Mapping the dynamics and nanoscale organization of synaptic adhesion proteins using monomeric streptavidin

Nat Commun. 2016 Mar 16;7:10773. doi: 10.1038/ncomms10773.

Abstract

The advent of super-resolution imaging (SRI) has created a need for optimized labelling strategies. We present a new method relying on fluorophore-conjugated monomeric streptavidin (mSA) to label membrane proteins carrying a short, enzymatically biotinylated tag, compatible with SRI techniques including uPAINT, STED and dSTORM. We demonstrate efficient and specific labelling of target proteins in confined intercellular and organotypic tissues, with reduced steric hindrance and no crosslinking compared with multivalent probes. We use mSA to decipher the dynamics and nanoscale organization of the synaptic adhesion molecules neurexin-1β, neuroligin-1 (Nlg1) and leucine-rich-repeat transmembrane protein 2 (LRRTM2) in a dual-colour configuration with GFP nanobody, and show that these proteins are diffusionally trapped at synapses where they form apposed trans-synaptic adhesive structures. Furthermore, Nlg1 is dynamic, disperse and sensitive to synaptic stimulation, whereas LRRTM2 is organized in compact and stable nanodomains. Thus, mSA is a versatile tool to image membrane proteins at high resolution in complex live environments, providing novel information about the nano-organization of biological structures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotinylation
  • Cell Adhesion Molecules, Neuronal / metabolism*
  • Green Fluorescent Proteins
  • HEK293 Cells
  • Hippocampus / cytology
  • Humans
  • Indicators and Reagents
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Knockout
  • Molecular Dynamics Simulation
  • Nanotechnology / methods*
  • Nerve Tissue Proteins / metabolism*
  • Neural Cell Adhesion Molecules / metabolism
  • Neurons / metabolism*
  • Rats
  • Staining and Labeling / methods*
  • Streptavidin
  • Synapses / metabolism*

Substances

  • Cell Adhesion Molecules, Neuronal
  • Indicators and Reagents
  • LRRTM2 protein, human
  • LRRTM2 protein, mouse
  • LRRTM2 protein, rat
  • Membrane Proteins
  • Nerve Tissue Proteins
  • Neural Cell Adhesion Molecules
  • neuroligin 1
  • Green Fluorescent Proteins
  • neurexin Ibeta
  • Streptavidin