Monitoring Notch signaling dynamics in real time using advanced microscopy techniques.
Final Report Abstract
The Notch signaling pathway plays fundamental roles during cell fate decisions in development and tissue homeostasis of most organs, and aberrant signaling in humans is associated with various diseases including cancer. The signaling is activated through ligand induced receptor proteolysis and subsequent release of receptors intracellular domain (NICD) into the nucleus where it acts as a transcriptional regulator. During my postdoctoral work, which was supported by the Walter-Benjamin Program, I have established a system to visualize the key Notch signaling events at physiological levels in real-time in living cells. Using CRISPR/Cas9 genome engineering we added fluorescent tags to endogenously expressed receptor and ligand proteins. We generated a microfluidics system to controllably pair ligand and receptor expressing cells while imaging and found that upon direct cell-cell contact of sender and receiver cell, ligands and receptors cluster and form Notch synapses with a stoichiometry of 1:1. We used a state-of-the-art lattice light-sheet microscope and visualized the spatial dynamics of these synapses with high resolution over time and spatiotemporally linked the order of signaling events. We defined that the formed synapses dissolve 15-20 minutes after formation and precede NECD trans-endocytosis and NICD entry in the nucleus. We further estimated the number of molecules involved in synapses and quantified nuclear NICD concentration after signal activation. This work describes with high resolution the spatiotemporal dynamics of Notch signaling activation and opens new avenues to study the dynamics of signaling components in real-time in living cells. The results of this work were published in Developmental Cell.
Publications
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A spatiotemporal Notch interaction map from plasma membrane to nucleus. Science Signaling, 16(796).
Martin, Alexandre P.; Bradshaw, Gary A.; Eisert, Robyn J.; Egan, Emily D.; Tveriakhina, Lena; Rogers, Julia M.; Dates, Andrew N.; Scanavachi, Gustavo; Aster, Jon C.; Kirchhausen, Tom; Kalocsay, Marian & Blacklow, Stephen C.
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Structural Requirements for Activity of Mind bomb1 in Notch Signaling.
Cao, Ruili; Gozlan, Oren; Tveriakhina, Lena; Zhou, Haixia; Jiang, Hanjie; Cole, Philip A.; Aster, Jon C.; Sprinzak, David & Blacklow, Stephen C.
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Temporal dynamics and stoichiometry in human Notch signaling from Notch synaptic complex formation to nuclear entry of the Notch intracellular domain. Developmental Cell, 59(11), 1425-1438.e8.
Tveriakhina, Lena; Scanavachi, Gustavo; Egan, Emily D.; Da Cunha Correia, Ricardo Bango; Martin, Alexandre P.; Rogers, Julia M.; Yodh, Jeremy S.; Aster, Jon C.; Kirchhausen, Tom & Blacklow, Stephen C.
