Gravitaxis of Bursaria truncatella: Electrophysiological and behavioural analyses of a large ciliate cell

Bursaria truncatella is a giant ciliate. Its volume of 3×10⁷ μm³ and a sedimentation rate of 923 μm s^?1 would induce the cell to rapidly sink to the bottom of a pond unless compensating mechanisms exist. The upward swimming behaviour of a cell population (negative gravitaxis) may be either a result of reorientations of the cells (graviorientation) and/or direction-dependent changes in propulsion rate (gravikinesis). The special statocyst hypothesis assumes a stimulation of mechanosensitive ion channels by forces of the cytoplasmic mass acting on the lower membrane. Here, we present basic electrophysiological data on B. truncatella. Investigation of the mechanosensitivity reveals a polar distribution of depolarising and hyperpolarising mechanosensitive channels at least on the dorsal membrane of the cell. Analysis of swimming behaviour demonstrates that Bursaria orients against the gravity vector (r_Oc=0.34) and performs a negative gravikinesis (?633 μm s^?1) compensating the sedimentation rate by 70%. Under hypergravity conditions gravitaxis in Bursaria is enhanced. Microgravity experiments indicate an incomplete relaxation of graviresponses during 4 s of weightlessness. Experimental data are in accordance with the special statocyst hypothesis of graviperception, as was demonstrated in other ciliates.     

Neutral gravitaxis of gliding Loxodes exposed to normal and raised gravity

Summary In the statocystoid-bearing, flat ciliate Loxodes, the peculiar steady locomotion on submersed substrates (called gliding) was investigated between 1 g and 5.4 g under controlled environmental conditions in a centrifuge microscope. Videorecordings of the movements of large cell populations were processed with an automated analysis procedure. At 1 g, possible sedimentation was fully compensated, and vertical shifts of the population were neutralized because upward and downward orientations of the cells occurred at equal proportions (neutral gravitaxis). With rising gravity the resultant velocity of upward-gliding cells remained unchanged, whereas the velocity of downward-gliding cells increased continuously. Long-term exposure to hypergravity did not generate detectable signs of adaptation. The bipolar orientation of Loxodes persisted even under fivefold normal gravity, but the axis of orientation rotated from the gravity axis in the counterclockwise direction. The data suggest that both gravikinesis and graviorientation of gliding Loxodes are instrumental in perfect neutralization of sedimentation at terrestrial conditions.     

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.     

Variable acceleration influences cyclic AMP levels in Paramecium biaurelia

Paramecium is used as a model system to analyse the gravity signal transduction pathway, that leads to gravitaxis and gravikinesis. In order to prove whether gravistimulation is coupled with second messenger production (cyclic AMP: hyperpolarization, cyclic GMP: depolarization) Paramecium was fixated under variable accelerations (1 x g, 9 x g and 10(-4) x g) on a centrifuge and during a sounding rocket flight (TEXUS 39). The analysis of cAMP and cGMP levels revealed an acceleration-dependent change in cAMP, while cGMP-levels showed gravity-independent variations. Hypergravity did not only induce an amplification of gravitaxis and gravikinesis, but also an increase in cAMP compared to the 1 x g-data. We conclude that the increased pressure of the cytoplasm on the lower membrane of upward swimming cells enhance the number of open K+(-)channels, thus causing hyperpolarization and change in cAMP concentration. Consequently, transition to microgravity declines gravitaxis and gravikinesis, and decreases cAMP concentration due to the loss of pressure on the cell membrane.     

超重状态出生大鼠转入正常重力状态后行为和Fos表达的改变

本工作观察了在超重（2G）环境中出生、生态4个月后返回正常（1G）环境的Long-Evans大鼠运动行为的改变和恢复,以及相关脑区Fos表达水平的变化,并与旋转刺激（重力变化的对照组）和去前庭传入大鼠相比较,结果表明：重力的变化导致实验大鼠表态和运行行为模式改变,伸肌张力增加,运动平衡、游泳定向和空中翻正的能力降低,对1G环境再适应的时程因行为的不同而异,其中游泳定向能力的恢复时间最长,长于1个月,旋转只对运动行为有短暂的影响。Fos表达被认为是揭示参与应答感觉刺激而功能活跃脑区的有用工具。结果显示,正常和毁迷路的对照大鼠的Fos呈低水平表达,减重刺激使上、下丘脑和围导水管灰质、中缝背核及孤束核的水平显著上调,相反,下橄榄、蓝斑和前庭核团的Fos水平没有明显改变,表明脑干对不同重力刺激存在不同的调控神经途径。     

A gravity-induced regulation of swimming speed in Euglena gracilis

We investigated the autotrophic flagellate Euglena gracilis for gravity-induced modulation of the speed of swimming as previously documented for larger protozoan cells. Methods of video-tracking of swimming and sedimenting cells under 1 g and hypergravity up to 2 g, and computer-assisted data processing were applied. The vertical and horizontal swimming speed, and sedimentation rates of immobilized cells, were found to be linear functions of acceleration. Accounting for sedimentation in the observed upward and downward movements of Euglena, the active component of speed (propulsion) rose in proportion to acceleration. No saturation of gravikinesis was seen within the g-range tested. Gravity-dependent augmentation of speed was maximal in upward swimmers and decreased continuously over horizontal to downward swimmers. Linear extrapolations of the data to zero-g conditions suggest the absence of a threshold of gravikinesis in Euglena. Energetic considerations indicate a high sensitivity of gravitransduction near the level of Brownian molecular motion. Accepted: 22 August 1999     

超重对前庭系统及相关体系结构和功能的影响

超重环境中怀孕、出生或生存的动物返回正常环境后,行为活跃、站立姿势夸张、空中翻正及游泳和在转动横梁上行走的平衡能力下降,超重增加伸肌的力、改变耳石的形态和分布、降低毛细胞和前神经元对重力的敏感性、相应增加和减少某些神经递质（如云甲肾上腺素和5－HT）的合成和分泌,脑干内不同的核团构成特异性应答超重和减重刺激的神经网络。     

Effects of gravity on proliferation and differentiation of adipose tissue-derived stem cells

Recent studies have demonstrated that culturing stem cells under altered gravity conditions modulates their proliferation and differentiation. In the current study we focused on osteogenesis. In an attempt to induce high proliferation rates and low differentiation of adipose tissue-derived stem cells (AT-MSCs), we exposed them to simulated microgravity (sim-microg) and hypergravity. We used the random positioning machine (RPM) to simulate microg and the medium sized centrifuge for acceleration research (MidiCAR) for hypergravity. AT-MSCs from different origin (human and goat) seeded in OptiCells were housed in the RPM and MidiCAR and compared with suitable controls cultured under static conditions (1 g). The experiments lasted 7 or 14 days. We report data on AT-MSCs proliferation as DNA content, and on the expression of specific osteogenic markers (cbfa-1, alkaline phosphatase activity and Van Kossa staining).     

Graviorientation in protists and plants.

Gravitaxis, gravikinesis, and gravitropism are different graviresponses found in protists and plants. The phenomena have been intensively studied under variable stimulations ranging from microgravity to hypergravity. A huge amount of information is now available, e.g. about the time course of these events, their adaptation capacity, thresholds, and interaction between gravity and other environmental stimuli. There is growing evidence that a pure physical mechanism can be excluded for orientation of protists in the gravity field. Similarly, a physiological signal transduction chain has been postulated in plants. Current investigations focus on the question whether gravity is perceived by intracellular gravireceptors (e.g. the Muller organelle of the ciliate Loxodes, barium sulfate vacuoles in Chara rhizoids or starch statoliths in higher plants) or whether the whole cell acts as a sedimenting body exerting pressure on the lower membrane. Behavioral studies in density adjusted media, effects of inhibitors of mechano-sensitive ion channels or manipulations of the proposed gravireceptor structures revealed that both mechanisms have been developed in protists and plants. The threshold values for graviresponses indicate that even 10% of the normal gravitational field can be detected, which demands a focusing and amplifying system such as the cytoskeleton and second messengers.     

Responses of the photosynthetic flagellate,Euglena gracilis,to hypergravity

Motility and orientation has been studied in the unicellular photosynthetic flagellate, Euglena gracilis, using real time image analysis capable of tracking up to 200 cells simultaneously in the slow rotating centrifuge microscope (NIZEMI) which allows one to observe the cells' swimming behavior during centrifugation accelerations between 1 g and 5 g. At 1 g the cells show a weak negative gravitaxis, which increases significantly at higher accelerations up to about 3 g. Though most cells were capable of swimming even against an acceleration of 4.5 g, the degree of gravitaxis decreased and some of the cells were passively moved downward by the acceleration force; this is true for most cells at 5 g. The velocity of cells swimming against 1 g is about 10% lower than that of cells swimming in other directions. The velocity decreases even more drastically in cells swimming against higher acceleration forces than those at 1 g. The degree of gravitactic orientation drastically decreases after short exposure to artificial UV radiation which indicates that gravitaxis may be due to an active physiological perception rather than a physical effect such as an asymmetry of the center of gravity within the cell. Offprint requests to: D.-P. Häder     

Graviperception in the flagellate Euglena gracilis during a shuttle space flight

During a recent space flight, gravitaxis of the unicellular photosynthetic flagellate, Euglena gracilis, was studied on board of the American shuttle Columbia. Accelerations were varied between 0 and 1.5 x g using a slow rotating centrifuge microscope (NIZEMI). The cells showed a sigmoidal response curve for the dependence of the precision of gravitaxis on acceleration which is indicative of the involvement of an active, physiological gravireceptor with a threshold at g-values < or = 0.16 x g and a saturation at g-values > or = 1 x g. No adaptation to microgravity was found during the prolonged space mission. After return the cells showed a normal gravitactic behavior at 1 x g. Since the cells are heavier than water, their swimming velocity is affected by sedimentation. The velocity distribution at different accelerations closely follows Stokes' law for sedimentation indicating that, in contrast to the ciliate Paramecium, E. gracilis, does not show any gravikinesis.     

How Much Gravity Is Needed to Establish the Perceptual Upright?

Might the gravity levels found on other planets and on the moon be sufficient to provide an adequate perception of upright for astronauts? Can the amount of gravity required be predicted from the physiological threshold for linear acceleration? The perception of upright is determined not only by gravity but also visual information when available and assumptions about the orientation of the body. Here, we used a human centrifuge to simulate gravity levels from zero to earth gravity along the long-axis of the body and measured observers'' perception of upright using the Oriented Character Recognition Test (OCHART) with and without visual cues arranged to indicate a direction of gravity that differed from the body''s long axis. This procedure allowed us to assess the relative contribution of the added gravity in determining the perceptual upright. Control experiments off the centrifuge allowed us to measure the relative contributions of normal gravity, vision, and body orientation for each participant. We found that the influence of 1 g in determining the perceptual upright did not depend on whether the acceleration was created by lying on the centrifuge or by normal gravity. The 50% threshold for centrifuge-simulated gravity''s ability to influence the perceptual upright was at around 0.15 g, close to the level of moon gravity but much higher than the threshold for detecting linear acceleration along the long axis of the body. This observation may partially explain the instability of moonwalkers but is good news for future missions to Mars.     

The effects of hypergravity on vestibular epithelia and behaviour of the rat

Researchers examined the vestibules of rats and hamsters raised in a centrifuge to determine the histology of the peripheral sensory system, vestibule-induced ocular reflexes, and vestibular-controlled behavior. Actin and tubulin structures were compared in animals raised in hypergravity and normal gravity. Air righting and swimming also were compared.     

Restoration of gravitropic sensitivity in starch-deficient mutants of Arabidopsis by hypergravity

Despite the extensive study of plant gravitropism, there have been few experiments which have utilized hypergravity as a tool to investigate gravisensitivity in flowering plants. Previous studies have shown that starch-deficient mutants of Arabidopsis are less sensitive to gravity compared to the wild-type (WT). In this report, the question addressed was whether hypergravity could restore the sensitivity of starch-deficient mutants of Arabidopsis. The strains examined include a WT, a starchless mutant and a reduced-starch mutant. Vertical orientation studies with dark-grown seedlings indicate that increased centrifugal acceleration improves orientation relative to the acceleration vector for all strains, even the WT. For starchless roots, growth of seedlings under constant 5 g acceleration was required to restore orientation to the level of the WT at 1 g. In contrast, approximately 10 g was required to restore the orientation of the starchless mutant hypocotyls to a WT level at 1 g. Examination of plastid position in root cap columella cells of the starchless mutant revealed that the restoration of gravitropic sensitivity was correlated with the sedimentation of plastids toward the distal cell wall. Even in WT plants, hypergravity caused greater sedimentation of plastids and improved gravitropic capability. Collectively, these experiments support the hypothesis of a statolith-based system of gravity perception in plants. As far as is known, this is the first report to use hypergravity to study the mechanisms of gravitropism in Arabidopsis.     

Impact of microgravity and hypergravity on free‐running circadian rhythm of the desert beetle Trigonoscelis gigas reitt.

Abstract

Free‐running circadian rhythms of locomotor activity of Tenebrionid beetles Trigonoscelis gigas Reitt., taken from the Turkmenian sand desert, were monitored in DD. The effects of microgravity (μG) ‐11 days in space flight aboard the Russian BION‐10 “COSMOS”; satellite, and of 2G hypergravity ‐ seven days on a centrifuge, were determined.

Two kinds of effects were found.

In stable 2‐peak records, there was a moderate decrease of τ in μG and an increase of τ in 2G, both of about 0.3 hr.

In unstable records, alterations of gravity caused drastic deviations of τ and ?. Remarkably, two peaks of the activity rhythm, which are supposed to be controlled by separate oscillators, responded to gravity transitions in different ways.

Gravity effects on the circadian system could be explained from a direct effect on the oscillator(s) itself or from a feed‐back by altered locomotion to the pacemaker.

Thus, for the first time the gravity dependence of a free‐running circadian rhythm was proved in a combination of real space flight and centrifuge experiments.     

Electric-field effects on gravikinesis in Paramecium

Equilibrated Paramecium caudatum cells exposed to a constant DC gradient reorient with their depolarized anterior ends toward the cathode (galvanotaxis). Voltage gradients were applied to cells swimming either horizontally or vertically. Their velocity and orientation were recorded and compared to unstimulated cells. The DC field increased the horizontal velocity (= reference) up to 175% (galvanokinesis). Swimming velocities saturated after 1 min and were unchanged during the following 4 min. The upward and downward swimming velocities of stimulated cells were below those of horizontal swimmers. The difference in vertical rates (determining gravikinesis) was independent of variations in absolute velocity. Normalization of vertical velocities to horizontal velocities (= 100%) separated DC-field dependent changes from gravity-induced changes in velocities. A weak voltage gradient (0.3 V/cm) was most effective in raising downward gravikinesis up to threefold (-202 m/s) above the unstimulated reference (-66 m/s) and to change sign of gravikinesis in upward swimmers (-43 m/s +33 m/s). We conclude that DC-field stimulation is equivalent to a depolarizing bias on gravikinetic responses of Paramecium. The stimulation does not directly interfere with mechanoreception, but modulates somatic Ca²⁺ entry to induce contraction of the cell soma. This presumably affects the gating of gravisensory transduction channels.     

Orientation of Paramecium under the conditions of weightlessness

A cell culture of Paramecium with a precise negative gravitaxis was exposed to 4 x l0(-6) g during a parabolic flight of a sounding rocket for 6 min. Computer image analysis revealed that without gravity stimulus the individual swimming paths remained straight. In addition, three reactions could be distinguished. For about 30 s, paramecia maintained the swimming direction they had before onset of low gravity. During the next 20 s, an approximate reversal of the swimming direction occurred. This period was followed by the expected random swimming pattern. Similar behavior was observed under the condition of simulated weightlessness on a fast-rotating clinostat. Control experiments on the ground under hyper-gravity on a low-speed centrifuge microscope and on a vibration test facility proved that the observed effects were caused exclusively by the reduction of gravity.     

Stimulation of DNA polymerase alpha by hypergravity generated by centrifugal acceleration.

Gravity alteration is known to influence cell proliferation. Here we tested the effects of hypergravity on the action of DNA polymerase alpha, one of the DNA replication enzymes in eukaryotes. Hypergravity was produced by horizontal centrifugal acceleration with a hand-made rotator. The reaction rate of DNA polymerase alpha in centrifuge tubes increased along with the acceleration up to 4g, when a plateau was reached. In contrast, no stimulation was observed with primase, DNA polymerase epsilon, and the E. coli DNA polymerase I Klenow fragment. Kinetic analysis of DNA polymerase alpha reactions revealed that, under high gravity conditions, the K(m) value for template DNA decreased while the V(max) stayed constant. In contrast, the centrifugal acceleration did not affect the K(m) values for deoxyribonucleoside triphosphates. These results suggest that the hypergravity enhances the activity of DNA polymerase alpha by increasing the affinity of the enzyme for template DNA. Such enhancement was more prominent with a low concentration of DNA polymerase alpha under low ionic conditions.     

Gravity-dependent changes in bioconvection of Tetrahymena and Chlamydomonas during parabolic flight: increases in wave number induced by pre- and post-parabola hypergravity

Bioconvection emerges in a dense suspension of swimming protists as a consequence of their negative-gravitactic upward migration and later settling as a blob of density greater than that of water. Thus, gravity is an important parameter governing bioconvective pattern formation. However, inconsistencies are found in previous studies dealing with the response of bioconvection patterns to increased gravity acceleration (hypergravity); the wave number of the patterns has been reported to decrease during the hypergravity phases of parabolic aircraft flight, while it increases in centrifugal hypergravity. In this paper, we reassess the responses of bioconvection to altered gravity during parabolic flight on the basis of vertical and horizontal observations of the patterns formed by Tetrahymena thermophila and Chlamydomonas reinhardtii. Spatiotemporal analyses of the horizontal patterns revealed an increase in the pattern wave number in both pre- and post-parabola hypergravity. Vertical pattern analysis was generally in line with the horizontal pattern analysis, and further revealed that hypergravity-induced changes preceded at the top layer of the suspensions while microgravity-induced changes appeared to occur from the bottom part of the settling blobs. The responses to altered gravity were rather different between the two sample species: T. thermophila tended to drastically modify its bioconvection patterns in response to changes in gravity level, while the patterns of C. reinhardtii responded to a much lesser extent. This difference can be attributed to the distinct physical and physiological properties of the individual organisms, suggesting a significant contribution of the gyrotactic property to the swimming behavior of some protists.     

Evidence of central and peripheral gravireception in the ciliate Loxodes striatus

Effects of the density of the external medium on gravireception in Loxodes striatus were investigated using Percoll solutions. With increasing density, the swimming rates changed from prevailing in the downward direction to prevailing in the upward direction. A cellular density of 1.036 g cm⁻³ was determined measuring direction and speed of sedimenting immobilized cells at different accelerations and medium densities. Viscosity increases by Percoll were measured and taken into account. At 30% air saturation Loxodes maintained a negative gravikinesis of approximately −27 μm s⁻¹ at external densities corresponding to cellular density (±0.02 g cm⁻³). Negative gravikinesis decreased gradually to −9 μm s⁻¹ with the density difference rising from 0.020 to 0.036 g cm⁻³ (=normal). The data indicate the existence of central gravireception, presumably by the Müller organelle, to generate in swimming Loxodes a constant value of gravikinesis and a bimodal gravitaxis. Peripheral gravireception occurs, in addition to central gravireception, when the transmembrane density difference exceeds 0.02 g cm⁻³. Peripheral gravireception can neutralize, in part, gravikinesis as raised by the central gravireceptor. We hypothesize that both central and peripheral gravireception of Loxodes guide vertical locomotion in gliding and swimming cells. Accepted: 26 May 1998