Quantum dot conjugated nanobodies for multiplex imaging of protein dynamics at synapses.

Neurons communicate with each other through synapses, which show enrichment for specialized receptors. Although many studies have explored spatial enrichment and diffusion of these receptors in dissociated neurons using single particle tracking, much less is known about their dynamic properties at synapses in complex tissue like brain slices. Here we report the use of smaller and highly specific quantum dots conjugated with a recombinant single domain antibody fragment (VHH fragment) against green fluorescent protein to provide information on diffusion of adhesion molecules at the growth cone and neurotransmitter receptors at synapses. Our data reveals that QD-nanobodies can measure neurotransmitter receptor dynamics at both excitatory and inhibitory synapses in primary neuronal cultures as well as in ex vivo rat brain slices. We also demonstrate that this approach can be applied to tagging multiple proteins to simultaneously monitor their behavior. Thus, we provide a strategy for multiplex imaging of tagged membrane proteins to study their clustering, diffusion and transport both in vitro as well as in native tissue environments such as brain slices.