A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Abstract

Notch signaling relies on ligand-induced proteolysis to liberate a nuclear effector that drives cell fate decisions. The location and timing of individual steps required for proteolysis and movement of Notch from membrane to nucleus, however, remain unclear. Here, we use proximity labeling with quantitative multiplexed mass spectrometry to monitor the microenvironment of endogenous Notch2 after ligand stimulation in the presence of a gamma secretase inhibitor and then as a function of time after inhibitor removal. Our studies show that gamma secretase cleavage of Notch2 occurs in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurs within 45 minutes of inhibitor washout. This work provides a spatiotemporal map of unprecedented detail tracking the itinerary of Notch from membrane to nucleus after activation and identifies molecular events in signal transmission that are potential targets for modulating Notch signaling activity.

Temporal Dynamics and Stoichiometry in Notch Signaling - from Notch Synaptic Complex Formation to NICD Nuclear Entry

“…The accumulation of steady-state levels of NICD in the nucleus by ~45 min is also in agreement with the observed timing for transcriptional induction of Notch target genes in Drosophila and cell culture systems (Falo-Sanjuan et al, 2019;Ilagan et al, 2011;Pillidge & Bray, 2019). The timing of these dynamics are also similar to that obtained by following the kinetics of proximity labeling of nuclear proteins associated with the Notch transcriptional response which become labeled within 30-45 min of release from GSI inhibition (Martin et al, 2023).…”

“…JAGGED1-Fc expression and purification. Human JAGGED1-Fc (Martin et al, 2023) was transfected into Expi293F cells (ThermoFisher, A14527) using FectroPro (Polyplus, 101000007). Secreted JAGGED1-Fc was recovered from the culture media on Protein A agarose (Millipore, and eluted with 100 mM glycine, pH 3.0.…”

“…NICD can access the nucleus within 30 min of g-secretase inhibitor removal (Martin et al, 2023) and can induce a transcriptional response in the nucleus within 60 min of Notch activation (Falo-Sanjuan & Bray, 2022;Ilagan et al, 2011). To quantify the amount of N2-C entering nuclei after cell-cell contact, we paired and imaged sender and receiver cells immediately (1-5 min) and 60 min after cell contact.…”

“…SVG-A immortalized fetal astrocytes met these criteria as a Notch-expressing (receiver) cell line. They have been previously successfully engineered using CRISPR/Cas9 (Chou et al, 2016), they express vastly more NOTCH2 than other Notch isoforms (as judged by analysis of mRNA abundance by quantitative reverse-transcriptase PCR (Martin et al, 2023)), and when co-cultured with U2OS cells ectopically expressing the DLL4 ligand, they exhibit strong induction of a Notch-responsive luciferase reporter gene (Figure S1A, related to Figure 1). The reporter response was blocked by treatment with a g-secretase inhibitor (GSI; Compound E) (Figure S1A, related to Figure 1) and was not observed in co-culture assays with parental U2OS cells.…”

Notch dimerization contributes to maintenance of intestinal homeostasis by a mechanism involving HDAC2

“…Endocytosis of Notch regulates protein recycling [44] and degradation [45]. Importantly, spatiotemporal analysis using pulses of APEX2 biotinylation [46] mapped discreate Notch interactomes during ligand-induced Notch proteolysis, endocytosis, and nuclear translocation. 696 of 1093 interacting proteins identified by Martin et al were also biotinylated in our experiment (202 of which were nuclear, all shown in S2 Table).…”