gravikinesis in stylonychia mytilusis based on membrane ... · 161 introduction during the past...

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INTRODUCTIONDuring the past decades, the sensation of gravity by cells has beeninvestigated in many systems (Bräucker and Hemmersbach, 2002;Häder et al., 2005; Hughes-Fulford and Lewis, 1996; Lewis, 2002;Sievers and Volkmann, 1979). By the end of the 19th century,negative gravitaxis in Paramecium had been described as amovement anti-parallel to the direction of gravity (Verworn, 1889).Gravitaxis consists of a directional component (graviorientation) anda kinetic component (gravikinesis). They act to oppose sedimentationof the cell. The density of the cytoplasm of Paramecium exceedsthe density of fresh water by at least 4% (Kuroda and Kamiya, 1989;Taneda, 1987; Watzke, 2000). Without a compensation ofsedimentation the cell will continuously sink to the ground, leavingpreferred conditions for food uptake and reproduction.

Behavioural experiments were designed to explain thegraviresponses in ciliates (Hemmersbach-Krause et al., 1991;Machemer et al., 1991; Machemer and Bräucker, 1992). Accordingto a common assumption, the cytoplasm surrounded by themembrane of the cell acts as a statocyst to generate an outward-directed force against the lower cell membrane, which opensmechanosensitive ion channels (Machemer et al., 1991). There aretwo types of these ion channels: depolarising Ca2+ channels(accumulating anteriorly) and hyperpolarising K+ channels(accumulating posteriorly). These channels show a polar distributionalong the antero-posterior cell-axis at the lateral membrane inStylonychia (de Peyer and Machemer, 1978) and over the wholesurface membrane of Paramecium (Ogura and Machemer, 1980).We show that reorienting the cell with respect to the gravity vectorleads to a modulation of the membrane potential, presumably dueto activation of these mechanosensitive ion channels. The couplingof the membrane potential to the direction and frequency of theciliary power stroke (Machemer, 1974; Machemer and de Peyer,1982) results in an increase in the forward locomotion rate in

upward-moving cells and in a decrease of the forward locomotionrate in downward-moving cells (gravikinesis). The molecularstructure and the mechanism of activation of the mechanosensitiveion channels are so far unknown. The involvement of secondmessengers, cAMP and cGMP, is still debated (Hemmersbach etal., 2001; Richter et al., 2002).

The effect of gravity on locomotion rates results from behaviouralanalysis. Measurements of the downward and upward locomotionrates (VD, VU), the sedimentation rate (S), and the gravity-independent rate of propulsion (P) determine the gravity-inducedcomponent of active locomotion (gravikinesis) in downward-moving (D) and upward-moving cells (U) (Machemer et al., 1991):

D VD – P – S, (1)

U P – VU – S. (2)

To exactly determine P, locomotion rates have to be measured underlong-term microgravity conditions. For a species with a polar, two-gradient distribution of Ca2+ and K+ mechanoreceptor channels(Paramecium, Stylonychia), it is acceptable to use the locomotionrate of horizontally moving cells (VH) as an approximation of thevalue of P. This is possible because depolarising and hyperpolarisingreceptor channels activate simultaneously in the horizontal positionof Stylonychia, which neutralises their effects on the Ca2+/K+

conductance ratio (Machemer, 1998b).P is cancelled from the equations by subtraction of terms (1) and

(2), giving the generalised value of gravikinesis () as the arithmeticmean of D and U:

(VD – VU) / 2 – S. (3)

Gravikinesis has been repeatedly established in protists investigatedso far: Paramecium caudatum (Machemer et al., 1991), Parameciumtetraurelia (Hemmersbach-Krause et al., 1993), Didinium nasutum

The Journal of Experimental Biology 213, 161-171Published by The Company of Biologists 2010doi:10.1242/jeb.030940

Gravikinesis in Stylonychia mytilus is based on membrane potential changes

Martin Krause1,*, Richard Bräucker2 and Ruth Hemmersbach3

1Department of General Zoology and Neurobiology, Ruhr-University Bochum, D-44780 Bochum, Germany, 2DLR_School_Lab Köln,Linder Höhe, D-51174 Köln, Germany and 3DLR, Institute of Aerospace Medicine, Linder Höhe, D-51174 Köln, Germany

*Author for correspondence ([email protected])

Accepted 14 September 2009

SUMMARYThe graviperception of the hypotrichous ciliate Stylonychia mytilus was investigated using electrophysiological methods andbehavioural analysis. It is shown that Stylonychia can sense gravity and thereby compensates sedimentation rate by a negativegravikinesis. The graviresponse consists of a velocity-regulating physiological component (negative gravikinesis) and anadditional orientational component. The latter is largely based on a physical mechanism but might, in addition, be affected by thefrequency of ciliary reversals, which is under physiological control. We show that the external stimulus of gravity is transformedto a physiological signal, activating mechanosensitive calcium and potassium channels. Earlier electrophysiological experimentsrevealed that these ion channels are distributed in the manner of two opposing gradients over the surface membrane. Here, weshow, for the first time, records of gravireceptor potentials in Stylonychia that are presumably based on this two-gradient systemof ion channels. The gravireceptor potentials had maximum amplitudes of approximately 4mV and slow activation characteristics(0.03mVs–1). The presumptive number of involved graviperceptive ion channels was calculated and correlates with the analysisof the locomotive behaviour.

Key words: Stylonychia mytilus, ciliate, behaviour, electrophysiology, mechanosensitivity, gravikinesis, gravitaxis, gravireceptor potential.

THE JOURNAL OF EXPERIMENTAL BIOLOGY

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(Machemer et al., 1993), Loxodes striatus (Neugebauer et al., 1998),Tetrahymena pyriformis (Kowalewski et al., 1998), Euglena gracilis(Machemer-Röhnisch et al., 1999) and Bursaria truncatella (Krauseand Bräucker, 2008).

In the ciliate Loxodes striatus, a spherical cell organelle – theMüller body – was considered as a putative gravisensor (Fencheland Finlay, 1986). The Müller body consists of a vacuole, containinga BaSO4 crystal, connected to a modified cilium. Cells whose Müllerbody had been destroyed by means of a laser did not show gravitaxis(Hemmersbach et al., 1997). Experiments in density-adjusted media(no density difference between cytoplasm and surrounding medium)revealed a reduced, but still persistent, gravikinesis, indicating theparticipation of mechanosensitive ion channels located in the outercell membrane in the gravitransduction process (Neugebauer et al.,1998). Loxodes and Stylonychia have a common habitat: thesubstrate of a pond. In the literature, Stylonychia has been describedas a ‘walking’ cell. In resting and slow ‘walking’ cells, it can beobserved that most of the ventral cirri have contact with the solidground (M.K., personal observations) (Machemer and Deitmer,1987). So far, there are no studies that describe whether this contactof all cirri still persists during ‘walking’. Furthermore, the paroralmembranelles are described to produce a negative pressure thatpresumably holds the cell close to the solid substrate (Machemer,1965). In our opinion, it is possible that during ‘walking’, severalcirri lose their contact with the substrate and that the cell glides ona small fluid film. A similar kind of locomotion can be observedin Loxodes and is more precisely described as ‘gliding’ locomotion(Bräucker et al., 1992; Machemer-Röhnisch et al., 1998). Stylonychiadiffers from Loxodes in the fact that an intracellular organelle forsensation of gravity has not been identified.

In the present study, we show for the first time that Stylonychiamytilus modulates the membrane potential in response to spatialorientation of the cell in the water column. Earlier investigations(Gebauer et al., 1999) in Paramecium had indicated the existenceof gravireceptor potentials of small amplitudes (<1.5mV). The ciliateStylonychia is highly sensitive to mechanical stimulation (de Peyerand Machemer, 1978; Machemer and Deitmer, 1987). Therefore, itwas likely that gravireceptor potentials in this ciliate would be moreprominent. The identification of electric membrane properties andmeasurements of the mechanosensitivity lead to calculations of thenumber of involved graviperceptive ion channels. Furtherbehavioural analyses reveal that Stylonychia mytilus performs anegative gravitaxis, as shown for other protists. The data are inaccordance with the special statocyst hypothesis (Machemer et al.,1991). The present data are part of a dissertation by Martin Krauseat the Faculty of Mathematics and Science, University of Bonn,Germany.

MATERIALS AND METHODSCulturing

The hypotrichous ciliate Stylonychia mytilus (Müller) was culturedin buffered Pringsheim solution (0.08mmoll–1 MgSO4,0.85mmoll–1 Ca(NO)3, 0.25mmoll–1 KCl, buffered with Sörensenbuffer at pH7) and fed with the mixotrophe flagellateChlorogonium elongatum three times a week. Cells were exposedto a 14h:10h light:dark regimen (maximum 3.5Wm–2); theculturing temperature was 20°C. Earlier experiments had shownthat the behaviour of Stylonychia relates to the time passed aftercell division (Machemer, 1965). To consider this and to minimisethe effect of other circadian rhythms, all experiments were doneat the same time of day with cells of the same age under definedconditions (temperature, light).

Determination of cell sizeThe cell size of Stylonychia was determined by video recording cellsat 150� magnification, followed by a computer-aided single-frameanalysis. Calibration of measured body length was achieved bycomparison of video records to scale paper under the samemagnification. Measurement of longitudinal and transversal axes ofcell bodies allows the calculation of cell volume and surface areaas described previously (Machemer and Deitmer, 1987). For thiscalculation, we assume the cell body to be a rectangular solid. Withthe optical magnification used, the preciseness of measurements was±3.6m. Double measurements of single cells were avoided bycontinuous resuspension of the population. The total recording timeof a sample was limited to 15min.

Behavioural analysisFor investigation of the behaviour, 150–200 cells were transferredto an experimental chamber as described elsewhere (Nagel et al.,1997). Stylonychia shows a long-lasting inactivation after transferfrom culture medium to an experimental chamber, as known fromother ciliates (Machemer-Röhnisch et al., 1998; Oka et al., 1986).Therefore, all behavioural experiments were carried out inPringsheim solution, giving an adaptation time of 4h. To preventinfluences of temperature changes, all experiments were done at21±1°C.

Experimental chambers were mounted on a platform, allowinga reorientation of chambers from horizontal to vertical position andvice versa. Locomotion of cells was recorded by a commercial CCDcamcorder with macro lens. We use a dark-field illumination by acircular arrangement of 48 green LED (565nm, 700lx) to avoidlight-dependent cues for cell orientation. Photostimulation at thewavelength of 565nm had been experimentally excluded for otherciliates such as Paramecium (Iwatsuki and Naitoh, 1982). The videoimage size was 9�7mm2 or 5% of the experimental chamber surfacearea. Locomotion rates, reversal frequencies, linearity of tracks andorientation of individual cells were analysed using computer-aidedimage processing (Häder and Vogel, 1991) and additional softwaredeveloped by R.B.

We visualise the graviresponses of Stylonychia using circularhistograms, which show the circular distribution of medianlocomotion rates and the percentage of cells found withinorientational sectors at defined angles (Batschelet, 1981; Machemerand Bräucker, 1992). For quantification of graviorientation of cells,the orientation coefficient rO (Machemer et al., 1991; Machemer etal., 1997) was used. This coefficient is +1 if all cells are strictlyoriented upwards and –1 if all cells are oriented downwards.Significance of orientation was tested applying the Rayleigh test(Batschelet, 1981).

For determination of direction-dependent locomotion rates, therecording area was subdivided into sectors of 90°. Locomotion ofupward, downward and horizontally walking cells (VU, VD, VH) wasdetermined by calculating the median locomotion rates within thesesectors.

For measurements of the sedimentation rate in Stylonychia, thecells were immobilised with 2mmoll–1 NiCl2 using proceduresdescribed for other ciliates (Nagel et al., 1997). Immediately afterimmobilisation, cells were infused into the experimental chamberand the sedimenting cells were recorded. Each sample wasmeasured four times after performance of a 180° turn of thechamber.

Non-parametric statistics (median values, 95% confidence range,U-test) were applied because Gaussian distribution of data was notassured.

M. Krause, R. Bräucker and R. Hemmersbach


163Graviresponses in Stylonychia

ElectrophysiologyIntracellular electrophysiological experiments in Stylonychia werecarried out applying methods described by Naitoh and Eckert(Naitoh and Eckert, 1972) and de Peyer and Machemer (de Peyerand Machemer, 1977). Two hours prior to experiments, starved cellswere washed in experimental solution (1mmoll–1 CaCl2, 1mmoll–1

KCl, 1mmoll–1 Tris-HCl, pH 7.0). An intact specimen was selectedfor measurements and placed on the lower side of a glass bridgeabove the experimental chamber (bath). Under microscopicobservation, a holding capillary was inserted into the cell to avoidmovements of the cell during the recordings. After this mechanicalfixation of the cell, the bath was filled with experimental solution(21±1°C). The membrane potential was measured differentiallybetween an intra- and an extracellular glass electrode.

The electrodes (borosilicate glass capillaries) were filled with1moll–1 KCl. Resistances were between 40MW (current electrodes)and 100MW (electrodes for potential measurement). The tips of theelectrodes and the holding capillary were bent twice by 30° each.For achievement of a long-lasting recording, the electrodespenetrated the anterior or posterior macronucleus of the cell fromventral. A second electrode was inserted for current injection.Measurements of input resistance and capacitance were performedusing small hyperpolarising current steps. For establishment ofcurrent–voltage relationships, injections of 80ms current-steps from–6 to +6nA were performed, and the resulting changes of membranepotential were measured. Voltage-clamp experiments wereperformed using a high-gain differential amplifier (Analog Devices,171K). The holding potential was set equal to the membrane restingpotential, and early and steady-state transmembrane currents weremeasured at voltage steps in the range of –70 to +90mV from theresting potential. All data from voltage-clamp experiments werecorrected for leakage current.

Mechanosensitivity in Stylonychia was analysed using a smallglass stylus mounted on a piezo-electric crystal to indent the cellmembrane (de Peyer and Machemer, 1977). The stylus was drivenby trapezoid voltage pulses, allowing a fast movement of thestimulator and avoiding its oscillation. The deflection of the stylusdepends on the voltage amplitude and was calibrated using an ocularmicrometer. For measurements of membrane potential changes afterstimulation of the lateral membrane, the tip of the stimulator wasoriented vertically. Stimulation of the ventral and dorsal cell surface

was performed using a horizontally oriented tip of the glass stylus.Stimulations were done in five segments of the longitudinal axis ofthe cell, and the median potential changes in each segment werecalculated.

For measurements of gravireceptor potentials, the electrodes wereinserted from lateral. To avoid mechanical stimulation, theseelectrodes were placed centrically between the anterior and posteriorcell poles. The cell was carefully reoriented by means ofmicromanipulators. Changes in resting potential were measuredbefore, during and after reorientation of the cell.

Four turning modes of the cell were applied: from horizontal toposterior down, from horizontal to anterior down, from anteriordown to posterior down and vice versa. For statistical evaluations,the recording time (maximum 120s) was divided into classes of 1s,and the median membrane potential change in each class wascalculated.

RESULTSCell size

For determinations of cell volume and membrane area, longitudinal(cell length) and transversal (cell width) axes of 486 cells weremeasured (Fig.1). Microscopic observations of individual cellsconfirmed that the dorso-ventral axis of the cell body measures 1/10of cell length (L). As has been shown by earlier analyses (Machemerand Deitmer, 1987), the width of the cell body corresponds to 0.4L.The median cell length of Stylonychia was found to be 236.1m(–3.9m/+2.9m). The median cell width was 97.2m(–3.6m/+1.0m). Considerations of the membranelles, undulatingmembrane and cirri of Stylonychia strongly affect calculations ofthe complete membrane area (2�10–3cm2) but not the calculatedcell volume (3.6�10–7cm3). The data and calculations aresummarised in Table1.

GravitaxisHistograms in Fig.2 and data in Table2 show orientation andlocomotion rate in Stylonychia in vertical and horizontalexperimental chambers. Turning of the experimental chamber intothe vertical position allows the cells to orient with respect to gravity.A statistically secured majority of cells walk antiparallel to thegravity vector although this behaviour is not predominant (negativegravitaxis; rO0.06; P≤5%). As might be expected, no preferential

0

10

20

30

40

50

Dimension (µm)

Transversal Longitudinal

140 30026022018040 80 120

Rel

ativ

e fr

eque

ncy

(%)

Fig.1. Frequency distribution histograms showing cell sizes of Stylonychiamytilus (N486). Median distances along the transverse axis were 97.2m,and 236.1m along the longitudinal axis. Insets illustrate cell axes. Celldrawings modified after Machemer and Deitmer (Machemer and Deitmer,1987).

Table 1. Geometry of Stylonychia: (A) median values of cell lengthand width; (B) calculations of cell geometry

Cell Cell Cell A length (m) width (m) height (m)

236.1(232, 239) 97.2 (93.6, 98.2) 23.6

B Membrane area (cm2) Volume (cm3)

Soma 6.1�10–4 3.55�10–7

Membranelles 8.8�10–4 5.5�10–9

Cirri 4.9�10–4 3.1�10–9

Undulating membrane 2.2�10–5 1.4�10–10

Complete 2�10–3 3.6�10–7

Median values of cell length and width (N486; limits of 95% confidenceinterval of the medians in parentheses). Calculations of cell geometrywere done according to the terms by Machemer and Deitmer (1987). Theheight of the cell body is estimated at 1/10 of the cell-length. The diameterof one single cilium is 0.25m. The membranelles and cirri of Stylonychiahave specific lengths and are composed of tens of independent cilia; here,their summed areas and volumes are given.


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orientation was observed in horizontally oriented chambers(rO–0.01). The measured locomotion rates of nearly 1mms–1 wereindependent of walking direction and position of the experimentalchamber.

SedimentationApplication of 2mmoll–1 NiCl2 completely arrested the ciliary beatin Stylonychia and led to passive sedimentation of the cells. Nodeformation of cells was observed within 45min. After that time,swelling of cell body occurred and some cells disintegrated or lost

their adoral membranelles. Considering these observations, the timefor sedimentation rate determination was restricted to 30min. Fig.3shows the relative frequency distribution of sedimentation rates atnormal gravity. The resulting median sedimentation rate was180ms–1 (N2549). Confidence ranges are small (–2/+3ms–1), andthe median coincides with the arithmetic mean, indicating a Gaussiandistribution. We analysed the orientation of the cell longitudinal axisfrom 835 sedimenting cells. In 519 cells (60.3%), the longitudinalaxis was aligned with the direction of the gravity vector (±45°) whereaswith the used magnification it was not possible to discriminate betweenanterior and posterior cell poles. 183 cells (22.8%) sank with theirlongitudinal axes transverse to the gravity vector. In 133 cells, thelongitudinal axis could not be identified clearly.

GravikinesisThe median direction-dependent locomotion rates (VD, VU and VH)of Stylonychia were determined using data from cells that orientedwithin 90° sectors. A general gravikinesis () of –178ms–1 wascalculated according to Eqn3, applying a median sedimentation rateof 180ms-1. It is seen that Stylonychia mytilus shows a value ofgeneral gravikinesis, which completely compensates thesedimentation rate.

The gravity independent propulsion rate (P) has so far not beendetermined experimentally. Assuming a fully balanced bipolardistribution of gravireceptors, P is estimated using velocities ofhorizontally walking cells (993±7mms–1). The direction-dependent gravikinesis (D, U) is then calculated to be –198ms–1

and –159ms–1, respectively (Eqns1,2; see Table2).According to the special statocyst hypothesis, the cytoplasmic

mass exerts an outward-directed force on the lower cell membrane,thereby causing channel activation. This force is calculated usingthe values of cell volume (soma) and mean density. In ciliates sofar investigated, a mean density difference between cytoplasm andsurrounding medium was determined to be 0.04gcm–3 (Taneda,1987; Kuroda and Kamiya, 1989; Neugebauer et al., 1998).Assuming this value and a volume of 3.6�10–7cm3 for Stylonychia,the mass of the cytoplasm is estimated at 1.4�10–8g. At normalgravity, this mass induces an effective downward force of1.39�10–10N on the lower membrane.


1.5 mm s–1

1.5 mm s–1

Orientation Locomotion rate

Ver

tical

Hor

izon

tal

rO=0.065%

rO=–0.01

5%

g

Fig.2. Polar histograms of orientation and locomotion rate of Stylonychiamytilus in a vertically and horizontally oriented experimental chamber. Inthe vertical chamber, a small but significant orientation antiparallel to thevector of gravity is shown (rO0.06). No preferred direction of locomotionwas seen in the horizontal chamber (rO–0.01). The locomotion rate isindependent of the direction of movement (number of data: vertical, 21,517;horizontal, 15,287). For exact values of locomotion rates, also see Table2.

Table 2. Graviresponses of Stylonychia mytilus

Velocity (m s–1) N

Downwards (VD) 975 (961, 987) 4840Upwards (VU) 972 (960, 981) 5818Horizontal (VH) 993 (982, 1000) 5484Sedimentation (S) 180 (178, 183) 2549

Gravikinesis (m s–1)

–178U –159D –198

Orientation Corresponding coefficients locomotion

(ro) rate (m s–1) N

Vertical plane (all directions) 0.06 983 (977, 989) 21,517Horizontal plane (all directions) –0.01 973 (966, 980) 15,287

The observed, direction-dependent rates of locomotion (VD, VH, VU) weredetermined using 90° sectors (N number of analysed tracks).Gravikinesis was calculated using Eqns 1–3. Negative graviorientation isrepresented by the orientation coefficient. Values in parentheses give thelimits of the 95% confidence interval of the medians.

0100

8

12

16

20

200 300 400 500 6000

4

Sedimentation rate (µm s–1)

Rel

ativ

e fr

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ncy

(%)

S=180 µm s–1

0

60

120

180

240

0 10 20 30 40

y=168.3+0.5x

Time (min)

Sed

imen

tatio

nra

te (

µm s

–1)

.

Fig.3. Frequency distribution of sedimentation rates (S) of Ni2+-immobilisedand free-floating Stylonychia mytilus specimen. The median sedimentationrate is 180ms–1. Data approximate a Gaussian distribution (N2549).Inset: sedimentation rate as a function of recording time. No significantchanges were observed within 30min after the immobilisation procedure.Shaded area represents limits of the confidence ranges.



Electrophysiological propertiesIntracellular recordings were performed to characterise passive andactive membrane properties of Stylonychia mytilus. The medianmembrane potential was –44.3mV (–0.7/+1.1mV, N45). Somecells show spontaneous spiking activity at an average frequency of1–2Hz. Changes in membrane potential were measured followinginjection of constant current pulses. The median input resistance,determined from the linear part of the current–voltage relationship,was found to be 21.3MW (–5.2/+2.2MW). Calculation of the inputcapacitance of the cell (1.8nF) was possible using the measuredmedian time constant of 38.9ms (–2.4/+1.9ms) obtained fromrecordings of small hyperpolarisations. For specific input resistance(Ri) and specific membrane capacitance (Ci), the value of thecomplete membrane area was taken into account. The measuredvalue of Ci was 0.9Fcm–2, which is near the value known forbiological membranes (1Fcm–2). This indicates a realistic estimateof the membrane area. The value for the specific membraneresistance was found to be 42.5kWcm2.

Measurements of membrane currents during voltage-clampexperiments reveal activation of voltage-dependent membranecurrents (not shown). It is evident that positive voltage steps activatetwo types of Ca2+-depending early inward currents (see also de Peyerand Machemer, 1977; Deitmer, 1986). The maximum amplitude ofthe low threshold current (activated with depolarisations above3mV) was –4nA. The amplitude of the late-activating Ca2+ currentwas –17nA at depolarisations of +55mV.

MechanosensitivityFig.4 gives examples of membrane potential changes aftermechanical stimulation of the dorsal, ventral and lateral membrane.Independent from the site of stimulation, latencies between firstcontact of the stimulating needle with the membrane and the onsetof the potential change were 3–4ms. A polar distribution ofdepolarising and hyperpolarising mechanoreceptor conductances inStylonychia applies to the whole cell body. A mechanical stimulationof the anterior part of the cell leads to a depolarisation and, becauseof a low threshold of voltage-activated Ca2+ channels, to actionpotentials. The amplitude of depolarisation decreases with moreposterior stimulation. Touching the cell membrane exactly halfwaybetween anterior and posterior cell ends does not elicit a membranepotential change; possibly, depolarisation and hyperpolarisationcompensate each other. Maximum hyperpolarisations were obtainedfollowing stimulation of the posterior cell pole.

Results of a quantitative analysis are shown in Fig.5. Largestamplitudes of membrane potential changes were measured afterstimulation of the lateral cell. Maximum depolarisations were+42mV and, in most cases, action potentials were superimposed.At the posterior cell pole, lateral stimulation led to hyperpolarisationsof –35mV. Stimulation of the anterior dorsal cell side elicitedmaximum receptor potentials (+22mV), and stimulation of theposterior dorsal cell side elicited –20mV. Note that pronounceddepolarisations were obtained from stimulation in an area at 80%of the distance from posterior (Fig.5). A possible explanation forthis result is that receptor channel abundance is decreased in themost anterior membrane segment. Amplitudes of potential changesfrom ventral stimulation exceed those obtained from dorsalstimulation. No significant difference was seen between stimulationof left and right lateral side. Likewise, no differences inmeasurements were seen after stimulation of the left or right dorsalside. A potential change after stimulation at the right anterior-ventral side confirms earlier observations suggesting thatmechanosensitivity in Stylonychia does not involve cilia or cirri (de

Peyer and Machemer, 1978). A participation of cilia and cirri inmechanosensation is unlikely in Stylonychia because stimulation ofcilia-free membrane segments lead to a membrane potential change.This finding supports results obtained in deciliated Parameciumcaudatum (Ogura and Machemer, 1980).

A mechanical stimulation of the ventral membrane was alsoperformed during voltage-clamp experiments (four cells; data notshown). A stimulation of the anterior cell pole induced an inwardcurrent (mean amplitude of –13.5nA); posterior stimulation resultedin outward currents (mean amplitude +19.6nA).

GravireceptionGravireceptor potentials are changes of the resting membranepotential induced by gravity-dependent increases in conductance ofmechanically sensitive ion channels. First measurements were longtime records of membrane potential without reorientation of the cell,to exclude a possible drift, which could mask the gravireceptorpotential. During a sampling period of 120s, the mean membranepotential was found to fluctuate (standard deviation ±0.8mV). Afterthat, starting from the horizontal position, the cell was reorientedparallel to the gravity vector either with the anterior or with theposterior cell pole downwards. Only those cells were selected forevaluation, which did not show fluctuations in membrane potentialprior to reorientation. Some cells fired repetitive action potentials

40 ms

40 mV

Lateral stimulation

Dorsal stimulation

Ventral stimulation

K

L

M

N

O

A

B

C

D

E

S

A

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C

D

E

K

L

M

N

O

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S

F

G

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I

J

F

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H

I

J

Fig.4. Examples of potential changes from the resting voltage after localmechanical stimulation at different positions of the dorsal (A–E), ventral(F–J) and lateral (K–O) membrane. All recordings show a bipolar pattern ofmembrane responses, indicating a gradient-type distribution ofantagonising mechanosensitive ion channels between the polar ends ofStylonychia. Trapezoid track marks the time of stimulation (S). Nodifferences were observed between responses evoked on the left and righthemispheres of the cell.


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before reorientation or after turning downwards. These cells werealso excluded from statistical analysis.

Fig.6A shows typical recordings of the membrane potentialbefore, during and after a reorientation of the cell. In Fig.6Ai, turningthe cell from ‘posterior down’ to ‘horizontal’ led to a depolarisation.The special statocyst hypothesis explains this observation becausedepolarising Ca2+ channels open near the anterior cell pole, andhyperpolarising K+ channels close near the posterior pole. Uponturning the horizontal cell with its anterior end downwards(Fig.6Aii), only depolarising channels are activated. After turningthe cell up by 180° from ‘posterior down’ to ‘anterior down’(Fig.6Aiii), similar amplitude of depolarisation was found comparedwith Fig.6Ai and Fig.6Aii. The special statocyst hypothesis alsopredicts that a gravity-induced activation of posterior receptorchannels leads to a hyperpolarisation. Fig.6B shows three kinds ofreorientations which result in such potential change: from ‘anterior

down’ to ‘horizontal’ (Fig.6Bi), from ‘horizontal’ to ‘posteriordown’ (Fig.6Bii) and from ‘anterior down’ to ‘posterior down’(Fig.6Biii).

A statistical analysis of the experiments (Fig.7) assured that thepotential changes measured are receptor potentials induced bygravity. The potential changes occur slowly (with a small timeconstant of 0.03mVs–1). In most cases, reorientation of the cellsresulted in a new steady-state potential after 60s. Turning the cellfrom ‘posterior down’ to ‘horizontal’ (Fig.7A), two depolarisationmaxima were measured near 40s and 80s after reorientation. Thereason for this observation is obscure; possibly, it is related to thesmall number (seven) of experimental cells. Table3 summarises allsteady-state potential changes as determined 90s after turning ofthe cell. Voltage-clamp experiments revealed no recognisablechanges in transmembrane current after turning the cell. Theamplitudes of expected currents would be smaller than 0.2nA. Suchcurrents were at the limit of separation from the current noise levelin our setup.

DISCUSSIONThe behavioural experiments to analyse gravireception ofStylonychia deal with the preferred locomotion of this ciliate, whichis ‘walking’ on surfaces of submersed substrates. Here, our datadiffer from earlier investigations on graviresponses obtained fromprotists that are generally free swimmers. Apart from that, theestablished methods of behavioural and electrophysiological analysisare easily applied to Stylonychia.

Analysis of sedimentationDetermination of sedimentation

The determination of sedimentation rates is essential for the analysisof gravireception in unicellular organisms. Sedimentation affectscell orientation because a high sedimentation rate, relative to thepropulsion rate, leads to a stronger deviation of cell orientation fromthe measured locomotion track (Machemer et al., 1997). In addition,the sedimentation rate is necessary for the calculation of gravikinesis(Eqns1,2). The sedimentation rate of a spheroid body dependsmainly on its weight and radius (Stokes’ law). Assuming a spherewith the volume of Stylonychia (3.6�10–7cm3) results in asedimentation rate of 170m s–1, which is close to the measuredvalue of 180m s–1.

Problem of sedimentation in walking cellsIn the experiments, S was determined in cells that were floating inthe experimental chamber. The sedimentation of cells sliding nearthe wall of the chamber cannot be assessed for technical reasons.Frictional forces reduce the sedimentation rate of a cell close to awall (Happel and Brenner, 1986). Eqn3 shows that, with the givendata, the absolute value of gravikinesis, , will decrease with adecrease in sedimentation rate. Thus, in Stylonychia, walking on avertical surface, is reduced by the same amount as sedimentationnear a wall decreases with respect to free sedimentation. The pointof this argument is that gravikinesis in Stylonychia can fullycompensate effects of sedimentation independent of its effectivevalue. The sedimentation rate measured in free-floating cells maybe overestimated for walking specimens. Consequently, we wouldoverestimate a gravikinesis. However, by means of statistical testswe could assure the existence of a gravikinesis as long as thesedimentation rate is larger than 8ms–1. Since such a strongdecrease of sedimentation rate by wall effects seems to be unlikely,we must assume a physiological component in gravitaxis ofStylonychia. In our lab, further experiments have been performed


Dorsal

–40

–20

0

20

40

Ventral

–40

–20

0

20

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Lateral

–40

–20

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0 0.2 0.4 0.6 0.8 1

Relative distance from posterior

Cha

nge

from

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e re

stin

g po

tent

ial (

mV

)

Fig.5. Analysis of mechanosensitivity in Stylonychia. Shown are themedian changes from the resting membrane potential after submaximumstimulation of the dorsal, ventral and lateral membrane (stimulator deflexionwas 8m). Abscissa represents the distance from posterior in relative units.Confidence ranges are shadowed in grey. Each data point representsmeasurements from at least four cells.



to study the locomotion behaviour of Stylonychia under conditionsof micro- and hypergravity. These, so far unpublished, observationsgave strong evidence that the sedimentation rate of walkingStylonychia cells is larger than 0.

First, the direction-dependent locomotion rates of Stylonychiawere measured after a step transition from 1g to microgravity inthe drop tower of ZARM (Centre of Applied Space Technologyand Microgravity, Bremen, Germany). It could be observed that,immediately after transition to microgravity, the locomotion rate ofdownward oriented cells decreased by 80ms–1 and that of upwardoriented cells increased by 70ms–1. This sudden change inlocomotion rate can only be explained by absence of sedimentationunder microgravity. This result indicates a sedimentation rate ingliding Stylonychia which is >0. However, our measured value ofsedimentation rate in free floating cells (and thereby the value ofgravikinesis) is possibly overestimated for walking specimens.

Further support for a gravikinetic component in Stylonychiacomes from experiments under raised gravity in a centrifuge. Here,the locomotion rates of upward and downward oriented cells were

found to be different functions of acceleration. The locomotion ratesof downward oriented cells increased with increased acceleration(and sedimentation rate) whereas the locomotion rate of upwardoriented cells remained more or less unchanged.

Mechanism of graviorientation in StylonychiaWe have demonstrated that Stylonychia is able to perceive andrespond to gravity. Stylonychia shows graviresponses thatcompensate sedimentation. The gravitaxis consists of a directingcomponent (graviorientation) and a velocity-regulating component(gravikinesis). In the vertical chamber, a small majority of cellswas oriented upwards. The orientation coefficient of 0.06 isstatistically significant but less prominent as compared with otherciliates so far investigated. Orientation coefficients of 0.2 wereobserved in Paramecium caudatum (Machemer et al., 1991) andwere even as large as 0.5 in Didinium nasutum (Machemer et al.,1993) and Bursaria truncatella (Krause and Bräucker, 2008).Assuming that orientation is based on exclusive physicalmechanisms, two hypotheses may be considered: (1) an uneven

10 s

10 mV

Vm

T

Ai

Vm

T

ii

Vm

T

iii

Vm

T

Bi

Vm

T

ii

Vm

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iii

g

Fig.6. Course of the membrane potential(Vm) before, during and after reorientationof typical recordings from different cells.The track (T) marks the time period ofreorientation. Cell positions relative to thevector of gravity are shown by insets.(A)Depolarisation was induced byreorientations from ‘posterior down’ tohorizontal (i), from horizontal to ‘anteriordown’ (ii) and from ‘posterior down’ to‘anterior down’ (iii). (B)Hyperpolarisationof the membrane potential occurred afterreorientation from ‘anterior down’ tohorizontal (i), from horizontal to ‘posteriordown’ (ii) and from ‘anterior down’ to‘posterior down’ (iii). The potentialchanges are explained by an activationand inactivation of topographicallyorganised gravisensitive Ca2+ and K+

channels corresponding to the specialstatocyst hypothesis.

–4

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0 10 20 30 40 50 60 70 80 90–10–20

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F

Time (s)

Med

ian

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e po

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ial c

hang

e (m

V)

Fig.7. Medians of changes of the membranepotential before, during and after reorientationsinducing a depolarisation (A) or hyperpolarisation (B).Shadowed areas mark the time period of turning thecell. Reorientations correspond to Fig.6. Confidenceranges were small (<0.5mV) and so have beenomitted for clarity. Maximum amplitudes with 95%confidence intervals are shown in Table3. Numbersof data: (A) 7 cells, 9 measurements; (B) 30 cells, 47measurements; (C) 12 cells, 17 measurements; (D)14 cells, 14 measurements; (E) 21 cells, 32measurements; (F) 12 cells, 22 measurements.


168

distribution of density in the cell (static hypothesis) (Verworn,1889) or (2) a passive orientation of the cell due to its shape(hydrodynamic hypothesis) (Roberts, 1970). Microscopic analysisof the cell body of Stylonychia shows that the anterior part is moreflattened and wider than the posterior part. The transversaldimensions shown in Fig.1 represent the maximum cell width andwere mostly found in the anterior part of the cell. The height ofthe cell is at maximum halfway between the anterior and posteriorend. According to the hydrodynamic hypothesis, this cell shapefavours a downward orientation with the anterior cell part down.This prediction does not agree with the experimental findings.Winet and Jahn (Winet and Jahn, 1974) had argued against thehydrodynamic hypothesis, showing that a negative gravitaxisoccurred in other ciliates shaped similar to Stylonychia.

The assumed density distribution gives an indication for the statichypothesis. It may be argued that density in the posterior part of thecell is increased, because the larger of the two macronuclei andremaining food particles are located at the posterior cell pole. Suchuneven distribution of weight induces a torque and would evoke anegative gravitactic orientation. An exclusive physical orientationimplies that all cells should be oriented upwards after some time sothat the orientation coefficient resembles the value +1. This does notagree with experimental findings in several ciliate species (Bräuckeret al., 1996; Krause and Bräucker, 2008), indicating contributions ofother, i.e. physiological, mechanisms to cell orientation. InStylonychia, it is likely that the comparatively high frequency ofreversals randomises the orientation of specimens in a cell population.Cells generate spontaneous action potentials, which are followed byrapid ‘back–forward’ movements (reversals). Reversals are shown inhorizontally and vertically oriented experimental chambers. Due torandomisation of orientations, a directed movement, which wouldcause a gravi-accumulation, is unlikely. Physiological contributionsto graviorientation in protists have long been a matter of controversy(Machemer and de Peyer, 1977). Häder et al. (Häder et al., 1995)postulated a physiological mechanism of graviorientation in theflagellate Euglena; the treatment of Euglena with UV radiation (Häderand Liu, 1990), application of various heavy metal ions (Stallwitzand Häder, 1994), and membrane-incorporated agents (Häder andHemmersbach, 1997) affected orientation in this species, suggestinga physiological control of gravitaxis without excluding physicalmechanisms. Investigations in Paramecium tetraurelia showed anincrease in the frequency of reversals in the downward swimmingcells as compared with the cells that favoured upward swimming(Nagel and Machemer, 2000).

GravikinesisGravity-dependent modulation of velocity (gravikinesis) is definitelybased on a physiological mechanism. In Stylonychia, the speed of

upward and downward walking is the same, and sedimentation rateis fully compensated by gravikinesis. Similar results were obtainedin gliding Loxodes (Bräucker et al., 1992). The degree ofcompensation of sedimentation by an opposing gravikinesis differsbetween the species. In the smallest ciliate investigated so far,Tetrahymena pyriformis, an overcompensating gravikinesis (130%of the value of S) was measured (Kowalewski et al., 1998). In thegiant ciliate Bursaria truncatella, the sedimentation rate of923ms–1 is compensated by 70% [gravikinesis –633ms–1 (Krauseand Bräucker, 2008)]. A similar ratio has been shown in Didiniumnasutum (Bräucker et al., 1994; Machemer et al., 1993). A smallercompensation of S has been measured in Paramecium tetraurelia[28% (Hemmersbach-Krause et al., 1993); 59% (Nagel andMachemer, 2000)]. Even within the same species – Parameciumcaudatum – experimental values of sedimentation and gravikinesis,and hence the degree of compensation of gravity, vary within limits[45% (Machemer et al., 1991); 42% (Watzke et al., 1998); 51%(Watzke, 2000)]. These data indicate that a correlation between cellsize and the amount of gravikinetic compensation is not systematic.

For the determination of a direction-dependent gravikinesis, thevalue of the gravity unrelated velocity of propulsion, P, is required.This value can be exactly determined in cells only after sufficienttime of adaptation to the weightless condition in space. As analternative to weightlessness, the rate of horizontally walkingStylonychia may be chosen as an approximation of the value of P.This is possible, because in horizontally oriented Stylonychia apossible gravistimulation is neutralised due to the bipolar, gradient-type distribution of mechanosensitive Ca2+ and K+ channels. Thisis similar to Paramecium (Machemer et al., 1991) and several otherciliates (Machemer and Teunis, 1996). The speed of horizontallywalking Stylonychia cells in the vertical chamber (993ms–1)exceeds VD, VU and locomotion rate in horizontally orientedchambers (973ms–1; Table2). This offset may be due to anaccumulation of hyperpolarising gravireceptors at the lateral surfacemembrane. We should mention that in the vertical chamber, cellsare oriented with their ventral side parallel to the gravity vector.Cells walking in a horizontally oriented chamber are oriented withtheir ventral or their dorsal side perpendicular to the gravity vector.

There are some indications that graviorientation and gravikinesisare subject to different mechanisms: long-term experiments withStylonychia exposed to different experimental solutions showed thatcells immediately oriented with respect to the gravity vector,whereas gravikinesis gradually rose to become maximum after 1–2h(data not shown). Previous experiments in Paramecium biaureliaadapted to low temperatures (4°C) established a gravikinesis but nosignificant graviorientation (Freiberger, 2004).

Electrophysiology and mechanosensitivityThe electrophysiological data in Stylonychia are based on a largenumber of cells to support a statistical analysis of the relationshipbetween behaviour and its electrophysiological basis. Numerouselectrophysiological data are in the literature (for a review, seeMachemer and Deitmer, 1987). Most of these data were collectedfrom different cell clones (Table4). Therefore, it was reasonable todetermine the electrophysiological properties of the present cellclone. Since we did not find differences in our current–voltagerelationships compared with earlier results (de Peyer and Machemer,1977), we conclude that the properties of voltage-dependent ionchannels are the same in actual and earlier cell lines.

Stylonychia mytilus is highly sensitive to mechanical stimulation.Membrane currents following stimulation at the anterior cell poleare much larger than observed in Paramecium (Ogura and


Table 3. Peak amplitudes of membrane potential changes (Vm)after reorientation of the cell as described in the Materials and

methods

Starting position End position Vm (mV)

Horizontal Posterior down –2.9mV (–2.8/–3.0)Posterior down Horizontal +3.9mV (3.6/4.1)Horizontal Anterior down +2.7mV (2.65/2.8)Anterior down Horizontal –3.0mV (–2.5/–3.3)Posterior down Anterior down +3.4mV (3.3/3.5)Anterior down Posterior down –3.1mV (–3.05/–3.2)

Each value was determined 70–90 s after cell reorientation. Values inparentheses give the limits of the 95% confidence interval of the medians.



Machemer, 1980). In the literature, the data on mechanosensitivityin Stylonychia were previously obtained from stimulation of thelateral membrane (de Peyer and Machemer, 1978). Our experimentsshow that the bipolar distribution of gradients of sensitivity appliesto the entire cell surface. At the same latitude of the cell, a stimulationof the lateral, ventral and dorsal regions evoked different amplitudesof receptor potentials. With identical stimulus velocity and distanceto the membrane, the differences in amplitude are probably basedon variations in channel density or cytoskeletal substructure at thesites of stimulation.

As a next step, we tested whether or not the mechanosensitivechannels can be activated by the cytoplasmatic force acting againstthe cell membrane in the outward direction, as predicted by thespecial statocyst hypothesis (Machemer et al., 1991).

Gravireceptor potentialsStylonychia is highly mechanosensitive and has a relative large cellmass, which is a favourable condition for gravitransductionexperiments. The obtained potential changes of 4mV after cellreorientation support the special statocyst hypothesis. Previousexperiments in Paramecium documented gravity-induced smallpotential changes of 1.5mV (Gebauer et al., 1999).

We have evidence that a gravireceptor potential of 4mV issufficient for a change in locomotion rate in Stylonychia. This hasbeen confirmed by application of different potassium solutions thatinfluence the locomotion rate as well as the membrane potential.De Peyer and Machemer (De Peyer and Machemer, 1977) observeda shift of the steady-state membrane potential in Stylonychia of about3.5mVmmoll–1 K+. Correspondingly, a variation of the external[K+] induced a change in locomotion rate of 196ms–1 per mmoll–1

K+ in Stylonychia (M.K., unpublished). Because the membranepotential and the frequency of the cirral beat are linearly correlatednear the resting potential (Machemer and Deitmer, 1987), the K+-dependent change in speed of locomotion (56ms–1 per mV)extrapolates to a speed change of 224ms–1 per 4 mV gravireceptorpotential, which reasonably corresponds to the calculatedgravikinesis.

From a physiological point of view, larger amplitudes of receptorpotentials would not be useful: in a downward oriented cell, a largerdepolarisation would lead to reversed movements of cilia and, incourse, to a continuously backward movement of the cell.

Ion channels involved in gravireceptionPotential changes after external mechanostimulation exceed theobserved gravireceptor potentials by a factor of 10. Such divergencemay be due to the high velocity of inward deformation of themembrane. Possibly, the number of channels involved in thegravitransduction chain is small. Gebauer et al. estimated that <20channels activated after 180° reorientation of Paramecium (Gebaueret al., 1999).

As in other ciliates, the membrane potential of Stylonychia isdetermined, in the first line, by the equilibrium potentials for Ca2+

and K+ (ECa, +116mV; EK, –90mV) and the conductances for theseions (de Peyer and Machemer, 1977). A membrane potential of–42mV corresponds to a gCa/gK conductance ratio of 0.3. The changein membrane potential following reorientation of the cell from‘anterior down’ to ‘posterior down’ is due to the increase inconductance for K+ ions and the decrease in Ca2+ ions conductance.The number of ion channels involved in gravitransduction may beroughly approximated using data from the literature. We tentativelyassume a mean channel conductance of 30pS [corresponding to10–50pS from cells of the amphibian inner ear (Howard et al., 1988)]and a resting input resistance of 21MW. For a 4mV shift inmembrane potential 40 Ca2+ channels may close or 150 K+ channelsmay open.

Does gravity-induced opening and closing of a few ion channelsagree with the physical limits of signal transduction in cells? Acomparison of the thermal noise level at 20°C (2�10–21Nm) withthe available energy for Stylonychia gravitransduction is possible,assuming a minimal gating distance of 3.5nm (Howard et al., 1988).With a cell volume of 3.6�10–7cm3, a density difference (cytoplasmover freshwater) of 0.048gcm–3 and a gating distance (ofmechanoreceptor channels at the bottom membrane) of 3.5nm, thegross energy available for gravitransduction is 6�10–19Nm, whichexceeds the thermal noise level by more than two orders ofmagnitude.

In the basidiomycete Flammulina velutipes, participation of thecomparatively heavy nucleus (1.22gcm–3) has been implied ingravitransduction (Monzer, 1996). The nucleus of this fungus isconnected to the cytoskeleton. It is assumed that actin filamentstransmit forces from sedimentation of the nucleus to the lowermembrane. In Stylonychia, no such repositioning of the anteriormacronucleus was observed after turning the cell from horizontalto ‘anterior down’ and back to horizontal. Instead, it was possibleto evoke changes in gravireceptor potential even though electrodesfixed the posterior macronucleus. We therefore believe thatparticipation of the nuclei in gravitransduction is unlikely inStylonychia. However, involvement of the cytoskeleton ingraviperception seems to be probable in ciliates (Machemer, 1998a).This hypothesis is supported by results from drop-tower experimentsindicating slow relaxation kinetics of graviresponses after steptransition from 1g to weightlessness (Bräucker et al., 1998; Krause,2003; Krause et al., 2006).

To safeguard our electrophysiological data on the effects ofgravity from misinterpretations, two parameters associated withmicroscope illumination should be mentioned: possible effects ofvectors or gradients of light and temperature.

Effects of illumination and temperatureEffects of illumination

Many ciliates react to light. In Chlorella-free Paramecium bursaria,the photosensitive pigment rhodopsin was described at the antero-ventral membrane of the cell (Nakaoka et al., 1987; Nakaoka, 1989;Nakaoka et al., 1991). Photoreceptor potentials of 0.5mV and

Table 4. Measured membrane properties compared with values from the literature

Parameters Value as measured Value from the literature References

Resting potential –44mV –48mV de Peyer and Machemer (1977)Input resistance 21MW 33–100MW Machemer and Deitmer (1987)Time constant 39ms 40–80ms Deitmer (1986)Input capacitance 1.9nF 1nF M.K., personal observationsSpec. membrane resistance 42.5 kWcm2 220 kWcm2 Machemer and Deitmer (1987)Spec. membrane capacitance 0.9Fcm–2 0.3Fcm–2 Machemer and Deitmer (1987)


170

amplitude peaks after 500ms were determined at 0.7Wm–2.Specimens of horizontally oriented Stylonychia reacted to a gradualincrease of light intensity (of >100Wm–2) with an increasedreversal rate, indicating a depolarisation (M.K., unpublished).Photoreactions in response to the direction of light require shadowingof the photosensitive area. The Lieberkühn’sche organelle inOphryoglena catenula (Kuhlmann, 1993) represents a structure forshadowing. In Stylonychia, no similar structure has been observed.All experiments were performed at the lowest light intensitypossible (0.5Wm–2) to minimise possible light effects. To estimatethe influence of light, long-time recordings of the membranepotential were done, with changes in light intensity between0.5Wm–2 and 10Wm–2. Directly after increasing the light intensity,a phasic depolarisation of the cell was measured (median value<2mV; M.K., unpublished). A light-dependent and persistentpotential shift in the low-intensity range was not seen.

Effects of temperatureThermoreception was investigated in Paramecium by severalauthors. Cooling of the anterior cell area led to a depolarisationof 10mV after lowering the temperature from 25°C to 20°C andevoked a transient Ca2+-dependent inward current (Kuriu et al.,1996). According to Tominaga and Naitoh (Tominaga and Naitoh,1994), warming of the anterior cell membrane results indepolarisation and in hyperpolarisation at the posterior cell pole.These findings suggest that thermosensitive ion channels have abipolar distribution on the surface membrane in ciliates, which ispossibly similar to the mechanosensitive channels. In Stylonychia,de Peyer and Machemer (de Peyer and Machemer, 1977) observedchanges of the resting potential of 1.5mV per 10°C. In ourexperiments, we minimised temperature effects. The bathtemperature was continuously monitored and measurements weredone at the smallest possible light intensity. Full opening of theluminous-field diaphragm led – after 30min of illumination – toa 0.3°C temperature difference between the cone of light and therest of the bath. After closure of the aperture, the temperaturedifference decreased beyond the sensitivity of the thermometer(<0.05°C). Within the cone of light, the temperature difference inthe experimental chamber over the 5mm distance between thebottom of the bath and the topping glass bridge was 0.1°C. Withthese results, we conclude that Stylonychia does not sense andrespond to the residual fluctuation of temperature.

GravitransductionThe gravireceptor potentials measured in this series of experimentssupport the special statocyst hypothesis. Cells that walk against thevector of gravity are hyperpolarised, and downward walking cellsare depolarised. The observed potential changes are persistent(tonic) with slow increasing rates (0.03mVs–1). This indicates afunctional difference between the gravisensitive and themechanosensitive ion channel, the latter being associated withvoltage shifts of 2–3mVs–1 and inactivation with slow timeconstants. It is possible that gravisensitive ion channels represent aspecialised subgroup of mechanosensitive channels, as discussed inother ciliate species (Krause, 2003; Machemer, 1998a). Statisticalanalyses of the amplitudes of gravireceptor potentials suggest thatthe membrane potential change does not code the angle by whichthe cell is turned: e.g. the sum of a reorientation from ‘anterior down’to ‘posterior down’ is not equal to the sum of ‘anterior down’ to‘horizontal’ and ‘horizontal’ to ‘posterior down’ (Fig.7). The exactmechanism of gravitransduction from the membrane potentialchange to a change in ciliary beating mechanism remains so far

unknown, but it seems likely that the change in gravipotential isonly a first step in a complex transduction chain. Viscoelasticelements of the cytoskeleton and/or second messengers like cAMPmay be involved in the gravitransduction pathway.

Mogami and Baba (Mogami and Baba, 1998) postulated acontinuous change of the membrane potential in swimming ciliatesduring a helical swimming track, eventually leading to gravitacticorientation. The observed very slow kinetics of gravireceptorpotentials in Paramecium and Stylonychia, however, suggest thatthis hypothesis seems to be unlikely. Stylonychia, in particular, isable to perform gravitaxis in the absence of helical movement. Inour definition, gravitaxis consists of a physical component(graviorientation) and a pure physiological component(gravikinesis). Measurements of kinetics of graviresponses inParamecium revealed that the orientation coefficient increasesimmediately after a turn of the experimental chamber from horizontalto vertical position, while the gravikinetic response becomesmaximal after 1min. At this point, gravikinesis compensates theeffects of sedimentation (Bräucker et al., 1998). So far, we have noknowledge about the velocity of a change of the gravikineticcomponent in Stylonychia, but we suggest that gravikinesis but notgraviorientation is coupled to the slow changes in membranepotential after reorientation of the cell.

ConclusionsGravity-compensating mechanisms have so far been well documentedin swimming ciliates such as Paramecium, Didinium, Bursaria andTetrahymena. The investigations on gravireception in the walkingciliate Stylonychia suggest a general principle of graviresponses inciliates consisting of a combination of gravikinesis andgraviorientation, with varying emphasis on the two working principles.Gravikinesis modulates the locomotion rate at a given orientation andcan be seen as a graviorthokinesis. Gravitaxis is based on physicalor physiological processes. There are indications that both mechanismsact together. The special statocyst hypothesis has gained support byexperiments in varying fields: increase of the cytoplasmatic load byfeeding with iron particles (Watzke, 2000), experiments in density-adapted media (Neugebauer et al., 1998), measurements of direction-depending locomotion rates (Machemer et al., 1991) and first evidenceof a gravireceptor potential in Paramecium (Gebauer et al., 1999).The present paper gives further evidence of the statocyst hypothesisin Stylonychia mytilus. To assess the relevance of gravity-dependingreactions in a ciliate species in terms of its ecology, it is not sufficientto study unimodal stimulation by gravity. It is more likely that theinteraction of different multimodal stimuli (light, gravity, chemicalstimuli) influences the behaviour of a ciliate. This happens in acomplex manner and may not be obvious but is the result of acomputation of all receptor inputs on the level of the membranepotential.

LIST OF SYMBOLS AND ABBREVIATIONS general gravikinesisU gravikinesis of cells oriented upwardlyD gravikinesis of cells oriented downwardlyCi specific input capacitance of the cellECa equilibrium potential for Ca2+

EK equilibrium potential for K+

gCa conductance for calcium ionsgK conductance for potassium ionsP gravity unrelated propulsion rateRi specific input resistance of the cellrO orientation coefficientS sedimentation rateVm membrane potential




VU locomotion rate of cells oriented upwardlyVH locomotion rate of cells oriented horizontallyVD locomotion rate of cells oriented downwardly

ACKNOWLEDGEMENTSFinancial support came from the Deutsches Zentrum für Luft- und Raumfahrt e.V.(DLR), grant 50WB9923, and the Minister für Bildung und Forschung of theFederal Republic of Germany. The authors wish to thank Hans Machemer forcritical reading of the manuscript and fruitful discussions.

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