MBoC | ARTICLE Substrate stiffness regulates solubility of cellular vimentin

ABSTRACT The intermediate filament protein vimentin is involved in the regulation of cell behavior, morphology, and mechanical properties. Previous studies using cells cultured on glass or plastic substrates showed that vimentin is largely insoluble. Although substrate stiff-ness was shown to alter many aspects of cell behavior, changes in vimentin organization were not reported. Our results show for the first time that mesenchymal stem cells (hMSCs), en-dothelial cells, and fibroblasts cultured on different-stiffness substrates exhibit biphasic changes in vimentin detergent solubility, which increases from nearly 0 to 67 % in hMSCs co-incident with increases in cell spreading and membrane ruffling. When imaged, the deter-gent-soluble vimentin appears to consist of small fragments the length of one or several unit-length filaments. Vimentin detergent solubility decreases when these cells are subjected to serum starvation, allowed to form cell–cell contacts, after microtubule disruption, or inhibi-tion of Rac1, Rho-activated kinase, or p21-activated kinase. Inhibiting myosin or actin assem-bly increases vimentin solubility on rigid substrates. These data suggest that in the mechani-cal environment in vivo, vimentin is more dynamic than previously reported and its assembly state is sensitive to stimuli that alter cellular tension and morphology