Physical Characterization of Gravitaxis in Euglena gracilis

J Plant Physiol. Vol. 155. pp. 338-343 (1999)

• • OUR.AL OF •

http://www.urbanfischer.de/journals/ jpp

Plani Pb,••

,I,.,

© 1999 URBAN & FISCHER

Physical Characterization of Gravitaxis in Euglena gracilis MICHAEL LEBERT, MARKUS PORST, PETER RrCHTER,

and

DONAT-PETER HADER*

Friedrich-Alexander-Universitat, Institut fur Botanik und Pharmazeutische Biologie, Staudtstr. 5, D-91058 Erlangen Received October 16, 1998 . Accepted February 2, 1999

Summary

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 densiry difference berween cell body and surrounding medium. In order to test the feasibiliry of the current model in terms of physical considerations the specific densiry of different Euglena gracilis cultures was determined. Depending on the culture conditions the specific densiry was in a range berween 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 berween force and gravitaxis kinetics was found. A comparison berween estimated activation energy of the proposed stretchsensitive ion channels and energy supplied by the displacement of the lower membrane by the sedimentation of the cell body revealed that a focussing, 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.

Key words: Euglena gracilis, activation energy, gravitaxis, integration period, specific density. Introduction

Euglena gracilis, a unicellular, photosynthetic flagellate orients itself in the water column by means of negative and positive phototaxes (orientation away or toward a light source, respectively, Hader, 1987) and negative gravitaxis (orientation away from the center of graviry, Hader, 1987). During its forward movement, the cell body rotates around its long axis at a frequency of 1- 2 Hz. Originally, some authors discussed gravitaxis as a pure physical phenomenon (Brinkmann, 1968). Later findings suggested the involvement of an active, physiological process in gravitaxis. These results include the inhibition of graviorientation by a short exposure to ultraviolet radiation (Gerber and Hader, 1995), positive gravitaxis in young cells and the change in sign of gravitaxis (positive to negative) by the addition of trace amounts of some heavy metals (Stallwitz and * Correspondence.

Hader, 1994) as well as the dose-response relationship of gravitaxis (Hader et al., 1995). Recently, considerable progress was made in the detailed understanding of the mechanism leading to graviorientation (Lebert and Hader, 1996; Lebert et al., 1997). The current model explains gravitaxis based on the finding of a specific densiry difference berween the cell body and the surrounding medium. The resulting sedimentation of the cell body applies a force to the lower membrane, which opens or closes (depending on the activation modus) mechano-sensitive ion channels in the membrane. The activation of ion channels modulates the membrane potential, which eventually triggers orientational movements of the trailing flagellum. Furthermore, the model postulates an asymmetrical distribution of stretch-activated ion channels in the membrane. In a symmetric distribution of ion channels every orientation of the cell would result in the activation of some stretch-activated ion channels. Further support for the hypothesis of an asymmetric distribution came from a detailed analysis of reorienta0176-1617/99/155/338 $ 12.00/0

Gravitaxis in Euglena II tional movements of Euglena cells during gravitactic stimulation (Hader et a!., 1997). While the specific density difference was identified as important for gravitaxis, specific density measurements and the relation to the precision of orientation are scarce. The present paper addresses the question of specific density and its relation to gravitaxis as well as a detailed analysis of the orientational movements leading to an orientation in the gravitational field.

339

possible gray levels each (Matrox digitizer card PIP-1024, Quebec, Canada) inserted in a PC type computer. Cell tracking was performed using a real time software described earlier (Hader, 1994).

Reorientation ofthe cells Both flight samples and ground samples were studied using long track analysis in order to follow the steering maneuvers during reorientation of the cells. For this purpose the cell tracks were recorded with a low magnification objective. In the ground experiments the cells were allowed to orient themselves gravitactically in a vertical glass cuvette (60 x60xO.2 mm) until they were aligned with the gravivector. Then the cuvette was turned by 90 or 1S0° around its short axis, and the cell tracks were followed during the subsequent reorientation period using frame-by-frame analysis. Tracks were recorded by marking the position of a cell evety second or every 0.5 second on an acetate foil covering the monitor screen. Marked positions were determined, and the obtained data were further analyzed with a spreadsheet program on an IBM-compatible computer. 0

Materials and Methods

Cell growth Euglena gracilis Z was obtained from the collection of algal cultures at the University of Gottingen (Schlosser, 1994) and used for all experiments. The cells were grown in a mineral medium, complex medium or tap water as described earlier (Starr, 1964; Checcucci et a!., 1976) in stationary cultures in 100-mL Erlenmeyer flasks at about 20°C under continuous light of about IS W m- 2 from mixed cool white and warm tone fluorescent lamps. Density determination Cell densities of the cultures were determined by counting in a Thoma chamber. Cultures were diluted by addition of the original medium or concentrated by centrifugation to a final cell density of approximately 3 to 7* 10 5 cells mL -I. Step gradients of Ficoll (Serva, Heidelberg, Germany) were formed in Ultra Clear tubes (14 X 95 mm, Beckman, Palo Alto, California, USA) using a Ficoll stock solution (20 % w/v) in distilled water. To determine the cell density range, step gradieFts. fro.m a density of 1.01 _~ mL -I to a density of 1.07 g mL- with Increases of 0.01 g mL were used. Every other step was colored by the addition of 0.01 % neutral red for better visibility. For the final determination a range from 1.04 g mL -I to 1.07 g mL -I with an increase in density of 0.005 g mL- 1 was used. Aliquots of 1 mL cell culture were loaded on top of the gradients. Gradients were centrifuged (Beckman LS-70M Ultracentrifuge, SW 40 swing bucket rotor) and, after centrifugation, the position of the cell band was recorded. Centrifugation was repeated at least S times to determine the final position of the cell band.

Space experiments The experiments were carried out in custom-made circular cuvettes (0.2 mm depth and 50 mm diameter) manufactured from stainless steel and glass viewing windows (Daimler-Benz Aerospace, Bremen, Germany). These cuvettes were inserted into the experimental module during late access (1 h before launch). This module consisted of a centrifuge that accommodated two cuvettes at different radii. During launch the cuvettes were rotated around their short axes at a frequency of 2 rpm in order to stabilize the position of the cells. Varying the rotational velocity of the centrifuge allowed to apply different centrifugal accelerations to the cells. In each cuvette the image of the swimming cells was recorded by a 2.5 X objective operating in dark field modus linked to a b/w CCD camera. Illumination was obtained from a 42 W halogen lamp, which irradiated the cells using a circular fiber optic array. The cuvette was oriented vertically in the rocket standing upright before launch. During the rocket flight the images of the moving cells were transmitted by a video transmitter module to the ground station. The transmitted video sequence was recorded on a S-VHS video recorder (Panasonic AC-7330) , and the video signal was digitized during playback at a spatial resolution of 512 x 512 pixels with 256

Results Earlier, the specific density of Euglena gracilis was determined to be 1.04gmL-1 (Hader et al., 1991). The determination was based on weight and volume of the cells in a 10-L culture. To determine the specific weight more accurately, isopygnic centrifugation in a Ficoll step gradient was used. Older cultures (10 days and older) gave one sharp band after one or rwo centrifugation steps (see Material and Methods) at a specific density of Ficoll. Dependinl? on the culture conditions a specific density of 1.046 gmL - (complex medium) to 1.056 g mL -I (mineral medium) was observed. Younger cultures gave rwo sharp bands: one corresponding to the specific density of the cells of the inoculum, the other approximately 0.005 g mL -I to 0.01 g mL -I lighter. Microscopic evaluation of the cells revealed that the cell size varied according to the culture conditions, too. Table 1 summarizes the results. The current model of gravitaxis (Lebert and Hader, 1996; Lebert et a!., 1997) predicts a force applied to the lower membrane, which activates stretch-sensitive ion channels as a result of the sedimenting cell body. This is assumed to be the primary event of gravisensing. The force applied on the lower membrane can be estimated by equation 1 (Bjorkman, 1992):

where F = Force [NJ, V = Volume [1], gn = acceleration [m s- 2J and Llp = specific density difference berween cell body and medium [kgL -IJ. Under terrestrial conditions the acceleration equals 9.81 m s-2 and is almost constant. Using volumes and specific densities given in Table 1 the resulting force on the lower membrane is in a range berween 0.57 pN and 1.13 pN. If the current model holds, a lower limit should exist, which does not allow to activate ion channels. In addition, the higher the force the faster the orientation should be. Cultures, shown in Table 1, were tested for their ability to orient in the gravitational field. No orientation was observed in the old tap water culture. All other cultures showed an orientation, but with different kinetics depending on the culture conditions. The

340

and

MICHAEL LEBERT, MARKUS PORST, PETER RICHTER,

DONAT-PETER HADER

0.3~--------------------------------------------------------~

·S= " -;" 0.2

.

i:.

=

.~

.:: ·c"= ;; ...... e

~

0.1

" '"=

~

O.U

+--------1I--....--------r----------,r---------; 0.4

0.8

0.6

1.0

1.2

Force [pNI Fig. 1: Kinetics of reorientation in dependence of the maximum force on the lower membrane. Only the linear part of the reorientation kinetics was used to determine the initial slope. For an example see Fig. 2. The estimation of the force is based on values given in Table 1 using equation I.

precision of orientation can be determined with a statistical test, the Raleigh test (Batschelet, 1981). The r-value, a number between 0 (no orientation) and 1 (optimal orientation) gives an indication of the degree of orientation. While the absolute precision of orientation was unchanged, the time dependence of the degree of orientation showed a clear relationship to the force applied by the cell body (Fig. 1). An estimation of the minimal force that is sufficient to cause oriented movement can be obtained by the extrapolation of the data points. This value is between 0.5 pN and 0.6 pN. Under terrestrial conditions the acceleration used in equation 1 is constant. In order to vary this factor space experiments must be performed. In a recent space experiment (TEXUS 35, sounding rocket program, DARA, Germany, which allows for up to 6 min Ilgn) cells were exposed to Ilgn-conditions for 110 s. Afterwards, cells were exposed to a stepwise

Table 1: Specific density and cell size in relation to culture conditions.

----------------------------------------

Culture condition

(age)

Specific density Length [lJm) [gmL- 1 j

Diameter [l-lIn]

Volume [LJ* Force [pNJ

Mineral medium (IOd).ligh[

1.049

55

84

2.04 10- 12

0.98

Mineral medium

1.053

55

84

2.0410- 12

1.06

Complex medium (42 d)

1.046

52

9.6

2.5 10

12

1.13

Mineral medium (GOd)

1.054

32

1.0810

12

0.57 0.87 0.82

(10 d), heavy

Top watt' (GO d)

1.053

50

1.6810- 12

Tap water (300 d)

1.054

46

1.55 10. 12

• Volume

Wd.\

determined as.mming that (he cell i.\ approximately a rotational ellipsoid.

increasing acceleration on a slow rotating centrifuge within the rocket. Figure 2 summarizes the results obtained. Even at the lowest acceleration applied (O.l2gn filled squares) the cells oriented with respect to the resulting vector. The time dependency of reorientation is compared to results obtained under 1gn conditions (Fig. 2, open squares). No difference in the kinetics can be observed. The results of the space experiment can be used to determine the minimal exposure time to gravity before a response can be observed in analogy to the gravitropic response of higher plants (Perbal and Driss-Ecole, 1994). The extrapolation of the percentage of reacting cells as a function of the applied stimulation dose (in g X min) to 50 % of upward moving cells (no reaction) gives a value of 3.4 s exposure time necessary to induce a reaction (Fig. 3). A comparable value was obtained when terrestrial experiments were analyzed the same way (5.7s ± 3.3s). To further analyze the gravitactic response additional experiments were performed. Cells were allowed to orient under 1 gn conditions in a rotatable cuvette, After full orientation was reached the cuvette was rotated 90" and the resulting reorientation of the cells was recorded and analyzed frame by frame. The coordinates were used to determine the angular deviation of cell tracks per second during reorientation, the results of which are shown in Fig. 4. Only cell tracks orienting clockwise were included. Even during reorientation the cells swam straight forward most of the time (maximum number of counts at zero degree deviation per second). Two of the recorded three maxima represent the deviation caused by the helical path of the cell during forward movement (+6" and _6"). One additional maximum can be observed at 10" per second .

341

Gravitaxis in Euglena II

100.------------------------------------------------------------.0.6

50+------,~--~~------.------.-------r------.-----_,------_,------+0

10

60

110

160

260

210 Time

310

360

410

460

lsi

Fig. 2: Kinetics of reorientation in a TEXUS 35 experiment (filled squares: percentage of upward moving cells; filled circles: acceleration applied. For details of the experiment see Materials and Methods). Open squares represent the result of a parallel ground control experiment with cells from the same culture. Every data point includes about 1000 analyzed tracks.

100.-------------------------------------------------------------------------------,

• ••• •••••

60

••

-1.244

(3f)

50

-1.5

-I

-0.5

0

0.5

Log quantity of stimulation [gn x min) Fig. 3: Determination of minimal exposure time. Data of the experiment shown in Fig. 2 were used. For details see Fig. 2.

1.5

342

MICHAEL LEBERT, MARKUS PORST, PETER RICHTER, and DONAT-PETER HADER 200~-------------------------------T-------------------------------'

180 160 140 120 .'!l

..

;:; 100 o.l

80 60 40 20

-40 -36 -32 -28 -24 -20 -16 -12

-8

-4

0

4

8

12

16

20

24

28

32

36

40

Deviation [0 5- 1) Fig.4: Change in movement direction per second. Cells were allowed to orient with respect to the gravity vector. Then cells were turned for 90', and reorientation was recorded. Following the experiment, cell orientation was calculated by determining cell position once per second. For experimental details see Materials and Methods.

Discussion Gravitaxis as a phenomenon has been known for more than 100 years. Until recently the underlying mechanisms were not known. The current model for gravitactic orientation of Euglena gracilis is based on the density difference between cell body and the surrounding medium. This difference was determined to be in the range 0.05 g mL -\ and was related to the culture conditions. Interestingly, in young cultures two distinct populations could be identified: One light, most likely of young cells and a heavier of older cells. A close relationship between the force that is applied to the lower membrane, and that depends on the size and the specific density of the cells, was found. In order to activate specific ion channels this force must lead to a displacement of the membrane. A rough estimate of the energy required to open an ion channel is based on the assumption that this energy must be at least equal to the thermal energy of 3*10- 21 J at room temperature. This energy can be supplied by a displacement of the membrane of 3 nm to 4 nm (force X distance = energy), a value also found for other systems (Howard et aI., 1988). This energy would be sufficient to open one channel. It seems unlikely that the opening of one channel could trigger a reorientational movement of the flagellum. In addition, gravitactic orientation can be observed at 0.12 gn with the same kinetics as at 1gn. In terms of energy only one eighth of the energy would be supplied. These conclusions and findings suggest a focussing, amplification and/or integration step in the signal transduction chain to utilize the low gravitational force as a guiding orientational vector. The analysis of reorientation tracks of Euglena gracilis

makes an initial integration period very likely. A period of 3-8 s is required before a first gravitactic reaction can be observed. A possible integration mechanism involves asymmetrically distributed stretch-sensitive ion channels. During the revolution of the cell only in one position an opening of the channels is triggered. The resulting change in the ion concentration is counteracted by the action of ion pumps with a longer time constant than that of the ion channels. When orientated horizontally this will lead to a stepwise buildup of the ion concentration as the cells rotate. When a proposed threshold concentration is reached reorientational movements statts. The proposed mechanism finally leads to an upward movement of the cell and allows, even at a low stimulus condition, to detect the up- or downward direction by stimulus integration. This mechanism could explain the observation that the reorientational kinetics at 1 gn is the same as at 0.12 gn. If the threshold criterion is fulfilled a reaction is triggered. As in many all-or-none reactions every reaction leads to distinct angular reorientation, which is independent of the stimulus amplitude. A change in direction of approximately 10' per cell rotation was observed. In rotation experiments cells were phototactically stimulated and rotated with a constant speed. In these experiments cells could reorient phototactically up to a rotational speed of 12' per second (Hader et aI., 1986). This value corresponds very well to the angle determined in the path analysis during reorientation. Interestingly, a change in direction could not be observed at every revolution. Nevertheless, a more specific analysis is necessary to reveal all details of the reorientational movements. The current experimental system does not allow to analyze the reorienta-

Gravitaxis in Euglena II

tional movements at a very high spatial and temporal resolution. This would be necessary to address questions regarding the angular dependent reaction frequency and position of a cell with respect to a reaction. Currently, a faster, computerbased system is being developed to allow to measure the reorientation at a higher spatial and temporal resolution.

Acknowledgements

This work was supported by DARA grant no. 50WB9406-ZA. We thank M. Dautz for skillful technical assistance and Harald Tahedl for critical discussions.

References BATSCHELET, E.: Circular Statistics in Biology. Academic Press, London, 1981. BJORKMAN, T.: Perception of gravity by plants. Adv. Space Res. 12, 195-201 (1992). BRINKMANN, K.: Keine Geotaxis bei Euglena. Z. Pflanzenphysio!. 59, 12-16 (1968). CHECCUCCI, A, G. COLOMBETTI, R. FERRARA, and F. LENCI: Action spectra for photoaccumulation of green and colorless Euglena: evidence for identification of receptor pigments. Photochem. Photobio!' 23, 51-54 (1976). GERBER, S. and D.-P' HADER: Effects of artificial UV-B and simulated solar radiation on the flagellate Euglena gracilis: physiological, spectroscopical and biochemical investigations. Acta Protozoo!' 34, 13-20 (1995).

343

HADER, D.-P., M. LEBERT, and M. R. DILENA: New evidence for the mechanism of phototactic orientation of Euglena gracilis. Curr. Microbio!. 14, 157-163 (1986). HADER, D.-P., M. PORST, H. TAHEDI, P. RICHTER, and M. LEBERT: Gravitactic orientation in the flagellate Euglena gracilis. Microgravity sci techno!. XII, 53-57 (1997). HADER, D.-P., E. REINICKE, K. VOGEL, and K. KREUTZBERG: Responses of the photosynthetic flagellate Euglena gracilis to hypergravity. Eur. Biophys. J. 20, 101-107 (1991). HADER, D.-P., A RosuM, J. SCHAFER, and R. HEMMERSBACH: Gravitaxis in the flagellate Euglena gracilis is controlled by an active gravireceptor. J. Plant Physio!. 146, 474-480 (1995). HADER, D.-P.: Polarotaxis, gravitaxis and vertical phototaxis in the green flagellate, Euglena gracilis. Arch. Microbio!. 147, 179-183 (1987). - Real-time tracking of microorganisms. Binary 6, 81-86 (1994). HOWARD, J., W M. ROBERTS, and A. J. HUDSPETH: Mechanoelectrical transduction by hair cells. Annu. Rev. Biophys. Chern. 17, 99-124 (1988). LEBERT, M. and D.-P' HADER: How Euglena tells up from down. Nature 379,590 (1996). LEBERT, M., P. RICHTER, and D.-P' HADER: Signal perception and transduction of gravitaxis in the flagellate Euglena gracilis. J. Plant Physio!. 150,685-690 (1997). PERBAL, G. and D. DRISS-EcOLE: Sensitivity to gravistimulus oflentil seedling roots grown in space during the IML-1 mission of Spacelab. Physio!' Plant. 90,313-318 (1994). SCHLOSSER, U. G.: SAG-Sammlung von Algenkulturen at the University of Gottingen. Catalogue of Strains 1994. Botanica Acta 107, 113-186 (1994). STALLWITZ, E. and D.-P. HADER: Effects of heavy metals on motility and gravitactic orientation of the flagellate, Euglena gracilis. Europ. J. Protisto!' 30,18-24 (1994). STARR, R. The culture collection of algae at Indiana University, Amer. J. Bot. 51, 1013-1044 (1964).

c.: