Neurexin/neuroligin interaction kinetics characterized by counting single cell-surface attached quantum dots.
We report what to our knowledge is a new method to characterize kinetic rates between cell-surface-attached adhesion molecules. Cells expressing specific membrane receptors are surface-labeled with quantum dots coated with their respective ligands. The progressive diminution in the total number of surface-diffusing quantum dots tracked over time collectively reflects intrinsic ligand/receptor interaction kinetics. The probability of quantum dot detachment is modeled using a stochastic analysis of bond formation and dissociation, with a small number of ligand/receptor pairs, resulting in a set of coupled differential equations that are solved numerically. Comparison with the experimental data provides an estimation of the kinetic rates, together with the mean number of ligands per quantum dot, as three adjustable parameters. We validate this approach by studying the calcium-dependent neurexin/neuroligin interaction, which plays an important role in synapse formation. Using primary neurons expressing neuroligin-1 and quantum dots coated with purified neurexin-1beta, we determine the kinetic rates between these two binding partners and compare them with data obtained using other techniques. Using specific molecular constructs, we also provide interesting information about the effects of neurexin and neuroligin dimerization on the kinetic rates. As it stands, this simple technique should be applicable to many types of biological ligand/receptor pairs.
Saint-Michel
Edouard
E
Physiologie Cellulaire de la synapse, Centre National de la Recherche Scientifique and University of Bordeaux, Bordeaux, France.
Giannone
Grégory
G
Choquet
Daniel
D
Thoumine
Olivier
O
D000818:Animals; D015816:Cell Adhesion Molecules, Neuronal; D003198:Computer Simulation; D004058:Diffusion; D006801:Humans; D007700:Kinetics; D008024:Ligands; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009457:Neuroglia / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D045663:Quantum Dots
D015816:Cell Adhesion Molecules, Neuronal; D008024:Ligands; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C093438:neuroligin 1; C086959:neurexin Ibeta