- Email: [email protected]
On the mechanism of cell lysis by deformation
ARTICLE IN PRESS
Journal of Biomechanics 38 (2005) 117–124
On the mechanism of cell lysis by deformation Hiroshi Takamatsua,*, Ryu Takeyab, Seiji Naitoc, Hideki Sumimotob,d a
Institute for Materials Chemistry and Engineering, Kyushu University, 6-1 Kasugakoen, Kasuga, Fukuoka 816-8580, Japan b Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan c Department of Urology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan d CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Accepted 14 March 2004
Abstract In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM>150 mM>75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton. r 2004 Elsevier Ltd. All rights reserved. Keywords: Cell viability; Mechanical stress; Cell compression; Osmotic volume change; Cytoskeleton
1. Introduction During freezing, mammalian cells are subjected to a mechanical stress by extracellular ice crystals. Although the increased solute concentration during freezing has been considered to be the major cause of cell injury, several experiments indicate that cell deformation by mechanical stress is important for cell lysis. The observation of the freezing of cell suspensions under a cryomicroscope shows that the cells are left in the unfrozen solution and appear to be compressed by the surrounding ice crystals (Nei, 1967a,b; Ishiguro and Rubinsky, 1994). Cell survival depends on the volume fraction of the unfrozen solution at the freezing temperature (Mazur et al., 1981; Mazur and Rigopoulos, 1983), suggesting that more cells are lysed in smaller spaces. The information about cell lysis by mechanical stress is therefore important in understanding the mechanism of cell injury during freezing. *Corresponding author. Tel.: +81-92-583-7788; fax: +81-92-5837882. E-mail address: [email protected] (H. Takamatsu). 0021-9290/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2004.03.011
There are a number of studies that deal with the effect of mechanical stress on cells (Evans and Skalak, 1980; Bloom et al., 1991; Hochmuth, 2000). Red blood cells have been studied extensively (for example, Rand and Burton, 1964; Evans et al., 1976; Chien et al., 1978; . Tozeren et al., 1982; Hochmuth, 1993) because their membrane complex is distinctively separated from their cytoplasm, and the cells are suitable not only for the modeling of the mechanical behavior of the cell membrane, but also for the application in blood rheology, such as in evaluating the relationship between pathology and cell morphology. Sea urchin eggs have also been the targets of membrane mechanical studies (for example, Hiramoto, 1963, 1970, 1976) because of the motivation, in part, to understand the fertilization and division of cells. These studies have been aimed at the determination of the membrane stress–strain relationship and the viscoelastic properties of cells, and the improvement of our understanding of the physical aspects of cells that are subjected to mechanical stresses. However, little is known about cell lysis by mechanical deformation. This was the motivation of our previous studies on the viability of deformed cells (Takamatsu and Rubinsky,
ARTICLE IN PRESS 118
H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
1999; Takamatsu and Kumagae, 2002), where cells were compressed by two parallel plates, which mimics the situation of freezing observed under a directional solidification microscope. Cell survival was determined as a function of gap size between two parallel plates by a new experimental technique. About one-half of the human prostate cancer cell population was destroyed when they were deformed in a gap size of about 30% of their original diameter. The other interesting result obtained from these studies was that the relationships between cell viability and gap size at 0 C and 23 C, i.e., above and below the lipid-phase-transition temperature of the cell membrane, respectively, were identical, while the cell ability to withstand deformation decreased at 37 C. This suggests that the deformation damage is related not strongly to the mechanical properties of membrane lipids but to the deformation of other components such as the cytoskeleton. However, more experiments are required to understand the role of the cytoskeleton in cell lysis by mechanical deformation. To this end, experiments using extracellular solutions of modified osmolality are designed. Since cell volume is a function of solution osmolality, cells are deformed to a different extent in a given gap size between two plates depending on the osmolality of the solution. Experiments under hypertonic conditions also have important implications regarding freezing because cells are exposed to the extracellular solution of increased osmolality during freezing. It is known that alterations in the osmotic environment induce many cellular responses concomitant with the change in cell volume. In many types of cells, osmotic stress alters the transmembrane mobilization of osmotically active solutes and cytoskeletal organization through a variety of events in signal transduction (Sarkadi and Parker, 1991; Beck et al., 1998; Pedersen et al., 2001). After hypotonic stress, F-actin, which is localized in a narrow region of the cell cortex under isotonic conditions, is distributed throughout the cell and F-actin content decreases (Cornet et al., 1994; Pedersen et al., 1999; Guilak et al., 2002; Erickson et al., 2003). In contrast, hypertonic NaCl induces an increase in F-actin content or the peripheral accumulation of Factin, leading to a higher actin filament density at the early stage after osmotic shrinkage (Pedersen et al., 1999; Di Ciano et al., 2002; Bustamante et al., 2003). However, changes in F-actin content and F-actin organization are time-dependent (Erickson et al., 2003; Bustamante et al., 2003) and are considered to be associated with cell volume regulation processes, such as the recovery of cell volume after hypotonic exposure by the processes of regulatory volume decrease and regulatory volume increase after hypertonic exposure. Probably due to the dissociation or redistribution of the F-actin cytoskeleton, the mechanical properties of cells are influenced by the osmotic environment (Guilak et al., 2002; Pritchard et al., 2002).
The objectives of this study are to quantify the dependence of the viability of deformed cells on extracellular osmolality and to examine the role of the cytoskeleton in cell lysis by deformation. Cultured human prostatic adenocarcinoma cells in a solution of increased or decreased NaCl concentration were deformed in a gap between two parallel plates and the cell viability was measured as a function of gap size.
2. Materials and methods 2.1. Sample preparation Human prostatic adenocarcinoma cells (PC-3) were cultured in 25 cm2 flasks using minimum essential medium (Gibco) with Eagles’ salt, 10% fetal bovine serum (Gibco), 100 units/ml penicillin (Gibco), and 100 mg/ml streptomycin (Gibco), as previously described (Takamatsu and Kumagae, 2002). The cells were harvested by treatment with trypsin (0.05% trypsin, 0.53 mM EDTA 4Na, Gibco), and resuspended in a solution containing the indicated concentration of NaCl and 20 mM HEPES, pH 7.4. 2.2. Deformation of cells The experimental technique was basically the same as that described in our previous paper (Takamatsu and Kumagae, 2002) except that propidium iodide (PI, Molecular Probes), instead of trypan blue, was used for the viability assay. First, a cell-glass bead suspension was prepared by mixing cells with glass beads of precisely known diameter in the NaCl solution that contained 15 mM PI. Then, one drop of the suspension was placed on a cover slip, and a glass plate was pushed toward the cover slip. A mechanical device with two parallel temperature-controlled stages was used to keep the two glass surfaces parallel during the compression process and to ensure that the procedure was performed at a precisely controlled temperature (Fig. 1). The glass beads, which were randomly distributed throughout the solution, behaved as spacers of precisely known dimensions. The cells became deformed between the two flat surfaces and the distance between these surfaces was determined from the known diameter of the glass beads. The glass plates with the compressed cells were then placed under a microscope (E600, Nikon). The stage of the microscope was also maintained at a controlled temperature. The phase-contrast and fluorescence images observed using a 10x objective at the same position were recorded using a digital camera (COOLPIX 995, Nikon). More than 10 pairs of images (between 10 and 15) were recorded from different positions.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
119
for 30 min at 23 C. PI was added to the cell-bead suspension 10 min prior to the deformation procedure. Experiments were performed at 23 C. 2.3. Determination of viability Cells were observed under the phase-contrast microscope, and dead cells were determined by staining with propidium iodide (PI) using the fluorescence microscope. The number of cells was counted from each image and the sum total of the cell number counted from all images was used to calculate cell viability. The ratio of the number of cells that have not taken up the dye to the total number of cells was calculated for five different gap sizes including that for the undeformed control. The value of the ratio at each gap size was normalized by the control value, which is the viability under iso-, hypo- or hypertonic condition depending on the solution used. 2.4. Measurement of cell size
Fig. 1. Experimental technique for observation of deformed cells. A drop of cell-glass bead suspension on a cover glass was compressed by a slide glass on a mechanical stage (a) and observed under a microscope (b).
Before the deformation experiment, cell size was determined as a function of osmolality. Cell size was measured by observing the cell suspension in NaCl solutions in the sandwich configuration with glass beads of 30.2 or 40.0 mm diameter, using a 40x objective. Five different concentrations of NaCl were tested: 500, 300, 150, 75 and 37.5 mM. Osmolality was measured using an osmometer (OM802, Vogel). A digital image analysis software (Image-Pro Plus, Media Cybernetics) was used to measure the projected area of living cells. The diameter and the volume of cells were calculated, assuming that the cells were spherical. 2.5. Observation of the actin cytoskeleton
In this study, glass beads of four different diameters (Duke Scientific Co.), i.e., 2.5 mm (standard deviation, SD 1.0 mm), 5.1 mm (SD 0.8 mm), 7.8 mm (SD 1.0 mm) and 10.4 mm (SD 1.0 mm), were used to give different gap sizes smaller than cell size. The viability of undeformed cells was measured with the glass beads 30.2 mm (SD 1.8 mm) in diameter at the beginning and the end of a series of experiments to ensure that the viability of the control did not decrease during the experimental procedure. Usually, the experiment with one size of beads was completed within 10 min after mixing the solution, and thus a series of experiments were finished within 1 h. Three different NaCl concentrations were tested: 150 mM NaCl for the isotonic condition, 75 mM for the hypotonic condition, and 700 mM for the hypertonic condition. To investigate the role of the actin cytoskeleton in cell lysis, 150 mM NaCl solution with 10 mM cytochalasin D (SIGMA) was also tested. In the experiment, cells were preincubated with each solution
PC-3 cells were trypsinized and resuspended in the 150 mM NaCl solution. Following the treatment with cytochalasin D (10 mM) for 30 min, the cells were fixed for 15 min in 3.7% formaldehyde. To visualize the actin cytoskeleton, phalloidin staining was performed as described previously (Noda et al., 2001). Briefly, the cells were permeabilized for 4 min in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) containing 0.1% Triton X-100, washed three times with PBS, and blocked with PBS containing 3% bovine serum albumin for 60 min. The samples were incubated with Texas RedX-conjugated phalloidin (Molecular Probes), and images were acquired using a laser confocal microscope (LSM5 PASCAL, Zeiss). 2.6. Statistical analysis A linear regression analysis was used to examine the relationship between the cell volume and the reciprocal
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
120
of the solution osmolality. A two-way analysis of variance (ANOVA) was used to test for the difference among the different osmolalities in the relationship between cell viability and gap size. 2.7. Calculation of cell strain The apparent surface area of the cell membrane increases as a response to the deformation to maintain cell volume. Cell strain was estimated with the assumption that a spherical cell is compressed by two parallel plates. When a cell of diameter d0 before deformation is squeezed in a gap of h (Fig. 2), increases in the surface area A and the length l along the cell perimeter of diameter d are estimated using 2 2 A A0 p 2 h 1 d0 ¼ þ þ 3 h A0 16 3 d0 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ( )ffi u 3 2 p h 2u p 2 d 0 t þ ð1Þ 1 1 þ 4 d0 16 3 h and l l 0 d d0 ¼ l0 d0 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 s p h p2 2 h 2 d0 ; ð2Þ ¼ 1 þ þ 4 d0 3 h 16 3 d0 respectively, where the subscript 0 denotes the value at the initial state before deformation. In the estimation, the vertical contour of the cells was assumed to be a semicircle of radius h=2: The initial diameter of the cells in different osmolalities was given by the regression line that correlates cell volume as a function of inverse osmolality.
3. Results Cell size decreased monotonically in response to an increase in extracellular osmolality. Cell volume followed the classical Boyle-van’t Hoff behavior and was expressed by a linear regression correlation between cell volume and inverse osmolality (R2 ¼ 0:99) (Fig. 3). The mean diameter under the isotonic condition (17.8 mm) agreed well with that determined in our previous paper (Takamatsu and Kumagae, 2002) from 1463 cells (17.5 mm). In the phase-contrast micrographs observed using a 10x objective, which were used to count cells, all cells were clearly distinguishable (Figs. 4(a) and (c)). Dead cells were identified in the fluorescence image, and the number of dead cells increased with decreasing gap size (Figs. 4(b) and (d)). Cell viability was characterized as a function of gap size. Following our previous studies (Takamatsu and Rubinsky, 1999; Takamatsu and Kumagae, 2002), nominal gap size was taken to be the sum of the mean glass-bead diameter and one SD, taking into account the scatter in the size of glass beads. There was no significant difference between the measured viability of the undeformed control of 150 mM NaCl solution (9173%, n ¼ 6) and those of 75 mM NaCl solution (8973%, n ¼ 6; p ¼ 0:37; t-test) and 700 mM NaCl solution (9075%, n ¼ 6; p ¼ 0:58; t-test). The measured viability of the deformed cells was normalized by that of the undeformed control to reveal only the effect of deformation, and the normalized value is referred to as ‘cell viability’ in the following results and discussion. It has been confirmed by the preliminary experiment that the viability assay for the 150 mM NaCl solution using PI reproduced almost the same result as that of the
8000
d0
d l0
l top view
Cell volume µm3
7000 6000 5000 4000 3000 2000 1000
d0 h side view Fig. 2. Model for estimating cell strain by deformation. A spherical cell of diameter d0 was compressed and held in gap h between two parallel plates. The diameter of the contour in the top view increased from d0 to d; inducing the strain on the cell surface because of the increase in peripheral length from l0 to l:
0 0
2
4 1/Osmolality
6
8
10
12
(Osm/kg-water)-1
Fig. 3. Cell volume as a function of the reciprocal of osmolalities. A linear relationship between cell volume and inverse osmolality (Boylevan’t Hoff relationship) was observed and correlated by a regression line (R2 ¼ 0:99). Each data point represents the mean SD of n=477– 584 cells.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
Fig. 4. Examples of micrographs used to count cells. Undeformed cells in the control (a,b) and deformed cells in a B6-mm gap (c,d). Micrographs (a) and (c) were observed under the phase-contrast microscope, and (b) and (d) under the fluorescence microscope. All micrographs were observed using a 10 objective.
Fig. 5. Relationship between cell viability and gap size for different NaCl concentrations. An increase in NaCl concentration caused an increase in the viability from 75 to 150 mM (po0:001; ANOVA) and from 150 to 700 mM (po0:001; ANOVA). Each data point represents the mean 7SD of the viability (n ¼ 6) and is plotted at the nominal gap size that was assumed to be the mean + SD of glass-bead diameter. Horizontal error bars indicate the SD of glass-bead diameter.
previous study (Takamatsu and Kumagae, 2002) using trypan blue (p > 0:09; ANOVA, data not shown). There was a significant increase in the viability with increase in NaCl concentration from 75 to 150 mM (po0:001; ANOVA) and from 150 to 700 mM (p o 0.001, ANOVA) (Fig. 5). The mean diameters of the cells obtained from the linear regression line in Fig. 3 were 20.5 mm in the 75 mM solution, 17.8 mm in the 150 mM solution, and 15.4 mm in the 700 mM solution. Therefore, the increase in the viability at higher tonicity was probably due to the decrease in cell diameter with
121
Fig. 6. Cell viability as a function of gap size normalized by cell diameter before deformation. The data for different NaCl concentrations fell within a narrow band of the relationship between the viability and the normalized gap size.
increasing NaCl concentration, because smaller cells were less deformed than larger cells in a given gap size. This was confirmed by Fig. 6, where the viability was replotted as a function of the gap size normalized by the mean diameter of cells in each solution. The data for different concentrations showed a similar relationship between cell viability and the normalized gap size. When a spherical cell is compressed by two parallel plates, cell surface area increases to maintain cell volume. The dilatation of the surface area and the strain on the cell perimeter, which were estimated by Eqs. (1) and (2), respectively, are functions of the normalized gap size as shown in Fig. 7. From this relationship, the change in the strain on the cell perimeter with gap size is estimated for the cells of different diameters in different osmolalities (Fig. 8). The surface of a cell 17.8 mm in diameter, which is typical under the isotonic condition, is stretched by 48% in a 6-mm gap that destroys about one-half of the cell population. The difference in the strain between the cells 15.4 and 20.5 mm in diameters, which represents cells in the 700 and 75 mM NaCl solution, respectively, is 18% in the 6-mm gap. If the viability was replotted against the strain on the cell surface, the experimental data for all the solutions correlated with the unique relationship between the viability and the strain irrespective of NaCl concentration (Fig. 9). Fig. 9 indicates that the increase in cell surface area is crucial to the damage caused by deformation. Our previous study suggests that the deformation damage is not strongly related to the mechanical properties of membrane lipids. This raises the possibility that other components of the plasma membrane or submembranous structures such as the cytoskeleton are involved in the deformation damage. To test the role of the actin cytoskeleton in cell damage, we used cytochalasin D, an
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
122
100
surface area
150
75 mM 150 mM 700 mM
80 Viability %
Increase by deformation %
200
100 peripheral length 50
60
40
20
0
0 0
0.2
0.4
0.6
0.8
1
0
Gap size / Cell diameter before deformation Fig. 7. Percent increase in the surface area and the length of the perimeter of a cell as a function of the normalized gap size that is defined by the gap size divided by the original cell diameter before deformation. The percent increase in the cell peripheral length is equivalent to the largest strain in length on the cell surface. Both the surface area and the peripheral length increased at an increasing rate with decreasing gap size.
20
40 60 80 Strain on cell surface %
100
120
Fig. 9. Cell viability as a function of the strain on the cell peripheral length. The viability decreased monotonically with increasing strain on the cell surface and correlated with the unique relationship irrespective of NaCl concentration.
Strain on cell surface %
200
cell diameter 150
20.5 µm (75 mM)) 17.8 µm (150 mM)
100 15.4 µm (700 mM) Fig. 10. Fluorescence images observed under a laser confocal microscope. Actin filaments were observed at the circumference of cells in the control suspension (a), but disappeared in the cells treated with cytochalasin D (b).
50
0 0
5
10
15
20
Gap size µm Fig. 8. Change in strain on the cell peripheral length with gap size. A larger strain was imposed on cells in the lower osmolality than in the higher osmolality in the same gap size because of the difference in initial cell volume.
inhibitor of actin polymerization, in the 150 mM NaCl solution. A laser confocal micrograph of a slice image at the center of PC-3 cells stained with phalloidin confirmed that the treatment of cells with cytochalasin D leads to a marked decrease in cortical actin filament density, while most actin filaments in untreated cells are located at the cell cortex, as known in other types of isolated cells (Fig. 10). The viability dropped significantly at a gap size lower than 6 mm in the presence of the reagent (po0:001; ANOVA) (Fig. 11). This indicates that the loss of cytoskeletal integrity plays a crucial role in cell damage by deformation.
4. Discussion The susceptibility of cells to mechanical stress is important in the field of biomechanics. Even if the mechanical properties of cells and tissues usually depend on the extracellular matrix and the cell-to-cell interaction, an isolated cell is a base of all cellular phenomena. While micropipette aspiration has often been used to apply force to a cell (Hochmuth, 2000), cell compression by two parallel plates has been used by Hiramoto (1963) to measure the mechanical properties of sea urchin eggs in the studies of cellular phenomena. They measured the force and the displacement of the plates to obtain the elastic moduli of the cell membrane. All the mechanical studies, including those of Hiramoto (1963), have aimed at the determination of the mechanical properties of a cell within the range of deformation that allows cells to recover, but provided no information about the irreversible deformation.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
100 n=6
Viability %
80
60
40
20
Cyto-D Cyto-D +
0 0
5
10
15
Gap size µm Fig. 11. Effect of cytochalasin D on the relationship between cell viability and gap size. The viability of cells treated with cytochalasin D dropped significantly particularly in the gap of B6 mm as compared with untreated cells (po0:001; ANOVA).
A series of our studies show that one-half of the cell population survives the deformation in a gap size of about 30% of the mean diameter of the cells (Takamatsu and Rubinsky, 1999; Takamatsu and Kumagae, 2002). This degree of deformation may lead to an increase of about 40% in cell surface area. Since the critical surface area increase of the lipid bilayer is only by a few percentage (Bloom et al., 1991), a much larger expansion of the cell surface by deformation might be due to microvilli, blebs or other types of reservoirs of membrane materials. The other interesting result of our previous study (Takamatsu and Kumagae, 2002) is that there is no significant difference in the viability of deformed cells between 0 C and 23 C, which are probably below and above the phase-transition temperature of the cell-membrane lipid bilayer, respectively (Crowe et al., 1999). This implies that the damage is related not to the mechanical properties of membrane lipids but to those of other components such as the cytoskeleton. The present finding of a significant drop in the viability by the addition of cytochalasin D (Fig. 11) supports the idea that the integrity of the actin cytoskeleton plays an important role in the deformation damage. From the present results, we may hypothesize that cells lyse when the cell surface expands beyond the allowable strain. The critical strain is that which is determined only by deformation and does not include that induced by swelling or shrinkage due to a change in osmolality. Figure 9 indicates that the strain on the cell surface that destroys one-half of the cell population is about 55%. The percent increase in the area of the cell surface shown in Fig. 7 is a quantitative index of surface expansion. It was estimated by neglecting the friction between the cell and the glass surface during the compression process, and assuming that the expansion
123
is uniform. However, the extent of actual expansion might vary depending on the position on the cell surface, and the largest degree of local expansion is probably larger than the average value. On the other hand, the strain on the cell surface, i.e., the percent increase in cell perimeter, represents the maximal stretching of the cell surface. Therefore, the strain on the cell perimeter rather than the dilatation of the surface area was used to examine the critical expansion that results in cell lysis in the present paper. However, the effect of the twodimensional stretching of the cell surface should be evaluated in a future work. NaCl concentration influences cell viability in terms of the change in cell volume during the freezing of cells. The present study shows that it is sufficient to simply consider the osmotic volume change for cell lysis by deformation. However, since cells are subjected to much higher tonicities than the present experimental conditions during freezing, further study using higher NaCl concentrations is required. The other important difference between the present experiment and the situation in the freezing process is the rate of deformation; the deformation is instantaneous in the present experiment but it is much slower in the freezing process. In our previous paper, we showed that the uniform expansion of cells under hypotonic solutions up to the same magnitude of surface area increase as that by deformation was much less crucial to cell lysis (Takamatsu and Kumagae, 2002). The increase in cell volume due to extracellular hypotonic solutions depends on the membrane permeability of cells, and is much slower than the compression in the present experiments. This suggests the possibility that the deformation damage is ratedependent, which is obviously an important issue to understand the mechanism of cell lysis by deformation.
Acknowledgements This study was partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 11650227 and 15360115). The authors thank Ms. Nichika Kouno and Ms. Yuko Fukuda for their contribution to the experiments.
References . Beck, F.X., Burger-Kentischer, A., Muller, E., 1998. Cellular response to osmotic stress in the renal medulla. Pflugers . Archiv—European Journal of Physiology 436, 814–827. Bloom, M., Evans, E., Mouritsen, O.G., 1991. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Quarterly Reviews of Biophysics 24, 293–397. Bustamante, M., Roger, F., Bochaton-Piallat, M.-L., Gabbiani, G., Martin, P.-Y., F!eraille, E., 2003. Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton
ARTICLE IN PRESS 124
H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
remodeling in rat kidney MTAL. American Journal of Physiology—Renal Physiology 285, F336–F347. . Chien, S., Sung, K.-L.P., Skalak, R., Usami, S., Tozeren, A., 1978. Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane. Biophysical Journal 24, 463–487. Cornet, M., Isobe, Y., Lemanski, L.F., 1994. Effects of anisosmotic conditions on the cytoskeletal architecture of cultured PC12 cells. Journal of Morphology 222, 269–286. Crowe, J.H., Tablin, F., Tsvetkova, N., Oliver, A.E., 1999. Are lipid transitions responsible for chilling damage in human platelets? Cryobiology 38, 180–191. Di Ciano, C., Nie, Z., Sz!aszi, K., Lewis, A., Uruno, T., Zhan, X., Rotstein, O.D., Mak, A., Kapus, A., 2002. Osmotic stress-induced remodeling of the cortical cytoskeleton. American Journal of Physiology—Cell Physiology 283, C850–C865. Evans, E.A., Skalak, R., 1980. Mechanics and Thermodynamics of Membranes. CRC Press, FL. Evans, E.A., Waugh, R., Melnik, L., 1976. Elastic area compressibility modulus of red-cell membrane. Biophysical Journal 16, 585–595. Erickson, G.R.D., Northrup, L., Guilak, F., 2003. Hypo-osmotic stress induces calcium-dependent actin reorganization in articular chondrocytes. OsteoArthritis and Cartilage 11, 187–197. Guilak, F., Erickson, G.R., Ting-Beall, H.P., 2002. The effect of osmotic stress on the viscoelastic and physical properties of articular chondrocytes. Biophysical Journal 82, 720–727. Hiramoto, Y., 1963. Mechanical properties of sea urchin eggs. I. Surface force and elastic modulus of cell membrane. Experimental Cell Research 32, 59–75. Hiramoto, Y., 1970. Rheological properties of sea urchin eggs. Biorheology 6, 201–234. Hiramoto, Y., 1976. Mechanical properties of sea urchin eggs. III. Visco-elasticity of cell surface. Development Growth and Differentiation 18, 377–386. Hochmuth, R.M., 1993. Measuring the mechanical properties of individual human blood cells. Journal of Biomechanical Engineering 155, 515–519. Hochmuth, R.M., 2000. Micropipette aspiration of living cells. Journal of Biomechanics 33, 15–22. Ishiguro, H., Rubinsky, B., 1994. Mechanical interactions between ice crystals and red blood cells during directional solidification. Cryobiology 31, 483–500.
Mazur, P., Rigopoulos, N., 1983. Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: influence of warming rates. Cryobiology 20, 274–289. Mazur, P., Rall, W.F., Rigopoulos, N., 1981. Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophysical Journal 36, 653–665. Nei, T., 1967a. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. I. Microscopic observation of hemolyzing erythrocytes during the freezing and thawing process. Cryobiology 4, 153–156. Nei, T., 1967b. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. II. Investigations of factors affecting hemolysis by freezing. Cryobiology 4, 303–308. Noda, Y., Takeya, R., Ohno, S., Naito, S., Ito, T., Sumimoto, H., 2001. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C. Genes to Cells 6, 107–119. Pedersen, S.F., Mills, J.W., Hoffmann, E.K., 1999. Role of the F-actin cytoskeleton in the RVD and RVI processes in ehrich ascites tumor cells. Experimental Cell Research 252, 63–74. Pedersen, S.F., Hoffmann, E.K., Mills, J.W., 2001. The cytoskeleton and cell volume regulation. Comparative Biochemistry and Physiology—Part A 130, 385–399. Pritchard, S., Erickson, G.R., Guilak, F., 2002. Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton. Biophysical Journal 83, 2050–2510. Rand, R.P., Burton, A.C., 1964. Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure. Biophysical Journal 4, 115–135. Sarkadi, B., Parker, J.C., 1991. Activation of ion transport pathways by changes in cell volume. Biochimica et Biophysica Acta 1071, 407–427. Takamatsu, H., Rubinsky, B., 1999. Viability of deformed cells. Cryobiology 39, 243–251. Takamatsu, H., Kumagae, N., 2002. Survival of biological cells deformed in a narrow gap. Journal of Biomechanical Engineering 124, 780–783. . Tozeren, A., Skalak, R., Sung, K.-L.P., Chien, S., 1982. Viscoelastic behavior of erythrocyte membrane. Biophysical Journal 39, 23–32.
Journal of Biomechanics 38 (2005) 117–124
On the mechanism of cell lysis by deformation Hiroshi Takamatsua,*, Ryu Takeyab, Seiji Naitoc, Hideki Sumimotob,d a
Institute for Materials Chemistry and Engineering, Kyushu University, 6-1 Kasugakoen, Kasuga, Fukuoka 816-8580, Japan b Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan c Department of Urology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan d CREST, JST, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan Accepted 14 March 2004
Abstract In this study, we identify the extent of deformation that causes cell lysis using a simple technique where a drop of cell suspension is compressed by two flat plates. The viability of human prostatic adenocarcinoma PC-3 cells in solutions of various concentrations of NaCl is determined as a function of the gap size between the plates. The viability declines with decreasing gap size in the following order: 700 mM>150 mM>75 mM NaCl. This is considered to be due to the difference in cell size, which is caused by the osmotic volume change before deformation; cell diameter becomes smaller in a solution of higher NaCl concentration, which appears to increase the survival ratio in a given gap size. The deformation-induced decrease in cell viability is correlated with the cell surface strain, which is dependent on the increase in surface area, irrespective of NaCl concentration. In addition, the treatment of cells with cytochalasin D results in the disappearance of cortical actin filaments and a marked drop in the viability, indicating that cell lysis is closely related to the deformation of the cytoskeleton. r 2004 Elsevier Ltd. All rights reserved. Keywords: Cell viability; Mechanical stress; Cell compression; Osmotic volume change; Cytoskeleton
1. Introduction During freezing, mammalian cells are subjected to a mechanical stress by extracellular ice crystals. Although the increased solute concentration during freezing has been considered to be the major cause of cell injury, several experiments indicate that cell deformation by mechanical stress is important for cell lysis. The observation of the freezing of cell suspensions under a cryomicroscope shows that the cells are left in the unfrozen solution and appear to be compressed by the surrounding ice crystals (Nei, 1967a,b; Ishiguro and Rubinsky, 1994). Cell survival depends on the volume fraction of the unfrozen solution at the freezing temperature (Mazur et al., 1981; Mazur and Rigopoulos, 1983), suggesting that more cells are lysed in smaller spaces. The information about cell lysis by mechanical stress is therefore important in understanding the mechanism of cell injury during freezing. *Corresponding author. Tel.: +81-92-583-7788; fax: +81-92-5837882. E-mail address: [email protected] (H. Takamatsu). 0021-9290/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jbiomech.2004.03.011
There are a number of studies that deal with the effect of mechanical stress on cells (Evans and Skalak, 1980; Bloom et al., 1991; Hochmuth, 2000). Red blood cells have been studied extensively (for example, Rand and Burton, 1964; Evans et al., 1976; Chien et al., 1978; . Tozeren et al., 1982; Hochmuth, 1993) because their membrane complex is distinctively separated from their cytoplasm, and the cells are suitable not only for the modeling of the mechanical behavior of the cell membrane, but also for the application in blood rheology, such as in evaluating the relationship between pathology and cell morphology. Sea urchin eggs have also been the targets of membrane mechanical studies (for example, Hiramoto, 1963, 1970, 1976) because of the motivation, in part, to understand the fertilization and division of cells. These studies have been aimed at the determination of the membrane stress–strain relationship and the viscoelastic properties of cells, and the improvement of our understanding of the physical aspects of cells that are subjected to mechanical stresses. However, little is known about cell lysis by mechanical deformation. This was the motivation of our previous studies on the viability of deformed cells (Takamatsu and Rubinsky,
ARTICLE IN PRESS 118
H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
1999; Takamatsu and Kumagae, 2002), where cells were compressed by two parallel plates, which mimics the situation of freezing observed under a directional solidification microscope. Cell survival was determined as a function of gap size between two parallel plates by a new experimental technique. About one-half of the human prostate cancer cell population was destroyed when they were deformed in a gap size of about 30% of their original diameter. The other interesting result obtained from these studies was that the relationships between cell viability and gap size at 0 C and 23 C, i.e., above and below the lipid-phase-transition temperature of the cell membrane, respectively, were identical, while the cell ability to withstand deformation decreased at 37 C. This suggests that the deformation damage is related not strongly to the mechanical properties of membrane lipids but to the deformation of other components such as the cytoskeleton. However, more experiments are required to understand the role of the cytoskeleton in cell lysis by mechanical deformation. To this end, experiments using extracellular solutions of modified osmolality are designed. Since cell volume is a function of solution osmolality, cells are deformed to a different extent in a given gap size between two plates depending on the osmolality of the solution. Experiments under hypertonic conditions also have important implications regarding freezing because cells are exposed to the extracellular solution of increased osmolality during freezing. It is known that alterations in the osmotic environment induce many cellular responses concomitant with the change in cell volume. In many types of cells, osmotic stress alters the transmembrane mobilization of osmotically active solutes and cytoskeletal organization through a variety of events in signal transduction (Sarkadi and Parker, 1991; Beck et al., 1998; Pedersen et al., 2001). After hypotonic stress, F-actin, which is localized in a narrow region of the cell cortex under isotonic conditions, is distributed throughout the cell and F-actin content decreases (Cornet et al., 1994; Pedersen et al., 1999; Guilak et al., 2002; Erickson et al., 2003). In contrast, hypertonic NaCl induces an increase in F-actin content or the peripheral accumulation of Factin, leading to a higher actin filament density at the early stage after osmotic shrinkage (Pedersen et al., 1999; Di Ciano et al., 2002; Bustamante et al., 2003). However, changes in F-actin content and F-actin organization are time-dependent (Erickson et al., 2003; Bustamante et al., 2003) and are considered to be associated with cell volume regulation processes, such as the recovery of cell volume after hypotonic exposure by the processes of regulatory volume decrease and regulatory volume increase after hypertonic exposure. Probably due to the dissociation or redistribution of the F-actin cytoskeleton, the mechanical properties of cells are influenced by the osmotic environment (Guilak et al., 2002; Pritchard et al., 2002).
The objectives of this study are to quantify the dependence of the viability of deformed cells on extracellular osmolality and to examine the role of the cytoskeleton in cell lysis by deformation. Cultured human prostatic adenocarcinoma cells in a solution of increased or decreased NaCl concentration were deformed in a gap between two parallel plates and the cell viability was measured as a function of gap size.
2. Materials and methods 2.1. Sample preparation Human prostatic adenocarcinoma cells (PC-3) were cultured in 25 cm2 flasks using minimum essential medium (Gibco) with Eagles’ salt, 10% fetal bovine serum (Gibco), 100 units/ml penicillin (Gibco), and 100 mg/ml streptomycin (Gibco), as previously described (Takamatsu and Kumagae, 2002). The cells were harvested by treatment with trypsin (0.05% trypsin, 0.53 mM EDTA 4Na, Gibco), and resuspended in a solution containing the indicated concentration of NaCl and 20 mM HEPES, pH 7.4. 2.2. Deformation of cells The experimental technique was basically the same as that described in our previous paper (Takamatsu and Kumagae, 2002) except that propidium iodide (PI, Molecular Probes), instead of trypan blue, was used for the viability assay. First, a cell-glass bead suspension was prepared by mixing cells with glass beads of precisely known diameter in the NaCl solution that contained 15 mM PI. Then, one drop of the suspension was placed on a cover slip, and a glass plate was pushed toward the cover slip. A mechanical device with two parallel temperature-controlled stages was used to keep the two glass surfaces parallel during the compression process and to ensure that the procedure was performed at a precisely controlled temperature (Fig. 1). The glass beads, which were randomly distributed throughout the solution, behaved as spacers of precisely known dimensions. The cells became deformed between the two flat surfaces and the distance between these surfaces was determined from the known diameter of the glass beads. The glass plates with the compressed cells were then placed under a microscope (E600, Nikon). The stage of the microscope was also maintained at a controlled temperature. The phase-contrast and fluorescence images observed using a 10x objective at the same position were recorded using a digital camera (COOLPIX 995, Nikon). More than 10 pairs of images (between 10 and 15) were recorded from different positions.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
119
for 30 min at 23 C. PI was added to the cell-bead suspension 10 min prior to the deformation procedure. Experiments were performed at 23 C. 2.3. Determination of viability Cells were observed under the phase-contrast microscope, and dead cells were determined by staining with propidium iodide (PI) using the fluorescence microscope. The number of cells was counted from each image and the sum total of the cell number counted from all images was used to calculate cell viability. The ratio of the number of cells that have not taken up the dye to the total number of cells was calculated for five different gap sizes including that for the undeformed control. The value of the ratio at each gap size was normalized by the control value, which is the viability under iso-, hypo- or hypertonic condition depending on the solution used. 2.4. Measurement of cell size
Fig. 1. Experimental technique for observation of deformed cells. A drop of cell-glass bead suspension on a cover glass was compressed by a slide glass on a mechanical stage (a) and observed under a microscope (b).
Before the deformation experiment, cell size was determined as a function of osmolality. Cell size was measured by observing the cell suspension in NaCl solutions in the sandwich configuration with glass beads of 30.2 or 40.0 mm diameter, using a 40x objective. Five different concentrations of NaCl were tested: 500, 300, 150, 75 and 37.5 mM. Osmolality was measured using an osmometer (OM802, Vogel). A digital image analysis software (Image-Pro Plus, Media Cybernetics) was used to measure the projected area of living cells. The diameter and the volume of cells were calculated, assuming that the cells were spherical. 2.5. Observation of the actin cytoskeleton
In this study, glass beads of four different diameters (Duke Scientific Co.), i.e., 2.5 mm (standard deviation, SD 1.0 mm), 5.1 mm (SD 0.8 mm), 7.8 mm (SD 1.0 mm) and 10.4 mm (SD 1.0 mm), were used to give different gap sizes smaller than cell size. The viability of undeformed cells was measured with the glass beads 30.2 mm (SD 1.8 mm) in diameter at the beginning and the end of a series of experiments to ensure that the viability of the control did not decrease during the experimental procedure. Usually, the experiment with one size of beads was completed within 10 min after mixing the solution, and thus a series of experiments were finished within 1 h. Three different NaCl concentrations were tested: 150 mM NaCl for the isotonic condition, 75 mM for the hypotonic condition, and 700 mM for the hypertonic condition. To investigate the role of the actin cytoskeleton in cell lysis, 150 mM NaCl solution with 10 mM cytochalasin D (SIGMA) was also tested. In the experiment, cells were preincubated with each solution
PC-3 cells were trypsinized and resuspended in the 150 mM NaCl solution. Following the treatment with cytochalasin D (10 mM) for 30 min, the cells were fixed for 15 min in 3.7% formaldehyde. To visualize the actin cytoskeleton, phalloidin staining was performed as described previously (Noda et al., 2001). Briefly, the cells were permeabilized for 4 min in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, and 1.5 mM KH2PO4, pH 7.4) containing 0.1% Triton X-100, washed three times with PBS, and blocked with PBS containing 3% bovine serum albumin for 60 min. The samples were incubated with Texas RedX-conjugated phalloidin (Molecular Probes), and images were acquired using a laser confocal microscope (LSM5 PASCAL, Zeiss). 2.6. Statistical analysis A linear regression analysis was used to examine the relationship between the cell volume and the reciprocal
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
120
of the solution osmolality. A two-way analysis of variance (ANOVA) was used to test for the difference among the different osmolalities in the relationship between cell viability and gap size. 2.7. Calculation of cell strain The apparent surface area of the cell membrane increases as a response to the deformation to maintain cell volume. Cell strain was estimated with the assumption that a spherical cell is compressed by two parallel plates. When a cell of diameter d0 before deformation is squeezed in a gap of h (Fig. 2), increases in the surface area A and the length l along the cell perimeter of diameter d are estimated using 2 2 A A0 p 2 h 1 d0 ¼ þ þ 3 h A0 16 3 d0 vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ( )ffi u 3 2 p h 2u p 2 d 0 t þ ð1Þ 1 1 þ 4 d0 16 3 h and l l 0 d d0 ¼ l0 d0 ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 2 s p h p2 2 h 2 d0 ; ð2Þ ¼ 1 þ þ 4 d0 3 h 16 3 d0 respectively, where the subscript 0 denotes the value at the initial state before deformation. In the estimation, the vertical contour of the cells was assumed to be a semicircle of radius h=2: The initial diameter of the cells in different osmolalities was given by the regression line that correlates cell volume as a function of inverse osmolality.
3. Results Cell size decreased monotonically in response to an increase in extracellular osmolality. Cell volume followed the classical Boyle-van’t Hoff behavior and was expressed by a linear regression correlation between cell volume and inverse osmolality (R2 ¼ 0:99) (Fig. 3). The mean diameter under the isotonic condition (17.8 mm) agreed well with that determined in our previous paper (Takamatsu and Kumagae, 2002) from 1463 cells (17.5 mm). In the phase-contrast micrographs observed using a 10x objective, which were used to count cells, all cells were clearly distinguishable (Figs. 4(a) and (c)). Dead cells were identified in the fluorescence image, and the number of dead cells increased with decreasing gap size (Figs. 4(b) and (d)). Cell viability was characterized as a function of gap size. Following our previous studies (Takamatsu and Rubinsky, 1999; Takamatsu and Kumagae, 2002), nominal gap size was taken to be the sum of the mean glass-bead diameter and one SD, taking into account the scatter in the size of glass beads. There was no significant difference between the measured viability of the undeformed control of 150 mM NaCl solution (9173%, n ¼ 6) and those of 75 mM NaCl solution (8973%, n ¼ 6; p ¼ 0:37; t-test) and 700 mM NaCl solution (9075%, n ¼ 6; p ¼ 0:58; t-test). The measured viability of the deformed cells was normalized by that of the undeformed control to reveal only the effect of deformation, and the normalized value is referred to as ‘cell viability’ in the following results and discussion. It has been confirmed by the preliminary experiment that the viability assay for the 150 mM NaCl solution using PI reproduced almost the same result as that of the
8000
d0
d l0
l top view
Cell volume µm3
7000 6000 5000 4000 3000 2000 1000
d0 h side view Fig. 2. Model for estimating cell strain by deformation. A spherical cell of diameter d0 was compressed and held in gap h between two parallel plates. The diameter of the contour in the top view increased from d0 to d; inducing the strain on the cell surface because of the increase in peripheral length from l0 to l:
0 0
2
4 1/Osmolality
6
8
10
12
(Osm/kg-water)-1
Fig. 3. Cell volume as a function of the reciprocal of osmolalities. A linear relationship between cell volume and inverse osmolality (Boylevan’t Hoff relationship) was observed and correlated by a regression line (R2 ¼ 0:99). Each data point represents the mean SD of n=477– 584 cells.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
Fig. 4. Examples of micrographs used to count cells. Undeformed cells in the control (a,b) and deformed cells in a B6-mm gap (c,d). Micrographs (a) and (c) were observed under the phase-contrast microscope, and (b) and (d) under the fluorescence microscope. All micrographs were observed using a 10 objective.
Fig. 5. Relationship between cell viability and gap size for different NaCl concentrations. An increase in NaCl concentration caused an increase in the viability from 75 to 150 mM (po0:001; ANOVA) and from 150 to 700 mM (po0:001; ANOVA). Each data point represents the mean 7SD of the viability (n ¼ 6) and is plotted at the nominal gap size that was assumed to be the mean + SD of glass-bead diameter. Horizontal error bars indicate the SD of glass-bead diameter.
previous study (Takamatsu and Kumagae, 2002) using trypan blue (p > 0:09; ANOVA, data not shown). There was a significant increase in the viability with increase in NaCl concentration from 75 to 150 mM (po0:001; ANOVA) and from 150 to 700 mM (p o 0.001, ANOVA) (Fig. 5). The mean diameters of the cells obtained from the linear regression line in Fig. 3 were 20.5 mm in the 75 mM solution, 17.8 mm in the 150 mM solution, and 15.4 mm in the 700 mM solution. Therefore, the increase in the viability at higher tonicity was probably due to the decrease in cell diameter with
121
Fig. 6. Cell viability as a function of gap size normalized by cell diameter before deformation. The data for different NaCl concentrations fell within a narrow band of the relationship between the viability and the normalized gap size.
increasing NaCl concentration, because smaller cells were less deformed than larger cells in a given gap size. This was confirmed by Fig. 6, where the viability was replotted as a function of the gap size normalized by the mean diameter of cells in each solution. The data for different concentrations showed a similar relationship between cell viability and the normalized gap size. When a spherical cell is compressed by two parallel plates, cell surface area increases to maintain cell volume. The dilatation of the surface area and the strain on the cell perimeter, which were estimated by Eqs. (1) and (2), respectively, are functions of the normalized gap size as shown in Fig. 7. From this relationship, the change in the strain on the cell perimeter with gap size is estimated for the cells of different diameters in different osmolalities (Fig. 8). The surface of a cell 17.8 mm in diameter, which is typical under the isotonic condition, is stretched by 48% in a 6-mm gap that destroys about one-half of the cell population. The difference in the strain between the cells 15.4 and 20.5 mm in diameters, which represents cells in the 700 and 75 mM NaCl solution, respectively, is 18% in the 6-mm gap. If the viability was replotted against the strain on the cell surface, the experimental data for all the solutions correlated with the unique relationship between the viability and the strain irrespective of NaCl concentration (Fig. 9). Fig. 9 indicates that the increase in cell surface area is crucial to the damage caused by deformation. Our previous study suggests that the deformation damage is not strongly related to the mechanical properties of membrane lipids. This raises the possibility that other components of the plasma membrane or submembranous structures such as the cytoskeleton are involved in the deformation damage. To test the role of the actin cytoskeleton in cell damage, we used cytochalasin D, an
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
122
100
surface area
150
75 mM 150 mM 700 mM
80 Viability %
Increase by deformation %
200
100 peripheral length 50
60
40
20
0
0 0
0.2
0.4
0.6
0.8
1
0
Gap size / Cell diameter before deformation Fig. 7. Percent increase in the surface area and the length of the perimeter of a cell as a function of the normalized gap size that is defined by the gap size divided by the original cell diameter before deformation. The percent increase in the cell peripheral length is equivalent to the largest strain in length on the cell surface. Both the surface area and the peripheral length increased at an increasing rate with decreasing gap size.
20
40 60 80 Strain on cell surface %
100
120
Fig. 9. Cell viability as a function of the strain on the cell peripheral length. The viability decreased monotonically with increasing strain on the cell surface and correlated with the unique relationship irrespective of NaCl concentration.
Strain on cell surface %
200
cell diameter 150
20.5 µm (75 mM)) 17.8 µm (150 mM)
100 15.4 µm (700 mM) Fig. 10. Fluorescence images observed under a laser confocal microscope. Actin filaments were observed at the circumference of cells in the control suspension (a), but disappeared in the cells treated with cytochalasin D (b).
50
0 0
5
10
15
20
Gap size µm Fig. 8. Change in strain on the cell peripheral length with gap size. A larger strain was imposed on cells in the lower osmolality than in the higher osmolality in the same gap size because of the difference in initial cell volume.
inhibitor of actin polymerization, in the 150 mM NaCl solution. A laser confocal micrograph of a slice image at the center of PC-3 cells stained with phalloidin confirmed that the treatment of cells with cytochalasin D leads to a marked decrease in cortical actin filament density, while most actin filaments in untreated cells are located at the cell cortex, as known in other types of isolated cells (Fig. 10). The viability dropped significantly at a gap size lower than 6 mm in the presence of the reagent (po0:001; ANOVA) (Fig. 11). This indicates that the loss of cytoskeletal integrity plays a crucial role in cell damage by deformation.
4. Discussion The susceptibility of cells to mechanical stress is important in the field of biomechanics. Even if the mechanical properties of cells and tissues usually depend on the extracellular matrix and the cell-to-cell interaction, an isolated cell is a base of all cellular phenomena. While micropipette aspiration has often been used to apply force to a cell (Hochmuth, 2000), cell compression by two parallel plates has been used by Hiramoto (1963) to measure the mechanical properties of sea urchin eggs in the studies of cellular phenomena. They measured the force and the displacement of the plates to obtain the elastic moduli of the cell membrane. All the mechanical studies, including those of Hiramoto (1963), have aimed at the determination of the mechanical properties of a cell within the range of deformation that allows cells to recover, but provided no information about the irreversible deformation.
ARTICLE IN PRESS H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
100 n=6
Viability %
80
60
40
20
Cyto-D Cyto-D +
0 0
5
10
15
Gap size µm Fig. 11. Effect of cytochalasin D on the relationship between cell viability and gap size. The viability of cells treated with cytochalasin D dropped significantly particularly in the gap of B6 mm as compared with untreated cells (po0:001; ANOVA).
A series of our studies show that one-half of the cell population survives the deformation in a gap size of about 30% of the mean diameter of the cells (Takamatsu and Rubinsky, 1999; Takamatsu and Kumagae, 2002). This degree of deformation may lead to an increase of about 40% in cell surface area. Since the critical surface area increase of the lipid bilayer is only by a few percentage (Bloom et al., 1991), a much larger expansion of the cell surface by deformation might be due to microvilli, blebs or other types of reservoirs of membrane materials. The other interesting result of our previous study (Takamatsu and Kumagae, 2002) is that there is no significant difference in the viability of deformed cells between 0 C and 23 C, which are probably below and above the phase-transition temperature of the cell-membrane lipid bilayer, respectively (Crowe et al., 1999). This implies that the damage is related not to the mechanical properties of membrane lipids but to those of other components such as the cytoskeleton. The present finding of a significant drop in the viability by the addition of cytochalasin D (Fig. 11) supports the idea that the integrity of the actin cytoskeleton plays an important role in the deformation damage. From the present results, we may hypothesize that cells lyse when the cell surface expands beyond the allowable strain. The critical strain is that which is determined only by deformation and does not include that induced by swelling or shrinkage due to a change in osmolality. Figure 9 indicates that the strain on the cell surface that destroys one-half of the cell population is about 55%. The percent increase in the area of the cell surface shown in Fig. 7 is a quantitative index of surface expansion. It was estimated by neglecting the friction between the cell and the glass surface during the compression process, and assuming that the expansion
123
is uniform. However, the extent of actual expansion might vary depending on the position on the cell surface, and the largest degree of local expansion is probably larger than the average value. On the other hand, the strain on the cell surface, i.e., the percent increase in cell perimeter, represents the maximal stretching of the cell surface. Therefore, the strain on the cell perimeter rather than the dilatation of the surface area was used to examine the critical expansion that results in cell lysis in the present paper. However, the effect of the twodimensional stretching of the cell surface should be evaluated in a future work. NaCl concentration influences cell viability in terms of the change in cell volume during the freezing of cells. The present study shows that it is sufficient to simply consider the osmotic volume change for cell lysis by deformation. However, since cells are subjected to much higher tonicities than the present experimental conditions during freezing, further study using higher NaCl concentrations is required. The other important difference between the present experiment and the situation in the freezing process is the rate of deformation; the deformation is instantaneous in the present experiment but it is much slower in the freezing process. In our previous paper, we showed that the uniform expansion of cells under hypotonic solutions up to the same magnitude of surface area increase as that by deformation was much less crucial to cell lysis (Takamatsu and Kumagae, 2002). The increase in cell volume due to extracellular hypotonic solutions depends on the membrane permeability of cells, and is much slower than the compression in the present experiments. This suggests the possibility that the deformation damage is ratedependent, which is obviously an important issue to understand the mechanism of cell lysis by deformation.
Acknowledgements This study was partly supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (Nos. 11650227 and 15360115). The authors thank Ms. Nichika Kouno and Ms. Yuko Fukuda for their contribution to the experiments.
References . Beck, F.X., Burger-Kentischer, A., Muller, E., 1998. Cellular response to osmotic stress in the renal medulla. Pflugers . Archiv—European Journal of Physiology 436, 814–827. Bloom, M., Evans, E., Mouritsen, O.G., 1991. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Quarterly Reviews of Biophysics 24, 293–397. Bustamante, M., Roger, F., Bochaton-Piallat, M.-L., Gabbiani, G., Martin, P.-Y., F!eraille, E., 2003. Regulatory volume increase is associated with p38 kinase-dependent actin cytoskeleton
ARTICLE IN PRESS 124
H. Takamatsu et al. / Journal of Biomechanics 38 (2005) 117–124
remodeling in rat kidney MTAL. American Journal of Physiology—Renal Physiology 285, F336–F347. . Chien, S., Sung, K.-L.P., Skalak, R., Usami, S., Tozeren, A., 1978. Theoretical and experimental studies on viscoelastic properties of erythrocyte membrane. Biophysical Journal 24, 463–487. Cornet, M., Isobe, Y., Lemanski, L.F., 1994. Effects of anisosmotic conditions on the cytoskeletal architecture of cultured PC12 cells. Journal of Morphology 222, 269–286. Crowe, J.H., Tablin, F., Tsvetkova, N., Oliver, A.E., 1999. Are lipid transitions responsible for chilling damage in human platelets? Cryobiology 38, 180–191. Di Ciano, C., Nie, Z., Sz!aszi, K., Lewis, A., Uruno, T., Zhan, X., Rotstein, O.D., Mak, A., Kapus, A., 2002. Osmotic stress-induced remodeling of the cortical cytoskeleton. American Journal of Physiology—Cell Physiology 283, C850–C865. Evans, E.A., Skalak, R., 1980. Mechanics and Thermodynamics of Membranes. CRC Press, FL. Evans, E.A., Waugh, R., Melnik, L., 1976. Elastic area compressibility modulus of red-cell membrane. Biophysical Journal 16, 585–595. Erickson, G.R.D., Northrup, L., Guilak, F., 2003. Hypo-osmotic stress induces calcium-dependent actin reorganization in articular chondrocytes. OsteoArthritis and Cartilage 11, 187–197. Guilak, F., Erickson, G.R., Ting-Beall, H.P., 2002. The effect of osmotic stress on the viscoelastic and physical properties of articular chondrocytes. Biophysical Journal 82, 720–727. Hiramoto, Y., 1963. Mechanical properties of sea urchin eggs. I. Surface force and elastic modulus of cell membrane. Experimental Cell Research 32, 59–75. Hiramoto, Y., 1970. Rheological properties of sea urchin eggs. Biorheology 6, 201–234. Hiramoto, Y., 1976. Mechanical properties of sea urchin eggs. III. Visco-elasticity of cell surface. Development Growth and Differentiation 18, 377–386. Hochmuth, R.M., 1993. Measuring the mechanical properties of individual human blood cells. Journal of Biomechanical Engineering 155, 515–519. Hochmuth, R.M., 2000. Micropipette aspiration of living cells. Journal of Biomechanics 33, 15–22. Ishiguro, H., Rubinsky, B., 1994. Mechanical interactions between ice crystals and red blood cells during directional solidification. Cryobiology 31, 483–500.
Mazur, P., Rigopoulos, N., 1983. Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: influence of warming rates. Cryobiology 20, 274–289. Mazur, P., Rall, W.F., Rigopoulos, N., 1981. Relative contributions of the fraction of unfrozen water and of salt concentration to the survival of slowly frozen human erythrocytes. Biophysical Journal 36, 653–665. Nei, T., 1967a. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. I. Microscopic observation of hemolyzing erythrocytes during the freezing and thawing process. Cryobiology 4, 153–156. Nei, T., 1967b. Mechanism of hemolysis of erythrocytes by freezing at near-zero temperatures. II. Investigations of factors affecting hemolysis by freezing. Cryobiology 4, 303–308. Noda, Y., Takeya, R., Ohno, S., Naito, S., Ito, T., Sumimoto, H., 2001. Human homologues of the Caenorhabditis elegans cell polarity protein PAR6 as an adaptor that links the small GTPases Rac and Cdc42 to atypical protein kinase C. Genes to Cells 6, 107–119. Pedersen, S.F., Mills, J.W., Hoffmann, E.K., 1999. Role of the F-actin cytoskeleton in the RVD and RVI processes in ehrich ascites tumor cells. Experimental Cell Research 252, 63–74. Pedersen, S.F., Hoffmann, E.K., Mills, J.W., 2001. The cytoskeleton and cell volume regulation. Comparative Biochemistry and Physiology—Part A 130, 385–399. Pritchard, S., Erickson, G.R., Guilak, F., 2002. Hyperosmotically induced volume change and calcium signaling in intervertebral disk cells: the role of the actin cytoskeleton. Biophysical Journal 83, 2050–2510. Rand, R.P., Burton, A.C., 1964. Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure. Biophysical Journal 4, 115–135. Sarkadi, B., Parker, J.C., 1991. Activation of ion transport pathways by changes in cell volume. Biochimica et Biophysica Acta 1071, 407–427. Takamatsu, H., Rubinsky, B., 1999. Viability of deformed cells. Cryobiology 39, 243–251. Takamatsu, H., Kumagae, N., 2002. Survival of biological cells deformed in a narrow gap. Journal of Biomechanical Engineering 124, 780–783. . Tozeren, A., Skalak, R., Sung, K.-L.P., Chien, S., 1982. Viscoelastic behavior of erythrocyte membrane. Biophysical Journal 39, 23–32.