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Ligand tail is required for activation of the FKBP-FRB synNotch (ff-synNotch) system. (A) Schematic of the ff-synNotch system. Here, FKBP and FRB replace the N-terminal portion of Dll4 and the Notch1 EGF-like repeats 1−23, respectively. 6 (B) Luciferase activity assay showing the activation of a Notch reporter cell line (U2OS-FRB-Notch1-Gal4 transfected with a UAS-Luciferase reporter) co-cultured with CHO-TetR cells expressing the indicated variants of FKBP-hDll4 ligand either with or without ligand ICD (FKBP-hDll4-ΔICD). (C) Images showing a coculture of inducible CHO-TetR cells expressing the FKBP-hDll4 or FKBP-hDll4-ΔICD (red), with U2OS cells expressing the FRB-Notch-citrine (Citrine tag is inserted in the ECD, green). Images were taken 10 h after the induction of ligand expression with 100 ng/mL dox followed by 1 h induction of binding with 250 nM rapamycin. TEC is observed in full-length ligands (white arrows). Accumulation on the boundaries, but no TEC are observed in ligands lacking their ICD (orange arrows). Experiments with ligands lacking the ICD show reverse TEC (blue triangle). Data points show mean values from n = 13 measurements from four independent experiments. Error bars represent S.E.M. ***p < 0.001. Scale bars-10 μm.
Source publication
The Notch pathway converts receptor-ligand interactions at the cell surface into a transcriptional response in the receiver cell. In recent years, synthetic Notch systems (synNotch) that respond to different inputs and transduce different transcriptional responses have been engineered. One class of synNotch systems uses antibody-antigen interaction...
Contexts in source publication
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... also fused mCherry fluorescent protein at the C-terminal end of the ligands as with the endogenous ligand experiments. On the receptor, the FRB domain replaces the first 23 EGF-like repeats of Notch1, but retains the original Notch core and an ICD in which the Gal4 DNA-binding domain is substituted in place of the ankyrin repeat domain of the receptor ( Figure 3A). The FKBP− rapamycin complex binds to FRB tightly with K D ∼12 nM. ...
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... To test activity in the ff-synNotch system, we co-cultured U2OS cells stably expressing FRB-Notch1-Gal4 with CHOTetR sender cell lines stably expressing either full-length FKBP ligands, or FKBP ligands lacking their ICD. We note that variants tested exhibited similar expression levels ( Figure S3). Consistent with previous work using this system, 6 we found that ligands lacking the ICD in this ff-synNotch system exhibited greatly reduced activity compared to the full-length ligands ( Figure 3B). ...
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... note that variants tested exhibited similar expression levels ( Figure S3). Consistent with previous work using this system, 6 we found that ligands lacking the ICD in this ff-synNotch system exhibited greatly reduced activity compared to the full-length ligands ( Figure 3B). We also performed a TEC assay with the ff-synNotch by inserting a citrine fluorescent tag into the ECD of the receptor ( Figure 3C). ...
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... with previous work using this system, 6 we found that ligands lacking the ICD in this ff-synNotch system exhibited greatly reduced activity compared to the full-length ligands ( Figure 3B). We also performed a TEC assay with the ff-synNotch by inserting a citrine fluorescent tag into the ECD of the receptor ( Figure 3C). As with the endogenous Notch system, we observed TEC with the ligands containing the ICD (white arrows in Figure 3C), but not with the ligands that lack the ICD. ...
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... also performed a TEC assay with the ff-synNotch by inserting a citrine fluorescent tag into the ECD of the receptor ( Figure 3C). As with the endogenous Notch system, we observed TEC with the ligands containing the ICD (white arrows in Figure 3C), but not with the ligands that lack the ICD. Ligands lacking the ICD showed accumulation at the cell contact boundary (orange arrows in Figure 3C) as well as reverse trans-endocytosis (marked by a blue triangle in Figure 3C). ...
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... with the endogenous Notch system, we observed TEC with the ligands containing the ICD (white arrows in Figure 3C), but not with the ligands that lack the ICD. Ligands lacking the ICD showed accumulation at the cell contact boundary (orange arrows in Figure 3C) as well as reverse trans-endocytosis (marked by a blue triangle in Figure 3C). Overall, our results show that in contrast to the aasynNotch system, the ligand ICD is required for activation in the ff-synNotch system despite both having comparable receptor−ligand affinities. ...
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... with the endogenous Notch system, we observed TEC with the ligands containing the ICD (white arrows in Figure 3C), but not with the ligands that lack the ICD. Ligands lacking the ICD showed accumulation at the cell contact boundary (orange arrows in Figure 3C) as well as reverse trans-endocytosis (marked by a blue triangle in Figure 3C). Overall, our results show that in contrast to the aasynNotch system, the ligand ICD is required for activation in the ff-synNotch system despite both having comparable receptor−ligand affinities. ...
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... test whether the different behavior of the two systems depends on the TM domain, we replaced the TM of the endogenous hDll4 and FKBP ligands with the PDGFR TM and compared activities with and without the ligand ICD ( Figure 4A). All variants tested exhibited similar expression levels ( Figure S3). Our results, both in the luciferase and TEC assays ( Figures 4B,C, respectively), showed that only the fulllength ligand can activate Notch receptors irrespective of the TM domain used. ...
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... test the role of the ligand EGF repeats, we created four ligand FKBP variants with the PDGFR TM region by either including or excluding EGF repeats in the ligand ECD in full-length or tailless-ligand versions ( Figure 5A). The variants tested exhibited similar expression levels ( Figure S3). Both luciferase and TEC assays with these ligands show that removing the EGF repeats from the ligands did not compensate for the lack of the ligand ICD ( Figure 5B,C). ...
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... we tested whether removing the EGF repeats from the synNotch receptor can compensate for the lack of ligand ICD ( Figure 5D). As with the previous experiment, both luciferase and TEC assays showed that removing the EGF repeats on the receptor side cannot compensate for the lack of ligand ICD ( Figures 5E,F and S3). These results show that neither the presence of the EGF repeats in the receptors nor in the ligands can account for the differences between ff-synNotch and aasynNotch systems. ...
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... αGFP-mCherry was generated by inserting the mCherry tag to αGFP-Gal4 between the αGFP domain and the NRR domain. The ff-synNotch constructs ( Figure 3) were developed according to ref 6. A mCherry was added to the C-terminus of the FKBP ligand, and citrine (for the TEC experiments) was added between the FRB domain and the EGF repeats. ...
Citations
... IF-THEN logic gate systems are another modification to CARs in which the CAR protein will only be expressed if it has been activated by specific combinations of priming antigens [103À110]. One promising IF-THEN gated system utilizes the mechanism found among endogenous Notch receptor proteins [107,111] and engineers it for specific modular functions [111,112]. Synthetic Notch systems, referred to as synNotch, were developed to allow for multiple signal inputs that initiate intracellular responses ( Figure 4F) [107,110,112]. ...
... One promising IF-THEN gated system utilizes the mechanism found among endogenous Notch receptor proteins [107,111] and engineers it for specific modular functions [111,112]. Synthetic Notch systems, referred to as synNotch, were developed to allow for multiple signal inputs that initiate intracellular responses ( Figure 4F) [107,110,112]. ...
... In synNotch receptors, the extracellular domain is swapped for a single-chain fragment variable (scFv) which can target a specific antigen [111,113]. Extracellular ligation induces a conformational change within the core regulatory region (CRR), exposing an ADAM/gammasecretase protease cleavage site [111,112]. Once cleaved, the intracellular synthetic transcription factor can enter the nucleus and initiate a downstream effect [106,111] ranging from CAR expression in the IF-THEN gated system to singular cytokines [104,114,115] and proapoptotic factor secretion [106]. ...
... However, the notion that a pulling-force is strictly necessary is complicated by other experiments that indicate long-range activation via secreted ligands 18,19 and by in vitro activation using beads or Fc-clustered ligands 20,21 as well as being complicated by recent results on synthetic Notch constructs [22][23][24] . There has also not been successful ...
... However, the notion that a pulling-force is strictly necessary is complicated by other experiments that indicate long-range activation via secreted ligands 18,19 and by in vitro activation using beads or Fc-clustered ligands 20,21 as well as being complicated by recent results on synthetic Notch constructs [22][23][24] . There has also not been successful targeting of the pulling mechanism itself in vivo or in therapeutics. ...
... These artificial Notch-like constructs are assumed to require cell-cell contact for activation, which is explained with a force model. Interestingly, a recent study investigating differences between SynNotch and WT Notch found that SynNotch, as opposed to WT Notch, does not require an intracellular domain on the ligand cell side to initiate activation 23 . Another type of synthetic Notch, called SNIPR, has recently been introduced where the NRR is replaced altogether 24 . ...
The Notch signaling pathway has fundamental roles in embryonic development and in the nervous system. The current model of receptor activation involves initiation via a force-induced conformational change. Here, we define conditions that reveal pulling force-independent Notch activation using soluble multivalent constructs. We treat neuroepithelial stem-like cells with molecularly precise ligand nanopatterns displayed from solution using DNA origami. Notch signaling follows with clusters of Jag1, and with chimeric structures where most Jag1 proteins are replaced by other binders not targeting Notch. Our data rule out several confounding factors and suggest a model where Jag1 activates Notch upon prolonged binding without appearing to need a pulling force. These findings reveal a distinct mode of activation of Notch and lay the foundation for the development of soluble agonists.
... The endocytosis of DSL proteins is thought to provide the necessary force to deform the trans-bound Notch NRR [16]. Although the ubiquitylation of the intracellular domains (icds) of Delta and Ser by both Neur and Mib1 has been demonstrated [9,[17][18][19], its role in ligand activation is not clear [20,21]. ...
... However, the specific requirements for signaling in the Drosophila wing disk cannot necessarily be extrapolated to other systems. Different synthetic ligand-Notch pairs tested in mammalian cell culture did not recapitulate the necessity of ligand endocytosis for signal generation [21,23]. Meanwhile, studies of developmental processes in different Drosophila tissues have provided clues on the context dependence of Notch signal emission. ...
The execution of a Notch signal at the plasma membrane relies on the mechanical force exerted onto Notch by its ligand. It has been appreciated that the DSL ligands need to collaborate with a ubiquitin (Ub) ligase, either Neuralized or Mindbomb1, in order to exert this pulling force, but the role of ubiquitylation per se is uncertain. Regarding the Delta–Neur pair, it is documented that neither the Neur catalytic domain nor the Delta intracellular lysines (putative Ub acceptors) are needed for activity. Here, we present a dissection of the Delta activity using the Delta–Notch-dependent expression of Hey in newborn Drosophila neurons as a sensitive in vivo assay. We show that the Delta–Neur interaction per se, rather than ubiquitylation, is needed for activity, pointing to the existence of a Delta–Neur signaling complex. The Neur catalytic domain, although not strictly needed, greatly improves Delta–Neur complex functionality when the Delta lysines are mutated, suggesting that the ubiquitylation of some component of the complex, other than Delta, can enhance signaling. Since Hey expression is sensitive to the perturbation of endocytosis, we propose that the Delta–Neur complex triggers a force-generating endocytosis event that activates Notch in the adjacent cell.
... Notably, DLL4 binds to the NOTCH1 extracellular domain (ECD) with higher affinity than either DLL1 or JAG1, potentially accounting for the divergent strength of activation [5,6]. Quantitative analyses have further confirmed that higher affinity ligands indeed lead to more potent activation [7]. ...
... In the core regulatory region of endogenous Notch receptors, a pulling force is generated upon ligand binding, which exposes a protease cleavage site for a protease that is constitutively active in the membrane; this cleavage then liberates the intracellular domain which is a transcription coactivator 26,62 . The mechanisms of activation of Notch and synNotch receptors via cell-presented ligands have been compared, individuating possible mechanisms of activation that distinguish different synthetic and natural receptor constructs 63 . Thus, the mechanism of activation of was not certified by peer review) is the author/funder. ...
Synthetic Notch (synNotch) receptors are modular synthetic components that are genetically engineered into mammalian cells to detect signals presented by neighboring cells and respond by activating prescribed transcriptional programs. To date, synNotch has been used to program therapeutic cells and pattern morphogenesis in multicellular systems. However, cell-presented ligands have limited versatility for applications that require spatial precision, such as tissue engineering. To address this, we developed a suite of materials to activate synNotch receptors and serve as generalizable platforms for generating user-defined material-to-cell signaling pathways. First, we demonstrate that synNotch ligands, such as GFP, can be conjugated to cell- generated ECM proteins via genetic engineering of fibronectin produced by fibroblasts. We then used enzymatic or click chemistry to covalently link synNotch ligands to gelatin polymers to activate synNotch receptors in cells grown on or within a hydrogel. To achieve microscale control over synNotch activation in cell monolayers, we microcontact printed synNotch ligands onto a surface. We also patterned tissues comprising cells with up to three distinct phenotypes by engineering cells with two distinct synthetic pathways and culturing them on surfaces microfluidically patterned with two synNotch ligands.
We showcase this technology by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined spatial patterns towards the engineering of muscle tissue with prescribed vascular networks. Collectively, this suite of approaches extends the synNotch toolkit and provides novel avenues for spatially controlling cellular phenotypes in mammalian multicellular systems, with many broad applications in developmental biology, synthetic morphogenesis, human tissue modeling, and regenerative medicine.
The Fat/Dachsous (Ft/Ds) pathway is a highly conserved pathway regulating planar cell polarity (PCP) across different animal species. Proteins from the Ft and Ds family are large transmembrane protocadherins that form heterophilic complexes on the boundaries between cells. Fat4 and Dchs1, the main mammalian homologues of this pathway, have been implicated in PCP in various epithelial tissues and were shown to form extremely stable complexes at the boundaries between cells. It is unclear however, what are the dynamics controlling such stable boundary complexes, and how the formation and turnover of these complexes is regulated. Here, we use quantitative live imaging to elucidate the role of the intracellular domains (ICD) of Fat4 and Dchs1 in regulating Fat4/Dchs1 complex dynamics. We show that removing the ICD of Fat4 results in a reduction of both Trans-endocytosis (TEC) of Dchs1 into the Fat4 cells and boundary accumulation, but does not affect the diffusion of the complexes at the boundary. We further show that the ICD of Fat4 controls the turnover rate of Fat4/Dchs1 complexes. Finally, we find that while actin polymerization is required for maintaining the boundary accumulation of Fat4/Dchs1 complexes, we do not identify correlations between Fat4/Dchs1 complexes and local actin accumulation. Overall, we suggest that the Fat4 ICD is important for the turnover and plasticity of the highly stable Fat4/Dchs1 complexes associated with PCP.
Statement of Significance
The purpose of this study is to elucidate the dynamics leading to the formation and maintenance of Fat4/Dchs1 complexes at the cell boundary and how it is affected by the ICD of Fat4 in mammals. The insights from this work are important for obtaining a mechanistic molecular framework for understanding formation of planar cell polarity as well as for highlighting how cell boundary membrane complexes are regulated.
