CALCIUM AND MICROTUBULE SLIDIN ING CILIARY ...CALCIUM AND MICROTUBULE SLIDIN ING CILIARY AXONEMES ISOLATED FROM PARAMECIUM CAUDATUM YOSHIHIRO MOGAM ANI KEIICHD I TAKAHASHI Zoological

J. Cell Sd. 61, 107-121 (1983) 107Printed in Great Britain © The Company of Biologists Limited 1983

CALCIUM AND MICROTUBULE SLIDING IN CILIARYAXONEMES ISOLATED FROM PARAMECIUMCAUDATUM

YOSHIHIRO MOGAMI AND KEIICHI TAKAHASHIZoological Institute, University of Tokyo, Hongo, Tokyo 113, Japan

SUMMARY

Microtubule sliding was induced in axonemes obtained from isolated cilia of Parameciumcaudatum when they were exposed to a reactivating solution containing ATP after mild treatmentwith trypsin. Over a very wide range of concentrations (lnM-4mM), Ca2+ in the reactivatingsolution had no effect on the proportion of axonemes that disintegrated as the result of microtubulesliding. Also, the velocity of sliding, determined by cinematography, and the polarity of the direc-tion of sliding-force generation, determined by electron microscopy with regards to the base-to-tipaxis of the cilium, were not affected by Ca2+. The results indicate that the Ca sensitivity, which isresponsible for the ciliary reversal response, was removed from the axoneme, possibly as the resultof trypsin treatment. It is thus unlikely that Ca sensitivity is attributable to the basic slidingmachinery that powers ciliary movement.

INTRODUCTION

In many organisms, Ca2+ has been known to play a key role in the regulation ofciliary and flagellar motility. Well-studied examples of Ca-dependent responses incilia and flagella include the ciliary arrest response of the bivalve gill (Murakami &Takahashi, 1975; Tsuchiya, 1976, 1977; Satir, 1975; Walter & Satir, 1978), theflagellar wave reversal in Crithidia (Holwill & McGregor, 1975, 1976), alteration offlagellar waveform in Chlamydomonas (Hyams & Borisy, 1978), and the quiescencein sea-urchin sperm flagella (Gibbons, 1980; Gibbons & Gibbons, 1980). However,the most intensive studies on the control of ciliary activity by calcium have beencarried out with the ciliary reversal response of Paramecium. Here, the classicalstudies of Naitoh, Eckert and their collaborators have established that the reversal isinduced by an increase in the intracellular Ca2+ concentration, which occurs as a resultof transmembrane Caz+ influx during the action potential (Eckert, 1972; Naitoh &Kaneko, 1972; Naitoh & Eckert, 1974). There is now substantial evidence that asimilar mechanism underlies the ciliary and flagellar responses in other organisms.

The intracellular site of action of Ca2+ and the mechanism by which Ca2+ causessuch a profound alteration of motility, as is found in the ciliary reversal and the arrestresponses, are, however, still unknown. The fact that Ca2+ induces the response inreactivated, detergent-demembranated cilia and flagella (Naitoh & Kaneko, 1972;Tsuchiya, 1976, 1977; Holwill & McGregor, 1975, 1976; Hyams & Borisy, 1978;Walter & Satir, 1978; Gibbons & Gibbons, 1980) indicates direct action of Ca2+ oncertain component(s) of the motile apparatus or a closely associated structure. The

108 Y. Mogami and K. Takahashi

presence of calmodulin in cilia (Jamieson, Vanaman & Blum, 1979; Gitelman& Witman, 1980; Walter & Schultz, 1981; Maihle et al. 1981; Ohnishi, Suzuki& Watanabe, 1982) indicates a possible role for calmodulin in mediating theCa-dependent ciliary responses.

In order to gain an insight into the mechanism of Ca2+-dependent control of ciliarymotion and to localize the site of action of Ca2+, we have studied the effect of Caz+

concentration on the ATP-induced sliding of microtubules in trypsin-treatedaxonemes oi Paramecium. The procedure used to induce microtubule sliding, origin-ally developed by Summers & Gibbons (1971) for sea-urchin sperm flagella, enabledus to test whether or not Caz+ acts directly on the basic sliding machinery thatprovides the motive force for ciliary movement. If ciliary reversal in Paramecium isbrought about by direct action of Ca2+ on the sliding system, we may expect thatCaz+ modifies certain parameters of sliding, such as the velocity and the direction, orpolarity, of active sliding between the adjacent doublet microtubules. In the gill ciliaof the freshwater mussel, Walter & Satir (1979) have shown that a high concentration(~10mM) of Ca2+ does not inhibit sliding disintegration of trypsin-treatedaxonemes, although Ca2+ at much lower concentrations completely inhibits ciliarybeat in axonemes not treated with trypsin. In Tetrahymena, too, active sliding intrypsin-treated axonemes is not affected by increased Ca2+ in the reactivation medium(Sale, 1977). Although these results indicate that the basic sliding mechanism of ciliais not directly controlled by Ca2+, it is desirable to confirm this in Paramecium, sinceso much attention has been paid to ciliary reversal in this organism (for a review, seeDoughty & Dryl, 1981).

MATERIALS AND METHODS

Isolation of axonemes

Paramecium caudatum (syngen 1, mating type I, II) were grown in hay infusion. Cells in the late-logarithmic phase were harvested by negative geotaxis. The cells (3-5 (X105) cells) were thenconcentrated by low-speed centrifugation (2200 g, 10-15 min) and washed thoroughly with washingsolution, containing 2mM-KCl, 2mM-CaCl2, 1 mM-4-(2-hydroxyethyl)-l-piperazine ethanesul-phonic acid (Hepes) buffered at pH7-5, by a second low-speed centrifugation (400-700jjf,2-5 min). Up to this step, the temperature during centrifugation was kept at about 8 °C. It was thenset at 0°C, from the next step.

Deciliation was carried out following the procedure developed by Satir, Sale & Satir (1975) forTetrahymena. The washed cells were concentrated and resuspended in 5ml of cold washingsolution. Then 0-5 ml of 12-5 min-dibucaineHCl was added to the suspension. After 3 min ofincubation on ice, the suspension was drawn several times through a pipette with a bore of about1 mm. The suspension was then diluted with cold washing solution. After removing cell bodies andthe large amount of discharged trichocysts by centrifugation at 720gfor 5 min, the supernatant wascentrifuged at 7700g for 10 min. The resultant pellet was suspended in solution A containing20mM-KCl, 4 mM-glycoletherdiamine-N, N, N', iV'-tetraacetic acid (EGTA), 1 mM-dithio-threitol, 30min-Hepes (pH7-5). Following the measurement of optical density of the suspensionat 350 nm, a sample corresponding to 0-4oD3so (containing about 200 fig of total ciliary protein) wascentrifuged (7700 gf, 10 min). To remove the ciliary membrane, the resultant pellet was treated with0-04% (w/v) Triton X-100 in solution A for 5 min on ice. The demembranated cilia (axonemes)were collected by centrifugation (7700 g, 10 min) and suspended in solution B containing 20 mM-KC1, 4mM-EGTA, 1 mM-dithiothreitol, 30mM-Hepes (pH 7-5). The suspension was stored on iceuntil used. All the experiments were carried out within 1 h of suspending the axoneme.

Microtubule sliding in Paramecium cilia 109

If proteolytic treatment was required beforehand, the above axonemal suspension was incubatedwith trypsin (from bovine pancreas, Sigma type III) at a concentration of 1 ^g/ml. Digestion wasterminated with an excess amount of soybean trypsin inhibitor (Sigma) after appropriate incuba-tion.

ReactivationThroughout the experiments, the basic reactivating solution consisted of 20niM-KCl, 4mM-

MgSO.), 0-1 mM-ATP, 1 min-dithiothreitol, 30mM-Hepes (pH7-5). We controlled the concentra-tion of free Ca2+ by adding CaCk with either EGTA or EDTA', at an appropriate ratio, to the abovesolution keeping the combined concentration of the two chemicals at 4mM. According to theirbuffering ability for Ca2+, the two chelating reagents were used for different ranges of Ca2+

concentrations: EGTA for 10"9 to 10"6M-Ca2+, and EDTA for lfT6 to 105M-Ca2+. Ca2+ con-centrations above 10~5M were prepared by adding appropriate amounts of CaCb without thechelator. The stabilized concentrations of free calcium ions in the buffer containing Mg2"1" werecalculated according to Goldstein (1979). The apparent stability constants corrected for the ionicstrength and pH were calculated from the true values given by Sillen & Martell (1964). Theconcentrations of Mg2+ and MgATP2" were 3-0±0-5 mM and 90±5JUM, respectively.

To reactivate the axonemes under a light microscope, a drop of sample was placed in a narrowspace between a slide and a coverslip, which were separated by two strips of thin adhesive tape. Atfirst, a solution of appropriate ionic composition was perfused through one of the narrow openingsat the edge of the coverslip while the excess fluid was drained from the opposite end by small piecesof filter paper. Then the axonemes attached to the glass were reactivated by perfusing the reactiva-ting solution in the same way. The reactivation was carried out at room temperature.

Observation and recording

Dark-field microscopic observation was made with a 100 W high-pressure mercury vapour lamp(Ushio, USH-101) as the light source. The proportion of axonemes that underwent disintegrationwas measured by direct observation and also by counting the images of disintegrated axonemes onphotomicrographs. In any one measurement, a total of about 300 axonemes, both intact anddisintegrated, were counted. To measure the velocity of microtubule sliding, the process ofmicrotubule extrusion from the axonemes was recorded with a 16 mm cine camera (Beaulieu R16Automatic, Beaulieu Cinema Co.) on Kodak Double-X film. The recording speed was set at 8frames/s with an exposure time of 25 ms for each frame. The frame interval was calibrated to aprecision of 10 ms by photographing a digital clock simultaneously with the axonemes through aspecially constructed optical system. The length of the extruded microtubules was measured frame-by-frame on a film motion analyser (Nac Model 160B, Nac Inc.).

Electron microscopy

A drop of sample that had been treated with trypsin was placed on a carbon-stabilized, Formvar-coated grid. After an appropriate time, the grid was drained and the axonemes attached to the gridwere reactivated by applying several drops of reactivating solution. After leaving the grid for about5 min to complete the reaction, the grid was drained again and the specimens were negatively stainedby successive application of several drops of 1-2% aqueous uranyl acetate. The specimens wereobserved with a Hitachi HS-9 electron microscope at 80 kV.

RESULTS

Reactivation of isolated axonemes

The isolated axonemes were not uniform in length. Most of them were shorter thanintact cilia. Short fragments 2—4/im long were commonly found. The proportion ofshort fragments increased after a vigorous mixing of the axonemal suspension, in-dicating that the short fragments were the result of breakage caused by mechanicalagitation during the course of preparation.


Perfusion with a reactivating solution caused a small proportion (approx. 10%) ofthe axonemes to disintegrate. Also, a small proportion (1—20%) of the axonemesshowed beating movements. This occurred only in those axonemes that were aboutthe same length as the intact cilia. Their beat form differed between individualaxonemes, apparently depending on the way they were attached to the glass surface.

The proportion of axonemes undergoing disintegration was increased by trypsintreatment. As shown in Fig. 1, a severalfold increase was observed after a 2minincubation with 1 pig trypsin/ml axonemal suspension on ice. Even after the trypticdigestion about 30 % of the axonemes did not disintegrate in response to perfusedreactivating solution. Apparently, the longer fragments of axonemes were more dif-ficult to disintegrate than the shorter ones. This was not due to insufficient digestion,since further proteolysis did not render the longer axonemes capable of disintegration;rather, it decreased the proportion of disintegrated axonemes. Therefore, the time fortrypsin treatment, when this was required before the reactivation experiment, was setat 3-5 min.

80 r

20 -

Incubation time (min)

Fig. 1. Effect of duration of trypsin digestion on the percentage of axonemes disintegratedon subsequent reactivation. Axonemes obtained from a turbidimetrically defined sampleof ciliary suspension were incubated with a fixed amount of trypsin (see text for detail).Digestion was carried out on ice and stopped after an appropriate time by adding soybeantrypsin inhibitor. Percentages of axonemes that had undergone disintegration were deter-mined after reactivation with Ca-free reactivation solution. Mean values and standarddeviations from four separate experiments are shown.


Disintegration of axonemes could also be induced by perfusion with a reactivatingsolution containing trypsin as well as MgATP. In this solution, disintegration ofaxonemes took place after the few minutes of exposure that were needed for sufficientdigestion to occur. Therefore, by perfusing the axonemes first with a reactivatingsolution without trypsin and then with the same solution to which trypsin had beenadded, it was possible to induce axonemal disintegration in strictly defined concentra-tions of MgATP and inorganic ions (Kamimura & Takahashi, 1979; Yano & Miki-Noumura, 1980; Takahashi, Shingyoji & Kamimura, 1982).

The ATP-induced disintegration of trypsin-treated axonemes observed by bothlight and electron microscopy is shown in Fig. 2. In most cases, microtubulestelescoped out in the reactivating solution. Banana-peel type images were rarelyobserved. This implies that the observed disintegration was caused by active slidingbetween the doublet microtubules.

The effect of Ca2+ on the active sliding of doublet microtubules was studied in threeaspects: the proportion of axonemes disintegrated by sliding, the velocity ofmicrotubule sliding, and the polarity of sliding force generated by the dynein arms.

Percentage of MgATP-induced disintegration in various concentrations ofCa2 +

The proportions of trypsin-treated axonemes that underwent disintegration onsubsequent exposure to reactivating solutions containing various concentrations ofCa2+ are shown in Fig. 3. The percentage did not change significantly over the widerange of Ca2+ concentrations used (< 10~8M to 4 X 10~4M), although precautionsmight be needed against possible effects of different chelating agents used for differentranges of Ca2+ concentrations. Over the entire range of Ca2+ concentrations, lightmicroscopy revealed that the axonemes disintegrated mostly by telescoping, indica-ting that microtubule sliding was not inhibited by the Ca2+ concentrations tested.

Electron micrographs of disintegrated axonemes also showed that the doublets slidin a telescopic manner regardless of the Caz+ concentration (data not shown). Sincethere were many images of axonemes that indicated that sliding had occurred at almostall of the interdoublet sites, it seems likely that all the doublets in the axoneme canslide, regardless of the Ca2+ concentration.

Sliding velocity

When axonemes previously untreated with trypsin were perfused, first with thereactivating solution containing a known concentration of Ca2+, and then with thesame solution to which trypsin (2 //g/ml) had been added, they underwent disintegra-tion regardless of the Ca2+ concentration. Observation under a dark-field microscoperevealed that the fragments of axonemes lengthened as microtubules were extrudedfrom their ends, with the length sometimes reaching several times the initial value,indicating that sliding took place in many interdoublet sites.

The sliding displacements of extruded microtubules with respect to the part of theaxonemes that remained fixed to the glass surface were measured and plotted againsttime. Examples of these measurements are given in Fig. 4, which shows linear rela-tions, i.e. constant speeds, for most of the processes. This is similar to results found

Y. Mogami and K. Takahashi

n r r r r r . i

2AFig. 2. A. Electron micrograph of negatively stained doublet microtubules disintegratedin the presence of 7-9 X 10~9M-Ca2+, showing telescoping-out of nine doubletmicrotubules together with the central tubules (ct). Bar, 1 ^m. B - D . Prints from dark-fieldcinematographic records showing sequences of the process of microtubule extrusion in-duced by reactivating solutions containing trypsin. Extruding microtubules are indicatedby arrowheads. Frame intervals, 125ms. Ca concentrations: 7 - 9 X 1 0 ~ 9 M , 2 5 X10~6M and 4-0 X 10~3M for B, C and D, respectively. Bar,


80 r

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20 -

0 L

pCa

Fig. 3. Percentage of axonemes disintegrated upon reactivation in the presence of variousconcentrations of Ca2+. The mean values and standard deviations, each obtained from10-20 measurements, are shown. I n all cases the concentration of MgATP was 90 ± 5 /IM .Different symbols represent different methods used to control the Ca2+ concentration:(O) buffered with EGTA; ( • ) buffered with EDTA; (3) addition of appropriate amountof CaCl2 without a chelating agent. Temperature, 25 ± 1 deg. C.

in reactivated, trypsin-treated axonemes of sea-urchin sperm (Yano & Miki-Noumura, 1980; Takahashiei al. 1982). In some cases, the apparent speed of slidingshowed a more or less sudden change. Examples of microtubule extrusion with chang-ing velocities are indicated by open circles in Fig. 4. These changes possibly arosefrom situations in which sliding took place at more than one interdoublet site in thetelescoping axonemes (Takahashi et al. 1982). If sliding occurs simultaneously atthese sites, the overall velocity of lengthening of the axoneme would be the sum of thesliding velocities of individual doublets relative to the adjoining doublets. Therefore,the overall velocity would increase as a result of recruitment of actively slidingdoublets and decrease as some doublets in the telescoping axoneme cease to slide. Asthe number of doublets in the extruded part of the axoneme could not be determinedwith certainty under the dark-field microscope, the number of doublets participatingin active sliding was unknown. In our analysis, therefore, the velocities of sliding weredetermined from curves such as those shown in Fig. 4, by simply measuring the slopesof the portions that showed reasonable linearity for more than 1 s.


E

c(D

JOQ.

c

Time(s)

Fig. 4. Time course of sliding extrusion of doublet microtubules in various concentrationsof Ca2+ (A, 7-9 X 1 ( T 9 M ; B, 3-2 X 1 0 " 6 M ; C, 2-5 X 1 0 " 5 M ) . Lengths of extruded portionsof microtubules were measured on tracings of enlarged cinematographic images. ( • ) Casesin which a linear relation indicating a constant speed was observed throughout the extrud-ing process; (O) those with a changing speed. The values given in the graphs indicatevelocities (in (ims"1) calculated from the slope of the linear portions. Active microtubulesliding was started by digestion of certain axonemal components with trypsin in thereactivating solution. Temperature, 23 ± 0-5 deg. C.


The sliding velocities in various Ca2+ concentrations are summarized in Fig. 5.Although the data show considerable scattering, most of them tend to be concentratedat approximately 2 jums"1, regardless of the Ca2+ concentration. If we assume that thehigher values represent those cases in which sliding took place simultaneously at morethan one interdoublet site and disregard them as not representing the basic slidingvelocity between two adjacent doublets (Takahashi et al. 1982), we may tentativelyconclude that, at any Ca2+ concentration between < 1 0 ~ 8 M and 4 x 10~3M, thevelocity of interdoublet sliding is about 2firas"'.

Polarity of force generation

Electron microscopy of negatively stained preparations of disintegrated axonemesshowed a configuration of spokes similar to that observed in other organisms (Warner& Satir, 1974; Sale & Satir, 1977). The spokes are grouped in triplets, which arerepeated regularly along one side of the A-tubule of each doublet at 96 nm intervals(Fig. 6A) . Within each triplet, the spokes were not equally spaced: one of the intervals(approx. 18nm) was narrower than the other (approx. 30nm). The polarity ofdoublets can be identified by using the spoke configuration.

The method of Sale & Satir (1977) was followed to identify the polarity of sliding-force generation. We selected electron-microscopic images showing two doublets thathad almost slid apart, leaving only a short overlapping region between them, and alsoboth spoke triplets and dynein arms. In these images, the dynein arms generating thesliding force between the two doublets were identified first. Then the polarity of force

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Fig. 5. Velocity of microtubule sliding in various Ca2+ concentrations. In all cases theconcentration of MgATP was 90 ± 5/iM. Symbols are used in the same way as in Fig. 7.Temperature, 23 ± 0-5 deg. C.


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generation was determined by using the spoke configuration as an indicator of direc-tion. Thus the direction of force generation was either towards the side of the narrowerinterval in the triplet (narrow side) or towards the opposite direction (wide side).

An electron micrograph of part of an axoneme disintegrated in reactivating solutioncontaining < 10~8 M-Ca2+ is shown in Fig. 6B. The spoke configuration visible on oneof the doublets is indicated by brackets. As can be judged from the position of the A-tubule, which can be distinguished from the B-tubule by its greater width, the dyneinarms that generated the sliding force between the two doublets are those on the A-tubule of the lower doublet. In this case, therefore, the dynein arms on the lowerdoublets pushed the upper doublet in the direction indicated by the arrow; in otherwords, the direction of force generation was towards the narrow side. The polaritythus determined was consistent in every case examined.

The polarity did not change when the Ca2+ concentration was increased above thecritical Ca2+ concentration (10~7 to 10~6M) for inducing ciliary reversal; throughoutthe range of Ca2+ concentrations tested (up to 4 HIM), the dynein arms always pushedthe adjacent B-tubule towards the narrow side. An electron micrograph of doubletsthat had slid in the presence of 10~3 M-Ca2+ is shown in Fig. 6c.

It is therefore concluded that Ca2+ does not affect the polarity of force generation.The results also show that in Paramecium, as in Tetrahymena (Sale & Satir, 1977),active sliding between adjacent doublets always occurs with the same polarity. InTetrahymena, the direction of force generation has been determined as being towardsthe distal end of the cilium. In other organisms whose cilia have spokes in asymmetrictriplet configurations, the narrower interval in the triplet has been reported to beoriented towards the distal end of the cilium (Warner & Satir, 1974; Olson & Linck,1977). If this is also the case in Paramecium, then the direction of force generationby the dynein arms is towards the distal end of the cilium (tipward). The relation ofthe spoke configuration to the base-to-tip axis of the cilium in Paramecium is, how-ever, still to be established.

DISCUSSION

Ciliary reversal in Paramecium is a classical example of calcium-dependent regula-tion of cell motility. In the living cell, it occurs as a response to membrane depolariza-tion caused by various stimuli, and plays an essential role in the well-known avoidingreaction that forms the basis of many kinds of tactic behaviour (for reviews, seeEckert, Naitoh & Machemer, 1976; Nelson & Kung, 1978; Doughty & Dryl, 1981).The coupling between the membrane excitation and the motile response of cilia is

Fig. 6. Electron micrographs of negatively stained doublet microtubules after slidingdisintegration of axonemes. A. A doublet showing a triplet configuration of radial spokes.B and C. Overlapping region between two doublets, which had slid in <10~8M and10~3M-Ca2+, respectively. The radial spoke triplets are indicated by brackets. In both Band C, the direction of force generation by the dynein arms is towards the side of the shorterinterval within the triplet as indicated by arrows. Bars, 0-1 jJn\.


brought about by Ca2+. However, although recent studies have indicated that Ca2+

concentration within individual cilia may be controlled by voltage-dependent Cachannels localized in the ciliary membrane (Dunlap, 1976, 1977; Ogura & Takahashi,1976; Machemer & Ogura, 1979), the mechanism of Ca2+ action within the cilium isnot well understood.

There are three possible motile mechanisms on which Ca2+ may act to cause theciliary reversal: (1) an independent motile system activated by CaATP to reorient theciliary beating; (2) a Ca-dependent control system that regulates the spatial andtemporal pattern of active sliding between the doublets; and (3) the sliding systemitself. This last possibility has been considered in the present study.

If Ca2+ were to act directly on the sliding system, it may affect either the rate or thepolarity of interdoublet sliding. The first of these two mechanisms has been suggestedby Machemer (1977). According to his hypothesis, Ca2+ would induce a ciliary rever-sal if different doublets were selectively activated at different Ca2+ concentrations toproduce effective strokes in different directions. In the present study, we found thatCa2+ does not affect the sliding in terms of both the percentage of disintegration andthe rate of microtubule extrusion. Although we were unable to determine whether thesliding activity differed among individual doublets within an axoneme, absence of anydetectable effect of Ca2+ on the sliding velocity, together with the light- and electron-microscopic observations that almost all doublets slide in a telescopic fashion, regard-less of the Ca2+ concentration, seems to indicate that in trypsin-treated axonemesCa2+ does not affect sliding in any doublet. Our results are consistent with those ofWalter & Satir (1979), who found that Ca2+ does not inhibit active sliding of doubletsin bivalve gill cilia that undergo ciliary arrest in response to an elevated intracellularCa2+ concentration (Walter & Satir, 1978).

The second of the possible mechanisms mentioned above has been discussed byHolwill & McGregor (1975) in their study of the Ca-dependent alteration of thedirection of wave propagation in the flagella of Crithidia. These authors suggested thepossibility that the effect of Ca2+ is to regulate the direction of swinging movementsof linkages (arms) that generate the sliding force. Our electron micrographs ofParamecium axonemes that had undergone sliding disintegration in various Ca2+

concentrations indicate that the direction of force generation by the dynein arms is notaltered by Ca2+. Thus, at least in trypsin-treated axonemes of Paramecium, Ca2+ atany concentration does not seem to cause backsliding of the doublets.

While our results indicate the absence of Ca sensitivity in the trypsin-treatedParamecium axonemes, motility-related effects of Ca2+ have been reported inaxonemes isolated from various sources. Thus, for example, Ca2+-dependent changesin the packed volume (pellet height response) of Tetrahymena axonemes have beenreported by Blum & Hayes (1977). The waveform of Chlamydomonas axonemes ismodified by Ca2+ (Hyams & Borisy, 1978; Bessen, Fay & Witman, 1980). In chemi-cally demembranated Paramecium, Ca2+ reverses the reactivated ciliary beatingabove about 10~6M, and also inhibits the ciliary beating above about 10~4M (Naitoh& Kaneko, 1972). It is therefore likely that Ca sensitivity, which originally existed inthe ciliary axoneme, was lost in the course of trypsin treatment. Although our results


tell us nothing about the nature of the change brought about by trypsin, we mayspeculate on certain axonemal components that might be involved in the change. Anobvious candidate is the Ca2+-dependent regulator protein calmodulin, since its exis-tence in cilia has been demonstrated in Paramecium (Walter & Schultz, 1981; Maihleet al. 1981), as well as Tetrahymena (Jamieson et al. 1979; Maihle & Satir, 1980;Ohnishi et al. 1982) and Chlamydomonas (Gitelman & Witman, 1980). InTetrahymena, calmodulin has been found to bind in a Ca-dependent manner to both14 S and 30 S dyneins and thus to confer Ca sensitivity on the dynein ATPase (Blum,Hayes, Jamieson & Vanaman, 1980). On the other hand, Ohnishi et al. (1982) foundno calmodulin or Ca-dependent binding of added calmodulin in Tetrahymena dyneinfractions, although Tetrahymena axonemes contained calmodulin and showed Ca-dependent binding to added calmodulin. They also showed by immunoelectronmicroscopy that calmodulin was localized along the doublet at 90 nm intervals andsuggested the interdoublet (nexin) links as possible calmodulin-binding sites. In viewof the present results, this last point is of particular interest since, in sea-urchin spermflagella, nexin links are highly susceptible to tryptic digestion (Summers & Gibbons,1973).

While the role of calmodulin in ciliary reversal remains speculative, a direct effectof Ca2+ on the conformation of doublets prepared from sea-urchin sperm flagella bytrypsin digestion of axonemes has been reported by Miki-Noumura & Kamiya (1979).These workers demonstrated, by dark-field microscopy, a reversible change in thecoiled configuration of isolated doublets induced by Ca2+, with a critical concentra-tion of about 10~7M, and suggested that this effect of Ca2+ might play a role in theCa-dependent changes in the asymmetry of flagellar wave-form found by Brokaw,Josslin & Bobrow (1974). However, the Ca-dependent asymmetrical beating of sea-urchin sperm flagella is abolished by mild digestion with trypsin (Brokaw & Gibbons,1975; Brokaw & Simonick, 1977), and so is the Ca-dependent quiescence, which isthought to be closely related to the asymmetry (Gibbons & Gibbons, 1980). Thesefindings suggest that both asymmetry and quiescence in sea-urchin sperm flagella are,like ciliary reversal in Paramecium, dependent on trypsin-sensitive axonemal com-ponents that are distinct from the trypsin-resistant sliding machinery.

Although much further study is needed to understand the role of Ca2+ in the controlof ciliary and flagellar motility, there seems to be emerging a consistent, although stillvague, picture for the mechanism of various Ca-dependent responses. In all theseresponses, Ca2+ seems to act not directly on the sliding system itself, but on either anindependent motile system or a Ca-dependent control system, which confers Casensitivity on the axonemal motile system. Identification and localization of the keycomponent in any one of the Ca-dependent ciliary or flagellar responses would greatlyincrease our understanding of the mechanism controlling ciliary and flagellar motilityin general.

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BROKAW, C. J., JOSSLIN, R. & BOBROW, L. (1974). Calcium ion regulation of flagellar beatsymmetry in reactivated sea urchin spermatozoa. Biochem. biophys. Res. Commun. 58, 795-800.

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DOUGHTY, M. J. & DRYL, S. (1981). Control of ciliary activity in Paramecium: An analysis ofchemosensory transduction in a eukaryotic unicellular organism. Progr. Neurophysiol. 16, 1-115.

DUNLAP, K. (1976). Ca channels in Paramecium confined to ciliary membrane (abstr.).Aw. Zool.16, 185.

DUNLAP, K. (1977). Localization of calcium channels in Paramecium caudatum. J. Physiol. 271,119-133.

ECKERT, R. (1972). Bioelectric control of ciliary activity. Science, N.Y. 176, 473-481.ECKERT, R., NAITOH, Y. & MACHEMER, H. (1976). Calcium in the bioelectric and motor functions

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{Received 22 September 1982-Accepted 18 November 1982)

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