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A spatiotemporal Notch interaction map from plasma membrane to nucleus.

Alexandre P Martin1, Gary A Bradshaw2, Robyn J Eisert1

  • 1Department of Biological Chemistry and Molecular Pharmacology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA.

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|August 1, 2023
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Summary

Notch signaling involves cleaving the Notch receptor to release a nuclear effector. This study maps the precise timing and location of Notch receptor proteolysis and nuclear translocation, revealing key signaling events.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Notch signaling is crucial for cell fate determination.
  • Ligand binding triggers Notch receptor proteolysis, releasing the Notch intracellular domain (NICD).
  • NICD translocates to the nucleus to regulate gene transcription.

Purpose of the Study:

  • To precisely map the spatiotemporal dynamics of Notch receptor processing.
  • To identify the intracellular location and timing of Notch cleavage events.
  • To understand the kinetics of Notch nuclear complex formation and target gene induction.

Main Methods:

  • Utilized proximity labeling techniques.
  • Employed quantitative, multiplexed mass spectrometry.
  • Monitored endogenous NOTCH2 interaction partners in response to ligand stimulation and gamma-secretase inhibition.

Main Results:

  • Gamma-secretase-mediated cleavage of NOTCH2 occurs in an intracellular compartment.
  • Nuclear complex formation and chromatin modifier recruitment happen within 45 minutes of inhibitor washout.
  • Detailed spatiotemporal map of Notch trafficking from plasma membrane to nucleus generated.

Conclusions:

  • Elucidated the step-by-step pathway of Notch receptor activation and nuclear translocation.
  • Identified specific signaling events amenable to therapeutic modulation.
  • Provided insights into the dynamic regulation of Notch signaling in cellular processes.