Mechano-ID: Proximity Labeling of Mechanically Active Receptors Reveals the Mechanome and Tags Mechanically Active Cells

A major challenge in the field of mechanobiology relates to the lack of methods that enable the identification of mechanically active receptors, associated proteins, and the individual cells that display enhanced force generation. For example, potent T cell activation requires the transmission of biophysical forces between the T cell receptor (TCR) and its peptide-loaded major histocompatibility (pMHC) complex antigens. Interestingly, TCR-antigen interactions are highly dynamic, displaying a broad range of force magnitudes between different cells and even within the same cell. Therefore, methods that can chemically tag mechanically active T cells, TCRs, and their associated proteomes, or mechanomes are highly desirable. Such techniques may enable a deeper understanding of the mechanisms governing immune responses and may also have broad applications in immunotherapy. Herein, we report a technique dubbed mechano-ID, which allows for mechanically selective proximity tagging by leveraging DNA-based molecular force probes that recruit proximity tagging enzymes. We demonstrate mechano-ID tagging of T cells using microscopy and flow cytometry, with further confirmation by proteomics and Western blotting of mechanically active T cell receptors.