Myomaker and Myomerger Work Independently to Control Distinct Steps of Membrane Remodeling during Myoblast Fusion

Classic mechanisms for membrane fusion involve transmembrane proteins that assemble into complexes and dynamically alter their conformation to bend membranes, leading to mixing of membrane lipids (hemifusion) and fusion pore formation. Myomaker and Myomerger govern myoblast fusion and muscle formation but are structurally divergent from traditional fusogenic proteins. Here, we show that Myomaker and Myomerger independently mediate distinct steps in the fusion pathway, where Myomaker is involved in membrane hemifusion and Myomerger is necessary for fusion pore formation. Mechanistically, we demonstrate that Myomerger is required on the cell surface where its ectodomains stress membranes. Moreover, we show that Myomerger drives fusion completion in a heterologous system independent of Myomaker and that a Myomaker-Myomerger physical interaction is not required for function. Collectively, our data identify a stepwise cell fusion mechanism in myoblasts where different proteins are delegated to perform unique membrane functions essential for membrane coalescence. [Display omitted] •Myomaker and Myomerger govern distinct phases of the fusion pathway in myoblasts•Myomaker is essential for hemifusion of the plasma membranes•Myomerger ectodomains drive fusion pore formation by generating membrane stresses•Fusogenic activities of these proteins do not require a direct interaction Myoblast fusion is essential for muscle development, regeneration, and growth. Leikina et al. reveal that myoblasts proceed through a fusion mechanism characterized by a division of labor between muscle fusion proteins. The independent and sequential membrane functions of different proteins (Myomaker and Myomerger) culminate in membrane coalescence.