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.     

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.     

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.     

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.     

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.     

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.     

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.     

Contribution of phosphoinositide-dependent signalling to photomotility of Blepharisma ciliate

The effect of experimental procedures designed to modify an intracellular phosphoinositide signalling pathway, which may be instrumental in the photophobic response of the protozoan ciliate Blepharisma japonicum, has been investigated. To assess this issue, the latency time of the photophobic response and the cell photoresponsiveness have been assayed employing newly developed computerized videorecording and standard macro-photographic methods. Cell incubation with neomycin, heparin and Li+, drugs known to greatly impede phosphoinositide turnover, causes evident dose-dependent changes in cell photomotile behaviour. The strongest effect on photoresponses is exerted by neomycin, a potent inhibitor of polyphosphoinositide hydrolysis. The presence of micromolar concentrations of neomycin in the cell medium causes both prolongation of response latency and decrease of cell photoresponsiveness. Neomycin at higher concentrations (> 10 microM) abolishes the cell response to light at the highest applied intensity. A slightly lower inhibition of cell responsiveness to light stimulation and prolongation of response latency are observed in cells incubated in the presence of heparin, an inositol trisphosphate receptor antagonist. Lithium ions, widely known to deplete the intracellular phosphoinositide pathway intermediate, inositol trisphosphate, added to the cell medium at millimolar level, also cause a slowly developing inhibitory effect on cell photoresponses. Mastoparan, a specific G-protein activator, efficiently mimics the effect of light stimulation. In dark-adapted ciliates, it elicits ciliary reversal with the response latency typical for ciliary reversal during the photophobic response. Sustained treatment of Blepharisma cells with mastoparan also suppresses the photoresponsiveness, as in the case of cell adaptation to light during prolonged illumination. The mastoparan-induced responses can be eliminated by pretreatment of the cells with neomycin. Moreover, using antibodies raised against bovine transducin, a cross-reacting protein with an apparent molecular mass of about 55 kDa in the Blepharisma cortex fraction is detected on immunoblots. The obtained results indirectly suggest that the changes in internal inositol trisphosphate level, possibly elicited by G-protein-coupled phospholipase C, might play a role in the photophobic response of Blepharisma. However, further experiments are necessary to clarify the possible coupling between the G-protein and the putative phospholipase C.     

PHOTODYNAMIC ACTION IN Stentor coeruleus SENSITIZED BY ENDOGENOUS PIGMENT STENTORIN

Abstract— Stentorin acts as the photoreceptor for the step-up photophobic and negative phototactic responses in Stentor coeruleus . The chromophore of stentorin appears to be hypericin which is linked to apoprotein. In addition to the photomovement responses of the organism, S. coeruleus was found to be photodynamically sensitive to light absorbed by the hypericin chromophore, as the apparent action spectrum for the photodynamic killing matches the absorption spectrum of stentorin. The protective effect of β-carotene and crocetin on the photodynamic killing of S. coeruleus suggests that singlet oxygen generated by the stentorin-sensitization plays an important role, according to the so-called Type II mechanism of photosensitization. The generation of singlet oxygen via hypericin triplet was confirmed by in vitro photooxidation of tryptophan as a substrate. The photodynamic killing was more effective in deuterium oxide than in H₂O in both the photosensitization by stentorin (endogenous) and added hypericin (exogenous). These results are consistent with the involvement of singlet oxygen in the photodynamic killing of S. coeruleus .     

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.     

PHOTORECEPTOR INTERACTION IN PLANT PHOTOMORPHOGENESIS: THE LIMITS OF EXPERIMENTAL TECHNIQUES AND THEIR INTERPRETATIONS

Abstract— Problems concerning the interpretation of interactions of higher plant photomorphogenetic receptors are discussed. The theory that action of a blue light photoreceptor serves only to maintain responsiveness to phytochrome (Responsiveness Theory) is demonstrated to be unable to be properly tested with present techniques. This theory is also unable to explain experimental results any better than an alternative theory that a blue light photoreceptor may require the presence of the active form of phytochrome to express its activity (Presence Theory). This tatter theory is also incapable of being fully tested. There does not appear to be an adequate current theory to explain photoreceptor interactions. Other issues discussed include the use of displacement transducers in growth studies, the induction of phytochrome-type responses by blue light, and the relative importance of the photoreceptors. New data are introduced on the effect of blue light in the end-of-day growth response to phytochrome of the light-grown Cucumis sativus L. hypocotyl, and on the light equivalence principle in the same species.     

Phototransduction: different mechanisms in vertebrates and invertebrates

The photoreceptor cells of invertebrate animals differ from those of vertebrates in morphology and physiology. Our present knowledge of the different structures and transduction mechanisms of the two animal groups is described. In invertebrates, rhodopsin is converted by light into a meta-rhodopsin which is thermally stable and is usually re-isomerized by light. In contrast, photoisomerization in vertebrates leads to dissociation of the chromophore from opsin, and a metabolic process is necessary to regenerate rhodopsin. The electrical signals of visual excitation have opposite character in vertebrates and invertebrates: the vertebrate photoreceptor cell is hyperpolarized because of a decrease in conductance and invertebrate photoreceptors are depolarized owing to an increase in conductance. Single-photon-evoked excitatory events, which are believed to be a result of concerted action (the opening in invertebrates and the closing in vertebrates) of many light-modulated cation channels, are very different in terms of size and time course of photoreceptors for invertebrates and vertebrates. In invertebrates, the single-photon events (bumps) produced under identical conditions vary greatly in delay (latency), time course and size. The multiphoton response to brighter stimuli is several times as long as a response evoked by a single photon. The single-photon response of vertebrates has a standard size, a standard latency and a standard time course, all three parameters showing relatively small variations. Responses to flashes containing several photons have a shape and time scale that are similar to the single-photon-evoked events, varying only by an amplitude scaling factor, but not in latency and time course. In both vertebrate and invertebrate photoreceptors the single-photon-evoked events become smaller (in size) and faster owing to light adaptation. Calcium is mainly involved in these adaptation phenomena. All light adaptation in vertebrates is primarily, or perhaps exclusively, attributable to calcium feedback. In invertebrates, cyclic AMP (cAMP) is apparently another controller of sensitivity in dark adaptation. The interaction of photoexcited rhodopsin with a G-protein is similar in both vertebrate and invertebrate photoreceptors. However, these G-proteins activate different photoreceptor enzymes (phosphodiesterases): phospholipase C in invertebrates and cGMP phosphodiesterase in vertebrates. In the photoreceptors of vertebrates light leads to a rapid hydrolysis of cGMP which results in closing of cation channels. At present, the identity of the internal terminal messenger in invertebrate photoreceptors is still unsolved.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

PHOTOCONTROL OF HYPOCOTYL ELONGATION IN DE-ETIOLATED Cucumis sativus L. RAPID RESPONSES TO BLUE LIGHT

Abstract— Rapid inhibition of hypocotyl extension of de-etiolated Cucumis sativus L. by blue light is described, and compared with responses to white fluorescent light. Rapid inhibition of hypocotyl growth by blue light via the specific blue light photoreceptor requires a minimum Buence rate. Above this minimum value rapid modulations of growth rate accompany changes in blue light fluence rate. An initial response to blue light, or to a step-up in blue fluence rate takes less than 5 min. A drop from high to low fluence rate blue light (or to darkness) is followed by a recovery of the growth rate after about 20 min. A change from a low fluence rate to darkness elicits a more rapid recovery of growth rate (within 7 min). Similar responses were obtained in seedlings de-etiolated for a few hours and for several days.     

DEPOLARIZATION OF MOUSE MYELOMA CELL MEMBRANES DURING PHOTODYNAMIC ACTION

The effect of photodynamic action on plasma membranes was examined using a fluorescent potentiometric indicator [di-SBA-C2(3)] to measure alterations in the plasma membrane potential of mouse myeloma cells treated with zinc phthalocyanine sulfonate and light. Plasma membrane depolarization was observed to be an early event in photodynamic action, showing both photosensitizer concentration and light dose dependence. Depolarization occurred while membrane integrity was retained and appears to be an early event preceding cell death.     

Phototaxis in the ciliated protozoan Ophryoglena flava: dose-effect curves and action spectrum determination

The sensitivity of positive phototactic orientation of cells of the ciliated protozoan Ophryoglena flava has been measured for white light, broad-band blue and red light, and narrow-band monochromatic light, using a laboratory-developed computer aided system. The white-light fluence rate-response curve shows that there is no negative phototaxis in the fluence rate range investigated (0-15 W/m2) and no adaptation phenomena; it is very well fitted by a hyperbolic function; the fluence rate curves under broad band blue and red light (full width at half maximum, FWHM= 100 nm) can be fitted by the same model. The saturation level is, within experimental errors, the same for the three curves, indicating that there are no chromaticity effects and that if there is more than one photoreceptor pigment, they act independently of each other. The fluence rate-response curves determined under narrow band monochromatic light (FWHM = 10 nm) can also be fitted by the same model and show, within experimental errors, the same saturation level. An action spectrum for positive phototaxis at 10-nm intervals has been calculated from fluence rate-response curves: it shows three maxima, at 420, 540 and 590 nm. This action spectrum is significantly different from the ones for photomotile responses in Blepharisma japonicum, Stentor coeruleus and Chlamydodon mnemosyne, whereas it resembles the ones of Paramecium bursaria and Fabrea salina.     

PHOTOTROPISM IN PHYCOMYCES MUTANTS LACKING β-CAROTENE

Abstract. β-carotene and riboflavin are considered as the major candidates for the photoreceptor for physiological responses to blue light in Phycomyces and a number of other organisms. Mutants of Phycomyces blocked in all six steps of the biosynthesis of β-carotene from phytoene contain no detectable β-carotene (less than 4 times 10^-5 of wild-type amount) but exhibit phototropic responses identical to wild-type. Moreover, wild-type Phycomyces , while abundant in trans -β-carotene, contains no detectable cis -β-carotene, sometimes proposed as a photoreceptor candidate on the basis of the close similarity of the cis -species absorption spectrum in the near UV region to many action spectra for blue-light responses. These results indicate that β-carotene cannot be the photoreceptor for phototropism in Phycomyces.     

Two types of kinetics of membrane potential of water plant leaves illuminated by ultraviolet light

The effects of ultraviolet-B (UV-B) radiation on plasma membrane of water plant (Elodea canadensis, Vallisneria spiralis) cells were investigated by using microelectrode methods. A fast and reversible depolarization of membrane potential occurs initially during exposure of leaf cells to UV on a white light background, after which a slow phase of depolarization sets in. On action series, UV is pulsed for 15 s, with dark interval of 3 min, no monotonous response of systems on the UV excitation is observed. The action spectrum of the fast UV response lies in the interval of 300-330 nm and that of the slow phase-in the interval of 280-300 nm. The input impedance of membranes remains unchanged during the period of exposure. It is concluded that the H(+)-extruding complex of plant cell plasma membranes really consists of two types of interrelated electronic H(+)-pumps: an H(+)-pump of redox-active nature and the H(+)-pump of the H(+)-ATPase enzyme complex. Clearly, during the exposure of leaf cells to UV light, initially, the H(+)-pump of redox-active nature and then H(+)-ATPase are inhibited. It is proposed that the initial chromophore of UV-B light on plasma membrane can be one of the components of H(+)-pump of redox-active nature. It is probably the molecular of quinone.     

ACTION SPECTRUM FOR THE PHOTOPERIODIC INDUCTION OF BULB FORMATION IN Allium cepa L.

Abstract— The action spectrum for the induction of bulb formation in Allium cepa L. was determined by continuous irradiation with monochromatic light for 3 h in the middle of the daily light period of the inductive photoperiod (18 h). The resultant action spectrum showed a single peak at 714 nm. Daily 3 h dark interruptions in the middle of inductive long photoperiods do not permit bulb formation. Bulb formation was inhibited also by monochromatic far red light (758 nm) applied at the beginning of the 3 h dark interruptions. The results of dichromatic irradiations indicate that phytochrome is the only photoreceptor involved in the photoperiodic regulation of bulbing in onion plants. The effect of far red light exhibits the characteristics (action spectrum and fluence rate dependency) of a classical "HIR" response.