Membrane Tethered Delta Activates Notch and Reveals a Role for Spatio-Mechanical Regulation of the Signaling Pathway

Short-range Notch receptor signaling is necessary for coordinating developmental activities in metazoa. To investigate this juxtacrine pathway, we mimic receptor-ligand binding within the cell-cell junction by engaging Notch1-eGFP expressing cells to a supported lipid membrane displaying Delta-like protein 4 (DLL4). DLL4-Notch1 binding, oligomerization, and transport were observed in real time, and the molecular density and stoichiometry of the complexes were determined using quantitative fluorescence imaging. A Notch transcriptional reporter readout was used to quantify how ligand lateral mobility, orientation, and density modulate receptor activation levels. These experiments demonstrate that limiting the lateral mobility of DLL4 can enhance Notch activation by 2.6-fold, thus supporting the existence of a spatio-mechanical mechanism of signal regulation.