Myoblast fusion is a fundamental process for proper skeletal muscle formation during development, regeneration, and exercise-induced adaptivity. In spite of the large number of studies on myoblast fusion, the molecular mechanisms that govern this highly-coordinated process have remained poorly understood. Elucidation of the molecular pathways that control fusion is a critical step for understanding muscle develop and to establish new therapeutic strategies to augment degenerative skeletal muscle diseases. We have recently provided new insights into the molecular control of myoblast fusion through the discovery of two muscle-specific membrane proteins (myomaker and myomerger). Both proteins are essential for myoblast fusion and normal muscle development and regeneration. Furthermore, co-expression of myomaker and myomerger induces fusion of non-fusogenic fibroblasts, establishing that these fusogens are both necessary and sufficient for fusion in mammalian cells. More recently, we have shown that myomaker and myomerger drive myoblast fusion by independently impacting distinct phases of membrane fusion. Within this unique mechanism, myomaker is required for initial mixing of the outer lipid leaflets (hemifusion) and myomerger completes the fusion reaction through generation of a fusion pore. While the facilitation of fusion pore formation has been attributed to the membrane permeabilization activity of myomerger, the mechanistic function of myomaker leading to membrane remodeling during hemifusion has remained elusive. The main goal of this proposal is to understand the mechanisms by which myomaker controls myoblast hemifusion, which will eventually be used to design strategies for cell-based therapies for muscle repair and degenerative disease. We will define the membrane- remodeling activities of myomaker during myoblast fusion by first testing whether myomaker functions in cis or in trans using co-immunoprecipitation of affinity-tagged myomaker. We will use an arsenal of fluorescent membrane dyes as well as FRAP analysis to measure membrane fluidity in myomaker-null and myomaker- overexpressing cells, and assess lipid content using large-scale lipidomics. Using mutagenesis, we will examine the roles of several distinct myomaker domains for membrane-remodeling and myomaker localization. To identify additional factors required for myoblast fusion, we will conduct immunoprecipitation studies of FLAG-tagged myomaker during myoblast fusion to assess direct binding partners through mass spectrometry. Furthermore, we will interrogate multiple candidates identified from our genome-wide loss-of-function screen through CRISPR/Cas9-mediated knockout analyses, and direct interactions with myomaker will be tested. Completion of these studies will lead to an understanding of the molecular function of myomaker during lipid mixing, as well as the identification and characterization of the ancillary factors that cooperate with myomaker for hemifusion- competence, both indispensable for the development of therapeutic strategies for muscle repair and disease.
The coordinated fusion of myoblasts is a fundamental process for the proper development and regeneration of skeletal muscle. Our lab?s recent discovery of two muscle-specific membrane proteins necessary for fusion, myomaker and myomerger, has provided new insights into the molecular control of myoblast fusion; however, the mechanistic function of myomaker during fusion remains poorly understood. The goal of this project is to explore the mechanisms of myomaker action during myoblast fusion and to use that knowledge to develop new treatment strategies for degenerative skeletal muscle diseases.
