Functional interaction of dysferlin, myoferlin and lipids in membrane repair and in pathogenesis of dysferlinopathies

• Applicant: Professor Dr. Sven Thoms
• Subject Area: Pediatric and Adolescent Medicine ; Biochemistry
• Term: since 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 437762782

Dysferlin and the paralogous myoferlin are involved in plasma membrane repair and in sculpturing of the T-tubule system. Both belong to the ferlin family of multi-C2-domain tail-anchored proteins. In a previous study, we have shown that dysferlin exerts its role in forming and maintaining the T-tubule system by direct interaction with membrane lipids (Hofhuis … Thoms, Journal of Cell Science 2017). Dysferlinopathies are muscular dystrophies characterised by mutations in the DYSF gene and severe reduction of the level of the corresponding protein – dysferlin, Neither the molecular function of full-length Dysferlin nor the pathomechanistic pathways leading to dysferlinopathies are understood. In this study, we will biochemically dissect the function and direct interactions of dysferlin, myoferlin and lipids in vitro and in vivo. We hypothesise that dysferlin is capable of forming heterodimers with myoferlin and that ferlins are fusogens. Heterodimer formation could explain why a point mutation in the murine dysferlin gene shows more severe disease phenotype than the complete loss of dysferlin expression. This ‘dominant’ phenotype could be caused by the degradation of heterodimers resulting in the simultaneous reduction of native dysferlin homologues. We will test these hypotheses by analysing myoferlin and dysferlin expression levels in a mouse model, and by expression and functional analyses of mutant dysferlin in mammalian cells and by production of potential heterodimers in baker’s yeast (Saccharomyces cerevisiae). Of note, we have for the first time been able to express full-length dysferlin for use in biochemical experiments. Our fusion assays with reconstituted liposomes suggest that ferlins can function as fusogens under defined conditions. To determine whether ferlins can merge biological membranes, ferlins will be directed to the cell surface and their capability to fuse cells in a calcium- and lipid-dependent manner will be analysed. This work will give detailed insight into ferlin function and contribute to understanding the pathomechanisms of dysferlinopathies and other ferlinopathies. The results of this study will also advance our knowledge on the mechanisms of membrane fusion and membrane repair.

• DFG Programme: Research Grants