Despite a gradually emerging comprehensive protein catalogue, we still lack basic information describing how the nanoscopic organisation of proteins gives rise to cell biological functions. In essence, this is due to the diffraction-limited resolution of conventional light microscopy, which has hindered access to the spatial nanodomain in a physiologically relevant context.

To take this next step, we focused on the organisation of an established major component at the synaptic active zone (AZ), Drosophila Bruchpilot (Brp)^1,2. Super-resolution fluorescence imaging was employed to test whether nanoscopic adjustments to the AZ ultrastructure encode information on synaptic plasticity. Crucially, the utilisation of direct stochastic optical reconstruction microscopy (dSTORM³) yielded not only a spatial resolution well below 20 nm in tissue of neuromuscular junctions, but its explicit single-molecule sensitivity also delivered an estimate of the actual number of endogenous Brp molecules assembled at the AZ. By applying electrophysiology to functionally calibrate super-resolution images, we find that transmitter release sites and release probability scale with Brp protein number, while the radial distribution of Brp within AZs reflects vesicle trafficking speed.

The results illustrate that the precise nanoscopic organisation of a specific molecule in individual AZs provides a prediction of short-term plasticity and distinguishes distinct mechanistic causes of synaptic depression.

1. Wagh, D.A. et al. Neuron 49, 833-844 (2006).

2. Kittel, R.J. et al. Science 312, 1051 (2006).

3. van de Linde, S. et al. Nat Protoc 6, 991-1009 (2011).