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Confocal endomicroscopy of neuromuscular junctions stained with physiologically inert protein fragments of tetanus toxin

Lookup NU author(s): Dr Veronika Boczonadi, Emeritus Professor Clarke Slater

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This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

Abstract

© 2021 by the authors. Licensee MDPI, Basel, Switzerland.Live imaging of neuromuscular junctions (NMJs) in situ has been constrained by the suitability of ligands for inert vital staining of motor nerve terminals. Here, we constructed several truncated derivatives of the tetanus toxin C-fragment (TetC) fused with Emerald Fluorescent Protein (emGFP). Four constructs, namely full length emGFP-TetC (emGFP-865:TetC) or truncations comprising amino acids 1066–1315 (emGFP-1066:TetC), 1093–1315 (emGFP-1093:TetC) and 1109–1315 (emGFP-1109:TetC), produced selective, high-contrast staining of motor nerve terminals in rodent or human muscle explants. Isometric tension and intracellular recordings of endplate potentials from mouse muscles indicated that neither full-length nor truncated emGFP-TetC constructs significantly impaired NMJ function or transmission. Motor nerve terminals stained with emGFP-TetC constructs were readily visualised in situ or in isolated preparations using fibre-optic confocal endomicroscopy (CEM). emGFP-TetC derivatives and CEM also visualised regenerated NMJs. Dual-waveband CEM imaging of preparations co-stained with fluorescent emGFP-TetC constructs and Alexa647-α-bungarotoxin resolved innervated from denervated NMJs in axotomized WldS mouse muscle and degenerating NMJs in transgenic SOD1G93A mouse muscle. Our findings highlight the region of the TetC fragment required for selective binding and visualisation of motor nerve terminals and show that fluorescent derivatives of TetC are suitable for in situ morphological and physiological characterisation of healthy, injured and diseased NMJs.

Publication metadata

Author(s): Roesl C, Evans ER, Dissanayake KN, Boczonadi V, Jones RA, Jordan GR, Ledahawsky L, Allen GCC, Scott M, Thomson A, Wishart TM, Hughes DI, Mead RJ, Shone CC, Slater CR, Gillingwater TH, Skehel PA, Ribchester RR

Publication type: Article

Publication status: Published

Journal: Biomolecules

Year: 2021

Volume: 11

Issue: 10

Online publication date: 12/10/2021

Acceptance date: 08/10/2021

Date deposited: 26/10/2021

ISSN (electronic): 2218-273X

Publisher: MDPI

URL: https://doi.org/10.3390/biom11101499

DOI: 10.3390/biom11101499

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