A General Strategy to Synthesize Ultrastable, Ligand-General Molecular Tension Probes for Visualizing Cell Forces

Abstract

Cells sense and transduce chemical and physical signals from their environment using a sophisticated set of mechanisms mediated by cell surface receptors such as integrins. Accordingly, there is significant interest in developing probes to investigate chemo-mechanical coupling in cell biology. Our lab has developed a suite of molecular probes that optically report forces exerted by cell surface receptors. These probes are comprised of a molecular spring flanked by a fluorophore-quencher pair and immobilized on a surface such that receptor-specific forces lead to dequenching of the fluorophore.There are a number of challenges pertaining to current molecular tension probe design: 1) immobilization is non-covalent, thus cell forces lead to detachment of probes, 2) protein ligand labeling is random, reducing probe sensitivity, and 3) peptide labeling uses Lys and Cys residues, constraining ligand design. Therefore, there is a pressing need to develop site-specific ligation strategies that are modular and well-suited for the diverse number of known or suspected mechano-receptors.In this work, we developed a “universal” strategy to generate molecular tension probes. The approach combines solid phase peptide synthesis along with native chemical ligation (NCL) to conjugate the C-terminus of virtually any biomolecule to fluorescently tagged tension probes. The role of linear PEG polymers (Mw = 2000 and 5000) and zwitterionic silanes were tested in blocking non-specific binding, and modulating sensor conformation. We generated a small library of peptide ligand modified tension probes and compared force response as a function of ligand-receptor interaction. Importantly, this new general strategy for generating molecular tension probes is facile and robust, and allows for long-term force imaging that extends for up to 3 days which is important in a range of processes including developmental biology and cell migration.