Quantitative super-resolution imaging of Bruchpilot distinguishes active zone states

Abstract

The precise molecular architecture of synaptic active zones (AZs) gives rise to different structural and functional AZ states that fundamentally shape chemical neurotransmission. However, elucidating the nanoscopic protein arrangement at AZs is impeded by the diffraction-limited resolution of conventional light microscopy. Here we introduce new approaches to quantify endogenous protein organization at single-molecule resolution in situ with super-resolution imaging by direct stochastic optical reconstruction microscopy (dSTORM). Focusing on the Drosophila neuromuscular junction (NMJ), we find that the AZ cytomatrix (CAZ) is composed of units containing ~137 Bruchpilot (Brp) proteins, three quarters of which are organized into about 15 heptameric clusters. We test for a quantitative relationship between CAZ ultrastructure and neurotransmitter release properties by engaging Drosophila mutants and electrophysiology. Our results indicate that the precise nanoscopic organization of Brp distinguishes different physiological AZ states and link functional diversification to a heretofore unrecognized neuronal gradient of the CAZ ultrastructure.

Transmission, Development, and Plasticity of Synapses

“…Two isoforms of Brp appear to be arranged in an alternating circular pattern that is important for normal T-bar structure and SV tethering (Matkovic et al 2013). Furthermore, quantitative inferences about CAZ substructure and stoichiometry were made using data from stochastic optical reconstruction microscopy (dSTORM), which can provide information on the distribution of single molecules (Ehmann et al 2014). The authors propose that Brp molecules are clustered into 10-nm filaments, consistent with structures defined by EM, and that an average CAZ contains 137 molecules of Brp.…”

Superresolution microscopy reveals spatial separation of UCP4 and F 0 F 1 -ATP synthase in neuronal mitochondria

“…In photoswitching microscopy, the number of obtained single-molecule localizations usually exceeds the number of colocalized protein copies, partly because of repeated appearances of the same chromophore (47) during the recorded stream of images and partly because of a priori unknown degrees of labeling (48). In general, the total number of single-molecule localizations L tot resulting from N copies of a protein is given by L tot = N × L sm × η prim Ab × η sec Ab × η access , with L sm being the number of counts per single-protein molecule at the applied labeling conditions, η prim Ab and η sec Ab being the degrees of saturation of primary and secondary antibody labeling normalized to the maximally achievable labeling, respectively, and η access being the accessibility of the protein to antibody labeling (48). Although it is possible to determine L sm , η prim Ab , and η sec Ab , the unknown accessibility factor η access renders calculation of the protein copy number N difficult.…”

Artifacts in single-molecule localization microscopy

“…Especially, two-dimensional imaging of intrinsically threedimensional membrane structures like microvilli, filopodia, overlapping membranes and vesicles with high local emitter densities is prone to generate artifacts. To judge the quality and reliability of super-resolution images, the can even give quantitative information about the distribution of proteins and the ratio of molecules residing inside and outside of subcellular compartments such as plasma membrane domains (Williamson et al 2011;Lando et al 2012;Bar-On et al 2012;Puchner et al 2013;Ehmann et al 2014;Letschert et al 2014;Löschberger et al 2014;Saka et al 2014;Honigmann et al 2014;Fricke et al 2014;Gao et al 2015;Chen et al 2015).…”