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.Over the past decade, single-molecule biomechanical analyses on single cells have enabled studies of the inner workings of adhesion and signaling receptors one at a time 1 . In physiological conditions, however, multiple receptor species are present and often work cooperatively rather than independently. Understanding how multiple receptor species crosstalk to each other is essential for determining the mechanisms underlying a wide range of biological processes related to human health and diseases. One of the model receptor systems that exhibit such signaling crosstalk with other receptors is the integrin family. For example, integrin α L β 2 , or lymphocyte function-associated antigen-1 (LFA-1), crosstalks with chemokine receptors and antigen receptors via an "inside-out" signaling process 2 , which is essential for lymphocyte trafficking and immunological synapse formation 3 . The interaction between LFA-1 on a lymphocyte and its ligand, intercellular adhesion molecule-1 (ICAM-1) on an adjacent cell, can be upregulated by signals induced by binding of chemokines and antigens to their respective receptors on the same lymphocyte, manifesting enhanced cell adhesion 4,5 . As another example, upon a rapid shear force increase caused by blood flow perturbations, the function of integrin α IIb β 3 , or glycoprotein (GP) IIb-IIIa, on a platelet surface is rapidly upregulated by signals induced by ligand engagement with the mechanoreceptor GPIb 6,7 , which promotes platelet adhesion at the site of vascular injury as part of the hemostatic and thrombotic processes 8 . The synergistic binding of GPIb and GPIIb-IIIa enables efficient platelet recruitment

Receptor-mediated cell mechanosensing

“…More recently, the dual biomembrane force probe (dBFP) has been developed, which allows examination of the signal reception, initiation, and transduction from one receptor to another on a single cell step by step in space and time. It has demonstrated that the mechano-signals induced by TCR–pMHC and GPIbα–VWF lead to the up-regulation of integrin functions on a T-cell and a platelet, respectively ( Ju et al. , 2017 ).…”

Shear-induced integrin signaling in platelet phosphatidylserine exposure, microvesicle release, and coagulation

“…The reporter BFP consisted of a micropipette aspirated human red blood cell with a glass bead coated with annexin V attached to its apex via biotin-streptavidin coupling. [41][42][43] An opposing micropipette aspirated a platelet and was driven by a piezoelectric translator to move forward/backward to get in/out of contact with the annexin V bead to test for binding. The horizontal position of the bead was tracked by a high-speed camera, which reflected the red blood cell axial deformation.…”

Biomechanical thrombosis: the dark side of force and dawn of mechano-medicine

“…Inhibition of the GPIb-triggered Ca 2+ prevents platelet firm adhesion, suggesting that Ca 2+ is an obligatory signaling molecule on the pathway to GPIIb/IIIa activation 8 9 61 62. Although how the transmission of the mechanical signal across the membrane and its transduction into cytoplasmic chemical signals are fulfilled remains elusive, a juxtamembrane mechanosensitive domain (MSD) in the GPIbα subunit was found to correlate with intraplatelet Ca 2+ triggering (figure 1): force-induced MSD unfolding is well correlated with an α-type Ca 2+ signal8 9 and the subsequent GPIIb/IIIa intermediate activation, whereas failing to unfold MSD will most likely result in β-type or null-type Ca 2+ signals and limit GPIIb/IIIa activation 60 63. Intracellularly, a scaffold protein that binds to GPIb cytoplasmic tail, 14-3-3ζ, was also found to play a pivotal role in GPIb mechanosignaling (figure 1), as the inhibition of 14-3-3ζ–GPIb association strongly inhibited GPIb-triggered Ca 2+ flux and subsequent GPIIb/IIIa activation 60 64.…”