Electric potentiation of gravikinesis in Paramecium is possibly mediated by filaments.

Sensitivity of Paramecium to mechanical stress including gravitational force is organized along two opposing gradients of membrane channel distribution: depolarizing Ca channels and hyperpolarizing K channels. Mechanoreceptor channels reside in the membrane of the cell soma and are activated, when the weight of the cytoplasm deforms the "lower" plasma membrane. Channel distribution is such as to generate ciliary activation which can counteract sedimentation of the cells: a reduction in downward swimming rate and an augmentation in upward swimming rate. Application of weak DC fields does not only induce the well-known cathodal orientation and swimming of Paramecium toward the cathode (galvano-taxis). We document that swimming velocity is augmented up to 175% as a function of the voltage gradient between 0.3 V/cm and 0.8 V/cm (galvanokinesis). A gradient of 0.3 V/cm was highly effective in raising the common negative gravikinesis of downward swimmers threefold. The gravikinesis of upward swimmers reversed polarity under field stimulation inducing cells to augment sedimentation effects (positive gravikinesis). Both effects of electric-field stimulation on ciliary activation are of the depolarizing type: reduction in the frequency of normally beating cilia. Analysis of the data shows that a voltage-sensitivity of gravireceptor channels would not account for the observed potentiation of negative gravikinesis. It is suggested that a previously described voltage-dependent Ca channel of the soma membrane interferes with a Ca(2+)-sensitive, peripheral filament system, which directly connects to gravireceptor channels.     

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.     

Gravitaxis in unicellular microorganisms.

Orientation of organisms with respect to the gravitational field of the Earth has been studied for more than 100 years in a number of unicellular microorganisms including flagellates and ciliates. Several hypotheses have been developed how the weak stimulus is perceived. Intracellular statoliths have been found to be involved in gravitaxis of Loxodes, while no specialized organelles have been detected in other ciliates, e.g. Paramecium. Also in the slime mold Physarum no specialized gravireceptors have been identified yet. In the flagellate Euglena gracilis the whole cell body, which is denser than the surrounding medium, seems to act as a statolith pressing onto the lower membrane where it activates mechanosensitive ion channels. Similar results were obtained for the ciliate Paramecium. In contrast to the flagellate Euglena, several ciliates have been found to show gravikinesis, which is defined as a dependence of the swimming velocity on the direction of movement in the gravity field.     

Super-helix model: a physiological model for gravitaxis of Paramecium.

A new model explaining the gravitactic behavior of Paramecium is derived on the basis of its mechanism of gravity sensing. Paramecium is know to have depolarizing mechanoreceptor ion channels in the anterior and hyperpolarizing channels in the posterior of the cell. This arrangement may lead to bidirectional changes of the membrane potential due to the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell with respect to the gravity vector; i.e., negative- and positive-going shifts of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential, in combination with the close coupling between the membrane potential and ciliary locomotor activity, may allow the changes in swimming direction along the otherwise simple helical swimming path in the following manner: an upward shift of the axis of helical swimming occurs by decreasing the pitch angle due to channel-dependent hyperpolarization in upward-orienting cells, and an upward shift of the swimming helix occurs by increasing the cell's pitch angle due to depolarization in downward-orienting cells. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically along an axis parallel to the gravity vector.     

How gravity acts on Paramecium: new insights from free-fall experiments.

The swimming behaviour of Paramecium is affected by media of various specific gravities. At 1g, the negative gravitaxis of Paramecium virtually disappears in solutions the specific gravity of which is about the same as that of the organism (1.04). In solutions with a higher specific gravity (1.08), Paramecium becomes positively gravitactic. We recorded the swimming tracks of Paramecium in these media on videotape before, during and after free-falls. The records show that the density-dependent differences in the swimming behaviour disappeared immediately following the onset of the free-fall. The recorded tracks and distributions of cells in the experimental chambers were compared with computer-simulated traces and distributions based on gravitactic and gravikinetic models proposed for Paramecium. Our preliminary analysis favors a novel gravitactic mechanism involving modification of the ciliary movement The drop shaft at the Japan Microgravity Center, Hokkaido (JAMIC) was used for the free-fall experiments.     

Short-term responses of gravitaxis to altered gravity in Paramecium.

Negative gravitaxis of Paramecium almost disappeared in solutions having specific gravity about the same as that of the organisms (1.04). The taxis turned to positive in solutions of specific gravity 1.08. Using a drop shaft at the Japan Microgravity Center, Hokkaido (JAMIC) we examined how swimming behaviour in these media was modified by changing gravitational conditions before, during and after free-fall. Tracks of swimming cells recorded on videotape indicate that the swimming cells continued upward and downward shift depending on the specific gravity of the external medium under 1-g conditions and these vertical displacements disappeared immediately after the moment of launch. The effectiveness of changing gravity to induce displacement of the cells seems to depend on the orientation of the cells to gravity. These results suggest a corelation between vertical displacement of the cell through the medium and a gravitactic mechanism in Paramecium.     

Swimming behavior of Paramecium--first results with the low-speed centrifuge microscope (NIZEMI).

The low-speed centrifuge microscope NIZEMI (= Nieder-Geschwindigkeits-Zentrifugen-Mikroskop) is an excellent tool with which to investigate the effects of slightly increased gravity in the fields of biology and material sciences. We investigated the swimming behavior of Paramecium in the NIZEMI, by aid of a computer-controlled image analysis system. In the range of acceleration (1 g to 5 g), cells retained their swimming capability, did not sediment, and even increased the precision of their negative gravitaxis but reduced their mean swimming velocity.     

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).     

Putative graviperception mechanisms of protists.

Many (if not all) free-living cells use the gravity vector for their spatial orientation (gravitaxis). Additional responses may include gravikinesis as well as changes in morphological and physiological parameters. Though using essentially different modes of locomotion, ameboid and ciliated cells seem to rely on common fundamental graviperception mechanisms. Uniquely in the ciliate family Loxodidae a specialized intracellular gravireceptor organelle has been developed, whereas in all other cells common cell structures seem to be responsible for gravisensing. Changes in direction or magnitude of acceleration (from 0 to 5 g) as well as experiments in density-adjusted media strongly indicate that either the whole cytoplasm or dense organelles like nuclei act as statoliths and open directly or via cytoskeletal elements mechano-sensitive ion channels in the cell membrane. A recent spaceflight experiment (S/MM-06) demonstrated that prolonged (9 d) actual weightlessness did not affect the ability of Loxodes to respond to acceleration stimuli. However, prolonged cooling (> or = l4 d, 4-10 degrees C) destroyed the ability for gravitactic orientation of Paramecium. This may reflect a profound effect either on the gravireceptor itself or on the gravity-signal processing. In gravity signalling the ubiquitous second messenger cAMP may be involved in acceleration-stimulus transduction.     

The identification of gravikinesis from ciliates: methods and experience.

Recent advances in the gravitational physiology of ciliates are reported: the theoretical and experimental assessment of gravikinesis and sedimentation, calculation of gravikinesis using slopes of observed swimming and sedimentation data under hypergravity, orientational distributions of gravikinesis, central and membrane-associated gravitransduction, and the kinetics of activation and relaxation of gravikinesis.     

Comparative studies of the graviresponses of Paramecium and Loxodes.

Gravitactic protozoa offer advantages in studying how the gravity stimulus is perceived on the cellular level. By means of a slow rotating centrifuge microscope in space the acceleration thresholds for gravitaxis of Loxodes striatus and Paramecium biaurelia were determined: < or = 0.15 x g for Loxodes and 0.3 x g for Paramecium, indicating different sensitivities of these species. Neutral-buoyant densities of immobilized cells were determined using media of different densities, revealing densities of 1.03 to 1.035 g/cm3 for Loxodes and 1.04 g/cm3 to 1.045 g/cm3 for Paramecium. Behavioral studies revealed that gravitaxis of Loxodes persisted independent of the density of the medium. In contrast, negative gravitaxis of Paramecium was no longer measurable if the density of the medium approached the density of the cell. The results suggest that in the case of Loxodes gravity is perceived by an intracellular receptor and, in the case of Paramecium by its own mass via the pressure on the lower cell membrane.     

Theory and experimental results on gravitational effects on monocellular algae.

The orientation of a body which has an anisotropic distribution of mass and which is suspended in water is biased by gravitational torque, so that the center of gravity lies below the center of buoyancy. Many species of unicellular swimming algae are gravitationally oriented in this manner. Their axis of propulsion is essentially fixed within their bodies, so that when the cells swim, they swim upwards. Gravitaxis is an exotaxis, which requires no sensory processing. Nevertheless, gravity affects the lives of these cells both individually and collectively. For single cells, gravity intervenes in the execution and mechanism of sense-dependent taxes, such as phototaxis, it provides for fail-safe locomotion toward the upper interface of their habitat, the source of light and air, and it may cause up-accumulation. Populations of single cells, swimming in the presence of gravity, are coupled through fluid-mechanical interactions which cause spatial and temporal patterns of fluid convection and cell concentration. These patterns modify the cell's environmental interactions, by facilitating downward migrations of cell populations, by mixing the embedding fluid and its contents, and by providing a collective mechanism for controlling light intensity at the individual cell level. Summarizing, gravity modulates the interaction of algal cells with each other and with their environment.     

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.     

Survey of the vestibulum, and behavior of xenopus laevis larvae developed during a 7-days space flight

Aquatic animals have almost no body weight related proprioception for spatial orientation. larvae, like fish, maintain their attitude in water by continuous correction with their fin(s). For these reasons a special performance of the equilibrium system compared to terrestrial animals is necessary. Evidently fish therefore have more compact (dense) otoliths; larvae have less dense otolith (membranes) similar to land vertebrates; but their sacculus-otoliths are vertically positioned, which also may lead to a higher g-sensitivity.

For plausibility reasons gravity should influence the embryonic development of gravity receptors. Yet, evaluations of photographs taken from the surface of cut deep-frozen objects by incident light show no aberration of the shape of the whole vestibulum and of the shape, density, size and position of the otolith membrane in larvae developed under near-zero g (NEXPA-BW-STATEX in D1-Mission).

The further evaluation of the “weightless-larvae” revealed a probably not yet described statolith-like formation in the dorsal wall of the vestibulum. In the weightless larvae this formation outnumbers, also qualitatively, strongly the 1-g controls.

An extra result is the lack of striking effects of cosmic radiation on the embryonic development of the flown eggs.

The swimming behavior of the larvae which was observed about one hour after landing of the Space Shuttle showed a typical anomaly (loop swimming), which is known from larvae developed on the clinostat or from fish flown aboard Apollo capsules.     

Genetic analysis of the gravitropic set-point angle in lateral roots of Arabidopsis.

Research on gravity responses in plants has mostly focused on primary roots and shoots, which typically orient to a vertical orientation. However, the distribution of lateral organs and their characteristically non-vertical growth orientation are critical for the determination of plant form. For example, in Arabidopsis, when lateral roots emerge from the primary root, they grow at a nearly horizontal orientation. As they elongate, the roots slowly curve until they eventually reach a vertical orientation. The regulation of this lateral root orientation is an important component affecting overall root system architecture. We found that this change in orientation is not simply due to the onset of gravitropic competence, as non-vertical lateral roots are capable of both positive and negative gravitropism. Thus, the horizontal growth of new lateral roots appears to be determined by what is called the gravitropic set-point angle (GSA). This developmental control of the GSA of lateral roots in Arabidopsis provides a useful system for investigating the components involved in regulating gravitropic responses. Using this system, we have identified several Arabidopsis mutants that have altered lateral root orientations but maintain normal primary root orientation.     

Sedimenting particles and swimming micro-organisms in a rotating fluid.

Experiments and calculations on the trajectories of micron-sized spheres, suspended in a fluid that fills a dosed container which rotates about an axis perpendicular to g, relate to the planning and interpretation of clinostat experiments. For low Reynolds number motion, the orbits are nearly circular, the radius being inversely proportional to the rotation rate. The swimming direction of micro-organisms can be affected by light, gravity, vorticity etc. The trajectories of algae swimming in steadily rotating environments have been observed and compared with theoretical predictions for ideal gyrotactic micro-organisms, thus providing some insights into the mechanisms of gravitaxis, gyrotaxis and the behaviour of the cells.     

Protozoa as model systems for the study of cellular responses to altered gravity conditions.

The orientation behavior of Paramecium changed in a similar way after transition to conditions of free-fall in a sounding rocket and after transition to conditions of simulated weightlessness on a fast rotating clinostat. After a period of residual orientation, Paramecium cells distributed themselves randomly 80 s (120 s) after onset of free-fall (simulated weightlessness). Swimming velocity increased significantly; however, the increase was transient and subsided after 3 min in the rocket experiments, while the velocity remained enhanced even during 2 h of rotation on a fast clinostat. Trichocysts were present and without morphological changes in Paramecium cells which had been exposed to a rocket flight, as well as to fast or slow rotation on a clinostat. Regeneration of the oral apparatus of Stentor and morphogenesis of Eufolliculina proceeded normally on the clinostat. The results demonstrate that the clinostat is a useful tool to simulate the conditions of weightlessness on earth and to detect gravisensitive cellular functions.     

Microtubule self-organisation depends upon gravity.

The molecular processes by which gravity is transduced into biological systems are poorly, if at all, understood. Under equilibrium conditions, chemical and biochemical structures do not depend upon gravity. It has been proposed that biological systems might show a gravity dependence by way of the bifurcation properties of certain types of non-linear chemical reactions that are far-from-equilibrium. We have found that in-vitro preparations of microtubules, an important element of the cellular cytoskeleton, show this type of behaviour. On earth, the solutions show macroscopic self-ordering, and the morphology of the structures that form depend upon the orientation of the sample with respect to gravity at a critical moment at an early stage in the development of the self-organised state. An experiment carried out in a sounding rocket, showed that as predicted by theories of this type, no self-organisation occurs when the microtubules are assembled under low gravity conditions. This is an experimental demonstration of how a very simple biochemical system, containing only two molecules, can be gravity sensitive. At a molecular level this behaviour results from an interaction of gravity with macroscopic concentration and density fluctuations that arise from the processes of microtubule contraction and elongation.     

Neuroplastic reactivity of fish induced by altered gravity conditions: a review of recent results.

A review is being presented concerning behavioural, biochemical, histochemical and electronmicroscopical data on the influence of altered gravitational forces on the swimming performance and on the neuronal differentiation of the brain of cichlid fish larvae and adult swordtail fish that had been exposed to hyper-gravity (3g in laboratory centrifuges), hypo-gravity (>10(-2) g in a fast-rotating clinostat) and to near weightlessness (10(-4) g aboard the Spacelab D-2 mission). After long-term alterations of gravity (and parallel light deprivation), initial disturbances in the swimming behaviour followed by a stepwise regain of normal swimming modes are induced. Parallel, neuroplastic reactivities on different levels of investigation were found, such as adaptive alterations of activities of various enzymes in whole brain as well as in specific neuronal integration centers and an intraneuronal reactivity on ultrastructural level in individual brain parts and in the sensory epithelia of the inner ear. Taken together, these data reveal distinct adaptive neuroplastic reactions of fish to altered gravity conditions.