Visualization of Prosomes (MCP-Proteasomes), Intermediate Filament and Actin Networks by ‘‘Instantaneous Fixation’’ Preserving the Cytoskeleton C. Arcangeletti Institut J. Monod, CNRS, Universite ´ Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France; and Istituto di Microbiologia, Universita ` degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italy R. Su ¨ tterlin and U. Aebi M. E. Mu ¨ ller Institute, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland F. De Conto and S. Missorini Institut J. Monod, CNRS, Universite ´ Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France; and Istituto di Microbiologia, Universita ` degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italy C. Chezzi Istituto di Microbiologia, Universita ` degli Studi di Parma, Via Gramsci 14, 43100 Parma, Italy and K. Scherrer 1 Institut J. Monod, CNRS, Universite ´ Paris 7, 2 place Jussieu, 75251 Paris Cedex 05, France Received January 6, 1997, and in revised form March 15, 1997 A new ‘‘instantaneous’’ fixation/extraction proce- dure, yielding good preservation of intermediate filaments (IFs) and actin filaments when applied at 37°C, has been explored to reexamine the relation- ships of the prosomes to the cytoskeleton. Prosomes are protein complexes of variable subunit composi- tion, including occasionally a small RNA, which were originally observed as trans-acting factors in untranslated mRNPs. Constituting also the proteo- lytic core of the 26S proteasomes, they are also called ‘‘multicatalytic proteinase (MCP) complexes’’ or ‘‘20S-Proteasomes.’’ In Triton X-100-extracted epi- thelial, fibroblastic, and muscle cells, prosome par- ticles were found associated primarily with the IFs (Olink-Coux et al., 1994). Application of ‘‘instanta- neous fixation’’ has now led to the new observation that a major fraction of prosome particles, com- posed of specific sets of subunits, is distributed in variable proportions between the IFs and the micro- filament/stress fiber system in PtK1 epithelial cells and human fibroblasts. Electron microscopy using gold-labeled antibodies confirms this dual localiza- tion on classical whole mounts and on cells exposed to instantaneous fixation. In contrast to the resis- tance of the prosome–IF association, a variable frac- tion of the prosome particles is released from the actin cytoskeleton by Triton X-100 when applied prior to fixation. Moreover, in vitro copolymeriza- tion of prosomes with G-actin made it possible to observe ‘‘ladder-like’’ filamentous structures in the electron microscope, in which the prosome par- ticles, like the ‘‘rungs of a ladder,’’ laterally cross- link two or more actin filaments in a regular pat- tern. These results demonstrate that prosomes are bound in the cell not only to IFs but also to the actin cytoskeleton and, furthermore, not only within large M r complexes (possibly mRNPs and/or 26S protea- somes), but also directly, as individual prosome particles. r 1997 Academic Press INTRODUCTION Cytological research progressed considerably 20 years ago by the advent of monoclonal antibodies and more recently by confocal microscopy, which 1 To whom correspondence should be addressed. Fax: 33 1 44 27 76 47. E-mail: scherrer@ijm.jussieu.fr. JOURNAL OF STRUCTURAL BIOLOGY 119, 35–58 (1997) ARTICLE NO. SB973871 35 1047-8477/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.