Characterization of the Vimentin Intermediate Filament Network on Substrates of Varying Stiffness

Abstract

Cells, in vivo, generally live in environments that are much softer (∼kPa stiffness) than tissue culture plastic or glass on which they are usually studied (∼GPa stiffness). While the response of the microtubule and the actin cytoskeletal networks to substrate stiffness has been previously studied, the response of the vimentin intermediate filament network to changing substrate stiffness is not known. When cells are grown on different stiffness polyacrylamide gels, there is no change in the total amount of vimentin protein. However, there is a significant change in the amount of vimentin protein that can be extracted by Triton-X 100 when the cells are grown on substrates of physiological stiffness. In human mesenchymal stem cells (hMSCs) cultured on glass, less than 5% of the vimentin is in the soluble pool. On substrates of physiological stiffness, the amount of soluble vimentin responds in a biphasic manner. The amount of soluble vimentin increases as stiffness decreases to a peak of about 65% soluble pool in cells cultured on a 5 kPa substrate. The amount of soluble vimentin then decreases again as substrate stiffness decreases to 0.2 kPa. This phenomenon appears dependent on contractility, as on stiff gels treated with blebbistatin or cytochalasin D the soluble vimentin pool increases and there is no change in soluble vimentin on 5 kPa gels. In addition, cells grown in a confluent monolayer on a 5 kPa gel show a significant decrease in the soluble pool. These observations can help to elucidate the function of the vimentin network, and suggest that in vivo cells maintain a much larger pool of dynamic vimentin than is seen under standard tissue culture conditions.