Vision Res. Vol. 32, No. 9, pp. 1583-1591, 1992 Printed in Great Britain. All rights rewved

Copyright

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0042~6989/92 $5.00 -I”om 1992 Pergamon Press Ltd

The Eyespot of Euglena gracilis: a Microspectrophotometric Study T. W. JAMES,* F. CRESCITELLI,

** E. R. LOEW,? W. N. McFARLANDS

Received 5 August 1991; in revisedform 4 March 199.2

The eyespots in cells of streptomycin-bleached strains and of dark-grown cultures of Euglena gracilis, were examined by means of fluorescence microscopy and microspectrophotometry. When viewed with light in the region of 380-500 run, the stigma appeared as a dark spot. Adjacent to this was a second spot, not seen with white light, but which was seen to fluoresce when excited with radiation at 370 + 20 nm. This fluorescence proved to be polarized in contrast to other fluorescing bodies in the cell. The absorption curves, obtained by microspectrophotometry of individual eyespots, were found to consist of two spectral maxima, an A-band in the blue and a B-band in the green. Unlike tbe A-band, the B-band provided evidence of o~gina~~ from an anisotropic structure. Relating these data to literature findings, we conclude that the B-band is the absorbance of a pigment in the quasi-crystalline paraflageliar body and the A-band perhaps a pigment in the orange-red stigma. The spectrum of the B-band does not appear to be that of a flavoprotein or of a free carotenoid but its resemblance to the spectrum of rhodopsin is significant in relation to published data for the Clhlamydontonuseyespot that suggests the presence of a ~~opsin-Ike pigment as the ~ot~~itive system responsible for phototaxis in this alga. Euglena

Eyespot

Rhodospin

Microspectrophotometry

*OBITUARY

INTRODUCTION

On 22 March 1992 Fred Crescitelli and his wife, Ezna, were killed in a tragic automobile incident. It was providential, that only last year an issue of this Journal (Vol. 3 1, No. 3) contained reports by Fred and many of his students and colleagues who had gathered in Sardinia the previous year to celebrate his approximately 60 years in science. Such events are usually staged posth~ously, or at a time when the person is in the twilight of activity. This was not the case for Fred. In the Preface to that issue, I spoke of Fred’s history, honours, accomplishments and energy. He was contin~ng his daily experiments at UCLA in much the same way as he had for the previous 45 years. In the last 5 years he was author or co-author of 15 papers concerned with such areas as the biophysical determinants of visual pigment &,,,,, the visual pigments of sharks and deep sea fish, and the two papers in this issue on non-visual photopigments. His recent review on visual pigments (Progress in Retinal Research, Vol. 11) attests to his clarity of thought and integrative abilities. How should Fred be remembered? Certainly his work will continue to be cited and his philosophy espoused by his students. But beyond the work is the kind, generous man with the twinkle in his eye and the whimsical ways that endeared him to so many. I will remember his sitting in front of his ~crosp~tropbotometer, telling jokes one minute and literally bouncing with excitement at the discovery of yet some other curiosity of gecko visual pigments. He will be missed.

The eyespots of euglenoid flagellates have been studied with the view of identifying the specific photosensitive structure involved in exciting the phototactic responses and also in identifying the nature of the relevant photopigment. The microanatomy, examined by light and electron microscopy, shows the eyespot to consist of the orange-red stigma, the quasi-c~stalline paraflagellar body (PFB), and the flagellar rod (Leedale, 1967; Buetow, 1968; Kivic & Vesk, 1972). In some of the literature (Wolken, 1961) the stigma is identified as the eyespot but here we include both the stigma and PFB under the designation eyespot. There is uncertainty as to whether the stigma or the PFB (or both) is (are) the structure (structures) housing the pigment responsible for initiating the photoactic responses of the cell and as to whether this pigment is a flavonoid or a carotenoid (Wolken, 1961, 1977; Leedale, 1967; Benedetti, Bianchini, Checcucci, Ferrara, Grassi & Percival, 1976; Diehn, 1969). Here we examine the eyespot of Euglena gracilis in an attempt to clarify these two questions.

ELLIS R.

*Department of Biology, University

of California, 405 Hilgard Avenue, Los Angeles, CA ~2~1~, U.S.A. ?Physiology Division of Biological Sciences and New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, U.S.A. SCatalina Marine Science Center, University of Southern California, LOEW P.O. Box 398, Avalon, CA 90704, U.S.A. 1583

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7‘. W. JAMES

The chloroplasts in green Eugiena complicate the recording of the eyespot spectrum so a streptomycinbleached Strain was made and used in our studies which employed fluorescence microscopy and microspectrophotometry. In addition, cultures kept in darkness for varying periods of time were used. These cultures were largely chlorophyll free, so served to supplement the data obtained with the streptomycin-bleached strain as well as acting as controls on possible effects of streptomycin. We also employed green cells of the Z-strain of Euglena gracilis. The primary aim of this investigation was to record the in situ absorption spectrum of the eyespot, to relate it to published findings of microscopy (Foster & Smyth, 1980), and to attempt to identify the photopigment.

MATERIALS

AND METHODS

Wild-type green Euglena cells were grown in a Euglena broth (Difco) and bleached for 3 days with streptomycin (200 pg/ml), after which they were subcultured every 3-4 days in fresh broth without streptomycin. This chlorophyll-free strain was used in the experiments to be described. The eyespot in living cells was first examined by light microscopy and photographs were taken at illuminations between 380 and 500 nm, a procedure that enhanced contrast. In addition, epi-illumination with light at short wavelength (370 + 20 nm) excited a fluorescence at a wavelength above 530 nm, and this emission was examined for possible polarization. The eyespot spectrum was obtained by means of microspectrophotometry, employing an instrument designed by Loew (1982). This microspectrophotometer (MSP) is employed to examine the optical absorption of structures of microscopic size. Unlike certain other instruments which use two beams, one outside the structure of interest, the other within this structure, our MSP uses a single beam, with this beam placed first off the eyespot, then on the eyespot, the resulting record being the difference between the two successive scans. The light source for the monochromator is a 12 V tungsten-iodine lamp powered by a current-regulated power supply. The optics are such that an image of the monochromator slit is projected on to the object plane of the microscope, thus providing the probe beam in focus on the eyespot. The width and length of the probe are adjustable to fit the varying size and shape of the eyespots. The smallest probe size that fails to produce a diffraction pattern against a dark background is a square with 3.0 pm sides. The probe image is produced using a substage condenser of N.A. 0.45. The N.A. of the objective lens is 0.85. For recordings of visual pigments these are the numerical apertures generally employed in microspectrophotometers (Harosi & Malerba, 1975). To accommodate the short focal length of the substage condenser a 0.17 mm cover slip enclosed in a metal frame was used to hold the algal cells which were lightly compressed by placing a second cover slip over the cells.

CI ~1.

To search and find a quiescent cell with appropriately placed eyespot the substage is fitted with a second light source with a filter that serves to restrict the field illumination to the I.R. region. Infrared images of the field are viewed on a monitor screen the source of these images being an infrared camera that receives the filtered second light. The image of the slit passes through a beam splitter at the level of the eyepiece and is projected on to a photomultiplier, the output of which integrates both the wavelength scan and the digitized signal (absorbance). The voltage of the photomultiplier is controlled with a separate power supply which is manually varied to a specific level in accord with the individual absorbance being recorded. The fidelity of the MSP was tested by recording the spectra of a didymium filter, a human red blood cell and the outer segments of a frog visual rod. In all cases the MSP recorded these known spectra within f 2 nm. For the experiments, cells were placed between two cover slips, lightly compressed, and then examined on the video monitor under infrared illumination. The finding of an immobile cell and its eyespot proved to involve a long and tedious search until it was found that with the 470 nm beam of the monochromator, the eyespot was readily seen as a single dense entity at the anterior pole close to the reservoir. To justify this procedure we had to show (which we did) that the eyespot was not bleached by this illumination. Once the eyespot was located, the field was returned to infrared illumination and the analysis was begun. The probe beam, varied in size in different experiments, the smallest being a square with 3.0hm sides. It was first placed in the medium outside the cell and a background spectral scan (reference scan) was made over the selected wavelength span. The data were stored in the computer, following which the probe was placed on the eyespot and a sample spectral scan was run. The difference spectrum of these two scans was called out, observed on the computer monitor, and then stored for later call-up and possible printing. Either the raw data curve or a computer smoothed curve was available for study and analysis. Some 900 recordings were made from which those with the lowest noise level, like those of Fig. 2, were accepted. Records were also obtained in which the reference scan was made with the probe inside the cell but off the eyespot.

RESULTS 1.

Microscopy

With the light microscope the eyespot appeared as a double entity in which a darker rod was seen with transmitted blue light which was distinguished from an adjacent fluorescent rod excited by light at 370 nm [Fig. l(A)]. On the basis of literature findings, cited later, we interpret these two rods to be the stigma (dark rod) and the PFB (fluorescent rod). This fluorescence could

EUGLENA EYESPOT

be alternately extinguished and brought out by rotating a Polaroid analyzer in the light path. This fluorescence was short-lived (3-4 min) and its duration, as well as its intensity, was such that its polarization orientation, somewhat variable from eyespot to eyespot, could not be determined with accuracy. The electric vector angle for maximum fluorescence, relative .to the rod axis, was 4.5 + 10”. The variability that was encountered was probably due to the random alignments of the cell, a variation that must also have been present in the orientation of the rod axis pictured in Fig. l(A). Here we suggest that the fluorescent rod is the PFB, comparable to the quasi-crystalline structure whose unit cell in the Z-strain of Euglena has the dimensions: a = 8.9 nm, b = 7.7 nm, c = 8.1 nm and B = 110” (Piccinni & Manni, 1978). If the rod long axis is parallel to the c-axis and rotated, it could also account for some of the variability in the angle that was observed. It is clear, in any case, that an anisotropic element is present in the eyespot region where, as will be shown, there is absorption with dichroic character. The fluorescent rod was readily distinguished from other fluorescent granules throughout the cell by rotating the analyzer. Unlike the rod, these structures, especially prominent in old cultures, did not change their fluorescent intensity with such rotation. Figure l(B) also includes a sketch of the anterior pole of E. grucilis showing the eyespot (stigma and PFB) in relation to the reservoir and to the two flagella (emergent and internal). Note that the eyespot is not within a chloroplast.

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(A)

(8)

2. Microspectrop~otometry On the monitor screen the eyespot appeared in variable form, often as two rods each about 3-4pm long and arrayed roughly in parallel, in V-shape, atop one another, or in various irregular forms. In young cultures they were usually the only visible dark bodies on a lighter background. The absorption spectrum recorded from the eyespot consisted of two well-defined bands, the A-band in the blue, the B-band in the green region of the spectrum. The spectral form varied from some in which the two bands were close together, to others in which a clear separation was present between these two absorptions (Fig. 2). The degree of separation varied with the size of the probing beam, increasing as this size was enlarged above 3.0pm. In addition, the maximal O.D. decreased as the probing beam was enlarged. This effect of probe size increase was probably due to dilution of absorption by the inclusion of cytoplasm outside the eyespot which does not have the eyespot pigment, an effect like the narrowing of visual pigment absorption curves with decrease in pigment concentration (Dartnall, 1957). Changes in light scattering with variations of probe beam size probably also had an effect on the density and separation of the two bands. This two-banded absorption spectrum was identified with the eyespot, for moving the probe from the eyespot by as little as 1.0 pm lowered the absorption and changed its form (Fig. 3). It was indeed a significant and

FIGURE 1. (A) A photomicrograph of the eyespot showing the dark rod (s) and the fluorescent rod (p) believed to be the stigma and PFB, respectively. The microscopy was carried out using a Zeiss Universal microscope modified for epi-iiiumination and polarization examination. Filtered tungsten light (380-500 nm) and filtered mercury-arc light (37Ok 20nm were employed. The calibration bar indicates 10pm. (B) A sketch of the anterior pole of E. gracilis showing the eyespot adjacent to the reservoir (R), with its stigma (S) and paraflagellar body (PFB) from which the emergent Ragellum (EF) extends. Also shown are the internal flagellum (IF), the paramyelin bodies (P) and vacuole (V). The Euglena eyespot is extrachloroplastic and this sketch should be compared with the sketch of the Chfamydomonas eyespot in the following paper. The bar indicates 1am.

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T. W. JAMES

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0 400

500

600

Wavelength

700

- nm

FIGURE 2. An eyespot absorption spectrum obtained with a square probe with 3.0 pm sides. The raw data as well as the computer-smoothed curve are shown. The lack of overlay between these two curves is due to the way the smoothing program is written. Each curve is normalized individually before they are displayed on the screen, so that the highest peak on the “noisy” curve is set to the same 100% level as the highest point on the smooth curve. These two highest points are usually at different levels so that a normalization error takes place that offsets the zero absorption regions differently for the two curves. The important point is that the form of the smoothed curve is not significantly different from the raw data curve. This figure illustrates the typical spectrum with the A/B height ratio positive, but records were also obtained with this ratio negative. The 100% optical density for the smoothed curve is 0.0796.

satisfying result to see the change or disappearance of both A- and B-bands when the probe was moved by as little as 1 ,um. Several attempts were made, when the two rods were seen as clearly defined separate elements, to record from each, using a probe beam 3.0 pm in size.

The same A-B absorption was found with each of the two rods although the A/B size ratio was not necessarily the same for each of the two recordings from the two rods. Scattering of light probably prevented us from the localization required for independent recording.

500 Wavelength

600 - nm

FIGURE 3. Curve (1) The A-B-spectrum with a 3.0 pm probe on the active region. Curve (2) Probe moved about 1.0 pm off the active region. Density increase below 400 nm, highly variable in different eyespots, was due to increased light scattering. The 100% 0.D.s are 0.0212 for curve (1) and 0.0255 for curve (2). Normalization of the two curves was made separately for the two, after which the normalized curves were printed together as shown.

EUGLENA

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EYESPOT

.0625

1

00

500

400

600

Wavelength

- nm

FIGURE 4. Spectra obtained first with the reference probe in the medium outside the cell [curve (l)] and then with the reference probe in the cytoplasm next to the same eyespot [curve (2)J. Probe size 3.0 pm sides. Curves are plotted with absolute optical densities.

Presently, we are unable to assign the A- or the B-band specifically to either one of the two rods seen on the monitor screen although a change in the polarization axis of the probe, to be described, will relate these bands to each of the two rods seen microscopically [Fig. l(A)]. We are confident of the eyespot origin of both A- and B-bands because when the reference scan was made with

450

the probe inside the cell, but off the eyespot, we obtained the same spectrum as with the probe outside the cell (Fig. 4). Apparently, the chloroplast-free cytoplasm contains no pigments concentrated enough to interfere with the dual eyespot spectrum. Considerable variation was encountered in the spectral maxima and the O.D. maxima of the A- and

500

Wavelength FIGURE 5. Cell anterior-posterior systematically in a ratio of 1.21. In the B-band 0.D.s

550

600

- nm

approximately horizontal and quiescent. Curve (1) A 3.0 pm probe with light polarized parallel axis. Curve (2) Probe light now polarized perpendicularly to this axis. The A-band did absolute height with this polarization change since the 0.D.s are 0.1262 [curve (I)] and 0.1040 terms of the variation of the A-band optical densities (Table 1) this is not a significant difference. changed from 0.0846 [curve (l)] to 0.0364 [curve (2)], a dichroic ratio of 2.32. The dichroic ratio B-band figures (Table 1) is 1.50 (see text).

to the cell not change [curve (2)], In contrast, of the mean

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I. W. JAMES

Spectral maximum A-band

(nm)

B-band

ct rrl.

O.D. maximum A-band

O.D. B-band

ratios

A,B

Polarization axis parallel wirh long axis of cell (axial recording) 414.3 + 7.4 495.6 & 1.6 0.1132 + 0.0408 0.0826 + 0.0343

I .37 i 0.27

Polarization axis perpendicular to long axis of cell 431.9 * 3.57 492.7 k 3.22 0.1179 k 0.0425

2.13 +0.31

B-bands. These variations involved both random variations in repeated placement recordings from the same eyespot and variations from eyespots in different cells. In the case of the same eyespot we found, in an experiment with 10 successive placements, a mean Aband maximum at 420.4 + 1.3 nm with mean O.D. of 0.092 + 0.006. The comparable figures for the B-band were 489.4 f 1.8 nm and 0.071 ) 0.005 (SD). This sequence of readings was made with the probe beam polarization oriented parallel to the long axis of the cell (axial). A sequence of 20 recordings from 20 dzjferent eyespots revealed a greater variability, the comparable figures for the A- and B-bands being, respectively, 414.3 + 7.4 nm, 0.113 f 0.041 O.D.; and 495.6 f 7.6 nm, 0.083 f 0.034 O.D. Of special significance were two selective effects of the B-band with a change in the plane of polarization of the probing beam (Fig. 5). One was the reduction in O.D. maximum of this band when the plane was shifted from the parallel to the perpendicular relative to the cell axis. This reduction revealed the anisotropic nature of the structure giving rise to the B-band. The second effect was the red shift of the A-band (Fig. 5). These two effects are summarized in Table 1 from an experiment with 20 different eyespots in cells grown in the dark for 70 days in a culture of 250 ml of Difco Euglena broth. The mean data and SDS listed in this table show the A-band red-shifted by some 18 nm with this polarization change while the B-band remained fixed, close to 494 nm. Simultaneously, the mean A-band maximum optical density changed insignificantly from 0.1132 to 0.1179 while that of the B-band went from 0.083 to 0.055, indicating a figure of 1.5 as the dichroic ratio for these mean data. A considerable dispersion of the figures for the individual dichroic ratios was found in the experiments as indicated in Table 1 but this is not surprising in view of the varied orientations of the eyespot that we encountered in the living cells, as seen on the monitor screen. Despite this variability, a selective and significant polarization reduction of the B-band is suggested by these results, indicating an anisotropic element as the source of the B-band. The A- and B-bands appear to be independent absorptions originating in 2 different pigment systems, for the height ratios of the A- and B-bands were not constant for the recordings from different eyespots as they would be expected to be if a single pigment with two spectral bands was the source. Typically, the A-band was the larger (Fig. 2) but occasionally the B-band had the greater optical density. In addition, the A-band did not

0.0552 + 0.0196

appear to arise from a structure with anisotropic properties for we never found a consistent change in the height of the A-band with a polarization change in the probe (Table l), as was the result with the B-band. A comparison of the B-band of E. gracilis with three rhodopsins from (a) the three-spined stickelback (Gasterosteus aculeatus), (b) the bat ray (A4yliohati.s calijiirnica) and (c) the frog (Ram pipiens) is seen in Fig. 6. In these comparisons the same MSP and the same conditions were employed in making the recordings from a single eyespot and a single visual rod for each of the three panels.

a

40 “\,; / \\\

,/ 20 c-----

i

o$. 400

450

03

” ” 400



I

,.;A

550

500 Wavelength

‘\

-.-F-

,,s,

,.,,,,,(..

660

(nm)

” I” 500 Wavelength

600

700

(nm)

FIGURE 6. The B-bands of E. gracilis (upper curves) compared to the spectra of rhodopsins (lower curves) of (a) the three-spined stickelback, (b) the bat ray and (c) the frog. Single retinal rods of these three vertebrates were recorded in the same manner as each of the three different eyespots in (a, b,c). Note that the same wavelength scale applies to (a, b) but a different scale is used in (c) to bring out the A-band, an absorption absent in rhodopsin.

EUGLENA EYESPOT

Because of the spectral similarity between the B-band and these rhodopsin rods we tested the stability of the eyespot response to various treatments known to effect vertebrate rhodopins. We discovered a system relatively inert to light, for repeated scans of an eyespot did not alter the form or magnitude of the spectrum and attempts to bleach the eyespot with light exposures up to 30 min, known by us to totally bleach rhodopsins, failed to alter the A-B spectrum. Light at both 500 and 420 nm was employed in these bleaching trials and neither the A- nor the B-band was altered. We also compared the eyespot spectra of Euglena kept in darkness for 2-3 hr before testing, with those from cells exposed continuously to room light. No modi~cations of the A-B spectrum was noted in such light-adapted cells. The effect of hydroxylamine in combining with retinal released in the bleaching of some rhodopsins is well known, so a possible action of this reagent on the eyespot was examined but no effect was noted. We employed this reagent, neutralized to pH 7.0 and at a concentration of 0.1 M. These are the conditions in which this reagent combines with retinal released by bleaching rhodopsin or in which it attacks certain visual pigments even without bleaching (Crescitelli, 1977). The failure of hydroxylamine to attack the pigment could have been a true failure or as a result of this reagent’s inability to penetrate the pellicle. Also, since the system did not bleach with light, the possible effect of added retinal in regeneration of photopi~ent could not be examined as it has been in the Chlamydomonas experiments (Foster, Saranak, Patel, Zarilli, Gkabe, Kline & Nakanishi, 1984). The A-E spectrum described above was recorded from only one spot in a cell and this was always located at the anterior pole adjacent to the reservoir and closely associated with the emergent flagellum when this could be seen on the monitor screen. We conclude that it was the eyespot from which our spectra were obtained. The results described above were obtained with chlorophyllfree cultures, but similar recordings were made with green cells of the Z-strain when the eyespot was clearly separated from chloroplasts.

DISCUSSZON Regarding the mo~hological structures giving rise to the A- and B-absorptions, we consider the B-spectrum to be due to a pigment in the paraflagellar body and this is based on the microscopically visible fluorescence dichroism in the rod {Fig_ l(A)] which agrees with the ciichroism of the B-spectrum (Fig. 5). It has already been shown that the paraflagellar body has a quasi-crystalline structure and its fluorescence was seen to be polarized (Benedetti & Checcucci, 1975), a polarization also observed by us as explained above. The location of the PFB adjacent to the flagellum and enclosed within its capsule, [Fig. l(B)] suggests that it may be the structure directly communicating with the flagellum in the phototransductions reaction. The anisotropic character of the PFB may

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have ecoiogical significance for it has been shown that Eugiena has a preferential response in its movements to the plane of linearly polarized light {Creutz & Diehn, 1976). Regarding the structure giving rise to the A-spectrum we can only speculate that it could be the stigma whose function is presently unknown. It could also serve as a light-detecting organelle, or act as a shading modulator for the PFB as has been suggested (Mast, 191 I), or perhaps may even serve to provide precursor substance to the “visual pigment” in the PFB in+the manner of the vertebrate pigment epithelial cell. In respect to the apparent relation of the A-spectrum to the stigma, there is the statement by Creutz and Diehn (1976) that the stigma is not dichroic and this is a property shared by the structure giving rise to the A-band (Fig. 5, Table I). Assignment of the A-band to the stigma agrees with the experimental dimensions, There can be no doubt that the probe beam explored the stigma as well as the paraflagellar body. The two rods seen on the video monitor were

The eyespot of Euglena gracilis: a microspectrophotometric study.

The eyespots in cells of streptomycin-bleached strains and of dark-grown cultures of Euglena gracilis, were examined by means of fluorescence microsco...
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