erologous chicken vimentin subunits expressed under the control of an inducible promoter in transfected mouse fibroblasts. Using RNase protection, metabolic protein pulse-chase and immunofluorescence

The cytoskeleton in higher eukaryotes is composed of three major filament classes: actin, microtubules and intermediate filaments (IFs). Together with associated proteins, these filaments establish the internal architecture of cells. Of these three filament systems, the least is known about IF assembly characteristics and functional roles in vivo (for reviews see Steinert and Roop, 1988; Klymkowsky et al., 1989; Stewart, 1990). Owing to their relative insolubility in physiological conditions and small soluble subunit pool in vivo, IFs are often depicted as inert stable cytoskeletal components. Recent work, however, has clearly demonstrated that IFs are, in fact, highly dynamic structures (reviewed by Steinert and Liem, 1990; Skalli and Goldman, 1991). In vitro fluorescence resonance energy-transfer studies have demonstrated that the neurofilament polymer is in dynamic equilibrium with a small but kinetically active unassembled subunit pool (Angelides et al., 1989). Other studies have suggested a dynamic phosphorylation-mediated reversible disassembly of a wide variety of IFs including: nuclear lamins (Gerace and Blobel, 1980), neurofilaments (Hisanaga et al., 1990), desmin (Evans, 1988a; Geisler and Weber, 1988; Inagaki et al., 1988) and vimentin (Inagaki et al., 1987; Evans, 1988a,b). Even though most of these aforementioned studies have been carried out in vitro, such a phosphorylation-mediated IF assembly/disassembly mechanism likely plays an important role in vivo as well. Relatedly, microinjection of A-kinase into fibroblasts causes phosphorylation of specific vimentin moieties coincident with vimentin network collapse (Lamb et al., 1989). Furthermore, several reports have implicated cdc2 (an enzyme playing a key role in the regulation of mitosis) as the kinase responsible for site-specific lamin phosphorylation (Heald and McKeon, 1990; Peter et al., 1990; Ward and Kirschner, 1990) and vimentin phosphorylation (Chou et al., 1990). Thus, both A-kinase and cdc2 kinase may regulate IF assembly dynamics in vivo through phosphorylation of specific residues. Further evidence demonstrating IF assembly dynamics comes from transfection studies in which genes encoding tagged IF subunits were mutated, transfected and the resultant mutant proteins were assayed for assembly competence by immunofluorescence. This basic approach has been used by several groups to delineate structural and sequence requirements for IF assembly. Assembly-incompetent mutant neurofilament (Monteiro and Cleveland, 1989; Gill et al., 1990; Wong and Cleveland, 1990), vimentin (Christian et al., 1990), desmin (Raats et al., 1990) and keratin (Albers and Fuchs, 1987, 1989; Lu and Lane, 1990) subunits caused the breakdown of the entire filament network, suggesting that newly synthesized mutant subunits enter the pre-existing endogenous polymer, thereby facilitating its 689 Journal of Cell Science 103, 689-698 (1992) Printed in Great Britain © The Company of Biologists Limited 1992