GFP Reporter Cell Line - U2OS

The Notch pathway elicits transcriptional responses in recipient cells through receptor-ligand interactions at the cell surface. In recent years, synthetic Notch systems (synNotch) have been designed to respond to different inputs and transduce distinct transcriptional responses. Using antibody-antigen interactions, researchers have induced a proteolytic cascade of the endogenous Notch autoregulatory core at the cell surface, releasing the synthetic Notch intracellular domain (ICD) that translates surface antigen detection into a cellular response. Unlike endogenous Notch, whose activation requires ubiquitination and endocytosis of the ligand ICD, these synNotch systems appear to require no such process. The researchers compared the behavior of antibody-antigen synNotch (aa-synNotch), endogenous Notch, and a synNotch system that dimerizes via rapamycin-induced FK506 binding protein (FKBP) and FKBP rapamycin binding (FRB) domains (ff-synNotch). The study found that neither receptor-ligand affinity, the identity of the transmembrane domain, or the presence or absence of extracellular epidermal growth factor repeat regions could explain the differences in ligand ICD requirements between aa-synNotch and endogenous Notch or ff-synNotch. In addition, the researchers found that, unlike endogenous Notch and ff-synNotch, the aa-synNotch system did not exhibit reverse endocytosis of the receptor's extracellular domain. These results suggest that the aa-synNotch system may bypass the ligand ICD requirement by promoting other adhesive cell-cell interactions through antigen-antibody pairs.

Figure 1. The researchers evaluated Notch activity by co-culturing U2OS cells expressing either GFP or GFP-ICD ligands. Images show that after co-culturing U2OS-GFP or GFP-ICD ligands with U2OS-αGFP-mCherry receptors, the ligands accumulated strongly at the cell borders without TEC phenomenon. (Khamaisi B, et al., 2022)