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The Journal of Experimental Biology 213, 161-171 Published by The Company of Biologists 2010 doi: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

SUMMARY The graviperception of the hypotrichous Stylonychia mytilus was investigated using electrophysiological methods and behavioural analysis. It is shown that Stylonychia can sense gravity and thereby compensates sedimentation rate by a negative gravikinesis. The graviresponse consists of a velocity-regulating physiological component (negative gravikinesis) and an additional orientational component. The latter is largely based on a physical mechanism but might, in addition, be affected by the frequency of ciliary reversals, which is under physiological control. We show that the external stimulus of gravity is transformed to a physiological signal, activating mechanosensitive calcium and potassium channels. Earlier electrophysiological experiments revealed that these ion channels are distributed in the manner of two opposing gradients over the surface membrane. Here, we show, for the first time, records of gravireceptor potentials in Stylonychia that are presumably based on this two-gradient system of ion channels. The gravireceptor potentials had maximum amplitudes of approximately 4mV and slow activation characteristics (0.03mVs–1). The presumptive number of involved graviperceptive ion channels was calculated and correlates with the analysis of the locomotive behaviour. Key words: Stylonychia mytilus, ciliate, behaviour, electrophysiology, mechanosensitivity, gravikinesis, gravitaxis, gravireceptor potential.

INTRODUCTION upward-moving cells and in a decrease of the forward locomotion During the past decades, the sensation of gravity by cells has been rate in downward-moving cells (gravikinesis). The molecular investigated in many systems (Bräucker and Hemmersbach, 2002; structure and the mechanism of activation of the mechanosensitive Häder et al., 2005; Hughes-Fulford and Lewis, 1996; Lewis, 2002; ion channels are so far unknown. The involvement of second Sievers and Volkmann, 1979). By the end of the 19th century, messengers, cAMP and cGMP, is still debated (Hemmersbach et negative gravitaxis in had been described as a al., 2001; Richter et al., 2002). movement anti-parallel to the direction of gravity (Verworn, 1889). The effect of gravity on locomotion rates results from behavioural Gravitaxis consists of a directional component (graviorientation) and analysis. Measurements of the downward and upward locomotion a kinetic component (gravikinesis). They act to oppose sedimentation rates (VD, VU), the sedimentation rate (S), and the gravity- of the cell. The density of the cytoplasm of Paramecium exceeds independent rate of propulsion (P) determine the gravity-induced the density of fresh water by at least 4% (Kuroda and Kamiya, 1989; component of active locomotion (gravikinesis) in downward- Taneda, 1987; Watzke, 2000). Without a compensation of moving (D) and upward-moving cells (U) (Machemer et al., 1991): sedimentation the cell will continuously sink to the ground, leaving D  VD – P – S, (1) preferred conditions for food uptake and reproduction. Behavioural experiments were designed to explain the U  P – VU – S. (2) graviresponses in (Hemmersbach-Krause et al., 1991; Machemer et al., 1991; Machemer and Bräucker, 1992). According To exactly determine P, locomotion rates have to be measured under to a common assumption, the cytoplasm surrounded by the long-term microgravity conditions. For a species with a polar, two- membrane of the cell acts as a statocyst to generate an outward- gradient distribution of Ca2+ and K+ mechanoreceptor channels directed force against the lower cell membrane, which opens (Paramecium, Stylonychia), it is acceptable to use the locomotion mechanosensitive ion channels (Machemer et al., 1991). There are rate of horizontally moving cells (VH) as an approximation of the two types of these ion channels: depolarising Ca2+ channels value of P. This is possible because depolarising and hyperpolarising (accumulating anteriorly) and hyperpolarising K+ channels receptor channels activate simultaneously in the horizontal position (accumulating posteriorly). These channels show a polar distribution of Stylonychia, which neutralises their effects on the Ca2+/K+ along the antero-posterior cell-axis at the lateral membrane in conductance ratio (Machemer, 1998b). Stylonychia (de Peyer and Machemer, 1978) and over the whole P is cancelled from the equations by subtraction of terms (1) and surface membrane of Paramecium (Ogura and Machemer, 1980). (2), giving the generalised value of gravikinesis () as the arithmetic We show that reorienting the cell with respect to the gravity vector mean of D and U: leads to a modulation of the membrane potential, presumably due (VD – VU) / 2 – S. (3) to activation of these mechanosensitive ion channels. The coupling of the membrane potential to the direction and frequency of the Gravikinesis has been repeatedly established in protists investigated ciliary power stroke (Machemer, 1974; Machemer and de Peyer, so far: Paramecium caudatum (Machemer et al., 1991), Paramecium 1982) results in an increase in the forward locomotion rate in tetraurelia (Hemmersbach-Krause et al., 1993), nasutum

THE JOURNAL OF EXPERIMENTAL BIOLOGY 162 M. Krause, R. Bräucker and R. Hemmersbach

(Machemer et al., 1993), striatus (Neugebauer et al., 1998), Determination of cell size pyriformis (Kowalewski et al., 1998), Euglena gracilis The cell size of Stylonychia was determined by video recording cells (Machemer-Röhnisch et al., 1999) and Bursaria truncatella (Krause at 150ϫ magnification, followed by a computer-aided single-frame and Bräucker, 2008). analysis. Calibration of measured body length was achieved by In the ciliate Loxodes striatus, a spherical cell organelle – the comparison of video records to scale paper under the same Müller body – was considered as a putative gravisensor (Fenchel magnification. Measurement of longitudinal and transversal axes of and Finlay, 1986). The Müller body consists of a vacuole, containing cell bodies allows the calculation of cell volume and surface area a BaSO4 crystal, connected to a modified . Cells whose Müller as described previously (Machemer and Deitmer, 1987). For this body had been destroyed by means of a laser did not show gravitaxis calculation, we assume the cell body to be a rectangular solid. With (Hemmersbach et al., 1997). Experiments in density-adjusted media the optical magnification used, the preciseness of measurements was (no density difference between cytoplasm and surrounding medium) ±3.6m. Double measurements of single cells were avoided by revealed a reduced, but still persistent, gravikinesis, indicating the continuous resuspension of the population. The total recording time participation of mechanosensitive ion channels located in the outer of a sample was limited to 15min. cell membrane in the gravitransduction process (Neugebauer et al., 1998). Loxodes and Stylonychia have a common habitat: the Behavioural analysis substrate of a pond. In the literature, Stylonychia has been described For investigation of the behaviour, 150–200 cells were transferred as a ‘walking’ cell. In resting and slow ‘walking’ cells, it can be to an experimental chamber as described elsewhere (Nagel et al., observed that most of the ventral cirri have contact with the solid 1997). Stylonychia shows a long-lasting inactivation after transfer ground (M.K., personal observations) (Machemer and Deitmer, from culture medium to an experimental chamber, as known from 1987). So far, there are no studies that describe whether this contact other ciliates (Machemer-Röhnisch et al., 1998; Oka et al., 1986). of all cirri still persists during ‘walking’. Furthermore, the paroral Therefore, all behavioural experiments were carried out in membranelles are described to produce a negative pressure that Pringsheim solution, giving an adaptation time of 4h. To prevent presumably holds the cell close to the solid substrate (Machemer, influences of temperature changes, all experiments were done at 1965). In our opinion, it is possible that during ‘walking’, several 21±1°C. cirri lose their contact with the substrate and that the cell glides on Experimental chambers were mounted on a platform, allowing a small fluid film. A similar kind of locomotion can be observed a reorientation of chambers from horizontal to vertical position and in Loxodes and is more precisely described as ‘gliding’ locomotion vice versa. Locomotion of cells was recorded by a commercial CCD (Bräucker et al., 1992; Machemer-Röhnisch et al., 1998). Stylonychia camcorder with macro lens. We use a dark-field illumination by a differs from Loxodes in the fact that an intracellular organelle for circular arrangement of 48 green LED (565nm, 700lx) to avoid sensation of gravity has not been identified. light-dependent cues for cell orientation. Photostimulation at the In the present study, we show for the first time that Stylonychia wavelength of 565nm had been experimentally excluded for other mytilus modulates the membrane potential in response to spatial ciliates such as Paramecium (Iwatsuki and Naitoh, 1982). The video orientation of the cell in the water column. Earlier investigations image size was 9ϫ7mm2 or 5% of the experimental chamber surface (Gebauer et al., 1999) in Paramecium had indicated the existence area. Locomotion rates, reversal frequencies, linearity of tracks and of gravireceptor potentials of small amplitudes (<1.5mV). The ciliate orientation of individual cells were analysed using computer-aided Stylonychia is highly sensitive to mechanical stimulation (de Peyer image processing (Häder and Vogel, 1991) and additional software and Machemer, 1978; Machemer and Deitmer, 1987). Therefore, it developed by R.B. was likely that gravireceptor potentials in this ciliate would be more We visualise the graviresponses of Stylonychia using circular prominent. The identification of electric membrane properties and histograms, which show the circular distribution of median measurements of the mechanosensitivity lead to calculations of the locomotion rates and the percentage of cells found within number of involved graviperceptive ion channels. Further orientational sectors at defined angles (Batschelet, 1981; Machemer behavioural analyses reveal that Stylonychia mytilus performs a and Bräucker, 1992). For quantification of graviorientation of cells, negative gravitaxis, as shown for other protists. The data are in the orientation coefficient rO (Machemer et al., 1991; Machemer et accordance with the special statocyst hypothesis (Machemer et al., al., 1997) was used. This coefficient is +1 if all cells are strictly 1991). The present data are part of a dissertation by Martin Krause oriented upwards and –1 if all cells are oriented downwards. at the Faculty of Mathematics and Science, University of Bonn, Significance of orientation was tested applying the Rayleigh test Germany. (Batschelet, 1981). For determination of direction-dependent locomotion rates, the MATERIALS AND METHODS recording area was subdivided into sectors of 90°. Locomotion of Culturing upward, downward and horizontally walking cells (VU, VD, VH) was The hypotrichous ciliate Stylonychia mytilus (Müller) was cultured determined by calculating the median locomotion rates within these –1 in buffered Pringsheim solution (0.08mmoll MgSO4, sectors. –1 –1 0.85mmoll Ca(NO)3, 0.25mmoll KCl, buffered with Sörensen For measurements of the sedimentation rate in Stylonychia, the –1 buffer at pH7) and fed with the mixotrophe flagellate cells were immobilised with 2mmoll NiCl2 using procedures Chlorogonium elongatum three times a week. Cells were exposed described for other ciliates (Nagel et al., 1997). Immediately after to a 14h:10h light:dark regimen (maximum 3.5Wm–2); the immobilisation, cells were infused into the experimental chamber culturing temperature was 20°C. Earlier experiments had shown and the sedimenting cells were recorded. Each sample was that the behaviour of Stylonychia relates to the time passed after measured four times after performance of a 180° turn of the cell division (Machemer, 1965). To consider this and to minimise chamber. the effect of other circadian rhythms, all experiments were done Non-parametric statistics (median values, 95% confidence range, at the same time of day with cells of the same age under defined U-test) were applied because Gaussian distribution of data was not conditions (temperature, light). assured.

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Electrophysiology was performed using a horizontally oriented tip of the glass stylus. Intracellular electrophysiological experiments in Stylonychia were Stimulations were done in five segments of the longitudinal axis of carried out applying methods described by Naitoh and Eckert the cell, and the median potential changes in each segment were (Naitoh and Eckert, 1972) and de Peyer and Machemer (de Peyer calculated. and Machemer, 1977). Two hours prior to experiments, starved cells For measurements of gravireceptor potentials, the electrodes were –1 –1 were washed in experimental solution (1mmoll CaCl2, 1mmoll inserted from lateral. To avoid mechanical stimulation, these KCl, 1mmoll–1 Tris-HCl, pH 7.0). An intact specimen was selected electrodes were placed centrically between the anterior and posterior for measurements and placed on the lower side of a glass bridge cell poles. The cell was carefully reoriented by means of above the experimental chamber (bath). Under microscopic micromanipulators. Changes in resting potential were measured observation, a holding capillary was inserted into the cell to avoid before, during and after reorientation of the cell. movements of the cell during the recordings. After this mechanical Four turning modes of the cell were applied: from horizontal to fixation of the cell, the bath was filled with experimental solution posterior down, from horizontal to anterior down, from anterior (21±1°C). The membrane potential was measured differentially down to posterior down and vice versa. For statistical evaluations, between an intra- and an extracellular glass electrode. the recording time (maximum 120s) was divided into classes of 1s, The electrodes (borosilicate glass capillaries) were filled with and the median membrane potential change in each class was 1moll–1 KCl. Resistances were between 40MW (current electrodes) calculated. and 100MW (electrodes for potential measurement). The tips of the electrodes and the holding capillary were bent twice by 30° each. RESULTS For achievement of a long-lasting recording, the electrodes Cell size penetrated the anterior or posterior macronucleus of the cell from For determinations of cell volume and membrane area, longitudinal ventral. A second electrode was inserted for current injection. (cell length) and transversal (cell width) axes of 486 cells were Measurements of input resistance and capacitance were performed measured (Fig.1). Microscopic observations of individual cells using small hyperpolarising current steps. For establishment of confirmed that the dorso-ventral axis of the cell body measures 1/10 current–voltage relationships, injections of 80ms current-steps from of cell length (L). As has been shown by earlier analyses (Machemer –6 to +6nA were performed, and the resulting changes of membrane and Deitmer, 1987), the width of the cell body corresponds to 0.4L. potential were measured. Voltage-clamp experiments were The median cell length of Stylonychia was found to be 236.1m performed using a high-gain differential amplifier (Analog Devices, (–3.9m/+2.9m). The median cell width was 97.2m 171K). The holding potential was set equal to the membrane resting (–3.6m/+1.0m). Considerations of the membranelles, undulating potential, and early and steady-state transmembrane currents were membrane and cirri of Stylonychia strongly affect calculations of measured at voltage steps in the range of –70 to +90mV from the the complete membrane area (2ϫ10–3cm2) but not the calculated resting potential. All data from voltage-clamp experiments were cell volume (3.6ϫ10–7cm3). The data and calculations are corrected for leakage current. summarised in Table1. Mechanosensitivity in Stylonychia was analysed using a small glass stylus mounted on a piezo-electric crystal to indent the cell Gravitaxis membrane (de Peyer and Machemer, 1977). The stylus was driven Histograms in Fig.2 and data in Table2 show orientation and by trapezoid voltage pulses, allowing a fast movement of the locomotion rate in Stylonychia in vertical and horizontal stimulator and avoiding its oscillation. The deflection of the stylus experimental chambers. Turning of the experimental chamber into depends on the voltage amplitude and was calibrated using an ocular the vertical position allows the cells to orient with respect to gravity. micrometer. For measurements of membrane potential changes after A statistically secured majority of cells walk antiparallel to the stimulation of the lateral membrane, the tip of the stimulator was gravity vector although this behaviour is not predominant (negative oriented vertically. Stimulation of the ventral and dorsal cell surface gravitaxis; rO0.06; P≤5%). As might be expected, no preferential

Table 1. Geometry of Stylonychia: (A) median values of cell length 50 Transversal Longitudinal and width; (B) calculations of cell geometry Cell Cell Cell 40 A length (m) width (m) height (m) 236.1(232, 239) 97.2 (93.6, 98.2) 23.6 30

B Membrane area (cm2) Volume (cm3) 20 Soma 6.1ϫ10–4 3.55ϫ10–7 Membranelles 8.8ϫ10–4 5.5ϫ10–9 10 tive freq u ency (%) Rel a tive Cirri 4.9ϫ10–4 3.1ϫ10–9 Undulating membrane 2.2ϫ10–5 1.4ϫ10–10 0 Complete 2ϫ10–3 3.6ϫ10–7 40 80 120 140 80 3002602201 Dimension (µm) Median values of cell length and width (N486; limits of 95% confidence interval of the medians in parentheses). Calculations of cell geometry were done according to the terms by Machemer and Deitmer (1987). The Fig.1. Frequency distribution histograms showing cell sizes of Stylonychia height of the cell body is estimated at 1/10 of the cell-length. The diameter mytilus (N486). Median distances along the transverse axis were 97.2m, of one single cilium is 0.25 m. The membranelles and cirri of Stylonychia and 236.1m along the longitudinal axis. Insets illustrate cell axes. Cell  have specific lengths and are composed of tens of independent cilia; here, drawings modified after Machemer and Deitmer (Machemer and Deitmer, their summed areas and volumes are given. 1987).

THE JOURNAL OF EXPERIMENTAL BIOLOGY 164 M. Krause, R. Bräucker and R. Hemmersbach

Orientation Locomotion rate 20

240 )

16 –1 180 120 y=168.3+0.5x 12 60

g a te ( µ m s r a l Vertic S ediment a tion 0 010203040 8 Time (min) 5% –1 rO=0.06 1.5 mm s

tive freq u ency (%) Rel a tive 4 . –1 rO=–0.01 S=180 µm s

0 0 100 200 300 400 500 600 Sedimentation rate (µm s–1)

2+ Horizont a l Fig.3. Frequency distribution of sedimentation rates (S) of Ni -immobilised and free-floating Stylonychia mytilus specimen. The median sedimentation 5% rate is 180 m s–1. Data approximate a Gaussian distribution (N 2549). 1.5 mm s–1    Inset: sedimentation rate as a function of recording time. No significant changes were observed within 30min after the immobilisation procedure. Fig.2. Polar histograms of orientation and locomotion rate of Stylonychia Shaded area represents limits of the confidence ranges. mytilus in a vertically and horizontally oriented experimental chamber. In the vertical chamber, a small but significant orientation antiparallel to the vector of gravity is shown (rO0.06). No preferred direction of locomotion their adoral membranelles. Considering these observations, the time was seen in the horizontal chamber (rO–0.01). The locomotion rate is for sedimentation rate determination was restricted to 30min. Fig.3 independent of the direction of movement (number of data: vertical, 21,517; shows the relative frequency distribution of sedimentation rates at horizontal, 15,287). For exact values of locomotion rates, also see Table2. normal gravity. The resulting median sedimentation rate was 180ms–1 (N2549). Confidence ranges are small (–2/+3ms–1), and the median coincides with the arithmetic mean, indicating a Gaussian orientation was observed in horizontally oriented chambers distribution. We analysed the orientation of the cell longitudinal axis –1 (rO–0.01). The measured locomotion rates of nearly 1mms were from 835 sedimenting cells. In 519 cells (60.3%), the longitudinal independent of walking direction and position of the experimental axis was aligned with the direction of the gravity vector (±45°) whereas chamber. with the used magnification it was not possible to discriminate between anterior and posterior cell poles. 183 cells (22.8%) sank with their Sedimentation longitudinal axes transverse to the gravity vector. In 133 cells, the –1 Application of 2mmoll NiCl2 completely arrested the ciliary beat longitudinal axis could not be identified clearly. in Stylonychia and led to passive sedimentation of the cells. No deformation of cells was observed within 45min. After that time, Gravikinesis swelling of cell body occurred and some cells disintegrated or lost The median direction-dependent locomotion rates (VD, VU and VH) of Stylonychia were determined using data from cells that oriented Table 2. Graviresponses of Stylonychia mytilus within 90° sectors. A general gravikinesis () of –178ms–1 was calculated according to Eqn3, applying a median sedimentation rate Velocity (m s–1) N of 180ms-1. It is seen that Stylonychia mytilus shows a value of Downwards (VD) 975 (961, 987) 4840 general gravikinesis, which completely compensates the Upwards (VU) 972 (960, 981) 5818 sedimentation rate. Horizontal (VH) 993 (982, 1000) 5484 Sedimentation (S) 180 (178, 183) 2549 The gravity independent propulsion rate (P) has so far not been determined experimentally. Assuming a fully balanced bipolar –1 Gravikinesis (m s ) distribution of gravireceptors, P is estimated using velocities of  –178 horizontally walking cells (993±7mms–1). The direction- –1 U –159 dependent gravikinesis (D, U) is then calculated to be –198ms D –198 and –159ms–1, respectively (Eqns1,2; see Table2). According to the special statocyst hypothesis, the cytoplasmic Orientation Corresponding mass exerts an outward-directed force on the lower cell membrane, coefficients locomotion thereby causing channel activation. This force is calculated using –1 (ro) rate (m s ) N the values of cell volume (soma) and mean density. In ciliates so Vertical plane (all directions) 0.06 983 (977, 989) 21,517 far investigated, a mean density difference between cytoplasm and Horizontal plane (all directions) –0.01 973 (966, 980) 15,287 surrounding medium was determined to be 0.04gcm–3 (Taneda,

The observed, direction-dependent rates of locomotion (VD, VH, VU) were 1987; Kuroda and Kamiya, 1989; Neugebauer et al., 1998). determined using 90° sectors (N  number of analysed tracks). Assuming this value and a volume of 3.6ϫ10–7cm3 for Stylonychia, Gravikinesis was calculated using Eqns 1–3. Negative graviorientation is the mass of the cytoplasm is estimated at 1.4ϫ10–8g. At normal represented by the orientation coefficient. Values in parentheses give the gravity, this mass induces an effective downward force of limits of the 95% confidence interval of the medians. 1.39ϫ10–10N on the lower membrane.

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Electrophysiological properties Dorsal stimulation Intracellular recordings were performed to characterise passive and K active membrane properties of Stylonychia mytilus. The median A A membrane potential was –44.3mV (–0.7/+1.1mV, N45). Some B L cells show spontaneous spiking activity at an average frequency of B 1–2Hz. Changes in membrane potential were measured following  C C M injection of constant current pulses. The median input resistance, determined from the linear part of the current–voltage relationship, D D N was found to be 21.3M (–5.2/+2.2M ). Calculation of the input Lateral stimulation  W  W E capacitance of the cell (1.8nF) was possible using the measured E O median time constant of 38.9ms (–2.4/+1.9ms) obtained from K recordings of small hyperpolarisations. For specific input resistance S L (Ri) and specific membrane capacitance (Ci), the value of the complete membrane area was taken into account. The measured M –2 value of Ci was 0.9Fcm , which is near the value known for N biological membranes (1Fcm–2). This indicates a realistic estimate Ventral stimulation of the membrane area. The value for the specific membrane O resistance was found to be 42.5kWcm2. F F Measurements of membrane currents during voltage-clamp G experiments reveal activation of voltage-dependent membrane G S currents (not shown). It is evident that positive voltage steps activate H H two types of Ca2+-depending early inward currents (see also de Peyer and Machemer, 1977; Deitmer, 1986). The maximum amplitude of I I 40 mV the low threshold current (activated with depolarisations above 40 ms J J 3mV) was –4nA. The amplitude of the late-activating Ca2+ current was –17nA at depolarisations of +55mV. S Mechanosensitivity Fig.4 gives examples of membrane potential changes after mechanical stimulation of the dorsal, ventral and lateral membrane. Fig.4. Examples of potential changes from the resting voltage after local Independent from the site of stimulation, latencies between first mechanical stimulation at different positions of the dorsal (A–E), ventral contact of the stimulating needle with the membrane and the onset (F–J) and lateral (K–O) membrane. All recordings show a bipolar pattern of of the potential change were 3–4ms. A polar distribution of membrane responses, indicating a gradient-type distribution of  antagonising mechanosensitive ion channels between the polar ends of depolarising and hyperpolarising mechanoreceptor conductances in Stylonychia. Trapezoid track marks the time of stimulation (S). No Stylonychia applies to the whole cell body. A mechanical stimulation differences were observed between responses evoked on the left and right of the anterior part of the cell leads to a depolarisation and, because hemispheres of the cell. of a low threshold of voltage-activated Ca2+ channels, to action potentials. The amplitude of depolarisation decreases with more posterior stimulation. Touching the cell membrane exactly halfway Peyer and Machemer, 1978). A participation of cilia and cirri in between anterior and posterior cell ends does not elicit a membrane mechanosensation is unlikely in Stylonychia because stimulation of potential change; possibly, depolarisation and hyperpolarisation cilia-free membrane segments lead to a membrane potential change. compensate each other. Maximum hyperpolarisations were obtained This finding supports results obtained in deciliated Paramecium following stimulation of the posterior cell pole. caudatum (Ogura and Machemer, 1980). Results of a quantitative analysis are shown in Fig.5. Largest A mechanical stimulation of the ventral membrane was also amplitudes of membrane potential changes were measured after performed during voltage-clamp experiments (four cells; data not stimulation of the lateral cell. Maximum depolarisations were shown). A stimulation of the anterior cell pole induced an inward +42mV and, in most cases, action potentials were superimposed. current (mean amplitude of –13.5nA); posterior stimulation resulted At the posterior cell pole, lateral stimulation led to hyperpolarisations in outward currents (mean amplitude +19.6nA). of –35mV. Stimulation of the anterior dorsal cell side elicited maximum receptor potentials (+22mV), and stimulation of the Gravireception posterior dorsal cell side elicited –20mV. Note that pronounced Gravireceptor potentials are changes of the resting membrane depolarisations were obtained from stimulation in an area at 80% potential induced by gravity-dependent increases in conductance of of the distance from posterior (Fig.5). A possible explanation for mechanically sensitive ion channels. First measurements were long this result is that receptor channel abundance is decreased in the time records of membrane potential without reorientation of the cell, most anterior membrane segment. Amplitudes of potential changes to exclude a possible drift, which could mask the gravireceptor from ventral stimulation exceed those obtained from dorsal potential. During a sampling period of 120s, the mean membrane stimulation. No significant difference was seen between stimulation potential was found to fluctuate (standard deviation ±0.8mV). After of left and right lateral side. Likewise, no differences in that, starting from the horizontal position, the cell was reoriented measurements were seen after stimulation of the left or right dorsal parallel to the gravity vector either with the anterior or with the side. A potential change after stimulation at the right anterior- posterior cell pole downwards. Only those cells were selected for ventral side confirms earlier observations suggesting that evaluation, which did not show fluctuations in membrane potential mechanosensitivity in Stylonychia does not involve cilia or cirri (de prior to reorientation. Some cells fired repetitive action potentials

THE JOURNAL OF EXPERIMENTAL BIOLOGY 166 M. Krause, R. Bräucker and R. Hemmersbach

down’ to ‘horizontal’ (Fig.6Bi), from ‘horizontal’ to ‘posterior down’ (Fig.6Bii) and from ‘anterior down’ to ‘posterior down’ (Fig.6Biii). A statistical analysis of the experiments (Fig.7) assured that the potential changes measured are receptor potentials induced by 40 gravity. The potential changes occur slowly (with a small time Dorsal constant of 0.03mVs–1). In most cases, reorientation of the cells 20 resulted in a new steady-state potential after 60s. Turning the cell from ‘posterior down’ to ‘horizontal’ (Fig.7A), two depolarisation maxima were measured near 40s and 80s after reorientation. The 0 reason for this observation is obscure; possibly, it is related to the small number (seven) of experimental cells. Table3 summarises all –20 steady-state potential changes as determined 90s after turning of the cell. Voltage-clamp experiments revealed no recognisable –40 changes in transmembrane current after turning the cell. The 40 amplitudes of expected currents would be smaller than 0.2nA. Such Ventral currents were at the limit of separation from the current noise level 20 in our setup.

DISCUSSION 0 The behavioural experiments to analyse gravireception of a ne re s ting potenti l (mV) Stylonychia deal with the preferred locomotion of this ciliate, which –20 is ‘walking’ on surfaces of submersed substrates. Here, our data differ from earlier investigations on graviresponses obtained from –40 protists that are generally free swimmers. Apart from that, the 40 established methods of behavioural and electrophysiological analysis Lateral are easily applied to Stylonychia. Ch a nge from mem b r 20 Analysis of sedimentation Determination of sedimentation 0 The determination of sedimentation rates is essential for the analysis of gravireception in unicellular organisms. Sedimentation affects –20 cell orientation because a high sedimentation rate, relative to the propulsion rate, leads to a stronger deviation of cell orientation from –40 the measured locomotion track (Machemer et al., 1997). In addition, 0 0.2 0.4 0.6 0.8 1 the sedimentation rate is necessary for the calculation of gravikinesis Relative distance from posterior (Eqns1,2). The sedimentation rate of a spheroid body depends mainly on its weight and radius (Stokes’ law). Assuming a sphere Fig.5. Analysis of mechanosensitivity in Stylonychia. Shown are the with the volume of Stylonychia (3.6ϫ10–7cm3) results in a median changes from the resting membrane potential after submaximum sedimentation rate of 170m s–1, which is close to the measured stimulation of the dorsal, ventral and lateral membrane (stimulator deflexion value of 180 m s–1. was 8m). Abscissa represents the distance from posterior in relative units.  Confidence ranges are shadowed in grey. Each data point represents measurements from at least four cells. Problem of sedimentation in walking cells In the experiments, S was determined in cells that were floating in the experimental chamber. The sedimentation of cells sliding near the wall of the chamber cannot be assessed for technical reasons. before reorientation or after turning downwards. These cells were Frictional forces reduce the sedimentation rate of a cell close to a also excluded from statistical analysis. wall (Happel and Brenner, 1986). Eqn3 shows that, with the given Fig.6A shows typical recordings of the membrane potential data, the absolute value of gravikinesis, , will decrease with a before, during and after a reorientation of the cell. In Fig.6Ai, turning decrease in sedimentation rate. Thus, in Stylonychia, walking on a the cell from ‘posterior down’ to ‘horizontal’ led to a depolarisation. vertical surface,  is reduced by the same amount as sedimentation The special statocyst hypothesis explains this observation because near a wall decreases with respect to free sedimentation. The point depolarising Ca2+ channels open near the anterior cell pole, and of this argument is that gravikinesis in Stylonychia can fully hyperpolarising K+ channels close near the posterior pole. Upon compensate effects of sedimentation independent of its effective turning the horizontal cell with its anterior end downwards value. The sedimentation rate measured in free-floating cells may (Fig.6Aii), only depolarising channels are activated. After turning be overestimated for walking specimens. Consequently, we would the cell up by 180° from ‘posterior down’ to ‘anterior down’ overestimate a gravikinesis. However, by means of statistical tests (Fig.6Aiii), similar amplitude of depolarisation was found compared we could assure the existence of a gravikinesis as long as the with Fig.6Ai and Fig.6Aii. The special statocyst hypothesis also sedimentation rate is larger than 8ms–1. Since such a strong predicts that a gravity-induced activation of posterior receptor decrease of sedimentation rate by wall effects seems to be unlikely, channels leads to a hyperpolarisation. Fig.6B shows three kinds of we must assume a physiological component in gravitaxis of reorientations which result in such potential change: from ‘anterior Stylonychia. In our lab, further experiments have been performed

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Ai Bi Fig.6. Course of the membrane potential 10 mV (Vm) before, during and after reorientation g 10 s of typical recordings from different cells. The track (T) marks the time period of Vm Vm reorientation. Cell positions relative to the vector of gravity are shown by insets. T T (A)Depolarisation was induced by reorientations from ‘posterior down’ to ii ii horizontal (i), from horizontal to ‘anterior down’ (ii) and from ‘posterior down’ to ‘anterior down’ (iii). (B)Hyperpolarisation of the membrane potential occurred after Vm Vm reorientation from ‘anterior down’ to T T horizontal (i), from horizontal to ‘posterior down’ (ii) and from ‘anterior down’ to ‘posterior down’ (iii). The potential iii iii changes are explained by an activation and inactivation of topographically organised gravisensitive Ca2+ and K+ channels corresponding to the special Vm Vm statocyst hypothesis. T T

to study the locomotion behaviour of Stylonychia under conditions found to be different functions of acceleration. The locomotion rates of micro- and hypergravity. These, so far unpublished, observations of downward oriented cells increased with increased acceleration gave strong evidence that the sedimentation rate of walking (and sedimentation rate) whereas the locomotion rate of upward Stylonychia cells is larger than 0. oriented cells remained more or less unchanged. First, the direction-dependent locomotion rates of Stylonychia were measured after a step transition from 1g to microgravity in Mechanism of graviorientation in Stylonychia the drop tower of ZARM (Centre of Applied Space Technology We have demonstrated that Stylonychia is able to perceive and and Microgravity, Bremen, Germany). It could be observed that, respond to gravity. Stylonychia shows graviresponses that immediately after transition to microgravity, the locomotion rate of compensate sedimentation. The gravitaxis consists of a directing downward oriented cells decreased by 80ms–1 and that of upward component (graviorientation) and a velocity-regulating component oriented cells increased by 70ms–1. This sudden change in (gravikinesis). In the vertical chamber, a small majority of cells locomotion rate can only be explained by absence of sedimentation was oriented upwards. The orientation coefficient of 0.06 is under microgravity. This result indicates a sedimentation rate in statistically significant but less prominent as compared with other gliding Stylonychia which is >0. However, our measured value of ciliates so far investigated. Orientation coefficients of 0.2 were sedimentation rate in free floating cells (and thereby the value of observed in Paramecium caudatum (Machemer et al., 1991) and gravikinesis) is possibly overestimated for walking specimens. were even as large as 0.5 in Didinium nasutum (Machemer et al., Further support for a gravikinetic component in Stylonychia 1993) and Bursaria truncatella (Krause and Bräucker, 2008). comes from experiments under raised gravity in a centrifuge. Here, Assuming that orientation is based on exclusive physical the locomotion rates of upward and downward oriented cells were mechanisms, two hypotheses may be considered: (1) an uneven

8 Fig.7. Medians of changes of the membrane A D potential before, during and after reorientations 4 inducing a depolarisation (A) or hyperpolarisation (B). Shadowed areas mark the time period of turning the 0 cell. Reorientations correspond to Fig.6. Confidence ranges were small (<0.5mV) and so have been –4 omitted for clarity. Maximum amplitudes with 95% 8 confidence intervals are shown in Table3. Numbers B E of data: (A) 7 cells, 9 measurements; (B) 30 cells, 47 4 measurements; (C) 12 cells, 17 measurements; (D) 0 14 cells, 14 measurements; (E) 21 cells, 32 measurements; (F) 12 cells, 22 measurements. a ne potenti l ch nge (mV) –4 8 C F 4

Medi a n mem b r 0

–4 –10–20 0102030405060708090 30 –10–20– 010203040506070 Time (s)

THE JOURNAL OF EXPERIMENTAL BIOLOGY 168 M. Krause, R. Bräucker and R. Hemmersbach

Table 3. Peak amplitudes of membrane potential changes (Vm) upward and downward walking is the same, and sedimentation rate after reorientation of the cell as described in the Materials and is fully compensated by gravikinesis. Similar results were obtained methods in gliding Loxodes (Bräucker et al., 1992). The degree of compensation of sedimentation by an opposing gravikinesis differs Starting position End position Vm (mV) between the species. In the smallest ciliate investigated so far, Horizontal Posterior down –2.9mV (–2.8/–3.0) Tetrahymena pyriformis, an overcompensating gravikinesis (130% Posterior down Horizontal +3.9mV (3.6/4.1) of the value of ) was measured (Kowalewski et al., 1998). In the Horizontal Anterior down +2.7mV (2.65/2.8) S Anterior down Horizontal –3.0mV (–2.5/–3.3) giant ciliate Bursaria truncatella, the sedimentation rate of –1 –1 Posterior down Anterior down +3.4mV (3.3/3.5) 923ms is compensated by 70% [gravikinesis –633ms (Krause Anterior down Posterior down –3.1mV (–3.05/–3.2) and Bräucker, 2008)]. A similar ratio has been shown in Didinium Each value was determined 70–90 s after cell reorientation. Values in nasutum (Bräucker et al., 1994; Machemer et al., 1993). A smaller parentheses give the limits of the 95% confidence interval of the medians. compensation of S has been measured in Paramecium tetraurelia [28% (Hemmersbach-Krause et al., 1993); 59% (Nagel and Machemer, 2000)]. Even within the same species – Paramecium distribution of density in the cell (static hypothesis) (Verworn, caudatum – experimental values of sedimentation and gravikinesis, 1889) or (2) a passive orientation of the cell due to its shape and hence the degree of compensation of gravity, vary within limits (hydrodynamic hypothesis) (Roberts, 1970). Microscopic analysis [45% (Machemer et al., 1991); 42% (Watzke et al., 1998); 51% of the cell body of Stylonychia shows that the anterior part is more (Watzke, 2000)]. These data indicate that a correlation between cell flattened and wider than the posterior part. The transversal size and the amount of gravikinetic compensation is not systematic. dimensions shown in Fig.1 represent the maximum cell width and For the determination of a direction-dependent gravikinesis, the were mostly found in the anterior part of the cell. The height of value of the gravity unrelated velocity of propulsion, P, is required. the cell is at maximum halfway between the anterior and posterior This value can be exactly determined in cells only after sufficient end. According to the hydrodynamic hypothesis, this cell shape time of adaptation to the weightless condition in space. As an favours a downward orientation with the anterior cell part down. alternative to weightlessness, the rate of horizontally walking This prediction does not agree with the experimental findings. Stylonychia may be chosen as an approximation of the value of P. Winet and Jahn (Winet and Jahn, 1974) had argued against the This is possible, because in horizontally oriented Stylonychia a hydrodynamic hypothesis, showing that a negative gravitaxis possible gravistimulation is neutralised due to the bipolar, gradient- occurred in other ciliates shaped similar to Stylonychia. type distribution of mechanosensitive Ca2+ and K+ channels. This The assumed density distribution gives an indication for the static is similar to Paramecium (Machemer et al., 1991) and several other hypothesis. It may be argued that density in the posterior part of the ciliates (Machemer and Teunis, 1996). The speed of horizontally cell is increased, because the larger of the two macronuclei and walking Stylonychia cells in the vertical chamber (993ms–1) remaining food particles are located at the posterior cell pole. Such exceeds VD, VU and locomotion rate in horizontally oriented uneven distribution of weight induces a torque and would evoke a chambers (973ms–1; Table2). This offset may be due to an negative gravitactic orientation. An exclusive physical orientation accumulation of hyperpolarising gravireceptors at the lateral surface implies that all cells should be oriented upwards after some time so membrane. We should mention that in the vertical chamber, cells that the orientation coefficient resembles the value +1. This does not are oriented with their ventral side parallel to the gravity vector. agree with experimental findings in several ciliate species (Bräucker Cells walking in a horizontally oriented chamber are oriented with et al., 1996; Krause and Bräucker, 2008), indicating contributions of their ventral or their dorsal side perpendicular to the gravity vector. other, i.e. physiological, mechanisms to cell orientation. In There are some indications that graviorientation and gravikinesis Stylonychia, it is likely that the comparatively high frequency of are subject to different mechanisms: long-term experiments with reversals randomises the orientation of specimens in a cell population. Stylonychia exposed to different experimental solutions showed that Cells generate spontaneous action potentials, which are followed by cells immediately oriented with respect to the gravity vector, rapid ‘back–forward’ movements (reversals). Reversals are shown in whereas gravikinesis gradually rose to become maximum after 1–2h horizontally and vertically oriented experimental chambers. Due to (data not shown). Previous experiments in Paramecium biaurelia randomisation of orientations, a directed movement, which would adapted to low temperatures (4°C) established a gravikinesis but no cause a gravi-accumulation, is unlikely. Physiological contributions significant graviorientation (Freiberger, 2004). to graviorientation in protists have long been a matter of controversy (Machemer and de Peyer, 1977). Häder et al. (Häder et al., 1995) Electrophysiology and mechanosensitivity postulated a physiological mechanism of graviorientation in the The electrophysiological data in Stylonychia are based on a large flagellate Euglena; the treatment of Euglena with UV radiation (Häder number of cells to support a statistical analysis of the relationship and Liu, 1990), application of various heavy metal ions (Stallwitz between behaviour and its electrophysiological basis. Numerous and Häder, 1994), and membrane-incorporated agents (Häder and electrophysiological data are in the literature (for a review, see Hemmersbach, 1997) affected orientation in this species, suggesting Machemer and Deitmer, 1987). Most of these data were collected a physiological control of gravitaxis without excluding physical from different cell clones (Table4). Therefore, it was reasonable to mechanisms. Investigations in Paramecium tetraurelia showed an determine the electrophysiological properties of the present cell increase in the frequency of reversals in the downward swimming clone. Since we did not find differences in our current–voltage cells as compared with the cells that favoured upward swimming relationships compared with earlier results (de Peyer and Machemer, (Nagel and Machemer, 2000). 1977), we conclude that the properties of voltage-dependent ion channels are the same in actual and earlier cell lines. Gravikinesis Stylonychia mytilus is highly sensitive to mechanical stimulation. Gravity-dependent modulation of velocity (gravikinesis) is definitely Membrane currents following stimulation at the anterior cell pole based on a physiological mechanism. In Stylonychia, the speed of are much larger than observed in Paramecium (Ogura and

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Table 4. Measured membrane properties compared with values from the literature Parameters Value as measured Value from the literature References Resting potential –44mV –48mV de Peyer and Machemer (1977) Input resistance 21MW 33–100MW Machemer and Deitmer (1987) Time constant 39ms 40–80ms Deitmer (1986) Input capacitance 1.9nF 1nF M.K., personal observations Spec. membrane resistance 42.5 kWcm2 220 kWcm2 Machemer and Deitmer (1987) Spec. membrane capacitance 0.9Fcm–2 0.3Fcm–2 Machemer and Deitmer (1987)

+ Machemer, 1980). In the literature, the data on mechanosensitivity and K (ECa, +116mV; EK, –90mV) and the conductances for these in Stylonychia were previously obtained from stimulation of the ions (de Peyer and Machemer, 1977). A membrane potential of lateral membrane (de Peyer and Machemer, 1978). Our experiments –42mV corresponds to a gCa/gK conductance ratio of 0.3. The change show that the bipolar distribution of gradients of sensitivity applies in membrane potential following reorientation of the cell from to the entire cell surface. At the same latitude of the cell, a stimulation ‘anterior down’ to ‘posterior down’ is due to the increase in of the lateral, ventral and dorsal regions evoked different amplitudes conductance for K+ ions and the decrease in Ca2+ ions conductance. of receptor potentials. With identical stimulus velocity and distance The number of ion channels involved in gravitransduction may be to the membrane, the differences in amplitude are probably based roughly approximated using data from the literature. We tentatively on variations in channel density or cytoskeletal substructure at the assume a mean channel conductance of 30pS [corresponding to sites of stimulation. 10–50pS from cells of the amphibian inner ear (Howard et al., 1988)] As a next step, we tested whether or not the mechanosensitive and a resting input resistance of 21MW. For a 4mV shift in channels can be activated by the cytoplasmatic force acting against membrane potential 40 Ca2+ channels may close or 150 K+ channels the cell membrane in the outward direction, as predicted by the may open. special statocyst hypothesis (Machemer et al., 1991). Does gravity-induced opening and closing of a few ion channels agree with the physical limits of signal transduction in cells? A Gravireceptor potentials comparison of the thermal noise level at 20°C (2ϫ10–21Nm) with Stylonychia is highly mechanosensitive and has a relative large cell the available energy for Stylonychia gravitransduction is possible, mass, which is a favourable condition for gravitransduction assuming a minimal gating distance of 3.5nm (Howard et al., 1988). experiments. The obtained potential changes of 4mV after cell With a cell volume of 3.6ϫ10–7cm3, a density difference (cytoplasm reorientation support the special statocyst hypothesis. Previous over freshwater) of 0.048gcm–3 and a gating distance (of experiments in Paramecium documented gravity-induced small mechanoreceptor channels at the bottom membrane) of 3.5nm, the potential changes of 1.5mV (Gebauer et al., 1999). gross energy available for gravitransduction is 6ϫ10–19Nm, which We have evidence that a gravireceptor potential of 4mV is exceeds the thermal noise level by more than two orders of sufficient for a change in locomotion rate in Stylonychia. This has magnitude. been confirmed by application of different potassium solutions that In the basidiomycete Flammulina velutipes, participation of the influence the locomotion rate as well as the membrane potential. comparatively heavy nucleus (1.22gcm–3) has been implied in De Peyer and Machemer (De Peyer and Machemer, 1977) observed gravitransduction (Monzer, 1996). The nucleus of this fungus is a shift of the steady-state membrane potential in Stylonychia of about connected to the cytoskeleton. It is assumed that actin filaments 3.5mVmmoll–1 K+. Correspondingly, a variation of the external transmit forces from sedimentation of the nucleus to the lower [K+] induced a change in locomotion rate of 196ms–1 per mmoll–1 membrane. In Stylonychia, no such repositioning of the anterior K+ in Stylonychia (M.K., unpublished). Because the membrane macronucleus was observed after turning the cell from horizontal potential and the frequency of the cirral beat are linearly correlated to ‘anterior down’ and back to horizontal. Instead, it was possible near the resting potential (Machemer and Deitmer, 1987), the K+- to evoke changes in gravireceptor potential even though electrodes dependent change in speed of locomotion (56ms–1 per mV) fixed the posterior macronucleus. We therefore believe that extrapolates to a speed change of 224ms–1 per 4 mV gravireceptor participation of the nuclei in gravitransduction is unlikely in potential, which reasonably corresponds to the calculated Stylonychia. However, involvement of the cytoskeleton in gravikinesis. graviperception seems to be probable in ciliates (Machemer, 1998a). From a physiological point of view, larger amplitudes of receptor This hypothesis is supported by results from drop-tower experiments potentials would not be useful: in a downward oriented cell, a larger indicating slow relaxation kinetics of graviresponses after step depolarisation would lead to reversed movements of cilia and, in transition from 1g to weightlessness (Bräucker et al., 1998; Krause, course, to a continuously backward movement of the cell. 2003; Krause et al., 2006). To safeguard our electrophysiological data on the effects of Ion channels involved in gravireception gravity from misinterpretations, two parameters associated with Potential changes after external mechanostimulation exceed the microscope illumination should be mentioned: possible effects of observed gravireceptor potentials by a factor of 10. Such divergence vectors or gradients of light and temperature. may be due to the high velocity of inward deformation of the membrane. Possibly, the number of channels involved in the Effects of illumination and temperature gravitransduction chain is small. Gebauer et al. estimated that <20 Effects of illumination channels activated after 180° reorientation of Paramecium (Gebauer Many ciliates react to light. In Chlorella-free Paramecium bursaria, et al., 1999). the photosensitive pigment rhodopsin was described at the antero- As in other ciliates, the membrane potential of Stylonychia is ventral membrane of the cell (Nakaoka et al., 1987; Nakaoka, 1989; determined, in the first line, by the equilibrium potentials for Ca2+ Nakaoka et al., 1991). Photoreceptor potentials of 0.5mV and

THE JOURNAL OF EXPERIMENTAL BIOLOGY 170 M. Krause, R. Bräucker and R. Hemmersbach amplitude peaks after 500ms were determined at 0.7Wm–2. unknown, but it seems likely that the change in gravipotential is Specimens of horizontally oriented Stylonychia reacted to a gradual only a first step in a complex transduction chain. Viscoelastic increase of light intensity (of >100Wm–2) with an increased elements of the cytoskeleton and/or second messengers like cAMP reversal rate, indicating a depolarisation (M.K., unpublished). may be involved in the gravitransduction pathway. Photoreactions in response to the direction of light require shadowing Mogami and Baba (Mogami and Baba, 1998) postulated a of the photosensitive area. The Lieberkühn’sche organelle in continuous change of the membrane potential in swimming ciliates Ophryoglena catenula (Kuhlmann, 1993) represents a structure for during a helical swimming track, eventually leading to gravitactic shadowing. In Stylonychia, no similar structure has been observed. orientation. The observed very slow kinetics of gravireceptor All experiments were performed at the lowest light intensity potentials in Paramecium and Stylonychia, however, suggest that possible (0.5Wm–2) to minimise possible light effects. To estimate this hypothesis seems to be unlikely. Stylonychia, in particular, is the influence of light, long-time recordings of the membrane able to perform gravitaxis in the absence of helical movement. In potential were done, with changes in light intensity between our definition, gravitaxis consists of a physical component 0.5Wm–2 and 10Wm–2. Directly after increasing the light intensity, (graviorientation) and a pure physiological component a phasic depolarisation of the cell was measured (median value (gravikinesis). Measurements of kinetics of graviresponses in <2mV; M.K., unpublished). A light-dependent and persistent Paramecium revealed that the orientation coefficient increases potential shift in the low-intensity range was not seen. immediately after a turn of the experimental chamber from horizontal to vertical position, while the gravikinetic response becomes Effects of temperature maximal after 1min. At this point, gravikinesis compensates the Thermoreception was investigated in Paramecium by several effects of sedimentation (Bräucker et al., 1998). So far, we have no authors. Cooling of the anterior cell area led to a depolarisation knowledge about the velocity of a change of the gravikinetic of 10mV after lowering the temperature from 25°C to 20°C and component in Stylonychia, but we suggest that gravikinesis but not evoked a transient Ca2+-dependent inward current (Kuriu et al., graviorientation is coupled to the slow changes in membrane 1996). According to Tominaga and Naitoh (Tominaga and Naitoh, potential after reorientation of the cell. 1994), warming of the anterior cell membrane results in depolarisation and in hyperpolarisation at the posterior cell pole. Conclusions These findings suggest that thermosensitive ion channels have a Gravity-compensating mechanisms have so far been well documented bipolar distribution on the surface membrane in ciliates, which is in swimming ciliates such as Paramecium, Didinium, Bursaria and possibly similar to the mechanosensitive channels. In Stylonychia, Tetrahymena. The investigations on gravireception in the walking de Peyer and Machemer (de Peyer and Machemer, 1977) observed ciliate Stylonychia suggest a general principle of graviresponses in changes of the resting potential of 1.5mV per 10°C. In our ciliates consisting of a combination of gravikinesis and experiments, we minimised temperature effects. The bath graviorientation, with varying emphasis on the two working principles. temperature was continuously monitored and measurements were Gravikinesis modulates the locomotion rate at a given orientation and done at the smallest possible light intensity. Full opening of the can be seen as a graviorthokinesis. Gravitaxis is based on physical luminous-field diaphragm led – after 30min of illumination – to or physiological processes. There are indications that both mechanisms a 0.3°C temperature difference between the cone of light and the act together. The special statocyst hypothesis has gained support by rest of the bath. After closure of the aperture, the temperature experiments in varying fields: increase of the cytoplasmatic load by difference decreased beyond the sensitivity of the thermometer feeding with iron particles (Watzke, 2000), experiments in density- (<0.05°C). Within the cone of light, the temperature difference in adapted media (Neugebauer et al., 1998), measurements of direction- the experimental chamber over the 5mm distance between the depending locomotion rates (Machemer et al., 1991) and first evidence bottom of the bath and the topping glass bridge was 0.1°C. With of a gravireceptor potential in Paramecium (Gebauer et al., 1999). these results, we conclude that Stylonychia does not sense and The present paper gives further evidence of the statocyst hypothesis respond to the residual fluctuation of temperature. in Stylonychia mytilus. To assess the relevance of gravity-depending reactions in a ciliate species in terms of its ecology, it is not sufficient Gravitransduction to study unimodal stimulation by gravity. It is more likely that the The gravireceptor potentials measured in this series of experiments interaction of different multimodal stimuli (light, gravity, chemical support the special statocyst hypothesis. Cells that walk against the stimuli) influences the behaviour of a ciliate. This happens in a vector of gravity are hyperpolarised, and downward walking cells complex manner and may not be obvious but is the result of a are depolarised. The observed potential changes are persistent computation of all receptor inputs on the level of the membrane (tonic) with slow increasing rates (0.03mVs–1). This indicates a potential. functional difference between the gravisensitive and the mechanosensitive ion channel, the latter being associated with LIST OF SYMBOLS AND ABBREVIATIONS voltage shifts of 2–3mVs–1 and inactivation with slow time  general gravikinesis gravikinesis of cells oriented upwardly constants. It is possible that gravisensitive ion channels represent a U  gravikinesis of cells oriented downwardly specialised subgroup of mechanosensitive channels, as discussed in D Ci specific input capacitance of the cell 2+ other ciliate species (Krause, 2003; Machemer, 1998a). Statistical ECa equilibrium potential for Ca + analyses of the amplitudes of gravireceptor potentials suggest that EK equilibrium potential for K the membrane potential change does not code the angle by which gCa conductance for calcium ions the cell is turned: e.g. the sum of a reorientation from ‘anterior down’ gK conductance for potassium ions to ‘posterior down’ is not equal to the sum of ‘anterior down’ to P gravity unrelated propulsion rate R specific input resistance of the cell ‘horizontal’ and ‘horizontal’ to ‘posterior down’ (Fig.7). The exact i  rO orientation coefficient mechanism of gravitransduction from the membrane potential S sedimentation rate change to a change in ciliary beating mechanism remains so far Vm membrane potential

THE JOURNAL OF EXPERIMENTAL BIOLOGY Graviresponses in Stylonychia 171

VU locomotion rate of cells oriented upwardly Lewis, M. L. (2002). The cytoskeleton, apoptosis, and gene expression in T lymphocytes and other mammalian cells exposed to altered gravity. In Cell Biology VH locomotion rate of cells oriented horizontally and Biotechnology in Space (ed. A. Cogoli), pp. 77-128. Amsterdam: Elsevier. VD locomotion rate of cells oriented downwardly Machemer, H. (1965). Analyse langzeitlicher Bewegungserscheinungen des Ciliaten Stylonychia mytilus Ehrenberg. Arch. Protistenkd. 108, 91-107. ACKNOWLEDGEMENTS Machemer, H. (1974). Ciliary activity and metachronism in protozoa. In Cilia and Flagella (ed. M. A. Sleigh), pp. 199-286. New York, London: Academic Press. Financial support came from the Deutsches Zentrum für Luft- und Raumfahrt e.V. Machemer, H. (1998a). The dealing with gravity at the unicellular level: concepts and (DLR), grant 50WB9923, and the Minister für Bildung und Forschung of the data. Int. School of Biophysics, Ischia 1996. 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