A monoclonal antibody study of protein distribution in the membrane skeleton of the ciliate Pseudomicrothorax

Abstract

The membrane skeleton, or epiplasm, of the ciliated protozoon Pseudomicrothorax dubius is a chemically and structurally complex layer. It is responsible for the cell shape and the positioning of some cortical organelles. One may expect that its possible morphogenetic role can be achieved only via a regional differentiation of the protein distribution in the epiplasm. We have tried to demonstrate such differentiation by preparing an epiplasm extract, which consists predominantly of concanavalin A (ConA)-positive glycoproteins. This fraction, either untreated or deglycosylated, was used to raise monoclonal antibodies (mAbs), whose specificity was tested on western blots of either untreated or deglycosylated epiplasm. The recognized polypeptides were then localized in situ by fluorescence and electron microscopic immunocytochemistry. Six mAbs are presented here. Four of them are directed against ConA-positive glycoproteins and show a localization of the latter on the outer surface of the epiplasm. The two others are directed against other epiplasmic polypeptides: one is specific for a common epitope shared by most of the epiplasmic proteins, but not by the glycoproteins, and labels the entire membrane skeleton, whereas the other recognizes three minor polypeptides, which seem localized to the inner part of the epiplasm.