Resting cyst formation of unicellular organism Colpoda and phosphorylation analyses by Phos-tag

Abstract

In the unicellular eukaryote Colpoda cucullus, resting cyst formation (encystment), which is a cellular morphogenetic process, is mediated by intracellular signaling pathways, which are triggered by an inflow of Ca2+ due to cell-to-cell mechanical contact. The enhanced chemiluminescence detection (ECL) for phosphorylated proteins using biotinylated Phos-tag showed that the phosphorylation level in several proteins was enhanced in Ca2+-dependent manner prior to the beginning of the cyst formation (within 1 h after the onset of encystment induction). The cAMP enzyme immunoassay (EIA) showed that the intracellular cAMP concentration was elevated in encystment-induced cells. The encystment induction and the phosphorylation of some proteins are slightly promoted by the addition of membrane-permeable derivatives of cAMP or non-selective phosphodiesterase inhibitor, while they tend to be suppressed by the addition of an inhibitor of cAMP-dependent kinase (PKA). These results suggest that a Ca2+-activated signaling pathway involving cAMP/PKA-dependent protein phosphorylation may be responsible for the encystment induction of C. cucullus. Encystment-specific phosphorylated proteins were isolated by phosphate-affinity chromatography using Phos-tag agarose beads, and some of them were tentatively identified by mass spectrometry analysis.