Direct measurement
of intracellular
pressure
S. M. KELLY AND P. T. MACKLEM Meakins-Christie Laboratories, McGill University Clinic and Royal Victoria Hospital, Montreal, Quebec HZX 2P2, Canada
KELLY, S. M., AND P. T. MACKLEM. Direct measurement of intracellular pressure. Am. J. Physiol. 260 (Cell Physiol. 29): C652-C657,1991.-The feasibility of using the servo-null technique for direct measurement of intracellular pressure (Pin) was investigated. A large cell, the Xenopus laevis oocyte, was chosen for study, and it was established that Pi, obtained with this method was both stable and accurate in these cells. Median resting Pi, in oocytes was found to be 0.27 cmH20, range 0.140.61 cmH20. During osmotic swelling Pi, increased, in a nonlinear fashion, to a value of 4.11 cmH20, range 2.61-8.91 cmHzO, with increases in cell volume (V,) of 24 t 3% (SE). This technique may be of use in the study of cellular mechanics. cell mechanics; oocytes
micropressure;
osmotic swelling;
Xenopus
laevis
PRESSURE (Pi,)can be estimatedindirectly by a number of techniques. These include aspiration of part of the cell into a pipette (18, 19, 23, 25-27), the sessile drop technique (16, 17), compression of the cell (3, 18, 31), and stretching (24) or centrifugation (15) of cells. These methods are time consuming, since Pi, is usually determined by extrapolation back to the nonstressed situation and, as a result, it is difficult to follow changes in Pi, that occur with time. The servo-null technique uses a micropipette as a pressure probe and thus provides a means of measuring pressure in small spaces. It has been used successfully to measure pressure in the microvasculature (1, 6, 28). The small size of the probe (1-5 pm) allows penetration into cells of reasonable size (>20 pm diameter) with negligible increase in cell volume (V,), and it has a virtually zero volume displacement coefficient. The dynamic response of the system is 90% within 20 ms to a square pressure change (World Precision Instruments, model 900, micropressure system specifications); therefore, rapid alterations in pressure can be followed faithfully. To examine the possibility of using this system to measure pressure in cells we made measurements of Pi, in oocytes of the anuran Xenopus laevis. These are large single cells, spherical in shape, with diameters of 1.0-1.3 mm. Thus V, is -6 orders of magnitude greater than the terminal 5 pm of the pipette, and no significant increase in V, results from insertion of the micropipette into the cell. INTRACELLULAR
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METHODS
The servo-null technique uses variations in micropipette resistance to measure pressure. It has been described in detail before (7, 8). Briefly, a null resistance is established in an electrolyte-filled micropipette having a small quantity of low conductivity fluid (buffer) in its tip. Fluctuations in pressure in the fluid surrounding the tip will tend to move liquid in or out of the micropipette. The resulting changes in resistance are opposed by applying a counter pressure to the micropipette which is measured as the output variable. Micropipettes, tips 2-5 pm in external diameter, bevelled to 35”, were backfilled with 2 M KC1 containing Lissamine green dye. Their resistance was, on average, 0.7 + - 0.3 MQ, range 0.3-1.7 MQ. The pipettes were electrically and hydraulically connected to the servo-null system (model 900, World Precision Instruments, New Haven, CT) and then positioned in a micromanipulator (model MO-102, Narashige, Tokyo, Japan) mounted on the stage of an inverted microscope (model OM2, Olympus Optical, Tokyo, Japan) with a video camera attachment. Cells were micropunctured singly in a glass-bottomed dish containing a standard amphibian buffer, modified Barth’s medium (MBS), composed of 88 mM NaCl, 1 mM KCl, 2.4 mM NaHC03, 0.3 mM Ca(NO&, 0.41 mM CaClz* 6Hz0, 0.82 mM MgSO,. 7H20, 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 10 pg/ml sodium penicillin, and 10 pg/ml streptomycin sulfate, pH 7.8 (12). The cell image, time, and the servo-null digital output were recorded on video tape for later analysis. V, was calculated from the average of two perpendicular diameters (D,) measured from the video image of the cell using: V,= T(D,)~/~. Pi, was calculated as the difference between the average of the pressures recorded in the buffer before and after micropuncture and the stable value obtained in the cell. Data were discarded if the buffer values differed by >0.3 cmHZO. To obtain oocytes, ovarian lobes were surgically removed from mature Xenopus females anesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester (triCaine). Cells were freed from investing follicular tissue by tearing the ovary into small pieces, which were incubated for 30-40 min in 0.2% collagenase (Sigma type II) dissolved in MBS. The tissue was then removed with
0 1991 the American
Physiological
Society
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forceps. Free oocytes were maintained in MBS. The stability of the probe was examined in five cells in which the micropipette was left in place for up to 50 min. Pressure readings were taken at regular intervals and plotted as a function of time. The ability of the servo-null system to respond to changes in pressure while inside the cell was also examined. Four cells were placed in MBS and micropunctured. The hydrostatic pressure outside the cell (P,,,) was increased and decreased four consecutive times by changing the level of the buffer outside the cell. The change in Pi, with respect to the initial value was then calculated for each increment of Pout and plotted against Pout. The feasibility of using the device in cells was tested by measuring the resting intracellular pressure and the pressure generated in response to osmotic swelling, an intervention which could reasonably be expected to increase Pi,. Resting Pi, was determined in 42 cells. In 10 cells, after measuring the initial Pi,, the buffer was replaced with MBS diluted to 10% with distilled water (DMBS) to induce osmotic swelling. The probe was left in the cell until cytoplasm began to leak out around the puncture site and, at regular intervals, video recordings were obtained for later analysis. Many of the measured parameters had a skewed frequency distribution. Where this was the case the median was chosen as a more accurate representation of the “average value.” The values at the 25th and 75th percentile were also calculated to give an index of the range of the data. When necessary, the Wilcoxin signed rank test was used to assess the significance of paired, nonnormally distributed data. Where the data were normally distributed, values were reported as means k SD.
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The median value of Pi, obtained from 43 cells in NIBS was 0.27 cmHzO, with a range of 0.14-0.61 cmH20* These cells had a D, of 1.17 t 0.07 mm and a calculated V, of 0.85 t 0.15 mm3. There was no apparent relation between D, and Pi,; however, the range of values of D, was small. Figure 2 illustrates the relationship we obtained between Pi, and V, during osmotic swelling, and Fig. 3 illustrates the relationship between circumferential tension (TJ, calculated from the Laplace Law TC = (Pin. 0,)/4, and the normalized surface area of the cell (A,/ AJ. Pi, in this equation is expressed as dynes per square centimeter. The initial diameter of these cells was 1.21 t 0.03 mm, the initial volume was 0.92 t 0.08 mm3, and the initial Pi, was 0.27 cmHz0, range 0.11-0.54 cmHzO. Pi, increased with cell volume to an average value of 4.11 cmH20, range 2.61-8.91 cmHzO, but the relationship was not linear, and a single value of elastance (dPiJdV,) could not be assigned to the cells. However, visual inspection of the data suggested that all cells followed the same pattern, i.e., an initial relatively flat relationship between Pi, and V, followed by a steep rise in Pi, for small changes in V,. Accordingly, we fitted an exponential equation of the form: V, = A + BewCXto the data (Fig. 4). The values for the coefficients A, B, and c were 1.12 t 0.14, -0.25 t 0.09, and 0.9 t 0.57, respectively. The average r value was 0.96 t 0.03. DISCUSSION
The cells that were selected for Pi, measurements in this study were between 1.0 and 1.3 mm in diameter, the majority being X.2 mm. According to the classification established by Dumont (4), the cells were of stages 5 and 6. The major morphological difference between these two groups is a reduction in the number and size of the surface microvilli in the stage 6 cells. We were able to demonstrate that the servo-null system was stable in cells for a prolonged period of time (Fig. IA), suggesting that progressive obstruction of the tip by cytoplasm did not take place. The noise level was low and there were no oscillations or shifts in the pressure, hence, long-term measurements of Pi, can be made without concern for variability of the baseline. The validity of Pi, measured by the servo-null system was assessed by applying a pressure to the cells equal to the height of the buffer above the cell. This increased Pi, by a known amount. The measured Pi, accurately reflected the applied Pout (Fig. lB), indicating that the
RESULTS
The micropipette maintained a stable value of Pi, for up to 50 min inside the frog oocyte (Fig. IA). The data points from the five cells could be described by the equation: Pi, = 0.188 + O.OOOlt(r = 0.834), where t is time and r is the regression coefficient. The deviation of the baseline after 1 h would then be -0.06 cmH20 and, within the time frame of our measurements, could be considered negligible. This deviation is similar to that expected in buffer, i.e., ~0.14 cmHzO/h. The change in Pi, in response to changes in Pout is shown in Fig. 1B. The relationship is described by Pi, = -0.005 t 1.02 Pout (r = 0.999) and is not significantly different from the line of identity.
FIG. 1. Test of validity of servo-null pressure in cells. A: intracellular pressure (Pin) as a function of time in 5 cells in isotonic media. B: in situ calibration of Pi, in 4 cells [on ordinate hydrostatic pressure in medium surrounding cell (P,,,)]. Regression line is shown and is not significantly different from the line of identity.
-1
0
1 PO (cm
2
3
4
5
H20)
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PRESSURE
500
B 400 E -0
131 1 -= z
9-D
lz
7-w
5
5-399
z a,
Ii I n
1
I 0 i: ’ B@ d .A
i ; i
1 -= -1q 09.
oo-o-o-“1 .
1 ,l
I 1
I
1.3
1.5
1 .o
1 .I
AC/AC
vc/vco 2. Pressure-volume (Pin-V,) relationships of oocytes in hypomedia (20-60 mosM, i.e., media somewhat different for different V, normalized by dividing by initial volume (V,,). A: Pin-V, curve cell. B and C: Pin-V, curves of 10 cells. Data are separated into 2 for greater clarity. Note that, initially, substantial cell swelling with no marked increase in Pi,. End point is cell rupture.
FIG.
tonic cells). for 1 panels occurs
system is able to measure pressure changes in cells. We obtained a value of 0.3 cmHzO for Pi, in oocytes. The only other values of Pi, obtained by micropuncture were reported a number of years ago by Kao et al. (22). These authors found Pin in unfertilized Fund&s eggsto be -5.0 mmHg (-6.8 cmH20) shortly after placing them in a hypertonic medium (seawater) and 150 mmHg (204 cmH20) after 30 min in the medium, by which time they had become activated. Although these results confirm that a measurable pressure can exist in some cells, the validity of the magnitude of the measurement is questionable, since the micropipette tip contained a short column of oil and the interface between the oil and the cytoplasm probably contributed significantly to the values obtained. In addition, it is likely that the apparatus had a large volume displacement coefficient, a factor which would also affect the magnitude of the measurements. Our values of Pi, are about an order of magnitude higher than those calculated for other cell types by measuring the force required to produce cell distortion (3, 14, 23, 25). The reasons for this discrepancy are not clear. However, oocyte cytoplasm is quite crowded. It
00
o-oo @
@ I
II
1.2
1.3
I
0
3. Calculated tension (T,)-surface area (A,) relationships oocytes. Surface area is expressed as a fraction of the initial area (A,J. A and B: same cells as in Fig. 2. FIG.
in 10 surface
8 i
w
E 0
6 --
VOLUME
(rnrn3)
4. Pressure-volume (Pin-V,) curve of single oocyte. Open cles, data points; line, exponential fitted to the points. Equation the exponential: V, = 1.21 - 0.36c-0.61P;,, r = 0.98. FIG.
cirfor
contains large quantities of yolk protein stored in small vesicles called yolk platelets as well as large numbers of mitochondria (30). Interference from these solid elements could have partially obstructed the pipette tip, resulting in a falsely high pressure reading; however, this is unlikely since changing the position of the pipette tip in the cell, even pulling back slightly, did not alter the
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measured pressure. In addition, if the pipette were obstructed one would not expect it to be able to follow changes in applied pressure as faithfully as it did (Fig. W One could imagine that a higher resting pressure could arise as an artifact of the servo-null technique. Because the system balances alterations in resistance by applying a pressure to the micropipette (see METHODS), addition of a series resistance, the cell membrane resistance, by micropuncture could result in an artificially high pressure reading. However, in this case, the pressure in the pipette would be greater than that in the cell. The resulting movement of fluid out of the pipette would reduce the pipette resistance and lower the applied pressure. This would continue until flow stopped. Consequently, while there might be a transient pressure increase due to the membrane resistance, the stable value should reflect true intracellular pressure. Given the extremely steep resistance-pressure relationship of these pipettes (7, 8), the movement of the interface required to compensate for cell resistance would be small and should involve displacement of only fractions of a nanoliter of volume. Another problem could arise because the oocyte cytoplasm is very structured. Large quantities of the cytoskeletal proteins actin, tubulin, cytokeratin, and vimentin are present both in soluble forms and arrayed as a cytoskeletal network. This structure consists of a submembraneous meshwork containing actin microfilaments, microtubules, vimentin, and cytokeratin (9-11, 20,2I). A second shell formed of cytokeratin and tubulin surrounds the nucleus (10, 11, 20). The yolk-free cytoplasm between these two shells contains radial arrays of cytokeratin and randomly arranged fibers of cytokeratin and vimentin (10, 11). This degree of organization could confer on the cytoplasm the properties of a gel or a solgel mixture. In this case the cytoplasm could be considered to be analogous to the interstitium. One could then argue, as has been done in the past (13), that since the size of the pipette tip is much larger than the free fluid spaces of the cytoplasm, separation and distortion of the cytoskeletal elements could lead to increases in Pi, above that normally present. However, more recently, it has been demonstrated (29) that micropipette pressures in hyaluronic acid gels are, if anything, more negative than those obtained by other techniques and, in addition, variations in applied pressure between -10 and +lO mmHg were accurately recorded by the pipette. In our case, the cytoplasmic milieu did not appear to adversely affect the ability of the micropipette to record increases in pressure (Fig. 1B) when the external pressure was increased nor did it affect its stability (Fig. 1A). A more likely explanation for the high values of Pi, we obtained lies in structural differences between frog oocytes and other cells. Most estimates of Pi, have been made in somatic cells that are enclosed only by the cell membrane. In contrast, frog oocytes are invested by several layers of tissue (5) outside the plasma membrane. In order, from inside out, these are 1) the vitelline envelope, a loose gel-like network of interwoven fibers; 2) a fenestrated monolayer of follicle cells which extend processes downward into the vitelline envelope; 3) the
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theta, a connective tissue layer composed of collagen fibers and containing fibroblasts and blood vessels; and 4) the inner ovarian epithelium. Incubation of the cells in collagenase enabled us to dissect away all of the epithelium and theta and at least part of the follicle cell layer. The vitelline envelope remained intact. Because this as well as the cell cortex and possibly the follicle cells may be under tension, Pi, in oocytes might be greater than in somatic cells. In support of this idea, it has been noted that oocytes flatten when the vitelline envelope is removed (2). An additional explanation of the discrepancy is that indirect methods depend, in general, on the quantification of the shape of the cell membrane when the cell is distorted. In cells where the membrane is redundant, quantification of membrane shape may not necessarily reflect the shape of the underlying cortical structures which may be an important source of cell wall tension. This could lead to an underestimation of Pi,. Furthermore, indirect techniques require extrapolation to an undistorted shape, which leaves room for error. The fact that spherical cells retain their shape and return to it after they are distorted indicates that there is tension somewhere within the cell wall conferring the minimum energy configuration on the cell. Thus the LaPlace law should apply and Pi, should exceed Pout by an amount directly proportional to wall tension and inversely proportional to the radius of curvature of the cell. Our measurements support this idea. Oocytes are known to swell in very hypotonic media (2), and most of them will ultimately burst under such conditions. This indicates that the stress induced in the cell membrane is sufficient to rupture it. This, in turn, implies a rise in intracellular pressure. The data presented here are the first measurement of such a response in a cell. However, the Laplace law would predict a fall in Pi, with cell swelling if Tc remained constant, clearly an unstable situation. The condition for stability (a positive slope dPi,/dV,) is that dT,/d& >l, a condition that was met (Fig. 3C). The nonlinearity of the Pin-V, curves must be due to a progressive increase in dT,/dDc with increases in V,. Thus the positive slope we found confers stability. The physiological significance of this is that it is possible for the cell to reach osmotic equilibrium for small changes in tonicity of the extracellular liquid by physical mechanisms alone that do not require the expenditure of metabolic energy. This would occur when Pi, exactly counterbalanced the osmotic pressure gradient. Where does the tension arise that results in the positive Pin and potential pressure-volume stability? The cell membrane stores surface area in numerous microvilli (30) and is not likely to come under tension until the cell has swollen considerably. The membrane redundancy suggests that interfacial tension between membrane lipids and extracellular liquid is negligibly small. Furthermore, the values of tension of -100 dyn/cm that we obtained in swollen cells are far greater than could be accounted for by interfacial tension. The nonlinear relationship we observed is consistent with stresses induced in viscoelastic structures. In oocytes the most important of these would be the cell cortex and the
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vitelline envelope. Our data indicate that it is possible to apply the servonull technique to measure Pin in cells and obtain new information about the cell’s mechanical properties and how these may be of physiological significance. For example, most of the cells studied in hypotonic buffer swelled by -14% of their initial volume with only a small increase in Pi,, i.e., in the range of the normal cell volume the cell membrane/cortex/vitelline envelope is highly compliant. This may relate to membrane redundancy in that the rather sudden decrease in compliance (increase in elastance, dPin/dV,) may occur when all membrane redundancies are smoothed out and the membrane comes under tension. If so, considerable volume change can occur without risk of membrane rupture. Of great practical importance with regard to species survival is how mature eggs survive when they are laid in pond water that has an osmolarity of a similar magnitude to the dilute buffer used in these experiments. If intracellular osmotic pressure remained constant, the translocation from an isotonic milieu to fresh water would create an osmotic pressure gradient of ~4,0005,000 cmH20. Our data suggest that it is unlikely that Pi, increases enough to oppose such a gradient. A previous report (2) indicates that little or no swelling occurs in ovulated eggs in hypotonic solution and that the cell membrane stiffness is increased by exposure to pond water. We have observed that mature eggs do swell in hypotonic media but appear to reach equilibrium after -1 h (unpublished observations). It is therefore unlikely that the membrane is impermeable to water. Presumably other mechanisms are invoked to prevent cell rupture under these circumstances. Measurement of mechanical properties of cells by conventional techniques has focused primarily on single cells (3,14,17,19,23-27). The servo-null technique offers the possibility of measuring the mechanical properties of the cells which form various tissues. Because the forces that are applied to these cells are different from those applied to single cells, it seems likely that their mechanical properties will differ as well. The major limitations to doing this would be related to the small size of these cells and the magnitude of their intracellular pressure. The former could be overcome by the use of pipettes with smaller tips; however, tip obstruction would be more of a problem. The latter would require an improvement in the sensitivity of the instrument. Finally, the question of how cells move is of great interest at present. If inactive motile cells are spherical because of circumferential tension, then the Laplace law applies. Such cells would have the equivalent of surface free energy potentially available to perform external work when they become activated and start to move. If wall tension were to decrease over a region of the surface, a pseudopod would be pushed out by the intracellular pressure. Furthermore, motile cells should have pressure gradients associated with cytoplasmic flow. Micropuncture with dual electrodes might provide an elegant way to measure such pressure differences.
PRESSURE
Address for reprint requests: S. M. Kelly, ratories, McGill T_Tniversity, 3626 St. Urbain H2X 2P2, Canada.
Meakins-Christie St., Montreal,
Received
form
26 February
1990; accepted
in final
21 October
LaboQuebec 1990.
REFERENCES 1. BHATTACHARYA, J., AND N. C. STAUB. Direct measurement of microvascular pressures in the isolated perfused dog lung. Science Wash. DC 210: 327-328, 1980. 2. BERNTSSON, K.-E., B. HAGLUND, AND S. LVTRUP. Osmoregulation in the amphibian egg. The influence of calcium. J. Cell. Comp. Physio2. 65: 101-112, 1965. 3. COLE, K. S. Surface forces of the Arbacia egg. J. CelZ. Comp. Physiol. 1: 1-9, 1932. 4. DUMONT, J. N. Oogenesis in Xenopus laeuis (Daudin). I. Stages of oocyte development in laboratory maintained animals. J. Morphol. 136: 153-180,1972. 5. DUMONT, J. N., AND A. R. BRUMMET. Oogenesis in Xenopus laeuis (Daudin). V. Relationships between developing oocytes and their investing follicular tissues. J. Morphol. 155: 73-98, 1978. 6. FALCHUK, K. H., AND R. W. BERLINER. Hydrostatic pressures in peritubular capillaries and tubules in the rat kidney. Am. J. Physiol. 220: 1422-1426,1971. 7. FEIN, H. Microdimensional pressure measurements in electrolytes. J. Appl. Physiol. 32: 560-564, 1972. 8. FOX, J. R., AND C. A. WIEDERHIELM. Characteristics of the servocontrolled micropipette pressure system. Microuasc. Res. 5: 324335, 1973. 9. FRANKE, W. W., P. C. RATHKE, E. SEIB, M. F. TRENDELENBURG, M. OSBORN, AND K. WEBER. Distribution and mode of arrangement of microfilamentous structures and actin in the cortex of the amphibian oocyte. Cytobiologie 14: 11 l-130, 1976. 10. GODSAVE, S. F., B. H. ANDERTON, J. HEASMAN, AND C. C. WYLIE. Oocytes and early embryos of Xenopus laeuis contain intermediate filaments which react with anti-mammalian vimentin antibodies. J. Embryol. Exp. Morphol. 83: 169-187, 1984. 11. GODSAVE, S. F., C. C. WYLIE, E. B. LANE, AND B. H. ANDERTON. Intermediate filaments in the Xenopus oocyte: the appearance and distribution of cytokeratin containing filaments. J. Embryol. Exp. Morphol. 83: 157-167, 1984. 12. GURDON, J. B., AND H. R. WOODLAND. Xenopus. In: Handbook of Genetics, edited by R. C. King. New York: Plenum, 1975, vol. 4, p. 35-50. A. C., H. J. GRANGER, AND A. E. TAYLOR. Interstitial 13. GUYTON, fluid pressure. Physiol. Rev. 51: 527-563, 1971. 14. HANSSON-MILD, K., S. LVTRUP, AND T. BERGFORS. On the mechanical properties of the vitelline membrane of the frog egg. J. Exp. Biol. 60: 807-820, 1974. 15. HARVEY, E. N. The tension at the surface of marine eggs, especially those of the sea urchin, Arbacia. Biol. Bull. 61: 273-279, 1931. 16. HARVEY, E. N. The flattening of marine eggs under the influence of gravity. J. Cell. Comp. Physiol. 4: 35-47, 1934. 17. HARVEY, E. N., AND G. FANKHAUSER. The tension at the surface of the eggs of the salamander Triturus (Diemyctylus) viridescens. J. Cell. Comp. Physiol. 3: 463-475, 1933. 18. HIRAMOTO, Y. Mechanical properties of sea urchin eggs. II. Changes in mechanical properties from fertilization to cleavage. Exp. Cell Res. 32: 76-88, 1963. 19. HIRAMOTO, Y. Determination of the mechanical properties of the egg surface by elastimetry. Methods Cell Biol. 27: 435-442, 1986. 20. JESSUS, C., C. THIBIER, D. H~CHON, AND R. OZON. Taxol reveals cortical sites of microtubule assembly in Xenopus oocytes. Role of the nucleus. Cell Differ. Dev. 25: 57-64, 1988. 21. JESSUS, C., C. THIBIER, AND R. OZON. Levels of microtubules during the meiotic maturation of the Xenopus oocyte. J. Cell Sci. 87: 705-712, 1987. 22. KAO, C.-Y., R. CHAMBERS, AND E. L. CHAMBERS. The internal hydrostatic pressure of the unfertilized FunduZus egg activated by puncture. BioZ. BULL. 101: 210-211, 1951. 23. MITCHISON, J. M., AND M. M. SWANN. The mechanical properties of the cell surface. I. The cell elastimeter. J. Exp. BioL. 31: 443460, 1954. 24. NORRIS, C. H. The tension at the surface, and other physical properties of the nucleated erythrocyte. J. Cell. Comp. Physiol. 14:
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117-133,1939. 25. RAND, R. P., AND A. C. BURTON. Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure. Biophys. J. 4: 115-135, 1964. 26. SELMAN, G. G., AND C. H. WADDINGTON. The mechanism of cell division in the cleavage of the newt’s egg. J. Exp. BioZ. 32: 700733,1955. 27. VLI~S, F. Recherches sur une deformation mechanique des oeufs d’oursin. Arch. ZooL. Exp. Gen. 75: 421-463, 1933. 28. WIEDERHIELM, C. A., J. W. WOODBURY, S. KIRK, AND R. F. RUSHMER. Pulsatile pressures in the microcirculation of the frog’s
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mesentery. Am. J. Physiol. 207: 173-176, 1964. 29. WIIG, H., R. K. REED, AND K. AUKLAND. Micropuncture measurement of interstitial fluid pressure in rat subcutis and skeletal muscle: comparison to wick-in-needle technique. Bibl. Anat. 20: 263-266,198l. 30. WISCHNITZER, S. The ultrastructure of the cytoplasm of the developing amphibian egg. In: Advances in Morphogenesis, edited by M. Abercrombie and J. Brachet. New York: Academic, 1966, vol. 5, p. 131-177. 31. YONEDA, M. The compression method for determining the surface force. Methods CeZZ BioZ. 27: 421-434, 1986.’
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of intracellular
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S. M. KELLY AND P. T. MACKLEM Meakins-Christie Laboratories, McGill University Clinic and Royal Victoria Hospital, Montreal, Quebec HZX 2P2, Canada
KELLY, S. M., AND P. T. MACKLEM. Direct measurement of intracellular pressure. Am. J. Physiol. 260 (Cell Physiol. 29): C652-C657,1991.-The feasibility of using the servo-null technique for direct measurement of intracellular pressure (Pin) was investigated. A large cell, the Xenopus laevis oocyte, was chosen for study, and it was established that Pi, obtained with this method was both stable and accurate in these cells. Median resting Pi, in oocytes was found to be 0.27 cmH20, range 0.140.61 cmH20. During osmotic swelling Pi, increased, in a nonlinear fashion, to a value of 4.11 cmH20, range 2.61-8.91 cmHzO, with increases in cell volume (V,) of 24 t 3% (SE). This technique may be of use in the study of cellular mechanics. cell mechanics; oocytes
micropressure;
osmotic swelling;
Xenopus
laevis
PRESSURE (Pi,)can be estimatedindirectly by a number of techniques. These include aspiration of part of the cell into a pipette (18, 19, 23, 25-27), the sessile drop technique (16, 17), compression of the cell (3, 18, 31), and stretching (24) or centrifugation (15) of cells. These methods are time consuming, since Pi, is usually determined by extrapolation back to the nonstressed situation and, as a result, it is difficult to follow changes in Pi, that occur with time. The servo-null technique uses a micropipette as a pressure probe and thus provides a means of measuring pressure in small spaces. It has been used successfully to measure pressure in the microvasculature (1, 6, 28). The small size of the probe (1-5 pm) allows penetration into cells of reasonable size (>20 pm diameter) with negligible increase in cell volume (V,), and it has a virtually zero volume displacement coefficient. The dynamic response of the system is 90% within 20 ms to a square pressure change (World Precision Instruments, model 900, micropressure system specifications); therefore, rapid alterations in pressure can be followed faithfully. To examine the possibility of using this system to measure pressure in cells we made measurements of Pi, in oocytes of the anuran Xenopus laevis. These are large single cells, spherical in shape, with diameters of 1.0-1.3 mm. Thus V, is -6 orders of magnitude greater than the terminal 5 pm of the pipette, and no significant increase in V, results from insertion of the micropipette into the cell. INTRACELLULAR
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$1.50 Copyright
METHODS
The servo-null technique uses variations in micropipette resistance to measure pressure. It has been described in detail before (7, 8). Briefly, a null resistance is established in an electrolyte-filled micropipette having a small quantity of low conductivity fluid (buffer) in its tip. Fluctuations in pressure in the fluid surrounding the tip will tend to move liquid in or out of the micropipette. The resulting changes in resistance are opposed by applying a counter pressure to the micropipette which is measured as the output variable. Micropipettes, tips 2-5 pm in external diameter, bevelled to 35”, were backfilled with 2 M KC1 containing Lissamine green dye. Their resistance was, on average, 0.7 + - 0.3 MQ, range 0.3-1.7 MQ. The pipettes were electrically and hydraulically connected to the servo-null system (model 900, World Precision Instruments, New Haven, CT) and then positioned in a micromanipulator (model MO-102, Narashige, Tokyo, Japan) mounted on the stage of an inverted microscope (model OM2, Olympus Optical, Tokyo, Japan) with a video camera attachment. Cells were micropunctured singly in a glass-bottomed dish containing a standard amphibian buffer, modified Barth’s medium (MBS), composed of 88 mM NaCl, 1 mM KCl, 2.4 mM NaHC03, 0.3 mM Ca(NO&, 0.41 mM CaClz* 6Hz0, 0.82 mM MgSO,. 7H20, 15 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES), 10 pg/ml sodium penicillin, and 10 pg/ml streptomycin sulfate, pH 7.8 (12). The cell image, time, and the servo-null digital output were recorded on video tape for later analysis. V, was calculated from the average of two perpendicular diameters (D,) measured from the video image of the cell using: V,= T(D,)~/~. Pi, was calculated as the difference between the average of the pressures recorded in the buffer before and after micropuncture and the stable value obtained in the cell. Data were discarded if the buffer values differed by >0.3 cmHZO. To obtain oocytes, ovarian lobes were surgically removed from mature Xenopus females anesthetized by immersion in 0.1% 3-aminobenzoic acid ethyl ester (triCaine). Cells were freed from investing follicular tissue by tearing the ovary into small pieces, which were incubated for 30-40 min in 0.2% collagenase (Sigma type II) dissolved in MBS. The tissue was then removed with
0 1991 the American
Physiological
Society
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forceps. Free oocytes were maintained in MBS. The stability of the probe was examined in five cells in which the micropipette was left in place for up to 50 min. Pressure readings were taken at regular intervals and plotted as a function of time. The ability of the servo-null system to respond to changes in pressure while inside the cell was also examined. Four cells were placed in MBS and micropunctured. The hydrostatic pressure outside the cell (P,,,) was increased and decreased four consecutive times by changing the level of the buffer outside the cell. The change in Pi, with respect to the initial value was then calculated for each increment of Pout and plotted against Pout. The feasibility of using the device in cells was tested by measuring the resting intracellular pressure and the pressure generated in response to osmotic swelling, an intervention which could reasonably be expected to increase Pi,. Resting Pi, was determined in 42 cells. In 10 cells, after measuring the initial Pi,, the buffer was replaced with MBS diluted to 10% with distilled water (DMBS) to induce osmotic swelling. The probe was left in the cell until cytoplasm began to leak out around the puncture site and, at regular intervals, video recordings were obtained for later analysis. Many of the measured parameters had a skewed frequency distribution. Where this was the case the median was chosen as a more accurate representation of the “average value.” The values at the 25th and 75th percentile were also calculated to give an index of the range of the data. When necessary, the Wilcoxin signed rank test was used to assess the significance of paired, nonnormally distributed data. Where the data were normally distributed, values were reported as means k SD.
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The median value of Pi, obtained from 43 cells in NIBS was 0.27 cmHzO, with a range of 0.14-0.61 cmH20* These cells had a D, of 1.17 t 0.07 mm and a calculated V, of 0.85 t 0.15 mm3. There was no apparent relation between D, and Pi,; however, the range of values of D, was small. Figure 2 illustrates the relationship we obtained between Pi, and V, during osmotic swelling, and Fig. 3 illustrates the relationship between circumferential tension (TJ, calculated from the Laplace Law TC = (Pin. 0,)/4, and the normalized surface area of the cell (A,/ AJ. Pi, in this equation is expressed as dynes per square centimeter. The initial diameter of these cells was 1.21 t 0.03 mm, the initial volume was 0.92 t 0.08 mm3, and the initial Pi, was 0.27 cmHz0, range 0.11-0.54 cmHzO. Pi, increased with cell volume to an average value of 4.11 cmH20, range 2.61-8.91 cmHzO, but the relationship was not linear, and a single value of elastance (dPiJdV,) could not be assigned to the cells. However, visual inspection of the data suggested that all cells followed the same pattern, i.e., an initial relatively flat relationship between Pi, and V, followed by a steep rise in Pi, for small changes in V,. Accordingly, we fitted an exponential equation of the form: V, = A + BewCXto the data (Fig. 4). The values for the coefficients A, B, and c were 1.12 t 0.14, -0.25 t 0.09, and 0.9 t 0.57, respectively. The average r value was 0.96 t 0.03. DISCUSSION
The cells that were selected for Pi, measurements in this study were between 1.0 and 1.3 mm in diameter, the majority being X.2 mm. According to the classification established by Dumont (4), the cells were of stages 5 and 6. The major morphological difference between these two groups is a reduction in the number and size of the surface microvilli in the stage 6 cells. We were able to demonstrate that the servo-null system was stable in cells for a prolonged period of time (Fig. IA), suggesting that progressive obstruction of the tip by cytoplasm did not take place. The noise level was low and there were no oscillations or shifts in the pressure, hence, long-term measurements of Pi, can be made without concern for variability of the baseline. The validity of Pi, measured by the servo-null system was assessed by applying a pressure to the cells equal to the height of the buffer above the cell. This increased Pi, by a known amount. The measured Pi, accurately reflected the applied Pout (Fig. lB), indicating that the
RESULTS
The micropipette maintained a stable value of Pi, for up to 50 min inside the frog oocyte (Fig. IA). The data points from the five cells could be described by the equation: Pi, = 0.188 + O.OOOlt(r = 0.834), where t is time and r is the regression coefficient. The deviation of the baseline after 1 h would then be -0.06 cmH20 and, within the time frame of our measurements, could be considered negligible. This deviation is similar to that expected in buffer, i.e., ~0.14 cmHzO/h. The change in Pi, in response to changes in Pout is shown in Fig. 1B. The relationship is described by Pi, = -0.005 t 1.02 Pout (r = 0.999) and is not significantly different from the line of identity.
FIG. 1. Test of validity of servo-null pressure in cells. A: intracellular pressure (Pin) as a function of time in 5 cells in isotonic media. B: in situ calibration of Pi, in 4 cells [on ordinate hydrostatic pressure in medium surrounding cell (P,,,)]. Regression line is shown and is not significantly different from the line of identity.
-1
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PRESSURE
500
B 400 E -0
131 1 -= z
9-D
lz
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oo-o-o-“1 .
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AC/AC
vc/vco 2. Pressure-volume (Pin-V,) relationships of oocytes in hypomedia (20-60 mosM, i.e., media somewhat different for different V, normalized by dividing by initial volume (V,,). A: Pin-V, curve cell. B and C: Pin-V, curves of 10 cells. Data are separated into 2 for greater clarity. Note that, initially, substantial cell swelling with no marked increase in Pi,. End point is cell rupture.
FIG.
tonic cells). for 1 panels occurs
system is able to measure pressure changes in cells. We obtained a value of 0.3 cmHzO for Pi, in oocytes. The only other values of Pi, obtained by micropuncture were reported a number of years ago by Kao et al. (22). These authors found Pin in unfertilized Fund&s eggsto be -5.0 mmHg (-6.8 cmH20) shortly after placing them in a hypertonic medium (seawater) and 150 mmHg (204 cmH20) after 30 min in the medium, by which time they had become activated. Although these results confirm that a measurable pressure can exist in some cells, the validity of the magnitude of the measurement is questionable, since the micropipette tip contained a short column of oil and the interface between the oil and the cytoplasm probably contributed significantly to the values obtained. In addition, it is likely that the apparatus had a large volume displacement coefficient, a factor which would also affect the magnitude of the measurements. Our values of Pi, are about an order of magnitude higher than those calculated for other cell types by measuring the force required to produce cell distortion (3, 14, 23, 25). The reasons for this discrepancy are not clear. However, oocyte cytoplasm is quite crowded. It
00
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II
1.2
1.3
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3. Calculated tension (T,)-surface area (A,) relationships oocytes. Surface area is expressed as a fraction of the initial area (A,J. A and B: same cells as in Fig. 2. FIG.
in 10 surface
8 i
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VOLUME
(rnrn3)
4. Pressure-volume (Pin-V,) curve of single oocyte. Open cles, data points; line, exponential fitted to the points. Equation the exponential: V, = 1.21 - 0.36c-0.61P;,, r = 0.98. FIG.
cirfor
contains large quantities of yolk protein stored in small vesicles called yolk platelets as well as large numbers of mitochondria (30). Interference from these solid elements could have partially obstructed the pipette tip, resulting in a falsely high pressure reading; however, this is unlikely since changing the position of the pipette tip in the cell, even pulling back slightly, did not alter the
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measured pressure. In addition, if the pipette were obstructed one would not expect it to be able to follow changes in applied pressure as faithfully as it did (Fig. W One could imagine that a higher resting pressure could arise as an artifact of the servo-null technique. Because the system balances alterations in resistance by applying a pressure to the micropipette (see METHODS), addition of a series resistance, the cell membrane resistance, by micropuncture could result in an artificially high pressure reading. However, in this case, the pressure in the pipette would be greater than that in the cell. The resulting movement of fluid out of the pipette would reduce the pipette resistance and lower the applied pressure. This would continue until flow stopped. Consequently, while there might be a transient pressure increase due to the membrane resistance, the stable value should reflect true intracellular pressure. Given the extremely steep resistance-pressure relationship of these pipettes (7, 8), the movement of the interface required to compensate for cell resistance would be small and should involve displacement of only fractions of a nanoliter of volume. Another problem could arise because the oocyte cytoplasm is very structured. Large quantities of the cytoskeletal proteins actin, tubulin, cytokeratin, and vimentin are present both in soluble forms and arrayed as a cytoskeletal network. This structure consists of a submembraneous meshwork containing actin microfilaments, microtubules, vimentin, and cytokeratin (9-11, 20,2I). A second shell formed of cytokeratin and tubulin surrounds the nucleus (10, 11, 20). The yolk-free cytoplasm between these two shells contains radial arrays of cytokeratin and randomly arranged fibers of cytokeratin and vimentin (10, 11). This degree of organization could confer on the cytoplasm the properties of a gel or a solgel mixture. In this case the cytoplasm could be considered to be analogous to the interstitium. One could then argue, as has been done in the past (13), that since the size of the pipette tip is much larger than the free fluid spaces of the cytoplasm, separation and distortion of the cytoskeletal elements could lead to increases in Pi, above that normally present. However, more recently, it has been demonstrated (29) that micropipette pressures in hyaluronic acid gels are, if anything, more negative than those obtained by other techniques and, in addition, variations in applied pressure between -10 and +lO mmHg were accurately recorded by the pipette. In our case, the cytoplasmic milieu did not appear to adversely affect the ability of the micropipette to record increases in pressure (Fig. 1B) when the external pressure was increased nor did it affect its stability (Fig. 1A). A more likely explanation for the high values of Pi, we obtained lies in structural differences between frog oocytes and other cells. Most estimates of Pi, have been made in somatic cells that are enclosed only by the cell membrane. In contrast, frog oocytes are invested by several layers of tissue (5) outside the plasma membrane. In order, from inside out, these are 1) the vitelline envelope, a loose gel-like network of interwoven fibers; 2) a fenestrated monolayer of follicle cells which extend processes downward into the vitelline envelope; 3) the
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theta, a connective tissue layer composed of collagen fibers and containing fibroblasts and blood vessels; and 4) the inner ovarian epithelium. Incubation of the cells in collagenase enabled us to dissect away all of the epithelium and theta and at least part of the follicle cell layer. The vitelline envelope remained intact. Because this as well as the cell cortex and possibly the follicle cells may be under tension, Pi, in oocytes might be greater than in somatic cells. In support of this idea, it has been noted that oocytes flatten when the vitelline envelope is removed (2). An additional explanation of the discrepancy is that indirect methods depend, in general, on the quantification of the shape of the cell membrane when the cell is distorted. In cells where the membrane is redundant, quantification of membrane shape may not necessarily reflect the shape of the underlying cortical structures which may be an important source of cell wall tension. This could lead to an underestimation of Pi,. Furthermore, indirect techniques require extrapolation to an undistorted shape, which leaves room for error. The fact that spherical cells retain their shape and return to it after they are distorted indicates that there is tension somewhere within the cell wall conferring the minimum energy configuration on the cell. Thus the LaPlace law should apply and Pi, should exceed Pout by an amount directly proportional to wall tension and inversely proportional to the radius of curvature of the cell. Our measurements support this idea. Oocytes are known to swell in very hypotonic media (2), and most of them will ultimately burst under such conditions. This indicates that the stress induced in the cell membrane is sufficient to rupture it. This, in turn, implies a rise in intracellular pressure. The data presented here are the first measurement of such a response in a cell. However, the Laplace law would predict a fall in Pi, with cell swelling if Tc remained constant, clearly an unstable situation. The condition for stability (a positive slope dPi,/dV,) is that dT,/d& >l, a condition that was met (Fig. 3C). The nonlinearity of the Pin-V, curves must be due to a progressive increase in dT,/dDc with increases in V,. Thus the positive slope we found confers stability. The physiological significance of this is that it is possible for the cell to reach osmotic equilibrium for small changes in tonicity of the extracellular liquid by physical mechanisms alone that do not require the expenditure of metabolic energy. This would occur when Pi, exactly counterbalanced the osmotic pressure gradient. Where does the tension arise that results in the positive Pin and potential pressure-volume stability? The cell membrane stores surface area in numerous microvilli (30) and is not likely to come under tension until the cell has swollen considerably. The membrane redundancy suggests that interfacial tension between membrane lipids and extracellular liquid is negligibly small. Furthermore, the values of tension of -100 dyn/cm that we obtained in swollen cells are far greater than could be accounted for by interfacial tension. The nonlinear relationship we observed is consistent with stresses induced in viscoelastic structures. In oocytes the most important of these would be the cell cortex and the
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vitelline envelope. Our data indicate that it is possible to apply the servonull technique to measure Pin in cells and obtain new information about the cell’s mechanical properties and how these may be of physiological significance. For example, most of the cells studied in hypotonic buffer swelled by -14% of their initial volume with only a small increase in Pi,, i.e., in the range of the normal cell volume the cell membrane/cortex/vitelline envelope is highly compliant. This may relate to membrane redundancy in that the rather sudden decrease in compliance (increase in elastance, dPin/dV,) may occur when all membrane redundancies are smoothed out and the membrane comes under tension. If so, considerable volume change can occur without risk of membrane rupture. Of great practical importance with regard to species survival is how mature eggs survive when they are laid in pond water that has an osmolarity of a similar magnitude to the dilute buffer used in these experiments. If intracellular osmotic pressure remained constant, the translocation from an isotonic milieu to fresh water would create an osmotic pressure gradient of ~4,0005,000 cmH20. Our data suggest that it is unlikely that Pi, increases enough to oppose such a gradient. A previous report (2) indicates that little or no swelling occurs in ovulated eggs in hypotonic solution and that the cell membrane stiffness is increased by exposure to pond water. We have observed that mature eggs do swell in hypotonic media but appear to reach equilibrium after -1 h (unpublished observations). It is therefore unlikely that the membrane is impermeable to water. Presumably other mechanisms are invoked to prevent cell rupture under these circumstances. Measurement of mechanical properties of cells by conventional techniques has focused primarily on single cells (3,14,17,19,23-27). The servo-null technique offers the possibility of measuring the mechanical properties of the cells which form various tissues. Because the forces that are applied to these cells are different from those applied to single cells, it seems likely that their mechanical properties will differ as well. The major limitations to doing this would be related to the small size of these cells and the magnitude of their intracellular pressure. The former could be overcome by the use of pipettes with smaller tips; however, tip obstruction would be more of a problem. The latter would require an improvement in the sensitivity of the instrument. Finally, the question of how cells move is of great interest at present. If inactive motile cells are spherical because of circumferential tension, then the Laplace law applies. Such cells would have the equivalent of surface free energy potentially available to perform external work when they become activated and start to move. If wall tension were to decrease over a region of the surface, a pseudopod would be pushed out by the intracellular pressure. Furthermore, motile cells should have pressure gradients associated with cytoplasmic flow. Micropuncture with dual electrodes might provide an elegant way to measure such pressure differences.
PRESSURE
Address for reprint requests: S. M. Kelly, ratories, McGill T_Tniversity, 3626 St. Urbain H2X 2P2, Canada.
Meakins-Christie St., Montreal,
Received
form
26 February
1990; accepted
in final
21 October
LaboQuebec 1990.
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117-133,1939. 25. RAND, R. P., AND A. C. BURTON. Mechanical properties of the red cell membrane. I. Membrane stiffness and intracellular pressure. Biophys. J. 4: 115-135, 1964. 26. SELMAN, G. G., AND C. H. WADDINGTON. The mechanism of cell division in the cleavage of the newt’s egg. J. Exp. BioZ. 32: 700733,1955. 27. VLI~S, F. Recherches sur une deformation mechanique des oeufs d’oursin. Arch. ZooL. Exp. Gen. 75: 421-463, 1933. 28. WIEDERHIELM, C. A., J. W. WOODBURY, S. KIRK, AND R. F. RUSHMER. Pulsatile pressures in the microcirculation of the frog’s
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mesentery. Am. J. Physiol. 207: 173-176, 1964. 29. WIIG, H., R. K. REED, AND K. AUKLAND. Micropuncture measurement of interstitial fluid pressure in rat subcutis and skeletal muscle: comparison to wick-in-needle technique. Bibl. Anat. 20: 263-266,198l. 30. WISCHNITZER, S. The ultrastructure of the cytoplasm of the developing amphibian egg. In: Advances in Morphogenesis, edited by M. Abercrombie and J. Brachet. New York: Academic, 1966, vol. 5, p. 131-177. 31. YONEDA, M. The compression method for determining the surface force. Methods CeZZ BioZ. 27: 421-434, 1986.’
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