From T-tubule to sarcolemma: damage-induced dysferlin translocation in early myogenesis.

The dysferlin gene is mutated in limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. In mature skeletal muscle, dysferlin is located predominantly at the sarcolemma, where it plays a role in membrane fusion and repair. To investigate the role of dysferlin during early muscle differentiation, its localization was studied at high resolution in a muscle cell line. This demonstrated that dysferlin is not expressed at the plasmalemma of myotubes but mostly localizes to the T-tubule network. However, dysferlin translocated to the site of injury and toward the plasma membrane in a Ca2+-dependent fashion in response to a newly designed in vitro wounding assay. This reaction was specific to the full-length protein, as heterologously expressed deletion mutants of distinct C2 domains of dysferlin did not show this response. These results shed light on the dynamics of muscle membrane repair and are highly indicative of a specific role of dysferlin in this process in early myogenesis.

Protein studies in dysferlinopathy patients using llama-derived antibody fragments selected by phage display.

Mutations in dysferlin, a member of the fer1-like protein family that plays a role in membrane integrity and repair, can give rise to a spectrum of neuromuscular disorders with phenotypic variability including limb-girdle muscular dystrophy 2B, Myoshi myopathy and distal anterior compartment myopathy. To improve the tools available for understanding the pathogenesis of the dysferlinopathies, we have established a large source of highly specific antibody reagents against dysferlin by selection of heavy-chain antibody fragments originating from a nonimmune llama-derived phage-display library. By utilizing different truncated forms of recombinant dysferlin for selection and diverse selection methodologies, antibody fragments with specificity for two different dysferlin domains could be identified. The selected llama antibody fragments are functional in Western blotting, immunofluorescence microscopy and immunoprecipitation applications. Using these antibody fragments, we found that calpain 3, which shows a secondary reduction in the dysferlinopathies, interacts with dysferlin.

The differential gene expression profiles of proximal and distal muscle groups are altered in pre-pathological dysferlin-deficient mice.

The selective pattern of muscle involvement is a key feature of muscular dystrophies. Dysferlinopathy is a good model for studying this process since it shows variable muscle involvement that can be highly selective even in individual patients. The transcriptomes of proximal and distal muscles from wildtype C57BL/10 and dysferlin deficient C57BL/10.SJL-Dysf mice at a prepathological stage were assessed using the Affymetrix oligonucleotide-microarray system. We detected significant variation in gene expression between proximal and distal muscle in wildtype mice. Dysferlin defiency, even in the absence of pathological changes, altered this proximal distal difference but with little specific overlap with previous microarray analyses of dysferlinopathy. In conclusion, proximal and distal muscle groups show distinct patterns of gene expression and respond differently to dysferlin deficiency. This has implications for the selection of muscles for future microarray analyses, and also offers new routes for investigating the selectivity of muscle involvement in muscular dystrophies.