In vitro assembly of homopolymer and copolymer filaments from intermediate filament subunits of muscle and fibroblastic cells.

Abstract

Evidence is presented that the intermediate filament (IF) subunits of [hamster stomach] muscle cells (skeleton or desmin) and fibroblastic cells (decamin or vimentin) separately form homopolymer IF in vitro and, when mixed, prefer to form copolymer IF in vitro. Because they coexist in cells, they may also form copolymers in vivo. The IF of baby hamster kidney fibroblasts (BHK-21) consist of a major subunit, decamin and 2 minor subunits which, on the basis of 2-dimensional gel and peptide mapping criteria, are identical to the .alpha.- and .beta.-subunits of smooth muscle desmin. The subunits differ only in their degrees of phosphorylation: .alpha.-desmin contained 2 mol/mol of O-phosphoserine whereas .beta.-desmin contained none. The decamin and desmin subunits assembled into homopolymer IF in vitro in high yield from purified denatured subunits under identical conditions of pH and ionic strength. Homopolymer decamin IF disassembled into soluble protofilaments in solutions of ionic strength < 0.05 mol/l whereas homopolymer desmin IF disassembled at ionic strength < 0.02 mol/l. When decamin and desmin were mixed together as denatured subunits or as soluble protofilaments, the IF assembled in vitro had solubility properties intermediate between those of the homopolymer IF, indicating that the 2 subunits had formed copolymer IF. The stoichiometry of copolymerization as determined in mixtures in which 1 subunit was present in excess was suggestive of the formation of 3 chain units. The possibility of nonspecific aggregation was eliminated by isolation of stable 3-chain .alpha.-helix-enriched particles from such IF. When tracer amounts of [35S]methionine-labeled decamin were mixed with desmin, labeled IF were obtained under conditions in which homopolymer decamin IF were soluble. These in vitro findings may be of physiological significance because native BHK-21 IF also had solubility properties similar to those of the copolymer IF.