Optical Tweezers Studies on Notch: Single-Molecule Interaction Strength Is Independent of Ligand Endocytosis

Abstract

SUMMARY Notch signaling controls diverse cellular processes critical to development and disease. Cell surface ligands bind Notch on neighboring cells yet require endocytosis to activate signaling. The role ligand endocytosis plays in Notch activation has not been established. Here we integrate optical tweezers with cell biological and biochemical methods to test the prevailing model that ligand endocytosis facilitates recycling to enhance ligand interactions with Notch necessary to trigger signaling. Specifically, single-molecule measurements indicate that interference of ligand endocytosis and/or recycling does not alter the force required to rupture bonds formed between cells expressing the Notch ligand Delta-like1 (Dll1) and laser-trapped Notch1-beads. Together, our analyses eliminate roles for ligand endocytosis and recycling in Dll1-Notch1 interactions, and indicate that recycling indirectly affects signaling by regulating the accumulation of cell-surface ligand. Importantly, our study demonstrates the utility of optical tweezers to test a role for ligand endocytosis in generating cell-mediated mechanical force.

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

“…In addition to controlling number of ligands and receptors on the cell surface, ligand endocytosis is required for receptor activation (41). Two separate models for the requirement of endocytosis have been suggested: the first model suggests that recycling is important for maturation of the ligands to become active, whereas according to the second model, transendocytosis of the extracellular domain of the Notch receptor (NECD) is needed to produce a strain on the receptor.…”

“…Two separate models for the requirement of endocytosis have been suggested: the first model suggests that recycling is important for maturation of the ligands to become active, whereas according to the second model, transendocytosis of the extracellular domain of the Notch receptor (NECD) is needed to produce a strain on the receptor. This strain is termed the pulling force (41,42,44,45) and is thought to reveal the cleavage site and initiate proteolytic processing of the receptor with subsequent release of the NICD.…”

“…by protein trafficking (41)(42)(43). In addition to controlling number of ligands and receptors on the cell surface, ligand endocytosis is required for receptor activation (41).…”

Rapid disorganization of mechanically interacting systems of mammary acini

“…Cells use multiple approaches for integrating mechanical cues with biochemical cues provided by soluble factors; for example, there is extensive multilayered cross-talk between integrin and TGF-β signaling (16,17). It is now also clear that key developmental regulators, such as Notch, can be directly activated by mechanical force (18,19).Less is known about the interactions of organized multicellular structures with the ECM and how those interactions affect tissue architecture, composition, and stability, as well as the molecular pathways by which these effects are mediated. In certain situations, contractile multicellular structures are able to mechanically reorganize biopolymer networks over long distances and in a highly directional manner, generating what are variously referred to as fibers, tracts, cables, straps, or lines.…”

“…Cells use multiple approaches for integrating mechanical cues with biochemical cues provided by soluble factors; for example, there is extensive multilayered cross-talk between integrin and TGF-β signaling (16,17). It is now also clear that key developmental regulators, such as Notch, can be directly activated by mechanical force (18,19).…”