Chapter 15 Live-Cell Single-Molecule Force Spectroscopy

Terrence M. Dobrowsky, Porntula Panorchan, Konstantinos Konstantopoulos, Denis Wirtz

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

We describe a method to measure the kinetics and micromechanical properties of individual receptor-ligand bonds formed between two living cells. Using living cells rather than recombinant proteins ensures that the orientation, surface density, and posttranslational modifications of the probed receptors are physiological and that their regulated attachment to the cytoskeleton can occur. A cell is tethered to a flexible cantilever and brought into contact with cells adherent to a substratum before being pulled at a controlled retraction velocity. Measurements of bond rupture forces and associated bond loading rates over an extended range of retraction velocities allow us to compute precisely the tensile strength, reactive compliance, lifetime, and dissociation rate of individual intercellular receptor-ligand bonds. We also describe tests of specificity and Monte Carlo simulations, which ensure that measurements obtained by this method correspond to a single type of intercellular adhesion bond. We illustrate this live-cell single molecule force spectroscopy assay by characterizing homotypic bonds composed of vascular endothelial -cadherin pairs formed between living endothelial cells. This versatile assay could be used to establish the molecular principles that drive a wide range of important physiological processes involving receptor-mediated intercellular adhesion, such as the immunological synapse between a lymphocyte and an antigen-presenting cell and synaptic interactions between neuron cells, and pathological processes resulting in altered intercellular adhesion.

Original languageEnglish (US)
Title of host publicationBiophysical Tools for Biologists, Volume Two
Subtitle of host publicationIn Vivo Techniques
EditorsJohn Correia, William Detrich III
Pages411-432
Number of pages22
DOIs
StatePublished - Dec 30 2008

Publication series

NameMethods in Cell Biology
Volume89
ISSN (Print)0091-679X

ASJC Scopus subject areas

  • Cell Biology

Fingerprint Dive into the research topics of 'Chapter 15 Live-Cell Single-Molecule Force Spectroscopy'. Together they form a unique fingerprint.

  • Cite this

    Dobrowsky, T. M., Panorchan, P., Konstantopoulos, K., & Wirtz, D. (2008). Chapter 15 Live-Cell Single-Molecule Force Spectroscopy. In J. Correia, & W. Detrich III (Eds.), Biophysical Tools for Biologists, Volume Two: In Vivo Techniques (pp. 411-432). (Methods in Cell Biology; Vol. 89). https://doi.org/10.1016/S0091-679X(08)00615-8