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RESEARCH ARTICLE 785 Cellular membranes that undergo cyclic changes in tension: Direct measurement of force generation by an in vitro contractile vacuole of Paramecium multimicronucleatum Tomomi Tani*, Richard D. Allen and Yutaka Naitoh‡ Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, Hawaii 96822, USA *Present address: Department of Molecular Physiology, Tokyo Metropolitan Institute of Medical Science, Honkomagome 3-18-22, Bunkyo-ku, Tokyo 113-8613, Japan ‡Author for correspondence (e-mail: [email protected]) Accepted 28 November 2000 Journal of Cell Science 114, 785-795 © The Company of Biologists Ltd SUMMARY The contractile vacuole of the fresh water protozoan Paramecium is a membrane-bound vesicle that expels excess cytosolic water, acquired osmotically, through its periodic exocytotic activity. The in vitro contractile vacuole, isolated in a small amount of cytosol from the Paramecium cell and confined under mineral oil, showed periodic rounding and slackening at regular intervals for an extended time. The contractile vacuole rounded against the cytosol-mineral oil boundary tension. The tension at the surface of the contractile vacuole is, therefore, assumed to increase during the rounding phase. We first estimated the tension relative to the boundary tension from the degree of compression of the contractile vacuole by the boundary. We then determined the absolute value for the tension at the surface of the contractile vacuole from the degree of bending of an elastic carbon fiber microcantilever (8 µm thick; 2 mm long), whose free end was placed at the surface of an in vitro contractile vacuole. The tension was found to increase to its maximum value of approximately 5 mN m−1 when the contractile vacuole rounded. This value was more than 35 times higher than that for the slackened contractile vacuole. Electron micrographs of conventional thin sections of chemically fixed in vitro contractile vacuoles as well as those of in vivo contractile vacuoles obtained from rapid frozen and cryosubstituted cells revealed the lack of any ultrastructural evidence for the presence of a fibrous network system surrounding the contractile vacuole. Thus we conclude that the mechanism(s) by which tension is developed at the surface of the contractile vacuole membrane resides in the contractile vacuole membrane itself. We propose a hypothesis that periodic changes in the spontaneous curvature of the contractile vacuole’s lipid bilayer membrane is involved in the periodic development of higher contractile vacuole membrane tension. The isolated CV promises to be an excellent model system for understanding the molecular mechanisms of the dynamics of biological membrane. INTRODUCTION demonstrated that the CV’s rounding began immediately before severing of the radial arms from the CV, which was detected by using a fine-tipped microelectrode inserted into the CV as a marked decrease in the input capacitance of the CV. The roundness of the CV reached a maximum immediately before fluid discharge, which was detected as the disappearance of the CV membrane potential. We also demonstrated (Tominaga et al., 1998a) that membrane-bound vesicles that were derived from the CVC of the ruptured cell underwent independent rounding-slackening cycles. Recently we also found (Tani et al., 2000) that an in vitro CV, that had been dissected out of the cell together with a small amount of cytosol and confined under mineral oil, would continue repeated rounding-slackening cycles at regular intervals for more than 30 minutes. These results suggest that the membrane of the CVC possesses a mechanism by which its rounding can be periodically initiated. Since the in vitro CV was compressed between the surface of The contractile vacuole complex (CVC) is a membrane-bound osmoregulatory organelle, through which excess cytosolic water, acquired osmotically, is expelled from the cell in order to maintain cytosolic osmolarity within a rather narrow range. In Paramecium the organelle is composed of a central contractile vacuole (CV) and 5-10 radial arms surrounding the CV. Each radial arm consists of a membrane-enclosed space divided into an ampulla adjacent to the CV, the collecting canal that is continuous with the ampulla, the smooth spongiome that branches from the collecting canal, and the decorated spongiome that is continuous with the smooth spongiome at its inner periphery and ends blindly at its outer periphery (Hausmann and Allen, 1977). It has been observed that the filled CV rounds up immediately before fluid discharge (Patterson, 1980; Patterson and Sleigh, 1976). We previously (Tominaga et al., 1998b) Key words: In vitro contractile vacuole, Vacuole rounding-slackening cycle, Membrane tension, Microcantilever, Membrane tubulation, Spontaneous curvature of lipid bilayer, Paramecium multimicronucleatum 786 JOURNAL OF CELL SCIENCE 114 (4) the coverslip, on which it was prepared, and the mineral oil by a force produced by the boundary tension between the cytosol and the mineral oil, the CV rounded against this force during its rounding phase. It can, therefore, be supposed that the tension at the surface of the CV increases during the rounding phase. We previously proposed a hypothesis (Tominaga et al., 1998a; Tominaga et al., 1999) that an increase in the tension at the surface of the CV would play an important role (1) in severing the radial arms from the CV, (2) in rounding of the CV, and (3) in the subsequent opening of the CV’s pore. In other words, the periodic development of tension in the CVC membrane governs its dynamics and leads to its periodic exocytotic activity. Previous electron-micrographs of thin sections of chemically fixed CVs (Allen and Fok, 1988; Tominaga et al., 1999) have failed to reveal convincing evidence for a contractile fibrous meshwork system surrounded the CV of Paramecium. It is, therefore, conceivable that the tension at the surface of the CV originates from the lipid bilayer itself. In order to understand the mechanism by which the tension is developed and released in the CV membrane, we must first know the absolute value of the CV’s tension. We, therefore, developed a novel device that could measure a force exerted by the membrane of an isolated CV. Our device is a smaller version of the devise developed by Hiramoto (Hiramoto, 1976) for measuring the surface force produced by a sea urchin egg. The device was composed of a microcantilever made of a fine elastic carbon fiber and a photoelectric position-sensor (Kamimura, 1987) that was used for detecting the displacement of the free end of the lever as it was moved by the CV. The force was then determined from the amount of displacement and the elastic coefficient of the lever. Tension at the surface of the CV was estimated from this force and the morphological parameters of the CV. In order to further confirm the absence of a contractile cytoskeletal system surrounding the CV, we examined thin sections (1) of isolated in vitro CVs that had been undergoing rounding-slackening cycles before they were chemically fixed, and (2) of in vivo CVs in cells that were quick frozen and then fixed by the cryosubstitution process. The mechanism by which increased CV membrane tension might be developed will be discussed in terms of a model in which spontaneous curvature of the membrane’s lipid bilayer increases. MATERIALS AND METHODS Cells Cells of Paramecium multimicronucleatum (syngen 2) (Allen and Fok, 1988) were grown in an axenic culture medium at 24°C (Fok and Allen, 1979) and were harvested at the mid-logarithmic growth phase. These cells were washed with a standard saline solution that contained (final concentration in mM) 4.0 KCl, 1.0 CaCl2 and 20 MOPS-KOH buffer (pH 7.0). Cells were equilibrated with the solution for at least 2 hours before experiments. Isolation of the CV and radial arms from the cell Procedures for isolating the CV and radial arms from the cell were essentially the same as those we employed previously (Tani et al., 2000). A saline droplet containing a single cell was injected into a layer of mineral oil on a coverslip. Then, most of the saline solution was pipetted out of the droplet so that the cell was compressed between the coverslip and a thin film composed of the saline-oil Fig. 1. Schematic drawing of the experimental chamber, a pair of dissection microneedles, a drain pipette and a rotating platform all arranged on the microscope stage. A Paramecium multimicronucleatum cell is dissected on the platform by using microneedles and its contractile vacuole (CV) is teased out of the ruptured cell. This isolated in vitro CV can be seen from its side by rotating the platform 90 degrees. boundary. The cell was then ruptured by tearing the cell membrane near the CV pore region with the tip of a fine dissection microneedle. The CV was then teased out of the cell together with a minute amount of the cytosol. The remaining portions of the ruptured cell were then removed from the droplet by using the microneedle. Thus the CV remained in a small droplet of the cytosol under mineral oil with or without some radial arms. Sometimes other organelles such as digestive vacuoles and mitochondria were also enclosed in the droplet. This isolated in vitro CV showed rounding-slackening cycles at regular intervals for more than 30 minutes when the preparation was kept at room temperature (24-27°C). Estimation of tension at the surface of an in vitro CV from the amount of compression produced by the cytosolmineral oil boundary tension In order to estimate tension at the surface of the CV from the amount of compression on the CV produced by the cytosol-mineral oil boundary tension, we video-recorded the in vitro CV during roundingslackening cycles both from its top and from its side. To see the CV from its side a strip of coverslip (0.3×10 mm), on which an in vitro CV was placed, was rotated 90 degrees around a horizontal axis that passed through the approximate center of the CV (Fig. 1). Images of the CV obtained through an objective lens (PL FLUOTAR ×63, numerical aperture 0.7; Leica Inc. Deerfield, IL, USA) with a correction ring were recorded on a VHS tape by using a video cassette recorder (AG 6300, Panasonic Industrial Co., Secaucus, NJ, USA) and a CCD camera (Sony, XC-75, Tokyo, Japan). The CV was video-recorded first from its side when it showed rounding, then the vacuole was rotated 90° so it could be seen and video-recorded from its top during the next rounding. Side-top alternative video-recordings of the CV were continued as long as regular rounding-slackening cycles continued. The recorded images of the CV were fed into a computer (Power Macintosh 7600/132, Apple Computer Inc., Cupertino, CA, USA) and analyzed for the degree of compression of the CV. Representative side and top views of an in vitro CV are shown in Fig. 2A (i; side view, ii; top view, see also Fig. 5). It is clear from the figure that the CV is compressed against the coverslip (S) by the cytosol-mineral oil boundary (C-M), so that the contour of its side view is flat at the side contacting with the coverslip and dome-shaped at the other side. We determined the equatorial radius of the compressed CV Force development by the contractile vacuole 787 A force exerted by the internal pressure of the CV (P) against an area of the circle of radius r1 is the sum of the force around the circle contributed by the tension at the surface of the CV (TCV) and the force F1, and can be written as: Pπr12 = 2πr1TCV+ 2πx12 R TCM . (2) Similarly, a force against an area within the circle x1 due to P can be formulated as: Pπx12 = 2πx1TCV cos θ + 2πx12 R TCM = 2πx12 R (TCV + TCM) . (3) TCV can be obtained by solving for TCV from the simultaneous equations composed of equations 2 and 3, and is written as: TCV = Fig. 2. Side (i) and top (ii) views of an in vitro contractile vacuole (CV) of P. multimicronucleatum surrounded by a small amount of the cytosol (C). The CV and the cytosol are confined under mineral oil (M). (A) Actual photographs of the isolated CV. White lines are added for clarifying the shape of the CV, the border between the cytosol and the mineral oil (C-M) and the position of the edge of the platform (S) on which the CV is placed. (B) Line drawings of the photographs shown in A. See the text for the morphological parameters of the CV, x1, r1 and R, the positions P and P′ and the angle θ. (r1) from its top view. The image of the coverslip in side view corresponding to its surface (S) was estimated as a line between the actual image of the CV (Fig. 2A, upper image) and its more or less fuzzy mirror image (Fig. 2A, lower image). The complete image was bilaterally symmetrical with its halves separated by this line. As shown in Fig. 2A, the CV was compressed by the C-M boundary film that was in tight contact with the upper portion of the vacuole. We estimated an imaginary sphere with a radius R that would share a surface area with that part of the CV that was in contact with the CM boundary film (Fig. 2Bi). The contour of the C-M boundary was traced on a monitor screen by using drawing software (Canvas Ver. 5.0, Deneva Software, Miami, Florida, USA). The trace was then transformed into sets of coordinates by using image-processing software (NIH Image, Ver. 1.6/ppc). By using these coordinates, the slope of the contour relative to S was calculated. It is obvious that the slope is maximum on a circle with the radius x1 where the CV comes in contact with the C-M boundary film. This is schematically drawn in Fig. 2Bi, where a great circle of the sphere with the radius R crosses the circle with radius x1, which is perpendicular to the great circle, at points P and P′. The slope of the C-M boundary is maximum at these points. A force exerted against the CV by the C-M boundary film (perpendicular to S), F1, can be written as: F1 = 2πx1TCM cos θ = 2πx12 R TCM , (1) where TCM is the tension in the C-M boundary and θ is the complementary angle of the maximum slope in the C-M boundary. r12 − x12 r1(R − r1) TCM . (4) Estimation of tension at the surface of the in vitro CV from bending of a microcantilever placed on the surface of the CV In order to estimate tension at the surface of an in vitro CV more directly, we developed a microcantilever with a minute truncated rod at the free end of the lever based on a design of Hiramoto (Hiramoto, 1976), who developed a lever for estimation of the surface force of a sea urchin egg, whose size was more than 5 times larger than that of the in vitro CV. As schematically shown in Fig. 3, the cantilever was a thin elastic carbon fiber (8 µm thick, 2 mm long), one end of which was fixed to a thick glass rod (100 µm thick). At the free end of the lever, a minute glass rod (2 µm thick, 20 µm long) was attached perpendicularly to the lever. An isolated CV surrounded by a thin layer of cytosol was held at the tip of a glass holding pipette (15 µm in outer diameter, 5 µm in inner diameter) in a droplet of mineral oil. The cantilever was placed perpendicularly to the holding pipette in a single plane so that the minute glass rod at the free end of the lever is in alignment with the axis of the holding pipette, and therefore with the center of the CV. The holding pipette with an in vitro CV at its tip was, then, moved toward the tip of the thin glass rod by using a piezoelectric actuator (Burleigh Instruments, NY, USA) until the tip penetrated the cytosolic solution through the cytosol-mineral oil boundary to reach the surface of the CV. The thin glass rod was previously coated with bovine albumin (Sigma, fraction V, St Louis, MO, USA) by treating it with 1% (by volume) bovine albumin solution and allowing the albumin to dry. The albumin concentration was adjusted so that bending of the lever was negligible when the glass rod passed through the cytosolmineral oil boundary. Further movement of the CV toward the glass rod caused a dent on the surface of the CV as well as a bending of the lever away from the CV. We halted the movement when the depth of the dent became approximately 5 µm. A representative side view of an indented CV is presented in Fig. 4. The depth was shallower when the degree of bending of the lever increased as tension at the surface of the CV increased. The degree of bending of the lever was determined as a displacement of the free end of the lever, which was detected by using a position-sensitive photodetector (Kamimura, 1987) arranged to detect displacement of the free end of the lever. The relationship between the degree of displacement of the free end of the cantilever and the output voltage of the position-sensor was obtained by reading the voltage when the free end of the lever was pushed perpendicularly to it with a calibrated piezoelectric actuator. The elastic coefficient of the cantilever was determined from the degree of a vertical displacement of the free end of a horizontally placed lever when a thin 788 JOURNAL OF CELL SCIENCE 114 (4) Fig. 3. (A) Schematic drawing of the microcantilever system for measuring the force generated by an in vitro CV of P. multimicronucleatum. The CV, surrounded by a small amount of the cytosol, was placed at the tip of the holding pipette by lowering the hydrostatic pressure inside the pipette. (B) The relationship between the displacement of the free end of the microcantilever and the output voltage of the position sensor used to detect the displacement of the free end. See the text for more details. copper wire (15 µm diameter) of known weight was suspended from the free end of the lever. The elastic coefficient was in a range of 79 nN µm−1. We used several pieces of the copper wire with different lengths (5-10 mm), therefore, different weights (0.1-0.2 µN) for determining the coefficient, and found that the force-displacement relationship was fairly linear in a range of the force we examined (Fig. 3B). The displacement of the free end of the lever by the gravitational force is assumed to be so small as it does not affect the linearity of the force-displacement relationship. Moreover, when the cantilever is in the mineral oil for measurement of a force exerted by the CV a buoyancy of the lever might reduce its gravitational displacement to an extent that we could neglect it. The output voltage of the position-sensor was fed into the computer through an AD/DA converter (ITC-16; Instrutech Corp. Great Neck, NY, USA). The software we used consisted of Igor Pro (WaveMetrics, Inc., Lake Oswago, OR, USA) and PulseControl XOP software packages (Herrington et al., 1995). The image of the CV was continuously video-recorded simultaneously with the recordings of the bending of the cantilever. The force exerted by the indented CV against the lever or the force exerted by the lever against the CV to cause its indentation, F2, was calculated from the degree of bending of the lever and the elastic coefficient of the lever. Tension at the surface of the CV, TCV, was calculated according to Hiramoto (Hiramoto, 1976). In the side view of a glass rod-indented CV shown in Fig. 4 (A; an actual image, B; a drawing of the contour of A), we determined two points on the contour of the CV (white arrow heads in Fig. 4A; black arrowheads in Fig. 4B) at which lines drawn perpendicular to the direction of the F2 force Fig. 4. A photograph (A) and its line drawing (B) of an in vitro CV of P. multimicronucleatum as seen from the side. This view was used to define the morphological parameters of the CV, x2 and r2, that are needed for estimation of the tension at the surface of the CV. Tension is derived from the force (F2), which is equal to the counter force generated by the CV pushing against the microcantilever. The CV is placed at the tip of the suction pipette and is compressed by the tip of a thin glass rod which extends perpendicularly from the free end of the microcantilever. Bar at the upper right of A, 10 µm. See the text for more details. came in contact with the CV. We then determined (1) the distance between the two points, 2x2 and (2) a distance between two lines drawn parallel to the F2 force and tangential to the contour of the CV, 2r2 (the equatorial diameter). The internal pressure of the indented CV, PCV, with reference to the pressure in the oil drop, where the cantilever is present, can be written as: F2 PCV = + PCM , (5) πx22 where PCM is a pressure caused by the cytosol-mineral oil boundary tension. PCV can also be written as: PCV = 2πr2TCV + F2 πr22 + PCM . (6) From equations 5 and 6, the tension at the surface of the CV, TCV, can be formulated as: TCV = r22 − x22 2πr2x22 F2 . (7) Preparation of the isolated CV for transmission electron microscopy The CV was isolated from the cell with a minute amount of cytosol and confined under mineral oil as previously mentioned. A micropipette whose tip was approximately 2-5 µm in inner diameter, was connected to a syringe (0.5 ml) and filled with fixative containing 2% glutaraldehyde in a saline solution that contained (final concentration in mM) 20 KCl, 1 MgCl2, 10 EGTA and 30 MOPS- Force development by the contractile vacuole KOH buffer (pH 7.0). The tip of the pipette was placed in the mineral oil at a distance of approximately 10 µm from the cytosol droplet. Approximately 0.1 µl (more than twenty times the volume of the cytosol surrounding the isolated CV) of fixative was injected into the cytosol droplet when the isolated CV in the droplet showed rounding. Injection was accomplished by pushing the syringe by hand. The CV was kept immersed in the fixative for 15 minutes, then washed 30 minutes in the saline solution without glutaraldehyde and post fixed in 1% osmium tetraoxide in the saline solution for 15 minutes. After a 15 minute wash in distilled water the CV was dehydrated in an ethanol series and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA, USA). The thin sectioned CV was stained in lead citrate and uranyl acetate and examined in a Zeiss 10 transmission electron microscope. Rapid freezing and cryosubstitution of the cells We employed rapid freezing followed by cryosubstitution to fix and dehydrate the Paramecium cells so that any fragile cytoskeletal structure(s) that surrounds the CV, if it exists, would more likely be preserved than during conventional chemical fixation. Paramecium cells equilibrated in standard saline solution were kept immersed in a 100 µM (final concentration) NEM (N-ethylmaleimide)-containing standard saline solution for 20 minutes. NEM inhibited CV pore opening allowing the CV to increase in size without affecting the ongoing rounding-slackening cycle of the CV (Tani, Allen and Naitoh; unpublished data). The cells were then spread into a single cell layer onto a sheet of cellophane, while still in the NEMcontaining saline solution. The cells were then ultrarapidly frozen on a polished copper surface that was cooled with liquid nitrogen (−195°C) (Reichert-Jung KF 80, C. Reichert Optische Werke AG, Wien, Austria). The cryofixed cells on cellophane were then transferred to a 1% osmium tetroxide-acetone solution at −195°C for 2 hours. The cells were then stored in a freezer at −80°C for 2 days. The cells were then exposed to the room temperature and their surrounding solution was replaced with a fresh 1% osmium tetroxideacetone solution twice. The cells were then infiltered through pure acetone to Epon 812 resin. Sectioning of the Epon-embedded cells and electron microscopy were performed as for isolated CVs. RESULTS Tension development at the surface of an in vitro CV compressed by a cytosol-mineral oil boundary film An in vitro CV isolated from a Paramecium cell along with a minute amount of cytosol and confined under mineral oil showed rounding-slackening cycles at fairly regular intervals for 20-30 minutes at room temperatures of 24-27°C. Representative consecutive images of an in vitro CV recorded under these conditions are shown in Fig. 5 (A; top view, B; side view). The time in seconds from the start of rounding when each image was taken is indicated by a number in each frame. Values for the morphological parameters, R, r1 and x1 (see Fig. 2 for definitions), were determined from nine pairs of top and side images recorded in a range of times between 1.5 Fig. 5. A representative series of video-recorded consecutive images of an in vitro CV of P. multimicronucleatum confined under mineral oil. The number at the lower right corner of each frame is the time in seconds from the start of the CV’s rounding when each image was taken. (A) Top view. (B) Side view. Images surrounded by thick solid rectangles were used for estimation of the morphological parameters of the CV (see Fig. 2 for their definitions). Bar, 10 µm. See the text for more details. 789 seconds to 3.5 seconds from the start of rounding (five pairs of images surrounded by thick rectangles in Fig. 5 and four other pairs of images taken midway between these five pairs as indicated in Fig. 6A). These times were selected for measurements as it was difficult to obtain accurate 790 JOURNAL OF CELL SCIENCE 114 (4) Fig. 7. Periodic changes in the force generated by an in vitro CV of P. multimicronucleatum against the microcantilever shown as changes in the output voltage of a position-sensor that detects the shift of the free end of the cantilever produced by the CV force. The unit for the ordinate is transformed from voltage into force (nN) based on the relationship between the output voltage and the shift (Fig. 3B) and the elastic coefficient of the lever. Fig. 6. (A) The time courses of changes in the morphological parameters of an in vitro CV of P. multimicronucleatum confined under mineral oil, R, r1 and x1 (see Fig. 2 for their definitions) during rounding. (B) The time course of change in the tension at the surface of the CV relative to the cytosol-mineral oil boundary tension. The relative tension was calculated according to equation 4 based on the values for the morphological parameters shown in A. measurements for x1 from images taken before 1.5 seconds and R approached r1 as rounding of the CV proceeded, so that a small error in determining R and r1 from images taken after 3.5 seconds caused a large error in the calculated values for tension (see equation 4). The parameters for each pair of images are plotted in Fig. 6A against the time that the recordings were made. The tension at the surface of the CV, TCV, relative to the cytosol-mineral oil boundary tension, TCM, was calculated according to equation 4, and plotted in Fig. 6B against time. It is clear from the figure that when rounding proceeded the tension at the surface of the CV had increased to a value of more than twice that for the cytosol-mineral oil boundary tension. The maximum tension during the rounding phase was more than ten-times that during the slackening phase. Measurement of a force exerted by the surface of an in vitro CV against a microcantilever In order to determine the tension at the surface of an in vitro CV, a shift of the free end of a microcantilever, that was placed against the surface of a CV, was continuously monitored and recorded when the vacuole exhibited rounding-slackening cycles. A representative trace for the output voltage from a photoelectric position-sensor is shown in Fig. 7. The output voltage corresponds, as previously mentioned, to the shift of the free end of the lever, and therefore, can be used to measure the timing and force exerted by the CV against the lever. The voltage changes periodically for up to 20 minutes or more. This indicates that the isolated CV generates a cyclic force against the lever. Cycles of force generation occurred at a frequency of 1.36±0.07 minute−1 (mean ± s.d. for the first 13 cycles shown in Fig. 7). The lowest force or the base line force during the force generation cycles was approximately 5 nN at the beginning of recording (time 0), while the base line force tended to increase with time to approximately 15 nN after 25 minutes. In contrast, the highest force or the peak force during a series of force generation cycles remained more-or-less unchanged during 25 minutes of recording. The mean value for the peak force was 34.5±1.0 nN (n=30). In order to examine the time course of force generation more precisely, a representative trace of a single force generation cycle and the corresponding consecutive images of the same in vitro CV are shown in Fig. 8 (A; force, B; images). Time 0 in Fig. 8A (abscissa) corresponds to the time when the force is at its peak. Lettered arrowheads in A correspond to the moments when the images in B were taken. Force increases rapidly to its peak level, then it rapidly decreases to a level slightly lower than the peak level where its rate of decrease slows but then increases again as it approaches the base line level. Overall the decreasing time course shows a slower rate of force reduction than the rate of force production. That is, the period of time when the force starts to increase and the time required to reach its peak was approximately 4 seconds, while the time required for the force to decrease and return to the original base line level was approximately 10 seconds. Indentation of the CV by the tip of a minute glass rod attached to the free end of the microcantilever was deepest when the force was the lowest (Fig. 8a,b,g,h), while it was the shallowest when the force was at its peak (Fig. 8d). Estimation of tension at the surface of the in vitro CV from a force generated by the vacuole and associated changes in the vacuole shape We determined values for the morphological parameters, x2 and r2 (see Fig. 4 for definitions), for each image of a sequence of CV images, similar to that shown in Fig. 8B. We also determined the value for the force generated by the CV (F2, see Fig. 4) at each time along the force generation curve as shown in Fig. 8A. We could then calculate the tension at the surface of the CV (TCV) at each time along the force generation curve by introducing corresponding values for x1, r1 and F2 into equation 7. The tension thus calculated was plotted in Fig. 9 against the corresponding time. The tension was Force development by the contractile vacuole 791 Fig. 9. A single cycle of development of tension at the surface of an in vitro CV of P. multimicronucleatum. The tension was calculated according to equation 7 based on the force generated by the CV against the microcantilever shown in Fig. 8A and on the morphological parameters obtained from the corresponding images of the CV shown in Fig. 8B. Time 0 corresponds to the time when the tension is at its peak. Each arrow head labeling a letter on the tension trace corresponds to that in Fig. 8. Fig. 8. A single force generation cycle produced by an in vitro CV of P. multimicronucleatum (A) and corresponding consecutive images of the CV (B). Each arrow head labeling a letter on the force trace in A corresponds to the moment when the corresponding labeled image of the CV was taken. Time 0 in A corresponds to the time when the force is at its peak. A dark image of a horizontal bar at the top of each frame is the free end region of the microcantilever, from which a small glass rod is seen to protrude vertically (downward) against the CV surface. Bar, 10 µm. approximately 5×10−3 N m−1 at its peak, while it was as low as 1×10−4 N m−1 at its base line. The time course of the tension development was essentially identical with that of the development of force (compare Fig. 8A with Fig. 9), i.e. tension rapidly increased to its maximum value, decreased rapidly to a level slightly lower than the maximum value, then decreased more slowly to its minimum level, so that the overall time course showed a slower rate of tension reduction than production. In order to examine the time course of the tension development more precisely, we chose a portion of a trace for the force developing cycles that includes five very regular successive cycles. These force developing cycles were transformed into the tension developing cycles according to the procedures employed for obtaining a tension developing cycle shown in Fig. 9. To average these five time courses, the time when the tension was at its peak was chosen as the reference time for each tension developing cycle and denoted by 0 as in Figs 8 and 9. The values for the tension corresponding to 11 different time points in the 4-second time span in each cycle were determined and averaged, since, as previously mentioned, the time needed to reach the maximum tension from the start of tension development was approximately 4 seconds. These obtained averaged values for the tension at eleven different time points were plotted against corresponding time to obtain the time course for tension development (Fig. 10). The mean value for the maximum tension was 4.7×10−3±0.3×10−3 N m−1 and that for the minimum tension was 1.3×10−4±1.1×10−4 N m−1. Electron micrographs of the CV A representative thin section of an isolated in vitro CV that was fixed when it showed rounding is shown in Fig. 11. Preparation for electron microscopy has distorted the rounded CV. It is clear from the photograph that the membrane of this CV was for the most part planar and no cytoskeletal meshwork surrounding the CV was observable. Some 40 nm diameter tubules could be seen, some as transverse sections, others as cross sections. Wispy remnants of preserved but unorganized cytosolic proteins were also present around the in vitro CV. Electron micrographs of thin sections of rapid frozen and cryosubstituted cells containing in situ CVs showed no sign of a well organized cytoskeletal meshwork system surrounding the CV (data not shown). DISCUSSION Determination of the tension at the surface of an in vitro CV We have determined the force exerted by a rounding in vitro 792 JOURNAL OF CELL SCIENCE 114 (4) Fig. 10. Time course of tension development at the surface of an in vitro CV of P. multimicronucleatum. Each point is a mean of five values obtained from five successive force developing cycles. The vertical bar in each point is the standard deviation. Time 0 corresponds to the time when the tension is at its peak. See the text for more details. CV by placing a microcantilever’s free end against the CV’s surface and measuring the amount of shift of this free end (Figs 3, 8B). The force periodically changes in strength from its starting value of approximately 2 nN to a peak value of approximately 35 nN and then back to its starting value (Fig. 8A). This occurs at regular intervals (44.1±2.3 seconds; n=10, Fig. 7). The increase in force is attributable to an increase in the tension at the surface of the in vitro CV. We calculated this tension from the force produced by the CV and the CV’s geometry according to Hiramoto (Hiramoto, 1976; Figs 4, 8). The highest tension corresponded to the time of the peak force and was approximately 5×10−3 N m−1, while the lowest tension corresponded to the least force and was approximately 1×10−4 N m−1. The time course of tension development was essentially identical with that of force generation (Figs 8A, 9). The lowest tension at the surface of the CV approximated the tension in the plasma membrane of a neuron (10−5-10−4 N m−1) estimated by using a laser tweezer technique to measure tether force (Dai and Sheetz, 1995; Dai et al., 1998). By employing a compression method (Cole, 1932) early workers (Dan, 1963; Hiramoto, 1963; Mitchison and Swann, 1954; Yoneda and Dan, 1972; Yoneda et al., 1978) demonstrated periodic changes in tension at the surfaces of sea urchin and starfish eggs that accompanied egg cleavage. They found them to be in a range of 10−4-10−3 N m−1. Earlier we estimated the tension at the surface of a CV in a mechanically ruptured cell of P. multimicronucleatum based on the rate of fluid discharge and the initial diameter of the spherical CV when fluid discharge began. We found this tension to be approximately 2.6×10−4 N m−1 (Tominaga et al., 1998a). The highest tension, 4.7×10−3 N m−1, appears to be unusually high when compared with tensions in other conventional biological membranes. However, this value is still lower than the tension needed to tear the membrane of a vesicle derived from rabbit sarcolemma when it is stretched (12.4×10−3 Nm−1; Nichol and Hutter, 1996). We conclude, therefore, that a tension of 4.7×10−3 N m−1 is below the tension needed to break the CV membrane by stretching. Fig. 11. Electron micrograph of an in vitro CV of P. multimicronucleatum. (A) Low magnification of isolated CV. Bar, 1 µm. (B) A 40 nm diameter tubule (arrow) arising from CV membrane at bracket b in A. (C) Two 40 nm tubules (arrow) in cross section at bracket c in A. Bar, 0.1 µm. A single pixel of digitized images obtained through our recording system corresponded to approximately 0.1 µm, which was approximately 1% of the shift observed at the free end of the microcantilever when a calibration weight was loaded onto the lever. This implies that the relative error in the elastic coefficient calculated from this shift and weight approximates 1%. The error in reading the shift of the microcantilever by using a position sensor is far less than 1%, so that the relative error in a force estimated from the shift and the elastic coefficient approximates 1%. Absolute error in the morphological parameters of the CV, i.e. r2 or x2, corresponds to the length for a single pixel of the CV image, i.e. 0.1 µm. Thus we conclude that the relative error in the tension at the surface of the in vitro CV calculated according to equation 7 will be less than 10%. The membrane tension of an in vitro CV relative to the cytosol-mineral oil boundary tension increased from its starting value of approximately 0.2 to more than 2 as the CV rounded (Fig. 6). If we consider the rather large unavoidable error in the value for the relative tension calculated according to equation 4 as the CV rounds, the time course of the membrane tension development of the CV when compressed by the cytosol-mineral oil boundary (Fig. 6B) could be said to be essentially identical with that when compressed by a microcantilever (Fig. 10). If we assume that the cytosol-mineral oil boundary tension is the same as a water-mineral oil boundary tension (5×10−2 N m−1; Israelachvili, 1988), the CV membrane tension would change within a range from 1×10−2 to 1×10−1 N m−1. This is a few orders of magnitude higher than the values obtained directly by using a microcantilever. Thus the cytosol-mineral oil boundary tension must be lower than the water-mineral oil boundary tension, although we do not know its actual value at present. The mechanism of tension development We previously demonstrated in P. multimicronucleatum Force development by the contractile vacuole (Naitoh et al., 1997) that the cell’s cytosolic pressure was primarily responsible for discharge of the fluid from the CV. However, we found that when the cytosolic pressure was eliminated by rupturing the cell, the CV could still discharge its fluid, if its fusion with the plasma membrane for opening the pore was not prevented. This suggests the presence of a tension at the surface of the CV membrane that is important for fluid discharge in the absence of cytosolic pressure. Our analysis of the time course of fluid discharge of the CV in ruptured cells revealed that this tension was proportional to the area of planar CV membrane (termed membrane areaproportional tension) (Naitoh et al., 1997). In normal cells this membrane tension is assumed to be responsible for controlling the membrane dynamics of the CVC that governs exocytotic cycles of the CV. That is, the severing of the radial arms from the CV at the end of the fluid filling phase and fusion of the CV membrane with the plasma membrane in the CV pore region at the end of the rounding phase are under the control of the periodic development of this tension (Tominaga et al., 1998a; Tominaga et al., 1998b). One would normally assume that a contractile network surrounding the CV would be responsible for the tension development at the surface of the CV. However, previous investigations in our laboratory (Allen and Fok, 1988; Naitoh et al., 1997) have failed to demonstrate the presence of fibrous networks surrounding the CV in electron micrographs of thin sections of chemically fixed CV. Immunocytochemistry by others has also failed to detect a contractile cytoskeleton around the CV (Cohen et al., 1984; Plattner et al., 1991). In the current study we first examined thin sections of chemically fixed in vitro CVs that were isolated according to the same technique used for obtaining in vitro CVs to measure tension. Using this method we were unable to demonstrate the presence of a fibrous cytoskeletal network surrounding the CV (Fig. 11). We then employed cryosubstitution following rapid freezing to examine the CV membrane. This technique is believed to be capable of preserving fibrous cytoskeletal structures better than chemical fixation by itself. However, we were again unable to demonstrate the presence of a fibrous network surrounding the CV. As previously reported (Tani et al., 2000) the in vitro CV continues to show rounding-slackening in the presence of either 10 mM EGTA or in 0.5 mM cytochalasin B. These results argue against the possibility that a Ca2+-mediated contractile protein such as spasmin (centrin) or an actomyosinbased contractile system is involved in the CV’s tension developing mechanism. These observations together with our previous conclusion (Naitoh et al., 1997) that the time course of the CV fluid discharge does not correspond to that which would be expected if a shear force between sliding filaments, such as would be produced in an actomyosin network surrounding the CV, support the idea that a contractile network is not present around the CV, and therefore, the mechanism for tension development must reside in the CV membrane itself. Allen and Fok reported the transformation of the planar CV membrane into 40 nm diameter tubules, that remain continuous with the CV membrane, during CV fluid discharge (Allen and Fok, 1988). This has been confirmed in more recent studies (Naitoh et al., 1997). Recently we have examined the CV membrane at the rounding phase (Tominaga et al., 1999). We fixed the CV precisely at the time when the CV exhibited 793 rounding by using a decrease in the electrical potential across the CV membrane (Tominaga et al., 1998b) to trigger the discharge of fixative against the cell from a microinjector. We found that when the CV showed rounding numerous 40 nm diameter tubules formed in the vicinity of the CV’s cytoskeleton of microtubular ribbons that originate at the CV pore and pass over the surface of the CV and out to the tips of the radial arms (Hausmann and Allen, 1977). Based on these observations we proposed a hypothesis for the mechanism of tension development in the CV membrane during the rounding phase (Tominaga et al., 1998a; Tominaga et al., 1998b; Tominaga et al., 1999). This hypothesis proposes that tension in the planar CV membrane surrounding a fixed amount of fluid at the rounding phase (fluid supply to the CV being cut off by the detachment of the radial arms from the CV at this phase; Tominaga et al., 1998b) increases because a part of the planar CV membrane is transformed into 40 nm tubules (tubulation) and the phospholipid bilayer is stretched as the number of lipid molecules that surround the fluid is reduced. Involvement of dynamin-like protein in this membrane tubulation process might be possible (Hinshaw and Schmid, 1995; Takei et al., 1995) but so far we have no evidence for such a protein. We have now found that the maximum tension developed in the CV membrane was approximately 5×10−3 N m−1 (Fig. 9). This tension is assumed to be far lower than that needed to tear the CV membrane. This implies that increased tension in the CV membrane can not be responsible for its detachment from the radial arm membrane as previously hypothesized (Tominaga et al, 1998a; Tominaga et al., 1999). We, therefore, propose an alternative hypothesis that an increase in the tendency of bending of the membrane may be responsible for the increase in the force exerted by the CV against the cantilever. This tendency of bending of the membrane corresponds to the density of bending energy stored when a spontaneously curved bilayer is flattened. The density of bending energy was first defined as energy stored when a spontaneously curved monolayer is flattened and it was proportional to the square of the spontaneous curvature and the bending modulus of the membrane (Hui and Sen, 1989). Since the bending modulus of the lipid bilayer was reported to be more-or-less the same (approximately 10−19 J) in vastly different kinds of membranes with different protein and lipid compositions (Evans and Skalak, 1979; Evans and Yeung, 1994; Evans, 1983; Sackmann et al., 1986; Schneider et al., 1984; Waugh and Hochmuth, 1987), the spontaneous curvature of the CVC membranes must somehow increase periodically. This increase in the spontaneous curvature of the membrane might explain the CV’s rounding accompanied by an increased membrane tension as well as the severing of the radial arms from the CV at the end of the fluid filling phase and the tubulation of the CV membrane along the microtubule ribbons during the rounding phase. A change in the spontaneous curvature of the CV membrane must be largely dependent on a change in the degree of asymmetry between the two lipid monolayers of the membrane. The asymmetry is assumed to be brought about by modifying the effective shapes of the component phospholipid molecules of the monolayers and/or changing the effective areas of the monolayers (Döbereiner et al., 1999; Hui and Sen, 1989; Israelachvili, 1988; Lipowsky, 1999). How phospholipid 794 JOURNAL OF CELL SCIENCE 114 (4) molecules in the CV membrane can be modified or how they are translocated between the two monolayers must be understood before the mechanism by which such a periodic change in the CV membrane force is brought about can be understood. We previously demonstrated (Tani et al., 2000) that an application of suction to any small portion of the membrane of the isolated CV through a suction micropipette during the CV’s slackening phase, i.e. its period of smallest spontaneous curvature, triggers an extra rounding of the CV and this resets the timing of subsequent rounding-slackening cycles. We argued that stretching the planar CV membrane by suction activates a hypothetical tension developing mechanism(s) that resides in the membrane. The membrane tension starts to develop from the stretched site and spreads regeneratively over the entire membrane, causing rounding of the CV. Previous researchers have demonstrated that stored tension in a planar lipid bilayer is correlated with the biological activity of the membrane, e.g. enzymatic activities as well as a passive transport, and these activities are generally higher when the stored tension is higher (Hui and Sen, 1989; Sackmann, 1994). It is also well known that a mechanical stress applied externally to a membrane causes activation of some biological activities in the membrane, such as mechanosensitive ion channel activity (Hille, 1992). It is, therefore, possible that stretching a CV membrane by a suction micropipette may cause an activation of a membrane enzyme(s) that might change the effective shapes of phospholipid molecules or change the relative areas of the two membrane monolayers, causing an increase in the spontaneous curvature of the bilayer, which, in turn, could lead to CV rounding. The time course of tension development and the change in spontaneous curvature of the CV membrane As clearly shown in Fig. 10, the membrane tension of the in vitro CV increased very little during the first 2 seconds (from −4 to −2) from the start of tension development, then reached its maximum level in the following 2 seconds (from −2 to 0) (see also Fig. 9). Analysis of the time course of the rapid phase of tension (Tr) development (from −2 to −0.5 seconds in Fig. 10) which corresponds to 2 to 3.5 seconds from the start of tension development (t), revealed that the time course could be formulated as: Tr = Atn , (8) where n approximated 4.1 and A approximated 1×10−5 N m−1, respectively. Similar analysis of the time course of tension development of an in vitro CV compressed by the cytosolmineral oil boundary shown in Fig. 6B revealed that n approximated 3.7 and A was 0.02 relative to the cytosolmineral oil boundary tension. We tentatively conclude that the CV membrane tension increases with the forth power of time in this period of time. Consecutive images of the side view of an in vitro CV compressed by the microcantilever (Fig. 8B) show that the overall profile of the CV did not change remarkably during a force generation cycle. This implies that the elastic coefficient of the cantilever is rather high so that the cantilever does not allow the CV to change its shape although the CV exerts a force against the cantilever. Therefore, it can be said that (1) the force exerted against the cantilever by the CV membrane, F2, is proportional to the CV membrane tension (F2∝Tr; compare Fig. 8 with Fig. 9) and (2) the CV membrane area that is compressed by the cantilever does not change during the force generation cycle. It can, therefore, be said that F2 is proportional to the tendency of bending of the CV membrane, i.e. the density of bending energy of the membrane (∆E) (Hui and Sen, 1989), and is written as: F2 = B∆E , (9) where B is a constant. Since the bending modulus of the membrane is assumed to be constant, ∆E can be written as: ∆E = CSo2 , (10) where So is the spontaneous curvature of the membrane and C is a constant (Hui and Sen, 1989). By introducing equation 10 to equation 9, F2 can be rewritten as: F2 = BCSo2 , (11) Therefore, the time course of change in So can be written as: So = Dt2 , (12) where D is a constant. Equation 12 indicates that the rate of change in the spontaneous curvature, dSo/dt, increases linearly with time. Physical and chemical significances of this equation in the process of increasing the spontaneous curvature of the CV membrane should be sought in the future. The base line force of the in vitro CV increases with time As clearly shown in Fig. 7, the minimum (base line) force exerted by the in vitro CV against the microcantilever during the slackening phase slowly increased with time from its early value of approximately 5 nN immediately after the CV was placed against the tip of a microcantilever. The in vitro CV ceased to show cyclic changes in its membrane force approximately 30 minutes after its isolation. At this point its force remained at its highest value, approximately 35 nN, until the CV disintegrated (data not shown). This implies that rigor occurs in the membrane when the spontaneous curvature is highest. We previously demonstrated (Tani et al., 2000) that the CV in the ruptured cell remained slackened when the cell was perfused with an ATP-free saline solution. This CV could round once upon application of ATP to the perfusion solution, thereafter it remained permanently rounded. This finding together with our present finding of a slow rise in base line force supports the idea that a factor(s) that is essential for slackening of the CV, i.e. for reducing the spontaneous curvature of the CV membrane, might reside in the cytosol and this factor seems to be easily washed away from the CV by dilution and it also deteriorates slowly in the isolated cytosol that is confined under mineral oil. In vitro organelle movements We also observed that the in vitro CV, when isolated from the cell and confined under mineral oil, sometimes becomes motile in the cytosol droplet in a seemingly amoeboid fashion (data not shown). This occurs most frequently when the CV begins to show irregular rounding-slackening cycles a relatively long time after its isolation. It is highly probable that loss of Force development by the contractile vacuole coordinated periodic change in the membrane’s spontaneous curvature, or localized changes in the membrane curvature are responsible for the movement of the isolated CV. The factors that produce this movement should be sought in the future. The physical background for understanding changes in shape and movement of membrane bound vesicles has recently been summarized by Lipowsky (1999). This work was supported by NSF Grant MCB 9809929. We thank Dr Y. Hiramoto and Dr M. Yoneda for their valuable comments. 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