Imaging and Calibration of Receptor-Specific Cellular Forces Using Biotinylated Ligands.

Covalent conjugation of ligand molecules to force sensors provides a powerful approach to image receptor-transmitted cellular forces. While this strategy is effective for small-molecule ligands, the conjugation process can damage large protein ligands. To address this challenge, we developed a modular mechano-fluorescent coating (mMFC) that enables receptor-specific force imaging using widely available biotinylated ligands. The mMFC construct consists of a poly-L-lysine (PLL) backbone for substrate adsorption, polyethylene glycol (PEG) grafts to prevent nonspecific binding, and DNA-based integrative tension sensors (ITS) as force-reporting units. Benefiting from copper-free click chemistry, the mMFC is synthesized via a simple one-pot reaction. During application, the mMFC is adsorbed onto substrates, and biotinylated ligands are subsequently linked to the mMFC construct via biotin-avidin interactions, where the ITS reports receptor-transmitted forces by fluorescence. By testing HeLa cells, platelets, and human T lymphocytes on mMFC platforms, we imaged cellular forces transmitted through RGD peptide, latency-associated peptide (LAP), ICAM-1, and anti-CD3, which target integrins, LFA-1, and T cell receptor (TCR) complex, respectively. We further demonstrated molecular force calibration and single-molecule force imaging of these receptors using the mMFC platform. Overall, the mMFC provides a modular, convenient, and robust platform for investigating mechanotransduction across diverse receptors in various cell types.