C. elegans Notch proteins are tuned to lower force thresholds than Drosophila Notch, bypassing the requirement for Epsin-mediated ligand endocytosis.
Authors: Paul Langridge 1,2; Alejandro Garcia-Diaz 2; Jessica Chan 2; Iva Greenwald 2; Gary Struhl 2
Affiliations: 1) Augusta University, Georgia, GA; 2) Columbia University, New York, NY
Keywords: d. Notch; g. wing disc
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 a protease cleavage site; ligand binding induces cleavage of the NRR, leading to Notch ectodomain shedding as the initiating step of signal transduction. In Drosophila and vertebrates, recruitment of transmembrane protein 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. Here, we show that Epsin-mediated endocytosis of transmembrane ligands is not required to activate the two C. elegans Notch proteins. Using an in vivo force sensing assay in Drosophila, we present evidence (i) that the C. elegans NRRs are tuned to lower force thresholds than the NRR of Drosophila Notch, and (ii), that this difference depends on the presence of a “Leucine plug” that occludes the cleavage site in the Drosophila and vertebrate Notch NRRs but is absent from the C. elegans Notch NRRs. 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 potential determinant.