Annexins cooperate with Myomaker to promote myoblast fusion

Myoblast fusion is essential for skeletal muscle development and regeneration. Myomaker is a muscle-specific fusogen indispensable for this process. Cryoelectron microscopy analysis has shown that Myomaker dimerization is required for myoblast fusion and muscle formation. However, the precise molecular mechanisms of Myomaker function remain unclear. Notably, the short extracellular domain of Myomaker suggests the involvement of additional proteins that cooperate with Myomaker to facilitate membrane fusion. To identify potential interacting partners of Myomaker, we employed an APEX2-based proximity labeling approach in a synchronized myoblast fusion and differentiation system. By comparing results with a fusion-deficient mutant form of Myomaker, we identified several members of the Annexin family of calcium-dependent phospholipid-binding proteins as prominent candidates for interaction, particularly under prefusion conditions. Among these, Annexins A2, A5, and A6 were found to colocalize with Myomaker at the myoblast plasma membrane. Proximity ligation assays further confirmed the close association of these Annexins with Myomaker at the cell surface. Functional analyses demonstrated that these Annexins can promote heterologous cell–cell fusion between fibroblasts and myoblasts. While single knockdown of Annexin A2, A5, or A6 had minimal effects, the simultaneous knockdown of all three impaired myoblast fusion, indicating functional redundancy among them. Together, these findings indicate that Annexins A2, A5, and A6 cooperate with Myomaker to promote myoblast fusion, providing a mechanistic link between the fusogenic machinery and membrane dynamics during myoblast fusion.