Identification of the epiplasmins, a new set of cortical proteins of the membrane cytoskeleton in Paramecium

Abstract

ABSTRACT In most ciliates, the epiplasm, a superficial cytoskeletal layer of variable thickness, both surrounds basal bodies and interacts tightly with adjacent membrane networks; it constitutes the predominant structure in Paramecium cell ghosts. Previous indirect data suggested several cortical proteins as potential constituents of the epiplasm. New sharp monoclonal antibodies presented in this paper, positive both on immunotransfers and in immunocytochemical tests carried out on permeabilized cells and ultrathin sections, definitively identify the epiplasmins: a set of about twenty protein bands ranging from 45 to 33 kDa and making up the bulk of the epiplasmic layer. The complete epiplasmin pattern characterized from gradient-purified cortex is also present in unfractionated whole cells, confirming that the pattern is not generated artifactually. Comparative one-step extractions, performed either in 1 M KI or in 4 M urea, solubilize the epiplasmins as a whole, indicating that all of them share very similar biochemical properties. Two-dimensional electrophoresis shows the great complexity of this epiplasmin group. Epiplasmin solu-bilization properties are discussed with respect to other models of membrane-cytoskeleton interaction developed among protists and metazoans and also to intermediate filaments, specially lamins. Immunofluorescent labelling combined with confocal microscopy permits a more detailed study of epiplasm formation at the level of the fission furrow, with new insights into two successive steps of epiplasm growth. A first series of interspecific reactions has been carried out with one of the anti-epiplasmin antibodies, yielding results which are discussed in an evolutionary framework.