Chapter 15: Live-cell single-molecule force spectroscopy.
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.
Dobrowsky
Terrence M
TM
Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Panorchan
Porntula
P
Konstantopoulos
Konstantinos
K
Wirtz
Denis
D
D015820:Cadherins / Q000502:physiology*; D002448:Cell Adhesion; D002460:Cell Line; D042783:Endothelial Cells / Q000502:physiology; D006801:Humans; D055595:Mechanical Phenomena; D009010:Monte Carlo Method; D013057:Spectrum Analysis / Q000379:methods*
D015820:Cadherins