Stanisław Fabczak

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. Role of intracellular pH and comparison between Stentor coeruleus and Blepharisma japonicum.

To test the hypothesis that light signal transduction in the unicellular ciliates Stentor coeruleus and Blepharisma japonicum involves a change in intracellular pH as an initial signal following photoexcitation, we studied the dependence of the photophobic responses of the cells to changes in extracellular pH and to reagents that specifically affect intracellular pH. The extracellular pH can modify not only the intracellular pH, but can even reverse the sign of the pH gradient across the cell membrane. Thus, as predicted by the hypothesis, low extracellular pH reversibly inhibited the photophobic response of the ciliates. The intracellular pH-modulating reagents tested included ammonium chloride, a membrane-permeable weak acid that lowers the intracellular pH, and the protonophores carbonylcyanide m-chlorophenyl-hydrazone (CCCP) and carbonylcyanide p-(trifluoromethoxy)-phenyl-hydrazone (FCCP), which collapse the pH gradient across the cell membrane. The low pH and protonophore treatments caused a gradual inhibition of the photophobic responses in both ciliates. The observed reduction of the responsiveness of the cells to visible light can be attributed to the alteration of the intracellular pH, which is suggested to play a specific role in the photosensory transduction in both Stentor coeruleus and Blepharisma japonicum.

A Centrin3-dependent, transient, appendage of the mother basal body guides the positioning of the daughter basal body in Paramecium.

Basal bodies are tightly controlled not only for their time of duplication but also for their movements, which ensure proper division and morphogenesis. However, the mechanisms underlying these movements only begin to be explored. We describe here a novel basal body appendage in Paramecium, the anterior left filament (ALF), which develops transiently from the mother basal body before duplication and disassembles once the new basal body is docked at the surface. By comparing the ultrastructure of dividing wild type cells to that of cells defective in basal body duplication, either by depletion of conserved proteins required for basal body assembly, or by mutation, we showed 1) that assembly of the ALF requires PtCen3p, one of the two basal body specific centrins and 2) that absence of the ALF correlates with a failure of the newly assembled basal bodies to tilt up to their docking site at the surface. This correlation suggests that the function of the ALF consists in anchoring centrin-containing contractile fibers which pull up the new basal body toward its site of docking. The presence in T. thermophila of an ALF-like appendage suggests the conservation of an ancestral mechanism ensuring the coupling of basal body duplication and cell morphogenesis.

Additional Evidence for the Cyclic GMP Signaling Pathway Resulting in the Photophobic Behavior of Stentor coeruleus

Abstract 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.

Identification of Possible Phosducins in the Ciliate Blepharisma japonicum

Examination of ciliate Blepharisma japonicum whole cell lysates with an antibody against phosphoserine and in vivo labeling of cells with radioactive phosphate revealed that the photophobic response in the ciliate is accompanied by a rapid dephosphorylation of a 28 kDa protein and an enhanced phosphorylation of a 46 kDa protein. Analysis with antibodies raised against rat phosducin or human phosducin-like proteins, identified one major protein of a molecular weight of 28 kDa, and two protein bands of 40 kDa and 93 kDa. While the identified ciliate phosducin is phosphorylated in a light-dependent manner, both phosducin-like proteins exhibit no detectable dependence of phosphorylation upon illumination. An immunoprecipitation assay also showed that the ciliate phosducin is indeed phosphorylated on a serine residue and exists in a phosphorylated form in darkness and that its dephosphorylation occurs in light. Immunocytochemical experiments showed that protozoan phosducin and phosducin-like proteins are localized almost uniformly within the cytoplasm of cells adapted to darkness. Cell exposure to light caused a pronounced displacement of the cell phosducin to the vicinity of the plasma membrane; however, no translocation of phosducin-like proteins was observed upon cell illumination. The obtained results are the first demonstration of the presence and morphological localization of a possible phosducin and phosducin-like proteins in ciliate protists. Phosducin and phosducin-like proteins were found to bind and sequester the betagamma-subunits of G-proteins with implications for regulation of G-protein-mediated signaling pathways in various eukaryotic cells. The findings presented in this study suggest that the identified phosphoproteins in photosensitive Blepharisma japonicum may also participate in the regulation of the efficiency of sensory transduction, resulting in the motile photophobic response in this cell.

Phosducin interacts with the G-protein βγ-dimer of ciliateprotozoan Blepharisma japonicum upon illumination

SUMMARY Immunological techniques and high-resolution FRET analysis were employed to investigate the in vivo colocalization and interaction of phosducin (Pdc) with the βγ-subunits of G-protein (Gβγ) in the ciliate Blepharisma japonicum. Immunological techniques revealed that illumination of cells resulted in a decrease in phosphorylation levels of Pdc and its colocalization with Gβγ. The observed light-induced Pdc dephosphorylation was also accompanied by significant enhancement of Gβγ binding by this molecule. Possible formation of the Pdc–Gβγ complex in cells exposed to light was corroborated by FRET between these proteins. Treatment of cells with okadaic acid, an inhibitor of phosphatase activity, entirely prevented Pdc dephosphorylation by light, colocalization of this phosphoprotein with Gβγ and generation of the Pdc–Gβγ complex. Cell fractionation and immunoblotting revealed that in cells exposed to light, the formation of Pdc–Gβγ complex and its translocation into the cytoplasm occur simultaneously with a change in the gel migration of Gβ. Moreover, a 33 kDa immunoanalog of 14-3-3 protein was identified and we showed that this protein is bound by phosphorylated Pdc in a cell adapted to darkness. The results of this study provide additional detailed characterization of the functional properties of the ciliate Pdc. The likely functional role of Pdc in Blepharisma is discussed.

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.

Visualization of the interaction between Gβγ and tubulin during light-induced cell elongation of Blepharisma japonicum

Blepharisma japonicum ciliates display reversible cell elongation in response to lasting bright illumination. This light-induced phenomenon has been ascribed to the active sliding of the cortical microtubules of the ciliate. The detailed intracellular signaling pathway that activates the microtubule network in response to light, resulting in cell elongation, is unknown. We have previously reported that light stimulation initiates sequential molecular events consisting of a decrease in the phosphorylation of ciliate Pdc, followed by increased binding of Pdc to membrane-localised Gbetagamma and the subsequent translocation of the Pdc-Gbetagamma complex to the cytoplasm. In this study, we used selected agents known to influence protein phosphorylation to test whether alterations in Pdc phosphorylation levels by light affect ciliate shape. Behavioural analysis indicated that cell treatment with okadaic acid, an inhibitor of protein phosphatase activity, heavily abolished the effect of light on cell elongation, whereas the presence of H-89, a specific inhibitor of cAMP-dependent protein kinase (PKA) activity, had no appreciable effect on the cell length. Phosphorylation assays showed that cell incubation with H-89 mimicked light by promoting Pdc dephosphorylation and its colocalization with Gbetagamma. However, as demonstrated by FRET-AP, Pdc-Gbetagamma complex formation and changes in the length of the cell did not occur under the same conditions. Moreover, fluorescence microscopy showed localization of Gbetagamma and beta-tubulin in the same cell compartment and demonstrated that a direct interaction between these proteins occurs in cells adapted to darkness or exposed to prolonged illumination (> or = 10 min). In contrast, an opposite effect, i.e. a transient decrease in the interaction between Gbetagamma and beta-tubulin and distinct Pdc dephosphorylation, was observed in cells illuminated for short time. Under these conditions, Pdc preferentially occupies the cell submembrane region and interacts with Gbetagamma. In cells illuminated for a longer time (> or = 10 min) and despite the constant light intensity, Pdc was progressively rephosphorylated and then dissociated from Gbetagamma, relocalizing within the cell cytoplasm. The results obtained in this study suggest that alterations in Pdc phosphorylation may be involved in light-induced elongation of the Blepharisma cell body, which affects the interaction of Gbetagamma with beta-tubulin and cell cytoskeleton remodelling.

Detection and localization of a putative cyclic-GMP-activated channel protein in the protozoan ciliate Stentor coeruleus

Summary.Immunoblotting and immunocytochemical assays were employed to identify and localize a channel protein activated by cyclic GMP (cGMP) in the protozoan ciliate Stentor coeruleus. Analysis of whole-cell homogenate with antibodies raised against the α-subunit of the cGMP-activated channel protein from bovine rod outer segments and against cGMP revealed four major protein bands with molecular masses of 40 kDa, 63 kDa, and over 120 kDa, which bound cGMP. However, only a cGMP-binding protein of 63 kDa, corresponding to the α-subunit of the cGMP-activated ion channel protein from bovine rod outer segments, was found in the ciliate cortex fraction. The functional cGMP-activated channel protein was also shown to be present in the cortex fraction of S. coeruleus by patch-clamp measurements of artificial liposomes. Incorporation of the cortex fraction into liposomes resulted in the appearance of ion channel activity related to cGMP. The reconstituted protein channels were strongly inhibited by l-cis-diltiazem, a known potent blocker of many types of cyclic-nucleotide-activated channels. The results presented here are the first demonstration of the existence and localization of a putative cGMP-activated channel protein in the ciliate S. coeruleus. Cyclic-nucleotide-activated channel proteins are nonspecific cation channels which mediate the receptor potentials in photoreceptor cells and in cells of the olfactory epithelium. On the basis of these data, we suggest that the 63 kDa protein identified in Stentor coeruleus is also a cGMP-activated ion channel and that it may be involved as an effector in the photosensory transduction pathway leading to the motile photophobic response in this ciliate protist.

Cell cycle-dependent modulations of fenestrin expression in Tetrahymena pyriformis.

In Tetrahymena, besides apparent cell polarity generated by specialized cortical structures, several proteins display a specific asymmetric distribution suggesting their involvement in the generation and the maintenance of cell polarization. One of these proteins, a membrane skeleton protein called fenestrin, forms an antero-posterior gradient, and is accepted as a marker of cell polarity during different cellular processes, such as cell division or oral replacement. In conjugating cells, fenestrin forms an intracytoplasmic net which participates in pronuclear exchange. The function of fenestrin is still unknown. To better understand the role of fenestrin we characterized this protein in an amicronuclear Tetrahymena pyriformis. We show that in this ciliate not only does fenestrin localization change in a cell division-dependent manner, but its mRNA and protein level is also cell cycle-regulated. We determine that the two available anti-fenestrin antibodies, 3A7 and 9A7, recognize different pools of fenestrin isoforms, and that 9A7 is the more general. In addition, our results indicate that fenestrin is a phosphoprotein. We also show that the level of fenestrin in the amicronuclear T. pyriformis and the amicronuclear BI3840 strain of T. thermophila is several times lower than in micronuclear T. thermophila.