Immobilization of Notch ligand, Delta-1, is required for induction of Notch signaling

Cell-cell interactions mediated by Notch and its ligands are known to effec t many cell fate decisions in both invertebrates and vertebrates, However, the mechanisms involved in ligand induced Notch activation are unknown, Rec ently it was shown that, in at least some cases, endocytosis of the extrace llular domain of Notch and ligand by the signaling cell is required for sig nal induction in the receptive cell. These results imply that soluble ligan ds (ligand extracellular domains) although capable of binding Notch would b e unlikely to activate it. To test the potential activity of soluble Notch ligands, we generated monomeric and dimeric forms of the Notch ligand Delta -1 by fusing the extracellular domain to either a series of myc epitopes (D elta-1(ext-myc)) or to the Fc portion of human IgG-1 Delta-1(ext-IgG)), res pectively. Notch activation, assayed by inhibition of differentiation in C2 myoblasts and by HES1 transactivation in U20S cells, occurred when either Delta-1(ext-myc) or Delta-1(ext-IgG) were first immobilized on the plastic surface. However, Notch was not activated by either monomeric or dimeric li gand in solution (non-immobilized). Furthermore, both non-immobilized Delta -1(ext-myc) and Delta-1(ext-IgG) blocked the effect of immobilized Delta. T hese results indicate that Delta-1 extracellular domain must be immobilized to induce Notch activation in C2 or U20S cells and that non-immobilized De lta-1 extracellular domain is inhibitory to Notch function. These results i mply that ligand stabilization may be essential for Notch activation.