Biochimica et Biophyslca Acta, 1093 (1991) 162-167 © 1991 Elsevier Science Publishers B.V. 0167-4889/91/$03.50 ADONIS 0167488991001968
162
BBAMCR 12925
Local deformation of human red blood cells in high frequency electric field G.V. Gass 1, L.V. Chernomordik i and L.B. Margolis 2 The A.N. Frumkin Institute of Electrochemistry, Academy of Sciences of the U.S.S.R., Moscow (U.S.S.R.) and ' A,N. Belozersky laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University. Moscow (U.S.S.R.)
(Received 10 January 1991)
Key words: Local deformation; Echinocyte; Dielectrophoresis
A method of local and general deformation of single erythrocytes by external forces in high-frequency electric field is described. The method allows the avoidance of any mechanical contact of the cell with electrodes. Under the action of the forces applied human erythrocytes change their shape and produce various membrane structures: long filopodia-like processes, retraction fibers and lamella-like structures. These structures are never formed by erythrocytes under normal conditions, but are typical for fibroblasts, macrophages and epithelium cells. By the method developed the elastic properties of spicules on the membranes of echinocytes were also studied. Deformation of echinocyte in high-frequency electric field leads to the smoothing out of spicules. However, after the electric field is turned off, echinocyte restores its initial forms including the number and localization of all initial spicules on the cell surface.
Introduction The organization of human red blood cells is comparatively simple. These cells do not have a nucleus, organelles or cytoplasmic skeleton. They do not spread on the substrates, do not form long processes, filopodia, lamellas or retraction fibers. Unfike many other cells their shape in vivo is very stable. Only a limited set of shapes acquired by erythrocytes under various conditions has been described [1-4]. Many studies have been published concerning the mechenical properties of erythrocytes [5-11], their shape [1,2], the structure and properties of membrane skeleton [12-17]. However, the reason why erythrocytes do not form structures typical for most other cells remains still unclear. Perhaps these cells are unable to form such structures due to mechanical restrictions of the membrane or they cannot be stabilized due to the absence of cytoskeleton and contact with substrate. In the present study we describe a method of local deformation of erythrocytes in a high-frequency electric field. The method allows to apply locally graded mechanical forces and to deform the cell membrane without
Correspondence: L.B. Margofis, A.N. Belozersky Laboratory of Molecular Biology and Bioo~ani¢ Chemistry, Moscow State University, Moscow 119899, U.S.S.R.
direct contact with its surface. The method was used to study the local mechanical properties of erythrocytes, their ability to form local structures typical for other cells, and the mechanism controlling the position and the number of spicules on the echinocyte surface.
Materials and Methods To obtain human erythrocytes a drop of blood of healthy donors was diluted by Hanks' balanced salt solution (HBSS) to hematocrit < 0.05. After 30 min incubation cells at 37°C on coverslips erythrocytes became attached to the glass. The cell population consisted of approx. 70-80~ of discocytes and 20-30~ of echinocytes. The physiological solution was then replaced by an isosmotic sucrose solution with low ionic strength (0.29 M of sucrose (Sigma), 1 mM Hepes-NaOH (Serva) (pH 7.4)) and coverslips were placed on the stage of inverted photomicroscope (Leitz Fluovert). Thin wolfram microelectrodes (diameter - 2 - 4 pm at the tip) were placed using Narishigi MO 303 micromanipulators 10-20/~m from the cell surface and an alternating electric field of I MHz frequency and 0-4000 V / c m intensity was applied. The external force pulling the cell membranes toward the electrodes (Fig. la) was proportional to the square of the field intensity [18-20]. Direct observation of the deformation process was carried out by a phase contrast microscope with oil immersion
163
A
B E
E 4~" Ib
Fig. 1. The external forces applied to the cell membrane under the action of high-frequency alternating electric field in the experimental system with two thin microelectrodes near cell (A) and in the chamber with two flat electrodes separated by 90/~m gap (B). E, electrodes; C, cells. The force densities for the different regions of membranes are shown by arrows.
d
e
(magnification x 1250 or x 2000). In some experiments thin aluminum layers on coverslips of 0.1/~m thickness separated by a 90 /~m gap (Fig. lb) were used as electrodes [21]. In this particular case some tens of erythrocytes that attached to the glass in the gap between electrodes were deformed by the field appfication. For scanning electron microscopy stretched erythrocytes were fixed for 5 rain in electric field under the microscope. We fixed single cells (one stretched cell per sample) if we used two thin microelectrodes, or we fixed a few tens of cells in the gap if we used a flat electrodes chamber. Fixation was performed in isoosmotic sucrose solution (see above) containing 2~ of glutaraldehyde (pH 7.4). Then the specimen was transferred to HBBS containing 2% glutaraldehyde (pH 7.4) and postfixed for another several hours at room temperature. The samples were dehydrated in acetone, dried in a critical
Fig. 2. The deformation of the erythrocyte attached to coverslip in alternating electric field applied to the microelectrodes (black tips at the edges of every picture). The field intensities used were 0, 1000, 2000, 2800 and 3800 V/cm for the Figs. a, b, c, d and e, correspondingly. Phase contrast microscopy. Bar corresponds to 8.5/~m.
point dryer (Hitachi), shadowed with gold and arialyzed with a scanning electron microscope PSEM-500x (Phillips). Results
Erythrocyte deformation in high frequency electric field Two microelectrodes were placed near the chosen cell and the alternating electric field was applied. Due to the attachment of the cell to the substrate its position remained non-altered. The cell became elongated in both directions and acquired a spindle shape (Fig. 2).
f Fig. 3. The reversible deformation of discocyte (a,b,c) and echinocyte (d,e,f). a, d, the cells before and c, f, 1 rain after field appfication; b, e, the cell in alternating field 3700 V/cm; 3 rain). Phase contrast microscopy. Bar corresponds to 8/tm (a,b,c) and 8.8/tm (d,e,f).
164 With increase of field intensity two thin processes with thickenings at the distal ends were formed towards electrodes. The process diameter as measured with scanning electron microscopy was the same for different erythrocytes ((~.1 +0.02 /~m) and remained constant during the entire process, the diameter of thickenings was approx. 0.2-0.3/Lm. The process was generated from the smooth surface of discocytes. In the case of echinocytes it grew as elongation of a single spicule. The average lengths of the processes at 2000, 3000 and 4000 V / c m were approx. 1, 3.5 and 7 #m, correspondingly. Increasing of field intensity up to 5000-5500 V / c m led to swelling and hemolysis of a considerable part of the cells. At these intensities the length of processes before swelling could achieve 12-15 ~tm. Short deformation of erythrocytes (for 5 rain or less) was reversible. After the field was switched off approx. 80~ of the cells (both discocytes and echinocytes) acquired their initial shape in 0.5-1.5 min (Fig. 3a-c and d-f). The only irregularities which remained were the long processes directed towards the tips of elec-
Fig. 3.~}G
trodes. They shortened after the field was switched off but usually did not disappear completely. When we used thin metal layers on the coverslip as electrodes (Fig. lb), the mode of cell deformation was dependent on the position of the cell. If it was located in the middle of the gap (at the equal distances to both electrodes) the cell body elongated in the electric field symmetrically and the cell acquires a spindle-like shape (Fig. 4a,b). If the cell was attached near (5-10/tin) the edge of one of the electrodes the deformation was asymmetrical. The cell acquires a triangular form (Fig. 4c,d). Such a cell has a flat broad lamellar-like protrusion directed toward the nearest electrode and a thin 'tail' in the direction of the opposite distant electrode. The deformation in this system was also reversible and the cells acquired their initial shape after the field was switched off.
Echinocyte deformation in alternating electric' field When echinocytes were stretched by an alternating electric field (3800-4000 V / c m ) the elongation of cells was
celd of .............................
~ .....e ~ c a l
triangular deformation of erythrocytes attached near the edge of one of the flat electrodes (¢~ashed line in the left top ~.orner of the pictures). Scanning electron microscopy. Bar corresponds to 2/~m.
165 accompanied by the smoothing of their surfaces observed by phase contrast microscopy (Fig. 3e). The scanning electron microscopy of the stretched cells has shown that for echinocytes with a small amount of large spicules (of 0.5-0.8 #m diameter), the surface looks smooth and no sign of spicules could be found (Fig. 5a). For echinocytes with many small spicules the smoothing was complete only for some cells (Fig. 5b). For others the transformation of spicules into wide protrusions during the field application was observed (Fig. 4c). Sometimes spicules became shorter and were shifted along the direction of erythrocyte stretching (Fig. 5d). In the latter case, the diameter of the spicules remained constant independently of their height. In all cases even after a series of three to five stretchings after the field was switched off and the cells acquired their initial shape, each of individual spicules reappears again in the initial position. In particular for all the cells in Fig. 5a-d it was tested that after the field was switched off the spicules arise again at their initial positions. Fixation of these single cells was performed during the repeat stretching by the same force (field intensity). Discussion
The alternating electric fields are nowadays widely applied in cell biology [9,11,!9,21,22]. In the present study we use a high-frequency electric field to investigate the local mechanical properties of cell membranes as well as some shape transformations of whole human red blood cells. The method developed has several advantages compared to the commonly used techniques [5-7,9,23]: (i) Depending on the arrangement of the electrodes we can deform the cells in different directions. (ii) The electrodes are used only to generate the force, cell deformation takes place without direct contact of electrodes with the cell surface. (iii) By changing the intensity of the field a n d / o r the distance between the cell surface and electrodes it is possible to control the force applied to cell membrane. The force density f can be estimated [21] using the expression: f = EEoE2/4
where e is the dielectric constant of the water solution, co is the electrical constant, E is the field intensity near the cell surface. It can be estimated as E ~- 12 U. r/L 2, where U is the amplitude of the voltage applied to the electrodes, r is the radius of electrode tip and L the distance between the electrodes. The values of force densities calculated for our system by the estimation considered were varied in the range 15-100 Pa as a function of field intensity and the distance of the membrane from the edge of electrode.
The voltage across the erythrocyte membrane in alternating electric field (4 kV/cm; 1 MHz) is estimated to be 80 mV (see Ref. 21 for details of the estimation). This can hardly cause the damage of cell membrane due to electroporation [19,22]. Moreover, the absence of colloid-osmotic cell swelling and the lack of hemolysis is another evidence against pore formation in our conditions at field intensities 0-4000 V/cm. The obvious disadvantage of the method is the requirement of a medium of low ionic strength. The asymmetry of the ionic environment on the different sides of the membrane as well as the disturbance of the electrostatic interactions between the membrane surface charges can modify the mechanical properties of the membranes [24]. However, the long processes obsel~,ed in the present paper look similar to the tethers formed by erythrocytes in the flow of physiological solution [23]. In addition, no significant changes of the mechanical properties of erythrocytes and other cells have been observed after the short-term incubation of cells in isoosmotic non-ionic medium [9,21]. It is known that heating at 4 5 - 5 0 ° C dramatically alters red blood cell deformability due to denaturation of membrane proteins [25,26]. However, this factor can hardly contribute to the effects described in the present paper since even the local increase of the temperature of the medium was shown to be negligible in the similar experimental system [19]. In vivo the shape of erythrocytes is quite stable. In contrast to many other cells (macrophages, fibroblasts, epithelia cells, etc.) they have no ability (and necessity) to spread, to form long processes, lamellae or microvilli and so on. The reason for their shape limitation is still unclear. We have shown that when external forces are properly arranged erythrocytes can be deformed in various ways to form shapes typical for fibroblasts and other cells. For example, by external forces it is possible to stretch out from erythrocytes long membrane processes with specific thickenings on the ends. The diameters of the processes and thickenings are similar to those of typical filopodia. Such processes were not observed at lower field intensities (up to 1500 V / c m ) used in Ref. 9 to deform single erythrocytes by high-frequency electric field. Thin membrane fibers from the sides of stretched erythrocytes to the substrate (Fig. 5) are similar to the retraction fibers of fibroblasts. They appear when the cell body is shifted (in our case narrowed due to stretching), but contact of the cell with substrate remains unbroken in some points. And, at last, it proved to be possible by external forces to transform erythrocyte into a polarized flat triangular cell of fibroblast-like shape with lamella-like structure (unstraight 'front edge') (Fig. 4c and d). It is known that the shape of the fibroblast is stabilized by the forces determined by the tension of
166
Fig. 5. The surface of echinccytes stretched by electric tiela toJJOO v/cmJ, a, me complete smootmng o~ me sunace o~ me ~mnc~yte with large spicules, b, c and d, the different evolutions of the spicules of the echinocytes with small thin spicules. Scanning electron microscopy. Bar corresponds to 2 ~tm.
actin filaments of the cytoskeleton bound to the sites of the cell-substrate contact [27]. For erythrocytes a similar shape coul~ b,~ ot~tained by the simultaneous application of external forc,~s of different amplitudes and directions lo the cell membrane. However, in this case the shape can not be stabilized. Switching off the field leads to a relatively fast (0.5-1.5 min) restoration of the initial cell shape. For all these kinds of shape transformarion the cell should be ~tttached to the substrate to provide the local membrr~, ueformation. It seems reasonable to conclude that the elastic membrane skeleton of erythrocyte prevents the cell from acquiring different shapes in viva. However, when mechanical resistance of the skeleton is overcome by external forces the obtained shape is not stable due to the lack of cytoskeleton and contact with substrate required to stabilize it. The method suggested was used also to study the elastic properties of the spicules of the echinocyte. The deformation of glass-attached cells by an electric field generated force results in not only the replacement of spicules along the surface but in the partial or complete smoothing of them. However, all spicules appear again in their initial positions when the cell acquires its original shape after the end of field application. This observation is in agreement with the ones in Ref. 28 where the contact of euthrocytes with glass was used to crenate the cells. The results show that the existence and
the position of spicules are determined somehow by the elastic membrane skeleton. Acknowledgments We express our deep gratitude to Drs. M.M. Kozlov, P.l. Kuzmin, V.S. Markin and Yu.A. Chizmadzhev for fruitful discussions and Dr. S.V. Popov for his critical remark. References 1 Bessis, M. (1973) Living Blood Cells and Their Ultrastructure, Springer-Verlag, Berlin. 2 Surgenor, D. (ed.) (1974) The Red Blood Cells, Academic Press, New York. 3 Sheetz, M.P. and Singer, S.J. (1977) J. Cell Biol. 73, 638-646. 4 Glaser, R. (1982) J. Membr. Biol. 66, 79-85. 5 Rand, R.P. and Burton, A.C. (1964) Biophys. J. 4, 115-127. 6 Evans, E.A. and Skalak, R. (1979) Mechanics and Termodynamics of Biomembranes, CRC Press, FL. 7 Hochmuth, R.M., Wiles, H.C., Evans, E.A. and McCown, J.T. (1982) Biophys. J. 39, 83-89. 8 Lerche, D. (1982) Biorheology 19, 587-590. 9 Engelhardt, E.H. and Sackmann, E. (1988) Biophys. J. 54, 495-5~8. 10 Bareford, D., Stone, P.C.W., Caldwell, N.M., Meiselma~, H.J. and Stunt, J. (1985) Clin. Hemorheol. 5, 311-322. 11 Gass, GN., Kuzmin, P.l., Chernomordik, L.V., Pastushenko, V.F. and Chizmadzhev, Yu.A. (1987) Biologicheskye Membrany 4, 1059-1072.
167 12 Sheets, M.P. (1983) Semin. Hematol. 20, 175-188. 13 Liu, S.C., Derich, L.N. and Palek, J. (1987) J. Cell Biol. 104, 527-536. 14 Waugh, R.E. (1987) Biophys. J. 51, 363-369. 15 Kozlov, M.M. and Markin, V.S. (1987) J. Theor. Biol. 129, 439452. 16 Tang, E.K.Y., Thompson, M.G. and Hichman, J.A. (1987) Biochem. Soc. Trans. 15, 862-863. 17 Stokke, B.T., Mikkelsen, A. and Elgaeter, A. (1986) Biophys. J. 49, 319-327. 18 Pohl, H.A. (1978) Dielectrophoresis, Cambridge University Press, Cambridge. 19 Zimmermann, U. (1982) Biochim. Biophys. Acta 694, 227-297. 20 Pastushenko, V.F., Kuzmin, P.l. and Chizmadzhev, Yu.A. (1985) Stud. Biophys. 110, 51-57. 21 Margolis, L.B. and Popov, S.V. (1988) Bioelectrochem. Bioenerg. 20, 143-t53.
22 Sowers, A.E. (1989) in Electroporation and Electrofusion in Cell Biology (Neumann, E., Sowers, A.E. and Jordan, C.A., eds.), pp. 229-256, Plenum Press, New York. 23 Hochmuth, R.M., Mohandas, N. and Blackshear, P.L (1973) Biophys. J. 13, 747-762. 24 Lerche, D., Kozlov, M.M. and Mark[n, V.S. (1987) Biorheology 24, 23-34. 25 Mohandas, N., Gxeenquist, A.C. and Shohet, S.B. (1976) Blood 48, 991-1004. 26 Deeley, J.O.Th., Clam, L.A. and Coaldey, W.T. (1979) Biochim. Biophys. Acta 554, 90-101. 27 Vasifiev, Yu.M. (1985) Biochim. Binphys. Acta 780, 21-65. 28 Bessis, M. and Prenant, M. (1972) Nouv. Rev. Franc. HematoL 12, 351-375.
162
BBAMCR 12925
Local deformation of human red blood cells in high frequency electric field G.V. Gass 1, L.V. Chernomordik i and L.B. Margolis 2 The A.N. Frumkin Institute of Electrochemistry, Academy of Sciences of the U.S.S.R., Moscow (U.S.S.R.) and ' A,N. Belozersky laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University. Moscow (U.S.S.R.)
(Received 10 January 1991)
Key words: Local deformation; Echinocyte; Dielectrophoresis
A method of local and general deformation of single erythrocytes by external forces in high-frequency electric field is described. The method allows the avoidance of any mechanical contact of the cell with electrodes. Under the action of the forces applied human erythrocytes change their shape and produce various membrane structures: long filopodia-like processes, retraction fibers and lamella-like structures. These structures are never formed by erythrocytes under normal conditions, but are typical for fibroblasts, macrophages and epithelium cells. By the method developed the elastic properties of spicules on the membranes of echinocytes were also studied. Deformation of echinocyte in high-frequency electric field leads to the smoothing out of spicules. However, after the electric field is turned off, echinocyte restores its initial forms including the number and localization of all initial spicules on the cell surface.
Introduction The organization of human red blood cells is comparatively simple. These cells do not have a nucleus, organelles or cytoplasmic skeleton. They do not spread on the substrates, do not form long processes, filopodia, lamellas or retraction fibers. Unfike many other cells their shape in vivo is very stable. Only a limited set of shapes acquired by erythrocytes under various conditions has been described [1-4]. Many studies have been published concerning the mechenical properties of erythrocytes [5-11], their shape [1,2], the structure and properties of membrane skeleton [12-17]. However, the reason why erythrocytes do not form structures typical for most other cells remains still unclear. Perhaps these cells are unable to form such structures due to mechanical restrictions of the membrane or they cannot be stabilized due to the absence of cytoskeleton and contact with substrate. In the present study we describe a method of local deformation of erythrocytes in a high-frequency electric field. The method allows to apply locally graded mechanical forces and to deform the cell membrane without
Correspondence: L.B. Margofis, A.N. Belozersky Laboratory of Molecular Biology and Bioo~ani¢ Chemistry, Moscow State University, Moscow 119899, U.S.S.R.
direct contact with its surface. The method was used to study the local mechanical properties of erythrocytes, their ability to form local structures typical for other cells, and the mechanism controlling the position and the number of spicules on the echinocyte surface.
Materials and Methods To obtain human erythrocytes a drop of blood of healthy donors was diluted by Hanks' balanced salt solution (HBSS) to hematocrit < 0.05. After 30 min incubation cells at 37°C on coverslips erythrocytes became attached to the glass. The cell population consisted of approx. 70-80~ of discocytes and 20-30~ of echinocytes. The physiological solution was then replaced by an isosmotic sucrose solution with low ionic strength (0.29 M of sucrose (Sigma), 1 mM Hepes-NaOH (Serva) (pH 7.4)) and coverslips were placed on the stage of inverted photomicroscope (Leitz Fluovert). Thin wolfram microelectrodes (diameter - 2 - 4 pm at the tip) were placed using Narishigi MO 303 micromanipulators 10-20/~m from the cell surface and an alternating electric field of I MHz frequency and 0-4000 V / c m intensity was applied. The external force pulling the cell membranes toward the electrodes (Fig. la) was proportional to the square of the field intensity [18-20]. Direct observation of the deformation process was carried out by a phase contrast microscope with oil immersion
163
A
B E
E 4~" Ib
Fig. 1. The external forces applied to the cell membrane under the action of high-frequency alternating electric field in the experimental system with two thin microelectrodes near cell (A) and in the chamber with two flat electrodes separated by 90/~m gap (B). E, electrodes; C, cells. The force densities for the different regions of membranes are shown by arrows.
d
e
(magnification x 1250 or x 2000). In some experiments thin aluminum layers on coverslips of 0.1/~m thickness separated by a 90 /~m gap (Fig. lb) were used as electrodes [21]. In this particular case some tens of erythrocytes that attached to the glass in the gap between electrodes were deformed by the field appfication. For scanning electron microscopy stretched erythrocytes were fixed for 5 rain in electric field under the microscope. We fixed single cells (one stretched cell per sample) if we used two thin microelectrodes, or we fixed a few tens of cells in the gap if we used a flat electrodes chamber. Fixation was performed in isoosmotic sucrose solution (see above) containing 2~ of glutaraldehyde (pH 7.4). Then the specimen was transferred to HBBS containing 2% glutaraldehyde (pH 7.4) and postfixed for another several hours at room temperature. The samples were dehydrated in acetone, dried in a critical
Fig. 2. The deformation of the erythrocyte attached to coverslip in alternating electric field applied to the microelectrodes (black tips at the edges of every picture). The field intensities used were 0, 1000, 2000, 2800 and 3800 V/cm for the Figs. a, b, c, d and e, correspondingly. Phase contrast microscopy. Bar corresponds to 8.5/~m.
point dryer (Hitachi), shadowed with gold and arialyzed with a scanning electron microscope PSEM-500x (Phillips). Results
Erythrocyte deformation in high frequency electric field Two microelectrodes were placed near the chosen cell and the alternating electric field was applied. Due to the attachment of the cell to the substrate its position remained non-altered. The cell became elongated in both directions and acquired a spindle shape (Fig. 2).
f Fig. 3. The reversible deformation of discocyte (a,b,c) and echinocyte (d,e,f). a, d, the cells before and c, f, 1 rain after field appfication; b, e, the cell in alternating field 3700 V/cm; 3 rain). Phase contrast microscopy. Bar corresponds to 8/tm (a,b,c) and 8.8/tm (d,e,f).
164 With increase of field intensity two thin processes with thickenings at the distal ends were formed towards electrodes. The process diameter as measured with scanning electron microscopy was the same for different erythrocytes ((~.1 +0.02 /~m) and remained constant during the entire process, the diameter of thickenings was approx. 0.2-0.3/Lm. The process was generated from the smooth surface of discocytes. In the case of echinocytes it grew as elongation of a single spicule. The average lengths of the processes at 2000, 3000 and 4000 V / c m were approx. 1, 3.5 and 7 #m, correspondingly. Increasing of field intensity up to 5000-5500 V / c m led to swelling and hemolysis of a considerable part of the cells. At these intensities the length of processes before swelling could achieve 12-15 ~tm. Short deformation of erythrocytes (for 5 rain or less) was reversible. After the field was switched off approx. 80~ of the cells (both discocytes and echinocytes) acquired their initial shape in 0.5-1.5 min (Fig. 3a-c and d-f). The only irregularities which remained were the long processes directed towards the tips of elec-
Fig. 3.~}G
trodes. They shortened after the field was switched off but usually did not disappear completely. When we used thin metal layers on the coverslip as electrodes (Fig. lb), the mode of cell deformation was dependent on the position of the cell. If it was located in the middle of the gap (at the equal distances to both electrodes) the cell body elongated in the electric field symmetrically and the cell acquires a spindle-like shape (Fig. 4a,b). If the cell was attached near (5-10/tin) the edge of one of the electrodes the deformation was asymmetrical. The cell acquires a triangular form (Fig. 4c,d). Such a cell has a flat broad lamellar-like protrusion directed toward the nearest electrode and a thin 'tail' in the direction of the opposite distant electrode. The deformation in this system was also reversible and the cells acquired their initial shape after the field was switched off.
Echinocyte deformation in alternating electric' field When echinocytes were stretched by an alternating electric field (3800-4000 V / c m ) the elongation of cells was
celd of .............................
~ .....e ~ c a l
triangular deformation of erythrocytes attached near the edge of one of the flat electrodes (¢~ashed line in the left top ~.orner of the pictures). Scanning electron microscopy. Bar corresponds to 2/~m.
165 accompanied by the smoothing of their surfaces observed by phase contrast microscopy (Fig. 3e). The scanning electron microscopy of the stretched cells has shown that for echinocytes with a small amount of large spicules (of 0.5-0.8 #m diameter), the surface looks smooth and no sign of spicules could be found (Fig. 5a). For echinocytes with many small spicules the smoothing was complete only for some cells (Fig. 5b). For others the transformation of spicules into wide protrusions during the field application was observed (Fig. 4c). Sometimes spicules became shorter and were shifted along the direction of erythrocyte stretching (Fig. 5d). In the latter case, the diameter of the spicules remained constant independently of their height. In all cases even after a series of three to five stretchings after the field was switched off and the cells acquired their initial shape, each of individual spicules reappears again in the initial position. In particular for all the cells in Fig. 5a-d it was tested that after the field was switched off the spicules arise again at their initial positions. Fixation of these single cells was performed during the repeat stretching by the same force (field intensity). Discussion
The alternating electric fields are nowadays widely applied in cell biology [9,11,!9,21,22]. In the present study we use a high-frequency electric field to investigate the local mechanical properties of cell membranes as well as some shape transformations of whole human red blood cells. The method developed has several advantages compared to the commonly used techniques [5-7,9,23]: (i) Depending on the arrangement of the electrodes we can deform the cells in different directions. (ii) The electrodes are used only to generate the force, cell deformation takes place without direct contact of electrodes with the cell surface. (iii) By changing the intensity of the field a n d / o r the distance between the cell surface and electrodes it is possible to control the force applied to cell membrane. The force density f can be estimated [21] using the expression: f = EEoE2/4
where e is the dielectric constant of the water solution, co is the electrical constant, E is the field intensity near the cell surface. It can be estimated as E ~- 12 U. r/L 2, where U is the amplitude of the voltage applied to the electrodes, r is the radius of electrode tip and L the distance between the electrodes. The values of force densities calculated for our system by the estimation considered were varied in the range 15-100 Pa as a function of field intensity and the distance of the membrane from the edge of electrode.
The voltage across the erythrocyte membrane in alternating electric field (4 kV/cm; 1 MHz) is estimated to be 80 mV (see Ref. 21 for details of the estimation). This can hardly cause the damage of cell membrane due to electroporation [19,22]. Moreover, the absence of colloid-osmotic cell swelling and the lack of hemolysis is another evidence against pore formation in our conditions at field intensities 0-4000 V/cm. The obvious disadvantage of the method is the requirement of a medium of low ionic strength. The asymmetry of the ionic environment on the different sides of the membrane as well as the disturbance of the electrostatic interactions between the membrane surface charges can modify the mechanical properties of the membranes [24]. However, the long processes obsel~,ed in the present paper look similar to the tethers formed by erythrocytes in the flow of physiological solution [23]. In addition, no significant changes of the mechanical properties of erythrocytes and other cells have been observed after the short-term incubation of cells in isoosmotic non-ionic medium [9,21]. It is known that heating at 4 5 - 5 0 ° C dramatically alters red blood cell deformability due to denaturation of membrane proteins [25,26]. However, this factor can hardly contribute to the effects described in the present paper since even the local increase of the temperature of the medium was shown to be negligible in the similar experimental system [19]. In vivo the shape of erythrocytes is quite stable. In contrast to many other cells (macrophages, fibroblasts, epithelia cells, etc.) they have no ability (and necessity) to spread, to form long processes, lamellae or microvilli and so on. The reason for their shape limitation is still unclear. We have shown that when external forces are properly arranged erythrocytes can be deformed in various ways to form shapes typical for fibroblasts and other cells. For example, by external forces it is possible to stretch out from erythrocytes long membrane processes with specific thickenings on the ends. The diameters of the processes and thickenings are similar to those of typical filopodia. Such processes were not observed at lower field intensities (up to 1500 V / c m ) used in Ref. 9 to deform single erythrocytes by high-frequency electric field. Thin membrane fibers from the sides of stretched erythrocytes to the substrate (Fig. 5) are similar to the retraction fibers of fibroblasts. They appear when the cell body is shifted (in our case narrowed due to stretching), but contact of the cell with substrate remains unbroken in some points. And, at last, it proved to be possible by external forces to transform erythrocyte into a polarized flat triangular cell of fibroblast-like shape with lamella-like structure (unstraight 'front edge') (Fig. 4c and d). It is known that the shape of the fibroblast is stabilized by the forces determined by the tension of
166
Fig. 5. The surface of echinccytes stretched by electric tiela toJJOO v/cmJ, a, me complete smootmng o~ me sunace o~ me ~mnc~yte with large spicules, b, c and d, the different evolutions of the spicules of the echinocytes with small thin spicules. Scanning electron microscopy. Bar corresponds to 2 ~tm.
actin filaments of the cytoskeleton bound to the sites of the cell-substrate contact [27]. For erythrocytes a similar shape coul~ b,~ ot~tained by the simultaneous application of external forc,~s of different amplitudes and directions lo the cell membrane. However, in this case the shape can not be stabilized. Switching off the field leads to a relatively fast (0.5-1.5 min) restoration of the initial cell shape. For all these kinds of shape transformarion the cell should be ~tttached to the substrate to provide the local membrr~, ueformation. It seems reasonable to conclude that the elastic membrane skeleton of erythrocyte prevents the cell from acquiring different shapes in viva. However, when mechanical resistance of the skeleton is overcome by external forces the obtained shape is not stable due to the lack of cytoskeleton and contact with substrate required to stabilize it. The method suggested was used also to study the elastic properties of the spicules of the echinocyte. The deformation of glass-attached cells by an electric field generated force results in not only the replacement of spicules along the surface but in the partial or complete smoothing of them. However, all spicules appear again in their initial positions when the cell acquires its original shape after the end of field application. This observation is in agreement with the ones in Ref. 28 where the contact of euthrocytes with glass was used to crenate the cells. The results show that the existence and
the position of spicules are determined somehow by the elastic membrane skeleton. Acknowledgments We express our deep gratitude to Drs. M.M. Kozlov, P.l. Kuzmin, V.S. Markin and Yu.A. Chizmadzhev for fruitful discussions and Dr. S.V. Popov for his critical remark. References 1 Bessis, M. (1973) Living Blood Cells and Their Ultrastructure, Springer-Verlag, Berlin. 2 Surgenor, D. (ed.) (1974) The Red Blood Cells, Academic Press, New York. 3 Sheetz, M.P. and Singer, S.J. (1977) J. Cell Biol. 73, 638-646. 4 Glaser, R. (1982) J. Membr. Biol. 66, 79-85. 5 Rand, R.P. and Burton, A.C. (1964) Biophys. J. 4, 115-127. 6 Evans, E.A. and Skalak, R. (1979) Mechanics and Termodynamics of Biomembranes, CRC Press, FL. 7 Hochmuth, R.M., Wiles, H.C., Evans, E.A. and McCown, J.T. (1982) Biophys. J. 39, 83-89. 8 Lerche, D. (1982) Biorheology 19, 587-590. 9 Engelhardt, E.H. and Sackmann, E. (1988) Biophys. J. 54, 495-5~8. 10 Bareford, D., Stone, P.C.W., Caldwell, N.M., Meiselma~, H.J. and Stunt, J. (1985) Clin. Hemorheol. 5, 311-322. 11 Gass, GN., Kuzmin, P.l., Chernomordik, L.V., Pastushenko, V.F. and Chizmadzhev, Yu.A. (1987) Biologicheskye Membrany 4, 1059-1072.
167 12 Sheets, M.P. (1983) Semin. Hematol. 20, 175-188. 13 Liu, S.C., Derich, L.N. and Palek, J. (1987) J. Cell Biol. 104, 527-536. 14 Waugh, R.E. (1987) Biophys. J. 51, 363-369. 15 Kozlov, M.M. and Markin, V.S. (1987) J. Theor. Biol. 129, 439452. 16 Tang, E.K.Y., Thompson, M.G. and Hichman, J.A. (1987) Biochem. Soc. Trans. 15, 862-863. 17 Stokke, B.T., Mikkelsen, A. and Elgaeter, A. (1986) Biophys. J. 49, 319-327. 18 Pohl, H.A. (1978) Dielectrophoresis, Cambridge University Press, Cambridge. 19 Zimmermann, U. (1982) Biochim. Biophys. Acta 694, 227-297. 20 Pastushenko, V.F., Kuzmin, P.l. and Chizmadzhev, Yu.A. (1985) Stud. Biophys. 110, 51-57. 21 Margolis, L.B. and Popov, S.V. (1988) Bioelectrochem. Bioenerg. 20, 143-t53.
22 Sowers, A.E. (1989) in Electroporation and Electrofusion in Cell Biology (Neumann, E., Sowers, A.E. and Jordan, C.A., eds.), pp. 229-256, Plenum Press, New York. 23 Hochmuth, R.M., Mohandas, N. and Blackshear, P.L (1973) Biophys. J. 13, 747-762. 24 Lerche, D., Kozlov, M.M. and Mark[n, V.S. (1987) Biorheology 24, 23-34. 25 Mohandas, N., Gxeenquist, A.C. and Shohet, S.B. (1976) Blood 48, 991-1004. 26 Deeley, J.O.Th., Clam, L.A. and Coaldey, W.T. (1979) Biochim. Biophys. Acta 554, 90-101. 27 Vasifiev, Yu.M. (1985) Biochim. Binphys. Acta 780, 21-65. 28 Bessis, M. and Prenant, M. (1972) Nouv. Rev. Franc. HematoL 12, 351-375.
