Cell-to-cell fusion is a fundamental biological process essential for the development, repair, and regeneration of tissues in eukaryotes. Myoblast fusion, which forms multinucleated myofibers, is critical for skeletal muscle development and adaptation. This process requires precise membrane remodeling and restoration of cellular homeostasis to maintain tissue functionality. Despite its importance, the mechanisms driving myoblast fusion remain incompletely understood, presenting a major barrier to advancing treatments for muscle-related diseases and understanding tissue regeneration.
Our research aimed to elucidate the molecular, structural, and dynamic mechanisms underlying myoblast fusion, establishing it as a model for cell-to-cell fusion in vertebrates. A key achievement was the development of methods for robust and synchronous myoblast differentiation and fusion, enabling high-resolution capture of dynamic fusion events for the first time. This breakthrough provided insights into collective myoblast migration and the formation of specialized membrane protrusions at fusion interfaces with mature muscle fibers.
To achieve these objectives, we implemented cutting-edge methodologies such as advanced cryo-FIB-SEM and cryo-FM workflows, enabling ultrastructural resolution of fusion events. These techniques were applied across diverse model systems, including Drosophila melanogaster and Caenorhabditis elegans, demonstrating their broad applicability. We also developed on-section correlative light and electron microscopy (CLEM) protocols to study skeletal muscle regeneration, capturing key cellular events critical for tissue repair through ERK1/2 inhibition-driven synchronization of differentiation and myotube formation. By integrating live-cell imaging with machine learning, we tracked dynamic state transitions of single myoblasts during differentiation and fusion. This revealed p38 kinase as a regulator coordinating the transition from terminal differentiation to fusion, with actin dynamics playing a pivotal role. Supported by mass spectrometry, we identified novel proteins regulating fusion and maturation. Additionally, we discovered a myotube-specific calcium-dependent pathway that links muscle fusion with excitation-contraction coupling, suggesting co-evolutionary adaptation in amniotes. This discovery has significant implications for understanding skeletal muscle adaptation and its vulnerabilities in disease. The findings and methodologies have been widely disseminated through high-impact publications, international conferences, and public outreach. They offer powerful tools and transformative knowledge for studying skeletal muscle biology. Our research provides a foundation for advancing regenerative medicine, treating muscle-related diseases, addressing aging-associated challenges, and developing sustainable solutions for cultivated meat production
