TGF-β regulates LARG and GEF-H1 during EMT to affect stiffening response to force and cell invasion.
Bottom Line: Recent studies implicate a role for cell mechanics in cancer progression.Previously, force application on integrins has been shown to initiate cytoskeletal rearrangements that result in increased cell stiffness and a stiffening response.Here we demonstrate that transforming growth factor β (TGF-β)-induced EMT results in decreased stiffness and loss of the normal stiffening response to force applied on integrins.
Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.Show MeSH
Related in: MedlinePlus
Mentions: To test whether EMT affects GEF recruitment to sites of force application, we developed a rotating permanent magnet device (Supplemental Figure S2A) to generate a force regimen consistent in magnitude, duty cycle, and frequency with that produced by the magnetic tweezers (Supplemental Figure S2B) and pulsatile forces to the cells via externally attached FN-coated beads. After force stimulation, we separated the bead fraction containing adhesion complex proteins from the whole lysate (Guilluy et al., 2011) and found that LARG and GEF-H1 were recruited in a time-dependent manner in epithelial-state NMuMG cells. In contrast, TGF-β–induced EMT abrogated this time-dependent recruitment of LARG and GEF-H1 (Figure 5A). Examination of p114, another RhoGEF, showed no recruitment to the adhesion complex and was unchanged after EMT (Figure 5A), suggesting that LARG and GEF-H1 have specific roles in force transduction during EMT. Similar loss of force-dependent recruitment of LARG and GEF-H1 was found in PANC-1 cells treated with BMP-2 (Figure 5B).
Affiliation: Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599.