- Email: [email protected]
The dependence of pinocytosis on temperature and aerobic respiration
Ribonuclease
and pyrophosphate
exchange
191
REFERENCES 1. VON DER DECKEN, A. and HULTIN, T., 4th Infernafl. Congr. Biochem. p. 40 (1958). 2. HOAGLAND, M. B., KELLER, E. U. and ZAMECNIK, I’. CI, J. Uiol. C&m. 2i8, 3:25 (1956). 3. HOAGLAND, M. B., STEPHENSON, M. L., SCOTT, J. F.. HECHT, L. I. and Z,4i%n%xrK. P. C., J. Biol. Chem. 231, 241 (1958). 4. HOAGLANI), M. B., ZAMECNIK, P. C. and STEPHENSOY, M. L., Biochim. et Biophgs. Acfa 24, 215 (1957). 5. HOI,LEY, R. W., J. Am. Gem. Sot. 79, 658 (1957). 6. HULTIN, T. and VON DEH DECKEN, A., Ezptl. Cell Research 15, 581 (1958). 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RASDALL, R. .J., J. Biof. Chem. 193, 265 (1951). 8. OGATA, I<. and NOIIARA, H., Uiocl~im. ef Biophys. Acfa 25, 659, 26, 656 (1957).
THE
DEPENDENCE
OF PINOCYTOSIS
AND N.
DE
AEROBIC TERRA’
ON TEMPERATURE
RESPIRATION1 and
R.
C. RUSTAD2,3
Department of Zoology, University of California, Berkeley, California, and Department of Zoology, University of Edinburgh, Edinburgh, Received
March
U.S.A., Scotland
2, 1959
Pmocxnws,
or cell-drinking, was first described in 1925 by Edwards [5] who used salt solutions to induce one form of this phenomenon in amoebae. Lewis [7] observed the ingestion of fluid by cells in tissue culture through fusion of cell processes to enclose vacuoles; he also gave the process its name. Mast and Doyle [S] made a detailed study of pinocytosis in amoebae. In amoeba, pinocytosis induced with 2 per cent albumin is characterized by a frilling of the cell membrane into many small pseudopodia; some of these contain channels which extend from the cell surface into the interior. Vacuoles of fluid are pinched off into the cell at the bottom of these channels. In many tissues, electron microscopy has revealed the presence of channels strongly reminiscent of those formed during pinocytosis [4, 9, 111. This suggests that cell-drinking may be a normal mechanism for the active uptake of molecules to which the cell membrane is impermeable, as was first proposed by Lewis [7]. Strong experimental evidence for this hypothesis was obtained by Chapman-Andresen and Holter [2] who found that radioactive glucose enters the normal metabolism of the cell following pinocytosis in glucose-albumin solutions. various
1 Supported in part by grants from the American Cancer Society, Inc., to Professor Daniel Mazia. ’ This work was oerformed under the tenure of a U.S. Public Health Service Research Fellowship of the National Cancer Institute. s Present address: Department of Biological Sciences, Florida State University, Tallahassee, Florida, U.S.A. Experimental
Cell Research
17
192
N. de Terra and R. C. Rustad
Chapman-Andresen and Prescott [3] have tested various chemicals for their ability to induce pinocytosis. So many different kinds of chemicals are active as inducers of pinocytosis in amoeba that perhaps substances such as glucose which are not inducers may ultimately prove to be of greater theoretical interest. An examination of the means by which pinocytosis may be delayed or inhibited might also be expected to provide information about its mechanism. We will therefore describe in this paper the results of some experiments on the delay of pinocytosis by low temperature and its inhibition by potassium cyanide and carbon monoxide. Materials and Methods.-Amoeba proteus grown on Tetrahymena geleii according to the methods of Prescott and James [la] were washed, then starved for 24-48 hours in the inorganic medium described by these workers. Pinocytosis was induced by transfer to freshly-prepared 2 percent solutions of bovine plasma albumin (Armour) in inorganic medium. In some experiments, the amoebae were placed in solutions of inorganic medium or of inorganic medium and albumin containing IO-’ M KCN. Using 0.1 M HCl and 0.1 M KOH, all of these solutions were adjusted to pH 6.0, the same pH as inorganic medium alone. In the low temperature experiments, all equipment was equilibrated for at least 24 hours in a constant temperature room; thick cells containing copper sulfate solution were used to filter infra-red light from the microscope lamp beams. Carbon monoxide inhibition was tested in a gas perfusion chamber illuminated with green light isolated from a 554 rnp interference filter [14]. Observations-If albumin is used as the inducing agent, the characteristic features of pinocytosis in Amoeba proteus are the frilled appearance caused by the projection of many small pseudopodia and the formation of channels on some of these pseudopodia [S]. In the following experiments, results were evaluated in terms of the absence or presence of these two distinguishing features. In order to examine the effects of cyanide on pinocytosis, groups of cells were transferred into the following solutions: 2 per cent albumin, 2 per cent albumin plus lo-& M KCN and 1O-4 M KCN alone. All groups of cells were observed over a 45 minute period. In all three trials, identical results were obtained. Cells transferred into 2 per cent albumin solution formed channels within lo-15 minutes and continued pinocytosis for approximately 20 minutes after the first channels could be detected. Cells transferred into KCN alone continued to stream, although streaming was observed to be abnormally sluggish in 2 out of 3 trials. Cells transferred into KCN plus albumin either streamed sluggishly or became immobile and partially contracted, with a small degree of frilling; no channels could be seen. The concentration of cyanide used in these experiments has been shown by Pace and Kimura [IO] to inhibit respiration in the closely related amoeba Pelomyxa carolinensis by a factor of 60 per cent at 20°C. The effect of carbon monoxide was also tested. Amoebae streamed sluggishly when exposed to CO for two hours. After pre-treatment with CO for 45 minutes, the amoebae in the chamber were introduced into 2 per cent albumin by mixing of two drops. Frilling did not occur in the experimental group and no channel formation took place within 30 minutes. However, after 45 minutes to 1 hour, some of the CXperimental cells developed a few fleeting channels in the uroid. Amoebae which had not been pre-incubated in CO frilled and formed channels within 224 minutes after transfer to 2 per cent albumin solution. Experimental
Cell Research
17
Pinocytosis,
temperature
and aerobic respiration
193
Attempts were made to measure the time of appearance of the first channels over the temperature range of 12°C to 22°C in hopes of obtaining an approximate measure of the Arrhenius constant of the process. Although the first channels generally appeared later at the lower temperatures, the summation of individual variability and experimental error was too great to permit numerical comparisons in this temperature range. However, the temperature dependence of pinocytosis is illustrated by the experiments described below.
TABLE
I. Time in minutes
before general occurrence of channel after exposure to 2 per cent albumin at 6°C.
Exp’t
#
First determination
23' 30' 42' 27'
1
2 3 4
5
over
40’
formation
Second determination
26' 30 31-36' over40 over 40'
Amoebae were incubated overnight in inorganic medium at 6°C and then transferred into 2 per cent albumin at 6°C. The results of 5 such experiments, performed in duplicate, are summarized below in Table I. At 22”C, channel formation almost always took place from 3-4 minutes after transfer into albumin. Occasionally, a certain amoeba culture may exhibit a negative or delayed pinocytosis reaction; the cause of this variability is not yet known. In a second group of experiments, cells equilibrated overnight at 6°C were incubated in 2 per cent albumin (6°C) for half the time necessary for induction of pinocytosis at the low temperature. Variability in the temperature dependence of pinocytosis in different groups of amoebae (see Table I) made it necessaryto vary the time of incubation in this manner. After incubation, the cells were washed three times in 6°C inorganic medium. Upon transfer into inorganic medium at 22°C strong channel formation occurred within l-3 minutes in three trials out of five. The initial formation of individual channels could be observed after the cell had been inspected in order to be certain that no previously formed channels were present. In all five trials, frilling occurred to a greater or lesserextent during the incubation at 6°C. Discussion.-The studies on inhibition of pinocytosis by cyanide and carbon monoxide provide evidence that channel formation is dependent in someway on reactions associated with the iron-containing compounds of the cell. The inhibition of channel formation is perhaps the results of a drop in the level of that energy which is normally made available to the cell through the functioning of the cytochrome system in respiration. Channel formation exhibits temperature dependence as well as sensitivity to Experimental
Cell Research
17
N. de Terra and R. C. Rustad cyanide and monoxide. Pace and Kimura [IO] have shown that a drop in temperature from 20°C to 10°C lowers the oxygen consumption of Pelomyxa carolinensis from 0.209 mm3/hr/mm3 cell substance to 0.052. Hence there is a possibility that all or part of the overall temperature dependence of channel formation may, like monoxide and cyanide inhibition, be the result of a reduction of energy available from the respiratory process. Since channel formation seems to be the actual mechanism by which vacuoles of fluid are transported into the cell, it is interesting to note that in its dependence on temperature and its sensitivity to respiratory inhibitors it resembles the class of permeability phenomena known as “active transport” systems. In three out of five trials,l cells exposed to albumin at 6°C for half the time required for induction of pinocytosis, then washed and returned to 22°C in inorganic medium formed channels within l-5 minutes. This finding suggests that pinocytosis consists of a minimum of two sequential steps. On the basis of gross population studies on the rate of uptake of radioactive protein, Schumaker [13] has postulated the existence of three discrete steps; the first two of these appear to be insensitive to temperature, cyanide and DNP. The first step must necessarily represent the primary reaction between cell and inducer. Brandt [l] has used protein labelled with a fluorescent dye to show that this interaction consists in the adsorption of protein to the cell membrane. Since pinocytosis can follow when the inducer is removed, this primary step and possibly others must have taken place in order to permit subsequent channel formation. There is yet another characteristic feature of pinocytosis in albumin solutions which must be considered in terms of the experimental results: frilling of the cell surface [S]. Although frilling cannot be observed when certain inducers are used [6] it normally occurs in a 2 per cent albumin solution, where it seems to be the first visible response of the cell to adsorption of protein onto its membrane. If frilling were the direct result of a protein adsorption purely physical in nature, one would expect it to occur in monoxide and cyanide, since the adsorption of protein onto the membrane would not be inhibited by these compounds. However, frilling does not occur in the presence of carbon monoxide, and although the cells exhibited tendendes toward frilling in cyanide, the reaction was very weak. The cell assumes its frilled position by streaming; the sluggishness of streaming in carbon monoxide and cyanide strongly suggest that these compounds inhibit respiration to the point where some reaction (such as streaming) necessary for the occurrence of frilling cannot take place normally. Frilling was noticeably delayed at 6°C; this finding is readily explained by the extreme sluggishness of cell streaming at this temperature. To summarize, it has been found that channel formation is delayed by low temperature and inhibited by cyanide and carbon monoxide. Frilling of the cell surface shows some variability of response to the three inhibitory agents. These differences would seem to reflect variations in the susceptibility of processes which are necessary for the accomplishment of frilling (such as cell streaming) to the different inhibitors. 1 The fact that two out of five trials were not successful in producing pinocytosis in the absence of inducer is not surprising if one considers the fact that different cultures of amoebae were found to exhibit large variations with respect to the time at which pinocytosis first occurred, especially at 6°C (see Table I). If this spread reflects a culture-to-culture variability in the speed of some reaction which must occur prior to channel formation, it would follow that the incubation times necessary for the completion of such a hypothetical reaction would vary. Experimental
Cell Research
17
Pinocytosis,
temperature
and aerobic respiration
195
Acknowledgements.--We wish to thank Professor Daniel Mazia for stimulating discussionsof the experimental results and for suggesting the carbon monoxide confirmation. We are grateful to Professor H. Holter for helpful discussions;one of us (R. C. R.) also wishesto thank Professor M. M. Swann for the use of the gasperfusion equipment while a guest in his laboratory. REFERENCES 1. BRANDT, P. W., Expfl. Cell Research 15, 300 (1958). C. and HOLTER, H., Exptl. Cell Research Suppl. 3, 52 (1955). 2. CHAPMAN-ANDRESEN, 3. CHAPMAN-ANDRESEN, C. and PRESCOTT, D. M., Compt. Rend. Lab. Carlsberg, sir. chim. 57 (1956). 4. DF. ROBERTIS, E. D. P. and BENNETT, H. S., Expfl. Cell Research 6, 543 (1954). 5. EDWARDS, J. G., Biol. Bull. 48, 236 (1925). H. and MARSHALL, J. M., Compt. Rend. Lab. Carlsberg, se?. chim. 29. 7 (1954). 6. HOLTER, 7. LEWIS, W. H., Johns Hopkins Hosp. Bull. 49, 17 (1931). 8. MAST, S. 0. and DOYLE, W. L., Protoplasma 20, 555 (1934). 9. ODOR, D. L., J. Biophys. Biochem. Cytol. Suppl, 2, 105 (1955). 10. PACE, D. M. and KIMURA, T. E., Proc. Sot. Exptl. Biol. Fed. 62, 223 (1946). 11. PALADE, G. E., J. Appl. Physics 24, 1424 (1953). 12. PRESCOTT, D. M. and JAMES, T. W., Exptl. Cell Research 8, 256 (1955). V. N., Exptl. Cell Research 15, 314 (1958). 13. SCHUMAKER, 14. SWANN, M. M., Quart. J. Microscop. Sci. 94, 369 (1953).
Experimental
Cell Research
30,
17
and pyrophosphate
exchange
191
REFERENCES 1. VON DER DECKEN, A. and HULTIN, T., 4th Infernafl. Congr. Biochem. p. 40 (1958). 2. HOAGLAND, M. B., KELLER, E. U. and ZAMECNIK, I’. CI, J. Uiol. C&m. 2i8, 3:25 (1956). 3. HOAGLAND, M. B., STEPHENSON, M. L., SCOTT, J. F.. HECHT, L. I. and Z,4i%n%xrK. P. C., J. Biol. Chem. 231, 241 (1958). 4. HOAGLANI), M. B., ZAMECNIK, P. C. and STEPHENSOY, M. L., Biochim. et Biophgs. Acfa 24, 215 (1957). 5. HOI,LEY, R. W., J. Am. Gem. Sot. 79, 658 (1957). 6. HULTIN, T. and VON DEH DECKEN, A., Ezptl. Cell Research 15, 581 (1958). 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RASDALL, R. .J., J. Biof. Chem. 193, 265 (1951). 8. OGATA, I<. and NOIIARA, H., Uiocl~im. ef Biophys. Acfa 25, 659, 26, 656 (1957).
THE
DEPENDENCE
OF PINOCYTOSIS
AND N.
DE
AEROBIC TERRA’
ON TEMPERATURE
RESPIRATION1 and
R.
C. RUSTAD2,3
Department of Zoology, University of California, Berkeley, California, and Department of Zoology, University of Edinburgh, Edinburgh, Received
March
U.S.A., Scotland
2, 1959
Pmocxnws,
or cell-drinking, was first described in 1925 by Edwards [5] who used salt solutions to induce one form of this phenomenon in amoebae. Lewis [7] observed the ingestion of fluid by cells in tissue culture through fusion of cell processes to enclose vacuoles; he also gave the process its name. Mast and Doyle [S] made a detailed study of pinocytosis in amoebae. In amoeba, pinocytosis induced with 2 per cent albumin is characterized by a frilling of the cell membrane into many small pseudopodia; some of these contain channels which extend from the cell surface into the interior. Vacuoles of fluid are pinched off into the cell at the bottom of these channels. In many tissues, electron microscopy has revealed the presence of channels strongly reminiscent of those formed during pinocytosis [4, 9, 111. This suggests that cell-drinking may be a normal mechanism for the active uptake of molecules to which the cell membrane is impermeable, as was first proposed by Lewis [7]. Strong experimental evidence for this hypothesis was obtained by Chapman-Andresen and Holter [2] who found that radioactive glucose enters the normal metabolism of the cell following pinocytosis in glucose-albumin solutions. various
1 Supported in part by grants from the American Cancer Society, Inc., to Professor Daniel Mazia. ’ This work was oerformed under the tenure of a U.S. Public Health Service Research Fellowship of the National Cancer Institute. s Present address: Department of Biological Sciences, Florida State University, Tallahassee, Florida, U.S.A. Experimental
Cell Research
17
192
N. de Terra and R. C. Rustad
Chapman-Andresen and Prescott [3] have tested various chemicals for their ability to induce pinocytosis. So many different kinds of chemicals are active as inducers of pinocytosis in amoeba that perhaps substances such as glucose which are not inducers may ultimately prove to be of greater theoretical interest. An examination of the means by which pinocytosis may be delayed or inhibited might also be expected to provide information about its mechanism. We will therefore describe in this paper the results of some experiments on the delay of pinocytosis by low temperature and its inhibition by potassium cyanide and carbon monoxide. Materials and Methods.-Amoeba proteus grown on Tetrahymena geleii according to the methods of Prescott and James [la] were washed, then starved for 24-48 hours in the inorganic medium described by these workers. Pinocytosis was induced by transfer to freshly-prepared 2 percent solutions of bovine plasma albumin (Armour) in inorganic medium. In some experiments, the amoebae were placed in solutions of inorganic medium or of inorganic medium and albumin containing IO-’ M KCN. Using 0.1 M HCl and 0.1 M KOH, all of these solutions were adjusted to pH 6.0, the same pH as inorganic medium alone. In the low temperature experiments, all equipment was equilibrated for at least 24 hours in a constant temperature room; thick cells containing copper sulfate solution were used to filter infra-red light from the microscope lamp beams. Carbon monoxide inhibition was tested in a gas perfusion chamber illuminated with green light isolated from a 554 rnp interference filter [14]. Observations-If albumin is used as the inducing agent, the characteristic features of pinocytosis in Amoeba proteus are the frilled appearance caused by the projection of many small pseudopodia and the formation of channels on some of these pseudopodia [S]. In the following experiments, results were evaluated in terms of the absence or presence of these two distinguishing features. In order to examine the effects of cyanide on pinocytosis, groups of cells were transferred into the following solutions: 2 per cent albumin, 2 per cent albumin plus lo-& M KCN and 1O-4 M KCN alone. All groups of cells were observed over a 45 minute period. In all three trials, identical results were obtained. Cells transferred into 2 per cent albumin solution formed channels within lo-15 minutes and continued pinocytosis for approximately 20 minutes after the first channels could be detected. Cells transferred into KCN alone continued to stream, although streaming was observed to be abnormally sluggish in 2 out of 3 trials. Cells transferred into KCN plus albumin either streamed sluggishly or became immobile and partially contracted, with a small degree of frilling; no channels could be seen. The concentration of cyanide used in these experiments has been shown by Pace and Kimura [IO] to inhibit respiration in the closely related amoeba Pelomyxa carolinensis by a factor of 60 per cent at 20°C. The effect of carbon monoxide was also tested. Amoebae streamed sluggishly when exposed to CO for two hours. After pre-treatment with CO for 45 minutes, the amoebae in the chamber were introduced into 2 per cent albumin by mixing of two drops. Frilling did not occur in the experimental group and no channel formation took place within 30 minutes. However, after 45 minutes to 1 hour, some of the CXperimental cells developed a few fleeting channels in the uroid. Amoebae which had not been pre-incubated in CO frilled and formed channels within 224 minutes after transfer to 2 per cent albumin solution. Experimental
Cell Research
17
Pinocytosis,
temperature
and aerobic respiration
193
Attempts were made to measure the time of appearance of the first channels over the temperature range of 12°C to 22°C in hopes of obtaining an approximate measure of the Arrhenius constant of the process. Although the first channels generally appeared later at the lower temperatures, the summation of individual variability and experimental error was too great to permit numerical comparisons in this temperature range. However, the temperature dependence of pinocytosis is illustrated by the experiments described below.
TABLE
I. Time in minutes
before general occurrence of channel after exposure to 2 per cent albumin at 6°C.
Exp’t
#
First determination
23' 30' 42' 27'
1
2 3 4
5
over
40’
formation
Second determination
26' 30 31-36' over40 over 40'
Amoebae were incubated overnight in inorganic medium at 6°C and then transferred into 2 per cent albumin at 6°C. The results of 5 such experiments, performed in duplicate, are summarized below in Table I. At 22”C, channel formation almost always took place from 3-4 minutes after transfer into albumin. Occasionally, a certain amoeba culture may exhibit a negative or delayed pinocytosis reaction; the cause of this variability is not yet known. In a second group of experiments, cells equilibrated overnight at 6°C were incubated in 2 per cent albumin (6°C) for half the time necessary for induction of pinocytosis at the low temperature. Variability in the temperature dependence of pinocytosis in different groups of amoebae (see Table I) made it necessaryto vary the time of incubation in this manner. After incubation, the cells were washed three times in 6°C inorganic medium. Upon transfer into inorganic medium at 22°C strong channel formation occurred within l-3 minutes in three trials out of five. The initial formation of individual channels could be observed after the cell had been inspected in order to be certain that no previously formed channels were present. In all five trials, frilling occurred to a greater or lesserextent during the incubation at 6°C. Discussion.-The studies on inhibition of pinocytosis by cyanide and carbon monoxide provide evidence that channel formation is dependent in someway on reactions associated with the iron-containing compounds of the cell. The inhibition of channel formation is perhaps the results of a drop in the level of that energy which is normally made available to the cell through the functioning of the cytochrome system in respiration. Channel formation exhibits temperature dependence as well as sensitivity to Experimental
Cell Research
17
N. de Terra and R. C. Rustad cyanide and monoxide. Pace and Kimura [IO] have shown that a drop in temperature from 20°C to 10°C lowers the oxygen consumption of Pelomyxa carolinensis from 0.209 mm3/hr/mm3 cell substance to 0.052. Hence there is a possibility that all or part of the overall temperature dependence of channel formation may, like monoxide and cyanide inhibition, be the result of a reduction of energy available from the respiratory process. Since channel formation seems to be the actual mechanism by which vacuoles of fluid are transported into the cell, it is interesting to note that in its dependence on temperature and its sensitivity to respiratory inhibitors it resembles the class of permeability phenomena known as “active transport” systems. In three out of five trials,l cells exposed to albumin at 6°C for half the time required for induction of pinocytosis, then washed and returned to 22°C in inorganic medium formed channels within l-5 minutes. This finding suggests that pinocytosis consists of a minimum of two sequential steps. On the basis of gross population studies on the rate of uptake of radioactive protein, Schumaker [13] has postulated the existence of three discrete steps; the first two of these appear to be insensitive to temperature, cyanide and DNP. The first step must necessarily represent the primary reaction between cell and inducer. Brandt [l] has used protein labelled with a fluorescent dye to show that this interaction consists in the adsorption of protein to the cell membrane. Since pinocytosis can follow when the inducer is removed, this primary step and possibly others must have taken place in order to permit subsequent channel formation. There is yet another characteristic feature of pinocytosis in albumin solutions which must be considered in terms of the experimental results: frilling of the cell surface [S]. Although frilling cannot be observed when certain inducers are used [6] it normally occurs in a 2 per cent albumin solution, where it seems to be the first visible response of the cell to adsorption of protein onto its membrane. If frilling were the direct result of a protein adsorption purely physical in nature, one would expect it to occur in monoxide and cyanide, since the adsorption of protein onto the membrane would not be inhibited by these compounds. However, frilling does not occur in the presence of carbon monoxide, and although the cells exhibited tendendes toward frilling in cyanide, the reaction was very weak. The cell assumes its frilled position by streaming; the sluggishness of streaming in carbon monoxide and cyanide strongly suggest that these compounds inhibit respiration to the point where some reaction (such as streaming) necessary for the occurrence of frilling cannot take place normally. Frilling was noticeably delayed at 6°C; this finding is readily explained by the extreme sluggishness of cell streaming at this temperature. To summarize, it has been found that channel formation is delayed by low temperature and inhibited by cyanide and carbon monoxide. Frilling of the cell surface shows some variability of response to the three inhibitory agents. These differences would seem to reflect variations in the susceptibility of processes which are necessary for the accomplishment of frilling (such as cell streaming) to the different inhibitors. 1 The fact that two out of five trials were not successful in producing pinocytosis in the absence of inducer is not surprising if one considers the fact that different cultures of amoebae were found to exhibit large variations with respect to the time at which pinocytosis first occurred, especially at 6°C (see Table I). If this spread reflects a culture-to-culture variability in the speed of some reaction which must occur prior to channel formation, it would follow that the incubation times necessary for the completion of such a hypothetical reaction would vary. Experimental
Cell Research
17
Pinocytosis,
temperature
and aerobic respiration
195
Acknowledgements.--We wish to thank Professor Daniel Mazia for stimulating discussionsof the experimental results and for suggesting the carbon monoxide confirmation. We are grateful to Professor H. Holter for helpful discussions;one of us (R. C. R.) also wishesto thank Professor M. M. Swann for the use of the gasperfusion equipment while a guest in his laboratory. REFERENCES 1. BRANDT, P. W., Expfl. Cell Research 15, 300 (1958). C. and HOLTER, H., Exptl. Cell Research Suppl. 3, 52 (1955). 2. CHAPMAN-ANDRESEN, 3. CHAPMAN-ANDRESEN, C. and PRESCOTT, D. M., Compt. Rend. Lab. Carlsberg, sir. chim. 57 (1956). 4. DF. ROBERTIS, E. D. P. and BENNETT, H. S., Expfl. Cell Research 6, 543 (1954). 5. EDWARDS, J. G., Biol. Bull. 48, 236 (1925). H. and MARSHALL, J. M., Compt. Rend. Lab. Carlsberg, se?. chim. 29. 7 (1954). 6. HOLTER, 7. LEWIS, W. H., Johns Hopkins Hosp. Bull. 49, 17 (1931). 8. MAST, S. 0. and DOYLE, W. L., Protoplasma 20, 555 (1934). 9. ODOR, D. L., J. Biophys. Biochem. Cytol. Suppl, 2, 105 (1955). 10. PACE, D. M. and KIMURA, T. E., Proc. Sot. Exptl. Biol. Fed. 62, 223 (1946). 11. PALADE, G. E., J. Appl. Physics 24, 1424 (1953). 12. PRESCOTT, D. M. and JAMES, T. W., Exptl. Cell Research 8, 256 (1955). V. N., Exptl. Cell Research 15, 314 (1958). 13. SCHUMAKER, 14. SWANN, M. M., Quart. J. Microscop. Sci. 94, 369 (1953).
Experimental
Cell Research
30,
17