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

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

Wang X1,2,3, Rahil Z1,4, Li IT1,5, Chowdhury F2,6, Leckband DE4, Chemla YR1, Ha T1,2,7,8,9.

Author information

1
Department of Physics, Center for the Physics of Living Cells, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
2
Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
3
Department of Physics and Astronomy, Iowa State University, Ames, Iowa 50011, USA.
4
Department of Chemistry, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
5
Department of Chemistry, University of British Columbia Okanagan, Kelowna, BC, V1V1V7, Canada.
6
Mechanical Engineering and Energy Processes, Southern Illinois University, Carbondale, Illinois 62901, USA.
7
Howard Hughes Medical Institute, Baltimore, MD, 21218, USA.
8
Department of Biophysics &Biophysical Chemistry, Johns Hopkins University, Baltimore, MD 21205, USA.
9
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.

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.

PMID:
26875524
PMCID:
PMC4753514
DOI:
10.1038/srep21584
[Indexed for MEDLINE]
Free PMC Article

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