A spatiotemporal Notch interaction map from plasma membrane to nucleus

Abstract

Notch signaling relies on ligand-induced proteolysis of the transmembrane receptor Notch to liberate a nuclear effector that drives cell fate decisions. Upon ligand binding, sequential cleavage of Notch by the transmembrane protease ADAM10 and the intracellular protease γ-secretase releases the Notch intracellular domain (NICD), which translocates to the nucleus and forms a complex that induces target gene transcription. To map the location and timing of the individual steps required for the proteolysis and movement of Notch from the plasma membrane to the nucleus, we used proximity labeling with quantitative, multiplexed mass spectrometry to monitor the interaction partners of endogenous NOTCH2 after ligand stimulation in the presence of a γ-secretase inhibitor and as a function of time after inhibitor removal. Our studies showed that γ-secretase–mediated cleavage of NOTCH2 occurred in an intracellular compartment and that formation of nuclear complexes and recruitment of chromatin-modifying enzymes occurred within 45 min of inhibitor washout. These findings provide a detailed spatiotemporal map tracking the path of Notch from the plasma membrane to the nucleus and identify signaling events that are potential targets for modulating Notch 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).…”

“…NICD can access the nucleus within 30 min of ψ-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.…”

“…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.…”

“…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.…”

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

“…Nuclear intensities corresponding to nascent mCherry transcripts were detected within 1-2 hours following ligandinduced ICD release (Supplementary Figure 3). These results show that 𝛼FITC(E2)-SynNotch activation and its downstream reporter gene expression proceed along timescales consistent with those reported for Notch nuclear complex assembly and gene transcription [45][46][47] . Together, these results confirm the fluorescein-dependent activities of our receptor designs and provide insight into the timescales of synthetic signaling-induced transcript formation.…”