In vivo photocycle of the Euglena gracilis photoreceptor.

Abstract

We present the light-induced photocycle of the paraflagellar swelling of Euglena gracilis. The kinetics of this process was reconstructed by sampling its fluorescence emission and switching the excitation light from 365 nm to 436 nm. Stable intermediates in the photocycle were manifested. The measured millisecond resolution kinetics best fits a Michaelis-Menten equation. The data provide strong evidence that the paraflagellar swelling, a three-dimensional natural crystal of a light-detecting protein, is the true Euglena photoreceptor.

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Biophysical Journal
Volume 72
February 1997545-553
In Vivo Photocycle of the Euglena gracilis Photoreceptor
Laura Barsanti,* Vincenzo Passarelli,* Patricia L. Walne,# and Paolo Gualtieri*
*Istituto di Biofisica, CNR, 1-56127 Pisa, Italy, and #Department of Botany, University of Tennessee, Knoxville, Tennessee 37996 USA
ABSTRACT We present the light-induced photocycle of the paraflagellar swelling of Euglena gracilis. The kinetics of this
process was reconstructed by sampling its fluorescence emission and switching the excitation light from 365 nm to 436 nm.
Stable intermediates in the photocycle were manifested. The measured millisecond resolution kinetics best fits a Michaelis-
Menten equation. The data provide strong evidence that the
light-detecting protein, is the true Euglena photoreceptor.
paraflagellar swelling, a three-dimensional natural crystal of a
INTRODUCTION
Since the turn of the century the photosynthetic and photo-
sensitive flagellate Euglena has provided an intriguing sub-
ject for photobiological studies. This flagellate dwells in
natural shallow ponds, and uses sunlight as source of energy
and information. Its chloroplasts are the energy-supplying
devices, whereas a simple but sophisticated system is used
as a light detector. Two flagella are inserted in a subapical
invagination of the cell termed the reservoir. The stigma,
composed of red-orange pigment granules, lies in the adja-
cent cytoplasm. Only one flagellum emerges from the cell
and consists of an axoneme, a paraxial rod running parallel
to
(Rosati et al., 1991). In fact the swelling, a three-dimen-
sional natural crystal whose dimensions are about 1 ,um X
0.7 ,um X 0.7 ,um, can be considered the first ciliary
photoreceptor (Walne and Gualtieri, 1994). According to
Eakin's theory (1968, 1972) the photoreceptor ciliary line of
evolution, which had its climax in the elaborate and remark-
ably complex vertebrate photoreceptor, originated from this
organelle.
As the cell rotates while swimming, the stigma comes
between the light source and the paraflagellar swelling, thus
modulating the light that reaches
steering of the locomotory flagellum (Jennings, 1906). This
configuration of stigma, swelling, and flagellum can be
considered a simple but complete visual system. The fre-
quency of the rotation is 2 Hz (Ascoli et al., 1978).
The three-dimensional paraflagellar swelling, a proteic
crystal (Gualtieri, 1993), can be interpreted as a 3D crystal
of type I (Michel, 1990), i.e., a stack of 2D crystal arrays
characterized by in-plane hydrophobic interactions and held
together by hydrophilic interactions. On the basis of optical
diffraction studies conducted on thin sections of the Eu-
glena paraflagellar swelling, Piccinni and Mammi (1978)
it, and a swelling (paraflagellar body) near its base
it, and regulating the
Receivedforpublication 29 July 1996 and infinalform 13 November 1996.
Address reprint requests to Dr. Paolo Gualtieri, CNR Istituto di Biofisica,
Via San Lorenzo, 26, 1-56127 Pisa, Italy. Tel.: +39-50-513111; Fax:
+39-50-553501; E-mail: mbxgualtieri@mail.cnuce.cnr.it.
1997 by the Biophysical Society
0006-3495/97/02/545/09
$2.00
(
measured its crystalline monoclinic cell unit, the dimen-
sions of which are a = 8.9 nm, b = 7.7 nm, c = 8.3 nm,
,B
1100.
Other examples of naturally occurring crystalline light-
detecting organelles are the 2D arrays of bacteriorhodopsin
in the plasma membrane of Halobacterium halobium; the
2D arrays of large membrane particles forming the reaction
centers of photosystem II in the photosynthetic membranes
of green plants; and the photosynthetic membranes of Rho-
dopseudomonas viridis and related purple bacteria (Kuhl-
brandt, 1992). Among these, the paraflagellar swelling of
Euglena acquires a special meaning because it is the only
crystal of a photodetecting protein consisting of about 100
layers (Gualtieri, 1993). Recent experimental results have
suggested that it uses a rhodopsin-based detecting system.
In 1989 Gualtieri and co-workers (Gualtieri et al., 1989)
and subsequently in 1992 Crescitelli and co-workers (James
et al., 1992) measured the absorption spectrum of a single
Euglena paraflagellar swelling. Because of the great simi-
larity between these spectra and the absorption curve of the
rhodopsin a-band centered at 500 nm, both research groups
suggested a pigment such as the rhodopsin-like protein in
the photosystem of Euglena. Successive experiments on
inhibition of the swelling formation (Barsanti et al., 1992)
and the extraction of retinal from intact and demembranated
cells (Gualtieri et al., 1992, and successive developments;
Barsanti et al., 1993) have provided data to further support
the rhodopsin-like protein hypothesis.
However, the possible photoreceptive roles of the cyto-
plasmic stigma and the paraflagellar swelling of E. gracilis
are still under debate, because of conflicting interpretations
of the results produced so far by the different research
groups working on this microorganism. In addition to rho-
dopsin, flavins and pterins have been suggested as photo-
receptive pigments in Euglena (Schmidt et
Brodhun and Hader, 1990), but their presence has been
detected only in isolated flagella and not in the swelling
(Galland et al., 1990; Sineshchekov et al., 1994). Recently,
Neumann and Hertel (1994) purified a riboflavin-binding
protein from isolated flagella of Euglena gracilis and doc-
umented the presence of these binding proteins in the entire
flagellar membrane and not in the paraflagellar swelling.
al.,
1990;
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Volume 72
February 1997
As a rule, prokaryotic and eukaryotic light-sensory pro-
teins are capable of a cyclic photoregeneration, with iden-
tified intermediates. Selected examples of these proteins are
the Halobacterium sensory rhodopsins (Marwan and Oes-
terhelt, 1990; Bogomolni and Spudich, 1991); the squid
rhodopsin (Hubbard and St. George, 1958); the fly rhodop-
sin (Hamdorf and Schwemer, 1975); and the human rho-
dopsin as well, which has the possibility of photoreversal,
but is commonly regenerated from separated components
(Knowles and Dartnall, 1977).
In the case of Euglena, to positively identify the true
photoreceptor, it is necessary to verify which endocellular
compartment undergoes structural reversible changes upon
illumination, to localize the photochromic proteins. Assum-
ing the paraflagellar swelling to be a light-sensing device, it
should show this characteristic, i.e., a cyclic photoreaction.
Because of the presence of a clearly detectable emission
in the Euglena paraflagellar swelling, we decided to inves-
tigate its photobehavior by analyzing changes in the in vivo
fluorescence. Although it is not possible to measure the
Amax of the new light-induced intermediates, these changes
provide important information on the series of structural
changes that Euglena photoprotein undergoes in response to
light.
MATERIALS AND METHODS
Cultures
Cultures ofEuglena gracilis strain Z cells (Sammlung von Algen Kulturen,
Gottingen, 1224-5/25) were grown axenically in autotrophic Cramer-
Myers medium (0.025 M) in sodium acetate (pH 6.8) (Cramer and Myers,
1952), under constant temperature (24°C) and continuous illumination
(500 lux).
Demembranation
The demembranating solution was prepared as follows. Triton X-100 was
dissolved in a HEPES-buffered solution (100 mM HEPES-KOH, pH 7.00;
20mM piperazine-N,N'-bis(2-ethanesulfonic acid); 10mM EDTA; 50mM
sucrose;
concentration of 4% v/v, filtered, and added to the cells (4:1), which were
previously suspended in 100 mM HEPES buffer (pH 7.00). Cells were
incubated overnight at room temperature and then washed twice with
HEPES buffer to remove the extracted chlorophyll.
1 mM dithiothreitol; 7.5% v/v glycerol) to a final detergent
Hardware
The hardware platform consists of a Zeiss Axioplan fluorescence micro-
scope (Zeiss, Oberkoehen, Germany) equipped with an epifluorescence
system, a IOOX (N.A. 1.3) and a 20X (N.A. 0.5) planapochromatic
objective, a 100-W mercury lamp, and a monochrome CCD camera (NXA
1011/01; CCIR Standard, Philips, Netherlands), which ensures geometric
accuracy, stability, linearity, and sensitivity. The video signal is used as the
input for the image system board (FG1OOAT; Image Technology), which is
plugged into the bus of a personal computer (IBM). The board consists of
a frame memory (1024 X 1024 x 12 bit), a multiplexer, a 4096 X 12 bit
feedback input look-up table (LUT), and an A/D converter. The multi-
plexer has 24 input bits: 12 from frame memory, 8 from the A/D converter,
and 4 from the LUT control register. The A/D converter samplestheanalog
video signal of the TV camera at 12.5 MHz. According to the CCIR
standard the size of the digitized image is 640 x 512 x 8 bits, with equal
vertical and horizontal spacing. A black and white monitor (BM 7542;
Philips, Netherlands) is used to display the signal output of the TV camera,
and an RGB high-persistence monitor (C-3479; Mitsubishi, Tokyo, Japan)
is used to display the digital image.
Fluorescence microscopy
Fluorescence images from Euglena cells were acquired with the following
filter combinations: a UV-blue set (8-nm band-pass excitation filter, 365
nm; chromatic beam splitter, 395 nm; barrier filter, 397 nm; 800 tLW/cm2)
and a blue-violet set (8-nm band-pass excitation filter, 436 nm; chromatic
beam splitter, 460 nm; barrier filter, 470 nm; 1100 ,uW/cm2). Because of
the spectral distribution of the high-pressure mercury lamp, these two
excitation wavelengths are the only two available for our measurements,
because any other excitation wavelength has a fluence rate lower than 50
,uW/cm2, and the induced photomodifications, if any, are below the detec-
tion limits of our visual and acquisition system. In only one experiment a
50-W high-pressure xenon lamp was used in combination with a wide-band
blue-violet set (400-440-nm wide-band excitation filter; chromatic beam
splitter, 460 nm; barrier filter, 470 nm; 800 tLW/cm2). Irradiances were
measured with a hand-held optical power meter (model 840; Newport). In
this particular case UV absorption microspectrophotometry proved to be
less useful than fluorescence microspectrophotometry for both the very
sophisticated instrumental set-up necessary for such a measurement and the
intrinsic fluorescence characteristics (Gualtieri, 1991).
Software
Fluorescence images were acquired and digitized every 40 ms. Each
digitized image, consisting of 320 X 200 pixels, represents the apical part
of the cell, where the paraflagellar swelling is located. Fifteen images for
each excitation wavelength were stored in the frame memory. Because of
our hardware facilities, the operations of panning, scrolling, and storing
were performed during the frame acquisition (Gualtieri and Coltelli, 1991).
Digital image techniques such as segmentation and labelization procedures
were applied to each fluorescence image to automatically select and
measure the integrated emission of the Euglena paraflagellar swelling
(Coltelli and Gualtieri, 1990). The resulting variation versus time was
plotted and analytically fitted using the Sigma Plot package (Jendel).
Photography
Fluorescence, bright-field, and phase-contrast photographs were taken with
a Minolta X-100 camera mounted on a Zeiss Axioplan microscope, and
recorded on Kodak Ektachrome 100 ASA color film.
RESULTS
The changes in fluorescence of the paraflagellar swelling of
Euglena gracilis, in response to two different excitation
lights (436 nm and 365 nm) are shown in Figs. 1 and 2. The
436-nm excitation light (blue-violet filter set) produces the
typical red fluorescence of chloroplasts, and faint or no
fluorescence in the paraflagellar swelling zone (Figs. 1 a
and 2, a and c). Fig. 2 b shows the effect of a prolonged
exposure (5 min) to the 436-nm light: apart from chloro-
phyll bleaching, no detectable modification of the swell-
ing's emission can be observed. Under the 365-nm excita-
tion light (UV-blue filter set), the paraflagellar swelling
undergoes an increase in its emission from zero (Fig. I b) to
a maximum (Fig. 2 d) in the green range. With the blue-
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Biophysical Journal

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Euglena Photoreceptor Photocycle
FIGURE
magnification (20x). Under the 436-nm excitation light the cells show the
typical red emission of the chlorophyll, and faint or no emission in the
paraflagellar swelling area (a). The 365-nm excitation light produces an
increase in the emission of the swelling from zero (b) to a maximum in the
green range. Under the 436-nm excitation light this emission appears
extremely bright (c) and then fades (d). Scale bar = 50 ,im.
1
Fluorescence micrographs of Euglena gracilis cells at low
violet filter inserted, the swellings now appear as bright
green bodies (Figs. 1 c and 2 e), whose emission gradually
fades (Figs. 1 d and 2 f). Visual observations do not detect
any chromatic difference in the emission hue of the swelling
under the two excitation lights. Moreover, the insertion of
dark periods during and between excitations does not mod-
ify the fluorescence intensity attained.
To reconstruct the kinetics of this photocycle we sampled
the fluorescence emission of the paraflagellar swelling, dig-
itizing at constant rate fluorescence images of 20 Euglena
cells. Two sequences were created, one for the UV-blue
excitation light and the other for the blue-violet excitation
light. Fig. 3 shows these two sequences of 15 320 X 200
FIGURE 2
cation (lOOX). (a and b) The same cell under the 436-nm excitation
light:only a very faint emission is detectable in the paraflagellar swelling area
Fluorescence micrographs of Euglena cells at high magnifi-
-wWww
547
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Volume 72
February 1997
mm-
mm
mm'
mml
mm
FIGURE 3
images show the variation of the emission of a swelling under the 365-nm
excitation light (upper 15 images) and under the 436-nm excitation light
(lower 15 images). Each image represents the apical part of a cell, where
the paraflagellar swelling is located.
Two sequences of black-and-white digitized fluorescence
(a), and a prolonged exposure (5 min) to this wavelength produces bleach-
ing of the chlorophyll, but no increase in the swelling's emission (b). (c)
Another cell under the 436-nm excitation light. The 365-nm excitation light
produces an increase in the green emission of the swelling from zero (cf.
Fig. 1 b) to a maximum in the green range (d). Under the 436-nm excitation
light (e) this bright emission gradually fades (f). The bright green appear-
ance of the microscopic image of the swelling becomes yellowish-green (e)
because of a local overexposure of the photographic film due to thehigh
intensity of the swelling's emission. In all of the figures the arrowheads
point to the swelling. Scale bar = 10 ,um.
pixel digital fluorescence images. The 30 black-and-white
digitized images represent the apical part of the same Eu-
glena cell, where the swelling is located. The upper 15
images, acquired every 400 ms (10 TV frames), show the
swelling emission under the 365-nm excitation
whereas the lower 15 images, acquired every 640 ms (16
TV frames), show the emission under the 436-nm excitation
light. From the top left corner to the bottom right corner the
successive images show the variation of the emission from
the lowest intensity to the highest and vice versa.
Segmentation and labelization techniques were applied to
each of the 30 digital fluorescence images. These proce-
dures select all of the pixels belonging to the swelling in the
apical region of Euglena, count them, and add up their
intensity values. The resulting values represent the inte-
grated emission of the swelling. The variation of these
values versus time was plotted after an analytical fit. This
kinetics clearly indicates the presence of a complex chain
photoreaction with at least three intermediates (Fig. 4). A
represents the parent form of the protein; B (and/or B') and
C (and/or C') are the two light-induced intermediates, which
revert to A at the end of the cycle. The formation of each
intermediate best fits with a Michaelis-Menten equation of
form ax/(x + b) (X< 1). Fig. 4 shows the fitted kinetics in
its upper part and the distribution of residues between the
row and the fitted data in its lower part.
In Fig. 5 the maximum emission of a paraflagellar swell-
ing (Fig. 5 a) is compared with the emission of a swelling
light,
120 -
100 -
c
80-
.-
cn
20 -
0-
A
A
7
-
0.4
75
0.0
-0.2-
-~-0.2-\wI\w
'~-0.6-
8sec
10 sec
365 nm
436 nm
FIGURE 4
presence of at least three forms, i.e., A, B, and C. The distribution of
residues between the row and the fitted data is shown in the lower part of
the figure.
Kinetics of the photocycle of the swelling indicates the
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Biophysical Journal
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Euglena Photoreceptor Photocycle
FIGURE 5
wide-band excitation light (i.e., simultaneously with the UV and the blue
excitations; a) is compared with the emission under the 436-nm light of a
swelling previously excited with the 365-nm light (b). The difference is
impressive. Scale bar = 10,uAm.
The emission of a flagellar swelling under a 400-440-nm
excited with a 400-440-nm wide-band filter (i.e., simulta-
neously with the UV and the blue excitation wavelengths)
(Fig.5b).
Repeated cycles can be observed in living cells, but under
prolonged microscopic observation the slides dry up and the
cells burst. This fact prevents the recording of many con-
secutive cycles in the same cell. To make the cells more
resistant to dehydration, we treated them with a detergent
solution that effects complete extraction, with the exception
of pellicle, axonemes, the paraflagellar rod, the paraflagellar
swelling, and the microtubular system of the reservoir-canal
region (refer also to figures 8-10 in Barsanti et al., 1993).
Three successive photocycles of the same swelling are
shown in Fig. 6; the bright-field and phase-contrast images
show the swelling as a gray or dark body, respectively (Fig.
6, a and b, arrowhead); its fluorescent image under the
436-nm light shows only a faint emission (Fig. 6 c), which
increases to a maximum under the 365-nm light (Fig. 6, d,
h, 1). Under the 436-nm excitation light this emission ap-
pears extremely bright (Fig. 6, e, i, m) and then gradually
fades (Fig. 6,4 g, j, k, n-p). Because of the almost complete
extraction of pigments, the 365-nm irradiation produces a
different cellular hue (Fig. 6, d, h, 1). The same intensity and
same time of irradiation at 365 nm (800 IXW/cm2) and 436
nm (1100 ,.W/cm2) were obviously adopted throughout the
three cycles.
This photocycle would thus include the photoconversion
under the 365-nm excitation light of a nonfluorescent form
(A) to two or more fluorescent intermediates, which remain
stable almost indefinitely (C); these in turn would regener-
ate the nonfluorescent form under the 436-nm excitation
light.
mophoric conformational changes, can enhance their quan-
DISCUSSION
We report the presence of a photochromic pigment in the
paraflagellar swelling of Euglena gracilis that undergoes
repeated and reversible fluorescence changes with a deter-
minate kinetics; this cycle is present not only in cells under
physiological conditions, but also in cells treated with a
detergent solution that completely extracts them (Fig. 6).
The paraflagellar swelling possesses optical bistability, i.e.,
the parent form of its molecules upon photoexcitation gen-
erates relatively stable intermediate(s) that can be photo-
chemically driven back to the parent form, and the quantum
yields of the forward and reverse reactions are almost the
same (Fig. 4). The A form is not fluorescent.
These data allow us to identify the true photoreceptor of
Euglena gracilis with its crystalline paraflagellar swelling,
and seem to us to support the hypothesis of a rhodopsin-
based photoreceptor.
Photochromism with nearly similar forward and reverse
reaction quantum yields, both close to unity, is peculiar to
rhodopsins (Govindjee et al., 1990; Marwan and Oesterhelt,
1990; Siebert, 1990; Birge et al., 1995). Stable intermedi-
ates are very often present in the photocycle of rhodopsins,
particularly
in invertebrate
Schwemer, 1975; Franceschini, 1983); and the bacteriorho-
dopsin itself offers a good example of a cycle with ther-
mally stable intermediates whose lifetime can be relatively
easily modified (Birge, 1995; Birge et al., 1995).
Data already cited provide an estimate of 2 X 107 mol-
ecules of rhodopsin-like proteins in the crystalline structure
of the paraflagellar swelling of Euglena gracilis (Gualtieri
et al., 1989, 1992). Considering that in the present experi-
ment about 108 photons impinge on the swelling area and
produce a saturation effect (maximum number of stable
intermediate(s)) and assuming that the Euglena photorecep-
tor operates via a one-photon absorption mechanism, we can
approximate the forward and reverse quantum yields of its
cycle kinetics to unity, as expected.
Having stated this, we can describe the cycle we recorded
in Euglena gracilis in the following way. The initial form A,
present in a very high percentage in this state of photoequi-
librium, has a very low absorbance at 436 nm and a higher
absorbance at 365 nm, and does not fluoresce. It could
resemble the resting state of a rhodopsin-like protein with
an a-band at 500 nm and a secondary ,B-band centered in the
UV-blue region (Stavenga et al., 1993), as suggested by
Gualtieri et al. (1989) and James et al. (1992).
The intermediates, B and C, emit in the green range; we
cannot visually detect any difference in the emission hue of
the paraflagellar swelling under the two excitation lights.
Because they produce a green emission, these intermediates
should have an absorption band bathochromically shifted in
comparison with the parent A, and absorb the incident light
at 365 nm and at 436 nm, respectively. Either a change in
the polarity of the binding pocket or the intrinsic constraints
of their excited state, both due to proteic and/or chro-
rhodopsins (Hamdorf and
549
Barsanti et al.