title: Dysferlin interacts with calsequestrin-1, myomesin-2 and dynein in human skeletal muscle.
abstract: Dysferlinopathies are a group of progressive muscular dystrophies characterized by mutations in the gene DYSF. These mutations cause scarcity or complete absence of dysferlin, a protein that is expressed in skeletal muscle and plays a role in membrane repair. Our objective was to unravel the proteins that constitute the dysferlin complex and their interaction within the complex using immunoprecipitation assays (IP), blue native gel electrophoresis (BN) in healthy adult skeletal muscle and healthy cultured myotubes, and fluorescence lifetime imaging-fluorescence resonance energy transfer (FLIM-FRET) analysis in healthy myotubes. The combination of immunoprecipitations and blue native electrophoresis allowed us to identify previously reported partners of dysferlin - such as caveolin-3, AHNAK, annexins, or Trim72/MG53 - and new interacting partners. Fluorescence lifetime imaging showed a direct interaction of dysferlin with Trim72/MG53, AHNAK, cytoplasmic dynein, myomesin-2 and calsequestrin-1, but not with caveolin-3 or dystrophin. In conclusion, although IP and BN are useful tools to identify the proteins in a complex, techniques such as fluorescence lifetime imaging analysis are needed to determine the direct and indirect interactions of these proteins within the complex. This knowledge may help us to better understand the roles of dysferlin in muscle tissue and identify new genes involved in muscular dystrophies in which the responsible gene is unknown.
authors.affiliation: Servei de Neurologia, Laboratori de Neurologia Experimental, Hospital de la Santa Creu i Sant Pau i Institut de Recerca de HSCSP, Barcelona, Spain.
authors.forename: Bàrbara
authors.initials: B
authors.lastname: Flix
authors.forename: Carolina
authors.initials: C
authors.lastname: de la Torre
authors.forename: Juan
authors.initials: J
authors.lastname: Castillo
authors.forename: Carme
authors.initials: C
authors.lastname: Casal
authors.forename: Isabel
authors.initials: I
authors.lastname: Illa
authors.forename: Eduard
authors.initials: E
authors.lastname: Gallardo
mesh_terms: D000293:Adolescent; D000328:Adult; D001706:Biopsy; D015153:Blotting, Western; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002155:Calsequestrin; D002352:Carrier Proteins / Q000378:metabolism; D051244:Caveolin 3 / Q000378:metabolism; D002648:Child; D064211:Connectin / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism*; D000073939:Dysferlin; D004591:Electrophoresis, Polyacrylamide Gel; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008875:Middle Aged; D024101:Mitochondrial Proteins / Q000378:metabolism*; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000473:pathology; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000378:metabolism* / Q000473:pathology; D009363:Neoplasm Proteins / Q000378:metabolism; D011485:Protein Binding; D016622:Silver Staining; D000070584:Tripartite Motif Proteins
chemical_list: C075176:AHNAK protein, human; C503129:CASQ1 protein, human; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002352:Carrier Proteins; D051244:Caveolin 3; D064211:Connectin; C114839:DYSF protein, human; D000073939:Dysferlin; C577287:MYOM2 protein, human; D008565:Membrane Proteins; D024101:Mitochondrial Proteins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; C558399:TRIM72 protein, human; D000070584:Tripartite Motif Proteins; D004398:Dyneins
keywords: Blue native; CALQ1; Cav-3; Cytoplasmic dynein; DHPR; DMD; DYNC1LI2; Duchenne muscular dystrophy; Dysferlin; FLIM–FRET; FLIM–FRET analysis; FSHD; HAC6; LGMD1C; LGMD2B; MM; MYOM2; Miyoshi myopathy; PTRF; RRC; SERCA; SR; Trim72/MG53; calsequestrin-1; caveolin-3; cytoplasmic dynein 1 light intermediate chain 2; dihydropyridine receptor; fascio-scapulo-humeral dystrophy; fluorescence lifetime imaging–fluorescence resonance energy transfer; histone deacetylase 6; limb girdle muscular dystrophy 1C; limb girdle muscular dystrophy 2B; myomesin-2; polymerase I and transcript release factor; receptor recycling compartment; sarcoplasmic reticulum; sarcoplasmic/endoplasmic reticulum calcium ATPase; trim72/MG53; tripartite containing motif 72/mitsugumin 53