Recording Notch signaling in real time.
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.
Hansson
Emil M
EM
Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, Stockholm, Sweden.
Teixeira
Ana I
AI
Gustafsson
Maria V
MV
Dohda
Takeaki
T
Chapman
Gavin
G
Meletis
Konstantinos
K
Muhr
Jonas
J
Lendahl
Urban
U
D020816:Amino Acid Motifs / Q000235:genetics; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002460:Cell Line; D002490:Central Nervous System / Q000166:cytology / Q000196:embryology*; D002642:Chick Embryo; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008024:Ligands; D008164:Luminescent Proteins; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D051880:Receptors, Notch / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D015398:Signal Transduction / Q000502:physiology*; D013194:Staining and Labeling / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D013997:Time Factors; D000097573:Red Fluorescent Protein
D049452:Green Fluorescent Proteins; D008024:Ligands; D008164:Luminescent Proteins; D051880:Receptors, Notch; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein