Variation in cytoskeletal assembly during spreading of progressively subcultivated human embryo fibroblasts (IMR-90)

The cytoskeletons of early and late passage IMR-90 human diploid fibroblasts have been directly imaged in replicas of Triton X-100 extracted cells during spreading following reseeding. All cells from both young and sensescent cultures exhibit a cytoskeletal network of actin microfilaments, intermediate (10 nm) filaments, microtubules, and interconnecting thin filaments (6-8 nm in diameter) which do not interact with heavy meromyosin. Early passage cells assemble linear aggregates of actin filaments within 1 h of spreading. By 4 h of incubation, these bundles establish a structural bond with the cell membrane which results in resistance by the plasmalemma to detergent extraction at these sites. Furthermore, these membrane regions are associated with developing stress fibers of well-spread cells. In contrast, late passage cells exhibit slower spreading which correlates with a retarded assembly of actin bundles. In addition, by 8 h of spreading, cells of older cultures do not exhibit the regions of membrane-actin interaction which impart detergent resistance to the plasmalemma. We conclude that the ability to reassemble actin-actin and actin-membrane association during cell spreading is reduced with increased serial subcultivation of cells.