JOURNAL OF EXPERIMENTAL ZOOLOGY 289:467–471 (2001)

© 2001 WILEY-LISS, INC.

RAPID COMMUNICATIONS

Photosensory Transduction in UnicellularEukaryote Blepharisma

TATSUOMI MATSUOKA1* AND HIYOSHIZO KOTSUKI2

1Department of Biology, Kochi University, Kochi 780-8520, Japan2Department of Chemistry, Kochi University, Kochi 780-8520, Japan

ABSTRACT In the ciliated protozoan Blepharisma, step-up photophobic response is mediatedby a novel type of photosensory complex of pink-colored pigment “blepharismins” and 200-kDamembrane protein contained in the pigment granules located just beneath the plasma membrane.We found that the fluorescence intensity of isolated blepharismins decreased prominently with adecrease of H+ concentration in the surrounding medium. In the present study, therefore, we uti-lized the endogenous pigment blepharismins as the pH indicator. Light stimulation evoked a sud-den decrease in fluorescence intensity in a photosensitive anterior portion of the cell, suggestingthat a drop in H+ concentration occurred in the anterior region. The result indicates that thephotosignal is transduced into cytoplasmic signaling of H+ translocation across the outer mem-brane surrounding the pigment granules, so that cytosolic H+ concentration in the vicinity of plasmamembrane might be increased. J. Exp. Zool. 289:467–471, 2001. © 2001 Wiley-Liss, Inc.

The pink-colored ciliated protozoan Blepharismaexhibits transient backward swimming (step-upphotophobic response) upon a relative increase inlight intensity (a step-up light stimulation) (Mat-suoka, ’83a,b; Kraml and Marwan, ’83), so thatthe cells accumulate in relatively dark regions(Matsuoka, ’83a). The pink-colored quinone pig-ment (Matsuoka, ’96; Checcucci et al., ’97; Maedaet al., ’97), called “blepharismin” (Giese, ’73) andlocated inside the pigment granules that are regu-larly arranged in the cortical region of the cell(see Fig. 1), is believed to be a photosensor formediating the step-up photophobic response ofBlepharisma (Scevoli et al., ’87; Matsuoka et al.,’92a,b, ’95; Checcucci et al., ’93). The Blepharismacell contains five types of blepharismin moleculeswith different functional groups (Maeda et al., ’97).

The step-up photophobic response of Blephar-isma is performed by a transient reversed beat-ing of the cilia (ciliary reversal), which is mediatedby inflowing Ca2+ through depolarization-sensitiveCa2+ channels located in the ciliary membrane(Fabczack et al., ’96). The depolarization-sensitiveCa2+ channels should be activated by membranedepolarization (generation of photoreceptor poten-tial) elicited through the photosignaling pathway.

Previous studies revealed that the blephar-ismins were bound to a 200-kDa membrane pro-tein and that the blepharismins–protein complexwas localized on or in the invaginated membranes(Matsuoka et al., ’93, ’94, ’97) inside the pigment

granules surrounded by the outer membrane(Matsuoka et al., ’94; Harumoto et al., ’98); thegranules are arranged regularly just beneath theplasma membrane (see Fig. 1). Despite these pre-vious findings, determining how the photosignalmight be transduced finally into a cytoplasmic sig-nal inducing depolarization of the plasma mem-brane is quite difficult, because even H+ or Ca2+

indicators with a hydrophobic group hardly pen-etrate across the membrane, and endogenousblepharismin fluorescence disturbs the fluores-cence assays using exogenous indicators for H+ orCa2+ if they are introduced into the cell. More-over, it is difficult to know whether the changesof fluorescence intensity might reflect changes inionic concentration inside the pigment granulesor the cytoplasmic sol. However, if the blephar-ismins are confirmed to be sensitive to H+ or Ca2+,this endogenous pigment can be utilized as a quiteuseful indicator determining ionic environmentsin the vicinity of membranes where photorecep-tor blepharismins/200-kDa protein is localized.Therefore, we expected that the fluorescence in-tensity of blepharismins would be changed in re-

Grant sponsor: Ministry of Education, Science and Culture, Ja-pan; Grant numbers: 08640869, 10640668, 10480151, 99132; Grantsponsor: Sumitomo Foundation.

*Correspondence to: T. Matsuoka, Department of Biology, KochiUniversity, Kochi 780-8520, Japan. E-mail: tmatsuok@cc.kochi-u.ac.jp

Received 14 June 2000; Accepted 28 September 2000.

468 T. MATSUOKA AND H. KOTSUKI

sponse to changes in H+ or Ca2+ concentrations.Notably, we found that the fluorescence intensityof blepharismins was prominently influenced onlyby changes in H+ concentrations.

Using endogenous pH indicator blepharismins,the present study revealed that the H+ concen-tration of the intermembrane space between thepigment granule and the surrounding vesicle wasdecreased in response to light stimulation.

MATERIALS AND METHODSCells of Blepharisma japonicum were cultured

at 23°C in the dark in a 0.1% cereal leaves infu-sion containing bacteria (Enterobacter aerogenes),which were supplied by the Institute for Fermen-tation, Osaka (IFO). Bacteria were cultured on1.5% agar plates containing 0.5% polypeptone, 1%meat extract, and 0.5% NaCl. In order to extractthe pigment, cells were collected (150g, 1 min),rinsed in distilled water by centrifugation (150g,1 min), and then suspended in acetone. After a 1-min extraction, cells were sedimented (150g, 5min), and then the supernatant was decanted. Thepigment preparation was dried by a rotary evapo-rator and then dissolved in a 10 mM sodium cho-late solution.

Pigment granules were isolated by cold shocks.Prior to application of the cold shock to the cells,the cells were kept for 2 days at 23°C. The cellswere resedimented (150g, 1 min) to obtain a loosepellet, and the cell pellet was quickly transferredinto cooled water (2°C). After gentle pipetting, thecells were resedimented by centrifugation (150g,1 min) to decant the supernatant containing thepigment granules. The supernatant was centri-fuged at 8,000g (10 min) to obtain the pellet ofpigment granules.

Image analysis of the fluorescence changes (H+

concentration changes) was performed using theARGUS-50/CA (Hamamatsu Photonics, Japan)image analysis system. Fluorescence images attime intervals of about 50 msec were stored in acomputer. In the image analysis, cells suspendedin the solution containing 1 mM CaCl2, 1 mM KCl,and 5 mM Tris-HCl (pH 7.4) were loaded on aslide glass so that a smaller depth of cell suspen-sion was possible, and kept for a few minutes un-der a dim white light (below 0.05 W/m2) forobservation. An accidentally stopped cell was cho-sen, 443-nm light (3.5 × 1019 quanta/m2·s) was ap-plied, and then fluorescence images were analyzedand processed as pseudo-color images.

Fig. 1. Photomicrographs: (A) cell of Blepharisma; (B) pig-ment granules regularly arranged in the cortical region ofthe cell body. Lower section: schematic diagram showing the

pigment granule and its surrounding vesicle, drawn on thebasis of both transmission (Matsuoka et al., ’94) and scan-ning (Harumoto et al., ’98) electron micrographs.

PHOTOSENSORY TRANSDUCTION IN BLEPHARISMA 469

RESULTS AND DISCUSSIONFigure 1 shows photomicrographs of a Blephar-

isma cell (A), pigment granules located in the cor-tical region of the cell body (B), and a schematicdiagram of the pigment vesicle (complex of pig-ment granule and surrounding vesicle) suspendedjust beneath the plasma membrane, drawn on thebasis of electron microscopic observations (Mat-suoka et al., ’94; Harumoto et al., ’98). If theblepharismin molecules located on or in the in-vaginated membranes of pigment granules aresensitive to the cation concentrations and changetheir fluorescence intensity in response to lightstimulation, the changes in the ionic environmentsof intermembrane space (Fig. 1) can be visualizedby an image analyzing system. Fortunately, wefound that the fluorescence intensity of isolatedblepharismins (Fig. 2A) and that of isolated pig-ment granules (Fig. 2B) was increased by de-creased pH. On the other hand, the fluorescenceintensity was hardly affected by changes in K+ orCa2+ concentrations; actually, only a slight de-crease (5%) in fluorescence intensity of isolatedblepharismins was observed in Ca2+ concentra-tions ranging from 0 (contaminated free Ca2+

eliminated with 1 mM EGTA) to 0.1 mM (datanot shown).

When the intact cell of Blepharisma was stimu-lated by 443-nm light, fluorescence intensity wasquickly decreased only in the anterior portion of

the cell (Fig. 3, left column A–C). In this case, thecell began to move backward about 800 msec af-ter light stimulation. Fluorescence intensity of ananterior fragment bisected with a glass needle wasalso decreased (Fig. 3, middle column) in responseto light stimulation, whereas that of a posteriorfragment was not decreased (Fig. 3, right column).The anterior fragment started to move backwardabout 1,300 msec after light stimulation. The pos-terior fragment did not show backward movement.The fluorescence intensity of anterior portion ofthe cell (Fig. 3, left column) had decreased about25% at 830 msec after light stimulation. The fluo-rescence intensity of isolated pigment granulesdecreased 28% when the pH of surrounding me-dium was changed from pH 7.1 to 8.5 (Fig. 2B). Ifthe pH of intermembrane space (Fig. 1) of the cellskept in darkness was maintained around neutral,light stimulation (443-nm light, 3.5 × 1019 quanta/m2·s) could evoke a sudden increase of pH up toabout 8.5. The fluorescence intensity of isolatedpigment granules was slightly decreased only 5% when the Ca2+ concentration was changed inthe physiological range from 0 mM to 0.1 mM.Therefore, the drastic decrease (25%) in in vivofluorescence caused by light stimulation (Fig. 3)should be attributed mainly to changes in H+ con-centration, although in vivo fluorescence changespossibly partially reflect changes in Ca2+ concen-tration. Matsuoka (’83b) has reported that only

Fig. 2. (A) Fluorescence emission spectra of the pink formof blepharismins dissolved in Tris-HCl buffers containing 10mM sodium cholate that were adjusted to different pH. (B)Fluorescence emission spectra of pigment granules (pink form)

suspended in the Tris-HCl buffers adjusted to different pH.The pigment granules were extruded by cold shocks in dis-tilled water. The pigment was excited by 443-nm light.

470 T. MATSUOKA AND H. KOTSUKI

the anterior region of the Blepharisma cell is pho-tosensitive for the step-up photophobic response.The fact that H+ response occurs only in the an-terior region of cell supports that such a H+ re-sponse is responsible for the step-up photophobicresponse.

The fact that the fluorescence intensity of iso-lated pigment granules was affected by changesin surrounding pH (Fig. 2) implies that blephar-ismin molecules could be located facing on theintermembrane space between the pigment gran-ule and surrounding membrane (see Fig. 1). De-crease in H+ concentration in the intermembranespace could be attributed to H+ translocationfrom the intermembrane space to the cytoplas-mic sol. An increase of H+ concentration in thevicinity of the plasma membrane could elicit de-polarization of the plasma membrane (genera-tion of photoreceptor potential). In Blepharisma,a depolarizing photoreceptor potential that acti-vates depolarization-sensitive Ca2+ channels andinflowing Ca2+, activates the following signaling

pathway, which leads to a reversed beating ofcilia (Fabczak et al., ’96). However, it cannot beconcluded that increase in H+ concentration inthe vicinity of the plasma membrane directly ac-tivates the depolarization-sensitive Ca2+ chan-nels (Fabczak et al., ’96) or indirectly activatesthem through the activation of Ca2+-dependentphotoreceptor channels.

In addition to the step-up photophobic response,Blepharisma shows acceleration in a steady-stateswimming velocity, which depends on an absolutelight intensity (Matsuoka, ’83a,b). Because thephotosensitivity for the step-up photophobic re-sponse is localized in the anterior end of the cell,while that for swimming response is distributedon entire cell, Matsuoka (’83b) proposed that thephotoreceptor system responsible for the swim-ming response is different from that responsiblefor the step-up photophobic response. The presentresult that only anterior pigment vesicles respondto light by H+ translocation strongly supports thisidea proposed nearly two decades ago, and we con-

Fig. 3. Decrease in fluorescence intensity in the anteriorportion of the cell just after light stimulation (443-nm light,3.5 × 1019 quanta/m2·s). Left column, intact cell; middle col-umn, anterior fragment of cell bisected with a glass needle;

right column, posterior fragment of cell bisected with a glassneedle. Arrows indicate the anterior (ant.) and posterior (post.)ends, respectively. Relative fluorescence intensity is expressedas pseudo-color images.

PHOTOSENSORY TRANSDUCTION IN BLEPHARISMA 471

clude that the photoreceptor pigment vesicles in-ducing the step-up photophobic response and thoseinducing the swimming response are functionallydifferentiated.

REFERENCESCheccucci G, Shoemaker RS, Bini E, Cerny R, Tao N, Hyon

J-S, Gioffre D, Ghetti F, Lenci F, Song P-S. 1997. Chemicalstructure of blepharismin, the photosensor pigment forBlepharisma japonicum. J Am Chem Soc 119:5762–5763.

Checcucci G, Damato G, Ghetti F, Lenci F. 1993. Action spec-tra of the photophobic response of blue and red forms ofBlepharisma japonicum. Photochem Photobiol 57:686–689.

Fabczak S, Fabczak H, Walerczyk M, Sikora J, GroszynskaB, Song P-S. 1996. Ionic mechanisms controlling photopho-bic responses in the ciliate Blepharisma japonicum. ActaProtozool 35:245–249.

Giese AC. 1973. Blepharisma. The biology of a light-sensitiveprotozoa. Stanford, CA: Stanford University Press.

Harumoto T, Miyake A, Ishikawa N, Sugibayashi R, ZenfukuK, Iio H. 1998. Chemical defense by means of pigmentedextrusomes in the ciliate Blepharisma japonicum. Eur JProtistol 34:458–470.

Kraml M, Marwan W. 1983. Photomovement response of theheterotrichous ciliate Blepharisma japonicum. PhotochemPhotobiol 37:313–319.

Maeda M, Naoki H, Matsuoka T, Kato Y, Kotsuki H, UtsumiK, Tanaka K. 1997. Blepharismin 1-5, novel photoreceptorfrom the unicellular organism Blepharisma japonicum. Tet-rahedron Lett 38:7411–7414.

Matsuoka T. 1983a. Negative phototaxis in Blepharismajaponicum. J Protozool 30:409–414.

Matsuoka T. 1983b. Distribution of photoreceptors inducing

ciliary reversal and swimming acceleration in Blepharismajaponicum. J Exp Zool 225:337–340.

Matsuoka T. 1996. Photomovements and photoreceptors inthe unicellular organism Blepharisma. Int Congr Photobiol,12th, Abstract of ICP 96, p 185.

Matsuoka T, Matsuoka S, Yamaoka S, Kuriu T, Watanabe Y,Takayanagi M, Kato Y, Taneda K. 1992a. Action spectra forstep-up photophobic response in Blepharisma. J Protozool39:498–502.

Matsuoka T, Murakami Y, Furukohri T, Ishida M, Taneda,K. 1992b. Photoreceptor pigment in Blepharisma: H+ re-lease from red pigment. Photochem Photobiol 56:399–402.

Matsuoka T, Murakami Y, Kato Y. 1993. Isolation of blephar-ismin-binding 200 kDa protein responsible for behavior inBlepharisma. Photochem Photobiol 57:1042–1047.

Matsuoka T, Sato M, Maeda M, Naoki H, Tanaka T, KotsukiH. 1997. Localization of blepharismin photosensors andidentification of a photoreceptor complex mediating the step-up photophobic response of the unicellular organism,Blepharisma. Photochem Photobiol 65:915–921.

Matsuoka T. Tsuda T, Ishida M, Kato Y, Takayanagi M, FujinoT, Mizuta S. 1994. Presumed photoreceptor protein and ul-trastructure of the photoreceptor organelle in the ciliatedprotozoan, Blepharisma. Photochem Photobiol 60:598–604.

Matsuoka T, Watanabe Y, Sagara Y, Takayanagi Y, Kato Y.1995. Additional evidence for blepharismin photoreceptorpigment mediating step-up photophobic response of uni-cellular organism, Blepharisma. Photochem Photobiol62:190–193.

Scevoli P, Bisi F, Colombetti G, Ghetti F, Lenci F, PassarelliV. 1987. Photomotile responses of Blepharisma japonicum.I. Action spectra determination and time-resolved fluores-cence of photoreceptor pigments. J Photochem Photobiol B1:75–84.