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Incorporation of 3H-glucosamine in HeLa cells as revealed by light and electron microscopic autoradiography
3H-glucosamine incorporation in HeLa cells
tion by mercury compounds indicates that the sensitive function in the cell is protected perhaps in a subsurface location, rather than superficial, as is the case with the regulatory factor for phenylalanine uptake [lo]. When the effects of the known Hg2+ impurity are eliminated from the experiments with organic mercurials by adjustment of concentration, only the effects of the latter are seen. The differential inhibition then is likely to be related to the ability of the active component to penetrate the cell and reach the active site. Further investigations are under way on the nature of the regulatory system and how it differs in normal and Py-transformed hamster cells [2]. This work has been supported by grants CA-07239 from the National Cancer Institute and USPHS and NIDR Training Grant, DE 00003-13.
REFERENCES 1. Kessel, D & Shurin, S B, Biochim biophys acta 163 (1968) 17. 2. Hare, J D, Cancer res. In press. 3. Breslow, R E & Goldsby, R A, Exptl cell res 55 (1969) 339. 4. Vansteveninck, J, Weed, R I & Rothstein, A, J gen physio148 (1965) 67. 5. Eagle, H, Science 730 (1959) 432. 6. Kabat & Mayer, Experimental immunochemistry (ed Thomas) p. 557. Springfield, Ill. (1961). 7. Clarkson, T W. Personal communication. 8. Cafruny, E J, Cho, K C & Gussin, R Z, Ann N Y acad sci 139 (1966) 362. 9. Mudge, G H & Weiner, I M, Ann N Y acad sci 71 (1958) 344. 10. Hare, J D, Cancer res 27 (1967) 2357.
16’7
cells interact to form tissues and organs of multicellular organisms [5]. Recent investigations indicate that several of the relevant surface properties are related to the “glycocalyx”, a carbohydrate-rich coating of most if not all cells [8]. In some cell types this coating is easily demonstrated by electron microscopy following conventional preparative procedures. In most cell types the coating can only be demonstrated indirectly by use of cytochemical staining methods [8, 91. These methods have serious shortcomings: The underlying chemical reactions are mostly unknown and the specificity questionable. In a few cell types, however, specialized in producing large amounts of coating material, the coating has been demonstrated by autoradiography following the administration of tritiated small molecular carbohydrates [4]. These autoradiographic investigations have confirmed biochemical observation on the importance of these carbohydrates in the synthesis of the glycocalyx-at least in some cells, and have presented observations on the route of synthesis. It is not self-evident, however, that the route of synthesis in these specialized cells is the same as in other cells. The aim of the present investigation was to study the route as well as the time sequence of synthesis of the glycocalyx in a non-specialized cell type. Tissue culture cells were used because of the high specific activity that can be obtained in vitro.
Received September 29, 1969
Materials and methods
INCORPORATION OF 3HGLUCOSAMINE IN HeLa CELLS AS REVEALED BY LIGHT AND ELECTRON MICROSCOPIC AUTORADIOGRAPHY A. REITH, R. OFTEBRO and R. SELJELID, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway
Properties of the cell surface are probably responsible for the high specificity with which
HeLa S3 cells from our laboratorv stock. cultivated as monolayers in nunclon plast Petri dishes in E2a medium 171 at 37°C and DH 7.2. were incubated for 5 min or for 5 h with medium containing *H-~-Dglucosamine (N) hydrochloride (1.6 mM, spec. act. 1100 mCi/mM, manufactured by New England Nuclear Corp.), and then for 45 min or for 6 h, respectively, in glucosamine-free medium. The cells were fixed in 2 % 0~0, in 0.1 M s-collidine buffer, pH 7.4, dehydrated in ethanol and embedded in Epon. Specimens for autoradiography with the light and the electron microscope were prepared according to von Gaudecker [12]. The autoradiographic preparations Exptl Cell Res 59
168 A. Reith et. al. were exposed for 19 days to light microscopy and for 31 days to electron microscopy. The electronmicroscopic autoradiographs were developed in a fine-grained developer after gold “latensification” [ll]. All the thin sections were placed on the same grid and were accordingly develotid under identical conditions. The specimens were examined in an Elmiskop Ia electron microscope after staining with lead [lo].
Results
Light-microscopic autoradiographs of cells which had been incubated for 5 min in 3Hglucosamine showed moderate radioactivity throughout the cell without any favoured localization. After 5 h incubation, the cells were heavily labelled with large amounts of radioactivity in the perinuclear zones. Stripes of silver grains seemed to coincide with the cell margins. By electron-microscopic examination of specimens incubated for 5 h, labelling was found in the Golgi zones (figs 1, 2). All elements of Golgi complexes seemed to be labelled, Dense, single membrane-limited organelles, probably of lysosomal nature, were also heavily labelled. Only a few grains were found over the ground cytoplasm. Grains were numerous over distinct parts of the cell periphery: (a) contact surfaces of adjacent cells, especially where they interlocked, (b) small surface protrusions and larger, flattened cell recesses, and (c) small vesicles in the peripheral cytoplasm. Electron-microscopic autoradiographs of cells after 5 min incubation showed very few silver grains. Discussion
The present results are in accordance with those obtained by Ito & Revel [4] in intestinal
epithelium and confirm that a carbohydraterich cell coating is a general occurrence. The amount of coating material seems to be far less in HeLa cells than in intestinal epithelium, and the use of serial sections was necessary to obtain reliable observations. It is notable that grains were found predominantly over specific sites in the cell periphery. It cannot be excluded that this selective localization reflects technical shortcomings: A substantial part of the glycocalyx is removed during dehydration [I] and it cannot be presumed that this extraction is equally effective in all parts of the cell. However, the selective localization may also be of biologic significance, indicating that the turnover of the glycocalyx is not even all over the cell during a limited time period. The localization of radioactive carbohydrates in the Golgi area is in accordance with observations in goblet cells [6]. It has been found by gradient centrifugation that tritiated glucosamine and fucose are incorporated in smooth microsomes [2] probably containing Golgi elements. Furthermore it has been shown that a Golgi apparatus-rich fraction from rat liver catalyzes the transfer of glucosamine from UDP-iV-acetylglucosamine to endogenous protein acceptors, while plasma membrane and rough microsomes were practically devoid of such catalytic activity [13]. Taken together these observations indicate that the synthesis of macromolecular carbohydrates takes place in Golgi structures. The small vesicles in the apical cytoplasm may be the transport vehicle from the Golgi area to the cell surface. The consistent labelling of dense bodies is
Figs I, 2. Electron-microscopic autoradiographs of serial sections of two HeLa cells. There is considerable autoradiographic reaction over the Golgi zone (G). Lysosomes are also heavily labelled (7,~). The radioactive material has accumulated over distinct parts of the cell periphery (4) such as (a) contact surfaces of adjacent cells especially where they interlock, (b) small surface protrusions, (c) small vesicles in the peripheral cytoplasm. x 11,000. Inset 1a and 2~: Higher magnification of the two Golgi zones with one heavily labelled dense body. y 20,000. Inset 26: Higher magnification of the cell periphery with autoradiographic grains and numerous small vesicles. x 20,oco. Exptl
Cell .Res 59
3H-glucosamine incorporation in HeLa cells
Exptl
Cell
169
Res 59
170 R. G. Summers also notable. This localization may be caused by fusion between dense bodies and labelled phagosomes or labelled Golgi vesicles. Another possibility is that tritiated glucosamine is incorporated in the lysosomal glycoproteins [3]. The close chemical and physical similarity between plasma membranes and lysosomal membranes [3] may be highly significant in this context. The authors thank Miss S. Fjeldsenden for skilled technical assistance. A. R. is a fellow of the Norwegian Research Council for Science and the Humanities,
REFERENCES 1. Behnke, 0, J ultrastr res 24 (1968) 51. 2. Bosmann, H B, Hagopian, A & Eylar, E H, Arch biochem biophys 130 (1969) 573. 3. De Duve, Lysosomes in biology and pathology (ed J. T. Dingle &H. B. Fell) p. 3. NorthHolland, Amsterdam (1969). 4. Ito, S & Revel, J P, Monogr on nuclear med and biol, no. 1, Amsterdam. Excerpta med 27 (1968). 5. Moscona, A A, Develop biol 18 (1968) 250. 6. Neutra, M & Leblond, C P, J cell biol 30 (1966) 119. 7. Puck, T T, Ciecura, S J & Fisher, H W, J exptl med 106 (1957) 145. 8. Rambourg, A & Leblond, C P, J cell biol 32 (1967) 27. 9. Reith, A & Oftebro, R, J ultrastruct res 24 (1968) 164. 10. Reynolds, E S, J cell biol 17 (1963) 208. 11. Salpeter, M M & Bachmann, L, J cell biol 22 (1964) 469. 12. Von Gaudecker, B, Z Zellforsch 82 (1967) 536. 13. Wagner, R R & Cynkin, M A, Biochem biophys res comm 35 (1969) 139. Received October 2. 1969
THE EFFECT OF ACTINOMYCIN D ON DEMEMBRANATED LYTECHZNUS VARZEGATUS EMBRYOS R. G. SUMMERS, Department of Anatomy, Tulane Medical School, New Orleans, La 70112, USA
Several workers [2, 31 using actinomycin D have reported that considerable development can occur in the absence of RNA synthesis in the sea urchin embryo. However, recently Exptl Cell Res 59
Thaler et al. [4] have questioned the validity of these experiments, and have suggested that actinomycin D is unable to penetrate the gelatinous envelopes of sea urchin eggs and embryos. Accordingly, a series of experiments was performed to test, in Lytechinus variegatus, the effects of actinomycin D upon embryos with and without jelly coats and fertilization membranes. Materials and methods Lytechinus variegatus were collected near the Bermuda Biological Station, St George’s W., Bermuda. Gametes were obtained by the injection of 0.53 M KCI. Eggs which had been deiellied in acid sea water for 30 min were then placed in glutathione (0.45 g/l in sea water adjusted to pH 7.9-8.0 with 0.1 N NaOH [l]). Following 20 min treatment with glutathione, eggs were passed twice through 16~ bolting cloth to remove fertilization membranes and washed with sterile sea water. At 40 min post-fertilization 300-400 of these embryos were then placed in each of the following solutions: (a) actinomycin D solution (20 pg/ml in sea water) and (b) in sterile sea water, and subsequent development was observed. Since embryos lacked their membranes, it was not possible to observe their hatching. In order to determine the presence of hatching enzyme, freshly fertilized eggs with membranes intact were added to high density experimental and control cultures at the normal hatching time (45 h post-fertilization). If hatching enzyme was present in these cultures, then the fertilization membranes of the freshly added embryos would be dissolved.
Results Approx. 70 % of L. variegatus embryos which were deprived of their jelly coats and fertilization membranes developed normally through the pluteus stage. Likewise, 70% of the embryos which were treated with 20 pg/ml actinomycin D appeared to develop normally to the mesenchyme blastula stage. However, in no case did an actinomycin D treated embryo gastrulate. Actinomycin D treated embryos formed cilia and were able to swim as well as controls. Both actinomycin D treated and control blastulae released hatching enzyme in sufficient quantity to dissolve the fertilization membranes from freshly added embryos. A
tion by mercury compounds indicates that the sensitive function in the cell is protected perhaps in a subsurface location, rather than superficial, as is the case with the regulatory factor for phenylalanine uptake [lo]. When the effects of the known Hg2+ impurity are eliminated from the experiments with organic mercurials by adjustment of concentration, only the effects of the latter are seen. The differential inhibition then is likely to be related to the ability of the active component to penetrate the cell and reach the active site. Further investigations are under way on the nature of the regulatory system and how it differs in normal and Py-transformed hamster cells [2]. This work has been supported by grants CA-07239 from the National Cancer Institute and USPHS and NIDR Training Grant, DE 00003-13.
REFERENCES 1. Kessel, D & Shurin, S B, Biochim biophys acta 163 (1968) 17. 2. Hare, J D, Cancer res. In press. 3. Breslow, R E & Goldsby, R A, Exptl cell res 55 (1969) 339. 4. Vansteveninck, J, Weed, R I & Rothstein, A, J gen physio148 (1965) 67. 5. Eagle, H, Science 730 (1959) 432. 6. Kabat & Mayer, Experimental immunochemistry (ed Thomas) p. 557. Springfield, Ill. (1961). 7. Clarkson, T W. Personal communication. 8. Cafruny, E J, Cho, K C & Gussin, R Z, Ann N Y acad sci 139 (1966) 362. 9. Mudge, G H & Weiner, I M, Ann N Y acad sci 71 (1958) 344. 10. Hare, J D, Cancer res 27 (1967) 2357.
16’7
cells interact to form tissues and organs of multicellular organisms [5]. Recent investigations indicate that several of the relevant surface properties are related to the “glycocalyx”, a carbohydrate-rich coating of most if not all cells [8]. In some cell types this coating is easily demonstrated by electron microscopy following conventional preparative procedures. In most cell types the coating can only be demonstrated indirectly by use of cytochemical staining methods [8, 91. These methods have serious shortcomings: The underlying chemical reactions are mostly unknown and the specificity questionable. In a few cell types, however, specialized in producing large amounts of coating material, the coating has been demonstrated by autoradiography following the administration of tritiated small molecular carbohydrates [4]. These autoradiographic investigations have confirmed biochemical observation on the importance of these carbohydrates in the synthesis of the glycocalyx-at least in some cells, and have presented observations on the route of synthesis. It is not self-evident, however, that the route of synthesis in these specialized cells is the same as in other cells. The aim of the present investigation was to study the route as well as the time sequence of synthesis of the glycocalyx in a non-specialized cell type. Tissue culture cells were used because of the high specific activity that can be obtained in vitro.
Received September 29, 1969
Materials and methods
INCORPORATION OF 3HGLUCOSAMINE IN HeLa CELLS AS REVEALED BY LIGHT AND ELECTRON MICROSCOPIC AUTORADIOGRAPHY A. REITH, R. OFTEBRO and R. SELJELID, Norsk Hydro’s Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo 3, Norway
Properties of the cell surface are probably responsible for the high specificity with which
HeLa S3 cells from our laboratorv stock. cultivated as monolayers in nunclon plast Petri dishes in E2a medium 171 at 37°C and DH 7.2. were incubated for 5 min or for 5 h with medium containing *H-~-Dglucosamine (N) hydrochloride (1.6 mM, spec. act. 1100 mCi/mM, manufactured by New England Nuclear Corp.), and then for 45 min or for 6 h, respectively, in glucosamine-free medium. The cells were fixed in 2 % 0~0, in 0.1 M s-collidine buffer, pH 7.4, dehydrated in ethanol and embedded in Epon. Specimens for autoradiography with the light and the electron microscope were prepared according to von Gaudecker [12]. The autoradiographic preparations Exptl Cell Res 59
168 A. Reith et. al. were exposed for 19 days to light microscopy and for 31 days to electron microscopy. The electronmicroscopic autoradiographs were developed in a fine-grained developer after gold “latensification” [ll]. All the thin sections were placed on the same grid and were accordingly develotid under identical conditions. The specimens were examined in an Elmiskop Ia electron microscope after staining with lead [lo].
Results
Light-microscopic autoradiographs of cells which had been incubated for 5 min in 3Hglucosamine showed moderate radioactivity throughout the cell without any favoured localization. After 5 h incubation, the cells were heavily labelled with large amounts of radioactivity in the perinuclear zones. Stripes of silver grains seemed to coincide with the cell margins. By electron-microscopic examination of specimens incubated for 5 h, labelling was found in the Golgi zones (figs 1, 2). All elements of Golgi complexes seemed to be labelled, Dense, single membrane-limited organelles, probably of lysosomal nature, were also heavily labelled. Only a few grains were found over the ground cytoplasm. Grains were numerous over distinct parts of the cell periphery: (a) contact surfaces of adjacent cells, especially where they interlocked, (b) small surface protrusions and larger, flattened cell recesses, and (c) small vesicles in the peripheral cytoplasm. Electron-microscopic autoradiographs of cells after 5 min incubation showed very few silver grains. Discussion
The present results are in accordance with those obtained by Ito & Revel [4] in intestinal
epithelium and confirm that a carbohydraterich cell coating is a general occurrence. The amount of coating material seems to be far less in HeLa cells than in intestinal epithelium, and the use of serial sections was necessary to obtain reliable observations. It is notable that grains were found predominantly over specific sites in the cell periphery. It cannot be excluded that this selective localization reflects technical shortcomings: A substantial part of the glycocalyx is removed during dehydration [I] and it cannot be presumed that this extraction is equally effective in all parts of the cell. However, the selective localization may also be of biologic significance, indicating that the turnover of the glycocalyx is not even all over the cell during a limited time period. The localization of radioactive carbohydrates in the Golgi area is in accordance with observations in goblet cells [6]. It has been found by gradient centrifugation that tritiated glucosamine and fucose are incorporated in smooth microsomes [2] probably containing Golgi elements. Furthermore it has been shown that a Golgi apparatus-rich fraction from rat liver catalyzes the transfer of glucosamine from UDP-iV-acetylglucosamine to endogenous protein acceptors, while plasma membrane and rough microsomes were practically devoid of such catalytic activity [13]. Taken together these observations indicate that the synthesis of macromolecular carbohydrates takes place in Golgi structures. The small vesicles in the apical cytoplasm may be the transport vehicle from the Golgi area to the cell surface. The consistent labelling of dense bodies is
Figs I, 2. Electron-microscopic autoradiographs of serial sections of two HeLa cells. There is considerable autoradiographic reaction over the Golgi zone (G). Lysosomes are also heavily labelled (7,~). The radioactive material has accumulated over distinct parts of the cell periphery (4) such as (a) contact surfaces of adjacent cells especially where they interlock, (b) small surface protrusions, (c) small vesicles in the peripheral cytoplasm. x 11,000. Inset 1a and 2~: Higher magnification of the two Golgi zones with one heavily labelled dense body. y 20,000. Inset 26: Higher magnification of the cell periphery with autoradiographic grains and numerous small vesicles. x 20,oco. Exptl
Cell .Res 59
3H-glucosamine incorporation in HeLa cells
Exptl
Cell
169
Res 59
170 R. G. Summers also notable. This localization may be caused by fusion between dense bodies and labelled phagosomes or labelled Golgi vesicles. Another possibility is that tritiated glucosamine is incorporated in the lysosomal glycoproteins [3]. The close chemical and physical similarity between plasma membranes and lysosomal membranes [3] may be highly significant in this context. The authors thank Miss S. Fjeldsenden for skilled technical assistance. A. R. is a fellow of the Norwegian Research Council for Science and the Humanities,
REFERENCES 1. Behnke, 0, J ultrastr res 24 (1968) 51. 2. Bosmann, H B, Hagopian, A & Eylar, E H, Arch biochem biophys 130 (1969) 573. 3. De Duve, Lysosomes in biology and pathology (ed J. T. Dingle &H. B. Fell) p. 3. NorthHolland, Amsterdam (1969). 4. Ito, S & Revel, J P, Monogr on nuclear med and biol, no. 1, Amsterdam. Excerpta med 27 (1968). 5. Moscona, A A, Develop biol 18 (1968) 250. 6. Neutra, M & Leblond, C P, J cell biol 30 (1966) 119. 7. Puck, T T, Ciecura, S J & Fisher, H W, J exptl med 106 (1957) 145. 8. Rambourg, A & Leblond, C P, J cell biol 32 (1967) 27. 9. Reith, A & Oftebro, R, J ultrastruct res 24 (1968) 164. 10. Reynolds, E S, J cell biol 17 (1963) 208. 11. Salpeter, M M & Bachmann, L, J cell biol 22 (1964) 469. 12. Von Gaudecker, B, Z Zellforsch 82 (1967) 536. 13. Wagner, R R & Cynkin, M A, Biochem biophys res comm 35 (1969) 139. Received October 2. 1969
THE EFFECT OF ACTINOMYCIN D ON DEMEMBRANATED LYTECHZNUS VARZEGATUS EMBRYOS R. G. SUMMERS, Department of Anatomy, Tulane Medical School, New Orleans, La 70112, USA
Several workers [2, 31 using actinomycin D have reported that considerable development can occur in the absence of RNA synthesis in the sea urchin embryo. However, recently Exptl Cell Res 59
Thaler et al. [4] have questioned the validity of these experiments, and have suggested that actinomycin D is unable to penetrate the gelatinous envelopes of sea urchin eggs and embryos. Accordingly, a series of experiments was performed to test, in Lytechinus variegatus, the effects of actinomycin D upon embryos with and without jelly coats and fertilization membranes. Materials and methods Lytechinus variegatus were collected near the Bermuda Biological Station, St George’s W., Bermuda. Gametes were obtained by the injection of 0.53 M KCI. Eggs which had been deiellied in acid sea water for 30 min were then placed in glutathione (0.45 g/l in sea water adjusted to pH 7.9-8.0 with 0.1 N NaOH [l]). Following 20 min treatment with glutathione, eggs were passed twice through 16~ bolting cloth to remove fertilization membranes and washed with sterile sea water. At 40 min post-fertilization 300-400 of these embryos were then placed in each of the following solutions: (a) actinomycin D solution (20 pg/ml in sea water) and (b) in sterile sea water, and subsequent development was observed. Since embryos lacked their membranes, it was not possible to observe their hatching. In order to determine the presence of hatching enzyme, freshly fertilized eggs with membranes intact were added to high density experimental and control cultures at the normal hatching time (45 h post-fertilization). If hatching enzyme was present in these cultures, then the fertilization membranes of the freshly added embryos would be dissolved.
Results Approx. 70 % of L. variegatus embryos which were deprived of their jelly coats and fertilization membranes developed normally through the pluteus stage. Likewise, 70% of the embryos which were treated with 20 pg/ml actinomycin D appeared to develop normally to the mesenchyme blastula stage. However, in no case did an actinomycin D treated embryo gastrulate. Actinomycin D treated embryos formed cilia and were able to swim as well as controls. Both actinomycin D treated and control blastulae released hatching enzyme in sufficient quantity to dissolve the fertilization membranes from freshly added embryos. A