Nanobodies reveal the secrets of neuronal communication

“There are hundreds of thousands of tiny proteins at the synapse moving fast and chaotically,” says Frederic Meunier, head of the Single Molecule Neuroscience Laboratory at Queensland Brain Institute, University of Queensland, Australia, “it is a very busy environment.” His main focus is to understand how neurons communicate with each other amid this chaos.

Visualizing the precise location and motion of proteins at the synapse is possible with super-resolution fluorescence microscopy, which, unlike conventional light microscopy, is not constrained by the diffraction limit of light of around 200 nm. Super-resolution methods allow visualization of two fluorophores separated by as little as 5 nm.

Protein targets are often labelled with primary antibodies and fluorophore-conjugated secondary antibodies. However, the distance between the target and the dye-molecule reporter can be up to 30 nm. Given that the gap between pre- and post-synaptic cells is only 20–40 nm, and the target proteins themselves are 2–5 nm in diameter and densely packed, conventional immunostaining reagents are simply too bulky.

To overcome these problems, researchers have turned to single-domain antibodies, also known as nanobodies. These are derived from the variable domains of heavy-chain-only antibodies (VHHs) — distinct immunoglobulin structures found naturally in sharks and camelids.

Nanobodies can be just as selective as whole antibodies but are a fraction of the size so they can reach sites that were thought to be inaccessible. “With fluorophore-conjugated nanobodies, the distance between the dye and the target, referred to as the linkage error, can be reduced to 1–2 nm,” says James Trimmer, distinguished professor and neuronal signalling expert at UC Davis Health and School of Medicine, California.

Shrinking labels

Trimmer and colleagues are working on developing, screening and validating novel nanobodies against neuronal proteins¹. “With nano-scale antibodies we can enhance penetration into tissue and increase labelling efficiency whilst preserving brain ultrastructure,” he says. His team have generated a toolbox of reagents for visualizing proteins with highly spatially restricted subcellular localization and key roles in synaptic plasticity². They have developed specific workflows for validating nanobodies for this purpose, and are screening large sets of candidate nanobodies for immunocytochemistry and immunohistochemistry on brain samples. “The enhanced imaging resolution with nanobodies, due to their reduced linkage error, means they are useful for interrogating the role of signalling molecules such as cAMP in synapse development.”

Meunier’s team, meanwhile, are examining synapses in living cells. They are using dye-conjugated intracellular nanobodies, known as intrabodies, which have been designed to be expressed in neurons and label endogenous proteins. “With single-molecule localization microscopy, we can literally see how specific proteins are trafficked to the synapse, and cluster in nanodomains at presynaptic terminals,” he says. Using nanobodies that target specific conformations of the endogenous β2-adrenoreceptors (β2-ARs), they were able to report differences in the mobility and clustering behaviour of active and inactive receptors for the first time³. “These tools can be applied to other synaptic proteins to further our understanding of the contribution of nanocluster dynamics to neuronal communication,” he adds.

Given the catastrophic consequences of synaptic dysfunction on essential neurological processes, there is great hope that the addition of nanobodies to the neuronal imaging toolbox will reveal new therapeutic opportunities. Trimmer concludes: “With new tools to visualize and analyse the molecular complexes that underlie neuronal activity, including their dynamics, we will gain a better understanding of brain function and provide insights into disease.”