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.     

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.     

Photosensory transduction in unicellular eukaryotes: a comparison between related ciliates Blepharisma japonicum and Stentor coeruleus and photoreceptor cells of higher organisms

Blepharisma japonicum and Stentor coeruleus are related ciliates, conspicuous by their photosensitivity. They are capable of avoiding illuminated areas in the surrounding medium, gathering exclusively in most shaded places (photodispersal). Such behaviour results mainly from motile photophobic response occurring in ciliates. This light-avoiding response is observed during a relatively rapid increase in illumination intensity (light stimulus) and consists of cessation of cell movement, a period of backward movement (ciliary reversal), followed by a forward swimming, usually in a new direction. The photosensitivity of ciliates is ascribed to their photoreceptor system, composed of pigment granules, containing the endogenous photoreceptor -- blepharismin in Blepharisma japonicum, and stentorin in Stentor coeruleus. A light stimulus, applied to both ciliates activates specific stimulus transduction processes leading to the electrical changes at the plasma membrane, correlated with a ciliary reversal during photophobic response. These data indicate that both ciliates Blepharisma japonicum and Stentor coeruleus, the lower eukaryotes, are capable of transducing the perceived light stimuli in a manner taking place in some photoreceptor cells of higher eukaryotes. Similarities and differences concerning particular stages of light transduction in eukaryotes at different evolutional levels are discussed in this article.     

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.     

Light Induces lnositol Trisphosphate Elevation in Blepharisma japonicum

Abstract— Photoinduced formation of inositol 1,4,5-trisphosphate (Ins[1,4,5]P₃) was examined using a specific radioimmu-noassay to investigate the molecular mechanisms of light signal transduction mediating photophobic responses in the ciliate Blepharisma japonicum. Application of light stimuli of moderate intensity to dark-adapted cells induced a rapid and significant increase in the basal level of Ins (1,4,5)P₃, with a peak at about 20 s. Thereafter, the level of Ins (1,4,5)P₃ declined to the resting value within the subsequent 100 s. Light stimuli of higher intensity raised the cell Ins (1,4,5)P₃ content to still higher levels within about 20 s, but the decaying time course was considerably prolonged. In ciliates incubated under dark conditions with agents interfering with the inositol signalling pathway, like neomycin and Li⁺ the basal levels of Ins (1,4,5)P₃ were lower than in control cells. A photoinduced rise of Ins (1,4,5)P₃, content in ciliates treated with neomycin or Li⁺ was significantly inhibited in a dose-dependent manner. Depolarizing ionic stimuli in dark-adapted ciliates induced no significant alterations of the resting Ins (1,4,5)P₃ level, indicating a lack of a contribution of this kind of stimulation to the inositol turnover. These studies are the first in vivo demonstration of a possible role for inositol trisphosphate as a second messenger in the light signal transduction process in the ciliate B. japonicum.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. I. AN ANALYSIS OF LIGHT-INDUCED ELECTRICAL AND MOTILE RESPONSES IN Stentor coeruleus

Abstract— Light-induced membrane potantial changes and motile responses have been studied in Stentor cells with intracellular microelectrodes and video microscopy, respectively. Intracellulae microelectrode showed that step-up increase in light induced an electrical membrane response which consisted of an initial membrane depolarization (photoreceptor potential) followed by an action potential and maintaining phase of depolarization (afterdepolarization). The amplitude of the receptor potetial is dependent on the intensity of light stimulus and the action potetials appears with a lag period (latency) after the onset of light stimuklus. The extent of the membrane established between the latency for te action poitential and the onset of ciliary reversal (stop responses). A time correlation was also observed between the duration of the membrane afterdepolarization and the duration of backward swimming. the action spectrum for the photoreceptor potential amplitude of Stentor resembled the action spectra for the latency of ciliary reversal and the photoresponsiveness, iondicating that the photomovement response and membrane potential changes are coupled through the same photosensor system. A hypothesis on the photosensory transduction chain in Stentor is discussed according to ehich the photoreceptors and the ciliary apparatus is mediated by the membrane potential canges.     

PHOTOSENSORY TRANSDUCTION IN CILIATES. II. POSSIBLE ROLE OF G-PROTEIN AND cGMP IN Stentor coeruleus

Abstract— The heterotrichous ciliate, Stentor coerulus , exhibits a welll defines photophobic response to a sudden increase in the intensity of visible light. the phobic reactions usually appear with a latency perios (i.e. a time delay between the onset of the stimulus and the stop response). This latency of phobic response was significatly increased when the cells werw incubated with 8-bromo-guanosine3',5'-cyclic monophospjhate. In the presence of this nucleotide, a reduction of cell responsiveness (i.e. the number of photophobically responding cells) was also observed. similar effects were observed when cells were treated with pertussis toxin, a G-protein activity modulator, and 3'-isobutyl-methylxanthine, an inhibitor of guanosine 3', 5'-cyclic monophosphate (cGMP) phosphodiesterase. the G-protein activator fluoroaluminate and 6-anilino-5,8-quinolinedione (LY 83583) (an effective agent for lowerin cellular cGMP levels) showed opposite effects on hte cell photophobic response. These result indirectly suggesnt that the level of cytoplamic cGMP, possibly modulated by a G-protein-coupled CGMP phosphodiesterase, plays a phototreasducing role in Stentor . In addition, using an antiserum raised against bovine transducin, a cross reacting protein with an apparent molecular mass of 39 kDa was detected on immunoblots. The α-subunits of a Stentor G-protein has also been partially cloned and sequenced. However, the possible coupling between the G-protein and the putative phosphodiesterase remains to be established.     

Additional evidence for the cyclic GMP signaling pathway resulting in the photophobic behavior of Stentor coeruleus.

We report that exo- and endogenous guanosine 3',5'-cyclic monophosphate (cGMP) specifically influenced the photophobic response. In behavioral experiments the slowly hydrolyzable and membrane-permeable analogs of cGMP (8-bromo-cGMP [Br-cGMP] and N6,2'-o-dibutyryl-cGMP) dramatically prolonged the time for ciliary stop response and decreased the duration of ciliary reversal in a dose-dependent manner. When analogs of adenosine 3',5'-cyclic monophosphate (cAMP) (8-bromo-cAMP or N6,2'-o-dibutyryl-cAMP) were used, no essential effects were detected on the kinetics of the photophobic response. Both nonspecific cyclic nucleotide phosphodiesterase (PDE) activity inhibitors (3-isobutyl-1-methylxanthine [IBMX] and 1,3-dimethylxanthine [theophylline]) and the highly specific cGMP-PDE activity inhibitor 1,4-dihydro-5-[2-propoxyphenyl]-7H-1,2,3-triazolo[4,5-d]pyrimidine-7-one (zaprinast) mimicked the effects of cGMP analogs. Treatment of cells with an inhibitor of guanylate cyclase activity (6-anilino-5,8-quinolinedione [LY 83583]) exerted an effect opposite to that of cGMP analogs and PDE activity inhibitors. The positive physiological effect of LY 83583 was significantly diminished in ciliates that were treated simultaneously with Br-cGMP. In an assay of cell cyclic nucleotide content, the exposure of dark-adapted Stentor to light evoked a transient decrease in the basal level of intracellular cGMP. Alterations in internal cGMP levels were more distinct when the intensity of applied illumination was increased. In the presence of IBMX or theophylline the basal content of cGMP was markedly enhanced, and the photoinduced changes in cGMP level were less pronounced. In this paper the possible whole molecular mechanism by which the ciliary orientation in Stentor is controlled by light is presented.     

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.     

Localization of Blepharismin Photosensors and Identification of a Photoreceptor Complex Mediating the Step-up Photophobic Response of the Unicellular Organism, Blepharisma

Abstract— Photosensitivity for the step-up photophobic response of Blepharisma is localized in the anterior 1/5 of the cell body. Blepharismin pigment, which is believed to be a photoreceptor pigment mediating the step-up photophobic response of the cells, was separated into five types of blepharismin (BL-1, -2, -3, -4 and -5). Blepharismin-1, -3, -4 and -5 were localized in the posterior 4/5, while BL-2 was located over the entire cell body; the anterior end, which is the photosensitive region, contained only BL-2. The results indicate that a functional photoreceptor pigment mediating the step-up photophobic response is BL-2. Hydroxylapatite chromatography revealed that BL-2 was bound to a 200 kDa membrane protein. We concluded that a photoreceptor mediating the step-up photophobic response was a BL-2/200 kDa protein complex.     

ACTION SPECTRUM AND ELECTROPHYSIOLOGICAL RESPONSES CORRELATED WITH THE PHOTOPHOBIC RESPONSE OF STENTOR COERULEUS

Abstract— The blue-green ciliate. Stentor coeruleus , is found predominantly in shady places. This concentration occurs because stentor responds when swimming from a shaded area to a lighted area by reversing the direction of its ciliary beat and reorienting its swimming direction until it once again is in the shaded area. A graded receptor potential is recorded from microelectrodes in vacuoles of stentor when the animal is photically stimulated. For all but very weak stimuli this receptor potential is sufficient to elicit a regenerative transmembrane response of variable amplitude in a swimming animal. Suprathreshold electrical stimuli also elicit this regenerative response. In turn the regenerative response is coupled to ciliary reversal. Thus ciliary reversal appears to be produced whenever the photic receptor potential crosses the threshold for elicitation of the regenerative response.
Using the threshold for production of ciliary reversal as a criterion response, an action spectrum was obtained. This action spectrum correlates well with the absorption spectrum of the major pigment of S. coeruleus , stentorin. Stentor bleached of pigment also have an elevated threshold for ciliary reversal. Thus stentorin seems to be the photosensitive pigment in stentor responsible for its photophobic behavior.     

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.     

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.     

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.     

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.     

Action Spectra for UVB Impacts on Blepharisma japonicum Motility and Photobehavior

Abstract— The ciliate Blepharisma japonicum was exposed to artificial polychromatic and monochromatic UV radiation to evaluate the relative roles of UVB (280–315 nm UV radiation) and UVA (315–400 nm UV radiation) in altering its motility and photobehavior and to determine absolute weighting coefficients for these effects in the UVB range. Under polychromatic UV irradiation B. japonicum cells showed a severe reduction of cell speed and of the capability to respond to light stimuli. At low doses, however, UV caused a significant increase in the average velocity of a cell population. The UVB exclusion experiments indicated that UVA does not significantly alter motility and photoresponsiveness. The increase and the subsequent decrease in cell velocity was observed also under monochromatic irradiation at 281, 290 and 300 nm, whereas at 310 nm cells swim faster up to the highest photon flux density used. The cell capability of reacting to photic stimuli, conversely, steadily declined with increasing photon flux density at all the tested UVB wavelengths. The action spectra for the alteration of cell velocity and the impairment of photoresponsiveness show that the lower the irradiation wavelength, the more remarkable are the UVB effects and suggest different targets for the increase and the decrease in cell velocity.     

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.     

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.     

EFFECTS OF K+ AND Ca2+ IONS ON MOTILITY AND PHOTOSENSORY RESPONSES OF STENTOR COERULEUS

Extracellular K⁺ ions above a critical concentration induce ciliary reversal in unstimulated Stentor coeruleus and suppress step-up photophobic response. This threshold concentration of K⁺ ions depends on the extracellular Ca²⁺ concentration, and the subsequent backward gyration and light-sensitivity suppression seem to depend on the relative concentrations of K⁺ and Ca²⁺. The concentration of Ca²⁺ necessary to overcome K⁺-mediated inhibition of phobic response and backward swimming increases non-linearly with increasing K⁺ concentration. The Ca²⁺-blocking agent. D-600, selectively inhibits photophobic responses of Stentor , thus further confirming the role of Ca²⁺ ions in photosensory transduction of this ciliate.     

ROLE OF ENDOSYMBIOTIC ALGAE IN PHOTOKINESIS AND PHOTOPHOBIC RESPONSES OF CILIATES

Abstract— The photobehavior of the ciliates Climacostomuum virens, Euplotes daidaleos and Paramecium bursaria associated with endosymbiotic green algae ( Chlorella sp.) was compared with that of aposymbiotic ciliates (ciliates free of algae) and with the always aposymbiotic Paramecium arcticum . The aposymbiotic ciliates showed step-up photophobic responses. The responses were more frequent and occurred at lower light intensities when the organisms contained endosymbiotic algae. In contrast, step-down photophobic responses and photoaccumulations, as well as photokinesis, were never observed in aposymbiotic cells. These responses could be found only in ciliates when endosymbionts were present with the exception of C. virens , which showed none of these photoresponses.