Characterization of the Biophysical Origins of Mechanical Homeostasis at Cellular Adhesions

Abstract

Epithelial cells transmit mechanical forces through E-cadherin-mediated intercellular contacts, and these dynamic complexes undergo force-dependent remodeling. At present, however, fundamental aspects of how cells detect and generate mechanical forces at intercellular junctions remain poorly understood. Here, we describe a FRET-based molecular force sensor that reports the forces exerted via single E-cadherin complexes in living cells. We have created single-molecule tension sensors (SMTS) that replace the fluorescent proteins in a previously reported FRET-based force probe (Grashoff et al., Nature 2010) with organic fluorophores that can be observed at the single-molecule level. SMTS attach to glass coverslips, and present domains from protein A to immobilize the antibody Fc domain of a fusion protein containing the E-cadherin extracellular domain (Ecad-Fc). Madin-Darby Canine Kidney (MDCK) epithelial cells adhere to surfaces functionalized with SMTS and Ecad-Fc, but not to surfaces exposed to either SMTS or Ecad-Fc alone, showing that cell attachment occurs through the Ecad-Fc/SMTS complex. Ensemble FRET measurements reveal that the force per individual E-cadherin complex is approximately 2 pN, an order of magnitude lower than previously reported AFM measurements of E-cadherin homophilic bond rupture forces (Zhang et al., Proc Natl Acad Sci USA 2009). This difference may indicate that the maximal load supported by individual E-cadherins is considerably greater than what is generated at equilibrium. Consistent with this interpretation, the forces we measure agree with the single-pN tensions inferred from ensemble FRET measurements using a genetically-encoded E-cadherin force sensor (Borghi et al., Proc Natl Acad Sci USA 2012). The ability of the SMTS described here to selectively recruit fusion proteins and antibodies via their Fc domains makes them a flexible and potentially powerful tool for studying cellular mechanotransduction.