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J R Soc Interface. 2014 Oct 6;11(99). pii: 20140570. doi: 10.1098/rsif.2014.0570.

Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.

Author information

1
School of Biomedical Science, The University of Queensland, Brisbane, Queensland, Australia College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK.
2
Department of Physiology, The University of Auckland, Auckland, New Zealand.
3
Department of Physiology, The University of Auckland, Auckland, New Zealand Department of Cell Biology, Yale University, New Haven, CT, USA.
4
School of Biomedical Science, The University of Queensland, Brisbane, Queensland, Australia.
5
College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK Department of Physiology, The University of Auckland, Auckland, New Zealand c.soeller@exeter.ac.uk.

Abstract

Localization microscopy is a fairly recently introduced super-resolution fluorescence imaging modality capable of achieving nanometre-scale resolution. We have applied the dSTORM variation of this method to image intracellular molecular assemblies in skeletal muscle fibres which are large cells that critically rely on nanoscale signalling domains, the triads. Immunofluorescence staining in fixed adult rat skeletal muscle sections revealed clear differences between fast- and slow-twitch fibres in the molecular organization of ryanodine receptors (RyRs; the primary calcium release channels) within triads. With the improved resolution offered by dSTORM, abutting arrays of RyRs in transverse view of fast fibres were observed in contrast to the fragmented distribution on slow-twitch muscle that were approximately 1.8 times shorter and consisted of approximately 1.6 times fewer receptors. To the best of our knowledge, for the first time, we have quantified the nanometre-scale spatial association between triadic proteins using multi-colour super-resolution, an analysis difficult to conduct with electron microscopy. Our findings confirm that junctophilin-1 (JPH1), which tethers the sarcoplasmic reticulum ((SR) intracellular calcium store) to the tubular (t-) system at triads, was present throughout the RyR array, whereas JPH2 was contained within much smaller nanodomains. Similar imaging of the primary SR calcium buffer, calsequestrin (CSQ), detected less overlap of the triad with CSQ in slow-twitch muscle supporting greater spatial heterogeneity in the luminal Ca2+ buffering when compared with fast twitch muscle. Taken together, these nanoscale differences can explain the fundamentally different physiologies of fast- and slow-twitch muscle.

KEYWORDS:

junctophilin; localization microscopy; ryanodine receptor; skeletal muscle

PMID:
25100314
PMCID:
PMC4233733
DOI:
10.1098/rsif.2014.0570
[Indexed for MEDLINE]
Free PMC Article

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