The involvement of a protein kinase in phototaxis and gravitaxis of <Emphasis Type="Italic">Euglena gracilis</Emphasis>

The unicellular flagellate Euglena gracilis shows positive phototaxis at low-light intensities (<10 W/m²) and a negative one at higher irradiances (>10 W/m²). Phototaxis is based on blue light-activated adenylyl cyclases, which produce cAMP upon irradiation. In the absence of light the cells swim upward in the water column (negative gravitaxis). The results of sounding rocket campaigns and of a large number of ground experiments led to the following model of signal perception and transduction in gravitaxis of E. gracilis: The body of the cell is heavier than the surrounding medium, sediments and thereby exerts a force onto the lower membrane. Upon deviation from a vertical swimming path mechano-sensitive ion channels are activated. Calcium is gated inwards which leads to an increase in the intracellular calcium concentration and causes a change of the membrane potential. After influx, calcium activates one of several calmodulins found in Euglena, which in turn activates an adenylyl cyclase (different from the one involved in phototaxis) to produce cAMP from ATP. One further element in the sensory transduction chain of both phototaxis and gravitaxis is a specific protein kinase A. We found five different protein kinases A in E. gracilis. The blockage of only one of these (PK.4, accession No. EU935859) by means of RNAi inhibited both phototaxis and gravitaxis, while inhibition of the other four affected neither phototaxis nor gravitaxis. It is assumed that cAMP directly activates this protein kinase A which may in turn phosphorylate a protein involved in the flagellar beating mechanism.     

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     

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 increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.     

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.     

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     

ORIENTATION OF SWIMMING FLAGELLATES BY SIMULTANEOUSLY ACTING EXTERNAL FACTORS1

The directionality of phototaxis combined with gravitaxis was investigated experimentally for populations of the swimming alga Euglena gracilis Klebs. Two irradiances were used: a “weak” irradiance to elicit positive phototaxis and a “strong” irradiance to elicit negative phototaxis. In addition, by changing the density of cells in the suspension, the number of collisions between cells was varied to determine the effects of these collisions on the distribution of swimming directions in both the absence and the presence of illumination. We found that positive phototaxis was associated with a broader distribution of swimming directions than was negative phototaxis. In the latter case, the effect of phototaxis dominated over that of gravitaxis. Experiments on another swimming alga, Chlamydomonas nivalis Wille, showed that collisions between cells degraded the directionality of gravitaxis.     

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.     

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.     

Gravitaxis in Chlamydomonas reinhardtii: characterization using video microscopy and computer analysis

We characterized the gravitactic behavior of Chlamydomonas reinhardtii, a unicellular green alga, using a computer-analysis system in order to study directional swimming. The effects of the calcium-channel inhibitors gadolinium and diltiazem on graviorientation and swimming speed were examined. In addition, we studied directional swimming in the ptx1 strain of C. reinhardtii, a flagellar dominance mutant. Results indicate that Chlamydomonas reorients for gravitactic swimming through a mechanism different from the calcium-mediated pathway believed to be involved in gravity transduction in higher plants. We suggest that calcium-mediated gravitaxis originated in an organism that was more evolutionarily advanced than Chlamydomonas.     

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.     

Sensitivity of various parameters in Euglena gracilis to short-term exposure to industrial wastewaters

Due to the presence of various potential pollutants industrial wastewaters pose considerable threats to natural waters and make it unfit for the aquatic biota. Analysis of wastewaters by chemical methods is a common practice; however, it does not reflect the toxic effects on living organism. Therefore, bioassessment is necessary for monitoring of wastewater quality. In the present study, the toxic effects of wastewater samples from different industries were evaluated using Euglena gracilis as a biotest organism. Various parameters of the freshwater flagellate E. gracilis like motility, swimming velocity, cell shape, gravitactic orientation (using the automatic biotest ECOTOX) and photosynthetic efficiency (using a pulse amplitude modulated fluorometer) were used as end points for toxicity assessment. In addition, the samples were analysed for some ecologically important physicochemical properties. With some exceptions, most of the physicochemical properties of the tested samples were within the acceptable range of national environmental quality standards for municipal and industrial effluents. However, all the water samples adversely affected different parameters in E. gracilis. This study led to the conclusion that different toxic substances present in wastewater, even at low concentrations, can be a possible threat to aquatic biota. The results of this study prove that ECOTOX is a sensitive, easy, and fast bioassay for monitoring of water and wastewater quality. Gravitactic orientation and cell compactness of E. gracilis were the most sensitive parameters to wastewater toxicity.     

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.     

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.     

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.     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

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.     

Evidence for gravitactic behaviour in benthic diatoms

Vertical migration by diatoms is a well-known phenomenon, occurring in intertidal and subtidal benthic biofilms. It is partially endogenously driven, as cell movements can be observed in the absence of external stimuli such as light, temperature or water cover. Although vertical migration of diatoms under constant conditions has often been attributed to geotactic orientation, this hypothesis has never been experimentally demonstrated. Our study tested the gravitactic nature of the vertical migratory behaviour of benthic diatoms in sedimentary biofilms, using an experimental setup designed to distinguish gravitaxis from surface-oriented cell movements. The hourly variation of surface diatom biomass during migratory cycles was compared in homogenized sediment samples kept facing upwards (surface-oriented and gravity stimuli coinciding; controls) and facing sideways or downwards (surface-oriented and gravity stimuli not coinciding). During the experiments, sediment samples were kept in complete darkness in custom-made, sealed measuring chambers designed to avoid any contact with atmospheric air and the formation of physico-chemical gradients near the surface. Microalgal biomass was monitored non-intrusively using PAM fluorometry, by measuring dark-level fluorescence, F_o. The results showed a clear effect of sample orientation in relation to the gravitational stimulus. In the controls, a biphasic pattern in surface biomass was observed, with the formation of a clear biomass peak (three- to six-fold increase) followed by a slower decrease. In contrast, in samples facing sideways or downwards, surface biomass also varied but to a much lesser extent (typically < two-fold). These results strongly suggest that, in the absence of light, upward vertical migration of benthic diatoms is mostly guided by negative gravitaxis, supporting the often hypothesized capacity of these cells to sense and use gravity to move vertically within the sediment.     

Extracellular calmodulin: A polypeptide signal in plants?

Traditionally, calmodulin (CaM) was thought to be a multi-functional receptor for intracellular Ca²⁺ signals. But in the last ten years, it was found that CaM also exists and acts extracellularly in animal and plant cells to regulate many important physiological functions. Laboratory studies by the authors showed that extracellular CaM in plant cells can stimulate the proliferation of suspension cultured cell and protoplast; regulate pollen germination and pollen tube elongation, and stimulate the light-independent gene expression of Rubisco small subunit (rbcS). Furthermore, we defined the trans-membrane and intracellular signal transduction pathways for extracellular CaM by using a pollen system. The components in this pathway include heterotrimeric G-protein, phospholipase C, IP₃, calcium signal and protein phosphorylation etc. Based on our findings, we suggest that extracellular CaM is a polypeptide signal in plants. This idea strongly argues against the traditional concept that there is no intercellular polypeptide signal in plants.