Size-dependent membrane remodelling at Myomerger clusters during myoblast fusion

Myomerger drives fusion pore formation after hemifusion of membranes during myogenic cell-cell fusion in myogenesis, but its mechanical impact has been characterised largely in liposomes. In differentiating C2C12 cells, hemifusion began at 8 hr and peaked at 24 hr, whereas the earliest content-mixing events appeared only at 24 hr - Myomerger is therefore present much before it is competent to complete fusion. Using Interference Reflection Microscopy (IRM), we found that basal membrane tension in hemifusing cells was indistinguishable from that of non-fusing cells at 8-24 hr, and became significantly higher by 48 hr. Knockdown showed that Myomerger switched across this window from tension-reducing to enhancing cell-scale tension, unlike Myomaker, which enhanced tension in both the early and late phases. Addressing the local impact, Stimulated Emission Depletion microscopy (STED) imaging resolved Myomerger clusters built from a ∼40 nm basic unit that aggregate further. Small clusters induced nanometric outward bulges, a topology rare outside Myomerger-enriched regions; these bulges damped membrane fluctuations and showed higher lipid compaction at the centre, as deciphered by Flipper-TR FLIM, at the lateral as well as the basal membrane, and may be central to the hemifusion-to-pore transition. Larger clusters indented the membrane, showed enhanced fluctuations and associated with the clathrin-coated pits, consistent with surface clearance by endocytosis. Myomerger colocalized poorly with actin fibres and cholesterol-enriched nanodmains despite lying close to both. Together, these findings establish size-based clustering of Myomerger as a determinant of membrane topology and lipid compaction when hemifusion peaks and content mixing begins.