Intracellular localization of dysferlin and its association with the dihydropyridine receptor

Abstract

Mutations in the dysferlin gene underlie two phenotypically distinct muscular dystrophies: Miyoshi myopathy and limb-girdle muscular dystrophy 2B. Dysferlin was proposed to have a putative functional role in mediating the fusion of intracellular vesicles to the sarcolemma during injury-induced membrane repair, but dysferlin has been found not only at the sarcolemma but also within the cytoplasm of skeletal muscle fibers by immunohistochemistry. In this study, we examined the subcellular localization of dysferlin in skeletal muscle by immunohistochemical and biochemical analyses to elucidate other functional roles of dysferlin. Immunohistochemistry confirmed granular cytoplasmic expression pattern of dysferlin in muscle fibers. Subcellular membrane fractionation revealed that a portion of dysferlin associated with a T-tubule-enriched intracellular membrane fraction as well as a sarcolemmal fraction. This indication was consistent with subsequent results that dysferlin coprecipitates by immunoprecipitation with the dihydropyridine receptor (DHPR), a protein complex localized in T-tubules. Moreover, both proteins were observed to partially colocalize by double immunofluorescent labeling in skeletal muscle fibers. We also found that caveolin-3, previously shown to interact with dysferlin, coprecipitates with DHPR. These results demonstrated that dysferlin may be involved in the formation of an oligomeric complex with DHPR and caveolin-3. Caveolin-3 has been also reported to participate in an insulin-regulated transport mechanism in muscle, and caveolin-3-containing vesicles might traffic between intracellular sites and target sites on the sarcolemma and T-tubules. Therefore, it is very intriguing to assume that dysferlin might be involved in the fusion of caveolin-3-containing vesicles with T-tubules.