Tatsuomi Matsuoka

Assays of protein contained in water-soluble fraction of encysting cells Colpoda cucullus Nag-1 by two-dimensional electrophoresis (2-D PAGE) and mass spectrometry (MS) revealed that the amount of β-tubulin abruptly increased in 2.5–10 h after encystment induction. Judging from the results that total α-tubulin content did not decrease much until 12 h after encystment induction, the result indicates that disassembly of microtubules may occur soon after encystment is induced. Therefore, we tried to visualize dynamics of microtubules. Immunofluorescence microscopy using anti-α-tubulin antibody indicated that disassembly of axonemal microtubules of cilia became within 1.5 h after encystment induction, and resorbed in 3 days. Although the cytoplasmic microtubules failed to be visualized clearly, encystmentdependent globulation of cells was promoted by taxol, an inhibitor of disassembly of microtubules. It is possible that a temporary formation of cytoplasmic microtubules may be involved in cell globulation.

The phosphorylation level of actin (43 kDa) became slightly elevated just after encystment induction. Lepidosomes, the sticky small globes surrounding encysting cells, were vividly stained with Acti-stain 555 phalloidin, suggesting that 43-kDa actin or its homologues may be contained in lepidosomes.

We found that the water-rich (osmolality below 0.052 Osm/l) wet resting cysts of the soil ciliate Colpoda cucullus Nag-1 were tolerant to extremely low temperature (−65℃). When cell fluid obtained from the resting cysts was cooled at −65℃, small particles of ice crystals did not grow into large ice crystals. At −65℃, the cysts shrank due to an outflow of water, because a vapor pressure difference was produced between the cell interior and freezing surrounding medium. The osmolality of these shrunk cells was estimated 0.55 Osm/l, and the freezing point depression of the shrunk cell fluid was estimated to be 1.02℃. Hence, the antifreeze ability of wet cysts at −65℃can not be explained by freezing point depression due to elevation of cytoplasmic osmolality.

The cytoplasm of resting cysts was vividly stained red with periodic acid-Schiff (PAS) and stained purple with toluidine blue. On the other hand, the excystment-induced cysts were not stained with PAS, and exhibited a loss of the antifreeze activity. PAS staining of SDSPAGE gel obtained from encysting Colpoda cells showed that a large amount of PAS-positive macromolecules accumulated as the encystment stage progressed. These results suggest that antifreeze polysaccharides may be involved in the antifreeze activity of C. cucullus Nag-1 dormant forms.

n Colpoda cucullus, the signaling pathways for encystment induction involving protein phosphorylation have been believed to be triggered by an increase in the intracellular Ca2+ concentration promoted by cell-to-cell mechanical contact due to overpopulation. By means of fura 2 ratiometry, the present study showed that the intracellular Ca2+ concentration was actually elevated when vegetative cells were induced to encyst by being suspended at a high cell density in the presence of external free Ca2+ and suppressed by chelating external Ca2+. This result strongly suggests that an increase in the intracellular Ca2+ concentration caused by an inflow of Ca2+ promoted by cell-tocell mechanical contact due to overpopulation enhances the rate of encystation in Colpoda cucullus.

We found that vegetative cells of Colpoda cucullus Nag-1 accumulated in shaded areas of a container when grown in the laboratory and then formed resting cysts. The photodispersal (negative photoaccumulation) of C. cucullus was mediated, at least in part, by a difference in forward swimming velocity between the illuminated region and the shaded area of the Petri dish (motion slowed or stopped in the shaded area). When C. cucullus was stimulated by continuous light irradiation, the forward swimming velocity increased and reached a steady state within 10 s. When the light intensity decreased, the forward swimming velocity gradually decreased, and eventually returned to its original level for approximately 1 min. The action spectrum of the photokinetic response (steady-state swimming acceleration driven by continuous light stimulation) implies the involvement of blue light receptors.

It has been suggested that encystment of Colpoda cucullus is mediated by intracellular Ca2+-activated signaling pathways involving an increase in the cAMP concentration. In the present study, cAMP enzyme immunoassay (EIA) and chemiluminescence detection for phosphorylated proteins using anti-phosphoserine antibody, anti-phosphothreonine antibody and biotinylated phosphate-binding tag molecules (Phos-tag) showed that the intracellular cAMP concentration in Colpoda cells was raised and the phosphorylation level of serine/threonine residues was elevated in many proteins prior to the cyst formation. Such encystment induction and protein phosphorylation were suppressed by the addition of an intracellular Ca2+ chelating reagent (BAPTA-AM) or the encystment inhibitor, chlorophyllin in all or most of these proteins, respectively. The phosphorylation level of some proteins was slightly elevated by the addition of IBMX, which tended to promote encystment induction, and tended to be slightly suppressed by the addition of H-89 (PKA inhibitor), which also suppressed encystment induction. These results suggest that Ca2+-activated signaling pathway involving cAMP/PKA-dependent protein phosphorylation may be responsible for the encystment induction of C. cucullus.

Resting cysts of the terrestrial ciliate Colpoda cucullus (Nag-1 strain) are highly resistant to UV light. It has been speculated that auto-fluorescent (blue fluorescent) particles surrounding the nuclei and yellowish fluorescent layers of the cyst wall are the candidate structures for the protection of the cellular components from UV light. The UV resistance of encysting cells was quickly acquired up to 5 h after the onset of encystment induction, and then gradually increased for several days. The less fluorescent ectocyst layer, yellowish fluorescent first-synthesized endocyst layer (en-1) and the NSPs were formed within 5 h after the onset of encystment induction, and thereafter endocyst layers became gradually thicker for several days. The cyst wall sample (ectocyst and endocyst layers) markedly absorbed a broad range of UV light. This result indicates that the cyst wall evidently has UV-cut function. These results support that the cyst wall and NSPs of C. cucullus play a role in the shielding of the cell components from UV light.