Dual Biomembrane Force Probe enables single-cell mechanical analysis of signal crosstalk between multiple molecular species

Abstract

Conventional approaches for studying receptor-mediated cell signaling, such as the western blot and flow cytometry, are limited in three aspects: 1) The perturbing preparation procedures often alter the molecules from their native state on the cell; 2) Long processing time before the final readout makes it difficult to capture transient signaling events (<1 min); 3) The experimental environments are force-free, therefore unable to visualize mechanical signals in real time. In contrast to these methods in biochemistry and cell biology that are usually population-averaged and non-real-time, here we introduce a novel single-cell based nanotool termed dual biomembrane force probe (dBFP). The dBFP provides precise controls and quantitative readouts in both mechanical and chemical terms, which is particularly suited for juxtacrine signaling and mechanosensing studies. Specifically, the dBFP allows us to analyze dual receptor crosstalk by quantifying the spatiotemporal requirements and functional consequences of the up- and down-stream signaling events. In this work, the utility and power of the dBFP has been demonstrated in four important dual receptor systems that play key roles in immunological synapse formation, shear-dependent thrombus formation, and agonist-driven blood clotting.

Compression force sensing regulates integrin αIIbβ3 adhesive function on diabetic platelets

“…5a,b ). Therefore, we combined the compression assay and a newly developed “switch assay” using a dual BFP setup 51 (Fig. 6a lower).…”

T-Cell Mechanobiology: Force Sensation, Potentiation, and Translation

“…The off-rate of single TCR-pMHC bond is extracted from the distribution of lifetime using a firstorder irreversible dissociation model [11,155]. A dual BFP has been developed to allow for multiple for temporally distinct ligand presentation to study crosstalk between surface receptors [156]. Finally, deformations of the aspirated cell morphology can be used to monitor pushing and pulling forces, which has been applied to studying T-cell responses to antibodies [157].…”

Illustrated State‐of‐the‐Art Capsules of the ISTH 2019 Congress in Melbourne, Australia

“…25 We also developed a dual biomembrane force probe to visualize integrin biomechanical activation on a living platelet. 26 We found that, upon mechano-signaling of GPIb, αIIbβ3 adopts the EC conformation and possesses affinity and bond lifetime that are intermediate between the well-characterized active and resting states. This inside-out mechano-signaling is distinct from the biochemical signaling by agonists and potentiates the outside-in mechano-signaling of αIIbβ3 for affinity maturation (B).…”

“…24 Our work highlighted the existence, genesis, and regulation of a semistable intermediate state of αIIbβ3 that is fundamental in mechanically driven thrombosis. 26…”