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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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Embryo

SUBMITTER: Marian Kalocsay

LAB HEAD: Steve Blacklow

PROVIDER: PXD039008 | Pride | 2023-07-03

REPOSITORIES: Pride

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			Action	DRS
	526501_AP_GSI_APEX_16plex_Oct2021_Fr1_12266_psc_20ppm.mzIdentML	Mzid
	526541_AP_GSI_APEX_16plex_Oct2021_Fr2_12267_psc_20ppm.mzIdentML	Mzid
	526543_AP_GSI_APEX_16plex_Oct2021_Fr6_12271_psc_20ppm.mzIdentML	Mzid
	526545_AP_GSI_APEX_16plex_Oct2021_Fr3_12268_psc_20ppm.mzIdentML	Mzid
	526548_AP_GSI_APEX_16plex_Oct2021_Fr4_12269_psc_20ppm.mzIdentML	Mzid

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A Spatiotemporal Notch Interaction Map from Membrane to Nucleus

Project description: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.

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