Annexins and Dynamin in Myoblast Fusion

Cell-to-cell fusion is a key stage in many developmental processes including fertilization and formation of bone, placenta, and muscles, as well as in mature organisms in muscle repair and in formation of multinucleated giant cells during inflammatory reactions. A major challenge in studying molecular mechanisms of membrane fusion is to isolate the actual fusion stage from the preceding stages that prime the cells to fusion. As a result, even for well characterized myoblast fusion in formation of muscle fibers, we still do not know the specific proteins that fuse lipid bilayers of two plasma membranes into one. In this study, we isolated fusion stage in myotube formation by murine myoblasts (C2C12 cells) labeled with different membrane and cytosolic probes by blocking myotube formation immediately prior to fusion with reversible hemifusion-inhibitor lysophosphatidylcholine. This approach accumulates cells at a ready-to-fuse stage and, thus, synchronizes fusion upon lifting the inhibitor. Isolation of the fusion stage allowed us to explore myoblast fusion pathway and identify annexins A1 and A5 as proteins that are present at cell surface at the time of fusion and play an important role in fusion. Based on the ability of these proteins to fuse liposomes (Yang et al.), we suggest that annexins directly mediate rather than regulate cell fusion. Annexin-dependent early stages of myoblast fusion are followed by expansion of fusion pores that we found to depend on cell metabolism and be blocked by dynasore and MitMAB, inhibitors of dynamin GTPase. We propose that annexins and dynamin are also involved in other cell-to-cell fusion processes and hope that better understanding of protein-lipid interactions underlying different stages of cell fusion will bring new ways of controlling membrane fusion in pathophysiology.