Quantitative DNA-PAINT imaging of AMPA receptors in live neurons

Abstract

Summary: DNA-point accumulation for imaging at nanoscale topography (DNA-PAINT) can image fixed biological specimens with nanometer resolution and absolute stoichiometry. In living systems, however, the usage of DNA-PAINT has been limited due to high salt concentration in the buffer required for specific binding of the imager to the docker attached to the target. Here, we used multiple binding motifs of the docker, from 2 to 16, to accelerate the binding speed of the imager under physiological buffer conditions without compromising spatial resolution and maintaining the basal level homeostasis during the measurement. We imaged endogenous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) in cultured neurons—critical proteins involved in nerve communication—by DNA-PAINT in 3-dimensions using a monovalent single-chain variable fragment (scFv) to the GluA1 subunit of AMPAR. We found a heterogeneous distribution of synaptic AMPARs: ≈60% are immobile, primarily in nanodomains, defined as AMPARs that are within 0.3 μm of the Homer1 protein in the postsynaptic density; the other ∼40% of AMPARs have restricted mobility and trajectory. Motivation: Single-molecule localization microscopy can provide nanoscale resolution of molecular structure of biological samples, yet the application of the technique in live samples is limited. Here, we developed a live-cell DNA-PAINT technique to image and quantify neuronal receptor molecules in live hippocampal neurons.

Keywords

• AMPAR
• DNA-PAINT
• live cell imaging