Graviperception and gravitaxis in algae.

Photosynthetic flagellates are among the most intensely studied unicellular organisms in the field of graviperception and gravitaxis. While the phenomenon of graviorientation has been known for many decades, only recently was the molecular mechanism unveiled. Earlier hypotheses tried to explain the precise orientation by a passive buoy mechanism assuming the tail end to be heavier than the front. In the photosynthetic flagellate Euglena gracilis, the whole cell body is denser than the surrounding medium, pressing onto the lower cell membrane where it seems to activate mechanosensitive ion channels specific for calcium. The calcium entering the cells during reorientation can be visualized by the fluorescence probe, Calcium Crimson. Cyclic AMP is likewise involved in the molecular pathway. Inhibitors of calcium channels and ionophores impair gravitaxis while caffeine, a blocker of the phosphodiesterase, enhances the precision of orientation.     

Gravitaxis and graviperception in Euglena gracilis.

Gravitactic orientation in the flagellate Euglena gracilis is mediated by an active physiological receptor rather than a passive alignment of the cells. During a recent space flight on the American shuttle Columbia the cells were subjected to different accelerations between 0 and 1.5 x g and tracked by computerized real-time image analysis. The dependence of orientation on acceleration followed a sigmoidal curve with a threshold at < or = 0.16 x g and a saturation at about 0.32 x g. No adaptation of the cells to the conditions of weightlessness was observed over the duration of the space mission (12 days). Under terrestrial conditions graviorientation was eliminated when the cells were suspended in a medium the density of which (Ficoll) equaled that of the cell body (1.04 g/ml) and was reversed at higher densities indicating that the whole cytoplasm exerts a pressure on the respective lower membrane. There it probably activates stretch-sensitive calcium specific ion channels since gravitaxis can be affected by gadolinium which is a specific inhibitor of calcium transport in these structures. The sensory transduction chain could involve modulation of the membrane potential since ion channel blockers, ionophores and ATPase inhibitors impair graviperception.     

Physiological characterization of gravitaxis in Euglena gracilis and Astasia longa studied on sounding rocket flights.

Euglena gracilis is a photosynthetic, unicellular flagellate found in eutrophic freshwater habitats. The organisms control their vertical position in the water column using gravi- and phototaxis. Recent experiments demonstrated that negative gravitaxis cannot be explained by passive buoyancy but by an active physiological mechanism. During space experiments, the threshold of gravitaxis was determined to be between 0.08 and 0.12 x g. A strong correlation between the applied acceleration and the intracellular cAMP and Ca2+ was observed. The results support the hypothesis, that the cell body of Euglena, which is denser than the surrounding medium exerts a pressure onto the lower membrane and activates mechanosensitive Ca2+ channels. Changes in the membrane potential and the cAMP concentration are most likely subsequent elements in a signal transduction chain, which results in reorientation strokes of the flagellum.     

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.     

Molecular analysis of the graviperception signal transduction in the flagellate Euglena gracilis: Involvement of a transient receptor potential-like channel and a calmodulin

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavior responses. The organism shows pronounced negative gravitaxis. This movement is based on physiological mechanisms, which in the past had been only indirectly assessed. It was shown that mechano-sensitive calcium channels are involved in the gravitaxis response. Recent studies have demonstrated that members of the transient receptor potential (TRP) family function as mechano-sensitive channels in several different cell types. We have sequenced part of a TRP gene in Euglena and applied RNA interference (RNAi) to confirm that these channels are involved in graviperception. It was found that RNAi against the putative TRP channel abolished gravitaxis. The genes of three calmodulins were sequences in Euglena, one of which was previously known in its protein structure (cal 1). The other two were unknown (cal 2 and cal 3). Cal 2 has been analyzed in detail. The biosynthesis of the corresponding proteins of cal 1 and cal 2 was inhibited by means of RNA interference to see whether this blockage impairs gravitaxis. RNAi of cal 1 leads to a long-term loss of free swimming in the cells (while euglenoid movement persists). It induced pronounced cell form aberrations and the division of cells was hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Thus cal 1 does not seem to be involved in gravitaxis. In contrast, the blockage of cal 2 has no pronounced influence on motility and cell form but leads to a complete loss of gravitactic orientation for more than 30 days showing that this calmodulin is an element in the signal transduction chain. The data are discussed in the context of the current model of the gravitaxis signal transduction chain in Euglena gracilis.     

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.     

Hze-particles measured during IML-1 mission

Three stacks of CR-39 plastic nuclear track detectors were exposed to cosmic radiation during IML-1 mission. They were mounted with their axes parallel to the shuttle's axes to allow measurements of LET-spectra for different impinging directions. First results of this experiment are reported.     

Skeletogenesis in sea urchin larvae under modified gravity conditions.

From many points of view, skeletogenesis in sea urchins has been well described. Based on this scientific background and considering practical aspects of sea urchin development (i.e. availability of material, size of larvae, etc.), we wanted to know whether orderly skeletogenesis requires the presence of gravity. The objective has been approached by three experiments successfully performed under genuine microgravity conditions (in the STS-65 IML-2 mission of 1994; in the Photon-10 IBIS mission of 1995 and in the STS-76 S/MM-03 mission of 1996). Larvae of the sea urchin Sphaerechinus granularis were allowed to develop in microgravity conditions for several days from blastula stage onwards (onset of skeletogenesis). At the end of the missions, the recovered skeletal structures were studied with respect to their mineral composition, architecture and size. Live larvae were also recovered for post-flight culture. The results obtained clearly show that the process of mineralisation is independent of gravity: that is, the skeletogenic cells differentiate correctly in microgravity. However, abnormal skeleton architectures were encountered, particularly in the IML-2 mission, indicating that the process of positioning of the skeletogenic cells may be affected, directly or indirectly, by environmental factors, including gravity. Larvae exposed to microgravity from blastula to prism/early pluteus stage for about 2 weeks (IBIS mission), developed on the ground over the next 2 months into normal metamorphosing individuals.     

Repair of radiation induced genetic damage under microgravity.

The influence of microgravity on the repair of radiation induced genetic damage in a temperature-conditional repair mutant of the yeast Saccharomyces cerevisiae (rad 54-3) was investigated onboard the IML-1 mission (January 22nd-30th 1992, STS-42). Cells were irradiated before the flight, incubated under microgravity at the permissive (22 degrees C) and restrictive (36 degrees C) temperature and afterwards tested for survival. The results suggest that repair may be reduced under microgravity.     

The effect of microgravity on the development of plant protoplasts flown on Biokosmos 9.

An experiment using plant protoplasts has been accepted for the IML-1 Space Shuttle mission scheduled for 1991. Preparatory experiments have been performed using both fast and slow rotating clinostats and in orbit to study the effect of simulated and real weightlessness on protoplast regeneration. Late access to the space vehicles before launch has required special attention since it is important to delay cell wall regeneration until the samples are in orbit. On a flight on Biokosmos 9 ("Kosmos-2044") in September 1989 some preliminary results were obtained. Compared to the ground control, the growth of both carrot and rapeseed protoplasts was decreased by 18% and 44% respectively, after 14 days in orbit. The results also indicated that there is less cell wall regeneration under micro-g conditions. Compared to the ground controls the production of cellulose in rapeseed and carrot flight samples was only 46% and 29% respectively. The production of hemicellulose in the flight samples was 63% and 67% respectively of that of the ground controls. In both cases all samples reached the stage of callus development. The peroxidase activity was also found to be lower in the flight samples than in the ground controls, and the number of different isoenzymes was decreased in the flight samples. In general, the regeneration processes were retarded in the flight samples with respect to the ground controls. From a simulation experiment for IML-1 performed in January 1990 at ESTEC, Holland, regenerated plants have been obtained. These results are discussed and compared to the results obtained on Biokosmos 9. Protoplast regeneration did not develop beyond the callus stage in either the flight or the ground control samples from the Biokosmos 9 experiment.     

NASDA aquatic animal experiment facilities for Space Shuttle and ISS.

National Space Development Agency of Japan (NASDA) has developed aquatic animal experiment facilities for NASA Space Shuttle use. Vestibular Function Experiment Unit (VFEU) was firstly designed and developed for physiological research using carp in Spacelab-J (SL-J, STS-47) mission. It was modified as Aquatic Animal Experiment Unit (AAEU) to accommodate small aquatic animals, such as medaka and newt, for second International Microgravity Laboratory (IML-2, STS-65) mission. Then, VFEU was improved to accommodate marine fish and to perform neurobiological experiment for Neurolab (STS-90) and STS-95 missions. We have also developed and used water purification system which was adapted to each facility. Based on these experiences of Space Shuttle missions, we are studying to develop advanced aquatic animal experiment facility for both Space Shuttle and International Space Station (ISS).     

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.     

Structural and functional organisation of regenerated plant protoplasts exposed to microgravity on Biokosmos 9.

Preparatory experiments for the IML-1 mission using plant protoplasts, were flown on a 14-day flight on Biokosmos 9 in September 1989. Thirty-six hours before launch of the biosatellite, protoplasts were isolated from hypocotyl cells of rapeseed (Brassica napus) and suspension cultures of carrot (Daucus carota). Ultrastructural and fluorescence analysis of cell aggregates from these protoplasts, cultured under microgravity conditions, have been performed. In the flight samples as well as in the ground controls, a portion of the total number of protoplasts regenerated cell walls. The processes of cell differentiation and proliferation under micro-g did not differ significantly from those under normal gravity conditions. However, in micro-g differences were observed in the ultrastructure of some organelles such as plastids and mitochondria. There was also an increase in the frequency of the occurrence of folds formed by the plasmalemma together with an increase in the degree of complexity of these folds. In cell cultures developed under micro-g conditions, the calcium content tends to decrease, compared to the ground control. Different aspects of using isolated protoplasts for clarifying the mechanisms of biological effects of microgravity are discussed.     

A theory of gravikinesis in Paramecium.

The archaic eukaryote unicellular microorganism, Paramecium, is propelled by thousands of cilia, which are regulated by modulation of the membrane potential. Ciliates can successfully cope with gravity, which is the phylogenetically oldest stimulus for living things. One mechanism for overcoming sedimentation is negative gravitaxis, an orientational response antiparallel to the gravity vector. We have postulated the existence of a negative gravikinesis in Paramecium, i.e. a modulation of swimming speed as a function of cellular orientation in space. With negative gravikinesis, an upward oriented cell actively augments the rate of forward swimming and depresses active locomotion during downward orientation. A brief outline of the gravikinesis hypothesis is given on a quantitative basis and experimental data are presented which have confirmed the major assumptions.     

Gravitational response of the slime mold Physarum.

The acellular slime mold Physarum polycephalum is used as a model system to investigate the graviresponse of single cells which possess no receptors specialized for the perception of gravity. To obtain insights into the gravity-signal transduction mechanism the light response of the cell is used: Macroplasmodia of the slime mold show clear geo- and phototaxes. Gravity increases and white light decreases transiently the contraction frequency of plasmodial strands whereby both responses follow the same time pattern. Since mitochondria play a major role in changing the contraction rhythm in response to light and gravity stimuli, the simultaneous and subsequent inductions of the opposing light and gravity responses and their mutual influences on one another were investigated. The experiments were performed in weightlessness (0 g)--simulated on the fast-rotating clinostat as well as in actual weightlessness during the IML-1 Space Shuttle mission. The results indicate that mitochondria (chondriome) are part of the acceleration-stimulus reaction chain in Physarum. Two models for a direct gravireceptor mechanism are discussed.     

Development of plant protoplasts during the IML-1 mission.

During the 8 day IML-1 mission, regeneration of cell walls and cell divisions in rapeseed protoplasts were studied using the Biorack microscope onboard the Space Shuttle "Discovery". Samples from microgravity and 1g protoplast cultures were loaded on microscope slides. Visual microscopic observations were reported by the payload specialist Roberta Bondar, by down-link video transmission and by use of a microscope camera. Protoplasts grown under microgravity conditions do regenerate cell walls but to a lesser extent than under 1g. Cell divisions are delayed under microgravity. Few cell aggregates with maximum 4-6 cells per aggregate are formed under microgravity conditions, indicating that microgravity may have a profound influence on plant cell differentiation.     

Retinal photoreceptor and related gene expression in normal and clinostat-treated fish embryos

Medaka fish had performed mating behavior successfully in space for the first time among vertebrate, and the eggs which were laid in space developed normally, and hatched during the space travel of 15 days aboard the space shuttle in the second International Microgravity Laboratory (IML-2) mission in 1994 (Ijiri 1994). But there has been few studies whether microgravity affects the development of rather complex tissues in this fish. Investigating this problem, we focused on the organogenetic events in the retina in developing Medaka under normal and simulated microgravity conditions (by a three-dimensional clinostat, 3D-clinostat). Our results showed that both normal and 3D-clinostat-treated Medaka embryos developed on almost equal time course. Moreover, we investigated the development of the retina in normal and 3D-clinostat-treated embryos, but there were no differences in organogenesis of their retina. Lamination of retina occurred almost at equal timing and the expressions of opsin genes in the 3D-clinostat-treated group also began almost at the same time as control. Our observations suggest that there were no definite effects of simulated microgravity on the organizations of a complex tissue such as retina in developing fish embryos.     

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.