Epsin-Dependent Ligand Endocytosis Activates Notch by Force

DSL ligands activate Notch by inducing proteolytic cleavage of the receptor ectodomain, an event that requires ligand to be endocytosed in signal-sending cells by the adaptor protein Epsin. Two classes of explanation for this unusual requirement are (1) recycling models, in which the ligand must be endocytosed to be modified or repositioned before it binds Notch and (2) pulling models, in which the ligand must be endocytosed after it binds Notch to exert force that exposes an otherwise buried site for cleavage. We demonstrate in vivo that ligands that cannot enter the Epsin pathway nevertheless bind Notch but fail to activate the receptor because they cannot exert sufficient force. This argues against recycling models and in favor of pulling models. Our results also suggest that once ligand binds receptor, activation depends on a competition between Epsin-mediated ligand endocytosis, which induces cleavage, and transendocytosis of the ligand by the receptor, which aborts the incipient signal. [Display omitted] •In vivo dissection of the mechanism of Notch activation by ligand endocytosis•Epsin/Clathrin-mediated endocytosis of the ligand exerts force on Notch•Force induces ectodomain cleavage of Notch to initiate signal transduction•Ligand engulfment by receiving cell in the absence of force aborts incipient signal Force exerted by Epsin-mediated ligand endocytosis induces ectodomain cleavage of Notch to initiate signaling.