Generation of cell processes in a high frequency electric field

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Bioelectrochemistry and Bioenergetics, 20 (1988) 143-153 A section of J. Electroanal. Chem., and constituting Vol . 254 (1988) Elsevier Sequoia S .A., Lausanne - Printed in The Netherlands

Generation of cell processes in a high frequency electric field Leonid B . Margolis A .N. Belozersky Laboratory of Molecular Biology and Bioorganic Chemistry, Moscow State University, Moscow, 119899 (US .S .R.)

Sergey V . Popov Laboratory of Bioelectrochemistry, A .N. Frumkin Institute of Electrochemistry of the U.S.S.R . Academy of Sciences, Leninsky Prospect, 31, Moscow, 117071 (U . S. S . R .) (Received 5 October 1987 ; in revised form 8 February 1988)

ABSTRACT

A new experimental system which allows one to apply force to the plasma membrane of substrate-attached cells is described . The force, which is generated in a special chamber by an alternating current electric field of high frequency, pulls the membrane outward, generating cell processes . The phenomenon of cell outgrowth formation by this membrane-applied force is investigated . The experimental system developed also provides an opportunity to study the mechanical properties of various parts of substrateattached cells.

INTRODUCTION

As a rule, cells in vivo are attached to each other or to non-cellular substrates by cell protrusions (processes) [1,2] . The formation of cell processes is a complex energy-dependent phenomenon which includes deformation of the plasma membrane, reorganization of the underlying cortical layer, rearrangement and/or polymerization of actin microfilaments, etc . [3,4] . To study the mechanism of cell process formation, it is necessary to develop an experimental system to simulate the force which propels the membrane outgrowth outward .

* Presented at the 9th BES Symposium on Bioelectrochemistry and Bioenergetics, Szeged (Hungary), 1-5 September 1987 . ** To whom correspondence should be addressed .

0302-4598/88/$03 .50

O 1988 Elsevier Sequoia S .A .

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Here, we describe a new experimental system based on the phenomenon of dielectrophoresis [5,6] which allows one to apply force directly to the plasma membrane of cells . This extrinsic mechanical force, generated by an ac electric field of high frequency, pulls the membrane outward, presumably without directly affecting the cytoplasm . MATERIALS ANS METHODS

Cells Mouse L929 cells were a generous gift from Dr . I .N . Bandrina (Institute of Molecular Biology, Academy of Sciences of the U .S .S .R., Moscow) . Mouse embryo fibroblasts (MEF) were prepared according to ref . 7 . Cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and were used 2-3 days after seeding . One to three passages of MEF were used . Before electrical treatment, the cells were washed once in physiological saline (140 mM NaCl, 5 mM KCI, 2 mM MgCl, 1 mM CaC1 2 , 10 mM HEPES-NaOH, pH 7 .4) and resuspended twice in a sucrose medium (290 mM sucrose, 1 mM HEPES-NaOH, pH 7 .4) (with indicated additions) at a concentration of 10 4 -10 5 cells/ml . Erythrocytes were obtained from one of the authors (S .V .P .), washed three times in physiological saline, resuspended in sucrose medium, and treated by an EF as described below . To obtain echinocytes, erythrocytes were incubated in sucrose medium for 3-5 h . The conductivity of the sucrose medium was 4 X 10 -4 S/cm .

Cell treatment by electric field A coverslip was covered by an aluminium film divided in two halves by a gap in the middle of the coverslip (Fig . 1) . The two part of the film were used as electrodes . A drop of cell suspension in sucrose medium was placed on the surface of such a

60-80 (Mm

S 6_8V Fig. 1 . The experimental chamber : a cover slip covered with aluminium films (E) - 0 .1 gm thick. G is the gap between the two films . The metal films were used as a pair of electrodes . The aluminium film was made by heat evaporation in vacuum at -10 -6 Torr .

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coverslip in the gap between the electrodes . The cells were treated with an ac EF (frequency 1 MHz, voltage 6-8 V, signal generator 13-7 A (U .S .S .R.)) at room temperature for 3-5 min . In one series of control experiments, cells were washed in physiological saline, resuspended in sucrose medium, placed between the electrodes but were not treated with an electric field (EF) . In another series of control experiments, cells were washed, resuspended in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and placed between the electrodes . No EF was applied . Electron microscopy

The cells were fixed for scanning electron microscopy by 2 .5% glutaraldehyde (Serva) in phosphate-buffered saline (PBS) . The specimens were dehydrated in acetone, critical point dried, coated with platinum and examined under a Cambridge Stereoscan S-4 or in a Hitachi 405 A scanning electron microscope . Pretreatment of cells with drugs

Cytochalasin B (Sigma) was dissolved in DMSO (Sigma) at a concentration of I mg/ml and added to the suspension of cells in a serum-supplemented medium at a final concentration of 10 µg/ml for 1 h . Colcemide (Merck) was added to the spread cells at a concentration of 1 ttg/ml for 16 h . Sodium azide (Serva) at a concentration of 1 mg/ml and 10 mM 2-deoxy-D-glucose (Serva) were added to the cell suspension for 1 .5 h. 10 µM carbonyl-m-chlorphenylhydrazone (CCCP) (Sigma) and 10 mM 2-deoxy-D-glucose were added to the cell suspension for I h . In all cases, the pretreatment was done at 37 ° C . Then the cells were washed twice in a sucrose medium containing these substances at the indicated concentrations and treated with an EF as described above . We checked that cytochalasin B in sucrose medium induced characteristic morphological changes of spread MEF and L cells . To test cell viability, MEF and L cells were stained with rhodamine 123 (Sigma) as in ref. 8 immediately before EF treatment . Cell staining by the dye was checked 10 min after EF treatment . In some experiments, after EF treatment MEF and L cells were incubated in serum-supplemented Dulbecco's modified Eagle's medium and the process of spreading was investigated . To study the effect of an EF on the morphology of the cells, 10-15 experiments were performed for each type of cells . In every experiment 2-3 coverslips containing 10 3 cells were analysed by scanning electron microscopy . To study the effects of an EF on drug-pretreated MEF and L cells, four to five separate experiments were performed for each protocol of treatment . Three to five coverslips were studied in each experiment . RESULTS AND DISCUSSION

Cell behaviour during EF treatment

Ten to fifty µl of the suspension of cells in sucrose medium was placed on the surface of the experimental chamber . After the EF was switched on, the sucrose-suspended cells concentrated along the edges of the metal layers (Fig . 2) .

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Fig . 2 . Phase microscopy of mouse embryo fibroblasts subjected to an electric field of high frequency in the experimental chamber (see Fig . 1) . The cells are attached to the edges of the electrodes (dark areas) . Bar = 20 Am. Fig. I Scanning electron photomicrographs of L cell subjected for 3 min to an electric-field-generated mechanical force . The force is directed to the electrode which occupies the lower part of the photograph . Bar = 3 µm .

This behaviour of cells is accounted for by the theory of dielectrophoresis (see ref . 5 and Appendix), which predicts that cells subjected to a high frequency EF in low-conducting media concentrate in the areas of maximal field strength . We have studied single cells in contact with the electrodes . Three min treatment of cells with an EF resulted in their firm attachment to the substrate . The cells were not washed away when the solution in the chamber was changed . The electric field did not affect cell viability . In fact : (1) The percentage of cells stained by trypan blue did not increase after EF treatment. (2) The mitochondria potential of MEF and L cells was not affected by the EF as demostrated by rhodamine 123 staining . (3) EF-treated L cells and normal fibroblasts spread after the medium was replaced with Dulbecco's modified Eagle's medium containing 10% calf serum . Thirty min and 3 h after EF treatment, the morphology of the cells did not differ from that of the cells not treated with an EF . As opposed to the dielectrophoretic systems used earlier, our experimental chamber allows the study of the reactions of substrate-attached cells to forces applied to the plasma membrane in an ac EF . Morphology of the EF-treated cells

The cells were treated with an EF for 3-5 min, then the field was switched off and the cells were fixed as described in the Materials and Methods section .

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Fig . 4 . (a) As Fig. 3, but MEF cell ; bar = 3 µm. (b) MEF ; lamella type of protrusion ; bar =Iµm .

The cells were attached to the surface of the substrate by various cell protrusions . For all the cells described below, the processes were formed only at the part of the cell facing the nearest electrode . The morphology of the cytoplasmic processes was different for different types of cells (Fig. 3-5) . L cells were attached to the edge of the metal film by long thin processes (up to 10 µm long and 0 .1 tm thick) (Fig . 3) . Their distal ends were flattened upon the substrate. Mouse embryo fibroblasts also formed protrusions similar to those of the L cells (Fig . 4a) . However, approximately 50% of the cells examined formed lamellae, i .e .

Fig . 5 . As . Fig . 3, but erythrocyte cell ; bar = 3

jLm .

Fig . 6 . L cells in the "pearl-chain" during EF treatment . Cell processes between the cells can be seen . Scanning electron microscopy . Bar = 3 µ m .

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flat protrusions . In some cases, the width of these lamellae was 10 µm . Usually lamellae protruded from the cell body (Fig . 4b) . Sometimes, small lamellae could be observed in the region of process bifurcation . Two types of outgrowths, long thin processes and lamellae, usually were found in different parts of the same cell . The protrusions formed by the two types of the cells in the EF resemble those formed under normal physiological conditions . Erythrocytes are not capable of producing membrane protrusions upon attachment to the substrate . They did not produce processes or other kinds of protrusions under EF treatment either (Fig . 5) . Since erythrocytes, unlike the other cells tested, have a smooth surface without any microvilli one can suppose that initial outgrowths are necessary for the formation of long processes under our conditions . However, echinocytes subjected to the dielectrophoretic force did not produce cell processes either . At higher cell densities, the so-called "pearl-chains" [9] were formed . In this case, cell processes were observed not only on the surface of electrode-attached cells but also between the cells (Fig . 6) . The phenomenon of intercellular process formation in "pearl-chains" was described earlier by Berg et al . [10] . Since in our work we studied the interaction of single cells with the planar substrate, we avoided the formation of "pearl-chains" by using cell suspensions of low concentration (concentrations lower than 10 5 /ml) . What are the possible mechanisms of EF-induced cell process formation?

A cell brought in proximity to the electrode is subjected to a mechanical force, non-uniformly distributed over the cell body ; the force is applied to the plasma membrane and a thin (1-2 nm) cytoplasmic layer . The force on every site of the membrane is perpendicular to the surface of the cell and is directed outward [6] (see also the Appendix) . The forces acting on the different parts of the cell are shown in Fig . 7 . Evaluation (see Appendix) of the pressure generated by the EF on the plasme membrane gives an approximate value of 10 3 N/m z at a distance of 1 ttm from the electrode edge . As described above, the cell protrusions are formed only at the electrode-facing part of the cell - that is, where the applied force is maximal . We think that this membrane-applied mechanical force generated in the dielectrophoretic chamber is the immediate cause of process formation .

E

Fig . 7 . Schematic diagram of the distribution of EF-generated mechanical forces over the surface of a cell attached in the gap (G) to the edge of the electrode (E) . The largest force is applied to the electrode-facing part of the cell. The force tends to pull on the cell membrane towards the electrode .

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Fig . 8 . Scanning electron micrographs of control MEF . 15 min incubation at 20 ° C in sucrose medium (a) and in serum-supplemented Dulbecco's modified Eagle's medium (b) on the surface of the chamber in the gap between the electrodes . No electric field was applied. Bar = 3 µm.

It is obvious that the cause of formation of cell processes is EF treatment . Indeed, in the absence of an EF, virtually no MEF, erythrocytes or L cells attached to the substrate in sucrose medium in the control experiments unless the incubation time was prolonged up to 60 min . MEF or L cells which could be found on the substrate during shorter periods of incubation remained spherical (Fig . 8a) and could be easily washed away . When studied under a scanning electron microscope, a few (2-3) short processes could be noticed . No preferential direction of the cell process formation was observed . The surface of these cells was covered with microvilli or with microblebs, as is observed normally in complete serum-supplemented medium (Fig. 8b) . While the topography of the cell surface might depend on the phase of the cell cycle, we used only density-arrested cells in the G,/G, phase . What other physical phenomena could mediate the observed effects?

It is well known that a constant EF can redistribute charged species (e .g. proteins) along the cell body [11] . However, this is highly improbable in our experiments due to the high frequency of the electric field [5] . The fields we used during EF treatment (- 1 kV/cm) are known to cause electrical breakdown of membranes and fusion of cells in "pearl-chains" [9,10] . However, this does not happen in our system . Because of the high frequency of the EF the actual voltage across the plasma membrane is much smaller than in the case of rectangular (or capacitor discharge) voltage pulses 100 µs-1 ms long [10] (see Appendix) .

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The heating of the sucrose medium in the chamber is negligible due to the low conductivity of the medium [6] . Since the EF does not penetrate the cells, the heating of the cell cytoplasm is also negligible . We attempted to use various treatments to determine whether the cytoskeleton and the cytoplasm activity contribute to the formation of protrusions . Experiments were performed with MEF and L cells . We treated the cells with cytochalasin B + colcemide to inhibit polymerization of actin and tubulin, and with sodium azide + 2-deoxy-D-glucose to deplete ATP . The drugs were used according to the protocols given earlier to produce maximal specific effects [7,12] .

Fig . 9. Mouse embryo fibroblasts subjected to electric field treatment . Cells were incubated with 10 µg/ml cytochalasin B+1 µg/ml colcemide (a,c) or with sodium azide+2-deoxy-D-glucose (b) . Bar = 3 µm . For details see the Materials and Methods section .

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As revealed by scanning electron microscopy (Figs . 9a and 9b), none of these treatments affected cell outgrowth formation induced by the EF . Cell-specific protrusions were formed in both these cases ; the outgrowths differed neither in morphology nor in distribution over the cell body from the protrusions produced by drug-untreated cells . The treatment of cells with colcemide, cytochalasin B or with CCCP (inhibitor of cell metabolism) + 2-deoxy-D-glucose also does not affect the cell process formation in an EF . In some cases, the diameter of the processes formed in the EF by cytochalasin B (or cytochalasin B together with colcemide)-pretreated MEF or L cells increased (Fig . 9c) . CONCLUDING REMARKS

The mechanism of the formation of cell protrusions during attachment to or movement along a solid substrate is far from being fully understood, partly because if is difficult to differentiate between the contributions of the cytoskeleton and the plasma membrane to the process . The results of our experiments show that the process formation in our system is presumably mediated by the mechanical force generated in the high frequency EF . This force pulls out the cell membrane, forming various kinds of cell-specific protrusions . In this sytem, there is no need for actin polymerization or other kinds of cytoplasmic activity for cell process formation . Thus, when a sufficient mechanical force is applied to the cell membrane typical cellular processes are formed . Although EF-generated forces similar to those we have produced do not exist in vivo, they might simulate hydrostatic forces in the cytoplasm which tend to pull the plasma membrane outward [1] . Extrapolation of our experimental results predicts that these membrane-applied forces can play a critical role in producing cell processes in vivo . The above system, based on the phenomenon of dielectrophoresis, provides a new opportunity to study the mechanical properties of various areas of substrate-attached cells. ACKNOWLEDGEMENTS

We thank Drs . Yu . A . Chizmadzhev, J .M . Vasiliev and L .V. Chernomordik for critical discussions of the work . APPENDIX

Here, we will describe briefly the behaviour of the cells in our experimental system, and evaluate the forces applied to the membrane and the voltage across the membrane of substrate-attached cells .

* The Appendix was prepared in collaboration with P .I . Kuzmin.



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The cell is considered to be a sphere with conductivity a ; - 1 S/m, surrounded by a thin (h = 5 nm) dielectric shell with permitivity E m = 1 . Under the action of an external electric field (Ee ), a charged ionic diffuse layer is formed close to the inner surface of the membrane . The thickness of this layer K -' is - 1 rim. [12] . The characteristic time of its formation is determined by the coefficient of diffusion of ions in water (D) . For typical values of D = 10 -9 /s, we obtain Ti =_ (K 2D) -1 10 -9 s . Close to the external surface of the cell membrane another layer of counter-ions is formed . The characteristic time constant of this process (Te ) is determined by the specific capacitance of the cell membrane (C m ), the conductivity of the external medium (ae ) and the radius of the cell (R) [12] :

m

Te - CmR/ae

( 1) -4 Given Cm =_ 1 µF/cm, R = 5 µm and ae 10 S/m (these values are typical for MEF), we obtain Te -= 10 -4 s . The period of the field (T) in our case is 10 -6 s . So, i u T << e , and during T the inner diffuse layer is formed instantly, while the external layer does not form, i .e . the external medium can be regarded as non-conducting . The behaviour of a conducting body in non-conducting media in a EF is well known [5,13] . In particular, (1) Ee is normal to the cell membrane . (2) The cell is pulled towards the region of maximal field strength . (3) The pressure exerted on the cell surface is given by T

T

P = E eE O Ee /2

(2)

where E e is the dielectric constant of the external medium (in our experiment E e = 80), Ee is the field strength near the cell surface and E O is the electrical constant . P is directed outward . (4) The field inside the membrane, Em , can be found from the continuity of dielectric displacement : Em m = EE, . So, the voltage across the cell membrane can be estimated from E

U--- Em h = Eeh(Ee/Em) (3) For E e - 1 kV/cm, we obtained U - 20 mV . This voltage is too low to cause electrical breakdown of the membrane [9] . The membrane-applied pressure is easily evaluated under the assumption that the cell does not perturb Ee . Ee (r) in our experimental chamber can be calculated for r >> t, r << 1 [13], where r is the distance from the edge of the electrode, 1 is the width of the gap and t is the thickness of the electrodes : _1/2 Ee (r) = 2U(irrl) (4) So, P

=

7

EeEOEe / 2 = 2 EeE0U 2/( Tr1)

For r --- 1 µm, we obtained p = 10 3 N/m2 .

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This pressure is enough to pull the cytoskeleton-free lipid protrusion out of the cell [14] . Owing to the complex topography of the cell surface, the actual pressure at some points of the cell membrane (e .g. near the microprojections) might be higher. REFERENCES 1 J.B . Trinkaus, Exp . Biol . Med ., 10 (1985) 130 . 2 J .M. Vasiliev, Biochim. Biophys . Acta, 780 (1985) 21 . 3 S .J . Singer and A . Kupfer, Annu . Rev . Cell Biol ., 2 (1986) 337 . 4 M . Schliwa, The Cytoskeleton . An Introductory Survey, Springer, Wien, New York, 1986 . 5 H .A . Pohl, Dielectrophoresis, Cambridge University Press, Cambridge, 1978 . 6 V.Ph . Pastushenko, P .I . Kuzmin and Y .A . Chizmadzhev, Stud . Biophys ., 110 (1985) 51 . 7 A.D . Bershadsky, V .I . Gelfand, T .M . Svitkina and I .S . Tint, Exp . Cell Res ., 127 (1980) 421 . 8 L .K. Johnes, M .L. Walsh and L .B . Chen, Proc . Natl . Acad . Sci . USA, 77 (1980) 990. 9 U . Zimmermann, Biochim . Biophys . Acta . 694 (1982) 227 . 10 H . Berg, K. Augsten, E . Bauer, W . Forster, H-E . Jacob, P . Muhlig and H . Weber, Bioelectrochem . Bioenerg., 12 (1984) 119 . 11 M-M Poo, Annu . Rev. Biophys . Bioeng., 10 (1981) 245 . 12 L .V . Domnina, V .I . Gelfand, O .Y . Ivanova, E .V. Leonova, O.Y . Pletyuschkina, J .M. Vasiliev and I .M . Gelfand, Proc . Nad . Acad . Sci . USA ., 79 (1982) 7754. 13 L .D. Landau and E.M . Lifshits, Electrodynamics of Continuous Media, Pergamon Press, New York, 1960. 14 E .A . Evans and R . Skalak, Mechanics and Thermodynamics of Biomembranes, CRC Press, Boca Raton, FL, 1980 .