Affinity‐Purification of Concanavalin A‐Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Abstract

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.

Purification and initial characterization of two pheromones from the marine Antarctic ciliate,Euplotes nobilii

“…Pursuing pheromone purification and characterization has been more successful in Blepharisma, Dileptus, and Euplotes, because these ciliates secrete and diffuse their pheromones constitutively in biologically detectable concentrations into the extracellular environment, unlike Paramecium and Tetrahymena which apparently retain these molecules bound to the cell surface (Hiwatashi Kitamura, 1985;Kitamura, 1988;Wolfe, 1993;Driscoll & Hufnagel, 1999). In the environment, pheromones have usually been revealed by mating-induction assays in which the formation of mating pairs is induced after suspension of a cell culture with cell-free supernatant of another culture of different mating type.…”

A Proteomics Approach to Cloning Fenestrin from the Nuclear Exchange Junction of Tetrahymena

“…It would be interesting to label co‐stimulated cells with monovalent TRITC‐ConA and pursue whether or not the streaming can be observed in the absence of cross‐linking. It has been reported that novel ConA receptors are not synthesized in response to mating induction, but rather that co‐stimulation leads to a global redistribution of existing receptors, resulting in their aggregation over the future mating junction at the anterior end of the cell (Driscoll and Hufnagel 1999; Van Bell 1983; Wolfe and Feng 1988). Perhaps these ConA “zippers” represent early patterns of receptor flow as this global reorganization is achieved.…”