Six minutes of free-fall conditions during the parabolic flights of the TEXUS 27-28 sounding rockets were sufficient to induce behavioural changes in Paramecium. The centrifugal spin during launch was perceived by Paramecium, resulting in a higher precision of the negative gravitaxis than in 1 g. After onset of microgravity, paramecia maintained their former swimming direction for about 30 s. The ensuing random swimming indicated that gravity was indeed the stimulus for the observed orientation at 1 g. The individual swimming tracks remained straight in the absence of gravity, indicating that no depolarisation of the membrane to the level of an action potential had occurred. The mean swimming velocity increased significantly, indicating hyperpolarisation of the membrane and/or changes in the amount of cAMP. The increase in velocity was transient and subsided after 3 min to the speed of the former horizontal swimming at 1 g. Electron microscopy revealed that trichocysts, organelles which may be extruded through mechanical stress, were still present within Paramecium after the experiment.
How is the gravity stimulus perceived on the cellular level? Gravitactic protozoa offer the advantage in studying how the physical signal of gravity is transformed into a physiological response. The freshwater ciliate Paramecium uses gravity as an environmental stimulus for its spatial orientation (gravitaxis) and for the control of its swimming velocity (gravikinesis). Though Paramecium is heavier than water, it is able to swim against the direction of gravity (negative gravitaxis). Measurement of the swimming velocities in the different swimming directions revealed that paramecia antagonise sedimentation by an active speed regulation, resulting in a faster upward swimming velocity than expected by calculations.1,2 In the absence of other stimuli, gravitaxis and gravikinesis help the cells to move and to stay in suitable habitats.
The phenomena of the graviresponses are well described; however, the basic mechanisms are still under discussion. Drawing on our current knowledge of the perception of other environmental stimuli such as light³ and chemicals,4 a physiological signal transduction chain can be postulated for gravitaxis and gravikinesis. Cellular elements are discussed as possible gravireceptors in Paramecium: the whole cytoplasm via opening of specific ion channels in the membrane of the cell or heavy cell organelles in interaction with the cytoskeleton and membranes.2, 5
Paramecium offers the advantage that changes in membrane potential and/or second messengers can be identified by corresponding changes in the swimming behaviour.6, 7 Hyperpolarisation and/or an increase in cAMP augments the ciliary beat, thus raising the forward swimming velocity. On the other hand, depolarisation to the level of an action potential and/or an increase in cGMP induces a reversal of the ciliary beat direction and thus backward swimming.
An organism such as Paramecium with clear graviresponses might change its behaviour after transition to weightlessness, thus providing insight into the mechanism of graviperception.
Paramecium biaurelia in their original culture medium (straw medium, pH 7.2) were enriched without centrifuging to exclude acceleration effects before the beginning of the experiment. Cells were placed in a temperature-controlled (via Peltier elements) observation chamber (diameter 22 mm; depth 0.5 mm) consisting of two titanium frames housing two pieces of glass (RG 645, Schott & Gen., Mainz, Germany). The sample was placed 45 min before launch on the stage of the horizontal microscope within the rocket's module (TEM 06-19). The swimming cells were observed in dark field mode with a 1.6x objective at 22° C. The cells were again observed 45 min after landing in a horizontal microscope and afterwards washed in EGTA (0.5 mM), fixed in 1% OsO4(0°C) for 30 min and further prepared for electron microscopy.8
Accelerations
A mean linear acceleration
of 5 g and a centrifugal (spin) acceleration acted during the
first 50 s of the rocket's boost phase. Only the spin
acceleration, peaking at 6.4 g, remained just before the onset
of µg, and was stopped within 1 s. When the video signal was
received 58 s after lift-off, the residual acceleration had
dropped to 2.6x10-4 g and declined to
<4x10-6g at the sample's position.
Vibration test
Cells were exposed to a
vibration test (5.5 g, 20-2000 Hz for 1 min) simulating the
worst-case Skylark rocket launch (MBB-ERNO, Bremen, Germany).
Slow rotating centrifuge microscope
Hypergravity experiments (up to 5 g) were performed on the
Niedergeschwindigkeits-Zentrifugenmikroskop (NIZEMI) rotating
centrifuge (Dornier, Friedrichshafen, Germany). The optical
conditions were as in the flight experiment.9
Fast-rotating clinostat
A fast-rotating
clinostat microscope, developed by Briegleb and co-workers, was
used to cancel the influence of gravity by reducing the
presentation time of the gravitational stimulus.9, 10
At 60 rpm, the centrifugal force amounts to 0 g in the centre and
to 4x10-2g at the border of an observation chamber of
15 mm radius.
Video analysis
Evaluation was performed
by computer-controlled realtime online image analysis as well as
using video-recorded sequences.11, 12 Data for cell
orientation, swimming velocities and linearity of individual
swimming tracks were obtained. The degree of orientation was
quantified by the Rayleigh test13 which yields a
statistical value (r-value) that runs from 0 (random) to 1
(perfect orientation of all organisms in the same direction).
For simulating the effect of linear acceleration and vibrations during rocket launch, negative gravitactic cultures of Paramecium were exposed to 5 g and a vibration profile for 1 min. Gravitactic behaviour was maintained in both cases. Reorientation under 5 g towards the resulting acceleration vector occurred within seconds. Orientation precision was higher than under 1 g. Exposure to hypergravity decreased the mean swimming velocity, while vibration had no effect. The relationship between the up, down and horizontal velocities remained unaffected.14, 9
Before lift-off, Paramecium showed a precise negative gravitaxis. Most of the cells swam upwards with a high precision in orientation (r-value = 0.55; theta = 9°) (Fig. 1a & 1d; Fig. 2). After 1 s of free-fall, when the video signal was received from the rocket, paramecia still had a residual orientation, but towards 90±30° (theta = 96.2°), that is, towards the centre of the rocket (Fig. 1b & 1e; Fig. 2). The degree of orientation was even higher (r-value = 0.89) in comparison to the behaviour at 1 g. Random swimming was registered after about 80 s in µg (r-value: 0.01) (Fig. 1c & 1f; Fig. 2). Paramecium again showed negative gravitaxis immediately after payload retrieval.9
During the whole µg period, the linearity of the individual swimming tracks was the same as under 1 g. Swimming direction reversals and cell movement cessation were not observed.9
Fig. 1. Representative circular histograms from TEXUS 27 (a-c)
and TEXUS 28 (d-f) showing the swimming directions of Paramecium
at different times: negative gravitaxis 5 min before lift-off (a,
d); residual orientation 1 s after µg onset towards the
centre of the rocket (originally induced by the centrifugal
launch accelerations) (b, e); random distribution 80 s after
µg onset. Each histogram represents >300 tracks.
The cells' mean swimming velocity measured during the total free-fall period was higher (4.3% TEXUS 28; 7.5% TEXUS 27), than under 1 g. However, the increase was transient: after 3 min in µg the mean swimming velocity approached the value of the former horizontal swimming velocity (Fig. 2). The differences between the upward, downward and horizontal swimming at 1 g before launch disappeared under free-fall conditions, but were re-established after landing.14
Experiments on a fast-rotating clinostat on the ground revealed similar changes in orientation behaviour, albeit with some temporal delay. After starting rotation, a residual orientation in the former upward direction was measured for about 120 s, followed by random distribution. Paramecium regained its negative gravitaxis in the first minute after stopping the clinostat, even after a 2 hr run. During the run, Paramecium exhibited an increased swimming velocity as in the rocket. However, the increase was not transient but stable.9, 14
Fig. 2. Degree of orientation (r-value) (bars) and
corresponding mean swimming velocities (line) before launch (1
g), during µg (0 g) and after landing (1 g). Measurements
were performed continuously at 20 s intervals (N = 5809).
Trichocysts in Paramecium were taken as a stress indicator during the experimental conditions. Exocytosis of trichocysts can be provoked, for example, by an increase in the permeability of the membrane to calcium, by shear forces or by cell damage.15 Paramecia exposed to sounding rocket flights with periods of accelerations contained trichocysts in their normal condensed morphology, still attached to the cellular membrane.8
Experiments under the conditions of brief weightlessness do provide sufficient time to observe behavioural changes of gravitactic unicellular organisms and to provide further clues on the nature of the receptor mechanism.
Random distribution of the paramecia in µg proved that gravity is indeed the stimulus for the observed orientation at 1 g. Before a random distribution of the cells was measured, paramecia showed a residual orientation, induced by the last effective acceleration. Results obtained on the NIZEMI slow- rotating centrifuge support the conclusion that the first observed swimming direction in the rocket was originally induced by the centrifugal spin acceleration during launch. The knowledge of this residual effect is important for the design of further experiments.
During the 6 min free-fall conditions of the TEXUS experiment, Paramecium showed a transient increase in the mean swimming velocity. As supported by simulation experiments, this is presumably not induced by acceleration or vibration during launch. The fact that gravikinesis was not switched off immediately indicates a slow relaxation time constant of the gravikinetic response. The temporal delay between the gravitactic and the gravikinetic response rekindles the question whether both graviresponses are controlled by the same mechanism. Concerning the mechanism of graviperception in Paramecium, it is proposed that the whole cytoplasm acts as a statolith by exerting a pressure of about 0.1 Pa on the lower cell membrane,2 thus initiating a signal transduction cascade by stimulation of ion channels in the membrane. Suitable candidates as primary gravireceptors might also be heavy cell organelles in interaction with the cytoskeleton and/or membranes as in plant systems.16, 17 Further studies using inhibitors and mutants might prove this hypothesis. Experiments on clinostats and centrifuges are necessary for the explanation and statistical security of data obtained from experiments under the conditions of real µg.
An increasing number of experiments have been performed in recent years on sounding rockets dealing with questions from gravitational biology. Fast biological responses are well suited for investigation aboard this facility. Late access and early retrieval make this programme attractive for biological experiments. MAXUS, providing 12 min of free-fall, offers further possibilities. A centrifuge microscope on MAXUS 2 will allow us to determine the threshold for graviperception in different unicellular specimens.
The authors would like to thank D. Franke and his TEXUS-team of MBB-ERNO, Bremen, Germany, and G. Schmitt and his team from Kayser-Threde, Munich, Germany, for their excellent work. Thanks are also expressed to the German Minister for Research and Technology (BMFT), the German space agency (DARA) and the European Space Agency (ESA) for support.
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