Yoichiro Sogame

Resting cyst formation is a crucial process of cryptobiosis in protists. In colpodid ciliates, cyst formation is accompanied by large-scale morphological changes such as changes of cell shape, resorption of cilia, and formation of a cyst wall; additionally, the cell cycle is arrested. These changes provide acquired tolerance against environmental stresses. During cyst formation, mitochondrial membrane potential is reduced and the level of the ATP synthase beta chain is suppressed, strongly indicating that metabolism has ceased. Here, however, we show that ATP levels are elevated during the initial phases of encystment implying that metabolism may not be completely suppressed. This finding suggests another aspect of resting cyst formation that is not applicable to cryptobiosis.

A new species of a testate amoeba, Matsakision ogawaraensis sp. nov., is described from Ogawara pond, Aomori Prefecture, Japan. This is the third species in the genus Matsakision. Matsakision ogawaraensis sp. nov. is distinguished from two other species by the specific form of the shell in apertural view, which is triangular (the shells of other species are circular or elliptic). In addition, it has a chitinous lip around the aperture. For the first time, long needle-like pseudopodia have been observed.

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.

In this study, we report that the unicellular free-living protist Colpoda cucullus in the resting cyst (cryptobiosis) repairs stress damage. We previously demonstrated that resting cysts of Colpoda cucullus have extreme tolerance to gamma irradiation and can revert to vegetative cells after irradiation. Such irradiated cysts gradually  excyst, suggesting that stress repair mechanisms are active during excystment or in the resting cyst. Herein we provide bioassay evidence that the rate of excystment of irradiated cysts is elevated by subsequent incubation, thereby indicating that cells injured by gamma irradiation can repair themselves in the resting cyst,  whereas irradiated dry cysts cannot.

The ciliate Colpoda cucullus forms resting cysts to survive unfavorable environmental stresses. In this study, we have shown that Colpoda resting cysts survived exposure to a gamma radiation dose of 4000 Gy, although vegetative cells were killed by 500 Gy. After 4000 Gy irradiation, more than 90% of resting cysts and approximately 70% of dry cysts could excyst to form vegetative cells. In both cases, the excystment gradually increased after the induction of excystment. In addition, we also showed that protein carbonylation level was increased by gamma irradiation, but decreased by incubation in the cyst state. These results indicated that cell damage was repaired in resting cysts. Colpoda probably developed tolerance to gamma radiation by forming resting cysts as a strategy for growth in terrestrial environments, as part of contending with the stress due to reactive oxygen species caused by desiccation.