Localization of actin in the Tetrahymena basal body-cage complex.

Abstract

In the ciliate cytoskeleton, basal bodies are contained within separate, filamentous cages which are closely associated with basal body microtubules. We have used two polyclonal anti-actin antibodies to localize actin within the basal body-cage complex of Tetrahymena. An antiserum against a Tetrahymena oral apparatus fraction enriched for basal body proteins was produced in rabbits. Agarose-linked chicken muscle actin was used to affinity-purify anti-Tetrahymena actin antibodies from the anti-oral apparatus antiserum. Agarose-linked chicken muscle actin was used to affinity-purify anti-chicken muscle actin antibodies from a commercially available antiserum against chicken muscle actin. Both affinity-purified antibodies were monospecific for Tetrahymena actin on immunoblots containing total oral apparatus protein. The anti-actin antibodies were localized to both somatic and oral basal bodies in Tetrahymena by immunofluorescence microscopy. At the ultrastructural level with the immunogold technique, these antibodies labeled actin epitopes in four distinct regions of the basal body-cage complex: (a) basal body walls, (b) basal plate filaments, (c) proximal-end filaments and (d) cage wall filaments. In addition, the antibody labeled filament bundles that interconnect groups of basal bodies (membranelles) within the oral apparatus. Identical labeling patterns were observed with basal bodies in the isolated oral apparatus, basal bodies in the in situ oral apparatus and somatic basal bodies in situ. Quantitative analysis of gold particle distribution was used to demonstrate the specificity of the antibodies for the basal body-cage complex and to show that non-specific binding of the antibodies was negligible. Preadsorption of the antibody with muscle actin effectively eliminated the capacity of the antibody to bind to proteins on immunoblots and to basal body structures with the immunogold labeling technique. These results provide evidence for actin in the basal body-cage complex and raise the possibility of a contractile system associated with basal bodies.