Microfilaments and tropomyosin of cultured mammalian cells: isolation and characterization

Abstract

Microfilaments were isolated from cultured mammalian cells, using procedures similar to those for isolation of native thin filaments from muscle. Isolated microfilaments from rat embryo, baby hamster kidney (BHK-21) and Swiss mouse [fibroblast] cells appeared structurally similar to muscle thin filaments, exhibiting long, 6 nm Diam profiles with a beaded, helical substructure. An arrowhead pattern was observed after reaction of isolated microfilaments with rabbit skeletal muscle myosin subfragment 1. Under appropriate conditions, isolated microfilaments will aggregate into a form that resembles microfilament bundles seen in situ in cultured cells. Isolated microfilaments represent a complex of proteins including actin. Some of these components have been tentatively identified, based on coelectrophoresis with purified proteins, as myosin, tropomyosin and a high MW actin-binding protein. The tropomyosin components of isolated microfilaments were unexpected; polypeptides comigrated on SDS[sodium dodecyl sulfate]-polyacrylamide gels with muscle and nonmuscle types of tropomyosin. To identify more specifically these subunits, tropomyosin from 3 cell types were isolated and partially characterized. BHK-21 cell tropomyosin was similar to other nonmuscle tropomyosins, as judged by several criteria. Tropomyosin isolated from rat embryo and 3T3 cells contained subunits that comigrated with skeletal muscle and nonmuscle types of myosin; the BHK cell protein consistently contained a minor muscle-like subunit. The array of tropomyosin subunits present in a cell culture was reflected in the polypeptide chain pattern seen on SDS-polyacrylamide gels of microfilaments isolated from that culture. These studies provide a starting point for correlating changes in the ultrastructural organization of microfilaments with alterations in their protein composition.