Little is known about the mechanisms of cell–cell fusion in development and diseases and, especially, about
fusion stages downstream of an opening of nascent fusion pore(s). Earlier works on different cell–cell fusion
reactions have indicated that cytoskeleton plays important role in syncytium formation. However, due to
complexity of these reactions and multifaceted contributions of cytoskeleton in cell physiology, it has
remained unclear whether cytoskeleton directly drives fusion pore expansion or affects preceding fusion
stages. Here we explore cellular reorganization associated with fusion pore expansion in syncytium
formation using relatively simple experimental system. Fusion between murine embryonic fibroblasts
NIH3T3-based cells is initiated on demand by well-characterized fusogen influenza virus hemagglutinin. We
uncouple early fusion stages dependent on protein fusogens from subsequent fusion pore expansion stage
and establish that the transition from local fusion to syncytium requires metabolic activity of living cells.
Effective syncytium formation for cells with disorganized actin and microtubule cytoskeleton argues against
hypothesis that cytoskeleton drives fusion expansion.