Periodic Reporting for period 2 - MYOCLEM (Elucidating the Molecular Mechanism of Myoblast Fusion in Vertebrates)
Période du rapport: 2021-05-01 au 2022-10-31
Cell-to-cell fusion is essential for development in most eukaryotes. A striking example is the fusion of myoblasts to form multinucleated myofibers during skeletal development and regeneration. During myoblast differentiation and fusion, membrane architecture undergoes radical remodeling not only to merge the cells but also to regain membrane- and cellular- homeostasis after the newly differentiated myoblast is introduced into the fully mature and contractile muscle fiber. We aim to elucidate the molecular mechanisms involved in myoblast fusion as a paradigm for cell-to-cell fusion in vertebrates. To achieve this goal, we dissect myoblast fusion on three levels: On the level of protein function, we aim to uncover proteins and pathways controlling membrane fusion. On the level of subcellular ultrastructure, we aim to reveal how membrane architecture and the localization of the fusion machinery change during cell-to-cell fusion. On the level of protein structure, we are developing the tools to resolve the structural organization of the fusion machinery using cryo-EM to define how the fusion machinery drives this process. However, defining molecules and mechanisms that regulate cell-to-cell fusion remains challenging owing to the difficulty distinguishing processes that regulate fusion from those that regulate myogenic differentiation. We recently overcame this significant barrier by developing a method to induce robust and synchronous differentiation and fusion in primary myoblasts. This breakthrough allowed us to capture a dynamic high-resolution view of mammalian myoblast fusion for the first time, showing that myoblasts migrate collectively and send membrane protrusions before fusing at a dynamic membrane interface that forms asymmetrically between the incoming myoblast and the receiving myotube. We also identified novel proteins and pathways that play a role in differentiation and fusion. Specifically, we found a calcium-dependent kinase that is activated upon early myotube formation and controls the activation of the fusion machinery in the myotubes, resulting in the expansion of the myotube at the expense of the mononucleated myoblasts. Our analysis suggests that cytosolic calcium (Ca2+) coordinates skeletal muscle differentiation, fusion, and contraction, specifically in the amniote lineage, to promote robust muscle regeneration, ensure muscles can recover efficiently after injury, and adapt to changing demands of life. These findings have profound implications for regenerative medicine, aging, and sustainable food production.
We developed a correlative light and electron microscopy workflow to visualize fusion events between primary mouse myoblasts using fluorescence (FM) and transmission electron microscopy (TEM). We also developed a correlative workflow combining focused ion beam milling followed by scanning electron microscopy (FIB-SEM) with confocal microscopy. We apply this workflow to various model organisms, including D. melanogaster (1) and C. elegans. Moreover, we recently demonstrated (2) a new method to induce synchronous differentiation and robust myotube formation. This discovery addressed a key fundamental challenge in the field: to capture myoblasts in the fusion process, which is essential for understanding how individual myoblasts fuse with existing multinucleated fibers and elucidating the fusion mechanism. Using this breakthrough, we uncovered a myotube-specific calcium-dependent pathway upstream of the fusion machinery, strongly suggesting that the mechanism of muscle fusion and excitation-contraction coupling (that also depends on calcium) co-evolved in amniotes (2). We also developed a correlative workflow combining cryo-FIB-SEM and cryo-FM on cultured myoblasts, which was published as a manuscript (3) and as a protocol (4).
(1) https://doi.org/10.1016/j.devcel.2021.05.004.
(2) https://doi.org/10.1016/j.devcel.2021.11.022.
(3) https://doi.org/10.1016/j.isci.2021.102714
(4) https://doi.org/10.1016/j.xpro.2022.101142
(1) https://doi.org/10.1016/j.devcel.2021.05.004.
(2) https://doi.org/10.1016/j.devcel.2021.11.022.
(3) https://doi.org/10.1016/j.isci.2021.102714
(4) https://doi.org/10.1016/j.xpro.2022.101142
Our holistic interdisciplinary experimental strategy combining the strengths of genetics, biochemistry, and multimodal correlative imaging has already generated an improved molecular and high-resolution quantitative view of myoblast fusion. We aim to reveal additional proteins that localize to the actual sites of fusion and determine their assembly kinetics and how membrane architecture changes due to their activity. We are working at the frontiers of tissue morphogenesis and homeostasis, which offers the unique opportunity to elucidate the role of the proteins and pathways we discover during development and disease.