Journal Article

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

Chamma, Ingrid, Tessier, Béatrice, Sainlos, Matthieu, Choquet, Daniel, Butler, Corey, Letellier, Mathieu, Lim, Kok-Hong, Gauthereau, Isabel, Sibarita, Jean-Baptiste, Thoumine, Olivier, Park, Sheldon

2016

Overview

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. The advent of fluorescence-based super-resolution microscopy has created a need for labeling strategies relying on small probes that minimally perturb protein function. Here the authors describe a labeling method that reduces protein tag and label sizes, allowing for accurate protein targeting and measurements of protein dynamics in tight cellular spaces.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 14/1 / 14/19 / 14/34 / 14/35 / 14/63 / 631/378 / 631/45/612/1237 / 631/80/2373 / 64/110 / 64/60 / 82/80 / 82/83 / Adhesion / Animals / Biotinylation / Cell Adhesion Molecules, Neuronal - metabolism / Green Fluorescent Proteins / HEK293 Cells / Hippocampus - cytology / Humanities and Social Sciences / Humans / Indicators and Reagents / Life Sciences / Membrane Proteins - metabolism / Mice / Mice, Knockout / Molecular Dynamics Simulation / multidisciplinary / Nanotechnology - methods / Nerve Tissue Proteins - metabolism / Neural Cell Adhesion Molecules - metabolism / Neurobiology / Neurons - metabolism / Neurons and Cognition / Proteins / Rats / Science / Science (multidisciplinary) / Staining and Labeling - methods / Streptavidin / Synapses - metabolism