Gravikinesis in Stylonychia mytilus is based on membrane potential changes

During the past decades, the sensation of gravity by cells has been investigated 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 a movement anti-parallel to the direction of gravity (Verworn, 1889). Gravitaxis consists of a directional component (graviorientation) and a kinetic component (gravikinesis). They act to oppose sedimentation of the cell. The density of the cytoplasm of Paramecium exceeds the density of fresh water by at least 4% (Kuroda and Kamiya, 1989; Taneda, 1987; Watzke, 2000). Without a compensation of sedimentation the cell will continuously sink to the ground, leaving preferred conditions for food uptake and reproduction.

Behavioural experiments were designed to explain the graviresponses in ciliates (Hemmersbach-Krause et al., 1991; Machemer et al., 1991; Machemer and Bräucker, 1992). According to a common assumption, the cytoplasm surrounded by the membrane of the cell acts as a statocyst to generate an outward-directed force against the lower cell membrane, which opens mechanosensitive ion channels (Machemer et al., 1991). There are two types of these ion channels: depolarising Ca²⁺ channels (accumulating anteriorly) and hyperpolarising K⁺ channels (accumulating posteriorly). These channels show a polar distribution along the antero-posterior cell-axis at the lateral membrane in Stylonychia (de Peyer and Machemer, 1978) and over the whole surface membrane of Paramecium (Ogura and Machemer, 1980). We show that reorienting the cell with respect to the gravity vector leads to a modulation of the membrane potential, presumably due to activation of these mechanosensitive ion channels. The coupling of the membrane potential to the direction and frequency of the ciliary 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 locomotion rate in downward-moving cells (gravikinesis). The molecular structure and the mechanism of activation of the mechanosensitive ion channels are so far unknown. The involvement of second messengers, cAMP and cGMP, is still debated (Hemmersbach et al., 2001; Richter et al., 2002).

In the ciliate Loxodes striatus, a spherical cell organelle – the Müller body – was considered as a putative gravisensor (Fenchel and Finlay, 1986). The Müller body consists of a vacuole, containing a BaSO₄ crystal, connected to a modified cilium. Cells whose Müller body 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 the participation of mechanosensitive ion channels located in the outer cell membrane in the gravitransduction process (Neugebauer et al., 1998). Loxodes and Stylonychia have a common habitat: the substrate of a pond. In the literature, Stylonychia has been described as a ‘walking’ cell. In resting and slow ‘walking’ cells, it can be observed that most of the ventral cirri have contact with the solid ground (M.K., personal observations) (Machemer and Deitmer, 1987). So far, there are no studies that describe whether this contact of all cirri still persists during ‘walking’. Furthermore, the paroral membranelles are described to produce a negative pressure that presumably holds the cell close to the solid substrate (Machemer, 1965). In our opinion, it is possible that during ‘walking’, several cirri lose their contact with the substrate and that the cell glides on a small fluid film. A similar kind of locomotion can be observed in Loxodes and is more precisely described as ‘gliding’ locomotion (Bräucker et al., 1992; Machemer-Röhnisch et al., 1998). Stylonychia differs from Loxodes in the fact that an intracellular organelle for sensation of gravity has not been identified.

In the present study, we show for the first time that Stylonychia mytilus modulates the membrane potential in response to spatial orientation of the cell in the water column. Earlier investigations (Gebauer et al., 1999) in Paramecium had indicated the existence of gravireceptor potentials of small amplitudes (<1.5 mV). The ciliate Stylonychia is highly sensitive to mechanical stimulation (de Peyer and Machemer, 1978; Machemer and Deitmer, 1987). Therefore, it was likely that gravireceptor potentials in this ciliate would be more prominent. The identification of electric membrane properties and measurements of the mechanosensitivity lead to calculations of the number of involved graviperceptive ion channels. Further behavioural analyses reveal that Stylonychia mytilus performs a negative gravitaxis, as shown for other protists. The data are in accordance with the special statocyst hypothesis (Machemer et al., 1991). The present data are part of a dissertation by Martin Krause at the Faculty of Mathematics and Science, University of Bonn, Germany.

The hypotrichous ciliate Stylonychia mytilus (Müller) was cultured in buffered Pringsheim solution (0.08 mmol l^–1 MgSO₄, 0.85 mmol l^–1 Ca(NO)₃, 0.25 mmol l^–1 KCl, buffered with Sörensen buffer at pH 7) and fed with the mixotrophe flagellate Chlorogonium elongatum three times a week. Cells were exposed to a 14 h:10 h light:dark regimen (maximum 3.5 W m^–2); the culturing temperature was 20°C. Earlier experiments had shown that the behaviour of Stylonychia relates to the time passed after cell division (Machemer, 1965). To consider this and to minimise the effect of other circadian rhythms, all experiments were done at the same time of day with cells of the same age under defined conditions (temperature, light).

The cell size of Stylonychia was determined by video recording cells at 150× magnification, followed by a computer-aided single-frame analysis. Calibration of measured body length was achieved by comparison of video records to scale paper under the same magnification. Measurement of longitudinal and transversal axes of cell bodies allows the calculation of cell volume and surface area as described previously (Machemer and Deitmer, 1987). For this calculation, we assume the cell body to be a rectangular solid. With the optical magnification used, the preciseness of measurements was ±3.6 μm. Double measurements of single cells were avoided by continuous resuspension of the population. The total recording time of a sample was limited to 15 min.

For investigation of the behaviour, 150–200 cells were transferred to an experimental chamber as described elsewhere (Nagel et al., 1997). Stylonychia shows a long-lasting inactivation after transfer from culture medium to an experimental chamber, as known from other ciliates (Machemer-Röhnisch et al., 1998; Oka et al., 1986). Therefore, all behavioural experiments were carried out in Pringsheim solution, giving an adaptation time of 4 h. To prevent influences of temperature changes, all experiments were done at 21±1°C.

Experimental chambers were mounted on a platform, allowing a reorientation of chambers from horizontal to vertical position and vice versa. Locomotion of cells was recorded by a commercial CCD camcorder with macro lens. We use a dark-field illumination by a circular arrangement of 48 green LED (565 nm, 700 lx) to avoid light-dependent cues for cell orientation. Photostimulation at the wavelength of 565 nm had been experimentally excluded for other ciliates such as Paramecium (Iwatsuki and Naitoh, 1982). The video image size was 9×7 mm² or 5% of the experimental chamber surface area. Locomotion rates, reversal frequencies, linearity of tracks and orientation of individual cells were analysed using computer-aided image processing (Häder and Vogel, 1991) and additional software developed by R.B.

We visualise the graviresponses of Stylonychia using circular histograms, which show the circular distribution of median locomotion rates and the percentage of cells found within orientational sectors at defined angles (Batschelet, 1981; Machemer and Bräucker, 1992). For quantification of graviorientation of cells, the orientation coefficient r_O (Machemer et al., 1991; Machemer et al., 1997) was used. This coefficient is +1 if all cells are strictly oriented 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, the recording area was subdivided into sectors of 90°. Locomotion of upward, downward and horizontally walking cells (, , ) was determined by calculating the median locomotion rates within these sectors.

For measurements of the sedimentation rate in Stylonychia, the cells were immobilised with 2 mmol l^–1 NiCl₂ using procedures described for other ciliates (Nagel et al., 1997). Immediately after immobilisation, cells were infused into the experimental chamber and the sedimenting cells were recorded. Each sample was measured four times after performance of a 180° turn of the chamber.

Non-parametric statistics (median values, 95% confidence range, U-test) were applied because Gaussian distribution of data was not assured.

Intracellular electrophysiological experiments in Stylonychia were carried out applying methods described by Naitoh and Eckert (Naitoh and Eckert, 1972) and de Peyer and Machemer (de Peyer and Machemer, 1977). Two hours prior to experiments, starved cells were washed in experimental solution (1 mmol l^–1 CaCl₂, 1 mmol l^–1 KCl, 1 mmol l^–1 Tris-HCl, pH 7.0). An intact specimen was selected for measurements and placed on the lower side of a glass bridge above the experimental chamber (bath). Under microscopic observation, a holding capillary was inserted into the cell to avoid movements of the cell during the recordings. After this mechanical fixation of the cell, the bath was filled with experimental solution (21±1°C). The membrane potential was measured differentially between an intra- and an extracellular glass electrode.

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

Mechanosensitivity in Stylonychia was analysed using a small glass stylus mounted on a piezo-electric crystal to indent the cell membrane (de Peyer and Machemer, 1977). The stylus was driven by trapezoid voltage pulses, allowing a fast movement of the stimulator and avoiding its oscillation. The deflection of the stylus depends on the voltage amplitude and was calibrated using an ocular micrometer. For measurements of membrane potential changes after stimulation of the lateral membrane, the tip of the stimulator was oriented 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 of the cell, and the median potential changes in each segment were calculated.

For measurements of gravireceptor potentials, the electrodes were inserted from lateral. To avoid mechanical stimulation, these electrodes were placed centrically between the anterior and posterior cell poles. The cell was carefully reoriented by means of micromanipulators. Changes in resting potential were measured before, during and after reorientation of the cell.

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

For determinations of cell volume and membrane area, longitudinal (cell length) and transversal (cell width) axes of 486 cells were measured (Fig. 1). Microscopic observations of individual cells confirmed that the dorso-ventral axis of the cell body measures 1/10 of cell length (L). As has been shown by earlier analyses (Machemer and Deitmer, 1987), the width of the cell body corresponds to 0.4L. The median cell length of Stylonychia was found to be 236.1 μm (–3.9 μm/+2.9 μm). The median cell width was 97.2 μm (–3.6 μm/+1.0 μm). Considerations of the membranelles, undulating membrane and cirri of Stylonychia strongly affect calculations of the complete membrane area (2×10^–3 cm²) but not the calculated cell volume (3.6×10^–7 cm³). The data and calculations are summarised in Table 1.

Histograms in Fig. 2 and data in Table 2 show orientation and locomotion rate in Stylonychia in vertical and horizontal experimental chambers. Turning of the experimental chamber into the vertical position allows the cells to orient with respect to gravity. A statistically secured majority of cells walk antiparallel to the gravity vector although this behaviour is not predominant (negative gravitaxis; r_O=0.06; P≤5%). As might be expected, no preferential orientation was observed in horizontally oriented chambers (r_O=–0.01). The measured locomotion rates of nearly 1 mm s^–1 were independent of walking direction and position of the experimental chamber.

Application of 2 mmol l^–1 NiCl₂ completely arrested the ciliary beat in Stylonychia and led to passive sedimentation of the cells. No deformation of cells was observed within 45 min. After that time, swelling of cell body occurred and some cells disintegrated or lost their adoral membranelles. Considering these observations, the time for sedimentation rate determination was restricted to 30 min. Fig. 3 shows the relative frequency distribution of sedimentation rates at normal gravity. The resulting median sedimentation rate was 180 μm s^–1 (N=2549). Confidence ranges are small (–2/+3 μm s^–1), and the median coincides with the arithmetic mean, indicating a Gaussian distribution. We analysed the orientation of the cell longitudinal axis from 835 sedimenting cells. In 519 cells (=60.3%), the longitudinal axis was aligned with the direction of the gravity vector (±45°) whereas with the used magnification it was not possible to discriminate between anterior and posterior cell poles. 183 cells (=22.8%) sank with their longitudinal axes transverse to the gravity vector. In 133 cells, the longitudinal axis could not be identified clearly.

The median direction-dependent locomotion rates (, and ) of Stylonychia were determined using data from cells that oriented within 90° sectors. A general gravikinesis (Δ) of –178 μm s^–1 was calculated according to Eqn 3, applying a median sedimentation rate of 180 μm s^-1. It is seen that Stylonychia mytilus shows a value of general gravikinesis, which completely compensates the sedimentation rate.

The gravity independent propulsion rate () has so far not been determined experimentally. Assuming a fully balanced bipolar distribution of gravireceptors, is estimated using velocities of horizontally walking cells (993±7 μm μm s^–1). The direction-dependent gravikinesis (Δ_D, Δ_U) is then calculated to be –198 μm s^–1 and –159 μm s^–1, respectively (Eqns 1, 2; see Table 2).

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 using the values of cell volume (soma) and mean density. In ciliates so far investigated, a mean density difference between cytoplasm and surrounding medium was determined to be 0.04 g cm^–3 (Taneda, 1987; Kuroda and Kamiya, 1989; Neugebauer et al., 1998). Assuming this value and a volume of 3.6×10^–7 cm³ for Stylonychia, the mass of the cytoplasm is estimated at 1.4×10^–8 g. At normal gravity, this mass induces an effective downward force of 1.39×10^–10 N on the lower membrane.

Intracellular recordings were performed to characterise passive and active membrane properties of Stylonychia mytilus. The median membrane potential was –44.3 mV (–0.7/+1.1 mV, N=45). Some cells show spontaneous spiking activity at an average frequency of 1–2 Hz. Changes in membrane potential were measured following injection of constant current pulses. The median input resistance, determined from the linear part of the current–voltage relationship, was found to be 21.3 MΩ (–5.2/+2.2 MΩ). Calculation of the input capacitance of the cell (1.8 nF) was possible using the measured median time constant of 38.9 ms (–2.4/+1.9 ms) obtained from recordings of small hyperpolarisations. For specific input resistance (R_i) and specific membrane capacitance (C_i), the value of the complete membrane area was taken into account. The measured value of C_i was 0.9 μF cm^–2, which is near the value known for biological membranes (1 μF cm^–2). This indicates a realistic estimate of the membrane area. The value for the specific membrane resistance was found to be 42.5 kΩ cm².

Measurements of membrane currents during voltage-clamp experiments reveal activation of voltage-dependent membrane currents (not shown). It is evident that positive voltage steps activate two types of Ca²⁺-depending early inward currents (see also de Peyer and Machemer, 1977; Deitmer, 1986). The maximum amplitude of the low threshold current (activated with depolarisations above 3 mV) was –4 nA. The amplitude of the late-activating Ca²⁺ current was –17 nA at depolarisations of +55 mV.

Fig. 4 gives examples of membrane potential changes after mechanical stimulation of the dorsal, ventral and lateral membrane. Independent from the site of stimulation, latencies between first contact of the stimulating needle with the membrane and the onset of the potential change were 3–4 ms. A polar distribution of depolarising and hyperpolarising mechanoreceptor conductances in Stylonychia applies to the whole cell body. A mechanical stimulation of the anterior part of the cell leads to a depolarisation and, because 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 between anterior and posterior cell ends does not elicit a membrane potential change; possibly, depolarisation and hyperpolarisation compensate each other. Maximum hyperpolarisations were obtained following stimulation of the posterior cell pole.

Results of a quantitative analysis are shown in Fig. 5. Largest amplitudes of membrane potential changes were measured after stimulation of the lateral cell. Maximum depolarisations were +42 mV and, in most cases, action potentials were superimposed. At the posterior cell pole, lateral stimulation led to hyperpolarisations of –35 mV. Stimulation of the anterior dorsal cell side elicited maximum receptor potentials (+22 mV), and stimulation of the posterior dorsal cell side elicited –20 mV. Note that pronounced depolarisations were obtained from stimulation in an area at 80% of the distance from posterior (Fig. 5). A possible explanation for this result is that receptor channel abundance is decreased in the most anterior membrane segment. Amplitudes of potential changes from ventral stimulation exceed those obtained from dorsal stimulation. No significant difference was seen between stimulation of left and right lateral side. Likewise, no differences in measurements were seen after stimulation of the left or right dorsal side. A potential change after stimulation at the right anterior-ventral side confirms earlier observations suggesting that mechanosensitivity in Stylonychia does not involve cilia or cirri (de Peyer and Machemer, 1978). A participation of cilia and cirri in mechanosensation is unlikely in Stylonychia because stimulation of cilia-free membrane segments lead to a membrane potential change. This finding supports results obtained in deciliated Paramecium caudatum (Ogura and Machemer, 1980).

A mechanical stimulation of the ventral membrane was also performed during voltage-clamp experiments (four cells; data not shown). A stimulation of the anterior cell pole induced an inward current (mean amplitude of –13.5 nA); posterior stimulation resulted in outward currents (mean amplitude +19.6 nA).

Gravireceptor potentials are changes of the resting membrane potential induced by gravity-dependent increases in conductance of mechanically sensitive ion channels. First measurements were long time records of membrane potential without reorientation of the cell, to exclude a possible drift, which could mask the gravireceptor potential. During a sampling period of 120 s, the mean membrane potential was found to fluctuate (standard deviation ±0.8 mV). After that, starting from the horizontal position, the cell was reoriented parallel to the gravity vector either with the anterior or with the posterior cell pole downwards. Only those cells were selected for evaluation, which did not show fluctuations in membrane potential prior to reorientation. Some cells fired repetitive action potentials before reorientation or after turning downwards. These cells were also excluded from statistical analysis.

Fig. 6A shows typical recordings of the membrane potential before, during and after a reorientation of the cell. In Fig. 6Ai, turning the cell from ‘posterior down’ to ‘horizontal’ led to a depolarisation. The special statocyst hypothesis explains this observation because depolarising Ca²⁺ channels open near the anterior cell pole, and hyperpolarising K⁺ channels close near the posterior pole. Upon turning the horizontal cell with its anterior end downwards (Fig. 6Aii), only depolarising channels are activated. After turning the cell up by 180° from ‘posterior down’ to ‘anterior down’ (Fig. 6Aiii), similar amplitude of depolarisation was found compared with Fig. 6Ai and Fig. 6Aii. The special statocyst hypothesis also predicts that a gravity-induced activation of posterior receptor channels leads to a hyperpolarisation. Fig. 6B shows three kinds of reorientations which result in such potential change: from ‘anterior 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 gravity. The potential changes occur slowly (with a small time constant of 0.03 mV s^–1). In most cases, reorientation of the cells 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 reason for this observation is obscure; possibly, it is related to the small number (seven) of experimental cells. Table 3 summarises all steady-state potential changes as determined 90 s after turning of the cell. Voltage-clamp experiments revealed no recognisable changes in transmembrane current after turning the cell. The amplitudes of expected currents would be smaller than 0.2 nA. Such currents were at the limit of separation from the current noise level in our setup.

The behavioural experiments to analyse gravireception of Stylonychia deal with the preferred locomotion of this ciliate, which is ‘walking’ on surfaces of submersed substrates. Here, our data differ from earlier investigations on graviresponses obtained from protists that are generally free swimmers. Apart from that, the established methods of behavioural and electrophysiological analysis are easily applied to Stylonychia.

The determination of sedimentation rates is essential for the analysis of gravireception in unicellular organisms. Sedimentation affects cell orientation because a high sedimentation rate, relative to the propulsion rate, leads to a stronger deviation of cell orientation from the measured locomotion track (Machemer et al., 1997). In addition, the sedimentation rate is necessary for the calculation of gravikinesis (Eqns 1, 2). The sedimentation rate of a spheroid body depends mainly on its weight and radius (Stokes' law). Assuming a sphere with the volume of Stylonychia (3.6×10^–7 cm³) results in a sedimentation rate of 170 μm s^–1, which is close to the measured value of 180 μm s^–1.

In the experiments, 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. Frictional forces reduce the sedimentation rate of a cell close to a wall (Happel and Brenner, 1986). Eqn 3 shows that, with the given data, the absolute value of gravikinesis, Δ, will decrease with a decrease in sedimentation rate. Thus, in Stylonychia, walking on a vertical surface, Δ is reduced by the same amount as sedimentation near a wall decreases with respect to free sedimentation. The point of this argument is that gravikinesis in Stylonychia can fully compensate effects of sedimentation independent of its effective value. The sedimentation rate measured in free-floating cells may be overestimated for walking specimens. Consequently, we would overestimate a gravikinesis. However, by means of statistical tests we could assure the existence of a gravikinesis as long as the sedimentation rate is larger than 8 μm s^–1. Since such a strong decrease of sedimentation rate by wall effects seems to be unlikely, we must assume a physiological component in gravitaxis of Stylonychia. In our lab, further experiments have been performed to study the locomotion behaviour of Stylonychia under conditions of micro- and hypergravity. These, so far unpublished, observations gave strong evidence that the sedimentation rate of walking Stylonychia cells is larger than 0.

First, the direction-dependent locomotion rates of Stylonychia were measured after a step transition from 1 g to microgravity in the drop tower of ZARM (Centre of Applied Space Technology and Microgravity, Bremen, Germany). It could be observed that, immediately after transition to microgravity, the locomotion rate of downward oriented cells decreased by 80 μm s^–1 and that of upward oriented cells increased by 70 μm s^–1. This sudden change in locomotion rate can only be explained by absence of sedimentation under microgravity. This result indicates a sedimentation rate in gliding Stylonychia which is >0. However, our measured value of sedimentation rate in free floating cells (and thereby the value of gravikinesis) is possibly overestimated for walking specimens.

Further support for a gravikinetic component in Stylonychia comes 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 rates of downward oriented cells increased with increased acceleration (and sedimentation rate) whereas the locomotion rate of upward oriented cells remained more or less unchanged.

We have demonstrated that Stylonychia is able to perceive and respond to gravity. Stylonychia shows graviresponses that compensate sedimentation. The gravitaxis consists of a directing component (graviorientation) and a velocity-regulating component (gravikinesis). In the vertical chamber, a small majority of cells was oriented upwards. The orientation coefficient of 0.06 is statistically significant but less prominent as compared with other ciliates so far investigated. Orientation coefficients of 0.2 were observed in Paramecium caudatum (Machemer et al., 1991) and were 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 physical mechanisms, two hypotheses may be considered: (1) an uneven 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 analysis of the cell body of Stylonychia shows that the anterior part is more flattened and wider than the posterior part. The transversal dimensions shown in Fig. 1 represent the maximum cell width and were mostly found in the anterior part of the cell. The height of the cell is at maximum halfway between the anterior and posterior end. According to the hydrodynamic hypothesis, this cell shape favours 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 the hydrodynamic hypothesis, showing that a negative gravitaxis occurred in other ciliates shaped similar to Stylonychia.

The assumed density distribution gives an indication for the static hypothesis. It may be argued that density in the posterior part of the cell is increased, because the larger of the two macronuclei and remaining food particles are located at the posterior cell pole. Such uneven distribution of weight induces a torque and would evoke a negative gravitactic orientation. An exclusive physical orientation implies that all cells should be oriented upwards after some time so that the orientation coefficient resembles the value +1. This does not agree with experimental findings in several ciliate species (Bräucker et al., 1996; Krause and Bräucker, 2008), indicating contributions of other, i.e. physiological, mechanisms to cell orientation. In Stylonychia, it is likely that the comparatively high frequency of reversals randomises the orientation of specimens in a cell population. Cells generate spontaneous action potentials, which are followed by rapid ‘back–forward’ movements (reversals). Reversals are shown in horizontally and vertically oriented experimental chambers. Due to randomisation of orientations, a directed movement, which would cause a gravi-accumulation, is unlikely. Physiological contributions to 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 the flagellate Euglena; the treatment of Euglena with UV radiation (Häder and Liu, 1990), application of various heavy metal ions (Stallwitz and Häder, 1994), and membrane-incorporated agents (Häder and Hemmersbach, 1997) affected orientation in this species, suggesting a physiological control of gravitaxis without excluding physical mechanisms. Investigations in Paramecium tetraurelia showed an increase in the frequency of reversals in the downward swimming cells as compared with the cells that favoured upward swimming (Nagel and Machemer, 2000).

Gravity-dependent modulation of velocity (gravikinesis) is definitely based on a physiological mechanism. In Stylonychia, the speed of upward and downward walking is the same, and sedimentation rate is fully compensated by gravikinesis. Similar results were obtained in gliding Loxodes (Bräucker et al., 1992). The degree of compensation of sedimentation by an opposing gravikinesis differs between the species. In the smallest ciliate investigated so far, Tetrahymena pyriformis, an overcompensating gravikinesis (130% of the value of ) was measured (Kowalewski et al., 1998). In the giant ciliate Bursaria truncatella, the sedimentation rate of 923 μm s^–1 is compensated by 70% [gravikinesis –633 μm s^–1 (Krause and Bräucker, 2008)]. A similar ratio has been shown in Didinium nasutum (Bräucker et al., 1994; Machemer et al., 1993). A smaller compensation of has been measured in Paramecium tetraurelia [28% (Hemmersbach-Krause et al., 1993); 59% (Nagel and Machemer, 2000)]. Even within the same species – Paramecium caudatum – 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 cell size and the amount of gravikinetic compensation is not systematic.

For the determination of a direction-dependent gravikinesis, the value of the gravity unrelated velocity of propulsion, , is required. This value can be exactly determined in cells only after sufficient time of adaptation to the weightless condition in space. As an alternative to weightlessness, the rate of horizontally walking Stylonychia may be chosen as an approximation of the value of . This is possible, because in horizontally oriented Stylonychia a possible gravistimulation is neutralised due to the bipolar, gradient-type distribution of mechanosensitive Ca²⁺ and K⁺ channels. This is similar to Paramecium (Machemer et al., 1991) and several other ciliates (Machemer and Teunis, 1996). The speed of horizontally walking Stylonychia cells in the vertical chamber (993 μm s^–1) exceeds , and locomotion rate in horizontally oriented chambers (973 μm s^–1; Table 2). This offset may be due to an accumulation of hyperpolarising gravireceptors at the lateral surface membrane. We should mention that in the vertical chamber, cells are oriented with their ventral side parallel to the gravity vector. Cells walking in a horizontally oriented chamber are oriented with their ventral or their dorsal side perpendicular to the gravity vector.

There are some indications that graviorientation and gravikinesis are subject to different mechanisms: long-term experiments with Stylonychia exposed to different experimental solutions showed that cells immediately oriented with respect to the gravity vector, whereas gravikinesis gradually rose to become maximum after 1–2 h (data not shown). Previous experiments in Paramecium biaurelia adapted to low temperatures (4°C) established a gravikinesis but no significant graviorientation (Freiberger, 2004).

The electrophysiological data in Stylonychia are based on a large number of cells to support a statistical analysis of the relationship between behaviour and its electrophysiological basis. Numerous electrophysiological data are in the literature (for a review, see Machemer and Deitmer, 1987). Most of these data were collected from different cell clones (Table 4). Therefore, it was reasonable to determine the electrophysiological properties of the present cell clone. Since we did not find differences in our current–voltage relationships compared with earlier results (de Peyer and Machemer, 1977), we conclude that the properties of voltage-dependent ion channels 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 pole are much larger than observed in Paramecium (Ogura and Machemer, 1980). In the literature, the data on mechanosensitivity in Stylonychia were previously obtained from stimulation of the lateral membrane (de Peyer and Machemer, 1978). Our experiments show that the bipolar distribution of gradients of sensitivity applies to the entire cell surface. At the same latitude of the cell, a stimulation of the lateral, ventral and dorsal regions evoked different amplitudes of receptor potentials. With identical stimulus velocity and distance to the membrane, the differences in amplitude are probably based on variations in channel density or cytoskeletal substructure at the sites of stimulation.

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

Stylonychia is highly mechanosensitive and has a relative large cell mass, which is a favourable condition for gravitransduction experiments. The obtained potential changes of 4 mV after cell reorientation support the special statocyst hypothesis. Previous experiments in Paramecium documented gravity-induced small potential changes of 1.5 mV (Gebauer et al., 1999).

We have evidence that a gravireceptor potential of 4 mV is sufficient for a change in locomotion rate in Stylonychia. This has been confirmed by application of different potassium solutions that influence the locomotion rate as well as the membrane potential. De Peyer and Machemer (De Peyer and Machemer, 1977) observed a shift of the steady-state membrane potential in Stylonychia of about 3.5 mV mmol l^–1 K⁺. Correspondingly, a variation of the external [K⁺] induced a change in locomotion rate of 196 μm s^–1 per mmol l^–1 K⁺ in Stylonychia (M.K., unpublished). Because the membrane potential and the frequency of the cirral beat are linearly correlated near the resting potential (Machemer and Deitmer, 1987), the K⁺-dependent change in speed of locomotion (56 μm s^–1 per mV) extrapolates to a speed change of 224 μm s^–1 per 4 mV gravireceptor potential, which reasonably corresponds to the calculated gravikinesis.

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

Potential changes after external mechanostimulation exceed the observed gravireceptor potentials by a factor of 10. Such divergence may be due to the high velocity of inward deformation of the membrane. Possibly, the number of channels involved in the gravitransduction chain is small. Gebauer et al. estimated that <20 channels activated after 180° reorientation of Paramecium (Gebauer et al., 1999).

As in other ciliates, the membrane potential of Stylonychia is determined, in the first line, by the equilibrium potentials for Ca²⁺ and K⁺ (E_Ca, +116 mV; E_K, –90 mV) and the conductances for these ions (de Peyer and Machemer, 1977). A membrane potential of –42 mV corresponds to a g_Ca/g_K conductance ratio of 0.3. The change in membrane potential following reorientation of the cell from ‘anterior down’ to ‘posterior down’ is due to the increase in conductance for K⁺ ions and the decrease in Ca²⁺ ions conductance. The number of ion channels involved in gravitransduction may be roughly approximated using data from the literature. We tentatively assume a mean channel conductance of 30 pS [corresponding to 10–50 pS from cells of the amphibian inner ear (Howard et al., 1988)] and a resting input resistance of 21 MΩ. For a 4 mV shift in membrane potential 40 Ca²⁺ channels may close or 150 K⁺ channels may open.

Does gravity-induced opening and closing of a few ion channels agree with the physical limits of signal transduction in cells? A comparison of the thermal noise level at 20°C (2×10^–21 Nm) with the available energy for Stylonychia gravitransduction is possible, assuming a minimal gating distance of 3.5 nm (Howard et al., 1988). With a cell volume of 3.6×10^–7 cm³, a density difference (cytoplasm over freshwater) of 0.048 g cm^–3 and a gating distance (of mechanoreceptor channels at the bottom membrane) of 3.5 nm, the gross energy available for gravitransduction is 6×10^–19 Nm, which exceeds the thermal noise level by more than two orders of magnitude.

In the basidiomycete Flammulina velutipes, participation of the comparatively heavy nucleus (1.22 g cm^–3) has been implied in gravitransduction (Monzer, 1996). The nucleus of this fungus is connected to the cytoskeleton. It is assumed that actin filaments transmit forces from sedimentation of the nucleus to the lower membrane. In Stylonychia, no such repositioning of the anterior macronucleus was observed after turning the cell from horizontal to ‘anterior down’ and back to horizontal. Instead, it was possible to evoke changes in gravireceptor potential even though electrodes fixed the posterior macronucleus. We therefore believe that participation of the nuclei in gravitransduction is unlikely in Stylonychia. However, involvement of the cytoskeleton in graviperception seems to be probable in ciliates (Machemer, 1998a). This hypothesis is supported by results from drop-tower experiments indicating slow relaxation kinetics of graviresponses after step transition from 1 g to weightlessness (Bräucker et al., 1998; Krause, 2003; Krause et al., 2006).

To safeguard our electrophysiological data on the effects of gravity from misinterpretations, two parameters associated with microscope illumination should be mentioned: possible effects of vectors or gradients of light and temperature.

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 0.5 mV and amplitude peaks after 500 ms were determined at 0.7 W m^–2. Specimens of horizontally oriented Stylonychia reacted to a gradual increase of light intensity (of >100 W m^–2) with an increased reversal rate, indicating a depolarisation (M.K., unpublished). Photoreactions in response to the direction of light require shadowing of the photosensitive area. The Lieberkühn'sche organelle in Ophryoglena catenula (Kuhlmann, 1993) represents a structure for shadowing. In Stylonychia, no similar structure has been observed. All experiments were performed at the lowest light intensity possible (0.5 W m^–2) to minimise possible light effects. To estimate the influence of light, long-time recordings of the membrane potential were done, with changes in light intensity between 0.5 W m^–2 and 10 W m^–2. Directly after increasing the light intensity, a phasic depolarisation of the cell was measured (median value <2 mV; M.K., unpublished). A light-dependent and persistent potential shift in the low-intensity range was not seen.

Thermoreception was investigated in Paramecium by several authors. Cooling of the anterior cell area led to a depolarisation of 10 mV after lowering the temperature from 25°C to 20°C and evoked 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 in depolarisation and in hyperpolarisation at the posterior cell pole. These findings suggest that thermosensitive ion channels have a bipolar distribution on the surface membrane in ciliates, which is possibly similar to the mechanosensitive channels. In Stylonychia, de Peyer and Machemer (de Peyer and Machemer, 1977) observed changes of the resting potential of 1.5 mV per 10°C. In our experiments, we minimised temperature effects. The bath temperature was continuously monitored and measurements were done at the smallest possible light intensity. Full opening of the luminous-field diaphragm led – after 30 min of illumination – to a 0.3°C temperature difference between the cone of light and the rest of the bath. After closure of the aperture, the temperature difference decreased beyond the sensitivity of the thermometer (<0.05°C). Within the cone of light, the temperature difference in the experimental chamber over the 5 mm distance between the bottom of the bath and the topping glass bridge was 0.1°C. With these results, we conclude that Stylonychia does not sense and respond to the residual fluctuation of temperature.

The gravireceptor potentials measured in this series of experiments support the special statocyst hypothesis. Cells that walk against the vector of gravity are hyperpolarised, and downward walking cells are depolarised. The observed potential changes are persistent (=tonic) with slow increasing rates (0.03 mV s^–1). This indicates a functional difference between the gravisensitive and the mechanosensitive ion channel, the latter being associated with voltage shifts of 2–3 mV s^–1 and inactivation with slow time constants. It is possible that gravisensitive ion channels represent a specialised subgroup of mechanosensitive channels, as discussed in other ciliate species (Krause, 2003; Machemer, 1998a). Statistical analyses of the amplitudes of gravireceptor potentials suggest that the membrane potential change does not code the angle by which the 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 exact mechanism of gravitransduction from the membrane potential change to a change in ciliary beating mechanism remains so far unknown, but it seems likely that the change in gravipotential is only a first step in a complex transduction chain. Viscoelastic elements of the cytoskeleton and/or second messengers like cAMP may be involved in the gravitransduction pathway.

Mogami and Baba (Mogami and Baba, 1998) postulated a continuous change of the membrane potential in swimming ciliates during a helical swimming track, eventually leading to gravitactic orientation. The observed very slow kinetics of gravireceptor potentials in Paramecium and Stylonychia, however, suggest that this hypothesis seems to be unlikely. Stylonychia, in particular, is able to perform gravitaxis in the absence of helical movement. In our definition, gravitaxis consists of a physical component (graviorientation) and a pure physiological component (gravikinesis). Measurements of kinetics of graviresponses in Paramecium revealed that the orientation coefficient increases immediately after a turn of the experimental chamber from horizontal to vertical position, while the gravikinetic response becomes maximal after 1 min. At this point, gravikinesis compensates the effects of sedimentation (Bräucker et al., 1998). So far, we have no knowledge about the velocity of a change of the gravikinetic component in Stylonychia, but we suggest that gravikinesis but not graviorientation is coupled to the slow changes in membrane potential after reorientation of the cell.

Gravity-compensating mechanisms have so far been well documented in swimming ciliates such as Paramecium, Didinium, Bursaria and Tetrahymena. The investigations on gravireception in the walking ciliate Stylonychia suggest a general principle of graviresponses in ciliates consisting of a combination of gravikinesis and graviorientation, with varying emphasis on the two working principles. Gravikinesis modulates the locomotion rate at a given orientation and can be seen as a graviorthokinesis. Gravitaxis is based on physical or physiological processes. There are indications that both mechanisms act together. The special statocyst hypothesis has gained support by experiments in varying fields: increase of the cytoplasmatic load by feeding 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 evidence of a gravireceptor potential in Paramecium (Gebauer et al., 1999). The present paper gives further evidence of the statocyst hypothesis in Stylonychia mytilus. To assess the relevance of gravity-depending reactions in a ciliate species in terms of its ecology, it is not sufficient to study unimodal stimulation by gravity. It is more likely that the interaction of different multimodal stimuli (light, gravity, chemical stimuli) influences the behaviour of a ciliate. This happens in a complex manner and may not be obvious but is the result of a computation of all receptor inputs on the level of the membrane potential.

 
• Δ

  general gravikinesis

 
• Δ_U

  gravikinesis of cells oriented upwardly

 
• Δ_D

  gravikinesis of cells oriented downwardly

 
• C_i

  specific input capacitance of the cell

 
• E_Ca

  equilibrium potential for Ca²⁺

 
• E_K

  equilibrium potential for K⁺

 
• g_Ca

  conductance for calcium ions

 
• g_K

  conductance for potassium ions

 
• 

  gravity unrelated propulsion rate

 
• R_i

  specific input resistance of the cell

 
• r_O

  orientation coefficient

 
• 

  sedimentation rate

 
• V_m

  membrane potential

 
• 

  locomotion rate of cells oriented upwardly

 
• 

  locomotion rate of cells oriented horizontally

 
• 

  locomotion rate of cells oriented downwardly