Notch Ligand Endocytosis Generates Mechanical Pulling Force Dependent on Dynamin, Epsins, and Actin

Abstract

SUMMARY Notch signaling induced by cell surface ligands is critical to development and maintenance of many eukaryotic organisms. Notch and its ligands are integral membrane proteins that facilitate direct cell-cell interactions to activate Notch proteolysis and release the intracellular domain that directs Notch-specific cellular responses. Genetic studies suggest Notch ligands require endocytosis, ubiquitylation and epsin endocytic adaptors to activate signaling, yet the exact role ligand endocytosis serves remains unresolved. Here we characterize a molecularly distinct mode of clathrin-mediated endocytosis requiring ligand ubiquitylation, epsins and actin for ligand cells to activate signaling in Notch cells. Using a cell-bead optical tweezers system, we obtained evidence for cell-mediated mechanical force dependent on this distinct mode of ligand endocytosis. We propose mechanical pulling force produced by endocytosis of Notch-bound ligand drives conformational changes in Notch that permit activating proteolysis.

Selective regulation of Notch ligands during angiogenesis is mediated by vimentin

“…S2G). During active signaling, Notch transendocytosis has been proposed to create a force on the receptor, known as the "pulling force," which is thought to reveal the cleavage site and promote receptor activation (42). A recent publication demonstrated that ligandmediated NECD transendocytosis during signal activation is mechanistically distinct from steady-state ligand endocytosis (55).…”

“…Vimentin interacts with membrane-tethered proteins through PDZ motifs (48) and Jagged and Dll carry distinct PDZ sequences in their ICDs (49)(50)(51). Whereas the ECDs are important for receptor binding, the ICDs play important roles in signal activation through posttranslational modifications (37,52) and interactions with endocytic regulators (41)(42)(43). To elucidate whether the ICDs of Dll4 and Jagged 1 influence vimentin binding and Notch signaling, we swapped the ICDs of Dll4 and Jagged 1 (53) to generate the chimeric variants: JaggedECDDll4ICD and Dll4ECDJaggedICD.…”

“…For immunofluorescence, the incubation was followed by fixation and immunocytochemistry was conducted as stated above. To produce resistance to Jagged-mediated endocytosis and mimic force-dependent ligand-mediated receptor endocytosis and Notch activation, the N1ECD was coupled to fluorescent protein A agarose beads (N1ECDPrtA) (42).…”

“…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).…”