Neurexin/Neuroligin Interaction Kinetics Characterized by Counting Single Cell-Surface Attached Quantum Dots

Abstract

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.

Neurexin-Neuroligin Adhesions Capture Surface-Diffusing AMPA Receptors through PSD-95 Scaffolds

“…1C,D). Intriguingly, the basal mobility of anti-GluA1-conjugated Qdots was twice lower than that of anti-GluA2-coated Qdots, reflecting either a valency difference between entire anti-GluA1 antibodies compared with anti-GluA2 Fabs (Saint-Michel et al, 2009), and/or an intrinsically higher diffusion of GluA2/GluA3 versus GluA1/GluA2 heteromers, as reported (Kropf et al, 2008).…”

“…The blinking behavior is a photo-physical characteristics of Qdots and a signature of the fact that they are isolated entities . However, even if Qdots form small aggregates, their diffusion coefficient should be reduced by a factor proportional to the logarithm of the aggregate size (Saint-Michel et al, 2009), thus making a small contribution to the measurement of AMPAR diffusion properties.…”

“…1C,D). Custom software provided calculation of the instantaneous diffusion coefficient for each trajectory (Saint-Michel et al, 2009), and the distributions of diffusion coefficients were plotted for hundreds of Qdots, allowing comparison between conditions. Global AMPAR diffusion was decreased by a factor of 2 upon overexpression of Nlg1WT, compared with control cells (Fig.…”

Assembly of Synapses: Biomimetic Assays to Control Neurexin/Neuroligin Interactions at the Neuronal Surface

“…This approach has been used, for instance, to reveal that Nlg1, like AMPARs, alternates between phases of free diffusion in extrasynaptic regions and phases of confinement to synapses, monitored as spots of Homer1c:GFP accumulation. In addition, Nrx1β-Fc-coated QDs have been observed to detach from the cell surface, reflecting occasional dissociation of individual Nrx1β/Nlg1 bonds (Saint-Michel et al, 2009). For this purpose, QDs were imaged at a frame rate of 0.5 Hz and the number of QDs remaining bound to the neuronal surface was measured over time (up to 30 min; Fig.…”

Tetraspanin-3 is an organizer of the multi-subunit Nogo-A signaling complex

“…Qdot imaging was conducted with Qdot ® -655 goat F(ab′) 2 anti-mouse IgG conjugates (1 μM stock solution, Life Technologies), and Qdots were prepared in a similar manner to that described before (Saint-Michel et al, 2009). Briefly, Qdots were mixed with anti-TSPAN3 antibodies (1 mg/ml, Ptglab) in 1× PBS for 20 min at a 3:1 dilution, blocked with 10% casein solution and centrifuged at 5000 g for 3 min prior to use.…”