membrane dynamics of the contractile vacuole

INTRODUCTION

The contractile vacuole complex (CVC) is an osmoregulatoryorganelle of fresh water protozoa. In Paramecium it consists ofa central contractile vacuole (CV) and 5-10 radial arms that fanout from the CV. A radial arm consists of (1) an ampullaadjacent to the CV, (2) the collecting canal which is continuouswith the ampulla, (3) the smooth spongiome that branches fromboth the ampulla and the collecting canal, and (4) the decoratedspongiome, which is continuous with the smooth spongiomeat its inner periphery and ends blindly in the cytosol at the

decorated spongiome’s outer periphery The decoratedspongiome is not usually found around ampullae. Allmembranes are organized around a cytoskeleton ofmicrotubular ribbons that originate at the CV pore. The ribbonspass in pinwheel fashion over the surface of the CV and out tothe tips of the radial arms, one ribbon passes along each arm(Hausmann and Allen, 1977).

Excess cytosolic water, acquired osmotically, is segregatedas a result of the activity of the decorated spongiome (Ishidaet al., 1993) and is transferred ultimately from the ampullaeinto the CV. The CV then expels the water to the exterior of

3733Journal of Cell Science 112, 3733-3745 (1999)Printed in Great Britain © The Company of Biologists Limited 1999JCS0790

The contractile vacuole complex of the fresh waterprotozoan Paramecium multimicronucleatum exhibitsperiodic exocytotic activity. This keeps cytosolic osmolarityat a constant value. The contractile vacuole, the centralexocytotic vesicle of the complex, becomes disconnectedfrom its surrounding radial arms and rounds before itsfluid content is expelled. We previously proposed ahypothesis that the rounding of the contractile vacuolecorresponds to an increase in its membrane tension andthat a periodic increase in membrane tension governs theexocytotic cycle. We also proposed a hypothesis thattransformation of excess planar membrane of thecontractile vacuole into 40 nm diameter tubules, thatremain continuous with the contractile vacuole membrane,is a primary cause for the tension development in theplanar membrane. In order to investigate tensiondevelopment further, we have examined electronmicroscopically the contractile vacuole membrane at therounding phase. To do this, we developed a computer-aidedsystem to fix the cell precisely at the time that thecontractile vacuole exhibited rounding. In this system adecrease in the electrical potential across the contractilevacuole membrane that accompanied the vacuole’srounding was monitored through a fine-tippedmicroelectrode inserted directly into the in vivo contractile

vacuole. A decrease in membrane potential was used togenerate an electric signal that activated an injector forinjecting a fixative through a microcapillary against the cellat the precise time of rounding. Subsequent electronmicrographs of the contractile vacuole membrane clearlydemonstrated that numerous ~40 nm membrane-boundtubules formed in the vicinity of the vacuole’s microtubuleribbons when the vacuole showed rounding. This findingsuggested that membrane tubulation was the cause fortopographical isolation of excess membrane from theplanar membrane during the periodic rounding of thecontractile vacuole. This together with stereo-pair imagesof the contractile vacuole complex membranes suggestedthat the microtubule ribbons were intimately involved inenhancing this membrane tubulation activity. Electronmicrographs of the contractile vacuole complexes alsoshowed that decorated tubules came to lie abnormally closeto the contractile vacuole in these impaled cells. Thissuggested that the contractile vacuole was capable ofutilizing the smooth spongiome membrane that lies aroundthe ampullae and the collecting canals to increase its size.

Key words: Contractile vacuole, Electrophysiologically controlledfixation, Membrane potential, Membrane tubulation, Membranetension, Microtubular ribbon, Paramecium

SUMMARY

A key function of non-planar membranes and their associated microtubular

ribbons in contractile vacuole membrane dynamics is revealed by

electrophysiologically controlled fixation of Paramecium

Takashi Tominaga*, Yutaka Naitoh and Richard D. Allen‡

Pacific Biomedical Research Center, Snyder Hall 306, University of Hawaii at Manoa, 2538 The Mall, Honolulu, Hawaii 96822,USA*Present address: Laboratory for Brain-Operative Devices, Brain Science Institute (BSI), The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa,Wako, Saitama 351-01, Japan‡Author for correspondence (e-mail: [email protected])

Accepted 25 August; published on WWW 18 October 1999

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the cell through the periodic opening of the pore. In a singleexocytotic cycle, the CV at first undergoes a relatively slowswelling process, as the segregated water enters the vacuole(the fluid filling phase). This is followed by a rapid roundingprocess (the rounding phase) and ends by a rapid shrinkage ofthe vacuole as the fluid is expelled to the cell’s exterior througha fixed pore (the fluid expulsion phase) (Allen and Fok, 1988;Kitching, 1967; Patterson, 1980; Wigg et al., 1967; Zeuthen,1992).

We previously found that an isolated in vitro CV alsoshowed rounding-slackening cycles (Tominaga et al., 1998a)without subsequent fluid expulsion. Such rounding withoutexpulsion can also occur in the in vivo CV (Patterson andSleigh, 1976; Tominaga et al., 1998a). Based on this finding,we proposed the hypothesis that the CV membrane possessesits own mechanism by which its tension is periodicallyincreased. The rounding phase thus corresponds to theincreased membrane tension phase of the CV. Such a periodicchange in membrane tension may also govern the exocytoticcycle of the in vivo CVC (see also Tani et al., 1999).

Previously we also found (Tominaga et al., 1998b) thatthe membrane potential and the input capacitance of thecontractile vacuole complex recorded through a fine-tippedmicroelectrode inserted into the in vivo CV markedlydecreased immediately after the start of the CV’s roundingphase. As input capacitance corresponds to the membrane area,the capacitance decrease implied that the CV has becomedisconnected from the radial arms during the rounding phase.Also, since the membrane potential is most likely generated inthe tubules of the decorated spongiome which are part of theradial arm (Tominaga et al., 1998b), the drop in membranepotential measured by the electrode indicates that the CV is nolonger connected to its electrogenic source (Giglione andGross, 1995) when the CV was disconnected from the radialarms.

Furthermore, we (Tominaga et al., 1998a) also demonstratedthat the roundness of the CV increased while the volume of theCV remained unchanged at its maximum level after the CV hadbeen disconnected from the radial arms during the roundingphase. This implies that the CV itself possesses a mechanismby which the amount of planar CV membrane (correspondingto the apparent surface area of the CV membrane) iseffectively reduced. In addition, we demonstrated electronmicroscopically that the CV membrane became transformedinto a network of 40 nm tubules (Allen and Fok, 1988; Naitohet al., 1997b) when the CV expels its content. We (Tominagaet al., 1998a), therefore, proposed the hypothesis that theapparent transformation of the CV membrane by tubulation(here termed ‘enhanced tubulation activity’) is the primarycause for the topographical isolation of excess membrane fromthe CV’s planar membrane during the rounding phase.

The primary objective of this paper is to determine electronmicroscopically whether membrane tubulation is enhanced inthe CV membrane when the CV undergoes rounding andwhether such tubulation is the primary cause of rounding. Inorder to fix the cell precisely in the rounding phase of the CV,we developed a mechanism to electrophysiologically detect adecrease in the CV’s membrane potential and to use thisdecrease to trigger the discharge of fixative against the cell.This technique has allowed us to detect the early stages of 40nm tubule formation of the CV membrane and to show that the

tubular enhancement begins adjacent to the microtubuleribbons when the CV is in its rounding phase. Only relaxedtubules are present along these ribbons in the late fluid fillingphase. We also observed that the rounded CV membrane awayfrom the microtubular ribbons does not tubulate. We concludethat enhanced membrane tubulation during the rounding phaseis somehow promoted by the association of the CV membranewith the CV’s cytoskeleton of microtubular ribbons.

It has been demonstrated, where laser tweezer techniqueswere used to measure the membrane tension produced bytethers, that the tension of the plasma membrane is animportant factor in controlling the shape of the cell and itsmotility (Dai et al., 1998; Sheetz and Dai, 1996). Visualizationof the membrane dynamics exhibited by intracellularorganelles and vesicles is also important for understandingcellular processes such as membrane recycling and surfacemembrane expansion as occurs, for example, in neurons(Ashery et al., 1996; Dailey and Bridgman, 1993). For suchstudies the electrophysiological trigger technique we describehere and use to fix Paramecium at a precise stage of CV activityshould be adaptable and highly useful to study membranedynamics at precise times in other cells and organelles.

MATERIALS AND METHODS

CellsCells of P. multimicronucleatum (syngen 2) (Allen et al., 1988) weregrown in an axenic culture medium at 24°C (Fok and Allen, 1979)and harvested at the mid-logarithmic growth phase. These cells werewashed with saline solution containing (final concentration in mM)1.0 KCl, 1.0 CaCl2 and 1.0 MOPS-KOH buffer (pH 7.0). The cellswere equilibrated in the solution for more than 4 hours prior toexperimentation (Naitoh et al., 1997b).

Electrophysiological control of the timing of fixationA diagram including a flow-chart for the computer-assisted control ofthe timing of fixation of the in situ CV during the rounding phase isshown in Fig. 1. An equilibrated cell was placed into a small dropletof the saline solution under silicone oil on a small (2 × 2 mm) sectionof a glass slide. Excess saline was pipetted out of the droplet until thecell was squeezed sufficiently by the saline-oil boundary to becomeimmobile.

The tip of a microcapillary electrode filled with 3 M KCl (e1;approximately 50 MΩ) was inserted into the cytosol and grounded. Afine-tipped microcapillary electrode filled with 3 M KCl (e2;approximately 100 MΩ) was inserted into one of the CVs (CV1) inorder to monitor the membrane potential across the CV membrane(ECVC). This electrode also served to inject square current pulses (0.3nA) used to monitor the input resistance of the organelle. Inputresistance increased 5 times over that of the cell when the electrodetip successfully entered the CV (Tominaga et al., 1998b). An electricoscillation generated by a temporary (approximately 20 milliseconds)overcompensation of the stray capacitance of the head amplifier wasindispensable for successful penetration of the electrode tip into theCV (Tominaga et al., 1998b).

The tip of a microcapillary (approximately 10 µm in innerdiameter) filled with fixative containing 2% glutarardehyde in 50 mMcacodylate buffer was placed in the silicone oil at a distance ofapproximately 50 µm from the saline droplet where the cell was held(fixative pipette). This pipette was connected to the pressure outlet ofan electronic microinjector (IM-200, Narishige USA, Inc., Greenvale,NY, USA) which was used to squirt the fixative against the cell whenthe injector was activated by a timing signal from an A/D-D/A

T. Tominaga, Y. Naitoh and R. D. Allen

3735Membrane dynamics of the contractile vacuole

converter (ITC 16, Instrutech Corp. Great Neck, NY, USA). Thetemperature of the experimental vessel was held at 17°C.

ECVC was fed into a computer (Power Macintosh 7600/136, AppleComputer Inc. Cupertino, CA, USA) through an A/D-D/A converter.The computer was programmed to send an electric signal to themicroinjector to activate it when ECVC decreased from its normalvalue maintained during the fluid filling phase (approximately 60 mV)(Tominaga et al., 1998b) to a predetermined lower value (thresholdlevel for activating the injector) that is achieved at the start of thesevering of the radial arms from the CV. The injector that expelled thefixative against the cell responded with some delay (approximately 90milliseconds) after it was activated by the signal from the computer

(Fig. 1B). The timing of fixation during the rounding phase wascontrolled by changing the threshold level. To fix the cell during theCV’s much longer fluid filling phase the injector was manuallyactivated to squirt the fixative against the cell. The hydrostaticpressure of the inside of the microcapillary had been kept slightlylower than the atmospheric pressure to prevent leakage of the fixativethrough the opening of the capillary before activating the injector.Also the oil prevented unintentional mixing of the fixative with thesaline solution before injector activation. The amount of fixativesquirted into the droplet was approximately 0.2 µl, which is 10 timesas large as that of the saline solution surrounding the cell. Softwarefor feeding the electrical signals into the computer and for generating

Fig. 1. Schematic representationof the procedures for theelectrophysiologically controlledfixation of a Parameciummultimicronucleatum cell.(A) Arrangement of theelectrodes and a flow-chart ofthe computer-assisted controls.e1; a grounded microcapillaryelectrode has its tip inserted intothe cytosol. e2; a fine-tippedmicrocapillary electrode has itstip inserted into one of the twoCVs (CV1; another is labeledCV2) through which thepotential difference across theCV membrane (ECVC) ismeasured. (B) Time course ofchange in ECVC. ECVC graduallydecreases as the CV enters therounding phase. A horizontalsolid line corresponds to thelevel for ECVC during the fluidfilling phase. The upperhorizontal dotted linecorresponds to the thresholdpotential level for triggering anelectric signal for activating thefixative injector. ∆E; thedifference between ECVC duringthe fluid filling phase and thethreshold level. The thresholdpotential was set by changing∆E in the control system. Thelower horizontal dotted linecorresponds to the reference(cytosolic) potential level. Thefixative is squirted against thecell with a certain delay after thetriggering signal is fed into theinjector.

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the electric signals required for the experiments was developed on thebasis of the IgorPro (WaveMetrics, Inc., Oswego, OR, USA) andpulseControl XOP software packages (Herrington et al., 1995).

Video microscopyImages of the cell obtained using Nomarski optics (×40 objective lenson a Leitz-DMIRB microscope, Leica Mikrosk. u. System. GmbH.Wetzlar, Germany) were monitored on a television screen after captureby a video camera (CCD-72, Dage MIT Inc., Michigan City, IN, USA;Fig. 1A) and were recorded continuously by a video cassette recorder(AG6300, Panasonic Indust. Co., Secaucus, NJ, USA) at 30 framessecond−1 during the experiments. The video images of the contractilevacuole were fed into the same computer using a frame grabber (LG-3, Scion Corp., Frederick, MD, USA) for determining the moment offixation of the cell.

Fixation of the ruptured cellsAn equilibrated cell was placed in a small drop of a saline solutionon a glass slide which was then covered with silicone oil. Excesssaline solution was removed through a micropipette until the pressureexerted by the oil-water interface ruptured the cell. The cells werefixed for varied times of not less than 15 minutes and not longer than

4 hours in 1% glutaraldehyde in 50 mM cacodylate buffer (pH 7.4).The fixative was microinjected against the cell under the oil. The cellwas video taped for future reference.

Electron microscopyFixed cells were washed 2 hours in 50 mM cacodylate buffer (pH 7.4)and post fixed in 1% osmium tetroxide in 50 mM cacodylate bufferfor 30 minutes. After a 30 minute wash in distilled water the cellswere prestained in a 4% aqueous solution of uranyl acetate for 1 hour,washed 10 minutes in water and dehydrated in an ethanol series.Embedment was in Epon 812. All the procedures of electronmicroscopic preparation, except curing the Epon 812, were performedat a room temperature of 24-26°C. Stereo pairs were taken by tiltingthe specimen stage in the electron microscope plus and minus 10°before taking pictures.

RESULTS

Fixation of the cell at the time of the CV’s roundingA series of consecutive images of a representative cell ofParamecium and an intermittent trace for the electric potential

of the CVC, with reference to the cytosolicpotential level (ECVC) as recorded simultaneouslybefore, during and after computer-aided fixation ofthe cell, are shown in Fig. 2 (A, ECVC; B, cellimages). ECVC had been almost constant atapproximately 60 mV during the fluid filling phase(frames 0-6). It then began to decrease as the CVbegan to round (frame 7). When ECVC decreasedto a threshold potential level, which was set at


Fig. 2. Changes in the membrane potential (A) and the shape (B) of a CV of a P. multimicronucleatum cell before, during and after fixation ofthe cell. Lower dotted line in A corresponds to the reference (cytosolic) potential level (0 for ECVC). Upper dotted line in A corresponds to thethreshold potential level for activating the fixative injector. Arrowheads in A correspond to the time when the images of the cell shown in Bwere taken. Numbers next to arrowheads correspond to the frame number in B. In frame 0 in B, the CV containing the fine-tippedmicroelectrode, e2, is labeled. This e2 electrode is not in sharp focus. e1 is the microelectrode whose tip is in the cytosol. e1 and e2 correspond toe1 and e2 in Fig. 1, respectively.

A

µ


approximately 50 mV in this case, the computer systemgenerated an electric signal to activate the injector after acertain delay (approximately 103 milliseconds). The momentof activation of the injector could be recognized by a downwardspike on the potential trace which occurs as an artifact signalfrom the injector. Approximately 97 milliseconds after the startof fixative injection into the saline droplet, the ECVC quicklyfell to 0. This moment corresponds to frame 8 where the cellimage is blurred by the cell’s displacement caused by thefixative current against the cell and/or cell contraction. The CVwas at the middle of its rounding phase when the fixative wasinjected. ECVC, then, began to increase to a steady level ofapproximately 35 mV that was reached in a second or two. Thissteady potential level varied case-by-case and is thought to bean artifactual tip potential caused by fixed membrane orcytosolic material forming a plug in the tip of themicroelectrode. CV activity ceased immediately after fixativeinjection, and the CV remained rounded, as is clearly seen inframes 9-11. The cell’s ciliary activity also ceased immediatelyafter fixative injection and ciliary orientation remained in areversed direction (cilia are poorly seen in Fig. 2B).

Electron micrographs of the CV of a cell thus fixedElectron microscopy of a serially sectioned CVC in a cell fixedas the CV had reached the late rounding phase (Fig. 3A)demonstrated that the CV’s pore was still closed but themembrane systems along the radial arms had become separatedfrom the CV. The CV was rounded following fixation (Fig. 3B)but due in part to the mechanical stress required to remove themicroelectrode that was used to monitor the CV’s membranepotential from the CV, its shape had become more oval thanspherical (Fig. 3C). A section passing tangentially through thepore region at the CV’s peripheral hemisphere is shown in Fig.3D. The membrane of this rounded CV is for the most partplanar (Fig. 3E) with no sign of extensive tubulation except inthe immediate vicinity of the microtubular ribbons.

These ribbons originate at the surface of the CV pore andpass over the peripheral hemisphere of the CV (Hausmann andAllen, 1977). Unlike the membrane that covers most of the CVthe CV membrane near the microtubules takes on a highlymodified topography (Fig. 3F,G). Here the membrane formsclumps of tubules of 40 nm or so in diameter or a network ofsuch tubules. It was impossible to determine the amount ofsuch tubulated membrane but the impression received is thatthe amount could represent a significant fraction of that makingup the planar CV membrane.

Against the rounded CV a microtubular ribbon lies flattenedin the plane of the spherical CV membrane. All excessmembrane extends as tubules that form along the edges of themicrotubular ribbons or that protrude from between adjacentmicrotubules where groups of microtubules of the ribbon areseparated by a narrow gap. These tubules often extend aroundto the cytosolic face of the ribbons where they becameassociated with the ribbons’ cytosolic side and, in effect,partially enclose the ribbon.

Such tubulation occurs all along the ribbons from the poreto the beginning of the radial arms (Fig. 3G). Such non-planarmembrane is even more pronounced along the extended radialarms where extensive networks of tubulated membranes occurdepending on the phase of fluid filling or expulsion in theampullae. These networks make up what has been called the

smooth spongiome (for terminology see Patterson, 1980). Evenwhere the ampullae are no longer continuous with the CVmembrane, sectioned tubules can be found along themicrotubular ribbons in the gap between the rounded CV andthe emptied and more-or-less collapsed ampullae.

Fig. 4 shows that the membrane of the ampulla/collectingcanal that is attached to the cytoskeleton of microtubularribbons along a radial arm is not continuous with the CVmembrane at the rounded phase. Images of three sections in aset of serial sections (Fig. 4A-C) of the collecting canal at itsjuncture with the CV show a small gap (Fig. 4B) between theCV and the canal bridged only by the microtubular ribbon(Fig. 4A and two additional sections not shown between A andB). As reported earlier (Allen and Fok, 1988) there is usuallyno special cytoskeletal material evident at this juncture evenin cells fixed at the rounding phase at the time this connectionis known to be severed. These serial sections, however, doshow that in cells that have had a microelectrode inserted intotheir CV the CVC organization can be altered. In Fig. 4 it isevident that the decorated tubules have come to lie against theCV membrane. In in vivo cells the decorated tubules are neverfound to lie around the ampullae (Allen and Fok, 1988) andso they are always kept a distance of a few micrometers (∼ 5µm) away from the CV membrane. Thus the time needed toinsert the microelectrode into the CV and to begin to obtainthe video images and electrophysiological recordings waslong enough for the CV to have gone through a few filling andexpulsion cycles. During this time the CV often becomesenlarged which requires significant amounts of additionalmembrane to be incorporated into the CV membrane. In Fig.4 the membrane of the ampulla has apparently been used toenlarge the CV.

In a cell fixed by activating the fixative injector manually atits CV’s fluid filling stage, when the CV membrane potentialremained at a relatively constant level of 60 mV or more, theserially sectioned CV was found to be attached to the radialarms, the CV pore was closed and the CV’s shape was less thanspherical (Fig. 5A). The microtubular ribbons near the porewere still attached to the CV membrane but the normal rigidityof these planar microtubular ribbons that arise from the side ofthe pore caused the membrane of the CV to assume a flutedcross-sectioned profile near the pore (Fig. 5B). The membraneof the CV appeared to be more relaxed, i.e. it did not have thesame extensive microtubule-associated tubulation seen in therounding phase (Fig. 5C,D). Tubules could still be seen nearthe microtubular ribbons but these were less extensive,particularly near the pore, wider in diameter and seemed to beunder less tension as though they might be in a state ofrelaxation.

Stereo images of tubulated membrane in rupturedcellsSome whole cells clearly show a close relationship between theCV membrane and the microtubular ribbons in the lateexpulsion phase (Naitoh et al., 1997b). However, cells rupturedand flattened at the end of the fluid expulsion phase (Fig. 6),clearly showed the CV membrane to be extensively tubulatedinto uniform 40 nm diameter tubules. These tubules assumedmostly a two-dimensional plane against the microtubularribbons where they came to lie oriented more-or-lessperpendicularly to the long axis of the ribbons. Stereo-pairs

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show this relationship best (Fig. 6, use a stereo viewer foreasier recognition). The tubules are spaced almost equally inthis stereo image. This spatial association is highly suggestivethat microtubules play some essential role during the collapseof the planar CV membrane into these tubules as their collapseseems to be centered on the ribbons.

Ruptured cells also clearly demonstrate the three-dimensional network of the non-planar smooth spongiomealong the radial arms. The three-dimensional lattice is mostevident in a stereo pair (Fig. 7). The membrane of the latticecan be described as having a cubic symmetry (seeDiscussion).


Fig. 3. A CVC of a P. multimicronucleatum cell fixed during the CV’s rounding phase. The cell was fixed when the membrane potential acrossthe CV membrane, ECVC, decreased to a threshold potential level set to trigger the discharge of the fixative against the cell. (A) The trace of theelectrical potential difference between e1 and e2, as shown in Fig. 1. The left open arrow indicates the moment when e2 was inserted into theCV. The potential, hereafter, corresponds to ECVC. A horizontal dotted line corresponds to the threshold potential (approximately 62 mV) usedfor triggering injection of the fixative. The right filled arrow indicates the moment when the fixative discharge started. (B) A light micrograph ofthe CV taken 500 milliseconds after the fixative discharge. Bar, 40 µm. (C) A section through the middle of the same CV as that shown in B.Area between brackets is enlarged in E. Bar, 10 µm. (D) A tangential section of the same CV showing the CV pore (p) and the attachment to aradial arm, boxed areas to left and right, respectively. (E) CV membrane away from microtubular ribbons is planar. Bar, 0.5 µm.(F) Enlargement of the CV through its surface next to the pore (boxed area to the right of D). Grazing sections of microtubular ribbons(arrowheads) that are attached to the CV membrane are interspersed with non-planar membrane tubules (arrows) lying to the sides of theribbons. Bar, 0.2 µm. (G) Membrane tubules (arrows) are also found along the microtubules (arrowhead) that pass from the CV to the radialarm (boxed area to the left of D). Non-planar, tubulated membrane is found on both sides of the microtubules. Bar, 0.2 µm.


Regeneration of the CVAttempts to push the tip of a microelectrode into the CV duringelectrical oscillation frequently caused the CV to rupture anddeflate. We observed that a new CV soon appeared (within afew seconds) after the original CV was ruptured. Moreover, athird CV could appear again soon after the second CV had beenruptured by further electrical oscillation. This renewal of theCV could be repeated several times until the cell deteriorated.

DISCUSSION

Computer-aided electrophysiological control of thetiming of fixationWe previously proposed a hypothesis that the periodicexocytotic activity of the CV in the Paramecium cell isprimarily governed by periodic development of tension in theCV membrane which is associated with the retrieval, i.e.topographical isolation of some of its membrane due to itstransformation into membrane-bound tubules that have adiameter of approximately 40 nm (enhanced tubulationactivity) (Tominaga et al., 1998a). In order to examine electronmicroscopically the beginning of the transformation of the CVmembrane into tubules, we needed to fix the CV precisely inthe very short rounding phase that occurs immediately prior tofluid expulsion.

At the start of the rounding phase the electrical potential,which is approximately 60 mV across the CV membrane withreference to the cytosolic potential, began to decrease toward0 (Tominaga et al., 1998b). We, therefore, utilized this decreasein the CV potential for triggering the generation of an electricsignal (controlled by a computer) to activate an injector toinject fixative into the small saline droplet that contained thecell (Figs 1, 2). The saline droplet was under silicone oil. Thetiming of fixation could be controlled by changing thethreshold potential level for generating the electric signal forfixative injection. When the threshold potential level waslowered the electric signal could be generated later fixing theCV at a later rounding phase.

We conclude that the computer-controlled injector triggeringsystem we developed required less than three seconds, possiblyas little as half of a second, to fix individual cells as the electronmicrographs show that the impaled CVs are all within the 1 to3 second window of the rounding phase, i.e. the CV pore isstill closed while the ampullae have already separated from theCV. This membrane potential-controlled fixation system isunique and is potentially useful for electron microscopicalexamination of cells in relation to a variety of morphologicaland physiological activities where physiological parameterssuch as membrane potential can be measured.

Tubulation of the CV membrane during the roundingphaseElectron microscopy of the CV membrane at very preciselytimed stages indicates that the rounding phase is accompaniedby an enhanced membrane tubulation of its excess membrane.This tubulation only occurs along the margins of themicrotubular ribbons (Fig. 3F,G) and does not occur over theplanar non-microtubule-associated parts of the CV (Fig. 3E).As tubulation progresses the tubules may collapse into a three-dimensional network of membranes that comes to cover the

Fig. 4. Sections of a serially sectioned CVC of P. multimicronucleatumthat show the relationship of a collecting canal (cc) with the CV (cv)during the rounding phase. Like the CVC in Fig. 3 this CVC was fixedafter the membrane potential across the CV membrane decreased tothe threshold level for triggering fixative discharge against the cell.Such microelectrode-impaled CVs tended to become abnormally large.(A) Section 1 shows the microtubular ribbon (arrowhead) that reachesfrom the CV membrane to pass along the collecting canal (cc) which isout of the plane of this section. The smooth spongiome (ss) thatencircles the collecting canal is present as is the peripherically placeddecorated spongiome (ds). (B) Section 4 is one of the first sectionsshowing the collecting canal (cc). Thus, the collecting canal is seen tobe separated from the CV by a short gap. (C) Section 14 in this seriesshows the collecting canal (cc) with its microtubular cytoskeleton (leftarrowheads) enclosed by the non-planar membranes of the smoothspongiome (ss) and, peripheral to this, the decorated spongiome (ds)that lies abnormally close to the membrane of the enlarged CV.Sectioned trichocysts (t) can be used to vertically align the serialsections. Bar, 0.5 µm.

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cytosolic side of the ribbon and thereby partially encloses theribbons. Such enclosing networks are commonly found allalong the ribbons of the radial arms.

This observation implies that rounding is brought about byan enhanced tendency of the membrane to tubulate and that themembranes assume non-planar symmetries. We now observe

that tubulation itself is produced mainly in association withmicrotubules. Such a mechanism in which the CV rounds as aresult of the tubulation of excess membrane does not require acontractile cytoskeleton covering the entire CV membrane,which we never see. However, a lack of a global cytoskeletonaround the CV does not rule out the possibility that an actin-myosin type of contractile process may indeed be associatedwith the microtubules and might be involved in a morerestricted membrane tubulation. In this regard, Doberstein etal. (1993) reported the presence of myosin I around thecontractile vacuoles of Acanthamoeba. We do sometimes seea zone of exclusion around the membrane tubules, whereribosomes and other small cytosolic components are preventedfrom approaching the tubules. What lies around the tubules andbetween the meshes of the tubular network is currentlyunknown.

The appearance of the CV membrane in the fluid fillingphase (Fig. 5) suggests a more relaxed membrane in which themembrane tubules near its microtubular ribbons are in theprocess of expanding into a more planar topography as the CVfills. This appearance fits a cyclic process of enhancedmembrane tubulation during the short period of rounding andfluid expulsion which is followed by a much longer period ofmembrane relaxation that covers most of the CV cycle.

The membranes of the radial arms also undergo fluid filling-expulsion cycles that occur out-of-phase with that of thetubulation-relaxation cycle of the CV membrane. The radialarm membranes tubulated after they had expelled their contentinto the CV when the CV membrane is relaxed. Thesemembranes then became planar as the ampullae, no longerconnected to the CV, now fill. If enhanced tubulation of thefilled ampullae occurs just before fluid expulsion as it does inthe CV, this might be manifested as a movement of tubulesalong the microtubular ribbons where the membranes ofampullae would be expected to encounter the collapsed CVmembrane bringing about fusion of these two membranes.Fusion would permit the release of the contents of the ampullaeinto the CV.

A role for microtubules in membrane tubulationElectron micrographs of serially sectioned CVs and,particularly, stereo images of ruptured cells make it clear thatthe collapse of the CV membrane is focused on sheets ofmicrotubules during the fluid expulsion phase. However, howmicrotubules promote this membrane tubulation is not known.


Fig. 5. A CVC of a P. multimicronucleatum cell fixed during theCV’s fluid filling phase. (A) Low magnification of the CV (cv) at thelevel of the CV pore (arrowhead). Bar, 5 µm. (B) The CV (cv)membrane is fluted near the pore (p) due to its regular associationwith the microtubular ribbons (arrowheads). The CV membraneshows little evidence of tubulation near the pore during the fluidfilling phase. Bar, 0.5 µm. (C) Junction of a collecting canal with theCV which is open to the CV (cv). The non-planar membrane(arrows) along the microtubular ribbons (arrowheads) at this junctionappears relatively relaxed compared to the tubules at a similarlocation in the rounded CV. Bar, 0.2 µm. (D) Another connection ofthe CV (cv) to a collecting canal showing the microtubularcytoskeleton (arrowhead) and related non-planar membranes (longarrows) next to the ribbon. Openings of tubular invaginations (shortarrows) appear in the gap between two parts of the ribbon in thissection. Bar, 0.2 µm.


What is known is that ER membranes can be pulled out intolong tubules as a result of their attachment to microtubules(reviewed by Schroer and Sheetz, 1991). In this case the ERmembrane is tethered to the microtubules and is presumablypulled along the microtubules by motors such as kinesin orcytoplasmic dynein that can bind the membranes to themicrotubules. It is possible in Paramecium that suchassociations may occur between the microtubular ribbons ofthe CV cytoskeleton and the CV membrane. Bridges ofunknown composition can always be seen between the ribbonsand the membrane (Hausmann and Allen, 1977) both duringthe fluid filling phase when tubulation is not enhanced as wellas in the rounding phase when tubulation is enhanced.

Enhanced tubulation activity begins at the rounding phaseand may persist to the end of the fluid expulsion phase. Thisenhanced tubulation activity is initially visualized as anincrease in membrane tubule activity along the microtubularribbons and the tightening of the CV membrane around itscontent to form a sphere. This precedes and leads to theelectrophysiologically detectable detachment of the radial armsfrom the CV membrane (Tominaga et al., 1998b). Thisdetachment is physically demonstrated in this study andis shown to occur before the CV pore opens. Theelectrophysiological triggering of fixation of the CV at aspecific time has allowed us to view the physical image of theCV at a very precise phase, a feat impossible to attain by

random sampling of fixed CVs. Thus we show that roundingis initiated at the exact time that the membrane of the CV showsenhanced tubulation activity along its microtubular ribbons.

The tubulation activity is initially exhibited as an array of 40nm tubules lying flat against the microtubular ribbons and atvarious angles to the long axis of the ribbon itself. In the radialarms this tubulation is further enhanced into the formation ofnetworks of membranes which exhibit cubic morphology suchas that discussed by Landh (1995) of which a bi-continuousform was recently described in mitochondria of an amoeba(Deng and Mieczkowski, 1998). Cubic membranes are nowpostulated to occur in caveolar membranes (Rietveld andSimons, 1998). Thus the smooth spongiome membranetogether with the CV membrane itself may fall into a categoryof biological membranes of growing interest and of potentialimportance that collapse into a membrane that exhibit tubularand/or cubic symmetry when their bending energy is released.We have already shown that the CV membranes ofParamecium potentially store enough bending energy toaccount for the work done by the in vitro CV during fluidexpulsion when their membrane reverts from a planar to atubular form (Naitoh et al., 1997a).

Utilization of the smooth spongiome for increasingthe CV’s sizeObservations of the displacement of the decorated spongiome

Fig. 6 Stereo pair of the CV pore (p) and microtubular ribbons in a section of a ruptured P. multimicronucleatum cell. The CV was at the end ofthe fluid expulsion phase. The CV membrane has collapsed into a complex tubular system (arrows) lying parallel to the microtubular ribbons(arrowhead). Some tubules lie free of microtubules. Bar, 0.5 µm.

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toward the CV membrane due to external manipulation of theCVC, such as is viewed in Fig. 4, or to naturally occurringconditions that prevent the CV pore from opening (Tominagaet al., 1998a) show that the smooth spongiome from around theampullae and the collecting canals can become incorporatedinto the CV membrane. Thus the CV membrane and thesmooth spongiome membrane appear to be interchangeable.However, we have no morphological or other evidence that thedecorated tubular membrane ever becomes continuous directlywith the CV membrane. The existence of a monoclonalantibody that we had raised against Paramecium protein thatlabels the membranes of the CV, collecting canals and smoothspongiome, along with the plasma membrane, but not thedecorated tubules (Fok et al., 1995; Naitoh et al., 1997b), hadalready suggested that the CV and smooth spongiomes weresimilar to each other but different from the decorated tubules.Thus, rapid growth of the CV appears to be possible byincorporation of the smooth spongiome into the CV membrane.

Based on their electron micrographs of the contractilevacuole complex, Fok et al. (1995) estimated the total surfacearea of the decorated spongiome of the two CVs of a singlecell with a standard size to be approximately 21×103 µm2. Wepreviously reported that the interiors of all parts of thecontractile vacuole complex during the fluid filling phase areelectrically isopotential (Tominaga et al., 1998b). The inputcapacitance of approximately 180 pF obtained from a singleCVC at this phase indicates the organelle’s total membranearea is approximately 18×103 µm2, which is calculated basedon the assumption that the specific membrane capacitance ofthe CV membrane approximates 1 µF cm−2 as is estimated forother conventional biomembranes (Cole, 1968). The area of thesmooth membrane of a single CVC, therefore, approximates

7.5×103 µm2. This area is sufficient to surround a CV of 49µm in diameter in its rounding phase. It has been reported thatthe CV, whose normal in vivo size is approximately 12 µmdiameter in standard saline solution, can become as large as 50µm in diameter when its CV is inhibited from expelling itscontent through its pore by exposing the cell to 33 µg l−1

cationized ferritin in the external saline solution (Naitoh et al.,1997b). This coincidence of the membrane area for a CV ofmaximum size with that for the total amount of smoothmembrane of the CVC also supports our idea that smoothmembranes of the contractile vacuole complex can be utilizedby the CV when it is needed for increasing CV size. It will benecessary to investigate the mechanism by which the smoothmembrane is differentiated into the CV, ampullae, collectingcanals and smooth spongiome, which may differ in theirfunction depending on their position within the CVC.Involvement of cytoskeletal factors, such as microtubularribbons, in this differentiation would be especially interesting.


Fig. 7. Stereo pair of the collecting canal(cc) and smooth spongiome (arrows) of aradial arm in a section of a ruptured P.multimicronucleatum cell. The CV was atthe end of the fluid expulsion phase. Thesmooth spongiome membrane exhibitscubic symmetry. Arrowheads, microtubularribbons. Bar, 0.5 µm.

Fig. 8. Schematic drawings of the CVC and its exocytotic cyclegiving special attention to the enhanced tubulation activity of theirmembranes along the cytoskeleton of the microtubule ribbons in P.multimicronucleatum. (A) The CVC. CV, the contractile vacuole; AP,an ampulla; CC, collecting canal; SS, smooth spongiome; DS,decorated spongiome; MTR, microtubular ribbon; P, pore of the CVthrough which its fluid content is expelled to the exterior of the cell.(B) The enhanced tubulation activity in association with anexocytotic cycle. The tubulation activity is indicated by themembrane projections that correspond to the tubules, T. The lengthof the projection corresponds to the relative degree of the tubulationactivity. Longer (shorter) projections correspond to higher (lower)activity. (C) More detailed membrane dynamics associated withclosure of the CV pore after fluid expulsion. See the text for details.


c b a

CVAP

a

MTRT

CV

T

AP

b

CVAP

CVAP

APCV AP

c

def

P

P

CV

APSS

DS

MTR

P

CC

A

B

C

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Exocytotic cycles of the CV with special attention tothe tubulation of the membraneBased on our present results together with previous publishedresults (Naitoh et al., 1997a,b; Tominaga et al., 1998a,b) wehave produced a schematic drawing of the exocytotic cycle ofthe CV and ampullae giving special attention to the enhancedtubulation activity of their membranes along the microtubuleribbons (MTR; Fig. 8B). A more realistic drawing of a portionof the contractile vacuole complex is presented in Fig. 8A.During the fluid filling phase (Fig. 8Ba) of the CV the tubulationactivity is high in the ampullary membrane (the degree of theactivity is shown as the relative length of the membraneprojections that represent the tubules) so that the ampullae arethin. Segregated water enters the CV from the ampullae so thatthe CV swells. The tubulation activity of the CV membrane ispredicted to be low in the fluid filling phase (reduced numberof membrane projections). At the end of the fluid filling phaseenhanced tubulation activity in the CV membrane becomeshigh, so that the CV membrane starts to actively tubulate. Thetubulation of the CV membrane results in an increase in thetension in the CV membrane, which leads to severing of theampullae from the CV and the rounding of the CV (the roundingphase; Fig. 8Bb). As the membrane tension reaches a maximumat the end of the rounding phase, the CV’s pore (p in Fig. 8Bc,d)opens, so that the segregated fluid in the CV is expelled to theoutside of the cell through the pore. Expulsion is due mainly tothe in vivo cytosolic pressure (Naitoh et al., 1997b) (fluidexpulsion phase; Fig. 8Bc,d). The CV membrane then continuesto tubulate as the segregated water is expelled, althoughenhanced tubulation activity may be reduced in this phase (Fig.8Bd). The pore closes at the end of fluid expulsion as themembrane tension decreases (Fig. 8Be). The ampullae begin toswell immediately after the separation of the ampullae from theCV, as segregated water continues to flow into the ampullaefrom the collecting canals (Fig. 8Bc,d,e). The swollen ampullaereattach to the tubulated CV membrane and fluid from theampullae enters the CV so that the portion of the tubulated CVattached again to ampullae (to the left in Fig. 8Bf) quicklyswells. The tubulation activity in this phase is apparently highin the ampullary membrane and low in the CV membrane. Withthe reattachment of additional ampullae to the CV additionaltubules of the CV swell and fuse with the previously swollenportion of the CV thus forming a single CV. Segregated watercontinues to flow into the CV through the ampullae which arenow thin due to their membrane being tubulated (back to Fig.8Ba).

Detailed membrane dynamics associated with closure of thepore, which corresponds to Fig. 8Bd,e, are shown in Fig. 8Cand are based on what normally occurs during fusion andfission of biological membranes. The plasma membrane,continuous with the CV membrane after pore opening (Fig.8Ca), fuses immediately after the CV collapses at the end offluid expulsion (Fig. 8Cb). To retain continuous membranes thelongitudinal section of the pore at this stage should show threelipid bilayers across the pore although we have yet to capturethis stage in our electron microscopic studies. Two of the lipidbilayer membranes, the two derived from the CV, should thenfuse. The tubulated CV may then be pulled apart along themicrotubular ribbons, so that the pore is now covered only bythe plasma membrane (Fig. 8Cc), the situation we usuallyobserve at this stage.

As is depicted in Fig. 8B, an enhanced tubulation cycle ofthe ampullary membranes may precede that of the CVmembrane. This phase difference in the tubulation cycles of theCV and the ampullae would require that the membranedynamics associated with the exocytotic activity of thecontractile vacuole be well coordinated. The mechanism bywhich the timing of tubulation in both membranes is controlledremains unknown. The possible involvement of themicrotubule ribbons in the timing mechanism should beinvestigated.

It is interesting to note here that severing of the ampullaefrom the CV, which can be detected as a sudden decrease inthe input capacitance of the contractile vacuole complex, takesplace even when the CV’s pore does not open for one reasonor another (Tominaga et al., 1998a). This implies that thecoordinated tubulation activity does not need to be directlyfollowed by fluid expulsion from the CV. Moreover, certainchemicals (such as cationized ferritin) or mechanical agitation(such as insertion of a microneedle) of the cell can causeprolongation of the period of the exocytotic cycle, although therate of water segregation which is at least partially under thecontrol of the decorated spongiome (Ishida et al., 1993) is notaffected (Naitoh et al., 1997b). This implies that the watersegregation activity of the contractile vacuole complex itself isnot dependent on the enhanced and coordinated tubulationactivity of the CVC membranes. Rather enhanced tubulationof the CVC membrane controls the gates in the plumbingsystem for moving the segregated water from its site of fluidcollection to the CV and ultimately out of the cell.

This work was supported by NSF Grants MCB 95 05910 and MCB98 09929. We thank Dr Tomomi Tani for his valuable comments. TheBiological Electron Microscope Facility is supported in part by NIHgrant RR-03061 and by NSF instrumentation grants

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