VISUALIZATION OF PROSOMES (MCP-PROTEASOMES), INTERMEDIATE FILAMENT AND ACTIN NETWORKS BY INSTANTANEOUS FIXATION PRESERVING THE CYTOSKELETON

A new ''instantaneous'' fixation/extraction procedure, yielding good p reservation of intermediate filaments (IFs) and actin filaments when a pplied at 37 degrees C, has been explored to reexamine the relationshi ps of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composition, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteolytic core of the 26S proteasomes, they are also called ''multicatalytic proteinase (MCP) complexes'' or ''20S-Proteasomes.'' In Triton X-100-extracted epithelial, fibroblast ic, and muscle cells, prosome particles were found associated primaril y with the IFs (Olink-Coux et al., 1994). Application of ''instantaneo us fixation'' has now led to the new observation that a major fraction of prosome particles, composed of specific sets of subunits, is distr ibuted in variable proportions between the IFs and the microfilament/s tress fiber system in PtK1 epithelial cells and human fibroblasts, Ele ctron microscopy using gold-labeled antibodies confirms this dual loca lization on classical whole mounts and on cells exposed to instantaneo us fixation. In contrast to the resistance of the prosome-IF associati on, a variable fraction of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation, Mor eover, in vitro copolymerization of prosomes with G-actin made it poss ible to observe ''ladder-like'' filamentous structures in the electron microscope, in which the prosome particles, like the ''rungs of a lad der,'' laterally cross-link two or more actin filaments in a regular p attern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, n ot only within large M-r complexes (possibly mRNPs and/or 26S proteaso mes), but also directly, as individual prosome particles. (C) 1997 Aca demic Press.