Eiji Kinoshita

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.

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.