Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus.

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca2+ to overpopulated vegetative cells. However, an increase in Ca2+ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca2+ concentrations is essential, but Ca2+ inflow does not necessarily occur during encystment. Ca2+ image analysis showed that Ca2+ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca2+ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca2+ from vesicles.

Early signaling pathways mediating dormant cyst formation in terrestrial unicellular eukaryote Colpoda.

Dormant (resting) cyst formation (encystment) in unicellular eukaryotes is the process of a large-scale digestion of vegetative cell structures and reconstruction into the dormant form, which is performed by cell signaling pathways accompanied by up- or down-regulation of protein expression, and by posttranslational modification such as phosphorylation. In this review, the author describes the morphogenetic events during encystment of Colpoda and the early molecular events in the Ca2+/calmodulin-triggered signaling pathways for encystment, based mainly on our research results of the past 10 years; especially, the author discusses the role of c-AMP dependently phosphorylated proteins (ribosomal P0 protein, ribosomal S5 protein, Rieske iron-sulfur protein, actin and histone H4) and encystment-dependently upregulated (EF-1α-HSP60, actin-related protein) and downregulated proteins (ATP synthase ß-chain). In addition, the roles of AMPK, a key molecule in the signaling pathways leading to Colpoda encystment, and differentially expressed genes and proteins during encystment of other ciliates are discussed.

Role of arginine kinase in Paramecium tetraurelia (Ciliophora, Peniculida): Subcellular localization of AK3 and phosphoarginine shuttle system in cilia.

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia.

Morphogenetic and molecular analyses of cyst wall components in the ciliated protozoan Colpoda cucullus Nag-1.

The cyst wall of the resting cyst of the ciliated protozoan Colpoda cucullus (Nag-1 strain) is composed of several layers of endocyst, a single layer of ectocyst associated with a mucous layer and lepidosomes composed of a fibrous or crystal-like structure. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the ectocyst associated with lepidosomes and mucous materials contained proteins corresponding to 27, 31, 45 kDa and smear bands ranging from 50 to 60 kDa. Liquid chromatography-tandem mass spectrometry of these proteins revealed that the 45-kDa protein (p45) was elongation factor Tu (EF-Tu). Immunofluorescence microscopy with an anti-EF-Tu polyclonal antibody showed that Colpoda EF-Tu (p45) was localized in the lepidosomes. The lepidosomes were stained vividly with Congo red, which is bound to the stacked ß-sheets of amyloid protofibrils. In the presence of puromycin, no cyst wall components including lepidosomes were formed, indicating that cyst wall formation requires synthesis of proteins including EF-Tu. Electron microscopy of encysting cells implied that vesicles which were presumably budded from endoplasmic reticula possibly fuse with a lepidosome-precursor vacuole containing electron-dense fine particles or fibrous structures, and followed by the subsequent fusion with other electron-lucent granules.

Identification of differentially expressed water-insoluble proteins in the encystment process of Colpoda cucullus by two-dimensional electrophoresis and LC-MS/MS analysis.

In the encystment process of the ciliate protist Colpoda cucullus, we observed that the cell total protein abundance was reduced at 12 h-1 d after the onset of encystment induction subsequent to the reduction in mRNA abundance. We analyzed the alteration of the expression levels of water-insoluble proteins by two-dimensional polyacrylamide gel electrophoresis using polyoxyethylene (20) sorbitan monooleate (Tween-80), and we identified proteins whose expression levels were altered in the encystment process by a liquid chromatography tandem mass spectrometry analysis. The expression level of a 60-kDa protein (p60; heat shock protein 60) was temporarily enhanced and that of a 55-kDa protein (p55; actin) and a 49-kDa protein (p49; actin) was enhanced in the Colpoda encystment process. In mature cysts, the expression level of p55 and p49 tended to be reduced, whereas the expression level of a 50-kDa protein (p50d; α-tubulin), a 25-kDa protein (p25; α-tubulin) and a 52-kDa protein (p52c; ß-tubulin) was enhanced.

Excystment-dependent alteration of protein expression in terrestrial ciliate Colpoda cucullus.

Protein expression during the excystment of Colpoda cucullus was studied by SDS-PAGE. The expression levels of 60-, 50- and 49-kDa proteins were markedly changed from the early to later stage of excystment. The 60-kDa protein (p60) was temporarily expressed first, and its expression was inhibited by actinomycin D. LC-MS/MS analysis showed that the amino acid sequences of p60 partially coincided with those of the Paramecium tetraurelia unnamed protein homologous to DEAD-box RNA helicase. These results suggest that p60 expression is enhanced by transcriptional regulation and may be involved in initiating the molecular events leading to cellular morphogenesis.

Emergence of the terrestrial ciliate Colpoda cucullus from a resting cyst: rupture of the cyst wall by active expansion of an excystment vacuole.

The first sign of excysting Colpoda cucullus cells is the initiation of the pulsation of a contractile vacuole, which is then replaced by a non-pulsating vacuole (excystment vacuole) that continues to expand and finally ruptures the outermost cyst wall (ectocyst) due to inner pressure. A ciliate surrounded by flexible membranes (endocyst) thus emerges. The osmolarity of the excysting cells is estimated to be 140 mOsm L(-1) from the relationship between the frequency of contractile vacuole pulsation and the external sucrose concentration. Both the expansion of the excystment vacuole and the emergence of ciliates occurred even when the cysts were immersed in hypertonic medium. In hypotonic medium containing sodium azide (NaN3, a cytochrome c oxidase inhibitor), the contractile vacuole of vegetative cells stopped pulsating and gradually expanded, causing cells to burst. When C. cucullus was induced to encyst in a hypotonic medium containing NaN3, the expansion of the excystment vacuoles was inhibited. These results suggest that the active uptake of water may be responsible for the expansion of the excystment vacuole required for the ectocyst to rupture.

EF-1α and mitochondrial ATP synthase ß chain: alteration of their expression in encystment-induced Colpoda cucullus.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the total proteins contained in encystment-induced Colpoda cucullus showed that a 50-kDa protein (p50) disappeared, whereas the expression of a 49-kDa protein (p49) was enhanced in early phase of morphogenetic transformation into the resting cyst (i.e. 2-5 h after the onset of encystment induction). Puromycin or actinomycin D inhibited the alteration in the expression of p50 and p49 by the induction of encystment. These results suggest that the encystment-specific alteration in expression of these proteins is performed by a transcriptional regulation. Liquid chromatography tandem mass spectrometry analysis revealed that p50 is mitochondrial ATP synthase ß chains, and that p49 is elongation factor 1α.

Protein phosphorylation in encystment-induced Colpoda cucullus: localization and identification of phosphoproteins.

In Colpoda cucullus, the morphogenetic transformation was preceded by an enhancement of the in vivo protein phosphorylation level. Immunofluorescence microscopy using antiphosphoserine antibody showed that these phosphorylated proteins were localized in the macronucleus and other cytoplasmic regions. Biotinylated Phos-tag/ECL assays of isolated macronuclei showed that a 33-kDa protein (p33) was localized within them. The p33 obtained from isolated macronuclei was tentatively identified as ribosomal P0 protein by LC-MS/MS analysis. In addition, among the encystment-specific phosphoproteins obtained by phosphate-affinity chromatography, the 29-, 31-, and 33-kDa proteins (p29, p31, and p33) were tentatively identified as ribosomal P0 protein, whereas the 24-kDa phosphoprotein (p24) was tentatively identified as ribosomal S5 protein.

Culture Age, Intracellular Ca(2+) Concentration, and Protein Phosphorylation in Encystment-Induced Colpoda cucullus.

In Colpoda cucullus, intracellular Ca(2+) mediates the encystment induction and protein phosphorylation that occur just prior to morphogenetic transformation into the resting form. When rapidly growing cells were stimulated to encyst, encystment was not readily induced, and the protein phosphorylation level was lower. On the other hand, in post-growing cells stimulated to encyst, the encystment rate and protein phosphorylation level were elevated. These results suggest that protein phosphorylation is closely linked to encystment induction. Why, then, are the protein phosphorylation level and encystment rate difficult to elevate in the rapidly growing cells? Fura 2 ratiometry showed that the intracellular Ca(2+) concentration (F340/F380 ratio) was raised in rapidly growing cells as well as in post-growing cells when the cells were stimulated to encyst. It is presumed that the Ca(2+)-mediated signal transduction pathways for protein phosphorylation and encystment may be triggered in rapidly growing cells, but downstream certain steps may be suppressed by certain intracellular components.

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus.

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca(2+) and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca(2+) or without the addition of Ca(2+). Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca(2+)/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca(2+) inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca(2+)-triggered signaling pathways, may be involved in encystment induction.

Light-induced and apoptosis-like cell death in the unicellular eukaryote, Blepharisma japonicum.

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300W m(-2)) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3-30W m(-2)) only induced pigment extrusion and photooxidation. We have found that moderate light (5W m(-2)) induced apoptosis-like cell death. Microscopically it was found that >3h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180-200 and 360-400bp, which were seen as apoptosis ladders.

Chromatin extrusion in resting encystment of Colpoda cucullus: a possible involvement of apoptosis-like nuclear death.

The extrusion of macronuclear chromatin is a remarkable characteristic during encystment in Colpoda, but the biological significance of this phenomenon has not been fully elucidated. Here we demonstrate that chromatin extrusion occurs with high frequency when encystment was induced by increasing Ca(2+) in growing cells in various stages of the cell cycle. The Feulgen-DNA reaction revealed that vegetatively growing cells have more macronuclear DNA than cells in the stationary phase, suggesting an association of macronuclear DNA content with the execution of chromatin extrusion. Using 4',6-diamidino-2-phenylindole (DAPI), we found that the size of the macronuclear extrusion body was reduced with time and eventually disappeared approximately 24h after encystment induction. In addition, oligonucleosome-sized DNA cleavage was confirmed to occur concomitant with the size reduction, suggesting that the extrusion body is selectively degraded, while the normal macronucleus remains alive. Combined use of acridine orange and Hoechst 33342 demonstrated that the extruded body was increasingly acidified before final resorption. These features are reminiscent of the nuclear degradation process in conjugating Tetrahymena, and therefore we conclude that chromatin extrusion in Colpoda might occur to adjust the macronuclear DNA content prior to encystment. In this way, it is similar to the apoptotic-like nuclear death that occurs during the conjugation of other ciliates.

Relationship between two tandemly arranged and light-induced glutathione S-transferase genes from the ciliated protozoa Blepharisma japonicum.

Recently we reported a light-induced cDNA encoding glutathione S-transferase (GST) from the ciliated protozoa Blepharisma japonicum, which possessed photosensitive pigments. In this study, a novel cDNA encoding GST was further isolated, and the two GSTs (BjGST1 and BjGST2) showed high sequence identity of 86%. Phylogenetic trees indicated that the BjGSTs were distantly related to known classes of GSTs, and they could form a protozoa-specific class. The recombinant proteins also existed as homo- or heterodimers that exhibited different enzyme activities, appreciating the functional differentiation. Furthermore, the transcription levels of BjGST genes were coordinately regulated in response to light stimulation. In addition, the genomic structure analysis revealed that the two genes were tandemly arranged through an approximately 500-bp spacer region of unusual DNA structure containing cis-acting elements related to oxidative stress response. These results demonstrate that the two BjGSTs are expressed simultaneously and act cooperatively against photooxidative stress.

Molecular cloning and characterization of a novel glutathione S-transferase gene induced by light stimulation in the protozoan Blepharisma japonicum.

A cDNA clone that is inducible by light stimulation was cloned by a differential screening method from a cDNA library of the protozoan Blepharisma japonicum, and the light-dependent expression was checked by semi-quantitative reverse transcription polymerase chain reaction analysis. Sequence analysis showed that the cDNA encodes a glutathione S-transferase (GST) that has not been characterized in the protozoa. Multiple alignment of B. japonicum GST (BjGST1), known protozoan, and mammalian alpha-, micro-, pi-, sigma-, theta-, zeta-, kappa-, and omega-class GSTs suggested that the BjGST1 may be a novel class GST. Furthermore, highly conserved amino acid residues among the GSTs and the substrate specificity of recombinant BjGST1 showed that BjGST1 is related to alpha-, micro-, pi-, and sigma-class GSTs rather than the other class of GSTs.