Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids unveils masked epitopes in live neurons

Diogo Bessa-Neto,Nicolas Chevrier,Daniel Choquet,Valeria Pecoraro,Markus Sauer,Alexander Kuhlemann,Natacha Retailleau,Gerti Beliu,David Perrais,Sören Doose

doi:10.1038/s41467-021-27025-w

Abstract

Progress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

Highlights

Progress in biological imaging is intrinsically linked to advances in labeling methods
To understand the lack of Transmembrane AMPAR regulatory protein (TARP) staining in neurons, we overexpressed in neurons γ2 fused to eGFP at the C-terminus (γ2::eGFP) or GluA2 tethered to γ2::eGFP (GluA2::γ2::eGFP) in which the GluA2 C-terminus is fused to the γ2 N-terminus by in-frame expression[37]
Structural data of γ2 or γ8 in complex with AMPA receptor (AMPAR) revealed the close proximity of both Ex1 and Ex2 to the AMPAR ligand-binding domain (LBD)[21,22,23], which likely results in epitope masking

Summary

Introduction

Progress in biological imaging is intrinsically linked to advances in labeling methods. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allows us to image the differential localization of two AMPA receptor (AMPAR) auxiliary subunits of the transmembrane AMPAR regulatory protein family in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy. Our antibodies directed against extracellular loops of γ2 and γ8 allow us to establish that there are virtually no free surface γ2 and γ8 in hippocampal neurons

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Conclusion