title: Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria.
abstract: Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles.
authors.affiliation: Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK.
authors.forename: Juliette A
authors.initials: JA
authors.lastname: Strauss
authors.affiliation: Centre for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong, Victoria, Australia.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Shaw
authors.affiliation: School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, Edgbaston, UK.
authors.forename: Helen
authors.initials: H
authors.lastname: Bradley
authors.affiliation: Institute for Sport, Physical Activity and Leisure, Carnegie Faculty, Leeds Beckett University, Leeds, UK.
authors.forename: Oliver J
authors.initials: OJ
authors.lastname: Wilson
authors.affiliation: Discovery Sciences, AstraZeneca R&D, Cambridge, UK.
authors.forename: Thierry
authors.initials: T
authors.lastname: Dorval
authors.affiliation: Discovery Sciences, AstraZeneca R&D, Cambridge, UK.
authors.forename: James
authors.initials: J
authors.lastname: Pilling
authors.affiliation: Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK a.j.wagenmakers@ljmu.ac.uk.
authors.forename: Anton J M
authors.initials: AJ
authors.lastname: Wagenmakers
mesh_terms: D002462:Cell Membrane / Q000737:chemistry*; D006801:Humans; D066292:Lipid Droplets / Q000737:chemistry*; D008297:Male; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000166:cytology*; D050766:Qb-SNARE Proteins / Q000032:analysis*; D050767:Qc-SNARE Proteins / Q000032:analysis*; D055815:Young Adult
chemical_list: D050766:Qb-SNARE Proteins; D050767:Qc-SNARE Proteins; C100493:SNAP23 protein, human
keywords: Glucose transporter 4; intramuscular triglyceride; lipid droplets; mitochondria; synaptosomal‐associated protein 23