Periodic Reporting for period 4 - MYOCLEM (Elucidating the Molecular Mechanism of Myoblast Fusion in Vertebrates)
Okres sprawozdawczy: 2024-05-01 do 2024-10-31
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
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
The research aimed to unravel the molecular mechanisms of myoblast fusion, a fundamental process in skeletal muscle development, regeneration, and adaptation. Our work yielded significant insights and innovations that have advanced cell and muscle biology. A key milestone was the development of a correlative light and electron microscopy (CLEM) workflow that combined fluorescence microscopy (FM) with transmission electron microscopy (TEM) to visualize myoblast fusion events in unprecedented detail. We further enhanced these methodologies by integrating focused ion beam milling (FIB-SEM) with confocal microscopy, enabling studies in model organisms such as Drosophila melanogaster and Caenorhabditis elegant. A pivotal breakthrough was establishing a method for robust and synchronous myoblast differentiation, enabling detailed observation of fusion events. This platform revealed a calcium-dependent pathway specific to myotubes, acting upstream of the fusion machinery, and highlighted co-evolution between myoblast fusion and excitation-contraction coupling in amniotes. Building on these advances, we developed cryo-FIB-SEM and cryo-FM workflows, providing high-resolution insights into cellular dynamics. These methodologies, published as both a research manuscript and protocol, now serve as benchmarks in the field. Using machine learning and live-cell imaging, we tracked state transitions in single myoblasts during differentiation and fusion, uncovering the regulatory role of p38 kinase and actin dynamics. Mass spectrometry further identified novel proteins involved in fusion and myotube maturation. To study muscle regeneration, we combined ERK1/2 inhibition with on-section CLEM to synchronize differentiation and achieve robust myotube formation within 24 hours. This technique allowed precise visualization of rare cellular events and provided insights into the molecular dynamics of muscle repair and related pathologies. Our findings, disseminated through publications, protocols, and presentations, have provided transformative insights into skeletal muscle biology and opened avenues for regenerative medicine, aging-related challenges, and sustainable food production solutions.
Our interdisciplinary approach, integrating genetics, biochemistry, and advanced imaging, has significantly advanced the understanding of myoblast fusion at molecular and ultrastructural levels. A major breakthrough was the development of methodologies for robust and synchronous myoblast differentiation and fusion, enabling the first detailed ex vivo recapitulation of the fusion process between myoblasts and mature myofibers. This achievement allowed us to observe dynamic interactions at the fusion interface, providing crucial insights into how mononucleated cells integrate into multinucleated fibers.
We identified novel proteins localized at fusion sites, revealing their assembly kinetics and roles in membrane remodeling and cytoskeletal reorganization. These findings, coupled with innovative correlative workflows such as cryo-FIB-SEM and cryo-FM, have provided unparalleled resolution of fusion events, deepening our understanding of the molecular machinery driving cell-to-cell fusion. Additionally, live-cell imaging and machine learning enabled continuous, single-cell-level analysis of differentiation and fusion dynamics, uncovering new regulatory mechanisms.
By linking myoblast fusion to broader processes such as tissue morphogenesis and regeneration, our work offers transformative insights into muscle biology with implications for regenerative medicine, therapeutic interventions, and sustainable food production. This progress lays the foundation for future breakthroughs in understanding and manipulating skeletal muscle development and adaptation.
We identified novel proteins localized at fusion sites, revealing their assembly kinetics and roles in membrane remodeling and cytoskeletal reorganization. These findings, coupled with innovative correlative workflows such as cryo-FIB-SEM and cryo-FM, have provided unparalleled resolution of fusion events, deepening our understanding of the molecular machinery driving cell-to-cell fusion. Additionally, live-cell imaging and machine learning enabled continuous, single-cell-level analysis of differentiation and fusion dynamics, uncovering new regulatory mechanisms.
By linking myoblast fusion to broader processes such as tissue morphogenesis and regeneration, our work offers transformative insights into muscle biology with implications for regenerative medicine, therapeutic interventions, and sustainable food production. This progress lays the foundation for future breakthroughs in understanding and manipulating skeletal muscle development and adaptation.