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Freeze-fracture and etching studies on membrane damage on human erythrocytes caused by formation of intracellular ice
CRYOBIOLOGY
17, 351-362
(1980)
Freeze-Fracture and Etching Studies on Membrane Damage on Human Erythrocytes Caused by Formation of Intracellular Ice SEIZO FUJIKAWA The Institute
of Low Temperature
Science, Hokkaido
Electron microscopists have widely employed a freezing technique to achieve ultrastructural preservation of biological materials, since cryofixation of specimens eliminates procedures of chemical fixation, dehydration, and embedding in ordinary electron microscopy which cause apprehensions about producing artifacts. It has been pointed out, however, that freezing procedures bring about new artifacts to biological specimens (20, 28). One of the most serious artifacts produced by freezing is a formation of intracellular ice, which results from the fact that the cooling rates obtained by methods used in the conventional procedures are too slow to convert intracellular water to amorphous ice (2, 24, 26). By displacing cellular organelles and cytoplasmic components from their true site, the formation of intracellular ice may give rise to morphological artifacts. In addition, the intracellular formation of ice implies the loss of viability of rapidly frozen cells associated with the presence of intracellular ice (1, 17, 19, 27). It is hypothesized that the cause of injury of rapidly frozen cells is a direct physical consequence of intracellular ice on membranes (3, 4, 16-18), but few studies have been reported on the ultrastructural alteration of membranes caused by formation of intracellular ice (21). In the present study, plasma membranes of human erythrocytes, in which intracellular ice of 0.2-2.0 pm in diameter were formed, were chosen as a model system, whereby a damaging effect Received 1980.
Spptember
5, 1979; accepted
February
University,
Sapporo 060, Japan
of intracellular ice on the stability of the membranes was examined on the basis of the membrane ultrastructural aspect as revealed by freeze-fracturing alone or followed by etching. MATERIALS
AND
METHODS
Materials Experimental materials used were human erythrocytes from whole blood collected in acid-citrate-dextrose (ACD). They were separated from plasma by centrifugation at 20008 for 10 min. After washing them twice with 0.15 M NaCl in a 0.005 M phosphate buffer solution, pH 7.4, packed cells were resuspended in the same solution (hematocrit = 50%). Freezing A small droplet (0.01 ml) of a specimen suspension was placed on a freeze-etching holder (1.5 mm thickness and 5 x 5 mm in size). Intracellular freezing was achieved by immersing the loaded holder abruptly into Freon 22 kept at - 160°C. The cooling rates were determined by a 41-gauge copper-constantan thermocouple fixed in the center of specimen suspension. The meaning cooling rate recorded by an electron magnetic oscillograph (Yokogawa Electric Works Ltd., Type 2901) was approximately 8000”C/min between 0 and -60°C. Meantime, extracellularly frozen cells were also prepared as control specimens, by abrupt immersion of a loaded holder into liquid nitrogen. The mean cooling rate was approximately 2400”C/min between 0 and
20,
351 OOll-2240/80/040351-12$02.00/O Copyright
0
1980 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
352
SEIZO FUJIKAWA
-60°C. The observation of fractured cyto- comparable to that of the intracellularly plasmic regions of erythrocytes indicated frozen ones. In the extracellularly frozen that at the cooling rate of 2400”C/min, al- erythrocytes, however, interruption of most all the cells were frozen extracellu- fracturing which was seen in the intracellularly . larly frozen ones was not found on the fracture faces (Fig. 4). Freeze-Fracture and Etching Electron After etching, the PF of intracellularly Microscopy frozen erythrocytes exhibited initially the A frozen specimen was transferred onto a altered membrane regions resembling cold stage of a freeze-etching apparatus “worm-eaten spots” which were slightly (JEOL AFE-I) and fractured at a tempera- depressed and wrinkled (Fig. 5, in circles of ture of - 100°C in a vacuum of 1 x 10m5tot-r. a and b) and membrane holes (Fig. 5, c). After fracturing, some specimens were im- The etched EF of intracellularly frozen mediately replicated at - 100°C without erythrocytes, on the other hand, exhibited etching, while other specimens were sub- membrane holes (Fig. 6, a) and adhered jected to etching by rewarming them to cytoplasmic materials (Fig. 6, b). While the -98°C or higher temperatures, at which ratio of number of worm-eaten spots (Fig. replicas were made. 5, a and b) to that of membrane holes (Fig. The replicas were observed with a JEM- 5, c) was about 9:l on the PF, the ratio of 1OOCelectron microscope operated at an number of membrane holes (Fig. 6, a) to accelerating voltage of 100 kV. that of adhered cytoplasmic materials (Fig. The nomenclature of Branton et al. (9) 6, b) was about 9:l on the EF. The worm-eaten spots on the etched PF was used to designate the fracture faces. The shadowing direction in all electron mi- corresponded to the regions where incrographs used herein was from the bottom tracellular ice crystals were in direct contoward the top of the page (except for tact with the membrane (Fig. 7). Furthermore, the worm-eaten spots were also obFig. 1). served on the etched exoplasmic surfaces RESULTS (ES) of intracellularly frozen erythrocytes, The freezing of human erythrocytes at which corresponded also to the regions the cooling rate of 8000”C/min resulted in where intracellular ice crystals were in diintracellular freezing. The ice crystals rect contact with membrane (Figs. 8 and 9). The size of worm-eaten spots both on the ranging from 0.2 to 2.0 pm in diameter were etched PF and ES depended closely upon formed in the cells (Fig. 1). In such intracellularly frozen erythro- the size of intracellular ice crystals being in cytes, freeze-fracturing without etching did direct contact with membrane. With the increase of the size of intracellular ice crysnot reveal any membrane ultrastructural alterations both on the protoplasmic frac- tals from 0.2 to 2.0 pm, the size of wormture face (PF) (Fig. 2) and the exoplasmic eaten spots increased also from 400 to 3000 fracture face (EF) (Fig. 3), but both the A in diameter. No perforations were usually detected on fracture faces were partially interrupted by the presence of smooth areas. For examin- the worm-eaten spots in specimens preing whether such a partial interruption of pared by slight etching (Fig. 10). Further fracturing was due to the presence of mere morphological alterations were, however, surface irregularity of membrane or not, observed on the worm-eaten spots followextracellularly frozen erythrocytes were ing the increase of etching. Even in slight chosen as control, because they had con- etching, small perforations were developed siderable surface irregularity of membrane artificially during etching on a very small
MEMBRANE
DAMAGE
BY INTRACELLULAR
ICE
FIG. 1. Low magnified figure of intracellularly frozen erythrocytes prepared by etching. Arrow indicates direction of shadowing. EI: extracellular ice. x6,200. FIG. 2. Protoplasmic fracture face (PF) of an intracellularly frozen erythrocyte prepared by freezefracturing without etching. Arrows indicate the smooth areas. ~83,000.
353
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SEIZO
FUJIKAWA
FIG. 3. Exoplasmic fracture face (EF) of an intracellularly frozen erythrocyte prepared by freezefracturing without etching. Arrows indicate the smooth areas. II: intracellular ice. CY: cytoplasmic region. X 52,000. FIG. 4. Protoplasmic fracture face (PF) of an extracellularly frozen erythrocyte prepared by freeze-fracturing without etching. ~83,000.
MEMBRANE
DAMAGE
FIG. 5. Protoplasmic fracture face (PF) of an Note that etching revealed altered membrane exoplasmic membrane leaflets, (b) worm-eaten leaflets, (c) membrane holes. Small perforation by etching. ES: exoplasmic surface. ~83,000.
BY INTRACELLULAR
ICE
intracellularly frozen erythrocyte prepared by etching. regions: (a) worm-eaten spots (in circles) with intact spots (in circles) without intact exoplasmic membrane (directed by an arrow) seem to be an artifact caused
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FUJIKAWA
MEMBRANE
DAMAGE
number of the worm-eaten spots (directed by an arrow of Fig. 5). Deep etching brought about conspicuous artifacts at the worm-eaten spots, whereby holes were left at places where membrane segments of the worm-eaten spots were completely lost (Fig. 11). For estimating the effect of deep etching to the other membrane being in direct contact with ice crystals, the EF of extracellularly frozen erythrocytes were chosen for control, because they were in direct contact with extracellular ice crystals. In the similar deep etching, however, the destruction of the EF was contined to the formation of small pores corresponding to the size of intramembrane particles (Fig. 12). DISCUSSION
Despite a number of studies indicating that the formation of intracellular ice is responsible for the loss of viability of frozen biological materials (4-6), the mechanism of damage on submicroscopic level is not yet clear. I have attempted in this study to investigate the damaging effect of intracellular ice on plasma membranes of rapidly frozen human erythrocytes by use of freeze-fracture and etching techniques. Membrane regions in direct contact with intracellular ice crystals exhibited ultrastructural alterations resembling “wormeaten spots” after etching, but no such alterations were observed on the fracture faces before etching. Figure 13 shows a schematical drawing suggesting the fracturing in the membrane of intracellularly frozen erythrocytes. It is suggested that
BY INTRACELLULAR
ICE
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the path of fracturing is deviated from the inner hydrophobic plane of membrane at the worm-eaten spots either extracellularly (Fig. 13a and b) or intracellularly (Fig. 13~). Therefore, freeze-fracturing without etching did not reveal worm-eaten spots, instead, it represented the smooth areas on the fracture faces. When extracellular deviation of fracturing occurred at the worm-eaten spots (Fig. 13a and b), etching exposed the wormeaten spots on the PF by removing the extracellular ice which was left on the freeze-fractured PF as smooth area. While the extracellular deviation of fracturing at the worm-eaten spots as shown in Fig. 13 a resulted in the structure as shown in Fig. 5 a after etching, the deviation of fracturing as shown in Fig. 13 b resulted in the structure as shown in Fig. 5 b after etching. In the case of Fig. 13 a, the deviation of fracturing from the inner hydrophobic plane of membrane is initiated at the worm-eaten spots and furthermore go over them for a short distance. In the case of Fig. 13 b, the deviation of fracturing from the membrane is restricted to the worm-eaten spots. After etching, the case of fracturing as shown in Fig. 13 b indicated the structure resembling intramembrane particles-free patches on the PF which might be induced by lateral translational motion of intramembrane particles through the membrane (13, 15, 30). This is not the case, however, because the freeze-fracturing without etching did not show such an ultrastructural alteration (Figs. 2 and 3). On the complementary EF, such extracellular deviation of fracturing (Fig. 13, a
FIG. 6. Exoplasmic fracture face (EF) of an intracellularly frozen erythrocyte prepared by etching. Note that etching revealed membrane holes (a) and adhered cytoplasmic materials (b). Small pores in (b) (directed by arrows) indicate the sublimation sites of intracellular ice. x 60,000. FIG. 7. A part of an intracellularly frozen erythrocyte prepared by etching. Note that the wormeaten spot on PF (directed by an arrow) appears at the region in direct contact with intracellular ice. x53,000. FIGS. 8 and 9. A part of an intracellularly frozen erythrocyte prepared by etching. Note that worm-eaten spots (directed by arrows) on the etched exoplasmic surface (ES) appear at the membrane regions in direct contact with intracellular ice. x53,000 (Fig. 8). x22,000 (Fig. 9).
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FUJIKAWA
FIG. 10. Exoplasmic surface (ES) and protoplasmic fracture face (PF) of an intracellularly frozen erythrocyte prepared by etching. Note that no perforations are formed in the worm-eaten spots (directed by arrows). ~69,000. FIG. 11. Similar to Fig. 10, except that it is prepared by deep etching. Note that deep etching brings about conspicuous artifacts at the worm-eaten spots. x 82,000. FIG. 12. Exoplasmic fracture face (EF) of an extracellularly frozen erythrocyte prepared by deep etching. Note that destruction of the EF is confined to the formation of small pores corresponding to the size and distribution of the intramembrane particles. x 82,000.
and b) resulted in the membrane holes as shown in Fig. 6 a. When intracellular deviation of fracturing occurred at the worm-eaten spots (Fig. 13, c), etching clarified the membrane holes on the PF as shown in Fig. 5 c by removing the intracellular ice crystals. On the complementary EF, such in-
tracellular deviation of fracturing brought about adhered cy toplasmic materials as shown in Fig. 6 b. In the adhered cytoplasmic materials, small pores indicating the sublimation sites of intracellular ice were observed. These deviation of fracturing from the membrane at the worm-eaten spots,did not
MEMBRANE Exterior
DAMAGE BY INTRACELLULAR
ICE
359
disorganization of the bilayer membrane caused by treatment with toluene, which removed the phospholipids from the cytoplasmic membrane. Furthermore, it has cytoplasm FIG. 13. Schematic drawing showing the cross sec- been reported that freeze-fracture of memtional view of membrane in the intracellularly frozen branes fixed with 0~0, resulted in marked erythrocyte. Dotted line indicates the path of fracture. reduction of fracture faces through the Black solid indicates the worm-eaten spot. For explainterior of the membranes as the result of nation, see the text. formation of bridges between the aliphatic chains of lipids (14, 22). result from mere surface irregularity of The deviation of fracture from the inner membrane, since such deviation did not hydrophobic plane of membrane at the occur in the membrane of extracellularly worm-eaten spots which were membrane frozen erythrocytes with considerable surregions in direct contact with intracellular face irregularity. The failure of fracturing ice crystals seems to be a reflection of the through the inner hydrophobic plane of occurrence of some alterations of the membrane at the worm-eaten spots strongly bilayer membrane, possibly at the molecuindicates the occurrence of membrane lar level. The physical force caused by the damage. formation of intracellular ice crystals is the It has now been generally conceived that, most likely factor that induces such altersince the bilayer membrane provides the ations. Figure 14 shows hypothetically the hydrophobic plane in the interior, which is molecular disorganization of bilayer memphysically weak in a frozen condition (7, 8, brane in direct contact with intracellular ice lo), the freeze-fracture occurs preferencrystals. It is suggested that in such a memtially through the inner hydrophobic plane. brane region, the physical force caused by It has also be indicated that some molecular growth of ice crystals brings about disoralteration of the bilayer membrane results ganization of molecular contigration of the in the reduction of fracturing through the membrane, and that consequently prefermembrane (11, 14, 22). De Smet ef al. (11) ential paths of fracture are lost. indicated that the freeze-fracturing no However, since this kind of membrane longer occurs through the cytoplasmic alteration does not increase membrane stamembrane in Escherichia coli which was bility against the physical force of fracturtreated with toluene in the presence of ing as in the case of 0~0, fixed membranes EDTA. They suggested (11) that the failure (14, 22), the other factors should be considof freeze-fracture was due to the molecular ered for explaining the cause of deviation of the fracture from the membrane at the worm-eaten spots. The extracellular and the intracellular deviation of the fracture at the worm-eaten spots seem to arise from the combined effects of surface irregularity of the membrane and membrane alteration. It is reasonable to conclude that, when the worm-eaten spots were depressed FIG. 14. Hypothetical scheme indicating the dam- below or elevated above the level of the age of membrane in direct contact with intracellular surrounding membrane where the fracture ice. The molecular organization of the bilayer memoccurred, the fracture will run along the brane in such a region is damaged physically by the plane corresponding to that of the undegrowth of ice crystal; consequently, the path of fracture is lost at the region. P: integral protein. viated surrounding membrane, seeking the a
b
c
360
SEIZO FUJIKAWA
path of minimum physical work. Since the worm-eaten spots were usually depressed structures toward the inside of the cell, the fracture in the majority of cases (90%) resulted in the extracellular deviation at the worm-eaten spots (Fig. 13b). Intracellular deviation occurred in the case in which the worm-eaten spots were elevated above the level of the surrounding membrane plane (Fig. 13~). Furthermore, the deviation of fracture which was initiated at the wormeaten spots was in some cases over the worm-eaten spots depending upon the surface irregularity of the membrane (Fig. 13a). Another supplementary aspect of membrane damage was that these worm-eaten spots were very sensitive to etching. It has been pointed out that a wind of subliming water molecules during etching brings about artificial destruction to the EF, which are in direct contact with extracellular ice crystals (23, 29). Destruction of the EF during etching was, however, proposed to be the result of the presence of pits which were made during membrane fracturing by the removal of intercalated intramembrane particles preferentially associated with the PF (23). The worm-eaten spots presented in this study are bilayer structure; nevertheless, the membrane destruction during etching was more serious in the worm-eaten spots than in the EF. Such sensitivity of the worm-eaten spots to etching may also indicate the occurrence of the disorganization of bilayer membrane. The previous observation by Nei (21) indicating that the formation of intracellular ice makes holes on membrane, therefore, can be referred to the result of artificial destruction caused by etching in the membrane regions in direct contact with intracellular ice crystals. In the rapid freezing of 8000”CYmin employed in this study, erythrocytes after rapid thawing hemolyzed completely (Fujikawa, unpublished data) (25). The
cause of hemolysis in intracellularly frozen erythrocytes can be related to the presence of membrane alterations caused by the formation of intracellular ice. Even in the rapid thawing which is a best way to minimize damaging during thawing in the rapidly frozen cells (3, 4), cells are obliged to subject themselves to a transmembrane osmotic gradient in the melting of intracellular ice (12). It is suggested that the altered membrane regions undergo further alterations during thawing and hemolysis occurs through these altered membrane regions. SUMMARY
The present study examined the damaging effect of intracellular ice on plasma membranes of human erythrocytes. Ice crystals of 0.2-2.0 pm in diameter were formed within the cells as the result of rapid freezing of erythrocytes at the cooling rates around 8000”C/min. Freeze-fracture and etching studies revealed the ultrastructural alterations of membranes caused by the formation of intracellular ice. In the membrane regions which were in direct contact with intracellular ice, depresspots” sions resembling “worm-eaten ranging from 400 to 3000 A in diameter were observed both on the etched protoplasmic fracture faces (PF) and the exoplasmic surfaces (ES); no perforations were detected in the worm-eaten spots as visualized by slight etching, but artificial destructions occurred on these worm-eaten spots following the increase of etching. The most important phenomenon concerning membrane damage was that in the wormeaten spots the fracture did not occur along the inner hydrophobic plane of membrane. It was suggested that the formation of intracellular ice in direct contact with a membrane brought about molecular disorganization of bilayer membrane. The presence of these altered membrane regions seems to be responsible for the postthawed hemolysis of the intracellularly frozen erythrocytes.
MEMBRANE
DAMAGE
BY
INTRACELLULAR
ACKNOWLEDGMENTS
The author is indebted to Emeritus Professor T. Nei for his many helpful suggestions during the course of this work, and to Professor S. Sagisaka and Dr. K. Shimada for their critical reading of the manuscript.
16.
REFERENCES
1. Asahina, E., Shimada, K., and Hisada, Y. A stable state of frozen protoplasm with invisible intracellular ice crystals obtained by rapid cooling. Exp. Cell Res. 59, 349-358 (1970). 2. Bachmann, L., and Schmitt, W. W., Improved cryofixation applicable to freeze-etching. Proc. Nat. Acad. Sci. USA 68, 2149-2152 (1971). 3. Bank, H. Visualization of freezing damage. II. Structural alterations during warming. Cryobiology
10, 157-
170 (1973).
4. Bank, H. Freezing injuly in tissue cultured cells as visualized by freeze-etching. Exp. Cell Rrs. 85, 367-376 (1974). 5. Bank, H., and Mazur, P. Relation between ultrastructure and viability of frozen-thawed Chinese hamster tissue-culture cells. Exp. Cell Res.
71, 441-454
(1972).
6. Bank, H., and Mazur, P. Visualization of freezing damage. J. Cell Biol. 57, 729-742 (1973). I. Branton, D. Fracture faces of frozen membranes. Prac. Nat. Accrd. Sci. (/SA 5.5, 1048-1056 (1966). 8. Branton, D., Membrane structure. Ann. Rev. Plant
Plqasiol.
20, 209-
238 (1969).
9. Branton, D., Bullivant, S., Gilula, N. B., Karnovsky, M., Moor, H., Mtihlethaler, K., Northtote, D. H., Packer, L., Satir, B., Satir, P., Speth, V., Staehlin, L. A., Steere, R. L., and Weinstein, R. S. Freeze-etching nomenclature. Scivncr
190, 54-56
(1975).
10. Deamer, D. W., and Branton, D. Fracture planes in an ice-bilayer model membrane system. Science
158, 655-657
15.
(1967).
11. De Smet, M. J., Kingma, J., and Witholt, B. The effect of toluene on the structure and permiability of the outer and cytoplasmic membranes of Escherichio co/i. Biochim. Biophys. Acto 506, 64-80 (1978). 12. Farrant, J. Water transport and cell survival in cryobiological procedures. Phil. Trans. Rag. Sac. Land. B 278, 191-205 (1977). 13. Hiichli, M., and Hackenbrock, C. R. Fluidity in mitochondrial membranes: Thermotropical lateral translational motion of intramembrane particles. Proc. Nat. Acad. Sci. USA 73, 1636- 1640 (1976). 14. James, R., and Branton, D. The correlation between the saturation of membrane fatty acids and the presence of membrane fracture faces
17. 18.
361
ICE
after osmium fixation. Biochim. Biophys. Acta 233, 504-512 (1971). Kleemann, W., and McConnell, H. M. Lateral phase separations in Escherichia co/i memBiophys. Acta 345, 220-230 branes. B&him. (1974). Mazur, P. Physical and chemical basis of injury in single-celled microorganisms subjected to freezing and thawing. In “Cryobiology” (H. T. Merymann, Ed.), pp. 213-315. Academic Press, New York, 1966. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). Mazur, P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology
14, 251-272
(1977).
19. Meryman, H. T. Mechanics of freezing in living cells and tissues. Science 124, 515-521 (1956). 20. Moor, H. Freeze fixation of living cells and its application in electron microscopy. Z. Zelljorsch.
Mikroscop.
Anat.
62,
546-580
(1964). 21. Nei, T. Freezing injury to erythrocytes. II. Morphological alterations of cell membranes. Cryobiology 13, 287-294 (1976). 22. Nermut, M. V., and Ward, B. J. Effect of fixatives on fracture plane in red blood cells. J. Microsc.
102, 29-39
(1974).
23. Pinto da Silva, P., Moss, P. S., and Fudenberg, H. H. Anionic sites on the membrane intercalated particles on human erythrocyte ghost membranes. Freeze-etch localization. Exp. Cell Res. 81, 127-138 (1973). 24. Plattner, H., Fischer, W. M., Schmitt, W. W., and Bachmann, L. Freeze-etching of cells without cryoprotectants. J. Cell Biol. 53, 116-126 (1972). 25. Rapatz, G., Sullivan, J., and Luyet, B. Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature. Cryobiology 5, 18-25 (1968). 26. Riehle, U., nique of Etching. Benedetti Soci&C tronique,
and Hiiechli, M. The theory and techhigh pressure freezing. In “FreezeTechniques and Applications” (E. L. and P. Favard, Ed.), pp. 31-62. Francaise de Microscopic ElecParis, 1973.
27. Sherman, J. K., and Kim, K. S. Correlation of cellular ultrastructure before freezing, while freezing, and after thawing in assessing freeze-thaw-induced injury. Cryobiology 4, 61-74 (1967). 28. Steere, R. L. Freeze-etching and direct observation of freezing damage. Cryobiology 6, 137- 150 (1969).
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29. Verkleij, A. J., and Ververgaert, P. H. J. Th. Freeze-fracture morphology of biological membranes. Biochim. Biophys. Acta 515, 303-327 (1978). 30. Wunderlich, F., Wallach, D. F. H., Speth, V.,
and Fischer, H. Differential effects of temperature on the nuclear and plasma membranes of lymphoid cells. A study by freeze-etch electron microscopy. Biochim. Biopys. Acta 373,34-43 (1974).
17, 351-362
(1980)
Freeze-Fracture and Etching Studies on Membrane Damage on Human Erythrocytes Caused by Formation of Intracellular Ice SEIZO FUJIKAWA The Institute
of Low Temperature
Science, Hokkaido
Electron microscopists have widely employed a freezing technique to achieve ultrastructural preservation of biological materials, since cryofixation of specimens eliminates procedures of chemical fixation, dehydration, and embedding in ordinary electron microscopy which cause apprehensions about producing artifacts. It has been pointed out, however, that freezing procedures bring about new artifacts to biological specimens (20, 28). One of the most serious artifacts produced by freezing is a formation of intracellular ice, which results from the fact that the cooling rates obtained by methods used in the conventional procedures are too slow to convert intracellular water to amorphous ice (2, 24, 26). By displacing cellular organelles and cytoplasmic components from their true site, the formation of intracellular ice may give rise to morphological artifacts. In addition, the intracellular formation of ice implies the loss of viability of rapidly frozen cells associated with the presence of intracellular ice (1, 17, 19, 27). It is hypothesized that the cause of injury of rapidly frozen cells is a direct physical consequence of intracellular ice on membranes (3, 4, 16-18), but few studies have been reported on the ultrastructural alteration of membranes caused by formation of intracellular ice (21). In the present study, plasma membranes of human erythrocytes, in which intracellular ice of 0.2-2.0 pm in diameter were formed, were chosen as a model system, whereby a damaging effect Received 1980.
Spptember
5, 1979; accepted
February
University,
Sapporo 060, Japan
of intracellular ice on the stability of the membranes was examined on the basis of the membrane ultrastructural aspect as revealed by freeze-fracturing alone or followed by etching. MATERIALS
AND
METHODS
Materials Experimental materials used were human erythrocytes from whole blood collected in acid-citrate-dextrose (ACD). They were separated from plasma by centrifugation at 20008 for 10 min. After washing them twice with 0.15 M NaCl in a 0.005 M phosphate buffer solution, pH 7.4, packed cells were resuspended in the same solution (hematocrit = 50%). Freezing A small droplet (0.01 ml) of a specimen suspension was placed on a freeze-etching holder (1.5 mm thickness and 5 x 5 mm in size). Intracellular freezing was achieved by immersing the loaded holder abruptly into Freon 22 kept at - 160°C. The cooling rates were determined by a 41-gauge copper-constantan thermocouple fixed in the center of specimen suspension. The meaning cooling rate recorded by an electron magnetic oscillograph (Yokogawa Electric Works Ltd., Type 2901) was approximately 8000”C/min between 0 and -60°C. Meantime, extracellularly frozen cells were also prepared as control specimens, by abrupt immersion of a loaded holder into liquid nitrogen. The mean cooling rate was approximately 2400”C/min between 0 and
20,
351 OOll-2240/80/040351-12$02.00/O Copyright
0
1980 by Academic
Press, Inc.
All rights of reproduction in any form reserved.
352
SEIZO FUJIKAWA
-60°C. The observation of fractured cyto- comparable to that of the intracellularly plasmic regions of erythrocytes indicated frozen ones. In the extracellularly frozen that at the cooling rate of 2400”C/min, al- erythrocytes, however, interruption of most all the cells were frozen extracellu- fracturing which was seen in the intracellularly . larly frozen ones was not found on the fracture faces (Fig. 4). Freeze-Fracture and Etching Electron After etching, the PF of intracellularly Microscopy frozen erythrocytes exhibited initially the A frozen specimen was transferred onto a altered membrane regions resembling cold stage of a freeze-etching apparatus “worm-eaten spots” which were slightly (JEOL AFE-I) and fractured at a tempera- depressed and wrinkled (Fig. 5, in circles of ture of - 100°C in a vacuum of 1 x 10m5tot-r. a and b) and membrane holes (Fig. 5, c). After fracturing, some specimens were im- The etched EF of intracellularly frozen mediately replicated at - 100°C without erythrocytes, on the other hand, exhibited etching, while other specimens were sub- membrane holes (Fig. 6, a) and adhered jected to etching by rewarming them to cytoplasmic materials (Fig. 6, b). While the -98°C or higher temperatures, at which ratio of number of worm-eaten spots (Fig. replicas were made. 5, a and b) to that of membrane holes (Fig. The replicas were observed with a JEM- 5, c) was about 9:l on the PF, the ratio of 1OOCelectron microscope operated at an number of membrane holes (Fig. 6, a) to accelerating voltage of 100 kV. that of adhered cytoplasmic materials (Fig. The nomenclature of Branton et al. (9) 6, b) was about 9:l on the EF. The worm-eaten spots on the etched PF was used to designate the fracture faces. The shadowing direction in all electron mi- corresponded to the regions where incrographs used herein was from the bottom tracellular ice crystals were in direct contoward the top of the page (except for tact with the membrane (Fig. 7). Furthermore, the worm-eaten spots were also obFig. 1). served on the etched exoplasmic surfaces RESULTS (ES) of intracellularly frozen erythrocytes, The freezing of human erythrocytes at which corresponded also to the regions the cooling rate of 8000”C/min resulted in where intracellular ice crystals were in diintracellular freezing. The ice crystals rect contact with membrane (Figs. 8 and 9). The size of worm-eaten spots both on the ranging from 0.2 to 2.0 pm in diameter were etched PF and ES depended closely upon formed in the cells (Fig. 1). In such intracellularly frozen erythro- the size of intracellular ice crystals being in cytes, freeze-fracturing without etching did direct contact with membrane. With the increase of the size of intracellular ice crysnot reveal any membrane ultrastructural alterations both on the protoplasmic frac- tals from 0.2 to 2.0 pm, the size of wormture face (PF) (Fig. 2) and the exoplasmic eaten spots increased also from 400 to 3000 fracture face (EF) (Fig. 3), but both the A in diameter. No perforations were usually detected on fracture faces were partially interrupted by the presence of smooth areas. For examin- the worm-eaten spots in specimens preing whether such a partial interruption of pared by slight etching (Fig. 10). Further fracturing was due to the presence of mere morphological alterations were, however, surface irregularity of membrane or not, observed on the worm-eaten spots followextracellularly frozen erythrocytes were ing the increase of etching. Even in slight chosen as control, because they had con- etching, small perforations were developed siderable surface irregularity of membrane artificially during etching on a very small
MEMBRANE
DAMAGE
BY INTRACELLULAR
ICE
FIG. 1. Low magnified figure of intracellularly frozen erythrocytes prepared by etching. Arrow indicates direction of shadowing. EI: extracellular ice. x6,200. FIG. 2. Protoplasmic fracture face (PF) of an intracellularly frozen erythrocyte prepared by freezefracturing without etching. Arrows indicate the smooth areas. ~83,000.
353
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FUJIKAWA
FIG. 3. Exoplasmic fracture face (EF) of an intracellularly frozen erythrocyte prepared by freezefracturing without etching. Arrows indicate the smooth areas. II: intracellular ice. CY: cytoplasmic region. X 52,000. FIG. 4. Protoplasmic fracture face (PF) of an extracellularly frozen erythrocyte prepared by freeze-fracturing without etching. ~83,000.
MEMBRANE
DAMAGE
FIG. 5. Protoplasmic fracture face (PF) of an Note that etching revealed altered membrane exoplasmic membrane leaflets, (b) worm-eaten leaflets, (c) membrane holes. Small perforation by etching. ES: exoplasmic surface. ~83,000.
BY INTRACELLULAR
ICE
intracellularly frozen erythrocyte prepared by etching. regions: (a) worm-eaten spots (in circles) with intact spots (in circles) without intact exoplasmic membrane (directed by an arrow) seem to be an artifact caused
355
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FUJIKAWA
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DAMAGE
number of the worm-eaten spots (directed by an arrow of Fig. 5). Deep etching brought about conspicuous artifacts at the worm-eaten spots, whereby holes were left at places where membrane segments of the worm-eaten spots were completely lost (Fig. 11). For estimating the effect of deep etching to the other membrane being in direct contact with ice crystals, the EF of extracellularly frozen erythrocytes were chosen for control, because they were in direct contact with extracellular ice crystals. In the similar deep etching, however, the destruction of the EF was contined to the formation of small pores corresponding to the size of intramembrane particles (Fig. 12). DISCUSSION
Despite a number of studies indicating that the formation of intracellular ice is responsible for the loss of viability of frozen biological materials (4-6), the mechanism of damage on submicroscopic level is not yet clear. I have attempted in this study to investigate the damaging effect of intracellular ice on plasma membranes of rapidly frozen human erythrocytes by use of freeze-fracture and etching techniques. Membrane regions in direct contact with intracellular ice crystals exhibited ultrastructural alterations resembling “wormeaten spots” after etching, but no such alterations were observed on the fracture faces before etching. Figure 13 shows a schematical drawing suggesting the fracturing in the membrane of intracellularly frozen erythrocytes. It is suggested that
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the path of fracturing is deviated from the inner hydrophobic plane of membrane at the worm-eaten spots either extracellularly (Fig. 13a and b) or intracellularly (Fig. 13~). Therefore, freeze-fracturing without etching did not reveal worm-eaten spots, instead, it represented the smooth areas on the fracture faces. When extracellular deviation of fracturing occurred at the worm-eaten spots (Fig. 13a and b), etching exposed the wormeaten spots on the PF by removing the extracellular ice which was left on the freeze-fractured PF as smooth area. While the extracellular deviation of fracturing at the worm-eaten spots as shown in Fig. 13 a resulted in the structure as shown in Fig. 5 a after etching, the deviation of fracturing as shown in Fig. 13 b resulted in the structure as shown in Fig. 5 b after etching. In the case of Fig. 13 a, the deviation of fracturing from the inner hydrophobic plane of membrane is initiated at the worm-eaten spots and furthermore go over them for a short distance. In the case of Fig. 13 b, the deviation of fracturing from the membrane is restricted to the worm-eaten spots. After etching, the case of fracturing as shown in Fig. 13 b indicated the structure resembling intramembrane particles-free patches on the PF which might be induced by lateral translational motion of intramembrane particles through the membrane (13, 15, 30). This is not the case, however, because the freeze-fracturing without etching did not show such an ultrastructural alteration (Figs. 2 and 3). On the complementary EF, such extracellular deviation of fracturing (Fig. 13, a
FIG. 6. Exoplasmic fracture face (EF) of an intracellularly frozen erythrocyte prepared by etching. Note that etching revealed membrane holes (a) and adhered cytoplasmic materials (b). Small pores in (b) (directed by arrows) indicate the sublimation sites of intracellular ice. x 60,000. FIG. 7. A part of an intracellularly frozen erythrocyte prepared by etching. Note that the wormeaten spot on PF (directed by an arrow) appears at the region in direct contact with intracellular ice. x53,000. FIGS. 8 and 9. A part of an intracellularly frozen erythrocyte prepared by etching. Note that worm-eaten spots (directed by arrows) on the etched exoplasmic surface (ES) appear at the membrane regions in direct contact with intracellular ice. x53,000 (Fig. 8). x22,000 (Fig. 9).
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FIG. 10. Exoplasmic surface (ES) and protoplasmic fracture face (PF) of an intracellularly frozen erythrocyte prepared by etching. Note that no perforations are formed in the worm-eaten spots (directed by arrows). ~69,000. FIG. 11. Similar to Fig. 10, except that it is prepared by deep etching. Note that deep etching brings about conspicuous artifacts at the worm-eaten spots. x 82,000. FIG. 12. Exoplasmic fracture face (EF) of an extracellularly frozen erythrocyte prepared by deep etching. Note that destruction of the EF is confined to the formation of small pores corresponding to the size and distribution of the intramembrane particles. x 82,000.
and b) resulted in the membrane holes as shown in Fig. 6 a. When intracellular deviation of fracturing occurred at the worm-eaten spots (Fig. 13, c), etching clarified the membrane holes on the PF as shown in Fig. 5 c by removing the intracellular ice crystals. On the complementary EF, such in-
tracellular deviation of fracturing brought about adhered cy toplasmic materials as shown in Fig. 6 b. In the adhered cytoplasmic materials, small pores indicating the sublimation sites of intracellular ice were observed. These deviation of fracturing from the membrane at the worm-eaten spots,did not
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disorganization of the bilayer membrane caused by treatment with toluene, which removed the phospholipids from the cytoplasmic membrane. Furthermore, it has cytoplasm FIG. 13. Schematic drawing showing the cross sec- been reported that freeze-fracture of memtional view of membrane in the intracellularly frozen branes fixed with 0~0, resulted in marked erythrocyte. Dotted line indicates the path of fracture. reduction of fracture faces through the Black solid indicates the worm-eaten spot. For explainterior of the membranes as the result of nation, see the text. formation of bridges between the aliphatic chains of lipids (14, 22). result from mere surface irregularity of The deviation of fracture from the inner membrane, since such deviation did not hydrophobic plane of membrane at the occur in the membrane of extracellularly worm-eaten spots which were membrane frozen erythrocytes with considerable surregions in direct contact with intracellular face irregularity. The failure of fracturing ice crystals seems to be a reflection of the through the inner hydrophobic plane of occurrence of some alterations of the membrane at the worm-eaten spots strongly bilayer membrane, possibly at the molecuindicates the occurrence of membrane lar level. The physical force caused by the damage. formation of intracellular ice crystals is the It has now been generally conceived that, most likely factor that induces such altersince the bilayer membrane provides the ations. Figure 14 shows hypothetically the hydrophobic plane in the interior, which is molecular disorganization of bilayer memphysically weak in a frozen condition (7, 8, brane in direct contact with intracellular ice lo), the freeze-fracture occurs preferencrystals. It is suggested that in such a memtially through the inner hydrophobic plane. brane region, the physical force caused by It has also be indicated that some molecular growth of ice crystals brings about disoralteration of the bilayer membrane results ganization of molecular contigration of the in the reduction of fracturing through the membrane, and that consequently prefermembrane (11, 14, 22). De Smet ef al. (11) ential paths of fracture are lost. indicated that the freeze-fracturing no However, since this kind of membrane longer occurs through the cytoplasmic alteration does not increase membrane stamembrane in Escherichia coli which was bility against the physical force of fracturtreated with toluene in the presence of ing as in the case of 0~0, fixed membranes EDTA. They suggested (11) that the failure (14, 22), the other factors should be considof freeze-fracture was due to the molecular ered for explaining the cause of deviation of the fracture from the membrane at the worm-eaten spots. The extracellular and the intracellular deviation of the fracture at the worm-eaten spots seem to arise from the combined effects of surface irregularity of the membrane and membrane alteration. It is reasonable to conclude that, when the worm-eaten spots were depressed FIG. 14. Hypothetical scheme indicating the dam- below or elevated above the level of the age of membrane in direct contact with intracellular surrounding membrane where the fracture ice. The molecular organization of the bilayer memoccurred, the fracture will run along the brane in such a region is damaged physically by the plane corresponding to that of the undegrowth of ice crystal; consequently, the path of fracture is lost at the region. P: integral protein. viated surrounding membrane, seeking the a
b
c
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path of minimum physical work. Since the worm-eaten spots were usually depressed structures toward the inside of the cell, the fracture in the majority of cases (90%) resulted in the extracellular deviation at the worm-eaten spots (Fig. 13b). Intracellular deviation occurred in the case in which the worm-eaten spots were elevated above the level of the surrounding membrane plane (Fig. 13~). Furthermore, the deviation of fracture which was initiated at the wormeaten spots was in some cases over the worm-eaten spots depending upon the surface irregularity of the membrane (Fig. 13a). Another supplementary aspect of membrane damage was that these worm-eaten spots were very sensitive to etching. It has been pointed out that a wind of subliming water molecules during etching brings about artificial destruction to the EF, which are in direct contact with extracellular ice crystals (23, 29). Destruction of the EF during etching was, however, proposed to be the result of the presence of pits which were made during membrane fracturing by the removal of intercalated intramembrane particles preferentially associated with the PF (23). The worm-eaten spots presented in this study are bilayer structure; nevertheless, the membrane destruction during etching was more serious in the worm-eaten spots than in the EF. Such sensitivity of the worm-eaten spots to etching may also indicate the occurrence of the disorganization of bilayer membrane. The previous observation by Nei (21) indicating that the formation of intracellular ice makes holes on membrane, therefore, can be referred to the result of artificial destruction caused by etching in the membrane regions in direct contact with intracellular ice crystals. In the rapid freezing of 8000”CYmin employed in this study, erythrocytes after rapid thawing hemolyzed completely (Fujikawa, unpublished data) (25). The
cause of hemolysis in intracellularly frozen erythrocytes can be related to the presence of membrane alterations caused by the formation of intracellular ice. Even in the rapid thawing which is a best way to minimize damaging during thawing in the rapidly frozen cells (3, 4), cells are obliged to subject themselves to a transmembrane osmotic gradient in the melting of intracellular ice (12). It is suggested that the altered membrane regions undergo further alterations during thawing and hemolysis occurs through these altered membrane regions. SUMMARY
The present study examined the damaging effect of intracellular ice on plasma membranes of human erythrocytes. Ice crystals of 0.2-2.0 pm in diameter were formed within the cells as the result of rapid freezing of erythrocytes at the cooling rates around 8000”C/min. Freeze-fracture and etching studies revealed the ultrastructural alterations of membranes caused by the formation of intracellular ice. In the membrane regions which were in direct contact with intracellular ice, depresspots” sions resembling “worm-eaten ranging from 400 to 3000 A in diameter were observed both on the etched protoplasmic fracture faces (PF) and the exoplasmic surfaces (ES); no perforations were detected in the worm-eaten spots as visualized by slight etching, but artificial destructions occurred on these worm-eaten spots following the increase of etching. The most important phenomenon concerning membrane damage was that in the wormeaten spots the fracture did not occur along the inner hydrophobic plane of membrane. It was suggested that the formation of intracellular ice in direct contact with a membrane brought about molecular disorganization of bilayer membrane. The presence of these altered membrane regions seems to be responsible for the postthawed hemolysis of the intracellularly frozen erythrocytes.
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ACKNOWLEDGMENTS
The author is indebted to Emeritus Professor T. Nei for his many helpful suggestions during the course of this work, and to Professor S. Sagisaka and Dr. K. Shimada for their critical reading of the manuscript.
16.
REFERENCES
1. Asahina, E., Shimada, K., and Hisada, Y. A stable state of frozen protoplasm with invisible intracellular ice crystals obtained by rapid cooling. Exp. Cell Res. 59, 349-358 (1970). 2. Bachmann, L., and Schmitt, W. W., Improved cryofixation applicable to freeze-etching. Proc. Nat. Acad. Sci. USA 68, 2149-2152 (1971). 3. Bank, H. Visualization of freezing damage. II. Structural alterations during warming. Cryobiology
10, 157-
170 (1973).
4. Bank, H. Freezing injuly in tissue cultured cells as visualized by freeze-etching. Exp. Cell Rrs. 85, 367-376 (1974). 5. Bank, H., and Mazur, P. Relation between ultrastructure and viability of frozen-thawed Chinese hamster tissue-culture cells. Exp. Cell Res.
71, 441-454
(1972).
6. Bank, H., and Mazur, P. Visualization of freezing damage. J. Cell Biol. 57, 729-742 (1973). I. Branton, D. Fracture faces of frozen membranes. Prac. Nat. Accrd. Sci. (/SA 5.5, 1048-1056 (1966). 8. Branton, D., Membrane structure. Ann. Rev. Plant
Plqasiol.
20, 209-
238 (1969).
9. Branton, D., Bullivant, S., Gilula, N. B., Karnovsky, M., Moor, H., Mtihlethaler, K., Northtote, D. H., Packer, L., Satir, B., Satir, P., Speth, V., Staehlin, L. A., Steere, R. L., and Weinstein, R. S. Freeze-etching nomenclature. Scivncr
190, 54-56
(1975).
10. Deamer, D. W., and Branton, D. Fracture planes in an ice-bilayer model membrane system. Science
158, 655-657
15.
(1967).
11. De Smet, M. J., Kingma, J., and Witholt, B. The effect of toluene on the structure and permiability of the outer and cytoplasmic membranes of Escherichio co/i. Biochim. Biophys. Acto 506, 64-80 (1978). 12. Farrant, J. Water transport and cell survival in cryobiological procedures. Phil. Trans. Rag. Sac. Land. B 278, 191-205 (1977). 13. Hiichli, M., and Hackenbrock, C. R. Fluidity in mitochondrial membranes: Thermotropical lateral translational motion of intramembrane particles. Proc. Nat. Acad. Sci. USA 73, 1636- 1640 (1976). 14. James, R., and Branton, D. The correlation between the saturation of membrane fatty acids and the presence of membrane fracture faces
17. 18.
361
ICE
after osmium fixation. Biochim. Biophys. Acta 233, 504-512 (1971). Kleemann, W., and McConnell, H. M. Lateral phase separations in Escherichia co/i memBiophys. Acta 345, 220-230 branes. B&him. (1974). Mazur, P. Physical and chemical basis of injury in single-celled microorganisms subjected to freezing and thawing. In “Cryobiology” (H. T. Merymann, Ed.), pp. 213-315. Academic Press, New York, 1966. Mazur, P. Cryobiology: The freezing of biological systems. Science 168, 939-949 (1970). Mazur, P. The role of intracellular freezing in the death of cells cooled at supraoptimal rates. Cryobiology
14, 251-272
(1977).
19. Meryman, H. T. Mechanics of freezing in living cells and tissues. Science 124, 515-521 (1956). 20. Moor, H. Freeze fixation of living cells and its application in electron microscopy. Z. Zelljorsch.
Mikroscop.
Anat.
62,
546-580
(1964). 21. Nei, T. Freezing injury to erythrocytes. II. Morphological alterations of cell membranes. Cryobiology 13, 287-294 (1976). 22. Nermut, M. V., and Ward, B. J. Effect of fixatives on fracture plane in red blood cells. J. Microsc.
102, 29-39
(1974).
23. Pinto da Silva, P., Moss, P. S., and Fudenberg, H. H. Anionic sites on the membrane intercalated particles on human erythrocyte ghost membranes. Freeze-etch localization. Exp. Cell Res. 81, 127-138 (1973). 24. Plattner, H., Fischer, W. M., Schmitt, W. W., and Bachmann, L. Freeze-etching of cells without cryoprotectants. J. Cell Biol. 53, 116-126 (1972). 25. Rapatz, G., Sullivan, J., and Luyet, B. Preservation of erythrocytes in blood containing various cryoprotective agents, frozen at various rates and brought to a given final temperature. Cryobiology 5, 18-25 (1968). 26. Riehle, U., nique of Etching. Benedetti Soci&C tronique,
and Hiiechli, M. The theory and techhigh pressure freezing. In “FreezeTechniques and Applications” (E. L. and P. Favard, Ed.), pp. 31-62. Francaise de Microscopic ElecParis, 1973.
27. Sherman, J. K., and Kim, K. S. Correlation of cellular ultrastructure before freezing, while freezing, and after thawing in assessing freeze-thaw-induced injury. Cryobiology 4, 61-74 (1967). 28. Steere, R. L. Freeze-etching and direct observation of freezing damage. Cryobiology 6, 137- 150 (1969).
362
SEIZO
FLJJIKAWA
29. Verkleij, A. J., and Ververgaert, P. H. J. Th. Freeze-fracture morphology of biological membranes. Biochim. Biophys. Acta 515, 303-327 (1978). 30. Wunderlich, F., Wallach, D. F. H., Speth, V.,
and Fischer, H. Differential effects of temperature on the nuclear and plasma membranes of lymphoid cells. A study by freeze-etch electron microscopy. Biochim. Biopys. Acta 373,34-43 (1974).