Membrane skeleton in Paramecium: a functional analysis of epiplasmins by RNAi

Abstract

The membrane skeleton, also called epiplasm, is a prominent cortical structure in the ciliate Paramecium. In the present study, we have developed RNAi of Ep31 and Ep34, two homologous epiplasmic proteins (epiplasmins) for a functional analysis of epiplasmic membrane skeleton in P. tetraurelia. A severe phenotype is evident 20 h after the induction of RNAi. In this phenotype, anlagen of daughter cells are formed but are unable to separate from each other at time of cytokinesis. This abortive division is repeated every 4–5 h leading to “monstrous” organisms that will die 70–75 h after induction. Immunofluorescence microscopy was performed in order to compare organization and dynamics of cytoskeletal systems in wild types and in cells processed for RNAi of epiplamins. Microtubule‐based systems are not affected as judged by examination of rows of basal bodies (bb) and bb‐associated fibers. Likewise, new oral apparatuses and pulsatile vacuoles are correctly positioned in anlagen of daughter cells. The main abnormalities concern the epiplasmic membrane skeleton. Immunolabeling of epiplasmins revealed significant disorganization of the epiplasm in regions close to aborted division furrows. Western blot analysis confirmed co‐silencing of Ep31 and Ep34 epiplasmins. These results suggest that epiplasmin‐mediated elongation of the cell body is essential for growth of the division furrow and accomplishment of binary fission in Paramecium tetraurelia.