Membrane Control of Ciliary Activity in the Protozoan Euplotes

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With 1 plate and 14 text-figures Printed in Great Britain

MEMBRANE CONTROL OF CILIARY ACTIVITY

IN THE PROTOZOAN EUPLOTES

BY MILES EPSTEIN* AND ROGER ECKERT

Department of Biology, University of California, Los Angeles, Calif. 90024, U.S.A.

(Received 17 August 1972)

INTRODUCTION

The locomotory behaviour of ciliated protozoa results from the activity of the cell's cilia. The major variables in the protozoan's ciliary activity are the orientation of the effective stroke and the beating frequency. Ciliary orientation as used here is the direction of ciliary beat, or more specifically, the direction of the effective or power stroke. The effective stroke is defined as that component of ciliary movement which transfers the maximum force to the medium. The effective and recovery phases of the beat can generally be distinguished unequivocally by differences in shape as revealed by visual inspection of high-speed cine pictures. However, the orientation or position in space of the power stroke is more difficult to ascertain and describe. The difficulty lies in documenting with two-dimensional photography the three-dimensional form of the ciliary beat (see Kinosita & Murakami, 1967). The cilia are said to beat in reverse

(ciliary reversal) when the power stroke is directed toward the anterior end of the

organism so as to propel it backwards (Kinosita & Murakami, 1967).' Normal' beating toward the posterior end causes the organism to swim forward. This will be termed

forward beating here. The ability to change ciliary orientation is found in protozoa,

the ciliated epithelia of some coelenterates and tunicate larvae.

There is evidence that ciliary orientation and frequency of beating are controlled independently. Cilia of Paramecium and Euplotes exposed to 1 mM-NiCl2 stop beating but respond to stimulation by shifting position so as to point toward the anterior end (Naitoh, 1966; Naitoh & Eckert, 1969; Eckert & Naitoh, 1970).

Further evidence indicates that Ca is involved in the regulation of ciliary orientation. Ca produces reversal in beating cilia of extracted models of Paramecium in which the cell membrane has been rendered non-selectively permeable with Triton X-100 (Naitoh & Kaneko, 1972). In glycerinated specimens of Paramecium, Naitoh (1969) showed a quantitative relationship between Ca and ciliary orientation in the presence of optimal amounts of ATP, Mg and Zn. Kinosita (1954) found in living Opalina that ciliary reversal in response to stimulation with isotonic-KCl required extracellular Ca.

Ciliary orientation varies with membrane potential. Kinosita (1954) found in

Opalina that the direction of ciliary beating shifts in a graded manner in response to

spontaneous and KCl-evoked changes in membrane potential; others have found ciliary reversal correlated with depolarization, and 'augmentation' (extreme forward

• Present address: Department of Zoology, University of Minnesota, Minneapolis, Minnesota S54SS, U.S.A.

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438 MILES EPSTEIN AND ROGER ECKERT

beating at high frequencies) with hyperpolarization in Paramecium (Yamaguchi, i960; Eckert & Naitoh, 1970) and in the hypotrichs Euplotes (Naitoh & Eckert, 1969) and

Stylonychia (Machemer, 1970).

It is well known that membrane potential is an important factor determining ionic movements across membranes. Eckert (1972) has proposed that ciliary reversal is coupled to membrane depolarization by an influx of Ca which occurs during a transient increase in Ca conductance of the membrane. The resulting rise in Ca concentration within the cilia is hypothesized as the immediate cause for activation of the mechanism producing the reversal (Eckert, 1972).

Much of the information about control of beating frequency comes from the work with reactivated extracted cellular models. ATP and Mg are necessary for reactivation of beating (Hoffmann-Berling, 1955; Bishop &Hoffmann-Berling, 1959; Bishop, 1962; Brokaw, 1961; Child, 1965; Seravin, 1961) and frequency has been found to vary proportionally with ATP concentration (Brokaw, 1967; Murakami & Eckert, 1972). In living cells changes in beating frequency are associated with changes in membrane potential as shown by Kinosita, Murakami & Yasuda (1965).

The first part of this present study investigates the relations of ciliary orientation and frequency to evoked changes in membrane potential. The results are discussed in the perspective of membrane-regulated Ca fluxes as the major regulatory mechanism for ciliary orientation. The second part examines the action of Ca and other ions on ciliary activity following the functional disruption of the cell membrane by extraction with the detergent Triton X. The hypotrich ciliate Euplotes was chosen for these studies because this cell is large enough for routine insertion of microelectrodes and its compound cilia (cirri) are large enough to photograph easily. Each of the cirri contains 40-120 '9 + 2' cilia packed together in a hexagonal array (Roth, 1956, 1957; Gliddon, 1966).

MATERIALS AND METHODS

A supply of Euplotes eurystomus was obtained from Connecticut Valley Biological Supply (Southampton, Mass.) and cultured in Chalkley's (1930) solution (solution A) containing wheat grains. The Chalkley's solution has the following composition: i*7mM-NaCl, 0-05 mM-KCl, 005 mM-CaCl2, and enough Na HCO3 to bring the solution to pH 7-0 (approximately 1 mM). Cells were removed from the culture, washed and equilibrated for 30 min in 2 mM-KCl, 1 mM-CaCl2 and 1 mM Tris-HCl

buffer pH 7-0-7-4 (solution B) prior to the beginning of experimentation.

A small drop of solution containing 3-10 cells was placed on the coverslip which was then inserted into the chamber. If the drop was large enough, the cells often moved only slightly. A microneedle was used to hold the cells with their ventral side against the coverslip. The chamber was then filled with solution B.

Voltage-recording and current-passing microelectrodes were inserted into the left margin of the cell in the area between the frontal and anal cirri. The holding needle was removed and a blunt-tipped glass needle of 2-10 /im diameter, held in a horizontal plane, was placed between the cell and the coverslip and slowly lowered vertically. This action rotated the cell so that the anal cirri were seen in profile. Often the ventral and frontal cirri were also in view in side profile.

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c

CRT

439

*Vm

Text-fig, i. Diagram of apparatus. The specimen was held against the lower surface of the coverslip by the voltage-recording and current-passing microelectrodes. The strobe lamp (S) passed light through the microscope (M) onto film in the cine camera (C). Membrane potential (Vm) was recorded on a CRT, whose face was projected onto the cine film through a prism (P). The bath was held at virtual ground byconnecting the indifferent electrode (I.E.) to thesumming junction of an operational amplifier (O.A.) used to monitor current intensity (/). The cell was bathed in various test solutions injected into the experimental chamber with a syringe (Sy) and removed with a suction pipette (SP). A, neutralized capacity amplifier; CAL, square wave calibrator; Mi, mirror.

interference optics (Text-fig, i). Light was provided by a strobe lamp (Strobex 164, Chadwick-Helmuth Co., Inc., Monrovia, Calif.). The image of the cell passed through a x 16 objective (N.A. = 0-35) and was projected with final magnification of x 80-128 onto 16 mm film (4-X Negative, Kodak) in a high-speed camera (164-4 DC Locam, Red Lake Laboratories, Santa Clara, Calif.). The frame speed was usually 200 frames/ sec. The face of a Tektronix 360 cathode-ray-tube was also projected onto the cine film by means of a prism housed in a Zeiss Basic Body II. After the camera was started and had reached the selected frame speed, a pulse of stimulating current was passed into the cell. Both the membrane potential displayed without time base on a cathode-ray-tube and the image of the cell were recorded simultaneously by superimposition on the cine film. The films were analysed with a projector equipped with single-frame advance. Membrane potential was recorded with o-1 M-KCl-filled microelectrodes (resistance, 100-500 MD) which were connected to a capacity-neutralized amplifier (NFi, Bio-electric Instruments) and displayed on both 561 and 360 Tektronix cathode-ray oscilloscopes. A square-pulse calibrator was used to check the amplifier response before each stimulus. The indifferent electrode was a 0-5 mm glass capillary rilled with

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44-O M I L E S E P S T E I N AND R O G E R ECKERT

2-8 M-KC1 or 0-5 M - K C I dissolved in 2 - 3 % agar. The bath was held at ground by connecting the indifferent electrode to the summing junction of a Philbrick P25 operational amplifier used to monitor current intensity. All liquid-wire interfaces were connected through Ag/AgCl electrodes. Current pulses for stimulation were provided by pulse generators in series with a io10 ohm resistor and were delivered through intracellular microelectrodes filled with either o-i or 0-5 M-KCI. Membrane potential and stimulus current intensity were photographed from the oscilloscope face in the conventional manner. A steady membrane potential and stable response to current pulses were required before cine photography was begun. Experiments were carried out at room temperatures of 17-20 °C.

For the reactivation of extracted models many variations of extraction and reactiva-tion solureactiva-tions were tried with the aim of maximizing the durareactiva-tion of beating. The composition of the solutions eventually selected for this purpose, similar to that used by Eckert & Murakami (1972) for Necturus oviduct, was as follows:

(1) Wash solution: KjSC^ 56 i m , sucrose 46 mM, Tris-maleate 20 mM, KOH 20 mM, EGTA Tris neutralized) 1 mM, MgSO4 o-1 mM, Tris base approximately 5 mM to bring to pH = 7-0.

(2) Extraction solution: o-oi%(w/v) Triton X-100 (Octyl Phenoxy Polyethoxy-ethanol; Sigma Chemical Company, St Louis, Mo.) plus wash solution. The Triton X-100 was stirred for a minimum of 30 min before use.

(3) Reactivation solution: ATP Nag 1 mM (Calbiochem) + wash solution + base to make pH 7-0.

Different Ca concentrations were made with EGTA-buffered solutions (EGTA, La Mont Laboratories), which were calculated from a dissociation constant of 4-83 x io6 (Hagiwara & Najakima, 1966). If the value of Ca in the double-distilled water was io~s M, then the free Ca in the presence of 1 mM EGTA would be 2 x io~9 M. However, since the value of free Ca was not determined and might vary, the concentra-tion of free Ca in soluconcentra-tions with 1 mM EGTA was taken to be icr8 M or less.

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Caudal

Posterior side view

Text-fig. 2. A tracing from a photomicrograph of Euplotes seen in side view. The various groups of compound cilia are indicated but not all cirri of a given group are visible.

RESULTS

(a) Description of the ciliary organelles and activity of the unstimulated cell

The movements of Euplotes are brought about by aggregates of cilia termed membra-nelles and cirri. The location of these orgamembra-nelles is seen in the side view of a cell as illustrated in Text-fig. 2. Going from the anterior to the posterior end, there is first a collar of membranelles, followed by seven frontal cirri, two ventral cirri, five anal cirri, and finally four caudal cirri. The activity of these different organelles varied in both free-swimming and impaled cells. The membranelles moved continuously, while the frontal cirri usually beat at a low frequency. Ventral, anal and caudal cirri were often motionless and showed only sporadic activity (see Taylor, 1920, for further details).

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Anal cirrus Forward

_ 1

-1

f\

I ^ t

\

Reversed

2

n

Forward

Frontal cirrus

A

A/\

Forward ₁ Reversed

r 1\

AA

o7'> 8 3 Membranelles

I Forward

Forward

/ Reversed

\ / V V \ /

^\ Forward

600

Text-fig. 3. Responses to outward current. A pulse of outward current (recorded in trace / at bottom) resulted in depolarization of the membrane potential (trace Vm) and changes in the direction and frequency of the beat of the frontal and anal cirri. This activity in time is plotted as extremes of inclination angle (a) for an anal cirrus (uppermost plot) and a frontal cirrus (second plot). Inclination angles of the membranelles (third plot) were not measured. The direction of beating is noted in each case. After the onset of the stimulus a reversal of the direction and increase in frequency occurred. The changes in shape of one anal and one frontal cirrus during a cycle of forward beating before the stimulus are shown in tracing no. i at the left. The numbered bars (<—•) over the plots of inclination angles indicate which beating cycles were traced. The recovery stroke is indicated by the dashed lines. Numbers identifying each position of the cirrus correspond to cine frames counting from the onset of the current pulse. The frame interval was 5 msec. The stimulus began at frame o, and frames before the stimulus are indicated by numbers 900 to 999. The changes in shape of the same cirrus during a cycle of reversed beating are shown in tracing no. 2 at the right side of the figure. There the power stroke is directed toward the front of the cell. Voltage calibration pulse was 20 mV, 50 msec. Current pulse was 1 x io~° A.

The following factors should be considered in the measurements of the inclination angle: (a) movements of the cirri were not limited to a single plane, but are somewhat three-dimensional; (b) the cell did not lie perfectly in the plane of focus; (c) the surface outline of the cell was often vague; and (d) slight movements of the cell resulted from changes in ciliary activity. It must be emphasized that the measurements were some-what arbitrary, and the values were most useful in describing changes in ciliary activity in a given cell.

(b) Response of ciliary organelles to current pulses

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443

Anal cirrus Forward

1

Frontal cirrus

Forward Forward 1 2

Forward 2

Forward

mmmmmi

Forward

\ M \ U '„ ^ I U U If 1/ '# \l U II I ' J U I < I J J ' J ' 1 I I » I J I J i i

Forward

21

14 15

300 600

Text fig. 4. Responses to inward current. A pulse of inward current (trace I) hyperpolarized the membrane potential (trace Vm). The activities of the anal and frontal cirri, expressed as extremes of inclination angle (a), are shown in the first and second plots. Inclination angles of membranelles were not measured. The beating direction of the cirri and membranelles is indicated. The frequency of beating increased during hyperpolarization and slowly decreased after the end of the 300 msec stimulus. A beat cycle of an anal (top) and frontal (bottom) cirrus before (tracing no. 1 at left) and during (tracing no. 2 at right) the stimulus are shown. The numbered bars (•—•) over the plots of inclination angles indicate which beating cycles were traced. The voltage calibration pulse in Text-fig. 3 applies to this figure also. The current pulse was 1 x io~'A.

(1) Outward current

The changes in the beat pattern of the anal and frontal cirri are shown before and during stimulation by pulses of current in Text-fig. 3. Prior to the stimulus, the beating of these cirri and membranelles was in the ' forward' (i.e. rearwardly-directed, forward-swimming) direction; the anal cirri beat sporadically at a low frequency while the frontal cirri and membranelles beat continuously. After application of the stimulus pulse, and membrane depolarization, the beating direction was reversed and the frequency increased.

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differences in shape in the extreme position and in the range between extremes for forward and reversed beating. Differences in extreme position of the reversed frontal cirri were less than the values recorded by Okajima & Kinosita (1966), which was consistent with the different methods of measuring. Although details of the beat cycle of the membranelles were not photographically resolved, changes in direction of beat and sometimes in frequency could be determined. As shown in Text-fig. 3 the duration of reversed beating was the same in all groups of cirri and was followed by forward beating although, in this case, the membranelles' reversal continued approximately ico msec longer. Reversal continued after the end of the pulse, and even after mem-brane repolarization; the duration of the reversal after the end of the stimulus depended on the amplitude of the depolarization as shown below (Text-fig. 11).

Beating frequency of the anal cirri increased from 5-5 cycles per second (c/s) in a single spontaneous cycle, to an average of 28-5 c/s within the stimulus period, while the frontal cirri increased from 182 to 368 c/s. The frequencies became steady after one cycle and declined following the end of the pulse. A smaller decrease in frequency occurred in the frontal cirri than in the anal cirri before forward beating resumed. In both frontal and anal cirri the frequency of forward beating immediately after the end of reversed beating was higher than the frequency prior to stimulation.

(2) Inward current

Inward current of sufficient intensity produced movement in the forward beating (normal) direction and an increase in the frequency of beating (Text-fig. 4). Upon stimulation the nearly quiescent anal cirri began beating at 33 c/s in the forward direction while the frontal cirri, which were spontaneously active prior to stimulation, increased their frequency from 15 to 25 c/s. The frequency increase began at approxi-mately the same time in all the ciliary organelles and persisted here beyond the end of the filming period (450 msec beyond end of stimulus pulse). The decline in frequency following stimulation was more rapid in the anal cirri than in the frontal cirri.

(c) Differences in responsiveness between groups of organelles

A small depolarization resulted in a reversal of some ciliary organelles while others continued to beat in a forward direction. This was seen in Text-fig. 5 where the anal and ventral cirri beat in reverse in response to a depolarization of about 6 mV while the frontal cirri and membranelles continued to beat in a forward direction. However, the frontal group showed a reduction in frequency and in amplitude of beating. In this case three of the five anal cirri beat once in reverse while the other two beat twice.

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445

0J

0

--*' »'

Anal cirrus

/

Ventral cirrus

Frontal cirrus Forward

'« A A A

\ / \ ^ : \ / \

Membranelles

Forward

Reversed

t *

S \ I

/

X

ft

Reversed

* '*.

Forward

n ,*, A^ /

V V v v

Forward

i i i

Forward

\ / \

V \

r

300 600 msec

Text-fig. 5. Responsiveness of anal, ventral, and frontal cirri to a small depolarization. A current pulse (at bottom of figure) evoked a small depolarization and consequent changes in the ciliary activity which is plotted as extremes of inclination angle (a). The anal and ventral cirri began beating in a reversed direction toward the end of the stimulus. The frontal cirri continued to beat in a forward direction. The membranelles showed no change in beat direction during the film sequence. The voltage calibration pulse was 10 mV, 50 msec. Current pulse was C25 x io~10 A.

Because the frontal cirri and membranelles were beating continuously and the anal cirri spontaneously underwent single forward beats, it was difficult to establish differ-ences of threshold between the various groups of cirri in response to hyperpolarization. Threshold for forward beating of the anal cirri varied between 15 and 29 mV in four different cells. Activation of the anal cirri was accompanied by an increase in frequency of the frontal cirri.

(d) Behaviour of the anal cirri

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[image:10.451.58.422.131.235.2]

446 M I L E S EPSTEIN AND ROGER ECKERT

Table 1. Differences in response to small depolarizations between groups of cirri

(BD = beat direction, F = forward, R = reversed; Freq. = frequency decrease; Amp = amplitude decrease; RP = resting potential; AVp = value of the peak change in membrane potential; No. cirri = number of cirri showing reversed beating.)

Date 14. x. 20. x. 20. x. 23.x. 1. ii. RP 2 6 26 23 3 ' 2 6

(10-10 A) 0 5 0 2 5 0 2 5 C 2 5 i - o

• No AVp (mV) 6 6 3 6 9 movement. Anal BD R R R R F cirri cirri 3 5 2 2 — + Could Ventral cim BD R R R • R Frontal cirri BD F F F F R not determine. Freq. Yes Yes Yes Yes — Amp. Yes Yes No No Membra nelles BD F F F F t

Table 2. Values of depolarization (as a peak value of membrane potential change)

necessary to produce ciliary reversal in all ciliary organelles in different cells

Date

14. x. (1) 14. x. (2) 18. x. 20. x. 23.x. 23.

i-3 - i i ( 0 3- ii- (2)

Maximum depolariza-tion that failed to evoke

ciliary reversal (mV) — 6 4 6 6 — — — Minimum depolariza-tion that evoked ciliary

reversal (mV) 7 1 1 S'5 8 1 0 4 4-5 3 Resting potential (mV) 23 26-27 33-28 20-31 3 i 2 0 17 2 0

the anal cirri were used for a more detailed analysis of the ciliary response to stimu-lation.

The five anal cirri were usually quiescent except for occasional slow forward beats. Upon activation these cirri beat out of phase with one another, and individual cirri showed differences in threshold, number of beats and frequency. These differences were most apparent with small depolarizations and became less noticeable as the maximal response was approached. Responses of these cirri were examined at different stimulus intensities of inward and outward current.

Text-fig. 6 shows responses of the cirri to pulses of outward current of increasing intensity. Extremes of inclination angle (a) of a single cirrus are plotted along with membrane potentials, and tracings are shown of the first and second beating cycles of that cirrus.

(a) Amplitude. By amplitude is meant the width of the envelope (formed by the

[image:10.451.84.383.276.393.2]

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447

0-150

l 23

300 msec

600 Cycle 1 Cycle 2

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448 MILES EPSTEIN AND ROGER ECKERT A

AKm

.41

AKm

A Km

8 x l 0 -1 0-u

Cycle 2

600

msec

Text-fig. 7. Activity of an anal cirrus in response to various intensities of inward current. The same cell and probably the same cirrus were used for figs. 6 and 7. Details are in Text-fig. 6. Beating was forward (toward the rear of the specimen) throughout. Note the differences in the voltage and current scales relative to those in Text-fig. 6.

anterior and a complete recovery stroke followed the initial effective stroke. Beyond this, the amplitude of the beat showed little increase with increasing depolarization.

(b) Extremes of inclination angle (a). When the extreme positions of the proximal

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449

A

o 8

CD

o o o

Cycles Forward "> T

1 4

12

1 0

8

6

4

-- 5 6 -- 4 8 X * * 1 X O A 6

-Re\erscd

DO A p /

A / X

An'x I

t x 2 - - GX'

-40 - 3 2 - 2 4 - 1 6 - 24 mV

Text-fig. 8. The number of evoked cycles of beating of anal cirri plotted against the steady-state shift in membrane potential produced by injected currents of 300 msec duration. Zero on the abscissa is the resting potential. Depolarizing changes, indicated on the right side of the abscissa, resulted in cycles of reversed beating. Hyperpolarizing changes, shown on the left side of the abscissa, evoked beating in a forward direction. Different symbols represent a single cirrus on different cells. The dashed line connects the values recorded for the specimen used for Text-figs. 6 and 7.

(c) Number of cycles. Cycles were counted starting from the position of the cirrus

at the onset of the stimulus; half cycles were counted as a whole cycle. The number of reversed beats increased with greater depolarization (Text-fig. 8). This resulted from a greater persistence of reversed beating beyond the stimulus (also see Text-figs. 6 and 11) and an increased frequency of beating.

(d) Frequency. Frequency was measured as the average for all the cycles, except

the first, falling within the stimulus period. If only one cycle of beat occurred, the reciprocal of its duration was taken as its frequency. After the first cycle a steady frequency was maintained for the stimulus duration. After the stimulus ended, frequency declined.

Beating frequency increased with membrane depolarization until a maximum was reached after a steady-state change of about 10-20 mV (Text-figs. 9 and 13). The maximum frequencies varied between 17 and 31 c/s. By comparison the frequency of cilia in Paramedum increased from 13 to 37 during a spontaneous Ba-induced action potential (Kinosita et al. 1965).

(e) Latency. As membrane depolarization increased, the time between onset of

stimulus and the first consistent movement of the cirrus decreased (Text-fig. 10). The minimum latency (not shown in Text-fig. 10) obtained with high current intensities was 5-10 msec. This value was consistent with the latencies of 22-36 msec (Kinosita et al. 1965) and 40 msec in Ni-paralysed preparations (Eckert & Naitoh,

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45°

MILES EPSTEIN AND ROGER ECKERT

Cycles/sec

Forward —.— 26 Reversed

o o

o

-f

k—.

-o-A-6-- -o-A-6-- 2 4

22

- - 2 0

- - 18

- - 16

- - 1 4

- - 12

- - 1 0

A A

D A •

u .

I

- - 6

2'

l

-aU- 1

- 4 4 - 4 0 - 3 6 - 3 2 - 2 8 - 2 4 - 2 0 - 1 6 - 1 2 - 8 - 4 0 8 12 16 20 mV

Text-fig. 9. The frequency of beating of anal cirri plotted against the steady-state shift in mem-brane potential produced by a 300 msec pulse of injected current. Reversed beating, due to depolarization, is shown on the right side of the abscissa; and forward beating, due to hyper-polarization, is shown on the left side. The dashed line connects the values recorded for the specimen used for Text-figs. 6 and 7.

(a) Amplitude. The amplitude of the forward beat showed little variation with

increasing hyperpolarization as indicated by the tracings of the first and second evoked cycles in Text-fig. 7. The amplitude was reduced slightly and the envelope directed more posteriorly in the second cycle. Okajima & Kinosita (1966) have reported a reduction in amplitude and a more posteriorly-inclined beating axis when the cirri beat at higher frequencies.

(b) Extremes of inclination angle. Little variation in the extremes was observed with

increasing membrane hyperpolarization (Text-fig. 7). Spontaneous cycles showed smaller extremes.

(c) Number of cycles. Increasing hyperpolarization produced an increase in the number of cycles (Text-fig. 8) which varied widely between different cells.

(d) Frequency. Frequency approached a maximum value as the hyperpolarizing

stimulus was increased (Text-fig. 9). The maximum values varied between 16 and 36 c/s for the different cells. The maximum frequency was reached with a 22-30 mV hyperpolarization and showed little gradation between quiescence and maximum frequency of beating.

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45*

Forward o

o

A

o

A

o

A t o '

A f>

1

A

/_{00/ o}

^-* o o

1 1 1 1 1 1 1 1 1 1 1 1 1

msec , ,s n Reversed A

D • 200

'- 140

- 120 \

\ - S0l_,A

- 60 \

X. \AX

• 40 \*A

nn it

• 20 **

1 1 1 1 1 1

- 5 6 - 4 8 - 4 0 - 3 2 - 2 4 -16 -i mV

16 24

Text-fig. 10. Latency of evoked beating of anal cirri as a function of the steady-state shift in membrane potential. Details as in Text-figs. 8 and 9. The dashed line connects the values recorded for the cell in Text-figs. 6 and 7.

(Text-fig. 10). High current intensities resulted in minimum latencies of 10-15 msec. This can be assumed to result from faster rates of hyperpolarization with higher stimulus currents.

(e) Large displacement of membrane potential

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b

- / c N

« i • 5 t

V ¥ V V !

i / ', /

/ V \ / \ ; \ ; \ / \ / \ / \ / \ / -I

o

0 L- 0

300 msec

600

Text-fig, I I . Suppression of the reversed beating response in a single cell with large positive shifts in membrane potential. A series of increasing internally positive polarizations resulted in ciliary activity, which is plotted as extremes of inclination angle (a). Beating was in reverse except when the direction was forward as indicated by the letter F. The number of beats in reverse increased with larger positive shifts. In sequence d, however, no reversed beating occurred during the shift in membrane potential to + 87 mV (value at the end of the pulse). Reversed beating was initiated with repolarizau'on after the end of the stimulus pulse. Smaller potential shifts (sequences e and/) were accompanied by ciliary reversal. The resting potential remained at about —25 mV as indicated by the voltage scale at the right side of sequence d.

positive internal potential and then declined towards zero, which was reached at a minimum positive shift of 107 mV. No response was seen for positive shifts greater than 107 mV. The suppression potential for these cells lay between +70 to +82 mV (resting potentials varied between 20 and 26 mV). Since the membrane potential sagged during stimulation, the suppression potential could not be more precisely determined.

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453

Cycles

Forward 10-p Reversed 9 - - o

A S - " A • X

6- - «* \ AA

\

4 - - ,

3 " /

2 - - /

i

• \

O \Cfc \ _{• A}

\ \

. 1 . I I I I | _ I l

- 1 4 0 - 1 2 0 - 1 0 0 - 8 0 - 6 0 - 4 0 - 2 0 0 20 40 60 80 100 120 140 160mV Text-fig. 12. The number of evoked cycles of beating within the 300 msec stimulus period plotted against large steady-state shifts in membrane potential resulting from injected current. Depolarizing changes, indicated on the right side of the abscissa, produced reversed beating. Hyperpolarizing changes are shown on the left side and resulted in beating in a forward direction. Zero on the abscissa is the resting potential. Dashed line represents the values obtained from the cell in Text-fig. 11.

Cycles/sec

+ Forward 36 T Reversed

- '

3 2

-28 • • * x T x . y Q

2 4

-

16--

12--8 - 1

1 1 1 1 1 I I I I I I I I I I I I ^ I I,,-, I I

- 1 6 0 - 1 2 0 - 8 0 - 4 0 0 20 40 60 80 100 120 140 160 - 1 4 0 - 1 0 0 - 6 0 - 2 0

mV

Text-fig. 13. The frequency of beating plotted against large steady-state shifts in membrane potential produced by 300 msec pulses of injected current. Depolarizing changes, resulting in reversed beating, are shown on the right side of the abscissa. Hyperpolarizing changes are shown on the left side. Dashed line represents the values obtained from the cell in Text-fig. 11.

the stimulus period. The intermediate frequency values arose when the membrane potential dropped below the suppression potential and reversed beating was initiated. On termination of the pulse the frequency of the first beat was greatest and declined in subsequent beats (Text-fig. 11).

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Using high intensities of inward current Naitoh (1958) evoked ciliary reversal in

Opalina. In the present study of Euphtes attempts to produce ciliary reversal with

inward current were unsuccessful despite hyperpolarizations of 100 mV, extended stimulus durations (several seconds without filming, 750 msec with filming) and elevation of the bath Ca to 10 mM. However, some aberrations in ciliary beating during strong hyperpolarizations were observed. These included a twisting and bending at two different points along the cirrus, usually in the first 1-2 cycles, and a sporadic stoppage of ciliary activity. In two cells reversal of the frontal cirri occurred, but this was not a consistent result. In general, the amplitude and extremes of inclination angle were similar to those in Text-fig. 7.

Prolonged and strong hyperpolarization (usually deflections of 100-200 mV for periods of greater than 300 msec) disrupted and eventually caused detachment of the anal cirri while the frontal cirri and membranelles continued to beat in a forward direction. With more stimulation the cell was killed.

The effect of large hyperpolarizations on the number of forward beats within the stimulus period is shown in Text-fig. 12. The number of beats reached a maximum and declined slightly as the magnitude of the hyperpolarization increased. Frequency also increased to a maximum and then decreased as the membrane was further hyper-polarized (Text-fig. 13).

(/) Reduced concentrations of external calcium

Exposure of the cell to a Ca-free solution for about 5-10 min resulted in the loss of the depolarization-evoked reversal response. After raising the external Ca to its previous level reversed beating could not be produced and the cell soon died. How-ever, immediately after the external Ca level was lowered to io~6 M with EGTA, large depolarizations failed to produce reversed beating. Upon reintroduction of io~3 M-Ca, depolarization again resulted in ciliary reversal (Text-fig. 14). These results indicated Ca was apparently required for reversal. Reversed beating could be evoked in IO~8 M-Ca, and the amplitude of depolarization needed to evoke a response was essentially unchanged after the Ca was reduced from io"3 M to io~B M.

Exposure of Euphtes to the low-Ca solution resulted in a decline in resting potential and input resistance. For the cell in Text-fig. 14 the resting potential fell from — 28 mV before addition of EGTA to —10 mV in io~3 M-Ca after removal of EGTA. Despite the reduction in resting potential, depolarizations (about 10 mV) still evoked the reversed beating response. On the other hand, large depolarizations (up to 30 mV) in io"8 M-Ca did not produce reversal even before the usual decline in resting potential occurred. Thus, a decline in resting potential was not necessary in order to observe a loss of the depolarization-evoked reversal.

(g) Extracted models

(1) Extraction with Triton X

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455

600

Text-fig. 14. Loss of depolarization-induced reversed beating response in reduced external Ca. Membrane depolarizations of a single cell, bathed in solution of various Ca concentrations, resulted in ciliary activity, which is plotted as extremes of inclination angle (a). When the free Ca concentration was reduced to io~* M with io~~* M-EGTA, a 30 mV depolarization did not produce reversed beating. After returning the Ca concentration to io~* M, a depolarization of about 10 raV resulted in reversed beating. The resting potential and K concentration associated with each Ca concentration were — 28 mV in io~* M-Ca, 2 x io"* M-K; —45 mV in io-'M-Ca, 2 X I O - * M - K ; - 3 5 to - i 6 m V in io"* M-Ca + EGTA, 6 X I O ~ * M - K ; and finally —10 mV upon return to io~* M-Ca and 2 x io~* M-K. Voltage calibration was 10 mV, 50 msec. The intensities of the current pulses were different.

and groups of cilia. These often interfered optically with one another, complicating the measurement of their movements.

After 1-2 min of extraction ciliary activity was absent. At this time no resting potential could be recorded, and the input resistance had dropped to less than 1 % of its value in the living cell. It is presumed that the membrane permeability was in-creased greatly by the extraction with Triton X so that the concentrations of small diffusible molecules within the cell had come into equilibrium with the extracellular medium. Thus, the influence of Ca on the ciliary apparatus could be directly evaluated.

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(2) Reactivation

Since the anal cirri were studied in the living cell, the reactivation of this group was the chief concern here. Reactivation of ciliary activity required ATP and Mg as previous workers found (Hoffrnann-Berling, 1955; Alexandrov & Arronet, 1956; Seravin, 1961; Child, 1965; Gibbons, 1965; Brokaw, 1967; Brokaw& Benedict, 1968; Eckert & Murakami, 1972). The duration of beating of the anal cirri was longest in I O ~8M - A T P and 10.* M-Mg. The membranelles beat longest in io~3M-Mg. No reactivation was observed in io~3 M-AMP or io~3 M - A D P in the presence of io~* M-Mg and io~9M-Ca. Ca and ATP alone did not reactivate beating. The differences in optimal Mg concentration for maximum beating duration of the anal cirri and mem-branelles have not been reported and may be peculiar to the present method of extraction. Seravin (1961) found the optimal Mg concentration to be between io~* and io~6 M in Euplotes extracted with saponin. Frequency increased upon raising the Mg from io~* to io~3 M in the presence of either icr8 or io"6 M-Ca. Reactivation with Mg and ATP are not enough evidence that the cell is freely permeable to ATP and not metabolizing. Eckert & Murakami (1972) found that small concentrations of ATP (icr8 M) stimulated beating in Necturus oviduct decalcified with EGTA.

Therefore, to insure that the ATP synthetic capacity of Euplotes was destroyed, extraction was carried out in the presence of icr3 M-iodoacetate, a glycolytic inhibitor. After such extraction the cilia were activated in a solution of icr* M-Mg, io~3 M - A T P and 1 o~3 M-iodoacetate. Unlike io~3 M-cyanide, which did not affect Euplotes, iodo-acetate quickly stopped movement of the living cell. The ability of externally supplied ATP to reactivate ciliary movement was further evidence that it freely entered the cell through the extracted membrane and acted as the source of energy for ciliary movement.

(3) Effect of Ca on reactivated cilia

There is a qualitative difference in the extreme positions of the beating cirrus in reactivation solutions containing io"8 M-Ca compared to io~* M-Ca. These differences are illustrated in Plate I, which shows the beating pattern of a cell exposed first to icr8 and then to icr6 M-Ca. In icr8 M-Ca the extreme anterior position of the clumps of cilia was approximately perpendicular to the cell, but in io~6 M-Ca it was approxi-mately parallel to the longitudinal axis of the cell. The beating pattern was more posteriorly-inclined in icr8 M-Ca, while the pattern showed a more anterior inclination and possibly larger amplitude in io"6 M-Ca. The pattern of movement in icr8 M-Ca resembled forward beating, and the pattern in io~* M-Ca reversed beating of the cirri in the living cell. However, no distinctive effective and recovery phases of the beating cycle were observed in the extracted cirri.

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Table 3. The beatmg direction of the anal cirri of extracted cell models after reactivation

in solutions of different Ca concentrations

(Each cell was first placed in reactivation solution plus io~* M-Ca and then exposed to reactiva-tion solureactiva-tions with a greater Ca concentrareactiva-tion. A majority of the cells were observed in only io~* and io~* M-Ca.)

Ca concentration io~* M 5 x io~* M 1 x io~7_{M 1 x io~* M}

Number cells with forward beating 12 5 2 1 Number cells with reversed beating 1 2 4 10

Concentration of Ca necessary for reversal

The Ca concentration was varied between io~8 and io"6 M. Table 3 shows the type of beating associated with the different Ca concentrations. Threshold differed between cells but most cells showed reversed beating between icr7 to io"8 M-Ca.

Gradations in degree of ciliary reversal as a function of free Ca were not observed. Gradations, if they occur, presumably extend over smaller increments of Ca than those covered in these experiments.

DISCUSSION

The results of this work support various lines of evidence that Ca plays an essential role in the reversal of ciliary beating. Evidence for this was obtained in living cells by Bancroft (1906) and Kinosita (1954). In extracted models of Paramecium Ca is required for a change in the orientation of non-beating cilia (Naitoh, 1969) and for reversed ciliary beating (Naitoh & Kaneko, 1972). In the latter study, the orientation, as judged by swimming direction and velocity, gradually reversed as the Ca concentration was raised from io~7 M to io~6 M and above. The maximum velocity of swimming in reverse occurred at 5 x io~6 M-Ca. The essential role of Ca in the reversal of ciliary beating was confirmed for Euplotes in the present work. The direction of beating of the extracted anal cirri reversed when the Ca concentration was raised above approxi-mately io~7 M. This is significant because it indicates an effective concentration sufficiently low so that transient Ca influx across the cell membrane may be adequate to produce reversal.

In unstimulated squid axons (Baker, Hodgkin & Ridgway, 1971) intracellular free Ca concentrations of 3 x io~7 M have been reported. Muscle contraction in barnacle fibres is initiated at free Ca concentrations of 4-8 x io~7 M (Hagiwara & Naka, 1964; Ashley, 1967). Syneresis of isolated myofibrils showed a threshold of io~7 M-Ca (Weber & Herz, 1963). The present data on extracted models of Euplotes show a similar degree of sensitivity of ciliary reversal (occurring between io~7 and io"6 M-Ca) to Ca, and is in the range reported for Paramecium (Naitoh & Kaneko, 1972).

No gradation in the direction of the effective stroke as reported for models of

Paramecium (Naitoh & Kaneko, 1972) and in living Opalina (Kinosita, 1954) was

observed in Euplotes. The effective stroke of the anal cirri is confined largely to a single plane for both forward and reversed beating, while the cilia of Paramecium and

Opalina beat in different planes perpendicular to the cell surface depending on the

extent of reorientation of the beat. The change in the beating direction of the anal cirri is expressed as a change of shape of the quasiplanar movements of the cirrus.

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of how the intracellular Ca concentration is controlled in the living cell. Ca conduct-ance changes, resulting from changes in membrane potential, could lead to an in-creased Ca influx and subsequent increase in intracellular Ca (Eckert, 1972). Naitoh, Eckert & Friedman (1972) have demonstrated in Paramecium graded, regenerative depolarizations, termed 'calcium responses' because their amplitudes increase with greater external Ca concentrations.

Increases in free intracellular Ca concentration accompanying depolarization have been demonstrated in barnacle muscle, where the peak tension developed during contraction is a function of the Ca concentration (Ashley & Ridgway, 1970).

Depolarizations of at least 3-11 mV (Table 2) were required to produce ciliary reversal. The component of depolarization due to Ca influx (as opposed to purely electrotonic depolarization) through postulated voltage-sensitive Ca channels is un-certain. However, calculations demonstrate that currents producing millivolt changes in membrane potential can make increments in intraciliary concentrations of Ca sufficient to evoke reversal of the ciliary beat (Eckert, 1972).

The changes in the response (reversed beating) with progressive depolarization were consistent with a progressive increase in intracellular Ca concentration. Reversed beating showed a threshold of membrane potential change and increased in frequency and duration with greater depolarizations (Text-fig. 6). Likewise, the intracellular Ca concentration probably increased after a given degree of depolarization and continued to increase to a maximum with greater depolarization, as has been found in barnacle muscle (Ashley & Ridgway, 1970). With greater depolarizations the reversed beating continued for some time beyond membrane repolarization (Text-fig. 11). This might result from a prolongation of the period of increased Ca concentration assuming a time-dependent process (e.g. Ca pump) for the reduction of the concentration below the threshold for reversed beating.

According to the ionic hypothesis (Hodgkin, 1964; Katz, 1969) the net flux of an ion will be reduced (at a given conductance) if the driving force on the ion is reduced (i.e. as the membrane potential approaches the equilibrium potential of the ion). The data from the extracted model suggests that the intracellular concentration of Ca in the unstimulated living cell is below io~* M. Assuming an intracellular Ca concentra-tion of io~7 M, and knowing the extracellular concentration to be io"3 M, the calculated value of the Ca equilibrium potential (i?ca) is in the vicinity of +116 mV. When the membrane potential was shifted by stimulus current to positive values less than + 70 mV, reversed beating occurred. However, further increase in positive potential produced a suppression of ciliary reversal (Text-figs. 11 and 12). This is to be expected if it is assumed that reversal requires a certain minimum increment in net Ca influx. As .E'en was approached, the influx of Ca presumably dropped below the rate required to produce the threshold concentration in the cilium and the stimulus, although suprathreshold, failed to produce ciliary reversal. The minimum suppression potential lay between + 70 and + 82 mV. A similar suppression of ciliary reversal was observed with long depolarizing currents in OpaJina (Naitoh, 1958). Katz & Miledi (1967) found no release of pre-synaptic transmitter in the squid stellate ganglion and Baker

et al. (1971) found Ca entry itself to be suppressed in squid giant axon when the

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Finally, the experiments in reduced external Ca demonstrate that no reversal occurs in response to depolarization in a solution where the external concentration of Ca is below a minimum (Text-fig. 14). This can be interpreted as the result of a reduction in the inward Ca current because of a reduced electrochemical gradient. Consequently, the change in the internal Ca concentration is insufficient for a reversal of beating direction. Small depolarizations (3-6 mV) of Euplotes produced reversed beating of the anal cirri while the frontal cirri continued to beat in a forward direction (Text-fig. 5, Table 1). Such differences in responsiveness of cilia have been observed in other preparations. Naitoh (1958) found regional differences in the current threshold for reversal beating in Opalina. The differences in threshold coincided topographically with the differences in membrane resistance over the cell surface. Thus, the anterior end showed both the lowest threshold and the lowest membrane resistance. Similarly, in

Paramecium Ni-paralysed cilia at the anterior end displayed a faster and greater

reorientation in response to depolarization (Eckert & Naitoh, 1970). The differences in beating direction in Euplotes are not the result of variations in the amplitude of depolarization between the anterior and posterior ends since the cell is isopotential (Naitoh & Eckert, 1969). The observed differences may reflect dissimilarities in the reorientation process, in the local intracellular concentrations of Ca required for reversal, or in the local increment in intracellular concentration of Ca produced by excitation. The last possibility seems most likely in view of local differences in resist-ance found in Opalina (Naitoh, 1958).

Hyperpolarization

Hyperpolarization activates the anal cirri to beat in a forward direction (Text-figs. 4 and 7). The stimulus-response characteristics of forward beating differ in some ways from those of depolarization-induced reversed beating. First, activation of forward beating required a greater shift (3—6 times) in membrane potential than did reversed beating (Text-fig. 8). Secondly, the latencies were generally greater than those found after depolarization (Text-fig. 10). Thirdly, the relation between amplitude of hyperpolarization and number of evoked beats appeared to be more discontinuous than the relation associated with depolarization (Text-fig. 8) although lack of data makes this only a preliminary finding. Finally, the forward beating evoked by hyperpolarization continues, in some cells, for a much longer period than reversed beating (Text-fig. 8).

Large hyperpolarizing stimuli were applied to Euplotes to determine whether an increased electrochemical gradient acting on Ca would increase Ca influx to the extent that reversed beating would result (Text-fig. 12). No reversed beating was observed, even with current intensities which caused the cell to deteriorate. This contrasts with results in Opalina (Naitoh, 1958) in which large inward currents produced reversed beating. The negative results with Euplotes may be due to a low level of Ca con-ductance with hyperpolarization or simply greater susceptibility to damage by hyper-polarization.

Beating frequency

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SUMMARY

1. Membrane control of ciliary activity in the protozoan Euplotes was investigated by a combination of electrophysiological and cinematographic techniques.

2. The anal cirri, which are quiescent in the absence of stimulation, were selected for this study.

3. Membrane depolarization by means of injected current produced a reversal of the direction of beating (i.e. towards the cell anterior so as to make the ciliate swim back-wards). Depolarization also increased the frequency of beating. Increasing depolariza-tions resulted in an increased number of reversed beats and increased frequency.

4. When the membrane potential was shifted beyond + 70 mV, reversed beating did not occur until after the current pulse ended.

5. Depolarization did not evoke reversed beating when the external calcium (Ca) concentration was reduced to io~* M with EGTA.

6. Hyperpolarization caused the cirri to beat in a normal direction (i.e. towards the rear of the ciliate so as to cause the animal to swim forward). Increasing hyperpolariza-tions resulted in an increased number of forward beats and an increased frequency. 7. The cell was treated with the detergent Triton X-100 to permit Ca, Mg and ATP direct access through the extracted membrane to the cell interior. At Ca concentrations below io~7 M, Mg-ATP-reactivated cilia of Triton-extracted cells beat normally. At Ca concentrations above approximately io~7 M the reactivated beat resembled the reversed beat in the living cell.

8. The evidence suggests that membrane-regulated concentrations of intracellular Ca control the direction of ciliary beating. Thus, stimuli which produce an adequate Ca influx lead to ciliary reversal.

Dr A. Murakami made indispensable suggestions and Drs H. Machemer, Y. Naitoh, and K. Friedman provided advice. Dr J. Frankel and J. Ruffolo, Department Zoology, University of Iowa identified the species of Euplotes. The authors are grateful to Dr T. L. Jahn for the use of his projector and to J. Fonseca for his assistance. Dr D. Junge and Dr J. Morin made suggestions on the early form and Dr J. Sheridan assisted in the final preparation of the manuscript.

This work was supported by a U.S.P.H.S. Training Grant 5TO1 GM00448 to the Physiology Department, U.C.L.A. and U.S.P.H.S. grant NS08364 and NSF grant GB-30499 to R. Eckert. Based on a dissertation submitted by Miles Epstein in partial fulfilment of the requirements for the Ph.D. to the Zoology Department, University of California, Los Angeles.

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EXPLANATION OF PLATE

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x

-55. fid. 65

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