Evolutionary plasticity in the requirement for force exerted by ligand endocytosis to activate C. elegans Notch proteins

The conserved transmembrane receptor Notch has diverse and profound roles in controlling cell fate during animal development. In the absence of ligand, a negative regulatory region (NRR) in the Notch ectodomain adopts an autoinhibited confirmation, masking an ADAM protease cleavage site;^1,2 ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the first step of signal transduction.^3,4 In Drosophila and vertebrates, recruitment of transmembrane Delta/Serrate/LAG-2 (DSL) ligands by the endocytic adaptor Epsin, and their subsequent internalization by Clathrin-mediated endocytosis, exerts a “pulling force” on Notch that is essential to expose the cleavage site in the NRR.^4–6 Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not essential to activate the two C. elegans Notch proteins, LIN-12 and GLP-1. Using an in vivo force sensing assay in Drosophila,⁶ we present evidence (1) that the LIN-12 and GLP-1 NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (2) that this difference depends on the absence of a “leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs.^1,2 Our results thus establish an unexpected evolutionary plasticity in the force-dependent mechanism of Notch activation and implicate a specific structural element, the leucine plug, as a determinant.