Saccharomyces cerevisiae Scc2 binds Scc4 to form an essential complex that loads cohesin onto chromosomes. The prevalence of Scc2 orthologs in eukaryotes emphasizes a conserved role in regulating sister chromatid cohesion, but homologs of Scc4 have not hitherto been identified outside certain fungi. Some metazoan orthologs of Scc2 were initially identified as developmental gene regulators, such as Drosophila Nipped-B, a regulator of cut and Ultrabithorax, and delangin, a protein mutant in Cornelia de Lange syndrome. We show that delangin and Nipped-B bind previously unstudied human and fly orthologs of Caenorhabditis elegans MAU-2, a non-axis-specific guidance factor for migrating cells and axons. PSI-BLAST shows that Scc4 is evolutionarily related to metazoan MAU-2 sequences, with the greatest homology evident in a short N-terminal domain, and protein–protein interaction studies map the site of interaction between delangin and human MAU-2 to the N-terminal regions of both proteins. Short interfering RNA knockdown of human MAU-2 in HeLa cells resulted in precocious sister chromatid separation and in impaired loading of cohesin onto chromatin, indicating that it is functionally related to Scc4, and RNAi analyses show that MAU-2 regulates chromosome segregation in C. elegans embryos. Using antisense morpholino oligonucleotides to knock down Xenopus tropicalis delangin or MAU-2 in early embryos produced similar patterns of retarded growth and developmental defects. Our data show that sister chromatid cohesion in metazoans involves the formation of a complex similar to the Scc2-Scc4 interaction in the budding yeast. The very high degree of sequence conservation between Scc4 homologs in complex metazoans is consistent with increased selection pressure to conserve additional essential functions, such as regulation of cell and axon migration during development.

Mutations in the dysferlin gene cause limb-girdle muscular dystrophy type 2B, Miyoshi myopathy, and distal anterior compartment myopathy. Dysferlin mainly localizes to the sarcolemma in mature skeletal muscle where it is implicated in membrane fusion and repair. In different forms of muscular dystrophy, a predominantly cytoplasmic localization of dysferlin can be observed in regenerating myofibers, but the subcellular compartment responsible for this labeling pattern is not yet known. We have previously demonstrated an association of dysferlin with the developing T-tubule system in vitro. To investigate the role of dysferlin in adult skeletal muscle regeneration, we studied dysferlin localization at high resolution in a rat model of regeneration and found that the subcellular labeling of dysferlin colocalizes with the developing T-tubule system. Furthermore, ultrastructural analysis of dysferlin-deficient muscle revealed primary T-tubule anomalies similar to those seen in caveolin-3-deficient muscle. These findings indicate that dysferlin is necessary for correct T-tubule formation, and dysferlin-deficient skeletal muscle is characterized by abnormally configured T-tubules.

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.

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 limbgirdle 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.

Dysferlin (DYSF) is a type II transmembrane protein implicated in surface membrane repair of muscle. Mutations in dysferlin lead to Limb Girdle Muscular Dystrophy 2B (LGMD2B), Miyoshi Myopathy (MM), and Distal Myopathy with Anterior Tibialis onset (DMAT). The DYSF protein complex is not well understood, and only a few protein-binding partners have been identified thus far. To increase the set of interacting protein partners for DYSF we recovered a list of predicted interacting protein through a systems biology approach. The predictions are part of a “reverse-engineered” genome-wide human gene regulatory network obtained from experimental data by computational analysis. The reverse-engineering algorithm behind the analysis relates genes to each other based on changes in their expression patterns. DYSF and AHNAK were used to query the system and extract lists of potential interacting proteins. Among the 32 predictions the two genes share, we validated the physical interaction between DYSF protein with moesin (MSN) and polymerase I and transcript release factor (PTRF) in mouse heart lysate, thus identifying two novel Dysferlin-interacting proteins. Our strategy could be useful to clarify Dysferlin function in intracellular vesicles and its implication in muscle membrane resealing.

The disease mechanisms underlying dystrophin-deficient muscular dystrophy are complex, involving not only muscle membrane fragility, but also dysregulated calcium homeostasis. Specifically, it has been proposed that calcium channels directly initiate a cascade of pathological events by allowing calcium ions to enter the cell. The objective of this study was to investigate the effect of chronically blocking calcium channels with the aminoglycoside antibiotic streptomycin from onset of disease in the mdx mouse model of Duchenne muscular dystrophy (DMD).Treatment in utero onwards delayed onset of dystrophic symptoms in the limb muscle of young mdx mice, but did not prevent degeneration and regeneration events occurring later in the disease course. Long-term treatment had a positive effect on limb muscle pathology, reduced fibrosis, increased sarcolemmal stability, and promoted muscle regeneration in older mice. However, streptomycin treatment did not show positive effects in diaphragm or heart muscle, and heart pathology was worsened. Thus, blocking calcium channels even before disease onset does not prevent dystrophy, making this an unlikely treatment for DMD. These findings highlight the importance of analyzing several time points throughout the life of the treated mice, as well as analyzing many tissues, to get a complete picture of treatment efficacy.

Ariel is a mouse mutant that suffers from skeletal muscle myofibrillar degeneration due to the rapid accumulation of large intracellular protein aggregates. This fulminant disease is caused by an ENU-induced recessive mutation resulting in an L342Q change within the motor domain of the skeletal muscle myosin protein MYH4 (MyHC IIb). Although normal at birth, homozygous mice develop hindlimb paralysis from Day 13, consistent with the timing of the switch from developmental to adult myosin isoforms in mice. The mutated myosin (MYH4(L342Q)) is an aggregate-prone protein. Notwithstanding the speed of the process, biochemical analysis of purified aggregates showed the presence of proteins typically found in human myofibrillar myopathies, suggesting that the genesis of ariel aggregates follows a pathogenic pathway shared with other conformational protein diseases of skeletal muscle. In contrast, heterozygous mice are overtly and histologically indistinguishable from control mice. MYH4(L342Q) is present in muscles from heterozygous mice at only 7% of the levels of the wild-type protein, resulting in a small but significant increase in force production in isolated single fibres and indicating that elimination of the mutant protein in heterozygotes prevents the pathological changes observed in homozygotes. Recapitulation of the L342Q change in the functional equivalent of mouse MYH4 in human muscles, MYH1, results in a more aggregate-prone protein.

Nipbl (Scc2) and Mau2 (Scc4) encode evolutionary conserved proteins that play a vital role for loading the cohesin complex onto chromosomes, thereby ensuring accurate chromosome segregation during cell division. While mutations in human NIPBL are known to cause the developmental disorder Cornelia de Lange syndrome, the functions of Nipbl and Mau2 in mammalian development are poorly defined. Here we generated conditional alleles for both genes in mice and show that neural crest cell-specific inactivation of Nipbl or Mau2 strongly affects craniofacial development. Surprisingly, the early phase of neural crest cell proliferation and migration is only moderately affected in these mutants. Moreover, we found that Mau2 single homozygous mutants exhibited a more severe craniofacial phenotype when compared to that of Nipbl;Mau2 double homozygous mutants. This raises the possibility that the Mau2/Nipbl protein interaction is not only required for cohesin loading, but may also be required to restrict the level of Nipbl involved in regulating gene expression. Together, the data suggest that proliferating neural crest cells tolerate a substantial reduction of cohesin loading proteins and we propose that the successive decrease of cohesin loading proteins in neural crest cells may alter developmental gene regulation in a highly dynamic manner.