Investigating the Physical Requirement of Ligand Endocytosis in the Notch Pathway Using Optical Tweezers

The Notch signaling pathway is mediated by cell-cell interactions and is used extensively throughout embryonic development and the maintenance of tissues and stem cells in adults. Notch signaling requires a series of proteolytic cleavage events to release the Notch intracellular domain that functions directly in signal transduction. Furthermore, ligand cells binding to Notch must be capable of ligand endocytosis to activate signaling. Two different ligand endocytic events---ligand recycling or the pulling of ligand on Notch---have been proposed to explain the requirement for ligand internalization. Our biochemical and cellular studies identify Notch-induced ligand ubiquitination that promotes interactions with epsin endocytic adaptors, which along with dynamin and actin allow ligand cells to activate signaling in Notch cells. In order to determine whether endocytosis enables ligand recycling to generate high-affinity Notch binding, we integrated optical tweezers with cell biological and biochemical methods. Results indicate blocking endocytosis and/or recycling does not alter the binding strength of ligand-expressing cells to laser-trapped Notch coated beads, however, blocking endocytosis does prevent ligand cells from physically pulling on laser-trapped Notch beads. Together these results propose Notch contact induces ligand cells to form clathrin-dependent endocytic structures specialized in mechanical force to physically pull on Notch and activate signaling.