Hydrostatic Pressure Has Different Effects on the Assembly of Tubulin, Actin, Myosin II, Vinculin, Talin, Vimentin, and Cytokeratin in Mammalian Tissue Cells

Abstract

Hydrostatic pressures in the range of hundreds of atmospheres are known to disrupt cytoskeletal organization in tissue culture cells, with profound changes in cell shape. The molecular mechanisms of these effects are poorly understood. To determine the effect of pressure on the cytoskeleton, and thus to provide better indicators of the molecular mechanisms, we used fluorescent antibody staining to compare the organizations of seven different cytoskeletal proteins in HeLa cells and rat osteosarcoma cells (ROS-17/2.8) subjected to different pressures up to 400 atm. Pressures of 300 atm or more caused cells of both lines to “round up” and to withdraw their lamellar extensions. However, this response varied within a population of cells, with some cells remaining spread at pressures that caused their neighbors to round up. The most resistant to rounding were those cells touching other cells, and the occasional giant cells. As expected, the rounded cells showed disruption of actin stress fibers and of vinculin and talin at focal contacts. The unrounded cells showed less disruption in the organization of these same proteins. Microtubules and myosin II filaments appeared resistant to 400 atm pressure in both cell types, whether rounded or unrounded. However, in HeLa cells, the intermediate filaments, vimentin and cytokeratin, depolymerized and formed small vesicles when pressures exceeded 200 atm, and this occurred in rounded as well as unrounded cells. In osteosarcoma cells, which do not have cytokeratin, vimentin did not depolymerize. We discuss different mechanisms that might explain these responses to pressure, including direct effects on the equilibria of protein polymerization and less direct effects on regulatory mechanisms, such as phosphorylation pathways, that control cytoskeletal organization. The latter type of explanation seems more consistent with both the variability of response within cell populations and the difference in vimentin's response in one cell line compared with the other.