Journal
INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
Volume 45, Issue 8, Pages 1927-1938Publisher
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.biocel.2013.06.007
Keywords
Dysferlin; FLIM-FRET analysis; Blue native; Cytoplasmic dynein; Trim72/MG53
Categories
Funding
- Fundacion Isabel Gemio
- Ministerio de Sanidad del Gobierno de Espana [FIS 09/1944]
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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 irnmunoprecipitations 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 IF 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. (C) 2013 Elsevier Ltd. All rights reserved.
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