FILENSIN AND PHAKININ FORM A NOVEL TYPE OF BEADED INTERMEDIATE FILAMENTS AND COASSEMBLE DE-NOVO IN CULTURED-CELLS

The fiber cells of the eye lens possess a unique cytoskeletal system k nown as the ''beaded-chain filaments'' (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) pr oteins. To examine the organization and the assembly of these heteropo lymeric IFs, we have performed a series of in vitro polymerization stu dies and transfection experiments. Filaments assembled from purified f ilensin and phakinin exhibit the characteristic 19-21-nm periodicity s een in many types of IFs upon low angle rotary shadowing. However, qua ntitative mass-per-length (MPL) measurements indicate that filensin/ph akinin filaments comprise two distinct and dissociable components: a c ore filament and a peripheral filament moiety. Consistent with a nonun iform organization, visualization of unfixed and unstained specimens b y scanning transmission electron microscopy (STEM) reveals the existen ce of a central filament which is decorated by regularly spaced 12-15- nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures wh en expressed transiently in cultured cells. Experiments in IF-free SW1 3 cells reveal that coassembly of the lens-specific proteins in vivo d oes not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nucle ar envelope. However, filament assembly in CHO and SV40-transformed le ns-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These ob servations document that the filaments formed by lens-specific IF prot eins are structurally distinct from ordinary cytoplasmic IFs. Furtherm ore, the results suggest that the spatial arrangement of filensin/phak inin filaments in vivo is subject to regulation by host-specific facto rs. These factors may involve cytoskeletal networks (e.g., vimentin IF s) and/or specific sites associated with the cellular membranes.