Gravitaxis and graviperception in flagellates.

There is strong evidence that gravitactic orientation in flagellates and ciliates is mediated by an active physiological gravireceptor rather than by passive alignment of the cells in the water column. In flagellates the threshold for graviorientation was found to be at 0.12 x g on a slow rotating centrifuge during the IML-2 mission on the Shuttle Columbia and a subsequent parabolic rocket flight (TEXUS). During the IML-2 mission no adaptation to microgravity was observed over the duration of the space flight, while gravitaxis was lost in a terrestrial closed environmental system over the period of almost two years. Sedimenting statoliths are not likely to be involved in graviperception because of the small size of the cells and their rotation around the longitudinal axis during forward locomotion. Instead the whole cytoplasmic content of the cell, being heavier than the surrounding aqueous medium (1.05 g/ml), exerts a pressure on the lower membrane. This force activates stretch-sensitive calcium specific ion channels which can be inhibited by the addition of gadolinium which therefore abolishes gravitaxis. The channels seem to mainly allow calcium ions to pass since gravitaxis is blocked by the addition of the calcium ionophore A23187 and by vanadate which blocks the Ca-ATPase in the cytoplasmic membrane. Recently, a gene for a mechanosensitive channel, originally sequenced for Saccharomyces, was identified in Euglena by PCR. The increase in intracellular free calcium during reorientation can be visualized by the fluorophore Calcium Crimson using laser excitation and image intensification. This result was confirmed during recent parabolic flights. The gated calcium changes the membrane potential across the membrane which may be the trigger for the reorientation of the flagellum. cAMP plays a role as a secondary messenger. Photosynthetic flagellates are suitable candidates for life support systems since they absorb CO2 and produce oxygen. Preliminary experiments are discussed.     

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.     

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 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.     

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.     

Calcium signaling in plant cells in altered gravity.

Changes in the intracellular Ca2+ concentration in altered gravity (microgravity and clinostating) evidence that Ca2+ signaling can play a fundamental role in biological effects of microgravity. Calcium as a second messenger is known to play a crucial role in stimulus-response coupling for many plant cellular signaling pathways. Its messenger functions are realized by transient changes in the cytosolic ion concentration induced by a variety of internal and external stimuli such as light, hormones, temperature, anoxia, salinity, and gravity. Although the first data on the changes in the calcium balance in plant cells under the influence of altered gravity have appeared in 80th, a review highlighting the performed research and the possible significance of such Ca2+ changes in the structural and metabolic rearrangements of plant cells in altered gravity is still lacking. In this paper, an attempt was made to summarize the available experimental results and to consider some hypotheses in this field of research. It is proposed to distinguish between cell gravisensing and cell graviperception; the former is related to cell structure and metabolism stability in the gravitational field and their changes in microgravity (cells not specialized to gravity perception), the latter is related to active use of a gravitational stimulus by cells presumebly specialized to gravity perception for realization of normal space orientation, growth, and vital activity (gravitropism, gravitaxis) in plants. The main experimental data concerning both redistribution of free Ca2+ ions in plant cell organelles and the cell wall, and an increase in the intracellular Ca2+ concentration under the influence of altered gravity are presented. Based on the gravitational decompensation hypothesis, the consequence of events occurring in gravisensing cells not specialized to gravity perception under altered gravity are considered in the following order: changes in the cytoplasmic membrane surface tension --> alterations in the physicochemical properties of the membrane --> changes in membrane permeability, --> ion transport, membrane-bound enzyme activity, etc. --> metabolism rearrangements --> physiological responses. An analysis of data available on biological effects of altered gravity at the cellular level allows one to conclude that microgravity environment appears to affect cytoskeleton, carbohydrate and lipid metabolism, cell wall biogenesis via changes in enzyme activity and protein expression, with involvement of regulatory Ca2+ messenger system. Changes in Ca2+ influx/efflux and possible pathways of Ca2+ signaling in plant cell biochemical regulation in altered gravity are discussed.     

Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level.

The changes of [Ca2+]i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station "Salyut 6". These results: 1) indicate that observed Ca(2+)-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca2+ influx through membranes. In model presented, I propose that Ca(2+)-activated channels in plasma membrane in response to microgravity allow the movement of Ca2+ into the root cells, causing a rise in cytoplasmic free Ca2+ levels. The latter, in its turn, may induce the inhibition of a Ca2+ efflux by Ca(2+)-activated ATPases and through a Ca2+/H+ antiport. It is possible that increased cytosolic levels of Ca2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca2+]i. Plant cell can response to such a Ca2+ rise by an enhancement of membranous Ca(2+)-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca(2+)-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca2+ to plant cell.     

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.     

Gravisensing: ionic responses, cytoskeleton and amyloplast behavior.

In Zea mays L., changes in orientation of stems are perceived by the pulvinal tissue, which responds to the stimulus by differential growth resulting in upward bending of the stem. Gravity is perceived in the bundle sheath cells, which contain amyloplasts that sediment to the new cell base when a change in the gravity vector occurs. The mechanism by which the mechanical signal is transduced into a physiological response is so far unknown for any gravity perceiving tissue. It is hypothesized that this involves interactions of amyloplasts with the plasma membrane and/or ER via cytoskeletal elements. To gain further insights into this process we monitored amyloplast movements in response to gravistimulation. In a pharmacological approach we investigated how the dynamics of plastid sedimentation are affected by actin and microtubule (MT) disrupting drugs. Dark grown caulonemal filaments of the moss Physcomitrella patens respond to gravity vector changes with a reorientation of tip growth away from the gravity vector. MT distributions in tip cells were monitored over time and MTs were seen to accumulate preferentially on the lower flank of the tip 30 min after a 90 degree turn. Using a self-referencing Ca2+ selective ion probe, we found that growing caulonemal filaments exhibit a Ca2+ influx at the apical dome, similar to that reported previously for other tip growing cells. However, in gravistimulated Physcomitrella filaments the region of Ca2+ influx is not confined to the apex, but extends about 60 micrometers along the upper side of the filament. Our results indicate an asymmetry in the Ca2+ flux pattern between the upper and side of the filament suggesting differential activation of Ca2+ permeable channels at the plasma membrane.     

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.     

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).     

The role of calcium accumulation and the cytoskeleton in the perception and response of Coprinus cinereus to gravity.

The role of Ca2+ in the gravitropic perception and/or response mechanism of Coprinus cinereus was examined by treating stipes with inhibitors of Ca2+ transport and calmodulin. Inhibitors had no effect on gravity perception but significantly diminished gravitropism. It is concluded that, under the conditions tested, Ca2+ is not involved in gravity perception by Coprinus stipes, but does contribute to transduction of the gravitropic impulse. The results would be consistent with regulation of the gravitropic bending process requiring accumulation of Ca2+ within a membrane-bound compartment. Treatment of stipes with an actin inhibitor caused a significantly delayed response, a result not observed with the Ca2+ inhibitors. This suggests that cytoskeletal elements may be involved directly in perception of gravity by Coprinus stipes while Ca(2+)-mediated signal transduction may be involved in directing growth differentials.     

Calcium balance in pea root statocytes under both clinorotation and Ca2+ channel blockers' influence.

We have previously demonstrated that space flight and clinorotation conditions increase cytoplasmic Ca2+ level in pea root statocytes. A rise in [Ca2+]i may be a serious problem for plants in microgravity environment. It is hypothesized that involvement of Ca2+ channel blockers in the growth medium may rescue a plant from abundance of Ca2+ ions. Indeed, combination of clinorotation (2 rpm, 5 days) and any Ca2+ channel blocker (1 micromole D600 or nicardipine, 12 hr) causes decreasing the Ca2+ concentration in pea root statocytes in comparison with clinorotation alone. Redistribution of Ca(2+)-ATPase activities observed under clinorotation comes to normal after D600 application whereas following by nicardipine action the pattern of the cytochemical staining is intermediate between those in stationary control and under clinorotation. Our data support the hypothesis that Ca2+ channel blockers may act as protectors for plants against rise in [Ca2+]i. The role for Ca2+ channels in graviperception and in microgravity effects as well as ways for stabilization of Ca2+ balance in plant cells in space flights are discussed.     

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.     

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.     

Lithium-induced changes in gravicurvature, statocyte ultrastructure and calcium balance of pea roots.

Calcium signaling has been implicated in plant graviperception. In order to investigate the role of intracellular calcium in the process, I used lithium ions (LiCl), which suppress inositol 1,4,5-trisphosphate (IP3) cycling and signaling by inhibiting inositol-1-phosphatase. After 4 h of gravistimulation, no curvature was observed in 81% of the roots of 5-day Pisum sativum seedlings pretreated with 5 mM LiCl. Structural features of statocyte ultrastructure in these roots were the following: loss of a cellular polarity, appearance of amyloplast clusters, condensed mitochondria, local dilations in a perinuclear space, increases in a relative volume of vacuoles. The intensity of a cytochemical reaction (pyroantimonate staining which detected Ca2+ ions) was moderate: the Ca2+ pyroantimonate deposits were observed in all organelles. There were few granules of this precipitate in a hyaloplasm of the statocytes. Mitochondria and vacuoles were found to contain more granules of the precipitate compared with the controls. Additionally, Ca(2+)-ATPase activity in the statocytes of pea roots pretreated with LiCl was approximately the same as in control roots. Data obtained by using inhibitor of inositol signaling suggest that the observed effects of LiCl on root gravicurvature and ultrastructure of root statocytes were due to effects on Ca2+ homeostasis, particularly on IP3-mediated release of intracellular Ca2+ which can be inhibited by inositol depletion. The work demonstrates the key role played by second messengers (Ca2+ and IP3) in a gravity perception and response.     

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.     

Evaluation of gravity-dependent membrane potential shift in Paramecium.

It is still debated whether or not gravity can stimulate unicellular organisms. This question may be settled by revealing changes in the membrane potential in a manner depending on the gravitational forces imposed on the cell. We estimated the gravity-dependent membrane potential shift to be about 1 mV G⁻¹ for Paramecium showing gravikinesis at 1–5 G, on the basis of measurements of gravity-induced changes in active propulsion and those of propulsive velocity in solutions, in which the membrane potential has been measured electrophysiologically. The shift in membrane potential to this extent may occur from mechanoreceptive changes in K⁺ or Ca²⁺ conductance by about 1% and might be at the limit of electrophysiological measurement using membrane potential-sensitive dyes. Our measurements of propulsive velocity vs membrane potential also suggested that the reported propulsive force of Paramecium measured in a solution of graded densities with the aid of a video centrifuge microscope at 350 G was 11 times as large as that for −29 mV, i.e., the resting membrane potential at [K⁺]_o = 1 mM and [Ca²⁺]_o = 1 mM, and, by extrapolation, that Paramecium was hyperpolarized to −60 mV by gravity stimulation of 100- G equivalent, the value corrected by considering the reduction of density difference between the interior and exterior of the cell in the graded density solution. The estimated shift of the membrane potential from −29 mV to −60 mV by 100- G equivalent stimulation, i.e., 0.3 mV G⁻¹, could reach the magnitude entirely feasible to be measured more directly.