Analyses of structure of photoreceptor organelle and blepharismin-associated protein in unicellular eukaryote Blepharisma

In the ciliated protozoan Blepharisma, step-up photophobic response is believed to be mediated by a novel type of photosensory pigment known as "blepharismins" (BL) that are contained in the pigment granules located just beneath the plasma membrane. We examined the ultrastructure of the pigment granules by freeze-fracture and thin-section electron microscopy and proposed a schematic diagram showing the granules' three-dimensional inner membranous structure. Some of the BL are suggested to be associated with 200 kDa membrane protein. High-pressure liquid chromatography analysis of pigment species associated with 200 kDa protein obtained from blue forms of Blepharisma (oxyblepharisma) revealed that the 200 kDa protein was associated with five types of oxyblepharismin. The fluorescence intensity was increased when the pigments were dissociated from the 200 kDa protein. The result supports the hypothesis that the pigment-200 kDa complex is able to transduce light energy into signals mediating the photobehavior of Blepharisma.     

ADDITIONAL EVIDENCE FOR BLEPHARISMIN PHOTORECEPTOR PIGMENT MEDIATING STEP-UP PHOTOPHOBIC RESPONSE OF UNICELLULAR ORGANISM, Blepharisma

Abstract— In the ciliated protozoan, Blepharisma japonicum, the pink-colored pigment (blepharismin) contained in the pigment granules is believed to be the photoreceptor pigment responsible for the step-up photophobic response. When the cells partially bleached by extrusion of the pigment granules caused by cold shocks were subsequently cultured under illuminated conditions, the pigment-less granules regenerated and the cells were further bleached (pigment content below 0.5%). The photosensitivity of such colorless cells disappeared completely. In contrast, the blepharismin pigment regenerated gradually when such colorless cells were transferred to darkness. The photosensitivity of the cells also recovered with regeneration of the pigment. We found that blepharismin pigment was not photobleached in the absence of O₂. The step-up photophobic response was also completely repressed in the absence of O₂. These results strongly confirm that blepharismin is a photoreceptor pigment mediating photobehavior of Blepharisma and that O₂ is required for the early step in the phototransduction of the light-excited pigment.     

Immunochemical analysis of a photoreceptor protein using anti-IP3 receptor antibody in the unicellular organism, Blepharisma

The blepharismin-200 kD protein complex of the ciliated protozoan Blepharisma is a novel type of photosensor responsible for the step-up 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.     

ISOLATION OF BLEPHARISMIN-BINDING 200 kDa PROTEIN RESPONSIBLE FOR BEHAVIOR IN Blepharisma

Abstract— The ciliated protozoan, Blepharisma, shows an avoidance reaction (step-up photophobic response) in response to light stimulation. A profile of a gel-permeation of a crude detergent-solubilized sample of the cells resulted in several red-colored fractions. Among these blepharismin-containing fractions, the fractions III-V did not contain amino acids. The peak of fraction II monitored by 580 nm absorbance was much smaller. A prominent peak appeared in fraction I, which contained a large amount of amino acids. The absorption spectrum of fraction I was well fitted to the action spectrum of the step-up photophobic response, although free pigment (blepharismin) also fitted. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction resulted in a thicker band corresponding to molecular mass of 200 kDa. These results suggest that the 200 kDa chromoprotein (blepharismin-protein complex) is responsible for the step-up photophobic response in Blepharisma. The absorption spectrum of free chromophore dissociated from the chromophore-protein complex was identical to free red pigment termed blepharismin. The absorption spectrum of the other fractions agreed with that of thin-layer chromatography-purified red pigment, indicating that the pigments contained in these fractions are free pigment dissociated from the chromophore-protein complex.     

CAFFEINE-ENHANCED PHOTOMOVEMENT IN THE CILIATE, STENTOR COERULEUS

The effects of caffeine, ionophores and calcium flux blockers on the step-up photophobic response, phototactic orientation and the intracellularly recorded, light-induced electrical action potential were studied in the ciliate, Stentor coeruleus . Caffeine alters the absorption and CD spectra and enhances the fluorescence of the photoreceptor pigment, stentorin. Independent of its effects on the spectroscopic properties of the photoreceptor pigment, caffeine shortens the photophobic response time by enhancing the Ca²⁺ conductivity of membranes, while Ca²⁺ flux blockers (LaCI₃ or ruthenium red) prolong it; both effects cancel each other. Evidence is presented that phototactic orientation is brought about by repetitive photophobic responses, since a change in the phobic response time results in a decreased accuracy of phototaxis.     

PHOTORECEPTOR PIGMENT IN Blepharisma: H+ RELEASE FROM RED PIGMENT

In faded cells of Blepharisma kept in a standard saline solution containing bacteria which had been cultured on agar plates containing glucose and polypepton, threshold light intensity for step-up photophobic response elevated. This result suggests that red pigment (blepharismin) contained in Blepharisma cells is involved in the step-up photophobic response. The pH of the aqueous solution of the red pigment was found to decrease when light was applied, indicating that the pigment releases H⁺ in response to light stimulation. However, faded pigment preparation by light irradiation did not show pH decrease. In the living cells faded by light irradiation, threshold light intensity for the step-up photophobic response was raised. Results suggest that H⁺ release from the red pigment induced by light irradiation might be responsible for the step-up photophobic response of the cells.     

PRESUMED PHOTORECEPTOR PROTEIN AND ULTRASTRUCI'URE OF THE PHOTORECEPTOR ORGANELLE IN THE CILIATED PROTOZOAN, Blepharisma

Abstract-The red pigment granule of Belpharisma japonicum is believed to be a photoreceptor organelle mediating photodispersal. Freeze-fracture and thin section electron microscopy revealed that the pigment granules contained a honeycomb-like structure constructed of folded membranes. In the fracture face of the honeycomb-like structure, small membrane particles were observed, which might correspond to pigment—protein complexes. The pigment granules were isolated and detergent-solubilized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the pigment granules mainly contained a 200 kDa membrane protein. The complex of red pigment and 200 kDa protein was isolated by gel-filtration chromatography of the detergent-solubilized components, and the protein was subjected to SDS-PAGE and amino acid analysis. The 200 kDa protein could not be dissociated into subunits by 2-mercaptoethanol, indicating that the protein is composed of a single polypeptide chain. Hydrophobic amino acids contained in the 200 kDa protein were not dominant, suggesting that only partial domains may extend across the membrane of the honeycomb-like structure.     

ACTION SPECTRA OF THE PHOTOPHOBIC RESPONSE OF BLUE AND RED FORMS OF Blepharisma japonicum

Abstract— When exposed, in the presence of molecular oxygen, to light intensities of the order of3–30 W m^-2, the ciliate Blepharisma japonicum changes its color from red to blue, because of the photooxidation of the photoreceptor pigment, blepharismin, to pxyblepharismin. Both red-and blue-pigmnentes cells show step-up photophobic responses. The action spectra f the light-dependent behaviour of the red and the blue form of Blepharisma have been determined; their structure is very similar to that the photosensing and phototransducing properties of blepharismin are maintained in its photooxidized form. oxyblepharismin.     

PHOTOMOVEMENT RESPONSES OF THE HETEROTRICHOUS CILIATE Blepharisma Japonicum

Light-induced movement responses of the heterotrichous ciliate Blepharisma japonicum were studied by physiological experiments. Two photosensory responses could be identified. A step-up photophobic response is observed as a very rapid backward movement. Microbeam irradiations of individual cells showed that only the anterior part of the ciliate is able to perceive the light stimulus that mediates the phobic reaction. The action spectrum peaks at approximately 400 nm, which indicates that a blue light receptor is involved.
Positive photokinesis of Blepharisma could be shown as a forward movement that is accelerated by increasing the applied photon fluence rate. The steady state level of the velocity depends highly on wavelength and photon fluence rate of the actinic light. After specific inhibition of the phobic reaction bv 1 m/W NH₄⁺, photokinesis can be induced by microbeam irradiation at any part of the cell.
We isolated two main pigments by thin layer chromatography and characterized them as hypericin-like compounds: a red pigment that is obviously responsible for the red color of the ciliates (= blepharismin). and a yellow one with maximal absorption near 420 nm. The possible photoreceptor functions of these pigments are discussed.
We could not find in Blepharisma a distinct phototactic behavior which is so typical for the related ciliate Stentor.     

Primary photoprocesses in oxyblepharismin interacting with its native protein partner

The primary photoprocesses in the photoreceptor for the step-up photophobic response of the light-adapted cells of Blepharisma japonicum (OBIP, OxyBlepharismin bInding Protein) have been studied by ultrafast UV–vis transient spectroscopy. The results are rationalized in terms of heterogeneity of the OBIP sample. Two independent classes of chromoprotein are proposed: a “reactive” species, which presents a specific 680-nm band decaying in 4 and 56 ps and a “non-reactive” one, which behaves like the free chromophore (OxyBP) in solution. A bimolecular photooxidation of OxyBP in the presence of 1,4-benzoquinone was performed to record the absorption spectrum of the OxyBP radical cation. Comparison with reactive OBIP suggests that an electron transfer could be involved in the primary photoprocesses of OBIP and possibly trigger the sensory transduction chain of B. japonicum. In addition, the specificity of the chromophore–protein interaction was investigated by studying the artificial complex that OxyBP forms with human serum albumin (HSA). OxyBP–HSA happens to be spectroscopically much closer to free OxyBP than to OBIP. This highlights the specific nature of the interaction between OxyBP and its native protein partner and further supports the proposal that OBIP is the actual photoreceptor for the photophobic response of B. japonicum.     

A videomicroscopic study of the effect of l-cis-diltiazem on the photobehavior of Stentor coeruleus

The protozoan ciliate Stentor coeruleus displays a step-up photophobic response to an increase in light intensity in its environment. The motile response consists of a delayed stop of ciliary beating and transient ciliary reversal period. Such light-avoiding behavior was significantly influenced by an incubation of cells with l-cis-diltiazem, a common blocker of cyclic guanosine monophosphate (cGMP)-gated ion channel conductance. The introduction of l-cis-diltiazem to the medium induced ciliary reversal in control cells, mimicking the step-up photophobic response. In light-stimulated ciliates, the presence of this inhibitor caused a substantial decrease of the latency of ciliary stop response, prolongation of the ciliary reversal duration and also an increase of cell photoresponsiveness in a dose- and time-dependent manner. The obtained behavioral results support the suggestion that the photosensitive ciliate S. coeruleus possesses cGMP-gated channels, which may be involved in the process of light signal transduction for the motile photophobic response.     

BEHAVIORAL RESPONSES IN Paramecium multimicronucleatum TO VISIBLE LIGHT

Specimens of colorless Paramecium multimicronucleatum were found to respond to visible light. They accumulated in the shaded region (photodispersal) of a half-shaded glass tube during 2 min exposure to visible light. The specimens showed avoiding reaction upon both spatial and temporal increase in light intensity (step-up photophobic response). Steady-state swimming velocity (orthokinesis) was higher, while steady-state frequency of spontaneous change in swimming direction (klinokinesis) was lower when the light intensity was kept higher. In a light with wavelength of 440 nm the velocity was highest, while the frequency was lowest. The specimens did not show phototaxis (light direction-oriented locomotion). Spectral sensitivity curves for both the photodispersal and the step-up photophobic response showed a major peak at 520 nm and a minor peak at 680 nm. The photodispersal seems to be caused mainly by the step-up photophobic response exhibited by the specimens at the dark-light border. The photokinetic responses enhance the degree of the photodispersal.     

Stentor coeruleus SHOWS POSITIVE PHOTOKINESIS

Abstract— Stentor coeruleus responds to a sudden increase in light intensity with a step-up photophobic response (avoiding reaction), and to collimated light with negative phototaxis. The peaks of the action spectra for the photophobic response and for the phototaxis are in common, 610 nm.
5. coeruleus showed changes in its steady-state swimming velocity induced with varying intensities of light (photokinesis). The cells swam fast in light regions but slowly in dark ones (positive photokinesis); the mean velocity of swimming was about 0.6 mm/s at 100 lx but reached about 1.0 mm/s at 50000 lx. The peak of the action spectrum for this photokinesis was about 680 nm.
The organism is the first protozoan cell reported to show three types of photoresponse: photophobic response, phototaxis and photokinesis.     

PHOTOTACTIC ORIENTATION BY THE CILIATE, STENTOR COERULEUS

Stentor coeruleus exhibits negative phototaxis to visible light, in addition to a step-up photophobic response. The negative phototaxis was established by demonstrating the swimming of Stentor toward a focused beam away from the light source. The action spectrum showed a maximum at 610–620 nm and is essentially identical to that of the step-up photophobic response. Proton uncouplers such as micromolar concentrations of FCCP and TPMP⁺ inhibited the negative phototaxis.     

9-cis Retinal increased in retina of RPE65 knockout mice with decrease in coat pigmentation

The protein RPE65 is essential for the generation of the native chromophore, 11-cis retinal, of visual pigments. However, the Rpe65 knockout (Rpe65-/-) mouse shows a minimal visual response due to the presence of a pigment, isorhodopsin, formed with 9-cis retinal. Isorhodopsin accumulates linearly with prolonged dark-rearing of the animals. The majority of Rpe65-/- mice have an agouti coat color. A tan coat color subset of Rpe65-/- mice was found to have an enhanced visual response as measured by electroretinograms. The enhanced response was found to be due to increased levels of 9-cis retinal and isorhodopsin pigment levels. Animals of both coat colors reared in cyclic light have minimal levels of regenerated pigment and show photoreceptor degeneration. On dark-rearing, pigment accumulates and photoreceptor degeneration is decreased. In the tan Rpe65-/- mice, the level of photoreceptor degeneration is less than in the agouti animals, which have an increased pigment and decreased free opsin level. Therefore, photoreceptor damage correlates with the amount of the apoprotein present, supporting findings that the activity from unregenerated opsin can lead to photoreceptor degeneration.     

PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF Euglena grcrcilis

Abstract —Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity Chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K_D= 4 n M ) as well as under reducing conditions in the presence of dithionite (K_d= 6 n M ). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately lo⁶ flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.     

Origin of the vertebrate visual cycle: III. Distinct distribution of RPE65 and beta-carotene 15,15'-monooxygenase homologues in Ciona intestinalis

We previously identified three genes that encode putative visual cycle proteins that are homologues of retinal G-protein coupled receptor (Ci-opsin3), cellular retinaldehyde-binding protein (Ci-CRALBP) and beta-carotene 15,15'-monooxygenase (Ci-BCO) in the ascidian Ciona intestinalis. Ci-opsin3 and Ci-CRALBP are localized in both ocellus photoreceptor cells and surrounding non-photoreceptor cells in the brain vesicle of the larva. In the present study, we investigated the possible role and evolutionary origin of the BCO/RPE65 family in the visual cycle by analyzing Ci-BCO localization by immunohistochemistry and by identifying a novel gene that encodes a homologue of retinal pigment epithelium-specific 65 kDa protein (Ci-RPE65) in C. intestinalis. In situ hybridization and expressed sequence tag (EST) profiles consistently suggest that Ci-RPE65 is not significantly expressed in the ocellus and brain vesicle of the larva. Ci-RPE65 is expressed in the neural complex, a photoreceptor organ of the adult ascidian, at a level comparable to that of Ci-opsin3 and Ci-CRALBP. Ci-RPE65 is also expressed in various adult tissues, including the gill, body wall and intestine, suggesting that Ci-RPE65 plays a role in addition to that in the visual cycle. In contrast, Ci-BCO is predominantly localized in ocellus photoreceptor cells of the larva. The larval visual cycle seems to use Ci-opsin3 as a photo-isomerase. Our results also suggest that the RPE65-dependent visual cycle is used in the adult photoreceptors of a primitive chordate.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. IV. MODULATION OF THE PHOTOMOVEMENT RESPONSE OF Blepharisma japonicum BY cGMP

Abstract— The effect of various modulators of cytoplasmic guanosine 3',5'-cyclic monophosphate (cGMP) level on the step-up photophobic responses in Blepharisma japonicum has been investigated to clarify the possible role of cGMP in the mechanism of photosensory signal transduction. Membrane-permeable analogs of cGMP, 8-bromo-guanosine 3',5'-cyclic monophosphate or dibutyryl cGMP, caused a marked dose-dependent prolongation of the latency for the photophobic response, resulting in inhibition of the photophobic response in Blepharisma japonicum. A similar effect was observed when cells were treated with 3'-isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, and pertussis toxin, a G-protein activity modulator. The G-protein activator, fluoroaluminate, and 6-anilino-5,8-quinolinedione (LY 83583), an agent which effectively lowers the cytoplasmic cGMP level, significantly enhanced the photoresponsiveness of these ciliates to visible light stimuli. These results suggest that cellular cGMP serves as a signal modulator in the photophobic response of Blepharisma japonicum.     

Maristentorin, a novel pigment from the positively phototactic marine ciliate Maristentor dinoferus, is structurally related to hypericin and stentorin

The photoreceptor pigment of the heterotrich ciliate, Maristentor dinoferus, has been characterized. It is structurally similar to those of Stentor coeruleus and Blepharisma japonicum but differs significantly in that it bears no aromatic hydrogens. The structure of the pigment, maristentorin, is based upon the hypericin skeleton, and its spectra are nearly identical to those of hypericin but shifted toward the red. Within experimental error, its fluorescence lifetime is identical to that of hypericin, approximately 5.5 ns in dimethylsulfoxide. It is remarkable that while the pigments are structurally similar in S. coeruleus and M. dinoferus, in the former there is an abrupt photophobic response, whereas in the latter there is a slow response toward light. The roles of the hypericin-like pigments in the heterotrich ciliates are discussed as potentially analogous in Maristentor.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. III. THE TEMPORAL RELATION BETWEEN MEMBRANE POTENTIALS AND PHOTOMOTILE RESPONSES IN Blepharisma japonic urn

Abstract— Blepharisma japonicum exhibits a step-up photophobic response when subjected to an increase in light stimulus intensity. This response is characterized by the stop reaction after a period of delay followed by backward swimming (lateral rotation). The latency of the stop response decreased and duration of the lateral rotation increased as the intensity of light stimuli was raised. A step-increase in light intensity elicited a graded membrane depolarization (photic receptor potential), as measured by intracellular microelectrode. When the amplitude of receptor potential exceeded a threshold depolarization for membrane excitation (15–25 mV), an all-or-none action potential of 50–65 mV in amplitude was evoked which also occurred with some latency. Light stimuli of higher intensity (suprathreshold) elicited action potential which was followed by a membrane after-depolarization. Increasing the intensity of stimuli caused generation of an action potential with shorter lag period and prolonged after-depolarization. The action spectra for the latency of stop reaction, receptor potential amplitude and cell photoresponsiveness showed maxima at 460, 530 and 580 nm. The analysis of temporal relationships between the electrophysiological responses and the motile events showed that latency of an action potential, induced by the receptor potential, correlates well with the latency of a cell stop response. Also the duration of membrane after-depolarization resembled the time period of the cell's backward swimming (cell rotation). The data obtained indicate that the primary reaction initiated by light absorption in the photoreceptor pigment (blepharismin) is converted into the observed electrical potential changes, which in turn results in the photomotile response of Blepharisma cells.