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.
Highlights
Spread more extensively as tension tolerance (Ttol) increases beyond 43 pN and that it is the molecular tension, not the molecular stiffness, that dictates the degree of cell spreading[14]
The general strategy of direct conjugation of a ligand to a tension gauge tether (TGT) or to other force-reporting tethers is not ideal for large protein ligands because the procedures used for conjugation, purification and storage may greatly reduce the ligand’s activity which was the case in our initial attempt to link epithelial cadherin (Ecad) directly to a DNA strand in a TGT
The new strategy of indirect binding of protein ligands to TGT through protein G was successful in maintaining the ligand activity for Ecad, P-selectin and Notch
Summary
Spread more extensively as Ttol increases beyond 43 pN and that it is the molecular tension, not the molecular stiffness, that dictates the degree of cell spreading[14]. DLD-1 cells expressing Ecad adhered and spread normally on the surface with physically adsorbed unconjugated Ecad whereas the same cells adhered less and spread poorly on the surface with physically adsorbed Ecad-ssDNA conjugates. We confirmed that Ecad activity was not reduced after binding to ProG as DLD-1 cells adhered and spread normally on a glass surface physically adsorbed with Ecad-Fc:ProG (SFig. 1). Because native ProG has an albumin binding domain and a membrane binding domain which can cause nonspecific binding[21], we used a ProG fragment, amino acids 190–384, which contains the IgG-Fc binding domain (ab49807, Abcam) We conjugated this ProG to our double stranded DNA-based TGT (Fig. 1B) using a bifunctional linker that targets a thiol on DNA and one of lysines on ProG as detailed in the Methods section. A biotin on TGT is used to immobilize a ligand on the surface in the format of ligand-Fc:ProG-TGT-biotin:neutravidin:biotin-surface
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