The C2A domain in dysferlin is important for association with MG53 (TRIM72)

In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild- type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP- tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair. Abstract In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild- type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP- tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair. Abstract In skeletal muscle, Mitsugumin 53 (MG53), also known as muscle-specific tripartite motif 72, reportedly interacts with dysferlin to regulate membrane repair. To better understand the interactions between dysferlin and MG53, we conducted immunoprecipitation (IP) and pull-down assays. Based on IP assays, the C2A domain in dysferlin associated with MG53. MG53 reportedly exists as a monomer, a homodimer, or an oligomer, depending on the redox state. Based on pull-down assays, wild- type dysferlin associated with MG53 dimers in a Ca2+-dependent manner, but MG53 oligomers associated with both wild-type and C2A-mutant dysferlin in a Ca2+-independent manner. In pull-down assays, a pathogenic missense mutation in the C2A domain (W52R-C2A) inhibited the association between dysferlin and MG53 dimers, but another missense mutation (V67D-C2A) altered the calcium sensitivity of the association between the C2A domain and MG53 dimers. In contrast to the multimers, the MG53 monomers did not interact with wild-type or C2A mutant dysferlin in pull-down assays. These results indicated that the C2A domain in dysferlin is important for the Ca2+-dependent association with MG53 dimers and that dysferlin may associate with MG53 dimers in response to the influx of Ca2+ that occurs during membrane injury. To examine the biological role of the association between dysferlin and MG53, we co-expressed EGFP-dysferlin with RFP- tagged wild-type MG53 or RFP-tagged mutant MG53 (RFP-C242A-MG53) in mouse skeletal muscle, and observed molecular behavior during sarcolemmal repair; it has been reported that the C242A-MG53 mutant forms dimers, but not oligomers. In response to membrane wounding, dysferlin accumulated at the injury site within 1 second; this dysferlin accumulation was followed by the accumulation of wild-type MG53. However, accumulation of RFP-C242A MG53 at the wounded site was impaired relative to that of RFP-wild-type MG53. Co-transfection of RFP-C242A MG53 inhibited the recruitment of dysferlin to the sarcolemmal injury site. We also examined the molecular behavior of GFP-wild-type MG53 during sarcolemmal repair in dysferlin-deficient mice which show progressive muscular dystrophy, and found that GFP-MG53 accumulated at the wound similar to wild-type mice. Our data indicate that the coordination between dysferlin and MG53 plays an important role in efficient sarcolemmal repair.