Structure of fibroblastic intermediate filaments: analysis of scanning transmission electron microscopy

The structure of fibroblastic intermediate filaments from Chinese hamster ovary cells has been investigated by scanning transmission electron microscopy. Freshly extracted (native) filaments were compared with filaments reassembled in vitro from purified decamin. From digital micrographs of unstained specimens, direct measurements of linear mass density were performed on many individual filaments. Native filaments beyond a certain minimal length constitute a homogeneous population, averaging 38 +/- 4 kilodaltons (kDal)/nm. A minor but distinct polymorphic variant (23 +/- 4 kDal/nm) was also present as very short filaments or end-segments; these may represent breakdown products or assembly intermediates. Analysis of reassembled filaments demonstrates that the in vitro assembly reaction is--in the main--faithful, although the distribution of their mass measurements is appreciably broader than that of the native data. In addition to the predominant type at 37 +/- 4 kDal/nm and a minor component at 26 +/- 4 kDal/nm, small amounts of a third, more massive, polymorphic variant at 52 +/- 5 kDal/nm were also present. Micrographs of negatively stained specimens clearly demonstrate that the filaments are composed of bundles of protofilaments--each 2-3 nm in diameter--and also reveal an axial periodicity of about 46 nm. The implications of these findings are discussed for three classes of model previously proposed for the structure of intermediate filaments.