Physical characterization of gravitaxis in Euglena gracilis.

Gravitaxis in unicellular microorganisms like Euglena gracilis has been known for more than 100 years. The current model explains this phenomenon on the basis of a specific density difference between cell body and surrounding medium. In order to test the feasibility of the current model in terms of physical considerations the specific density of different Euglena gracilis cultures was determined. Depending on the culture conditions the specific density was in a range between 1.046 g mL-1 and 1.054 g mL-1. Size and gravitaxis measurements were performed in parallel, which allowed to relate the force applied to the lower membrane to the kinetic properties of gravitactic reorientation. A linear relationship between force and gravitaxis kinetics was found. A comparison between estimated activation energy of the proposed stretch-sensitive ion channels and energy supplied by the displacement of the lower membrane by the sedimentation of the cell body revealed that a focusing, an amplification and/or an integration period over time must be involved in the gravitactic signal transduction chain. Analysis of stimulus-response curves revealed an integration period of about 5 seconds before a gravitactic reorientation starts. The kinetics of gravitaxis at 1 x gn, and 0.12 x gn, was found to be similar. A hypothesis is presented that explains this finding on the basis of a combination of an integration period and an all-or-none reaction during gravitactic reorientation.     

Effects of heavy metals on motility and gravitactic orientation of the flagellate, Euglena gracilis

The effects of copper, mercury, cadmium and lead on the gravitactic orientation of the photosynthetic flagellate Euglena gracilis were investigated. The first two heavy metals reverse the direction of downward swimming (positive gravitaxis) in young cultures (up to 8 days) to an upward swimming (negative gravitaxis); cadmium produced a less pronounced effect. Higher concentrations of heavy metals decrease the precision of orientation as compared to the control due to frequent deviations of the cells from straight paths. Higher concentrations also decrease the swimming velocity of the populations. When the cells were growing in the presence of the heavy metal, copper was effective at > or = 50 microM, cadmium at > or = 3 microM and mercury at > or = 1 microM. Since lead formed insoluble precipitations with the acetate in the growth medium it was tested after the cells were transferred into Tris buffer. Under these conditions lead did not affect the direction of movement or the precision of orientation up to a concentration of 300 microM in the time up to 24 h after the addition of the heavy metal. However, high concentrations of lead strongly decreased the swimming speed of the cells, which was partially reversed with time.     

Gravitaxis in the flagellate Euglena gracilis--results from NiZeMi, clinostat and sounding rocket flights

Many motile microorganisms including flagellates such as the green Euglena gracilis move up and down within the water column and use a number of external clues for their orientation, the most important of which may be light and gravity. The cells use positive phototaxis and negative gravitaxis to move closer to the surface of the water column which for energetic reasons is vital for their survival. However, most phytoplankton organisms cannot tolerate the bright irradiance of unfiltered solar radiation at the surface which also bleaches the photosynthetic pigments, disables the photosynthetic apparatus and impairs phototaxis, gravitaxis and motility in Euglena. Thus, it is not surprising that at higher irradiances negative phototaxis operates antagonistically to the responses described above to guide the cells into deeper water where they are protected from excessive radiation. Phototaxis and gravitaxis are not independent from one another: in a vertically positioned cuvette negative gravitaxis can be "titrated" by light impinging from above and is compensated at about 30 W m-2. While the photoreceptor for phototaxis has been identified in Euglena gracilis biochemically and spectroscopically, the gravireceptor is not yet known. Young cultures of Euglena gracilis show a positive gravitaxis, the ecological signficance of which is not yet understood while older cultures show negative gravitaxis. One hypothesis concerning the nature of graviperception is based on a passive physical process such as an asymmetric distribution of the mass within the cell. However, the observation that short term UV irradiation decreases the precision of negative gravitaxis rather indicates the involvement of an active physiological gravireceptor. Furthermore, some heavy metal ions have been found to change the direction of movement from positive to negative gravitaxis in young cells.     

Physiological characterization of gravitaxis in Euglena gracilis

The motile, unicellular freshwater flagellate Euglena gracilis uses external stimuli, like gravity, light or oxygen pressure in order to orient itself in its natural habitat. In the darkness the cells normally show a negative gravitactic behavior, that means they swim upward in the water column, Many ground and space experiment revealed that gravitaxis is most likely based on active physiological mechanisms (involvement of calcium, cAMP, membrane potential and other parameters).     

Molecular characterization of a calmodulin involved in the signal transduction chain of gravitaxis in Euglena gracilis

The unicellular flagellate Euglena gracilis shows a negative gravitactic behavior. This is based on physiological mechanisms which in the past have been indirectly assessed. Meanwhile, it was possible to isolate genes involved in the signal transduction chain of gravitaxis. The DNA sequences of five calmodulins were found in Euglena, one of which was only known in its protein structure (CaM.1); the other four are new. The biosynthesis of the corresponding proteins of CaM.1–CaM.5 was inhibited by means of RNA interference to determine their involvement in the gravitactic signal transduction chain. RNAi of CaM.1 inhibits free swimming of the cells and pronounced cell-form aberrations. The division of cells was also hampered. After recovery from RNAi the cell showed precise negative gravitaxis again. Blockage of CaM.3 to CaM. 5 did not impair gravitaxis. In contrast, the blockage of CaM.2 has only a transient and not pronounced influence on motility and cell form, but leads to a total loss of gravitactic orientation for more than 30 days. This indicates that CaM.2 is an element in the signal transduction chain of gravitaxis in E. gracilis. The results are discussed with regard to the current working model of gravitaxis in E. gracilis.     

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.     

Gravitaxis in the flagellate Euglena gracilis is controlled by an active gravireceptor

Gravitactic orientation was investigated in the unicellular photosynthetic flagellate, Euglena gracilis, under different accelerations between 0 and 1.5 x g during a recent space flight on board the American shuttle Columbia. The threshold for gravitaxis was found at < or = 0.16 x g. Above the threshold the precision of orientation increased with acceleration in a sigmoidal fashion and reached saturation at about 0.32 x g, a behavior typical for physiological receptors. At accelerations above the saturation point the cells were closely aligned with the gravity vector (negative gravitaxis) and deviated more and more as the acceleration decreased. Obviously the gravireceptor responds to an error signal that elicits a course correction, again indicating the involvement of an active physiological gravireceptor. No adaptation of the cells to the conditions of weightlessness could be observed over the duration of the space mission (12 days). After landing, the cells showed a normal gravitactic behavior at 1 x g.     

Behavioral mutants of Euglena gracilis: functional and spectroscopic characterization

Three mutant strains of the phytoflagellate Euglena gracilis Z have been characterized in order to analyze the signal perception and signal transduction pathways involved in photo- and gravitaxis. Using the fluorescence of the chromophoric groups believed to be involved in photoperception (flavins and pterins) a method was developed for an in situ and in vivo detection of the paraxonemal body, the proposed location of the photoreceptor molecules. Two of the mutant strains, 1224-5/9f and 1224-5/1f, do not possess a stigma and also lack a paraxonemal body, as indicated by fluorescence measurements. The third strain, FB, has a small stigma, but only some cells contain a paraxonemal body. In contrast to the present hypothesis on photoorientation of Euglena, all strains were able to orient with respect to the light direction. However, the mutant strains did not show any orientation at low irradiances. At medium and high irradiances the strains 1224-5/9f and 1224-5/1f oriented perpendicular to the light direction (diaphototaxis) while cells of strains of FB showed partly negative phototaxis and partly diaphototaxis. Diaphototaxis was never observed in the wild type strain. Strains 1224-5/9f and 1224-5/1f showed normal graviresponses compared with the wild type. Astasia longa, a nonphtototactic relative of E. gracilis, as well as strain FB were both negative and positive gravitactic at all culture ages tested. This result confirmed the hypothesis that the paraxonemal body is not directly involved in graviperception.     

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis     

Effects of hypergravity on the photosynthetic flagellate, Euglena gracilis

Euglena gracilis, a unicellular, photosynthetic flagellate, orients itself by means of gravi- and phototaxis to reach and stay in regions optimal for survival and growth. An improved version of the slow rotating centrifuge microscope, NIZEMI, was used to test wild type and mutant strains for their responses to hypergravity. Wild type cells could actively move against the acceleration vector up to 8.5 gn and were centrifuged down at higher rates. Even at 10.5 gn, the highest value tested, cells were still negative gravitactically oriented as shown by video images. In contrast, all mutant strains as well as Astasia longa, a close relative of Euglena, could move against the acceleration vector under all conditions tested. With increasing accelerations the mean orientation of the populations shifted according to a vectorial addition of gravity and acceleration. The r-value, a statistical measure of the orientation of a population, increased with moderately increased acceleration rates and decreased at higher values. While wild type Euglena and two of the three mutant strains tested were exclusively negative gravitactically, in the third strain as well as in Astasia longa half of the population reacted negative gravitactically and the other half positive gravitactically. This variation of the wild type behavior was observed at moderate acceleration rates. At high accelerations the cells became exclusively positive gravitactic. The obtained results are discussed on the basis of the current model explaining gravitaxis.     

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis.     

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     

Circadian rhythm of gravitaxis in Euglena gracilis.

Euglena gracilis, a unicellular, photosynthetic flagellate is a model system for environmentally controlled behavioral reactions. One pronounced reaction is the orientation with respect to gravity. In synchronized cultures with no cell growth a distinct circadian rhythm of negative gravitactic orientation could be observed. The main maximum of sensitivity was detected 5 h after the beginning of the subjective day, the main minimum 5 h before the beginning of the subjective day. Transferring synchronized cultures to continuous light resulted in an almost instantaneous loss of rhythmicity. In contrast, after transfer to permanent darkness cells exhibited a circadian rhythm with a progressive shortening of the period for more than 5 days. These findings are in contrast to the circadian rhythm of phototaxis in Euglena, where a free-running period of 24 h was observed. Parallel measurements of negative gravitactic orientation, velocity, cell shape as well as cAMP concentration in synchronized cultures revealed a circadian rhythm of all reactions. The results are discussed with regard to the possible role of cell shape and cAMP in gravitactic orientation.     

High light exposure leads to a sign change in gravitaxis of the flagellate Euglena gracilis

In the absence of other external stimuli the motile, unicellular freshwater flagellate Euglena gracilis normally swims upward in the water column (negative gravitaxis). This behavior is most likely triggered by active physiological orientation mechanisms. Recently it was found that negative gravitaxis often inverts to a positive one upon high light exposure. This response is not mediated by the photoreceptor (the paraxonemal body - PAB), because PAB-free mutants do also show this response after high radiation. It is very likely that the phenomenon is triggered by reactive oxygen species, because in the absence of oxygen no gravitaxis sign change was observed. Also increased salinity inverses the sign of gravitaxis, leading to the assumption that environmental stressors induce the formation of reactive oxygen species, serving as signal molecules.     

Calcium is involved in the gravitactic orientation in colorless flagellates

The colorless flagellate Astasia longa shows a pronounced negative gravitaxis. The calcium fluorescence indicator Calcium Crimson was used to detect changes of the intracellular calcium concentration during gravitactical orientation. Astasia shows an increase of the fluorescence after a lag phase of about 10 s, a maximum after about 30 s and a decrease to the basic level within 60 s during gravitactic reorientation. The observed change in fluorescence corresponds to an almost doubling of the initial free calcium concentration. The influence of inhibitors, known to impair gravitaxis, on the calcium concentration of Astasia longa was tested. Addition of caffeine, an inhibitor of phosphodiesterase, increases, while addition of gadolinium, an inhibitor of mechanosensitive ion channels decreases the fluorescence signal. While gravitactic stimulation of caffeine-treated cells resulted in a kinetics of fluorescence intensity changes comparable to control cells the addition of gadolinium inhibited any calcium concentration change. Dynamic fluorescence imaging was used during a sounding rocket experiment (MAXUS 3 campaign). Different accelerations interrupted by microgravity intervals were applied to Astasia cells. The cells show an increase in the calcium signal upon acceleration and a decrease during the microgravity state. The results strongly reemphasize the working model of gravitaxis which is based on the activation of mechano-sensitive ion channels as one of the primary events in signal perception.     

Indications for acceleration-dependent changes of membrane potential in the flagellate Euglena gracilis

Summary. The effects of the calcium sequester EGTA on gravitactic orientation and membrane potential changes in the unicellular flagellate Euglena gracilis were investigated during a recent parabolic-flight experiment aboard of an Airbus A300. In the course of a flight parabola, an acceleration profile is achieved which yields subsequently about 20 s of hypergravity (1.8 g _n), about 20 s of microgravity, and another 20 s of hypergravity phases. The movement behavior of the cells was investigated with real-time, computer-based image analysis. Membrane potential changes were detected with a newly developed photometer which measures absorption changes of the membrane potential-sensitive probe oxonol VI. To test whether the data obtained by the oxonol device were reliable, the signal of non-oxonol-labelled cells was recorded. In these samples, no absorption shift was detected. Changes of the oxonol VI signals indicate that the cells depolarize during acceleration (very obvious in the step from microgravity to hypergravity) and slightly hyperpolarize in microgravity, which can possibly be explained with the action of Ca-ATPases. These signals (mainly the depolarization) were significantly suppressed in the presence of EGTA (5 mM). Gravitaxis in parallel was also inhibited after addition of EGTA. Initially, negative gravitaxis was inverted into a positive one. Later, gravitaxis was almost undetectable. Correspondence and reprints: Department of Plant Ecophysiology, University of Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Federal Republic of Germany.     

Motility and gravitactic orientation of the flagellate,Euglena gracilis,impaired by artificial and solar UV-B radiation

The effects of ultraviolet radiation on the gravitactic orientation of the freshwater flagellate,Euglena gracilis, were determined by a real time image analysis system. Both artificial UV radiation and solar radiation in a temperature-controlled growth chamber were employed. Histograms of gravitaxis showed that the degree of orientation decreased with increasing exposure time; this can be quantified using the Rayleigh test and upper quadrant summation. The effects of artificial UV radiation on the orientation are considerably stronger than those of solar radiation, probably because the radiation source emits higher fluence rates below 300 nm than found in solar radiation. The effects of monochromatic ultraviolet radiation on motility have been determined, and an action spectrum has been calculated.     

Physiological parameters of gravitaxis in the flagellate Euglena gracilis obtained during a parabolic flight campaign

The unicellular freshwater flagellate Euglena gracilis and its close relative Astasia longa show a pronounced negative gravitaxis. Previous experiments revealed that gravitaxis is most likely mediated by an active physiological mechanism in which changes of the internal calcium concentration and the membrane potential play an important role. In a recent parabolic flight experiment on board an aircraft (ESA 29th parabolic flight campaign), changes of graviorientation, membrane potential and the cytosolic calcium concentration upon changes of the acceleration (between 1 x g(n), 1.8 x g(n), microgravity) were monitored by image analysis and photometric methods using Oxonol VI (membrane potential) and Calcium Crimson (cytosolic calcium concentration). The parabolic flight maneuvers performed by the aircraft resulted in transient phases of 1.8 x g(n) (about 20 s), microgravity (about 22 s) followed by 1.8 x g(n) (about 20 s). A transient increase in the intracellular calcium concentration was detected from lower to higher accelerations (1 x g(n) to 1.8 x g(n) or microgravity to 1.8 x g(n)). Oxonol VI-labeled cells showed a signal, which indicates a depolarization during the transition from 1 x g(n) to 1.8 x g(n), a weak repolarization in microgravity followed by a rapid repolarization in the subsequent 1 x g(n) phase. The results show good coincidence with observations of recent terrestrial and space experiments.     

Gravitaxis in Chlamydomonas reinhardtii studied with novel mutants

Many free-swimming unicellular organisms show negative gravitaxis, i.e. tend to swim upward, although their specific densities are higher than the medium density. To obtain clues to the mechanism of this behavior, we examined how a mutation in motility or behavior affects the gravitaxis in Chlamydomonas. A phototaxis mutant, ptx3, deficient in membrane excitability showed weakened gravitaxis, whereas another phototaxis mutant, ptx1, deficient in regulation of flagellar dominance displayed normal gravitaxis. Two mutants that swim backwards only, mbo1 and mbo2, did not show any clear gravitaxis. We also isolated two novel mutants deficient in gravitaxis, gtx1 and gtx2. These mutants displayed normal motility and physical characteristics of cell body as assessed by the behavior of anesthetized cells. However, these cells were found to have defects in physiological responses involving membrane excitation. These observations are consistent with the idea that the gravitaxis in Chlamydomonas involves a physiological signal transduction system, which is at least partially independent of the system used for phototaxis.     

Phototaxis, gravitaxis and vertical migrations in the marine dinoflagellate Prorocentrum micans

Abstract The phototactic orientation of the marine dinoflagellate Prorocentrum micans was studied at three different ages and at several light intensities. High irradiances caused the cells to show negative phototaxis and low irradiances caused positive phototaxis. The precision of negative phototaxis reached a maximum in the early afternoon, while the precision of positive phototaxis was found to peak in the morning and at night. The cells also showed a pronounced negative gravitactic orientation, which had a maximum in precision in the early afternoon. The degree of gravitaxis was found to be constant over time when the cells were confined to a closed cuvette for up to 9 h. As a consequence of the orientation strategies, populations of Prorocentrum micans showed daily vertical migrations in a 3-m Plexiglas column. They accumulated in the top layers in the afternoon and were almost randomly distributed during the rest of the day.