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

Roesl, C. et al. (2021) Confocal endomicroscopy of neuromuscular junctions stained with physiologically inert protein fragments of tetanus toxin. Biomolecules, 11(10), 1499. (doi: 10.3390/biom11101499) (PMCID:PMC8534034)

Abstract

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.

• Item Type: Articles
• Additional Information: Funding: This research was principally funded by the Motor Neurone Disease Association (UK), grant number 838-791, and by the Medical Research Council (MRC), grant number MR/M024075. Additional support was obtained from the Biotechnology and Biological Research Council (BBSRC), grant numbers BBS/E/D/10002071 and BB/P007996/1; the Engineering and Physical Sciences Research Council (EPSRC) grants EP/R00661X/1, EP/S019367/1, EP/P02470X/1 and EP/P025285/1; and the Wellcome Trust, 208339/Z/17/Z.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Hughes, Dr David I

Creator Roles

Hughes, D. I.: Methodology, Investigation, Resources, Writing – review and editing, Funding acquisition

• Authors: Roesl, C., Evans, E. R., Dissanayake, K. N., Boczonadi, V., Jones, R. A., Jordan, G., Ledahawsky, L., Allen, G. C.C., Scott, M., Thomson, A., Wishart, T. M., Hughes, D. I., Mead, R. J., Shone, C. C., Slater, C. R., Gillingwater, T. H., Skehel, P. A., and Ribchester, R. R.
• College/School: College of Medical Veterinary and Life Sciences > School of Psychology & Neuroscience
• Journal Name: Biomolecules
• Publisher: MDPI
• ISSN: 2218-273X
• ISSN (Online): 2218-273X
• Published Online: 12 October 2021
• Copyright Holders: Copyright © 2021 The Authors
• First Published: First published in Biomolecules 11(10):1499
• Publisher Policy: Reproduced under a Creative Commons Licence

Project Code	Award No	Project Name	Principal Investigator	Funder's Name	Funder Ref	Lead Dept
174050		Determining the role of calretinin-RorB spinal interneurons in modulating mechanical pain	David I Hughes	Biotechnology and Biological Sciences Research Council (BBSRC)	BB/P007996/1	Institute of Neuroscience & Psychology