Graviperception in ciliates: steps in the transduction chain.

Ciliates represent suitable model systems to study the mechanisms of graviperception and signal transduction as they show clear gravity-induced behavioural responses (gravitaxis and gravikinesis). The cytoplasm seems to act as a "statolith" stimulating mechanosensitive ion channels in the cell membrane. In order to test this hypothesis, electrophysiological studies with Stylonychia mytilus were performed, revealing the proposed changes (de- or hyperpolarization) depending on the cell's spatial orientation. The behaviour of Paramecium and Stylonychia was also analyzed during variable acceleration conditions of parabolic flights (5th German Parabolic Flight Campaign, 2003). The corresponding data confirm the relaxation of the graviresponses in microgravity as well as the existence of thresholds of graviresponses, which are found to be in the range of 0.4xg (gravikinesis) and 0.6xg (gravitaxis).

Paramecium--a model system for studying cellular graviperception.

Experiments under varied gravitational accelerations as well as in density-adjusted media showed that sensation of gravity in protists may be linked to the known principles of mechanosensation. Paramecium, a ciliate with clear graviresponses (gravitaxis and gravikinesis) is an ideal model system to prove this hypothesis since the ciliary activity and thus the swimming behaviour is controlled by the membrane potential. It has also been assumed that the cytoplasmic mass causes a distinct stimulation of the bipolarly distributed mechano-sensitive K+ and Ca2+ ion channels in the plasma membrane in dependence of the spatial orientation of the cell. In order to prove this hypothesis, different channel blockers are currently under investigation. Gadolinium did not inhibit gravitaxis in Paramecium, showing that it does not specifically block gravireceptors. Further studies concentrated on the question of whether second messengers are involved in the gravity signal transduction chain. Exposure to 5 g for up to 10 min led to a significant increase in cAMP.

Relaxation and activation of graviresponses in Paramecium caudatum.

The kinetics of gravitaxis and gravikinesis in Paramecium caudatum were investigated by employing (1) step transitions from normal gravity (1 g) to weightlessness (microgravity) and (2) turns of the experimental chambers from the horizontal to the vertical position at 1 g. The transition to microgravity left existing cell orientations unchanged. Relaxation of negative gravitaxis under microgravity took longer than 10 s and may be described by the time constant of the decay of orientation coefficients. Gravitaxis was started at 1 g by turning the experimental chamber from a horizontal to a vertical position. Gravitaxis activated rapidly during the turning procedure and relaxed to an intermediate level after the turning had stopped. Gravity-induced regulation of swimming speed (gravikinesis) at 1 g had reached a steady state after 1 min; at this point, gravikinesis counteracted the effects of sedimentation (negative gravikinesis). A step transition to microgravity initially reversed the sign of the gravikinesis (positive gravikinesis). The relaxation of this kinetic response was not completed during 10 s of microgravity. The data suggest that gravikinesis is functionally unrelated to gravitaxis and is strongly affected by the rate of change in acceleration. We present a model explaining why gravikinesis reverses sign upon the onset of a step from 1 g to microgravity.

Graviresponses of gliding and swimming Loxodes using step transition to weightlessness.

Cells of Loxodes striatus were adjusted to defined culturing, experimental solution O2-supply, temperature, and state of equilibration to be subjected to step type transition of acceleration from normal gravity, (1 g) to the weightless condition (microgravity) during free fall in a 500 m drop shaft. Cellular locomotion inside a vertical experimental chamber was recorded preceding transition and during 10 s of microgravity. Cell tracks from video records were used to separate cells gliding along a solid surface from free swimmers, and to determine gravitaxis and gravikenesis of gliding and swimming cells. With O2 concentrations > or = 40% air saturation gliders and swimmers showed a positive gravitaxis. In microgravity gravitaxis of gliders relaxed within 5 s whereas gravitaxis relaxation of swimmers was not completed even after 10 s. Rates of horizontal gliders (319 micrometers/s) exceeded those, of horizontal swimmers (275 micrometers/s). Relaxation of gravikinesis was incomplete after 10 s of microgravity. Analysis of the locomotion rates during the g-step transition revealed that gliders sediment more slowly, than swimmers (14 versus 45 micrometers/s). The gravikinesis of gliders cancelled sedimentation effects during upward and downward locomotion tending to maintain cells at a predetermined level inside sediments of a freshwater habitat. At > or = 40% air saturation, gravikinesis of swimmers augmented the speed of the majority of cells during gravitaxis, which favours fast vertical migrations of Loxodes.

Assessment of g-dependent Cellular Gravitaxis: Determination of Cell Orientation from Locomotion Track

Movement of cells in the gravity field is principally affected in two ways: velocity and orientation. Experimental observation of gravitaxis in large cell populations can document the velocity and orientation of swimming tracks, but orientations of individual cells are not represented at low magnifications. Cell orientations may depart from track orientations due to superposition of sedimentation on cellular propulsion. Here, we show that determination of the sedimentation rate in addition to cell track parameters allows a reconstitution of cell orientation employing geometric principles. Published and original cellular data indicate that gravitactic orientation of cells swimming in the gravity field is superior to that suggested from the experimental tracks. Similar conclusions apply to cells which walk or glide along substrate surfaces. Calculation of cell orientation coefficients provides a basis for determinations of the acceleration-dependence of gravitaxis and for quantitative tests on physical and/or physiological principles of cellular gravitaxis. Copyright 1997 Academic Press Limited

Gravitaxis screened for physical mechanism using g-modulated cellular orientational behaviour.

Advanced methods of recording cellular orientation with respect to the gravity vector are yielding increasingly wellfounded data on gravitaxis. The present study introduces a quantitative method which allows us to predict the precision of orientational behaviour as a function of acceleration assuming static buoyancy as a hypothetical physical principle of gravitaxis. The precision of orientation is expressed by the orientation coefficient as derived from circular statistics. Orientation coefficients calculated from experimental data at various g-values are tested for fit with a sigmoidal orientation coefficient-g-transfer function including a proportionality factor k. Residual orientation values in the low-hypogravity range obey a reciprocal function between k and g. Intersection of this residual-g function with the orientation coefficient-g-relationship gives the minimal acceleration to generate cellular orientation. Those data which clearly diverge from the orientation coefficient-g-curve bear some probability that the observed gravitaxis was guided in part by a physiological mechanism of gravireception and active graviorientation. Data which fit the orientation coefficient-g-curve qualify as being in agreement with a mechanical basis of cellular gravitaxis. Examples from the literature are presented and discussed in the light of our scheme of gravitaxis screening.

Behavioural changes in Paramecium and Didinium exposed to short-term microgravity and hypergravity.

The swimming behaviour of two ciliate species, Paramecium caudatum and Didinium nasutum was analyzed under microgravity and hypergravity. In Paramecium the differences between former upward and downward swimming rates disappeared under weightlessness. At microgravity the swimming rates equalled those of horizontally swimming cells at 1g. In contrast, the swimming rates of Didinium increased under microgravity conditions, being larger than horizontal swimming rates at 1g. These findings are in accordance with a hypothesis of gravireception in ciliates based on electrophysiological data, which considers the different topology of mechanoreceptor channels in theses species. The hypothesis received further support by data recorded under hypergravity conditions.

Short-term microgravity to isolate graviperception in cells.

In the fall of 1991 a series of drop-tower experiments in ZARM (Bremen) was devoted to behavioural responses of unicellular organisms to step-type transition from normal gravity to microgravity. Modules for simultaneous 4-fold video-recording were incorporated into the flight capsule. In the course of 25 flights, 100 sets of experiments, each holding 100 to 200 cells, were flown under various conditions with a technical success rate of 94% and about 80% of the cells accessible to evaluation in the laboratory. A major goal of the experiments was the assessment of parameters of locomotion (velocity, orientation) in the absence of the gravity vector. The data show that in two species, Paramecium and Loxodes, the properties of steady-state microgravity-swimming correspond to horizontal swimming under 1g-conditions. In a third species, Didinium, microgravity-swimming velocity exceeds 1 g-horizontal rates. The data are in agreement with an electrophysiological hypothesis of graviperception in cells.

Gravireception and graviresponses in ciliates.

An account is given of approaches to gravireception, terminology mechanisms of responses to gravity as investigated and documented in the literature, and sensorimotor coupling properties in ciliates. Current theories and methods are discussed, and previously published experimental data on graviresponses are reviewed.

Gravity-controlled gliding velocity in Loxodes.

The locomotion of Loxodes as controlled by the natural gravity vector was investigated employing a mass-cell approach. Samples of cells were incubated for 4 hours in a 1.6 mm deep well (41 × 85 mm) filled with defined experimental solution. Their gliding locomotion on surfaces inclined between 0° and 90° was recorded by videocamera. Steady gliding rates (median of total: 206 µm/s; n = 12711) were evaluated by calculating the vertical components from observed tracks. At a given inclination the rates of downward and upward gliding were similar. The sedimentation rate of freely suspended nickel-immobilized specimens (S = 49 µm/s), and vertical rates of displacement at 6 differently inclined planes with cells in "sitting" posture as well as 'hanging' (upside down) were used to determine the gravity-dependent component (Δ = - 49 µm/s) during gliding motion. Numerical equality of the gravity force to produce S and of the counterforce to produce Δ follows from the observed constancy of gliding rates (V) and identical medians of vertical displacements during gliding along vertically oriented surfaces (169 µm/s). It is suggested that neutralization of sedimentation might be a favourable condition for migration and accumulation of Loxodes along vertical O(2) gradients in its freshwater habitat.