Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Yunfeng Chen; Baoyu Liu; Lining Ju; Jinsung Hong; Qinghua Ji; Wei Chen; Cheng Zhu

doi:10.3791/52975

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Method Article

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

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DOI:

10.3791/52975

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August 4th, 2015

Yunfeng Chen^*¹ , Baoyu Liu^*² , Lining Ju^*³ , Jinsung Hong^*¹ , Qinghua Ji⁴^,⁵ , Wei Chen⁶ , Cheng Zhu¹

¹Woodruff School of Mechanical Engineering, Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, ²Coulter Department of Biomedical Engineering, Georgia Institute of Technology, ³Charles Perkins Centre, The University of Sydney, ⁴Institute of Biophysics, Laboratory of RNA Biology, Chinese Academy of Sciences, ⁵University of Chinese Academy of Sciences, ⁶School of Medicine and Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang University

^* These authors contributed equally

Summary

We describe a technique for concurrently measuring force-regulated single receptor-ligand binding kinetics and real-time imaging of calcium signaling in a single T lymphocyte.

Abstract

Membrane receptor-ligand interactions mediate many cellular functions. Binding kinetics and downstream signaling triggered by these molecular interactions are likely affected by the mechanical environment in which binding and signaling take place. A recent study demonstrated that mechanical force can regulate antigen recognition by and triggering of the T-cell receptor (TCR). This was made possible by a new technology we developed and termed fluorescence biomembrane force probe (fBFP), which combines single-molecule force spectroscopy with fluorescence microscopy. Using an ultra-soft human red blood cell as the sensitive force sensor, a high-speed camera and real-time imaging tracking techniques, the fBFP is of ~1 pN (10^-12 N), ~3 nm and ~0.5 msec in force, spatial and temporal resolution. With the fBFP, one can precisely measure single receptor-ligand binding kinetics under force regulation and simultaneously image binding-triggered intracellular calcium signaling on a single live cell. This new technology can be used to study other membrane receptor-ligand interaction and signaling in other cells under mechanical regulation.

Introduction

Cell-to-cell and cell-to-extracellular matrix (ECM) adhesion is mediated by binding between cell surface receptors, ECM proteins, and/or lipids¹. Binding allows cells to form functional structures¹, as well as recognize, communicate, and react to the environment^1-3. Unlike soluble proteins (e.g., cytokines and growth factors) that bind from a three-dimensional (3D) fluid phase onto the cell surface receptors, cell adhesion receptors form bonds with their ligands across a narrow junctional gap to bridge two opposing surfaces that constrain molecular diffusion in a two dimensional (2D) interface^4-7. In contrast to 3D ....