title: Observation of the molecular organization of calcium release sites in fast- and slow-twitch skeletal muscle with nanoscale imaging.
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.
authors.affiliation: School of Biomedical Science, The University of Queensland, Brisbane, Queensland, Australia College of Engineering, Mathematics and Physical Sciences, University of Exeter, Exeter, UK.
authors.forename: Isuru D
authors.initials: ID
authors.lastname: Jayasinghe
authors.affiliation: Department of Physiology, The University of Auckland, Auckland, New Zealand.
authors.forename: Michelle
authors.initials: M
authors.lastname: Munro
authors.affiliation: Department of Physiology, The University of Auckland, Auckland, New Zealand Department of Cell Biology, Yale University, New Haven, CT, USA.
authors.forename: David
authors.initials: D
authors.lastname: Baddeley
authors.affiliation: School of Biomedical Science, The University of Queensland, Brisbane, Queensland, Australia.
authors.forename: Bradley S
authors.initials: BS
authors.lastname: Launikonis
authors.affiliation: 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.
authors.forename: Christian
authors.initials: C
authors.lastname: Soeller
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism; D007091:Image Processing, Computer-Assisted; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018656:Muscle Fibers, Fast-Twitch / Q000502:physiology; D018657:Muscle Fibers, Slow-Twitch / Q000502:physiology; D018482:Muscle, Skeletal / Q000378:metabolism*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism*
chemical_list: D002155:Calsequestrin; D008565:Membrane Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C412507:junctophilin; D002118:Calcium
keywords: junctophilin; localization microscopy; ryanodine receptor; skeletal muscle
references.citation: Mol Cell. 2000 Jul;6(1):11-22
references.pmid: 10949023
references.citation: Microsc Microanal. 2010 Feb;16(1):64-72
references.pmid: 20082730
references.citation: Pflugers Arch. 2010 Oct;460(5):813-23
references.pmid: 20577885
references.citation: J Physiol. 2012 Dec 1;590(23):6199-212
references.pmid: 23027818
references.citation: Biophys J. 2012 Mar 7;102(5):L19-21
references.pmid: 22404946
references.citation: PLoS One. 2011;6(5):e20645
references.pmid: 21655189
references.citation: Biophys J. 1978 May;22(2):145-54
references.pmid: 306839
references.citation: J Gen Physiol. 2007 Oct;130(4):365-78
references.pmid: 17846166
references.citation: Am J Physiol. 1993 Jun;264(6 Pt 1):C1587-93
references.pmid: 8333507
references.citation: Nat Cell Biol. 2000 Sep;2(9):669-71
references.pmid: 10980710
references.citation: Physiol Rev. 1972 Jan;52(1):129-97
references.pmid: 4256989
references.citation: J Cell Biol. 1999 Aug 9;146(3):621-30
references.pmid: 10444070
references.citation: J Biol Chem. 1995 Dec 22;270(51):30787-96
references.pmid: 8530521
references.citation: J Physiol. 2014 Sep 1;592(17):3727-46
references.pmid: 24973406
references.citation: J Biol Chem. 2011 Dec 23;286(51):43717-43725
references.pmid: 22020936
references.citation: J Anat. 1984 Mar;138 ( Pt 2):297-308
references.pmid: 6715252
references.citation: J Muscle Res Cell Motil. 1984 Jun;5(3):333-47
references.pmid: 6746893
references.citation: Nat Methods. 2008 Feb;5(2):159-61
references.pmid: 18176568
references.citation: PLoS One. 2012;7(7):e39962
references.pmid: 22768324
references.citation: J Physiol. 2009 Jul 1;587(Pt 13):3101-11
references.pmid: 19403601
references.citation: J Cell Biol. 1984 Nov;99(5):1735-42
references.pmid: 6386826
references.citation: J Cell Biol. 1970 Nov 1;47(2):488-99
references.pmid: 19866746
references.citation: Biophys J. 1997 Jul;73(1):97-111
references.pmid: 9199775
references.citation: Physiol Rev. 1997 Jul;77(3):699-729
references.pmid: 9234963
references.citation: Nat Commun. 2013;4:2805
references.pmid: 24241282
references.citation: Biochem Biophys Res Commun. 1993 Dec 30;197(3):1303-11
references.pmid: 8280147
references.citation: J Physiol. 1995 Dec 1;489 ( Pt 2):349-62
references.pmid: 8847631
references.citation: J Cell Biol. 1965 Jun;25(3):Suppl:209-31
references.pmid: 5840799
references.citation: J Muscle Res Cell Motil. 1992 Aug;13(4):394-405
references.pmid: 1328292
references.citation: J Cell Biol. 2001 Sep 3;154(5):1059-67
references.pmid: 11535622
references.citation: J Physiol. 2003 Aug 15;551(Pt 1):125-38
references.pmid: 12813151
references.citation: J Physiol. 1996 Jun 1;493 ( Pt 2):357-70
references.pmid: 8782101
references.citation: Chromosome Res. 2008;16(3):523-62
references.pmid: 18461488
references.citation: Tissue Cell. 1989;21(5):783-94
references.pmid: 2617518
references.citation: J Physiol. 2009 Jan 15;587(2):443-60
references.pmid: 19029185
references.citation: J Ultrastruct Res. 1975 May;51(2):176-87
references.pmid: 1127796
references.citation: J Biophys Biochem Cytol. 1957 Mar 25;3(2):269-300
references.pmid: 13438910
references.citation: J Physiol. 2013 Feb 1;591(3):719-29
references.pmid: 23148318
references.citation: Biophys J. 1999 Sep;77(3):1528-39
references.pmid: 10465763
references.citation: Proc Natl Acad Sci U S A. 1982 Dec;79(23):7243-7
references.pmid: 6961404
references.citation: Physiol Rev. 2011 Oct;91(4):1447-531
references.pmid: 22013216
references.citation: J Membr Biol. 1989 Jul;109(1):73-83
references.pmid: 2769737
references.citation: Int J Biochem Cell Biol. 2014 Mar;48:28-38
references.pmid: 24374102
references.citation: J Biol Chem. 2003 May 2;278(18):16176-82
references.pmid: 12594204