Immunofluorescence Localization of a 23-kDa Tetrahymena Calcium-Binding Protein, TCBP-23, in the Cell Cortex

Abstract

Previously, we succeeded in cloning a cDNA of the 23-kDa Tetrahymena Ca²⁺-binding protein, designated TCBP-23. Analysis of the deduced amino acid sequences showed that TCBP-23 is a member of the EF-hand family of Ca²⁺-binding proteins. However, its physiological function was not elucidated. In the studies reported here, recombinant TCBP-23 was expressed in Escherichia coli and purified. Since recombinant TCBP-23 binds Ca²⁺ in vitro, Ca²⁺-binding domains of the protein are likely to be functional in vivo. Rabbit antibodies against TCBP-23 were raised and used to determine the intracellular localization of the protein in Tetrahymena cells by indirect immunofluorescence. The antibodies strongly stained the whole cell cortex except for the oral apparatus and around the basal bodies. TCBP-23 remained in detergent-extracted cells, suggesting that it is associated with the epiplasm, the membrane skeleton of Tetrahymena. These results suggest that TCBP-23 may mediate Ca²⁺-regulated processes in the cell cortex.