Constructing modular and universal single molecule tension sensor using protein G to study mechano-sensitive receptors

Sci Rep. 2016 Feb 15;6:21584. doi: 10.1038/srep21584.

Abstract

Recently a variety of molecular force sensors have been developed to study cellular forces acting through single mechano-sensitive receptors. A common strategy adopted is to attach ligand molecules on a surface through engineered molecular tethers which report cell-exerted tension on receptor-ligand bonds. This approach generally requires chemical conjugation of the ligand to the force reporting tether which can be time-consuming and labor-intensive. Moreover, ligand-tether conjugation can severely reduce the activity of protein ligands. To address this problem, we developed a Protein G (ProG)-based force sensor in which force-reporting tethers are conjugated to ProG instead of ligands. A recombinant ligand fused with IgG-Fc is conveniently assembled with the force sensor through ProG:Fc binding, therefore avoiding ligand conjugation and purification processes. Using this approach, we determined that molecular tension on E-cadherin is lower than dsDNA unzipping force (nominal value: 12 pN) during initial cadherin-mediated cell adhesion, followed by an escalation to forces higher than 43 pN (nominal value). This approach is highly modular and potentially universal as we demonstrate using two additional receptor-ligand interactions, P-selectin &PSGL-1 and Notch &DLL1.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Biomechanical Phenomena
  • Biosensing Techniques / instrumentation*
  • Biosensing Techniques / methods*
  • CHO Cells
  • Cadherins / physiology
  • Cell Adhesion
  • Cricetulus
  • DNA / physiology
  • Humans
  • Mechanoreceptors / physiology*
  • Recombinant Proteins / metabolism

Substances

  • Cadherins
  • Recombinant Proteins
  • DNA