Combined structural and biochemical studies of dysferlin-dependent membrane repair mechanisms

• Applicant: Constantin Cretu, Ph.D.
• Subject Area: Structural Biology ; Biochemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542658629

The C2 domain protein dysferlin plays a crucial role in maintaining the integrity of muscle cell membranes, and recessive mutations in the large ferlin have been associated with muscle dystrophies and various complex human disorders. Despite extensive studies, our understanding of how dysferlin functions as the main calcium sensor of the repair pathway, particularly within multi-subunit repair complexes to effectively reseal injured membranes, remains limited. Moreover, although structures of individual subunits or domains have been reported, the spatial organization of dysferlin's membrane-bound higher-order complexes is not well understood. To develop a detailed molecular model of dysferlin in its active lipid-bound state, I will employ a bottom-up experimental approach that integrates cutting-edge high-resolution imaging and structural biology techniques (such as single-particle cryo-EM, cryo-ET, and nanoscopy) with careful in vitro investigations using model membrane systems. In my first specific objective, building on my recent cryo-EM structure of a human ferlin, I will focus on obtaining high-resolution structural models of dysferlin in its active membrane-bound states. This includes its complexes with endosomal SNAREs STX4-SNAP-23 and the membrane repair factor MG53, as well as reconstituted in surrogate lipid membranes (liposomes and nanodiscs). Simultaneously, I will employ a multidisciplinary, hypothesis-driven approach to systematically characterize the dysferlin-dependent membrane binding and fusion machinery in vitro. This will involve using recombinantly expressed repair factors, structure-guided domain mutants, and carefully designed liposome-based assays. Finally, by building on the recent advances in super-resolution light microscopy, as part of a collaborative effort, we will attempt to image human dysferlin in its complex cellular environment, following laser-induced membrane injury, as a proof of concept for future in situ cryo-electron tomography (cryo-ET) investigations.

• DFG Programme: Research Grants