Notch engagement by Jag1 nanoscale clusters indicates a force-independent mode of activation

Abstract

The Notch signaling pathway is a cell-cell communication system with fundamental roles in embryonic development and the nervous system. The model of Notch receptor activation that is currently most accepted, involves a force-induced conformation change at the negative regulatory region of the receptor, the subsequent recruitment of ADAM metalloproteases and a cleavage cascade that releases the Notch intracellular domain. Here, we define conditions that enable force-independent Notch activation through the formation of soluble, long-lived, multivalent ligand-receptor complexes. To investigate how ligand valency affects activation of Notch receptors, we treated iPSc-derived neuroepithelial stem-like (lt-NES) cells with different spatially defined, molecularly precise ligand nanopatterns on DNA origami nanostructures. Our data indicate that Notch signaling is activated via stimulation with multivalent clusters of the ligand Jag1, and even multivalent chimeric structures where some Jag1 proteins are replaced by other binders that do not target Notch. The findings are corroborated by systematic elimination, through experimental control, of several confounding factors that potentially could generate forces, including electrostatic interactions, endocytosis and non-specific binding. Taken together, our data suggest a model where Jag1 ligands are able to activate Notch receptors upon prolonged binding, which subsequently triggers downstream signaling in a force independent manner. These findings reveal a distinct mode of activation of Notch and could lay the foundation for the development of soluble Notch agonists that currently remain elusive.