Alterations of ciliate phosducin phosphorylation in Blepharisma japonicum cells

We have previously reported that motile photophobic response in ciliate Blepharisma japonicum correlates with dephosphorylation of a cytosolic 28 kDa phosphoprotein (PP28) exhibiting properties similar to those of phosducin. Here we demonstrate in in vivo phosphorylation assay that the light-elicited dephosphorylation of the PP28 is significantly modified by cell incubation with substances known to modulate protein phosphatase and kinase activities. Immunoblot analyses showed that incubation of ciliates with okadaic acid and calyculin A, potent inhibitors of type 1 or 2A protein phosphatases, distinctly increased phosphorylation of PP28 in dark-adapted cells and markedly weakened dephosphorylation of the ciliate phosducin following cell illumination. An enhancement of PP28 phosphorylation was also observed in dark-adapted ciliates exposed to 8-Br-cAMP and 8-Br-cGMP, slowly hydrolysable cyclic nucleotide analogs and 3-isobutyryl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiesterase (PDEs) inhibitor. Only slight changes in light-evoked dephosphorylation levels of PP28 were observed in cells treated with the cyclic nucleotide analogs and IBMX. Incubation of ciliates with H 89 or KT 5823, highly selective inhibitor of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, decreased PP28 phosphorylation levels in dark-adapted cells, whereas the extent of light-evoked dephosphorylation of the phosphoprotein was only slightly influenced. Cell treatment with higher Ca2+ concentration together with ionophore A23187 in culture medium resulted in marked increase in PP28 phosphorylation levels, while quite an opposite effect was observed in cells exposed to Ca2+ chelators, EGTA or BAPTA/AM as well as calmodulin antagonists, such as trifluoperazine (TFP), W-7 or calmidazolium. Light-dependent dephosphorylation was not considerably affected by these treatments. The experimental findings presented here suggest that an endogenous light-dependent protein kinase-phosphatase system may be engaged in the alteration of phosducin phosphorylation in ciliate B. japonicum thereby to modulate the cell motile photophobic behavior.     

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.     

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.     

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.     

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 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. 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.     

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.     

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.     

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.