rm eharHderistic ellipsoidal stn\('t urt's a dht'ring the protophtsm ic s urfa ce of the plasma m embrane.
The shape of the electron-dense deposit s observed in calcium-loaded and freeze-substituted ceBs slightly differs from that characteristic for the deposits form ed in the presence of glutaraldehyde used in conventional procedure. Only in the eaEe when formaldehyde is appli ed instead of glutaraldehyde, did the cakium-dependent depoRits appear as tiny plaques like in the calcium-loaded and shock-frozen cells (Fig., 4) . It seelllS that the morphological character of t he deposits is influenced by the (;onditions of their form ation. During fixation with glutaraldehyde-CaCl 2 solution, conventional Oschman and Wall' procedure (OseRMAN et al. 1972) calcium enters the cell together with glutaraldehyde. The known ability of this bifunctional fixative to react with various chemically active groups of cell const-it uents induces crosslinking of the la tter (HOPWOOD 197:3) what may prevent shifting of the molecules engaged in the formation of calcium deposits. As the result, the deposits form characteristic semispherical ellipsoid struct ures, dist inctly circumscribed in t he surrounding protoplasm (Fig. 5). l!'ixation of cells with fo rmaldehyde having only one fu nctional group is apparently less accurate than fixation with glutaraldehyde lasting equally long. Hence during dehydration of the formaldehyde-fixed cells in a graded series of ethanol some rearrangements of cell structure at the molecular level due to diffusion, may occur. Such
Visualization of calcium-binding sites
129
rearrangements may be responsible for the appearance of deposits in the form of plaques tightly adhering to the cell membrane. The similar appearance of the deposits in shock-frozen and freeze-substituted cells, forming plaques with even diffuse contours, may be caused by organic solvents: hexane and/or propane used as substitution medium. In eonclusion, it seems that the calcium-related deposits appearing in calcium preloaded and shock-frozen Acanthamoeba cells, as well as in aldehyde-CaCl 2 mixture fixed cells, reflect a physiological phenomenon of binding the excess of inflowing calcium at the plasma membrane. Cells may differ in the intensity of this process, what probably is the reason why the resulting deposits may not be visualized at the electron microscope level in all of them. This suggestion coincides well with the observations done on Acanthamoeba castellanii in which the calcium-related deposits are particularly large and abundant in cells descending from the advanced exponential growth phase, whereas they are quite minute and hardly distinguishable in cells from the early exponential growth phase and do not appear at all in cells undergoing encystment (in preparation), Visualization of calcium binding at the cell membrane by the method in which calcium inflows into the cell simultaneously with the chemical fixative may be regarded only as more convenient and easier preparatory technique than the freeze-substitution.
Acknowledgements The skillful technical assistance of Mrs. K. MROZINSKA is greatly appreciated.
Literature HOPWOOD, D., Theoretical and practical aspects of glutaraldehyde fixation. Fixation in Biochemistry (P.L. Stoward, eeL), pp. 47-83. Chapman & Hall, London 1973. NEUMANN, D., Zur Darstellung pflanzlicher Gewebe nach Gefriersubstitution unter besonderer Beriicksichtigung der Strukturerhaltung der Plastiden. Acta Histochem. 47, 278-288 (1973). OSCRMAN, L., and VVALL, B. F., Calcium binding to intestinal membranes ..J. Cell BioI. 55, 58-73 (1972). - HALL, T. A., l'ETEltS, P. D., and WALL, B ..J., Association of calcium with membfanes of squid axon .•J. Cell BioI. 61, 156-165 (1974). PRZELECKA, A., and SOBOTA, A., Calcium-dependent deposits at the plasma membrane during development of the oocyte of Galleria mellonella. Cyto biologie 13, 182 -190 (1976). SOBOTA, A., HREBENDA, B., and I'RZELECKA, A., Formation of calcium-dependent deposits at the plasma membrane of Acanthamoeba castellanii. Cytobiologie 15, 259-268 (1977). - PRZELECKA, A., and JAXOSSY, A. G. S., X-ray microanalysis of calcium-dependent deposits at the plasma membrane of Acanthamoeba castellanii. Cytobiologie 17, 464-469 (1978). STOCKEM, \V., and KLEIN, H. P., Pinoeytosis and locomotion in Amoeba. XV. Demonstration of Ca++-binding sites during pinocytosis in Amoeba proteus. Protoplasm a 100, 33-43 (1979). Address: Dr. A. SOBOTA, Department of Cell Biology, M. Nencki Institute of Experimental Biology, 3 Pasteur Street, PL - 02-093 vVarsaw.
9 Acta histochem. Bd. 68
M. N encki Instit ute of Experimental Biology, W arsaw, Poland
Visualization of calcium-binding sites at plasma membrane of shock-frozen Acanthamoeba cells By A"NDRZEJ SOBOTA and ALEKSANDRA PRZELEOKA With 5 figures (Recei ve d June 2, 1980)
Summary Elect ron·dense deposits appeal' at the protoplasmic side of plasma membrane in Acanthamoeba log· phase cells when fixed either with glutaraldehyde or formaldehyde supplemented with Ca 2 +. Similar deposits appear when the cells are preloaded wit h Ca2+ and thereafter shock· frozen and prepared for electron microscopic examina tion b y free ze·substitution t echnique. This suggests that their form ation reflects the presen('e of a physiologically active system involved in capturing excess of inflowing ('alcium .
Introduction Electron-dense calcium-dependent deposits appear at the protoplasmic side of the plasma membrane of some cells when they are fi xed in glutaraldehyde supplemented with calcium ions. The deposits are observed independently of whether the cells are subsequently postfixed with an osmium tetroxide-CaCl2 mixture, or not. They were found at the plasma membrane of cells active in food absorption, like insect intestinal epithelium (OSOHMAN et al. 1972), insect oocytes, but only at the stage of vitellogenesis (PRZELEOKA et al. 1976), and in amoebae (SOBOTA et al. 1977, STEOKEM et al. 1979), all highly active in pinocytosis. Moreover, the deposits were found in nerve cells the physiological functioning of which is virtually dependent on the calcium level. The presence of calcium in t he deposits was shown by use of the electron microprobe X-ray microanalysis (OSOHMA"N et a!. 1974, SOBOTA et al. 1978). It has been suggested that the appearence of the Ca-deposits reflects the presence of calcium-binding sites involved in regulating the intracellular calcium level. However, as the deposits are formed in the presence of glutaraldehyde, the question arises whether they might be a kind of a rtifacts induced by chemical fixation . To solve this question experiments were done in which Acanthamoeba cells were loaded with calcium under physiological conditions and prepared thereafter for electron microscope examination by shock-freezing and freeze-substitution technique.
Material and Methods Acantha11loebu castellanii cells (Neff strain) were grown a xenically, without aeration , at 28°C in darkness, as described previously (SOBOTA et al. 1977). Cells fro m log· phase cultures (5 to
126
A. SOBOTA and A. PRZELECKA
7 days) were collected by centrifugation at 600 g for 2 min. In order to load the cells with ~al('iulll they were resuspended in 100 mM HEPES buffer, pH = 7.4 containing either 10 or 50 m"~1 C"CI 2 • Cells at a final concentration of about 4 J 0 5 cells/ml were incubated in this medium for 120 min at 5 to 8°C. Under these conditions, it was shown by radio assay that the Acanthamoeba cells a('('uIllulate about 15 nIlloles Ca2+ per 1 mg cell dry weight per h. After incubation the cells were harvested by eentrifugation in tubE'S the bottoms of which were coverE'd with thin aluminium foil. Subsequently, the aluminium eups ('ontaining the cell pellet, removed from the tubes, were rapidly dried with filter paper and quenched eithE'!' in isopE'ntane or in propane both previously cooled to - H;O °C by liquid nitrogen. Freeze substitution of isoppntane quenehed samples was carried out in dry hexane containing 0.1 % osmium tetr'oxide (w/v), at about - ti5 °C for three weeks. The temperature was maintained by a solid carbon dioxide-ethanol bath. DUl'ing substitution the hexane-Os0 4 solution was continuously dried by means of a molecular sieve type 4 A. After fulfilling the substitution the tubes containing the samples were slowly warmed gradually up to 0 DC. Then the samples were transferred through a propylene oxide-Epon 812 mixture and embedded in Epon 812 (NEUMAXN 1973). The propane-gu(,Il(·hed samples were transferred into another portion of propane, cooled with liquid nitrogen as well and dried eontinuously by means of a molecular sieve, type 4A. In this freeze-substitution media the "Pll samples were maintained fol' 5 days at about -150°C. Ther'eatter, the samples were transferred into a eopper ehamber, also cooled with liquid nitrogen. Subsequently, the {'hamber was placed in vacuum and slowly, over 24 h, warmed up to room temperature. During this procedure propane evaporated slowly and the cells, devoided of this substitution fluid, were transferred dire<:tly into low-viseosity Spurr resin used as embedding media (Be-ROVINA, personal (·ommunieation). For comparison, some Acanthamoeba cells were fixed either in 2.5% glutaraldehyde or in 4% formaldehyde, both supplemented with 10 mM CaCl2 , and postfixed in 1 % OsO. containing 10 mM CaCI 2 , ac('ording to the routine procedure (OSCRMAN et al. 1972). Ultrathin cell sections were cut with an LKB II Ultratome using glass knives and examined in a JEM 100 B electron microscope after contrasting them with uranyl and lead salts, or without any additional contrast. The control samples were prepared for electron microseope examination in a similar mamwr as the experimental ones only without any exposure of the cells to the excess of calcium ions, either before, or during the fixation.
Results and Discussion In Acanthamoeba cells which have been loaded with calcium and prepared by the freeze-substitution technique, without any further exposure to calcium ions, numerous electron-dense deposits are observed at the protoplasmic side of their plasma membrane. The deposits form thin plaques adhering to the plasma membrane. They are well visible in uncontrasted sections. They appear both after using propane or hexaneosmium mixture as substitution media (Figs. 1 to 3). The presence of osmium tetroxide in the substitution media enhances only the electron contrast of the sample, without influencing the appearance of the deposits. A similar effect was obtained when osmium tetroxide was applied as postfixing agent for visualization of the calcium-dependent deposits by the conventional OSCIIMAN and WALL procedure (OSCIIMAN et al. 1972). It should be added that the poor preservation of the cell interior seen on the presented electron micrographs (Figs. 1 to 3) could be forseen. We consciously avoided soaking the cells before freezing in a cryoprotective fluid, as it is done in routinc practice. The rational was to prevent any chemical disturbances which could impair formation of the deposits. Glycerol which is most fre-
127
VislH,liz" l ion of calcium-binding sites
0.1.
0.1pm
I
i.1 ./
0.1 IJ m
11l1m
O.~m
2
3
Fig.!. Cr yofixed, he xane-osmium freeze-~ubstitute d Acanthamoeba cell. Before eryo[ixation the cells wt're in cubated in medium containing 50 111M C"CI2 fmd ]00 mM HE PES buffer, pH = 7.4 at 5 to il DC. Insert: Fragment of the cell sho wn in Fig. 1. - 0.1., i.l. - oute r and inner plasma membrane It'aflet. Uneontl'aste
quent.Jy applied as a cryoprotecting agent in Acanthamoeba cells was found to prevent formation of the calcium-related deposits, most probably owing to the extraction of the low molecular weight phosphat.ase responsible for their formation (SOBOTA et al. 1977, 1978). Another cryoprotector, namely DMSO (dimethylsulphoxide) apparently affects protein-lipid association in the cell membrane and this may also prevent formation of the deposits, as has been shown previously by treating the cells with Triton X 100, acting similarly in this respect (SOBOTA et al. 1977). The lack of any electron-dense structures in control unloaded cells examined without additional staining, independently of the Stl bstitution medium used for their preparation, confirmed the calcium relation of the deposits found in the cells loaded with calcium.
128
A.
SOBOTA
a nd A.
FRZELECKA
-
0.1 JJm
4
Fi g. 4. Acanthamopba cell fix ed in formaldehyde contain in g 10 mM CaCl z and postfixed in oSl1lium·CaCl 2 solution; d ectron·df'nse calcium-related deposits form tiny plaques adherin g the prot,oplasmic surface of the pl"811»\ membrane. Fig . 5. Acantharnoeba eell fix ed in glutaraldehyde containing 10 mM CaCl 2 and post{ixed in osmium-CaCl z solution; ,,1f'(,t ron-denRf' ealcium-rdated deposit'" fo rm eharHderistic ellipsoidal stn\('t urt's a dht'ring the protophtsm ic s urfa ce of the plasma m embrane.
The shape of the electron-dense deposit s observed in calcium-loaded and freeze-substituted ceBs slightly differs from that characteristic for the deposits form ed in the presence of glutaraldehyde used in conventional procedure. Only in the eaEe when formaldehyde is appli ed instead of glutaraldehyde, did the cakium-dependent depoRits appear as tiny plaques like in the calcium-loaded and shock-frozen cells (Fig., 4) . It seelllS that the morphological character of t he deposits is influenced by the (;onditions of their form ation. During fixation with glutaraldehyde-CaCl 2 solution, conventional Oschman and Wall' procedure (OseRMAN et al. 1972) calcium enters the cell together with glutaraldehyde. The known ability of this bifunctional fixative to react with various chemically active groups of cell const-it uents induces crosslinking of the la tter (HOPWOOD 197:3) what may prevent shifting of the molecules engaged in the formation of calcium deposits. As the result, the deposits form characteristic semispherical ellipsoid struct ures, dist inctly circumscribed in t he surrounding protoplasm (Fig. 5). l!'ixation of cells with fo rmaldehyde having only one fu nctional group is apparently less accurate than fixation with glutaraldehyde lasting equally long. Hence during dehydration of the formaldehyde-fixed cells in a graded series of ethanol some rearrangements of cell structure at the molecular level due to diffusion, may occur. Such
Visualization of calcium-binding sites
129
rearrangements may be responsible for the appearance of deposits in the form of plaques tightly adhering to the cell membrane. The similar appearance of the deposits in shock-frozen and freeze-substituted cells, forming plaques with even diffuse contours, may be caused by organic solvents: hexane and/or propane used as substitution medium. In eonclusion, it seems that the calcium-related deposits appearing in calcium preloaded and shock-frozen Acanthamoeba cells, as well as in aldehyde-CaCl 2 mixture fixed cells, reflect a physiological phenomenon of binding the excess of inflowing calcium at the plasma membrane. Cells may differ in the intensity of this process, what probably is the reason why the resulting deposits may not be visualized at the electron microscope level in all of them. This suggestion coincides well with the observations done on Acanthamoeba castellanii in which the calcium-related deposits are particularly large and abundant in cells descending from the advanced exponential growth phase, whereas they are quite minute and hardly distinguishable in cells from the early exponential growth phase and do not appear at all in cells undergoing encystment (in preparation), Visualization of calcium binding at the cell membrane by the method in which calcium inflows into the cell simultaneously with the chemical fixative may be regarded only as more convenient and easier preparatory technique than the freeze-substitution.
Acknowledgements The skillful technical assistance of Mrs. K. MROZINSKA is greatly appreciated.
Literature HOPWOOD, D., Theoretical and practical aspects of glutaraldehyde fixation. Fixation in Biochemistry (P.L. Stoward, eeL), pp. 47-83. Chapman & Hall, London 1973. NEUMANN, D., Zur Darstellung pflanzlicher Gewebe nach Gefriersubstitution unter besonderer Beriicksichtigung der Strukturerhaltung der Plastiden. Acta Histochem. 47, 278-288 (1973). OSCRMAN, L., and VVALL, B. F., Calcium binding to intestinal membranes ..J. Cell BioI. 55, 58-73 (1972). - HALL, T. A., l'ETEltS, P. D., and WALL, B ..J., Association of calcium with membfanes of squid axon .•J. Cell BioI. 61, 156-165 (1974). PRZELECKA, A., and SOBOTA, A., Calcium-dependent deposits at the plasma membrane during development of the oocyte of Galleria mellonella. Cyto biologie 13, 182 -190 (1976). SOBOTA, A., HREBENDA, B., and I'RZELECKA, A., Formation of calcium-dependent deposits at the plasma membrane of Acanthamoeba castellanii. Cytobiologie 15, 259-268 (1977). - PRZELECKA, A., and JAXOSSY, A. G. S., X-ray microanalysis of calcium-dependent deposits at the plasma membrane of Acanthamoeba castellanii. Cytobiologie 17, 464-469 (1978). STOCKEM, \V., and KLEIN, H. P., Pinoeytosis and locomotion in Amoeba. XV. Demonstration of Ca++-binding sites during pinocytosis in Amoeba proteus. Protoplasm a 100, 33-43 (1979). Address: Dr. A. SOBOTA, Department of Cell Biology, M. Nencki Institute of Experimental Biology, 3 Pasteur Street, PL - 02-093 vVarsaw.
9 Acta histochem. Bd. 68