Loss of giant obscurins alters breast epithelial cell mechanosensing of matrix stiffness.

ABSTRACT

Obscurins are a family of RhoGEF-containing proteins with tumor and metastasis suppressing roles in breast epithelium. Downregulation of giant obscurins in normal breast epithelial cells leads to reduced levels of active RhoA and of its downstream effectors. Herein, we elucidate how depletion of giant obscurins affects the response of breast epithelial cells to changes in the mechanical properties of the microenvironment. We find that knockdown of obscurins increases cell morphodynamics, migration speed, and diffusivity on polyacrylamide gels of ≥ 1 kPa, presumably by decreasing focal adhesion area and density as well as cell traction forces. Depletion of obscurins also increases cell mechanosensitivity on soft (0.4-4 kPa) surfaces. Similar to downregulation of obscurins, pharmacological inhibition of Rho kinase in breast epithelial cells increases migration and morphodynamics, suggesting that suppression of Rho kinase activity following obscurin knockdown can account for alterations in morphodynamics and migration. In contrast, inhibition of myosin light chain kinase reduces morphodynamics and migration, suggesting that temporal changes in cell shape are required for efficient migration. Collectively, downregulation of giant obscurins facilitates cell migration through heterogeneous microenvironments of varying stiffness by altering cell mechanobiology.