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Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma
www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 54 (2000) 131–135
Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma Tatsuomi Matsuoka a,*, Naoko Moriyama a, Akemi Kida a, Kazuo Okuda a, Tomohiko Suzuki a, Hiyoshizo Kotsuki b a b
Department of Biology, Kochi University, Kochi, Japan Department of Chemistry, Kochi University, Kochi, Japan Received 8 September 1999
Abstract The blepharismin–200 kD protein complex of the ciliated protozoan Blepharisma is a novel type of photosensor responsible for the stepup photophobic response of the cell. In immunoblotting assays, the 200 kD protein is weakly cross-reacted with anti-inositol triphosphate receptor antibody (anti-IP3 R antibody). Indirect immunofluorescence assays show that the pigment granules in which the blepharismin–200 kD protein complex is localized are labelled by anti-IP3 R antibody. When the anti-IP3 R antibody or antisense oligonucleotide for IP3 receptor is introduced into the living cells of Blepharisma, both the photosensitivity of the cells and content of blepharismin–200 kD protein are reduced. The results suggest that the photoreceptor 200 kD protein is possibly an IP3 receptor-like protein. q2000 Elsevier Science S.A. All rights reserved. Keywords: Anti-IP3 R antibody; Blepharisma; Immunochemistry; Photoreceptor proteins
1. Introduction Rhodopsin-like molecules, where seven-transmembrane proteins are bound to the photosensor pigment retinal, function not only in animals [1,2] but also in unicellular eukaryotes [3] or even in prokaryotes [4]. On the other hand, evidence showing that novel types of molecules, such as blepharismin of Blepharisma [5–8], stentorin of Stentor coeruleus [9,10], flavin and pterin of Euglena [11–13] or yellow protein of purple bacteria [14], function as the photosensors responsible for the photobehaviour of unicellular organisms has been reported. In Blepharisma japonicum a pink-coloured pigment, called blepharismin [15], is contained in the pigment granules located just beneath the plasma membrane [16]. The cells of Blepharisma show step-up photophobic response (temporal backward swimming or rotating movement due to ciliary beating reversal induced by a sudden increase in light intensity) [17–19]. Such a photoresponse aids the cells to avoid strongly illuminated regions and to accumulate in shaded regions [18]. Blepharismin has been demonstrated to function as the photosensor molecule mediating the step-up photophobic response [5–8]. Recently, the complete molecular structure of blepharismin-2 was deter* Corresponding author. Fax: q81-888-44-8356.
mined [20]; thereafter those of blepharismin-3 [21] and other types of blepharismins were determined [22]. Moreover, it has been revealed that blepharismin is associated with a 200 kD membrane protein [16,23], which is embedded in the membrane constructing the ‘honeycomb-like structure’ inside the pigment granules [16]. Inositol triphosphate receptor (IP3 receptor), which is located in the Ca2q channels gated by IP3, activates intracellular Ca2q-storing sites to generate intracellular Ca2q signals [24–27]. Recently, Fabczak et al. reported that IP3 receptorlike protein occurs in the cortical region of Blepharisma cells [28,29]. The present immunochemical assay agrees with the results obtained by Fabczak et al. [28,29]. The present study also suggests that Blepharisma photoreceptor 200 kD protein is possibly related to IP3-like protein.
2. Materials and methods 2.1. Cell culture Blepharisma japonicum was cultured at 238C in the dark in 0.1% cereal leaf infusion containing bacteria (Enterobacter aerogenes) as food. The bacteria, which were supplied
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by the Institute for Fermentation, Osaka, Japan (IFO), were cultured on 1.5% agar plates containing 0.5% polypeptone, 1% meat extract and 0.5% NaCl. For assays of photobehaviour, cells were collected by gentle centrifugation (150g, 1 min), resuspended in a saline solution containing 5 mM Tris– HCl (pH 7.4), 1 mM CaCl2 and 1 mM KCl, and then kept at 23–258C overnight. Photobehaviour of the cells was measured according to Ref. [6]. 2.2. Extraction and assay of free blepharismin pigment To extract the pigment, the cells were collected, rinsed with distilled water and then suspended in acetone. After a 1 min extraction, the cells were resedimented (8000g, 1 min), and the supernatant containing extracted pigment was decanted. The absorption spectra of the pigments were measured with a Hitachi 220 A spectrophotometer.
2.5. Immunofluorescence staining For indirect immunofluorescence assays, Blepharisma was fixed with a fixative containing 3% paraformaldehyde, 1% glutaraldehyde, 1 mM EGTA, 1 mM KCl and 10 mM PIPES (pH 7.4), and subsequently the cells were permeabilized for 1 h in 0.1% Triton X-100. The cells were incubated sequentially with rabbit anti-IP3 receptor polyclonal antibody (2 mg mly1; Calbiochem) for 2 h at 378C, followed by FITClabelled goat anti-rabbit Ig G (2 mg mly1; Sigma) for 2 h at 378C.
2.3. Isolation of blepharismin–protein complex and SDS–PAGE The cells were frozen once at y208C and remelted to disrupt cell structure. The disrupted cells were sedimented (8000g, 10 min) to remove water-soluble components. In order to solubilize blepharismin–membrane protein complexes, the pellet of cells was resuspended in a solution containing 60 mM sodium cholate, 10 mM Tris–HCl (pH 7.4) and 0.5 mM PMSF (phenylmethylsulfonyl fluoride), and kept for 24–48 h at 48C. After incubation, the cell debris was sedimented (8000g, 10 min). After the pink-coloured supernatant was decanted, a three-fold volume of sample buffer containing 2% SDS, 30 mM Tris–HCl (pH 6.8) and 10% glycerol was added, and then incubated at 238C for 24–48 h. Prior to a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the preparation was concentrated through a 10 kD cut-off membrane filter (Millipore UFP1 LGC BK). Gels were run at 150 V, stained with 0.2% Coomassie Brilliant Blue R-250 (CBB) in 45% (v/v) methanol, 10% (v/v) glacial acetic acid solution for 10 min at 408C. For standard proteins, a molecular standards kit (Bio-Rad) was employed. 2.4. Immunoblotting The detergent-solubilized proteins were electrophoresed on a 10% polyacrylamide gel, followed by transfer to a ProBlott membrane (Applied Biosystems). In order to detect the proteins immunologically related to the IP3 receptor, the blots were immunostained with rabbit anti-IP3 receptor polyclonal antibody (2 mg mly1; Calbiochem) for 1 h at 378C, followed by incubation with peroxidase-labelled goat antirabbit Ig G (2 mg mly1; Kirkegaard & Perry Laboratories) for 1 h at 378C. The IP3 receptor-like protein was detected on the enhanced chemiluminescence detection system (Amersham).
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Fig. 1. (a) SDS–PAGE (silver staining) of the electrophoretically purified 200 kD protein. In order to obtain 200 kD protein, detergent-solubilized components of the cells were analysed by SDS–PAGE. The gel bands corresponding to molecular mass of 200 kDa were cut out, and electrophoretically eluted in a buffer (0.01% SDS, 2.5 mM Tris, 19 mM glycine). Eluted samples were concentrated, subsequently diluted 1:1 with solvent containing 2% SDS, 30 mM Tris–HCl (pH 6.8) and 10% glycerol and finally kept for 24 h at 238C (lane 1). The samples were boiled for 3 min (lane 2), treated with 2% 2-mercaptoethanol at room temperature (lane 3), or were treated with 2% 2-mercaptoethanol and then boiled for 3 min (lane 4). (b) SDS– PAGE (lane 1) of detergent-solubilized components of the cells and immunoblot (lane 2) of 200 kD protein with anti-IP3 R antibody.
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2.6. Electroporation Prior to electroporation, the cells were transferred to 10 mM KCl solutions containing antisense or sense oligonucleotide (3.2 nmol mly1 each), or anti-IP3 R antibody (20 mg mly1). Antisense or sense oligonucleotides (54 nucleotides; putative mixed oligonucleotides) corresponding to the preserved 18-amino-acid sequences (579–596, NH2-TITALLHNNRKLLEKHIT) of Xenopus IP3 receptor were electroporated into the cells. Electroporation was performed at 125 V cmy1 with a 250 mF capacitor (time constant, 70 ms), using a Gene Pulser (Bio-Rad). 3. Results and discussion To elucidate whether or not the 200 kD protein is composed of subunits, we completely purified electrophoretically the
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pigment-dissociated 200 kD protein (Fig. 1(a), lane 1). Boiled (Fig. 1(a), lane 2) or 2-mercaptoethanol-treated (Fig. 1(a), lanes 3, 4) purified 200 kD protein, from which pigment was completely dissociated, did not dissociate into subunits. This result indicates that the 200 kD protein is composed of a single polypeptide chain, like the IP3 receptor [27]. In immunoblotting assays, anti-IP3 R antibody weakly cross-reacted with the 200 kD protein of Blepharisma (Fig. 1(b)). Indirect immunofluorescence assays showed that the pigment granules (Fig. 2(a)) in which the blepharismin–200 kD protein complex is localized [16] were labelled by antiIP3 R antibody (Fig. 2(b)). On the other hand, secondary antibody alone could not label the pigment granules (Fig. 2(c)). This means that the pigment granules are specifically labelled by a primary antibody. Depressions of the cell sur-
Fig. 2. Immunofluorescence staining of the cells labelled with anti-IP3 R antibody: (a) normal living cells showing location of photosensory pigment granules; (b) a cell labelled with primary and secondary antibodies; (c) a cell labelled with only secondary antibody.
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face (Fig. 2(a), arrowheads), where ciliary lines existed, were also labelled (Fig. 2(b), arrowheads). Presumably, another type of IP3 R-like protein might be present along the ciliary line. When anti-IP3 R antibody (Fig. 3, top) or antisense oligonucleotide (Fig. 3, bottom) complementary to mRNA of IP3 receptor was electroporated into the cell, the step-up photophobic response of the cells was inhibited. The contents of blepharismin pigment (Fig. 4(a)) and 200 kD protein (Fig. 4(b)) of the cells into which the antisense oligonucleotide was introduced were reduced by about 20%. These results suggest that Blepharisma 200 kD protein is possibly related to an IP3 receptor-like protein. The result that the amino-acid composition [16] of the 200 kD protein resembled human (protein database search; A55713) and Xenopus (protein database search; A40743) IP3 receptor also supports this idea (data not shown). In Fig. 3, even in the control cells (top figure), the response was extremely repressed. This might be attributed to damage caused by electroporation, because the response is examined 1 h after electroporation. In contrast, the cells into which sense or antisense oligonucleotides are introduced might recover normal responsibility and motility, because the response of the cells is examined 24 h after electroporation. In the present immunoblotting assays (Fig. 1(b)), the cross-reaction between the 200 kD protein and anti-IP3 R antibody was very weak. It is likely that the conformation of the 200 kD protein is changed somewhat by SDS treatment. In the indirect immunofluorescence assays, on the other hand, pigment granules containing the 200 kD protein were prom-
Fig. 3. Repression of step-up photophobic response by anti-IP3 R antibody (top) or antisense oligonucleotides (bottom). The degree of the step-up photophobic response is expressed as the percentage of the total number of the cells (ns50) showing the response in 5 s. Columns and bars correspond to the means of four identical runs and standard errors. The step-up photophobic response was significantly repressed by anti-IP3 R antibody or antisense oligonucleotides (P-0.01 each). The step-up photophobic response of the cells into which IP3 R antibody was introduced was measured 1 h after electroporation. The photoresponse of the cells into which sense or antisense oligonucleotides were introduced was measured 24 h after electroporation. For light stimulation, 580 nm light (7.5=1018 quanta my2 sy1) was used.
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Fig. 4. (a) Absorption spectra (in acetone) of crude blepharismin contained in the cells in which antisense or sense oligonucleotides were electroporated. The pigment was extracted 24 h after sense or antisense oligonucleotides were introduced into the cells. (b) Comparison of 200 kD protein contents of the cells electroporated with antisense (1, 3) and sense oligonucleotides (2, 4). 1, 2: SDS–PAGE (CBB staining) of detergent-solubilized components of the cells. The cell debris disrupted by freeze and remelting was sedimented, and water-soluble components were partially removed. 3, 4: densitograms of CBB-stained gels obtained with NIH image (Yodo Co. Ltd.).
inently stained (Fig. 2). In this case, the 200 kD protein might still retain normal conformation. Fabczak et al. found that Blepharisma cells contained IP3 R-like proteins of molecular mass above 200 kD, using immunoblotting assays [28]. Our results are not inconsistent with the report of Fabczak et al., although it cannot be ascertained whether or not the protein they detected is identical with the photoreceptor 200 kD protein. They also reported that only the cortex region of Blepharisma cells is labelled by anti-IP3 R antibody [28], and the concentration of IP3 is changed by light irradiation of the cells [29]. They did not mention whether the pigment granules or other regions of cortex are stained [28]. The indirect immunofluorescence assays showed that the FITC fluorescence was detected in the pigment granules (Fig. 2). It is likely that the IP3 R-like protein detected by Fabczak et al. differs from the photoreceptor 200 kD protein analysed here. In the present study, the repression of step-up photophobic response of the cells that are electroporated with antisense oligonucleotide for IP3 receptor might be attributed at least to a decrease of
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T. Matsuoka et al. / J. Photochem. Photobiol. B: Biol. 54 (2000) 131–135
both pigment content and 200 kD protein. However, if the ordinal IP3 receptor, Ca2q channels gated by IP3, occurs in the photosignal transduction for the step-up photophobic response of Blepharisma cell, introduction of anti-IP3 R antibody and antisense oligonucleotide into the cell might more or less suppress photosignal transduction, and thereby the step-up photophobic response is suppressed. Ca2q-dependent membrane depolarization (generation of photoreceptor potential), which is caused by light stimulation [30], triggers a generation of Ca2q action potential related to the step-up photophobic response. It is possible, therefore, that Ca2q is translocated across the membrane surrounding the pigment granules so that plasma membrane depolarization may be evoked. However, in the present study, translocation of Ca2q from or into isolated pigment granules was not observed (data not shown).
Acknowledgements This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan (nos. 08640869, 10640668, 10480151, 99132) and was also supported financially by the Sumitomo Foundation.
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StyleTag -- Journal: JPB (J. Photochem. Photobiol. B: Biol.)
Article: 7920
Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma Tatsuomi Matsuoka a,*, Naoko Moriyama a, Akemi Kida a, Kazuo Okuda a, Tomohiko Suzuki a, Hiyoshizo Kotsuki b a b
Department of Biology, Kochi University, Kochi, Japan Department of Chemistry, Kochi University, Kochi, Japan Received 8 September 1999
Abstract The blepharismin–200 kD protein complex of the ciliated protozoan Blepharisma is a novel type of photosensor responsible for the stepup photophobic response of the cell. In immunoblotting assays, the 200 kD protein is weakly cross-reacted with anti-inositol triphosphate receptor antibody (anti-IP3 R antibody). Indirect immunofluorescence assays show that the pigment granules in which the blepharismin–200 kD protein complex is localized are labelled by anti-IP3 R antibody. When the anti-IP3 R antibody or antisense oligonucleotide for IP3 receptor is introduced into the living cells of Blepharisma, both the photosensitivity of the cells and content of blepharismin–200 kD protein are reduced. The results suggest that the photoreceptor 200 kD protein is possibly an IP3 receptor-like protein. q2000 Elsevier Science S.A. All rights reserved. Keywords: Anti-IP3 R antibody; Blepharisma; Immunochemistry; Photoreceptor proteins
1. Introduction Rhodopsin-like molecules, where seven-transmembrane proteins are bound to the photosensor pigment retinal, function not only in animals [1,2] but also in unicellular eukaryotes [3] or even in prokaryotes [4]. On the other hand, evidence showing that novel types of molecules, such as blepharismin of Blepharisma [5–8], stentorin of Stentor coeruleus [9,10], flavin and pterin of Euglena [11–13] or yellow protein of purple bacteria [14], function as the photosensors responsible for the photobehaviour of unicellular organisms has been reported. In Blepharisma japonicum a pink-coloured pigment, called blepharismin [15], is contained in the pigment granules located just beneath the plasma membrane [16]. The cells of Blepharisma show step-up photophobic response (temporal backward swimming or rotating movement due to ciliary beating reversal induced by a sudden increase in light intensity) [17–19]. Such a photoresponse aids the cells to avoid strongly illuminated regions and to accumulate in shaded regions [18]. Blepharismin has been demonstrated to function as the photosensor molecule mediating the step-up photophobic response [5–8]. Recently, the complete molecular structure of blepharismin-2 was deter* Corresponding author. Fax: q81-888-44-8356.
mined [20]; thereafter those of blepharismin-3 [21] and other types of blepharismins were determined [22]. Moreover, it has been revealed that blepharismin is associated with a 200 kD membrane protein [16,23], which is embedded in the membrane constructing the ‘honeycomb-like structure’ inside the pigment granules [16]. Inositol triphosphate receptor (IP3 receptor), which is located in the Ca2q channels gated by IP3, activates intracellular Ca2q-storing sites to generate intracellular Ca2q signals [24–27]. Recently, Fabczak et al. reported that IP3 receptorlike protein occurs in the cortical region of Blepharisma cells [28,29]. The present immunochemical assay agrees with the results obtained by Fabczak et al. [28,29]. The present study also suggests that Blepharisma photoreceptor 200 kD protein is possibly related to IP3-like protein.
2. Materials and methods 2.1. Cell culture Blepharisma japonicum was cultured at 238C in the dark in 0.1% cereal leaf infusion containing bacteria (Enterobacter aerogenes) as food. The bacteria, which were supplied
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by the Institute for Fermentation, Osaka, Japan (IFO), were cultured on 1.5% agar plates containing 0.5% polypeptone, 1% meat extract and 0.5% NaCl. For assays of photobehaviour, cells were collected by gentle centrifugation (150g, 1 min), resuspended in a saline solution containing 5 mM Tris– HCl (pH 7.4), 1 mM CaCl2 and 1 mM KCl, and then kept at 23–258C overnight. Photobehaviour of the cells was measured according to Ref. [6]. 2.2. Extraction and assay of free blepharismin pigment To extract the pigment, the cells were collected, rinsed with distilled water and then suspended in acetone. After a 1 min extraction, the cells were resedimented (8000g, 1 min), and the supernatant containing extracted pigment was decanted. The absorption spectra of the pigments were measured with a Hitachi 220 A spectrophotometer.
2.5. Immunofluorescence staining For indirect immunofluorescence assays, Blepharisma was fixed with a fixative containing 3% paraformaldehyde, 1% glutaraldehyde, 1 mM EGTA, 1 mM KCl and 10 mM PIPES (pH 7.4), and subsequently the cells were permeabilized for 1 h in 0.1% Triton X-100. The cells were incubated sequentially with rabbit anti-IP3 receptor polyclonal antibody (2 mg mly1; Calbiochem) for 2 h at 378C, followed by FITClabelled goat anti-rabbit Ig G (2 mg mly1; Sigma) for 2 h at 378C.
2.3. Isolation of blepharismin–protein complex and SDS–PAGE The cells were frozen once at y208C and remelted to disrupt cell structure. The disrupted cells were sedimented (8000g, 10 min) to remove water-soluble components. In order to solubilize blepharismin–membrane protein complexes, the pellet of cells was resuspended in a solution containing 60 mM sodium cholate, 10 mM Tris–HCl (pH 7.4) and 0.5 mM PMSF (phenylmethylsulfonyl fluoride), and kept for 24–48 h at 48C. After incubation, the cell debris was sedimented (8000g, 10 min). After the pink-coloured supernatant was decanted, a three-fold volume of sample buffer containing 2% SDS, 30 mM Tris–HCl (pH 6.8) and 10% glycerol was added, and then incubated at 238C for 24–48 h. Prior to a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the preparation was concentrated through a 10 kD cut-off membrane filter (Millipore UFP1 LGC BK). Gels were run at 150 V, stained with 0.2% Coomassie Brilliant Blue R-250 (CBB) in 45% (v/v) methanol, 10% (v/v) glacial acetic acid solution for 10 min at 408C. For standard proteins, a molecular standards kit (Bio-Rad) was employed. 2.4. Immunoblotting The detergent-solubilized proteins were electrophoresed on a 10% polyacrylamide gel, followed by transfer to a ProBlott membrane (Applied Biosystems). In order to detect the proteins immunologically related to the IP3 receptor, the blots were immunostained with rabbit anti-IP3 receptor polyclonal antibody (2 mg mly1; Calbiochem) for 1 h at 378C, followed by incubation with peroxidase-labelled goat antirabbit Ig G (2 mg mly1; Kirkegaard & Perry Laboratories) for 1 h at 378C. The IP3 receptor-like protein was detected on the enhanced chemiluminescence detection system (Amersham).
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Fig. 1. (a) SDS–PAGE (silver staining) of the electrophoretically purified 200 kD protein. In order to obtain 200 kD protein, detergent-solubilized components of the cells were analysed by SDS–PAGE. The gel bands corresponding to molecular mass of 200 kDa were cut out, and electrophoretically eluted in a buffer (0.01% SDS, 2.5 mM Tris, 19 mM glycine). Eluted samples were concentrated, subsequently diluted 1:1 with solvent containing 2% SDS, 30 mM Tris–HCl (pH 6.8) and 10% glycerol and finally kept for 24 h at 238C (lane 1). The samples were boiled for 3 min (lane 2), treated with 2% 2-mercaptoethanol at room temperature (lane 3), or were treated with 2% 2-mercaptoethanol and then boiled for 3 min (lane 4). (b) SDS– PAGE (lane 1) of detergent-solubilized components of the cells and immunoblot (lane 2) of 200 kD protein with anti-IP3 R antibody.
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T. Matsuoka et al. / J. Photochem. Photobiol. B: Biol. 54 (2000) 131–135
2.6. Electroporation Prior to electroporation, the cells were transferred to 10 mM KCl solutions containing antisense or sense oligonucleotide (3.2 nmol mly1 each), or anti-IP3 R antibody (20 mg mly1). Antisense or sense oligonucleotides (54 nucleotides; putative mixed oligonucleotides) corresponding to the preserved 18-amino-acid sequences (579–596, NH2-TITALLHNNRKLLEKHIT) of Xenopus IP3 receptor were electroporated into the cells. Electroporation was performed at 125 V cmy1 with a 250 mF capacitor (time constant, 70 ms), using a Gene Pulser (Bio-Rad). 3. Results and discussion To elucidate whether or not the 200 kD protein is composed of subunits, we completely purified electrophoretically the
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pigment-dissociated 200 kD protein (Fig. 1(a), lane 1). Boiled (Fig. 1(a), lane 2) or 2-mercaptoethanol-treated (Fig. 1(a), lanes 3, 4) purified 200 kD protein, from which pigment was completely dissociated, did not dissociate into subunits. This result indicates that the 200 kD protein is composed of a single polypeptide chain, like the IP3 receptor [27]. In immunoblotting assays, anti-IP3 R antibody weakly cross-reacted with the 200 kD protein of Blepharisma (Fig. 1(b)). Indirect immunofluorescence assays showed that the pigment granules (Fig. 2(a)) in which the blepharismin–200 kD protein complex is localized [16] were labelled by antiIP3 R antibody (Fig. 2(b)). On the other hand, secondary antibody alone could not label the pigment granules (Fig. 2(c)). This means that the pigment granules are specifically labelled by a primary antibody. Depressions of the cell sur-
Fig. 2. Immunofluorescence staining of the cells labelled with anti-IP3 R antibody: (a) normal living cells showing location of photosensory pigment granules; (b) a cell labelled with primary and secondary antibodies; (c) a cell labelled with only secondary antibody.
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face (Fig. 2(a), arrowheads), where ciliary lines existed, were also labelled (Fig. 2(b), arrowheads). Presumably, another type of IP3 R-like protein might be present along the ciliary line. When anti-IP3 R antibody (Fig. 3, top) or antisense oligonucleotide (Fig. 3, bottom) complementary to mRNA of IP3 receptor was electroporated into the cell, the step-up photophobic response of the cells was inhibited. The contents of blepharismin pigment (Fig. 4(a)) and 200 kD protein (Fig. 4(b)) of the cells into which the antisense oligonucleotide was introduced were reduced by about 20%. These results suggest that Blepharisma 200 kD protein is possibly related to an IP3 receptor-like protein. The result that the amino-acid composition [16] of the 200 kD protein resembled human (protein database search; A55713) and Xenopus (protein database search; A40743) IP3 receptor also supports this idea (data not shown). In Fig. 3, even in the control cells (top figure), the response was extremely repressed. This might be attributed to damage caused by electroporation, because the response is examined 1 h after electroporation. In contrast, the cells into which sense or antisense oligonucleotides are introduced might recover normal responsibility and motility, because the response of the cells is examined 24 h after electroporation. In the present immunoblotting assays (Fig. 1(b)), the cross-reaction between the 200 kD protein and anti-IP3 R antibody was very weak. It is likely that the conformation of the 200 kD protein is changed somewhat by SDS treatment. In the indirect immunofluorescence assays, on the other hand, pigment granules containing the 200 kD protein were prom-
Fig. 3. Repression of step-up photophobic response by anti-IP3 R antibody (top) or antisense oligonucleotides (bottom). The degree of the step-up photophobic response is expressed as the percentage of the total number of the cells (ns50) showing the response in 5 s. Columns and bars correspond to the means of four identical runs and standard errors. The step-up photophobic response was significantly repressed by anti-IP3 R antibody or antisense oligonucleotides (P-0.01 each). The step-up photophobic response of the cells into which IP3 R antibody was introduced was measured 1 h after electroporation. The photoresponse of the cells into which sense or antisense oligonucleotides were introduced was measured 24 h after electroporation. For light stimulation, 580 nm light (7.5=1018 quanta my2 sy1) was used.
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Fig. 4. (a) Absorption spectra (in acetone) of crude blepharismin contained in the cells in which antisense or sense oligonucleotides were electroporated. The pigment was extracted 24 h after sense or antisense oligonucleotides were introduced into the cells. (b) Comparison of 200 kD protein contents of the cells electroporated with antisense (1, 3) and sense oligonucleotides (2, 4). 1, 2: SDS–PAGE (CBB staining) of detergent-solubilized components of the cells. The cell debris disrupted by freeze and remelting was sedimented, and water-soluble components were partially removed. 3, 4: densitograms of CBB-stained gels obtained with NIH image (Yodo Co. Ltd.).
inently stained (Fig. 2). In this case, the 200 kD protein might still retain normal conformation. Fabczak et al. found that Blepharisma cells contained IP3 R-like proteins of molecular mass above 200 kD, using immunoblotting assays [28]. Our results are not inconsistent with the report of Fabczak et al., although it cannot be ascertained whether or not the protein they detected is identical with the photoreceptor 200 kD protein. They also reported that only the cortex region of Blepharisma cells is labelled by anti-IP3 R antibody [28], and the concentration of IP3 is changed by light irradiation of the cells [29]. They did not mention whether the pigment granules or other regions of cortex are stained [28]. The indirect immunofluorescence assays showed that the FITC fluorescence was detected in the pigment granules (Fig. 2). It is likely that the IP3 R-like protein detected by Fabczak et al. differs from the photoreceptor 200 kD protein analysed here. In the present study, the repression of step-up photophobic response of the cells that are electroporated with antisense oligonucleotide for IP3 receptor might be attributed at least to a decrease of
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T. Matsuoka et al. / J. Photochem. Photobiol. B: Biol. 54 (2000) 131–135
both pigment content and 200 kD protein. However, if the ordinal IP3 receptor, Ca2q channels gated by IP3, occurs in the photosignal transduction for the step-up photophobic response of Blepharisma cell, introduction of anti-IP3 R antibody and antisense oligonucleotide into the cell might more or less suppress photosignal transduction, and thereby the step-up photophobic response is suppressed. Ca2q-dependent membrane depolarization (generation of photoreceptor potential), which is caused by light stimulation [30], triggers a generation of Ca2q action potential related to the step-up photophobic response. It is possible, therefore, that Ca2q is translocated across the membrane surrounding the pigment granules so that plasma membrane depolarization may be evoked. However, in the present study, translocation of Ca2q from or into isolated pigment granules was not observed (data not shown).
Acknowledgements This work was supported in part by Grants-in-Aid from the Ministry of Education, Science and Culture, Japan (nos. 08640869, 10640668, 10480151, 99132) and was also supported financially by the Sumitomo Foundation.
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