The transition from lipid mixing to syncytium formation by C2C12 cells depends on PtdIns(4,5)P content. (A) Images of the fusion-committed cells accumulated by LPC block treated with PtdIns(4,5)P-binding PBP10 (7.5 µM) applied at the time of LPC removal (right) or the untreated cells released from LPC block (left). Images show DiI (red) and green cell tracker fluorescence and were taken 30 min after LPC removal. Notched arrows mark the multinucleated cells and smaller arrows mark the colabeled mononucleated cells. Bar, 50 µm. (B and C) Reagents lowering the concentration of accessible PtdIns(4,5)P2 in the plasma membrane inhibit fusion of C2C12 myoblasts assayed as lipid mixing (green) and myotube formation (red). We applied 5 and 7.5 µM PBP10 (B, 2 and 3) as well as 1% 1-butanol and, in the control experiments, its inactive isomers 2-butanol and t-butanol (C, 2, 3, and 4, respectively) at the time of LPC removal. Lipid mixing and syncytium formation extents were normalized to those for the untreated cells released from LPC block (B and C, 1; presented as means ± SEM; n ≥ 3). Levels of significance relative to controls (1) are shown: **, P < 0.01; *, P < 0.05. (D) The proposed pathway of the fusion stage of myotube formation. Anx A1 and A5 at the PS-exposing surface of fusion-committed myoblasts directly or via other proteins initiate membrane merger. Subsequent stages of syncytium formation are controlled by DNM- and PtdIns(4,5)P2-dependent intracellular protein machinery.