Visualization of Dysferlin Self-Interaction Using Fluorescence Resonance Energy Transfer and Fluctuation Correlation Spectroscopy

Abstract

Mutations in the dysferlin gene cause progressive muscle weakness and muscle wasting. Previously dysferlin was found to play a key role in the Ca2+-activated membrane repair process of striated myocytes. However, it remains to be explored as to how dysferlin is involved in muscle membrane repair. Here we report for the first time that dysferlin forms a homodimer in living cells as well as in detergent-solubilized cell lysates. Sucrose gradient fractionation experiments showed that endogenous dysferlin from digitonin-solubilized skeletal muscle membranes migrated into heavy fractions. A self-associated fluorescent protein labeled dysferlin (ECFP-dysferlin and EYFP-dysferlin) complex in living AAV293 cells and adult skeletal muscle fibers was demonstrated by fluorescence resonance energy transfer (FRET) assay. Importantly, fluorescent protein tag at the N terminus of dysferlin did not impair the function of dysferlin in restoring the membrane repair efficiency in dysferlin-deficient skeletal muscle fibers. FRET between ECFP-dysferlin and EYFP-dysferlin in skeletal muscle fibers showed hyperbolic dependence on protein concentration, with a maximum efficiency of 35.3% and a very low KD. The observed FRET corresponds to a probe separation distance of 54.4 A, according to a computational model of dimeric FRET. Dysferlin dimerization was further confirmed by using fluorescence fluctuation spectroscopy with photon counting histogram analyses. Moreover, myoferlin, a mammalian orthologue of dysferlin, also forms dimer. The heterodimeric affinity between dysferlin and myoferlin is weaker than that of either homodimer. Finally, the transmembrane domain was identified to mediate the dimerization of dysferlin. Therefore, our data demonstrate that dysferlin forms a homodimer in cells, which may be a common feature for ferlin-1-like protein family.