Confocal Endomicroscopy of Neuromuscular Junctions Stained with Physiologically Inert Protein Fragments of Tetanus Toxin.

Autor: Roesl C; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Evans ER; Public Health England, National Infection Service, Porton Down, Salisbury SP4 0JG, UK., Dissanayake KN; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Boczonadi V; Applied Neuromuscular Junction Facility, Bio-Imaging Unit, Biosciences Institute, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK., Jones RA; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Jordan GR; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Ledahawsky L; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Allen GCC; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Scott M; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Thomson A; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Wishart TM; The Roslin Institute, Royal (Dick) School of Veterinary Studies, College of Medicine and Veterinary Medicine, University of Edinburgh, Easter Bush, Edinburgh EH25 9RG, UK., Hughes DI; Spinal Cord Research Group, Institute of Neuroscience and Psychology, University of Glasgow, Glasgow G12 8QQ, UK., Mead RJ; Sheffield Institute for Translational Neuroscience (SITraN), University of Sheffield, Glossop Road, Sheffield S10 2HQ, UK., Shone CC; Public Health England, National Infection Service, Porton Down, Salisbury SP4 0JG, UK., Slater CR; Applied Neuromuscular Junction Facility, Bio-Imaging Unit, Biosciences Institute, University of Newcastle-upon-Tyne, Framlington Place, Newcastle-upon-Tyne NE2 4HH, UK., Gillingwater TH; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Skehel PA; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK., Ribchester RR; Centre for Discovery Brain Sciences and the Euan MacDonald Centre for Motor Neurone Disease Research, University of Edinburgh, George Square, Edinburgh EH8 9XD, UK.
Jazyk: angličtina
Zdroj: Biomolecules [Biomolecules] 2021 Oct 12; Vol. 11 (10). Date of Electronic Publication: 2021 Oct 12.
DOI: 10.3390/biom11101499
Abstrakt: 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 Wld S 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.
Databáze: MEDLINE
Externí odkaz:
Nepřihlášeným uživatelům se plný text nezobrazuje