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

// Kimberly M. Stroka 1 , Bin Sheng Wong 2, 3, 4 , Marey Shriver 5 , Jude M. Phillip 2, 3, 4 , Denis Wirtz 2, 3, 4 , Aikaterini Kontrogianni-Konstantopoulos 5, 6 and Konstantinos Konstantopoulos 2, 3, 4 1 Fischell Department of Bioengineering, University of Maryland, College Park, MD, 20742, USA 2 Johns Hopkins Institute for NanoBioTechnology, The Johns Hopkins University, Baltimore, MD, 21218, USA 3 Johns Hopkins Physical Sciences-Oncology Center, The Johns Hopkins University, Baltimore, MD, 21218, USA 4 Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD, 21218, USA 5 University of Maryland School of Medicine, Department of Biochemistry and Molecular Biology, Baltimore, MD, 21201, USA 6 University of Maryland School of Medicine, Marlene and Stewart Greenebaum National Cancer Institute Cancer Center, Baltimore, MD, 21201, USA Correspondence to: Kimberly M. Stroka, email: kstroka@umd.edu Konstantinos Konstantopoulos, email: konstant@jhu.edu Keywords: matrix stiffness, mechanosensitivity, cell migration, obscurin, RhoA Received: June 07, 2016      Accepted: July 20, 2016      Published: August 01, 2016 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.