Mechanical Allostery: Evidence for a Force Requirement in the Proteolytic Activation of Notch

• Published in: Developmental cell Vol. 33; no. 6; pp. 729 - 736
• Main Authors: Gordon, Wendy R., Zimmerman, Brandon, He, Li, Miles, Laura J., Huang, Jiuhong, Tiyanont, Kittichoat, McArthur, Debbie G., Aster, Jon C., Perrimon, Norbert, Loparo, Joseph J., Blacklow, Stephen C.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 22.06.2015
• Subjects: ADAM Proteins - metabolism; ADAM17 Protein; Allosteric Regulation; Animals; Artificial Cells; Biomechanical Phenomena; Cell Line; Endocytosis; HEK293 Cells; Humans; Ligands; Mechanotransduction, Cellular; Models, Biological; Proteolysis; Receptors, Notch - chemistry; Receptors, Notch - metabolism; Signal Transduction
• ISSN: 1534-5807; 1878-1551; 1878-1551
• DOI: 10.1016/j.devcel.2015.05.004

Ligands stimulate Notch receptors by inducing regulated intramembrane proteolysis (RIP) to produce a transcriptional effector. Notch activation requires unmasking of a metalloprotease cleavage site remote from the site of ligand binding, raising the question of how proteolytic sensitivity is achieved. Here, we show that application of physiologically relevant forces to the Notch1 regulatory switch results in sensitivity to metalloprotease cleavage, and bound ligands induce Notch signal transduction in cells only in the presence of applied mechanical force. Synthetic receptor-ligand systems that remove the native ligand-receptor interaction also activate Notch by inducing proteolysis of the regulatory switch. Together, these studies show that mechanical force exerted by signal-sending cells is required for ligand-induced Notch activation and establish that force-induced proteolysis can act as a mechanism of cellular mechanotransduction. [Display omitted] •Development of single-molecule assays to monitor Notch proteolysis under force•Exposure of the Notch1 masked proteolytic site occurs in a physiological force regime•Dll4-induced activation of Notch1 requires the application of mechanical force•Non-native tethering can substitute for receptor-ligand complexes in signaling Gordon et al. develop and apply single-molecule and high-throughput magnetic tweezers assays to show that mechanical force is needed to unmask the metalloprotease site in the Notch “activation switch” and induce Notch signaling in cells. Synthetic signaling systems also show a ligand endocytosis requirement even without native ligand-receptor interactions.