AbstractThe fusion of mononuclear myoblasts into multinucleated myofibers is essential for the formation and growth of skeletal muscle. Myoblast fusion follows a well‐defined sequence of cellular events, from initial recognition and adhesion, to alignment, and finally plasma membrane fusion. These processes depend upon coordinated remodeling of the actin cytoskeleton. Our recent studies suggest diacylglycerol kinase‐zeta (DGK‐ζ), an enzyme that metabolizes diacylglycerol to yield phosphatidic acid, plays an important role in actin reorganization. Here, we investigated whether DGK‐ζ has a role in the fusion of cultured C2C12 myoblasts. We show that DGK‐ζ and syntrophins, scaffold proteins of the dystrophin glycoprotein complex that bind directly to DGK‐ζ, are spatially regulated during fusion. Both proteins accumulated with the GTPase Rac1 at sites where fine filopodia mediate the initial contact between myoblasts. In addition, DGK‐ζ codistributed with the Ca2+‐dependent cell adhesion molecule N‐cadherin at nascent, but not previously established cell contacts. We provide evidence that C2 cells are pulled together at cell‐cell junctions by N‐cadherin‐containing filopodia reminiscent of epithelial adhesion zippers, which guide the advance of lamellipodia from apposing cells. At later times, vesicles with properties of macropinosomes formed close to cell‐cell junctions. Reconstruction of confocal optical sections showed these form dome‐like protrusions from the dorsal surface of contacting cells. Collectively, these results suggest DGK‐ζ and syntrophins play a role at multiple stages of the fusion process. Moreover, our findings provide a potential link between changes in the lipid content of the membrane bilayer and reorganization of the actin cytoskeleton during myoblast fusion. Cell Motil. Cytoskeleton 2007. © 2007 Wiley‐Liss, Inc.