Characterization of FER1L5, a novel dysferlin myoferlin related protein

Abstract

The ferlins are mammalian homologues of the C-elegans sperm vesicle fusion protein FER-1 characterised by multiple C2 domains and a C-terminal anchor. To date three human ferlin proteins, dysferlin, myoferlin and otoferlin have been characterized. Dysferlin and myoferlin have a role in muscle membrane fusion. Dysferlin mutations cause muscular dystrophy and are associated with defective sarcolemmal repair. Myoferlin has a role in myogenesis. Through bioinformatic analysis our group identified a novel ferlin, FER1L5, which by homology modelling and sequence analysis showed highest similarity with dysferlin and myoferlin. Previously, FER1L5 gene expression was shown to be elevated during C2C12 myoblast fusion. This led to the hypothesis that FER1L5 may have a role in muscle membrane fusion. In this study this was investigated in the C2C12 cell line using a specific polyclonal antibody to FER1L5. FER1L5 was shown to be present in vesicles. By biochemical fractionation FER1L5 was detected in low density vesicles the membranes of which were non-resistant to non-ionic detergent. The fractionation data showed that FER1L5, dysferlin and myoferlin vesicles have similar properties. By immunolabelling FER1L5 was shown to be present in distinct vesicles in myotubes compared to dysferlin and myoferlin respectively. During myoblast fusion FER1L5 expression was detected at all stages and high expression was observed at membrane fusion sites of fusing myoblasts. FER1L5 (but also dysferlin and myoferlin) showed a nuclear distribution during myoblast fusion, hi myotube nuclei FER1L5 was localized to subnuclear bodies sensitive to actinomycin D. Using the C3H-ER-MyoD cell line, I have shown that FER1L5 is not an early marker of muscle differentiation. The role of FER1L5 in muscle membrane fusion was investigated by examining the fusion index and membrane repair ability of C2C12 cells following FER1L5 inhibition. FER1L5 was shown to be required for myoblast fusion and membrane repair. In this study, I have also shown that C2C12 myotubes are active in membrane shedding. Using live cell imaging and confocal analysis, MVB like structures containing ferlin vesicles were shown to be released extracellularly from C2C12 myotubes. EM studies confirmed these findings. Overall my data shows that FER1L5 has a role in muscle membrane fusion. My work highlights that further studies on FER1L5 are required for example to determine if FER1L5 a therapeutic target in dysferlinopathy and to identify the mechanisms involved in FER1L5 mediated membrane fusion