Recording Notch signaling in real time

Developmental Neuroscience
Emil M HanssonUrban Lendahl

Abstract

Notch signaling is a highly conserved signaling pathway, which is critical for many cell fate decisions. Ligand activation of Notch leads to cleavage of the Notch receptor and liberation of the Notch intracellular domain (ICD) from the membrane-tethered receptor. After translocation to the nucleus, the Notch ICD interacts with the DNA-binding protein CSL to activate gene transcription. To better understand the temporal and spatial aspects of Notch signaling, we here describe a fluorescent protein-based reporter assay that allows Notch activation to be followed in real time in individual cells. We have generated a reporter construct composed of 12 CSL-binding motifs linked to fluorescent proteins with different half-lives: a stabler red fluorescent protein (DsRedExpressDR) and a destabilized form of green fluorescent protein (d1EGFP). The fluorescent reporters reflect the activation status of Notch signaling with single-cell resolution. The reporters rapidly respond to various forms of Notch activation, including ligand activation of full-length Notch receptors. Finally, we use this assay to gain insights into the level of Notch signaling in CNS progenitor cells in culture and in vivo.

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Citations

May 11, 2010·Cellular and Molecular Life Sciences : CMLS·Kerstin HuennigerChristoph Kaether
Mar 4, 2008·Nature Cell Biology·Paola Scaffidi, Tom Misteli
Feb 25, 2011·Molecular Biology of the Cell·Daniel J García-DomínguezJosé M Frade
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Apr 14, 2016·Stem Cell Reports·Eike-Benjamin BrauneUrban Lendahl
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Jan 11, 2019·ELife·Nagarajan NandagopalMichael B Elowitz
Feb 28, 2008·Neuroreport·Christoffer TammSandra Ceccatelli
Aug 20, 2010·Journal of Cell Science·Emil M HanssonUrban Lendahl
Sep 23, 2014·Journal of Cell Science·Kasper Kjaer-SorensenClaus Oxvig

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