Temporal dynamics and stoichiometry in human Notch signaling from Notch synaptic complex formation to nuclear entry of the Notch intracellular domain

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.

doi:10.1016/j.devcel.2024.03.021

Cited by 4 publications

(1 citation statement)

References 70 publications

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“…Nuclear intensities corresponding to nascent mCherry transcripts were detected within 1-2 hours following ligand-induced ICD release ( Supplementary Figure 3 ). These results show that α FITC(E2)-SynNotch activation and its downstream reporter gene expression proceed along timescales consistent with those reported for Notch nuclear complex assembly and gene transcription 45–47 . Together, these results confirm the fluorescein-dependent activities of our receptor designs and provide insight into the timescales of synthetic signaling-induced transcript formation.…”

Section: Resultssupporting

confidence: 88%

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

Tran,

Kuffner,

Marzilli

et al. 2024

Preprint

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We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of ano-nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

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Section: Resultssupporting

confidence: 88%

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

Tran,

Kuffner,

Marzilli

et al. 2024

Preprint

View full text Add to dashboard Cite

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Design of a Soluble Multivalent Notch Agonist

Mout,

Jing,

Egan

et al. 2024

Preprint

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Designed protein agonists can enhance the efficiency of endogenous signaling pathways, and provide a powerful means to control cellular functions and develop disease therapeutics. Designing a soluble cytokine-like agonist for Notch signaling, an evolutionarily conserved pathway that regulates cell fate in embryonic and adult development, is especially challenging because Notch receptor activation requires a mechanical force that is typically mediated by cell-associated transmembrane ligands at sites of cell-cell contact. Moreover, free soluble Notch ligand is signal inhibitory. Here, we exploit computationally designed protein oligomers with precise geometries and valencies to generate cytokine-like, ‘protein only,’ multivalent soluble Notch agonists. These tools promote cell-cell contact, cluster Notch proteins in synapses at the cell surface, and activate Notch signaling in reporter cell lines and cells expressing endogenous receptors. We demonstrate the utility of these soluble Notch agonists in T cell differentiation from cord blood (CB) and human induced pluripotent stem cells (iPSCs), and in bioreactor production of T cells in liquid suspension. Soluble multivalent Notch agonists can be applied broadly toin vitrocellular differentiation methods to generate clinical cell products and to develop immunotherapies.

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Synchronization of the segmentation clock using synthetic cell-cell signaling

Isomura,

Asanuma,

Kageyama

2024

Preprint

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Tight coordination of cell-cell signaling in space and time is vital for self-organization in tissue patterning. During vertebrate development, the segmentation clock drives oscillatory gene expression in the presomitic mesoderm (PSM), leading to the periodic formation of somites. Oscillatory gene expression is synchronized at the cell population level; inhibition of Delta-Notch signaling results in the loss of synchrony and the fusion of somites. However, it remains unclear how cell-cell signaling couples oscillatory gene expression and controls synchronization. Here, we report the reconstitution of synchronized oscillation in PSM organoids by synthetic cell-cell signaling with designed ligand-receptor pairs. Optogenetic assays uncovered that the intracellular domains of synthetic ligands play key roles in dynamic cell-cell communication. Oscillatory coupling using synthetic cell-cell signaling recovered the synchronized oscillation in PSM cells deficient for Delta-Notch signaling; non-oscillatory coupling did not induce recovery. This study reveals the mechanism by which ligand-receptor molecules coordinate the synchronization of the segmentation clock, and provides direct evidence of oscillatory cell-cell communication in the segmentation clock.

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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

Cited by 4 publications

References 70 publications

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues

Design of a Soluble Multivalent Notch Agonist

Synchronization of the segmentation clock using synthetic cell-cell signaling

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