/ . exp. Biol. (1976), 65, 229-342 2 2 9With 2 figures

Printed in Great Britain

EFFECTS OF CALCIUM ON FLAGELLAR MOVEMENT INTHE TRYPANOSOME CRITHIDIA ONCOPELTI

BY M. E. J. HOLWILL AND J. L. M C G R E G O R

Department of Physics, Queen Elizabeth College, London W8 7AH

(Received 6 February 1976)

SUMMARY

1. The effects of calcium on the motility of different preparations of flagellafrom Crithidia oncopelti were studied using stroboscopic and high-speed cinephotographic techniques.

2. By varying the concentration of calcium in suspensions of chemicallytreated samples of the organism it was found that changes occurred inbend shape, wave direction and frequency.

3. Waves on the flagellum of the organism in vivo possess the unusualability to propagate from tip to base, but reverse in direction during an avoid-ing response. In chemically extracted and reactivated preparations tip to basepropagation was observed only at low concentrations (< io~* mol mr3) ofcalcium ion; at high concentrations base to tip propagation only was seen. Incells treated with ionophores known to carry divalent ions across membranes,tip to base propagation was seen only in the presence of EGTA; when calciumwas added the majority of organisms propagated waves from base to tip.

4. At certain values (ca. io"3 mol m""3) of the calcium concentration thewave shape had meander-like characteristics, whereas at higher and lowerconcentrations it was more sinusoidal. At high calcium concentrations onlyone wave appeared on the flagellum whereas at low values two or three wereobserved.

5. A reduction in frequency at high calcium concentrations was probablydue to competitive inhibition of magnesium ions.

6. The results suggest that wave reversal in living Crithidia is induced bythe release of calcium ions within the flagellum following stimulation of themembrane. In terms of the sliding filament model of flagellar activity theeffects of calcium suggest that the ion is effective in modifying the interactionbetween the spoke head and central sheath and may control the relative direc-tion of microtubular sliding.

INTRODUCTION

The flagellum of the protozoan Crithidia oncopelti is capable of propagating wavesdistally or proximally; this property is shared with other trypanosomes (Holwill, 1965)Jahn & Bovee, 1968) but not with the majority of protozoa and spermatozoa, whichpropagate waves only from base to tip. Freely swimming Crithidia maintain proximallydirected waves unless stimulated mechanically, chemically or electrically, when atransient reversal of the wave direction occurs (Holwill & McGregor, 1974 and un-published results). If the distal portion of a flagellum is amputated using a microneedle

230 M. E. J. HOLWILL AND J. L. MCGREGOR

the proximal part still retains the capacity for wave reversal, although this capacity islost if the amputation is performed using a laser microbeam. The amputated part ofthe flagellum is also able to reverse its wave direction whichever method of amputationis used (Goldstein, Holwill & Silvester, 1970; Holwill & McGregor, 1974).

It has been possible to extract cells of Crithidia in glycerol- or detergent-basedsolutions and subsequently to reactivate them using adenosine triphosphate (ATP)(Douglas & Holwill, 1972; Holwill & McGregor, in preparation). These preparationsappear to have lost the capacity for wave reversal and propagate waves only from thebasal end of the flagellum. A similar effect is found when the cell body of an organismin a solution containing ATP is dissected to leave the flagellum intact with the basalend exposed to the external medium (Holwill & McGregor, 1974).

In a large number of excitable plant and animal cells the mechanical response to astimulus is associated with the production of bioelectric potentials (Eckert & Sibaoka,1967; Giese, 1962). Recent evidence strongly suggests that a variation of membranepotential precedes ciliary reversal in the ciliate Paramecium (Naitoh & Eckert, 1974)and that the potential is associated with the passage of calcium ions from the environ-ment across the cell membrane. It has also been shown that the calcium concentrationsurrounding detergent-extracted cells of this organism can be so adjusted as to inducereversal (Naitoh & Kaneko, 1973).

The small cell size of Crithidia (8 /Am long and 5 /im in diameter) makes it difficultto implant intracellular electrodes for electrophysiological studies and the behaviourof the system must at present be studied by different methods, such as the effects ofions on motility. A preliminary account of the effects of calcium ions on the flagellarmotility of various preparations of Crithidia has been presented elsewhere (Holwill &McGregor, I97S)> a nd here we present further results which show that this ion is acontrolling influence in the phenomenon of wave reversal and also has an effect onflagellar wave shape. To obtain information about the effect of calcium ions on motilityit is necessary to expose the intraflagellar mechanochemical system to these ions. Inthis work three preparations were used to achieve this objective, chemically extractedcells, mechanically detached flagella, and cells treated with ionophores known totransport divalent cations across membranes.

MATERIALS AND METHODS

Cultures of Crithidia oncopelti were grown in 10 cm3 aliquots in the syntheticmedium described by Newton (1957). After 5 or 6 days growth the organisms weretreated in one of the various ways to be described.

Preparation and reactivation of extracted cells

The techniques used in these procedures are based on those described by Douglas& Holwill (1972) and by Gibbons & Gibbons (1972). Two solutions were used to pre-pare the extracted organisms, one to wash the cells and the second to extract them,while a third was required for reactivation. The extraction techniques used eitherglycerol-based or detergent-based solutions and their composition is shown in Table 1.To make the extraction solution, a non-ionic detergent, Nonidet P.42 (B.D.H.), orglycerol was added to the wash solution. The concentration of free calcium in the

Effects of calcium onflagellar movement in C. oncopelti 231

Table 1. Composition of wash, extraction and reactivation solutions

(All concentrations in mol m~* ( = min).)

DetergentSolution [MgClJ [KC1] [EDTA]* [MCE]f [TRIS-TGA] J [ATP]§ Nonidet P.42 Glycerol

Wash andextraction

Reactiva-tion

4

4

ISO

50

o-5

OS

o-s

0

0

1 0 0

0

i-o

0.1 %ll(w/v)

0

60-70 %ll(v/v)

0

• EGTA and CaCl, were used to control the free calcium ion concentration and in many cases (seetext) no EDTA was added. The basic extraction and reactivation solutions contained no added EGTA orCaCl,.

t MCE •= mercaptocthanol.j TGA = thioglycollic acid.§ ATP = adcnosine triphosphate.|| Extraction solutions contained either detergent or glycerol (see text). The detergent or glycerol

was not present in the wash solution.

solution was controlled by adding EGTA in appropriate quantities and estimated bythe method of Portzehl, Caldwell & Ruegg (1964; see Results). In the preparation ofall solutions, reagents of analytical quality and double distilled, deionized water wereused. All processes involving the detergent-extracted preparations were carried out atroom temperature (18-22 °C), while those involving glycerol were maintained in thelower temperature range of 5-7 °C.

Detergent treatment. After centrifuging 10 cm3 of culture, the organisms were re-suspended in 5 cm3 of the wash solution and the amount of protein present in thepreparation was determined by optical absorption at 720 run following addition ofFolins phenol reagent as described by Lowry et al (1951). Calibration curves wereobtained using bovine serum albumen as the standard. The protein concentration wasfound to lie in the range c-5~i-o mg cm~3. Knowledge of the protein concentration wasrequired in order that an optimal ratio of protein to detergent could be obtained inthe extraction mixture. The optimal proportions were found to be o-i mg of protein to1 cm3 of the o-1 % detergent solution and with this ratio the cell membrane wasremoved in a reasonable time (see below) but no other structural damage could beobserved in the electron microscope (Fig. 4). If an insufficient amount of detergentwas present, extraction was incomplete, in that many cells retained their membraneseven after prolonged exposure to the solution, whereas an excess of detergent produceda very rapid extraction which could not be controlled and caused damage to theaxonemal components. Optimal extraction, as judged by later reactivation of theflagella, was achieved in a time which depended on the ambient temperature, being45 s at 18 °C and decreasing to 10 s at 22 °C. After the appropriate time had elapsedthe extraction process was terminated by cooling the preparation to o °C, at whichtemperature it was maintained until required.

Glycerol treatment. After centrifuging, the pellet from a 10 cm3 suspension oforganisms was resuspended in 1 cm3 of the wash solution. A small quantity of thissuspension was mixed with the extracting solution to give a final glycerol concentrationbetween 60 and 70% (w/v). The mixture was stored at a temperature of —20 °C andcould be reactivated satisfactorily for a period of several days. The extent of the extrac-

232 M. E. J. HOLWILL AND J. L. MCGREGOR

tion could be controlled by varying the amount of glycerol in the preparation. Electrormicroscope examination of the cells revealed membranes which were disrupted tovarying degrees but were never completely absent (Fig. 3).

Reactivation. To reactivate the cells, a drop (5 x io"3 cm3) of treated organisms wasadded to 0-5 cm3 of reactivating solution in a viewing chamber which consisted of aperspex ring, 3-4 mm deep and with an inner diameter of 15 mm, cemented onto acavity slide. The two solutions were mixed by diffusion or by gentle agitation with adrawn pasteur pipette. For visual observation an open viewing chamber was used butto obtain photographs a coverslip was needed for optimum optical performance of thesystem. To prevent the cells adhering to the surfaces of the chamber a coating of eggwhite was used.

Observation techniques

Reactivated flagella were observed using dark-field optics at magnifications of be-tween 120 x and 300 x . Beat frequency measurements were made using stroboscopicillumination which was provided by a Chadwick Helmuth point source Strobex unitdriven by an oscillator of variable frequency. The frequency was determined by meansof a pulse counter with a print-out facility. Wave-shapes were recorded on 16 mmfilm by high speed cine-photomicrography (100 pictures/s) using a Stalex camera anda 200 W mercury arc lamp (Holwill, 1965).

Micromanipulation techniques

Micromanipulation of cells was carried out on a Research Instruments TVC 500Bio-medical Micromanipulator using techniques described by Holwill & McGregor(i974)-

Ionophore preparationStock solutions of the ionophores X537A (Roche Products Ltd) and A23187 (Lilly

Research Centre Ltd) were prepared in dimethyl sulphoxide at a concentration of10 mg cm"3 and stored at 4 °C until required for use. The organisms were centri-fuged, washed and resuspended in a modified Ringer solution (100 mol m"8 NaCl,3 mol m"8 KC1, 30 mol m~3 glucose, 10 mol m"3 tris-maleate buffer, pH adjusted to7-0), a tris HC1 buffered solution or distilled water to which was added CaCl2 or EGTAat concentrations between 1 and 5 mol m"8. The ionophore solution was diluted to theappropriate strength (10 fig cm"3 for X537A and 2-3 fig cm"3 for A23187) with themodified Ringer solution before being added to the suspension of cells.

Determination of calcium concentration in solutions

The amount of calcium in the extracting and reactivating solutions was determinedusing an atomic absorption spectrophotometer after calibration with standard solu-tions containing calcium in the concentration range 2-5-100 fig cm"9. The amount offree calcium in the various solutions was then estimated using the method described byPortzehl et al (1964).

RESULTS

Estimation of free calcium in solutions

The total amounts of calcium in the various solutions were determined with thecalibrated atomic absorption spectrophotometer (Table 2). In the absence of EGTA,

Journal of Experimental Biology, Vol. 65, No. 1 Figs. 1-4

Figs. 1—4. Electron micrographs showing transverse sections of flagella from Crithidia oncopelti.Figs, i, 2 from living cells; Fig. 3 from glycerol treated cells; Fig. 4 from detergent treated

[. E. J. HOLWILL AND J. L. McGREGOR (Facing p. 232)

Journal of Experimental Biology, Vol. 65, No. 1 Figs. 5-32

131

17

21

25I

29

50 fim

Figs. 5-32. Sequences from high speed cine films showing the effects of calcium on wave shapein Critliidia oncopelti. Figs. 5-28 are all of detergent treated cells and Figs. 5-24 were alltreated with the 'basic' extracting solution (see text). The calcium concentration in the reacti-vating solution was controlled as follows: Figs 5-8, 2 mol m"1 CaClt; Figs. 9-12, 5 x io"1 molm-s CaCl,; Figs. 13-16 basic reactivating solution (see text); Figs. 17-20, 2 mol m~3 EGTA;Figs. 21-24, 4 mol m"1 EGTA. In Figs. 25-28 the 4 mol m~3 EGTA was added to both theextracting and the reactivating solutions. Figs. 29—32 show wave movement from base to tipon a detached flagellum.

M. E. J. HOLW1LL AND J. L. M C G R E G O R

Effects of calcium onflagellar movement in C. oncopelti 233

Table 2. Concentrations of calcium in the preparative solutions

(All concentrations in mmol m"* (= /*M).)

Culture Modified RingerSolution medium Extracting solution Reactivating solution solution

Total [Ca1*] iso±8 a-s±o-i ro±oi 0-501003Estimated free [Ca14] — 2-s±o-i I-O,±O-I —Estimated free [Ca1+]

(2 mol m~* EGTA) — ioo±os x io"1 iao±6xio-* —

i.e. in the basic preparative solutions, free calcium concentration is virtually the sameas the total calcium concentration, since EDTA has a greater affinity for magnesiumthan for calcium, and, at the concentrations of the ligand and ions used, estimatesbased on the method of Portzehl et al. (1964) indicate that very little free EDTA isavailable to bind calcium. In the presence of 2 mol m~3 EGTA the calculations indi-cate that the free calcium ion concentrations in both extraction and reactivation solu-tions are considerably reduced, and that the free calcium concentration in the reactiva-tion solution is much less than that in the extraction medium.

Calculations of this type must, however, be used with caution in media as complexin composition as those used in this study. For example it is difficult to assess accuratelythe combined effects of ATP and TGA on the free calcium ion concentration, althoughthe presence of these substances was allowed for in the calculations. For this reason,the concentrations quoted in this paper will, in general, be the total amounts ofcalcium or of EGTA added to a solution, and only in a situation where a criticalbehaviour pattern emerges will an estimate of free calcium concentration be made.

Chemically extracted organisms

As noted in Materials and Methods, organisms are chemically extracted by exposureto one solution and reactivated by the addition of a second solution. The calcium ionconcentration in both of these solutions affects the performance of the reactivated system.

Cells treated with the basic detergent-based extraction medium and subsequentlyreactivated using 1 mol m"3 ATP in the basic reactivating solution sustained flagellarwaves which were initiated at the basal end of the flagellum and propagated towardsthe tip (Figs. 13-16). In each of these basic media no EGTA or CaClj was added, butthe calcium ion concentration, although low (Table 2), was significant due to impuritiesin the other added chemicals. These media are therefore similar to those used in previ-ous experiments on Crithidia cells (Douglas & Holwill, 1972). The beat frequencies ofthe reactivated detergent-extracted cells lay between 17 and 20 Hz. In many cases theflagellar waves were initially of large amplitude and had strong meander-like shapes,such that adjacent loops almost touched (Fig. 13). Some cells with waves of this typewere pulled flagellum-first through the fluid, and the distal region of the flagellumappeared refractive to bending. After a period of 4-5 min the waves became smaller inamplitude and had a more sinusoidal appearance. The mean frequencies of the largeand small amplitude waves were 17*0 ± i-o Hz and 19*0 ± 1*5 Hz respectively.

When cells treated with the basic extraction solution were exposed to a reactivationsolution containing 2 mol m"3 CaCla and 1 mol m~i ATP, the movement of theflagella consisted of localized oscillations and bend propagation with decreasing ampli-

234 M. E. J. HOLWILL AND J. L. MCGREGOR

tude as a wave progressed (Figs. 5-8). The frequency of the movements v/ai10*5 ± 2-6 Hz and the motion was three-dimensional. The presence of the calcium ac-celerated the chemical degradation of the cell, which was reduced to a small sphereabout 1 /an in diameter within 1 min. When the concentration of calcium in the re-activating solution was reduced, using EGTA where appropriate, bend propagationof larger amplitude was observed and the beat frequency increased (Figs. 9-24). Thenumber of complete waves on a flagellum increased with decreasing calcium concen-tration, being about one at the high concentrations (Fig. 7) and three or more in thepresence of EGTA (Fig. 20). At a concentration of 2 mol m~3 CaCl2 a reactivatedsample would remain active for about 10 min, whereas in the presence of 2 mol m~3

EGTA motility was sustained for periods of up to 1 h. When movement had ceasedin the latter case it could be reinstated by adding further reactivating solution, but thiseffect was not observed in the presence of the higher concentrations of calcium.

The addition of between 1 and 5 mol m"3 CaCla to the extracting solution pro-duced, on reactivation with the basic solution, effects similar to those described abovefor reactivation by a solution containing a high calcium concentration. Local oscillationand propagated waves of decreasing amplitude with a frequency of about 10 Hz wereobserved in the presence of 1 mol rrr3 ATP. The rate of extraction was increased andthe time for which a preparation retained the capacity for reactivation was reduced to2 h from the 8 h typical of the basic reactivation solution. Cells extracted in solutionscontaining CaCl2 but no magnesium ions did not propagate meander-shaped waves onreactivation in a solution containing magnesium ions. The presence of Mga+ in theextracting solution was essential for the appearance of meanders on the flagella.Extraction and reactivation in solutions containing no magnesium induced roughlysinusoidal waves of frequency 3 or 4 Hz at an ATP concentration of 1 mol m"3.

When between 2 and 10 mol m~3 EGTA was added to both the extracting and thereactivation solutions, flagellar waves were observed to propagate from tip to base witha frequency of between 20 and 24 Hz at an ATP concentration of 1 mol m"3 (Figs.21-24). The conditions under which tip to base propagation was observed correspondto a maximum free calcium ion concentration of between io~* and io"5 mol m"3.Above this concentration only distally directed waves were observed. With a free calciumconcentration below io"8 mol m"3 the proximally directed waves were maintainedfor a period of between 2 and 4 min, after which the number of complete waves on theflagellum increased from one to two, the frequency fell and simultaneous propagationof waves from both ends of the flagellum was observed. Subsequently waves werepropagated from the base with a frequency of about 20 Hz and an individual flagellumcontained up to three complete waves. The above results are summarized in Table 3.

As mentioned earlier, an alternative procedure for the extraction of Crithidia in-volves the use of glycerol. When this is used instead of the detergent-based solutions,essentially the same results were obtained as those described above, with the excep-tion that cells extracted and reactivated in glycerol solutions containing EGTA propa-gated bends from tip to base for up to 20 min after reactivation and intermittentlyreversed their direction of propagation.

Individual organisms obtained using the glycerol-based extraction procedure withEGTA added to the solution were suspended in a micro-drop and were made motileby releasing reactivating solution containing 4 mol m~3 EGTA from a neighbouring

Effects of calcium onflagellar movement in C. oncopelti 235

Table 3. Effect of calcium ions onflagella activity in chemicallytreated Crithidia oncopelti

[Cal+] reactivatingsolution

[Ca*+] extractionsolution

High (2 mol m~*)

Normal (no Cat+ addedto basic solution)

Low (4 mol m-*EGTA)

High(2 mol m-*)

Local oscillationand base to tip

1 0

Local oscillation10-5 ±z-6

Base to tip

1 0

'Normal'(no Ca1+addedto basic soln.)

Local oscillationand base to tip

1 0

Base to tip17-20

Base to tip

2 0

Low(4 mol m"1 EGTA)

Base to tip

1 0

Base to tip2 0

Tip to base becom-ing base to tip

20-24

Wave direction

frequency (Hz)

Wave directionfrequency (Hi)Wave direction

frequency (Hi)

micropipette. As described above, flagella maintained proximally directed waves forsome time. If, during this period, the organisms were washed in reactivating solutioncontaining 5 mol m~3 CaCl2, wave propagation ceased and was replaced by a localoscillation lasting for less than one minute, after which the flagella became stationary.On washing the flagella again with reactivating solution containing EGTA, wavepropagation was again observed but was initially directed distally, i.e. in the oppositedirection to that obtaining before the calcium-rich solution was added. For the majorityof flagella this direction of propagation persisted for the duration of motility, butin several cases the direction of propagation reversed after the flagella had sustainedsimultaneous waves propagating in both directions. When the glycerol-extractedflagella in the micro-drop were initially reactivated with a solution contining 5 molm~3 CaCl2 instead of EGTA, some organisms maintained a local oscillation, otherspropagated waves from base to tip, while a number exhibited a tonsate (oar-like)movement. On washing with a solution containing EGTA, distally directed wavespersisted.

A few intact organisms suspended in reactivating solution containing EGTA wereadded, using a micropipette, to a drop of detergent-based extracting solution contain-ing EGTA. The extraction process was rapid, often taking only a few seconds, and wasmarked by a change in cell shape from the normal pear-shape to spherical and theappearance of particles ejected from the surface of the flagella which became quiescent.Resumption of bend propagation occurred after about 30 s and was initially from tipto base, as noted earner, but was replaced by distally directed waves after the briefappearance of simultaneous waves propagating in both directions. A second micro-pipette was used to introduce fresh extracting solution around a few organisms, thusreducing the ATP level in their immediate vicinity. This caused all flagellar move-ment to cease, with bends still on the flagella. After a period which depended on theoriginal ATP concentration, being about 30 s for high values and 1 min for low ones,wave propagation again resumed and was from base-to-tip. During the period ofinactivity, ATP was presumably diffusing into the diluted region. This procedurecould be repeated a number of times, although after several dilutions it became neces-sary to add a further amount of ATP.

236 M. E. J. HOLWILL AND J. L. MCGREGOR

Mechanically detached flageUa

Intact cells of Crithidia were suspended in a reactivating solution containing 5-10mol m"3 EGTA and 1 mol m~3 ATP. By careful micro-manipulation the cell wasquickly dissected leaving the flagellar system intact and presumably in contact with theexternal medium through the basal end (Holwill & McGregor, 1974). Flagella whichwere beating from tip to base before dissection continued to do so afterwards for aperiod of 1-2 min, after which bends were propagated from the basal end for less than1 min (Figs. 29-32). Bend propagation was then again reversed so that waves wereinitiated at the flagellar tip, with a frequency of about 12 Hz, and this lasted for between10 and 30 min, after which coordinated flagellar movements ceased.

Many flagella appeared to suffer mechanical damage at the basal end immediatelyafter dissection. The external appearance was of a lump or loop which was able to movedistally along the flagellum. The two portions of the flagellum separated by the loopeach propagated waves towards the abnormal region.

When the dissection procedure described above was carried out in a reactivationmedium containing 1-5 mol m~3 CaCla and 1 mol m"3 ATP, tip-to-base wave propa-gation ceased abruptly and was replaced by distally propagating bends with a frequencyof about 3 Hz. In some cases these bends gave way to a local oscillatory movement orto a tonsate motion. Little structural damage or deformation was observed when dis-section was carried out in calcium rich medium. On washing the calcium-treated cellswith a reactivating solution containing 4 mol m"3 EGTA, waves were propagatedfrom the flagellar tip with a frequency of between 11 and 15 Hz.

Effect of ionophores on intact cells

To find convenient working concentrations for the ionophore solutions, micro-organisms were exposed to various concentrations of X537A or of A23187 in thepresence of constant levels of CaCl2 or of EGTA. At concentrations of 10 /*g cm"3 forX537A and between 2 and 3 /ig cm"3 for A23187 similar and reproducible results wereobtained with both ionophores.

When either of the ionophores and between 1 and 5 mol m"3 CaCla were added toa suspension of cells in modified Ringer's solution, in a tris-HCl buffered solution, orin distilled water, the majority of organisms in the preparation propagated meander-shaped waves from base to tip at frequencies between 5 and 10 Hz. Subsequently, thedistal region of the flagellum appeared stiff and only the proximal region was seen topropagate waves from the base. The cells became spherical and there was a strongtendency for cells to clump together or to attach themselves to a glass surface or to thefluid meniscus. A few cells in a preparation were seen to propagate waves from tip tobase, but these organisms had prolonged periods during which the waves were propa-gated towards the tip. Occasionally, flagellar waves were observed to propagatesimultaneously from both ends of a single organelle. The combined presence of iono-phore and calcium chloride caused the flagella to shorten in length and the majorityof flagella were half the normal in vivo length after exposures of between 10 and 30 min.When the suspension was examined after two hours or more, a large number of cellshad disintegrated.

The addition of ionophores with 5-10 mol m"3 EGTA instead of the CaCl2 to a

Effects of calcium onflagellar movement in C. oncopelti 237

Suspension of living Crithidia caused a few organisms to exhibit distally directedflagellar waves, but the majority had waves which propagated from tip to base. Theaddition of CaCla in a concentration between 2 and 10 mol mr3 to this suspensioncaused a rapid increase in the number of cells propagating waves from base to tip andinduced cells to clump together or to adhere to glass surfaces or to the meniscus.Experiments in which EGTA was added to the ionophore preparations containingCaCl2 did not give clear results because the cells remained in the clumps or attachedto surfaces and it was not possible to determine unequivocally the predominant direc-tion of bend propagation. The addition to the living organisms of dimethyl sulphoxide,CaCl2 or EGTA separately in the concentrations used above caused no abnormalflagellar movements: the majority of cells propagated waves from tip to base with afew showing intermittent wave reversal.

DISCUSSION

The results presented in this paper indicate that calcium ions control the direction,form and to some extent the frequency of flagellar waves in various preparations ofCrithidia. It must be emphasized that the experiments described here were performedon in vitro systems and that the control mechanisms may be different in the livingorganism. Nevertheless, many features of the motility of the systems used are similarto those in vivo and it is tempting to speculate that adjustment of the calcium concen-tration within a living cell is used to alter the form and direction of the flagellar waves.It is reported in a recent paper (Hovi, Williams & Allison, 1975) that the ionophoreA23187 may produce effects other than that of increasing the permeability of mem-branes to divalent cations. In the present work, however, the evidence from otherexperiments indicates strongly that the primary effect of this ionophore is to transportcalcium ions across the flagellar membrane.

It seems likely that the forces required to bend flagella are derived from activesliding of the outer filaments (Satir, 1965) and that the energy needed is obtained fromthe hydrolysis of ATP by the enzyme dynein (e.g. Gibbons & Fronk, 1972). Thisreaction is activated strongly by magnesium ions and, at least for Crithidia flagellaand sea urchin sperm, to a lesser extent by calcium ions (Douglas & Holwill, 1972;Holwill & McGregor, in preparation; Gibbons & Fronk, 1972; Gibbons & Gibbons,1972). In the presence of 1 mol m~3 ATP and a similar concentration of magnesiumthe flagellar beat frequency is in the region of 20 Hz, which is close to the in vivofrequency. When the magnesium is replaced by a similar concentration of calcium thefrequency is about 3 or 4 Hz. The observed reduction in beat frequency in the pres-ence of calcium may therefore be the result of competitive inhibition of magnesium bycalcium ions (see also Douglas & Holwill, 1972).

The behaviour of the systems studied strongly favours the idea that the membraneis intimately involved in the control of the direction of wave propagation in Crithidia,as suggested by Holwill (1965). In those preparations where the membrane is com-pletely dissolved, namely the detergent extracted systems, reactivation induces prox-imally directed wave propagation for a few moments only, after which waves are initi-ated from the base of the organelle. It has not been possible to treat the flagellum sothat the wave propagation reverts to its initial direction. The other preparations arecharacterized by the presence of a membrane, albeit modified in the case of glycerol

238 M. E. J. HOLWILL AND J. L. MCGREGOR

extraction and ionophore treatment, and are able to sustain proximally directed wave^for prolonged periods and the direction of propagation may be reversed several timesfor a given organism. A possible interpretation of these results is that the membraneacts as a receptor for extra-cellular events and regulates the intraflagellar calciumconcentration which, in turn, controls the reaction to determine the direction inwhich waves will be propagated. An alternative interpretation lies in the differenteffects which detergent and glycerol may have on the structural integrity of the motilesystem. It is well known that glycerol-based solutions are used to preserve spermato-zoa, which contain axonemes, whereas detergents can have a disruptive effect on pro-tein systems. Organisms treated with glycerol may therefore contain a more completemotility mechanism than those exposed to detergent, and this could explain thedifferent properties of the two systems.

It is also significant that once chemically treated cells of Crithidia have been exposedto calcium at concentrations greater than io~* mol rcr3, waves propagate from base totip even if EGTA is added to the solution. Only in mechanically detached flagella,which have an intact membrane, can wave reversal be induced by alternately addingcalcium ions and EGTA to the preparation. The presence of the membrane thusprovides a binding site for which, under the appropriate conditions, calcium hasgreater affinity than for the site involved in wave reversal. In the absence of a mem-brane, EGTA cannot reverse the effects of calcium which is already present, so theaffinity of calcium seems to be less for EGTA than for the active site.

The ability of the Crithidia flagellum to reverse its direction of wave propagation hasimplications for the internal sliding filament mechanism which is responsible forproducing the motion. To allow the forces developed during the sliding to produceflagellar bending, it is necessary to have within the flagellum a shear-resistant systemintimately associated with the outer fibrils. In the system envisaged by Warner &Satir (1974), the resistance to shear is provided by the central sheath-radial spokecomplex. The radial spokes are permanently connected to the outer doublets, buttransient attachments appear to be possible between the inner end of each spoke - thespoke head - and the central sheath. By joining the central sheath to an outer doubletby a radial spoke relative sliding is restricted locally and, provided sufficient force isavailable, the structure will bend. Consideration of the geometry and mechanics of thesituation allows the prediction of the orientation of spokes relative to the doubletsthrough a bend, and an appropriate arrangement has been observed in cilia fromElliptio (Warner & Satir, 1974). Assuming independent sliding of microtubules onopposite sides of a flagellum, this mechanism could generate a wave with oppositecurvatures at different regions of the flagellum.

Once stress is generated within the flagellum by active filament sliding, the forma-tion of a bend and its direction of propagation would be controlled by the pattern ofthe spoke head-central sheath interactions. Active relative doublet sliding could occurin one direction only, as found in the relative sliding of actin and myosin filaments inmuscle, and, in Crithidia and similar organisms, the reversal of wave propagationwould arise by a reversal of the direction along the filament in which successive spokehead attachments to the central sheath are made. Alternatively wave reversal couldarise because of a change in the relative direction of doublet sliding together with analteration in the direction of propagation of the spoke head attachments.

Effects of calcium onflagellar movement in C. oncopelti 239

The directional control in Crithidia could thus involve the action of calcium on thesliding mechanism or on the spoke head-central sheath interaction. The likely involve-ment in wave reversal of the membrane and its close proximity to the outer fibrils mightfavour the former mechanism, namely that calcium alters the polarization of the slidingsystem thereby reversing the direction of sliding and consequently of wave move-ment. If sliding is induced by active swinging movements of the dynein arms (cf. theactin-myosin interaction in muscle), calcium could be instrumental in controlling thedirection of swing and hence of filament sliding. The active site for directional controlwould, presumably, be different from that involved in the magnesium-activated break-down of ATP which is believed to supply the energy for bending. Calcium could,alternatively or additionally, affect the behaviour of the spoke head-central sheathinteraction so that its direction of propagation along the flagellum is reversed.

On a sliding filament model, in which shear resistance is provided by the radialspoke-central sheath complex, the waveshape adopted by a flagellum would bedetermined by the relationship between sliding and spoke attachment in the flagellum.The meander shapes observed on the Crithidia flagellum under certain conditionscontain longer than normal curved regions and therefore imply a greater than usualamount of sliding with an associated resistance to shearing within the flagellum. If thespoke attachment is influenced by the presence of calcium ions a certain concentrationof such ions may favour attachment and, by feedback, filament sliding, leading to themeander shapes observed. With this mechanism, one might expect to observe flagellabent into complete loops with curvature of one sense when an appropriate quantity ofcalcium is present. This is not seen perhaps, because the mechanism of bend initiation,according to Goldstein (1975), requites the production of two partial bends of oppositecurvature to minimise sliding at the flagellar base, where structural features limit thepossible amount of sliding.

Calcium has been found to have an effect on ciliary reversal in Paramecium (Eckert,1972; Naitoh & Kaneko, 1973), on wave shapes of detergent extracted sea urchinspermatozoa (Brokaw, Josslin & Bobrow, 1974), and on wave shape and orientation ofChlamydomonas flagella (Hyams & Borisy, 1975). In the case of living and extractedParamecium, ciliary reversal can be induced by raising the concentration of externalcalcium. The mechanism of ciliary reversal may be the same as that of wave reversal inCrithidia, since the two activities could result from a changed sliding direction ofouter fibrils. These behavioural characteristics of the two organisms have the commonfeatures of being avoiding reactions in response to external stimuli; thus, like Para-mecium (Naitoh, 1973), Crithidia may have separate systems for propagating bendsand for its escape mechanism, both deriving energy from ATP hydrolysis, but theformer activated by magnesium and the latter by calcium. It is of interest that magne-sium- and calcium-activated ATPases have been reported in the flagella of Chlamy-domonas (Watanabe & Flavin, 1973), and it may be relevant to earlier discussion aboutspoke head interactions that there is histochemical evidence for ATPase activity in thecentral regions of certain sperm flagella (Burton, 1970).

By altering the calcium ion concentration in the solutions used to extract and toreactivate sea-urchin sperm, the symmetry of the propagated waves is affected(Brokaw et ah 1974). When the calcium ion concentration is increased in the reactiva-tion solution or omitted from the extracting solution, the waves become asymmetric

240 M. E. J. HOLWILL AND J. L. MCGREGOR

and some flagella exhibit local flexing. This behaviour is similar in certain respects tothat of Crithidia, although the effects on wave symmetry of calcium in the extractingsolutions for the two systems are different. The addition of calcium to the extractingsolution for the sea-urchin sperm results in more symmetric patterns on the reactivatedsystem, whereas the reverse is true for Crithidia. Further studies of the two types ofsystem are needed to determine the reasons for this behavioural difference.

Many flagellated micro-organisms have avoiding reactions or other controlledresponses in which the flagellar activity is modified, and in certain cases, it has beenshown that calcium is required if the response is to occur. For example, the chemotacticresponses of bracken spermatozoids and of Tubularia sperm require calcium to bepresent in the external medium (Brokaw, 1974; Miller, 1975), and results obtainedby Satir (1975) and Motokawa & Satir (1975) support the hypothesis that a local rise ininternal calcium concentration causes ciliary arrest in lateral cilia of freshwater musselgills. The avoiding response of Chlamydomonas is imitated by chemically extractedcells when the calcium ion concentration is lowered from above io~2 mol m"3 tobetween io"3 and io"6 mol m"3 (Hyams & Borisy, 1975).

During the chemotactic response of the male gametes of Allomyces and of Blasto-cladiella the flagellum stops beating before re-orientation occurs (Miles & Holwill,1969); a similar movement is reported by Miller & Brokaw (1970) for Tubularia sperm.It would be of interest to investigate the effects of calcium on various preparations ofthese and other organisms to discover whether calcium is universally involved in thecontrol of flagellar activity.

In several of the systems described by other authors (Naitoh & Kaneko, 1973;Brokaw, 1974; Miller, 1975; Satir, 1975) the calcium required to mediate the appro-priate response is derived from the external medium. It is believed that suitable mem-brane channels open in response to a particular signal so that calcium can reach the cellinterior, and when the appropriate manoeuvre is completed, the ion is pumped out ofthe cell. In Crithidia, the calcium needed for in vivo wave reversal is apparently alreadywithin the cell, since this activity occurs when EGTA is present externally. If thistentative conclusion is correct, there must exist within the flagellum calcium storagesites which are able to release and sequester the metal as required. It is of interest tonote that in Paramecium aurelia sites with calcium affinity have been located in theproximal portion of the cilia, notably on the cytoplasmic surface of the ciliary mem-brane in the region of the transverse plate (Fisher & Kaneshiro, 1975; Plattner, 1975).

It is convenient at this point to summarize the sequence of events which we con-sider leads to wave reversal in Crithidia. An organism propagating waves from tip tobase receives a membrane stimulus (not necessarily directly to the flagellar membrane)which may be tactile, electrical or chemical. The stimulus induces bioelectric mem-brane activity which triggers the release of calcium from storage sites within the cell.Calcium modifies the interaction between the spoke head and the central sheath andpossibly alters the direction of microtubule sliding so that wave reversal and bendshape changes occur. After a few reversed beats, calcium is again sequestered and theflagellar motion resumes its normal pattern.

The systems described in this paper provide experimental preparations whichallow the controlled investigation of various aspects of flagellar movement. Forinstance the ability to stop and re-establish waves by reducing and subsequently

Effects of calcium onflagellar movement in C. oncopelti 241

Increasing the ATP concentration will be useful in studies of the characteristics ofwave initiation. The stationary bent state of Crithidia flagella is similar to the rigorwaves described by Gibbons & Gibbons (1974) for sea urchin spermatozoa; bothsystems should be valuable in studying the action of the dynein arms and of the radialspokes. The simultaneous propagation of waves in both directions along the Crithidiaflagellum was reported earlier by Holwill (1965), but it is not a commonly observedfeature of motility in the intact cells. Since this phenomenon can be produced rou-tinely on chemically treated cells a detailed study is possible and should provide infor-mation about the sliding filament mechanism in this and other cilia and flagella with a9 + 2 axonemal structure.

This work is supported in large part by the Science Research Council, to whom weexpress our thanks. We are grateful to Dr M. N. Hughes for discussions and assistancein the use of the atomic absoiption spectrophotometer. We thank Roche Products Ltd.,and Lilly Research Centre Ltd, for respective gifts of the ionophores X537A andA23187. The assistance of Mrs J. Williams, who took the electron micrographs andhelped to prepare the figures is gratefully acknowledged.

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