Abstract
Simple SummaryFer1L5 is a dysferlin and myoferlin homologue and has been implicated in muscle membrane fusion events; myoblast fusion and membrane repair respectively during C2C12 skeletal muscle development. The role of Fer1L5 was analyzed by immunoblot analysis, biochemical fractionation, confocal microscopy and electroporation method. We demonstrated that Fer1L5 is present in low density vesicles and resistant to non-ionic detergent and shows overlapping properties with dysferlin and myoferlin. The expression of Fer1L5 was highly observed at the fusing myoblasts membranes and its expression level is gradually increase at the early stages multinucleated myotube formation. Fusion defects were observed in the Fer1L5 deficient C2C12 cells. Fer1L5 shows impaired membrane repair. Our data provide evidence that Fer1L5 is involved in aligning the adjacent myotubes close to each other for membrane—membrane fusion to increase the muscle mass for contraction during muscle development. Our data for Fer1L5 will be of great importance in the dysferlinopathy research in near future.Fer1L5 is a dysferlin and myoferlin related protein, which has been predicted to have a role in vesicle trafficking and muscle membrane fusion events. Mutations in dysferlin and otoferlin genes cause heredity diseases: muscular dystrophy and deafness in humans, respectively. Dysferlin is implicated in membrane repair. Myoferlin has a role in myogenesis. In this study, we investigated the role of the Fer1L5 protein during myoblast fusion and membrane repair. To study the functions of Fer1L5 we used confocal microscopy, biochemical fractionation, Western blot analysis and multiphoton laser wounding assay. By immunolabelling, Fer1L5 was detected in vesicular structures. By biochemical fractionation Fer1L5 was observed in low density vesicles. Our studies show that the membranes of Fer1L5 vesicles are non-resistant to non-ionic detergent. Partial co-staining of Fer1L5 with other two ferlin vesicles, respectively, was observed. Fer1L5 expression was highly detected at the fusion sites of two apposed C2C12 myoblast membranes and its expression level gradually increased at D2 and reached a maximum at day 4 before decreasing during further differentiation. Our studies showed that Fer1L5 has fusion defects during myoblast fusion and impaired membrane repair when the C2C12 cultures were incubated with inhibitory Fer1L5 antibodies. In C2C12 cells Fer1L5 vesicles are involved in two stages, the fusion of myoblasts and the formation of large myotubes. Fer1L5 also plays a role in membrane repair.
Highlights
The mammalian ferlins are evolutionary conserved vesicle fusion proteins composed of at least4-7tandem C2 domains and a C-terminal single pass transmembrane anchor which are implicated in membrane fusion events [1]
We suggest that the T-tubule membrane structures are the source of the dysferlin vesicles, and for Fer1L5 vesicles [10]
We have demonstrated that Fe1L5 is involved in myoblast fusion, and its role extends to membrane repair, similar to dysferlin and myoferlin [11,12,29]
Summary
The mammalian ferlins are evolutionary conserved vesicle fusion proteins composed of at least4-7tandem C2 domains and a C-terminal single pass transmembrane anchor which are implicated in membrane fusion events [1]. Dysferlin, otoferlin and myoferlin have been well characterized in myoblast fusion. Dysferlin and otoferlin mutations cause genetic diseases in humans: muscular dystrophy and deafness, respectively [6,7]. Dysferlin is the top characterized protein of the ferlin family, and the developed mouse models recapitulate human diseases [8]. Dysferlins contain 220–250 kDa sized proteins, which are highly expressed during skeletal muscle development: dysferlin in mature myofibers and myoferlin in myoblasts, respectively [8,9,10,11]. Loss or deficiency of these proteins mainly shows defects in myoblast fusion, resulting in immature myotubes and defects in membrane repair in patient muscle and mouse models (despite their other functions) [9,10,11]. The enhanced muscular dystrophy phenotype of the dysferlin and myoferlin suggested the pathogenicity of these two proteins [8,9,11,12]
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