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Real-Time Imaging of Notch Activation with a Luciferase Complementation-Based Reporter

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Sci. Signal.  12 Jul 2011:
Vol. 4, Issue 181, pp. rs7
DOI: 10.1126/scisignal.2001656

Abstract

Notch signaling regulates many cellular processes during development and adult tissue renewal. Upon ligand binding, Notch receptors undergo ectodomain shedding followed by γ-secretase–mediated release of the Notch intracellular domain (NICD), which translocates to the nucleus and associates with the DNA binding protein CSL [CBF1/RBPjκ/Su(H)/Lag1] to activate gene expression. Mammalian cells contain four Notch receptors that can have both redundant and specific activities. To monitor activation of specific Notch paralogs in live cells and in real time, we developed luciferase complementation imaging (LCI) reporters for NICD-CSL association and validated them as a specific, robust, and sensitive assay system that enables structure-function and pharmacodynamic analyses. Detailed kinetic analyses of various mechanistic aspects of Notch signaling, including nuclear translocation and inhibition of the activities of γ-secretase and ADAM metalloproteases, as well as agonist- and ligand-dependent activation, were conducted in live cells. These experiments showed that Notch-LCI is an effective approach for characterizing modulators that target Notch signaling and for studying pathway dynamics in normal and disease contexts.

Introduction

The Notch pathway is a short-range signal transducer that is broadly and reiteratively used during embryonic development and adult tissue renewal to regulate cell proliferation and apoptosis, fate specification, differentiation, and stem cell maintenance. Mammalian cells contain four Notch receptors and at least five cognate ligands (Dll1, 3, and 4 and Jag1 and 2). Most Notch-mediated cellular decisions use a canonical or core pathway (1) (Fig. 1A). Ligand binding leads to a conformational change allowing ADAM-mediated ectodomain shedding (S2 cleavage) and subsequent γ-secretase–mediated proteolysis within the transmembrane domain (TMD) (S3 cleavage) to release the Notch intracellular domain (NICD). NICD translocates to the nucleus and associates with the DNA binding protein CSL [named after CBF1/RBPjκ in mammals, Su(H) in flies, and Lag1 in worms] to form a composite interface where the coactivator Mastermind [Mastermind-like (MAML) in mammals] binds, leading to the recruitment of the transcription machinery and target gene expression (Fig. 1A) (1, 2). The biological consequences of Notch signaling are dependent on dose, timing, and cellular context, and, therefore, the core pathway is highly regulated. Indeed, abnormal loss or gain in Notch signaling has been associated with various human developmental disorders, late-onset diseases, and cancers, leading to its emergence as a key therapeutic target (36).

Fig. 1

The core Notch pathway and the various approaches for monitoring Notch activity. (A) Schematic of the canonical Notch pathway. Ligand binding leads to ectodomain shedding due to cleavage at the S2 site by ADAM, followed by intramembrane proteolysis at the S3 site by γ-secretase. The released Notch intracellular domain (NICD) translocates to the nucleus and interacts with the DNA binding protein CSL to recruit MAML proteins and other coactivators to activate gene expression. (B) Overview of the current methods for monitoring Notch pathway activation. Left: Antibodies, such as V1744, can specifically recognize the N terminus of NICD that is exposed only after γ-secretase cleavage. Middle: Overall pathway activity can be monitored by reporter proteins, such as luciferases (Luc) or fluorescent proteins (FP), controlled by promoters of endogenous target genes, such as Hes, or multimerized CSL-binding sites, such as 4XCSL and TP1. Right: Regulated proteolysis of Notch fusion proteins releases various heterologous proteins that can be monitored by either transcriptional reporters or nuclear translocation of fluorescent proteins. (C) The luciferase complementation imaging (LCI)–based approach for monitoring Notch activation in real time takes advantage of the specific interactions between a particular NICD and CSL (RBPjκ in our studies) to reconstitute activity between fused luciferase fragments (NLuc and CLuc). Variants of the reporter system can be used to interrogate different mechanistic aspects of Notch signaling.

Understanding the molecular mechanisms regulating the dose and timing of Notch activity in both normal and disease contexts depends on sensitive methods for monitoring Notch activity in real time and is emerging as a critical need. One strategy uses antibodies to detect the γ-secretase–cleaved and, hence, the activated form of Notch (Fig. 1B). Although this has been useful in assessing Notch cleavage in lysates, fixed cells, and tissues (7, 8), it is not a dynamic real-time reporting system. Another approach is based on the use of Notch-responsive promoters to drive fluorescent or bioluminescent reporters in cells or animals (Fig. 1B) (912). These widely used transcriptional reporters allow sensitive and dynamic, qualitative, or quantitative reporting of overall pathway activity. However, these reporters can also record input from other signaling pathways and cannot distinguish which paralog is active in cells where multiple Notch paralogs are present. Fusing heterologous proteins, for example, Gal-VP16 or Cre (1315), to a particular receptor can help boost sensitivity, specificity, or both (Fig. 1B). Because of the time needed for transcription and translation of downstream reporters, a delay is inherent in all promoter-reporter gene approaches. Nuclear translocation assays with Notch fused to green fluorescent protein (GFP) (16) can address some of these issues, but their dynamic range is limited due to cellular autofluorescence. Moreover, the need to distinguish the nuclear-associated versus the membrane-tethered fluorescence presents a technical challenge for high-throughput screening (HTS) approaches.

We developed an alternative reporter system for canonical Notch signaling based on optimized luciferase complementation imaging (LCI) (17), a protein fragment complementation assay (PCA) system that permitted us to directly monitor interactions between a particular NICD and RBPjκ in real time (Fig. 1C). The key advantages of LCI compared to other PCA or protein interaction–based methods include (i) the inherent sensitivity of luminescent enzymatic reporters due to signal amplification and the lack of background cellular luminescence; (ii) the negligible binding energy between the luciferase fragments, making complementation reversible and enabling accurate quantification of protein interactions; (iii) the ability to detect protein interactions in any subcellular compartment; (iv) the capability for noninvasive, repeated imaging in live cells and in vivo; (v) the easy deployment with relatively inexpensive, commercially available reagents; and (vi) the amenability to HTS applications (18, 19). Notably, LCI does not depend on activation of downstream reporters for readout, thereby allowing measurement of reconstituted luciferase activity in near real time and enabling kinetic analyses of pathway activation.

Here, we report the extensive validation of the Notch-LCI reporter technology. We confirmed its ability to faithfully reproduce known Notch biochemistry and demonstrated its use as a noninvasive, real-time reporter of Notch activation. We monitored the formation and turnover of the NICD-RBPjκ complex, measured the pharmacodynamics of different γ-secretase and metalloprotease inhibitors, compared the activation kinetics of receptors presented with ligands or agonists in different paradigms, and quantified responses to known modifiers. In addition, we generated several stable cell lines expressing variants of the reporter and validated their robustness for HTS. Thus, the Notch-LCI reporters represent HTS-ready tools for characterizing cellular activities and pharmacological agents that modulate Notch activation.

Results

Robust and specific complementation between NICD1-NLuc and CLuc-RBPjκ

Complementary luciferase fragments (NLuc and CLuc), which have no activity on their own, can reassemble into an active enzyme when brought into close proximity by the interacting proteins to which they are fused (17). To create a real-time assay system for Notch1 activation, we fused NLuc to several Notch1 variants and CLuc to RBPjκ (Figs. 1C and 2A). Notch1-NLuc variants consisted of a full-length receptor (NotchFL) and truncated or mutant derivatives with varying activities (Fig. 2A). NotchFL has a large extracellular domain (ECD) composed of 36 epidermal growth factor (EGF) repeats, some of which mediate ligand binding, and the negative regulatory region (NRR), which can be further subdivided into the Lin-Notch repeats (LNRs) and the heterodimerization domain (HD). Mature Notch receptors have been furin-processed at the S1 site within the HD to generate the NECD and Notch transmembrane and intracellular (NTMIC) polypeptides, which are held together by interactions between the N- and the C-terminal halves of the HD. The NRR domain keeps the receptor in the “off” state in the absence of ligand. NLNG molecules, which lack the EGF repeats but contain the NRR, cannot respond to ligand and are inactive unless mutations that lead to ligand-independent activation are present (for example, the C1675S/C1682S double mutant NLNGCC>SS) (20). Notch molecules that either lack the entire ECD (NΔE) or contain only the intracellular domain (NICD) are constitutively active (Fig. 2A). The intracellular domain contains the RAM (RBPjκ association module), ANK (seven ankyrin repeats), and PEST (proline-, glutamic acid-, serine-, and threonine-rich) domains (Fig. 2A). Although Notch activity is retained after addition of heterologous tags or molecules to the Notch C terminus (21), we included a short flexible Gly-Ser linker between the luciferase fragment and the fused protein to minimize potential steric hindrance (17).

Fig. 2

Development and validation of the Notch1/RBPjκ LCI reporters. (A) Domain organization of Notch1, RBPjκ, and their respective luciferase fragment fusions. The full-length receptor (NotchFL) has a large ECD composed of 36 EGF repeats and the NRR, which can be subdivided into the LNRs and the HD. During maturation, Notch receptors are processed at the S1 site within HD to generate the NECD and Notch transmembrane and intracellular (NTMIC) polypeptides, which remain associated. Following the TMD is a large intracellular region that carries the RAM, ANK, and PEST domains. Red arrowheads indicate the different mutations used for assay validation. The activity profile of the Notch variants used in this study is summarized. RBPjκ is composed of the N-terminal domain (NTD), β-trefoil domain (BTD), and C-terminal domain (CTD). (B) Western blot analysis of the Notch-NLuc proteins and production of NICD. The mN1A antibody recognizes the ANK domain within Notch-NLuc. NICD production from the constitutively active constructs was detected with V1744. (C) The transactivation profile of the Notch-NLuc variants is consistent with previous studies, with the constitutively active NΔE and NICD exhibiting the highest activity. (D) Complementation profile of the different Notch-NLuc fusions with CLuc-RBPjκ. Constitutively active forms exhibit the highest complementation activity. Bioluminescence images of 3T3 cells expressing different Notch-LCI pairs and the cotransfected Renilla Luc (RLuc) are shown. RLuc confirmed equivalent transfection efficiency. (E) NICD-NLuc produces robust complementation with CLuc-RBPjκ, but not with CLuc alone or a nuclear-targeted CLuc (nls-CLuc). RAM mutations diminished complementation.

The integrity of all Notch-NLuc proteins was confirmed by Western blot with a Notch1-specific monoclonal antibody, mN1A, and, where appropriate, γ-secretase–dependent proteolysis was confirmed with the V1744 antibody, which recognizes the cleaved free N terminus of NICD (22) (Fig. 2B). Although detectable with mN1A, NICDΔRAM molecules, which were either released by NΔEΔRAM cleavage or produced from a NICDΔRAM expression vector, were not recognized well by some lots of the V1744 antibody, highlighting the shortcoming of antibody-based detection of activated Notch proteins.

For comparison of the signaling properties of Notch1-NLuc and CLuc-RBPjκ fusion proteins to their untagged counterparts, each protein was individually evaluated for its ability to activate downstream transcriptional reporters based on multimerized RBPjκ binding sites (TP1-luciferase) (23) or endogenous Notch target promoters (Hes1- and Hes5-luciferase) (10). The functionality of CLuc-RBPjκ was examined in RBPjκ-null (OT11) cells. The fusion of CLuc to RBPjκ did not change its activity on either the TP1-luciferase or the Hes1-luciferase reporter (fig. S1). We tested the function of Notch-NLuc fusion proteins in unstimulated 3T3 cells. The NLuc fusion reduced the overall transactivation ability of Notch on promoters with multimerized sites (TP1-luciferase; fig. S2, A and B), but had less of an effect on Hes1-luciferase and no effect on Hes5-luciferase activation (fig. S2C). These results suggest that the tag can cause steric hindrance on reporters with tandem repeats of RBPjκ binding sites in head-to-tail orientation. Despite this effect on the artificial promoter with multimerized sites, overall, the data confirmed that the addition of luciferase fragments at the C terminus of Notch did not greatly alter its signaling properties. The relative activities of the NLuc-tagged Notch proteins were the same as those of the untagged molecules (Fig. 2C and fig. S2A).

TP1-luciferase activation was not seen with NotchFL molecules or with NLNG proteins unless they contained amino acid substitutions that relax Notch’s autoinhibition (NLNGCC>SS) (20) (Fig. 2C). The constitutively active forms, NΔE-NLuc and NICD-NLuc, both activated TP1-luciferase robustly (Fig. 2C). We also tested three types of biochemically characterized, inactivating mutations that are known to diminish or abolish Notch transcriptional activity (Fig. 2C): (i) a TMD mutant (NΔEV1744G) that produces a destabilized NICD (24, 25); (ii) mutations targeting the RAM domain that abolish interactions with RBPjκ, including a 27–amino acid deletion (ΔRAM) (22) and the WFP-to-LAA amino acid substitution (NΔEWFP>LAA and NICDWFP>LAA) (26); and (iii) mutations destabilizing the ANK domain (NICDM1 and NICDM2) (27, 28) that permit RBPjκ interactions but eliminate transcriptional activity due to failure in recruiting MAML.

After confirming the proteolytic processing and transcriptional function of Notch-NLuc fusion proteins, we tested their ability to reconstitute luciferase activity with CLuc-RBPjκ. Notch-NLuc variants were coexpressed with CLuc-RBPjκ along with Renilla luciferase (as a transfection control), and then bioluminescence imaging was performed in live cells (Fig. 2, D and E). A strong signal was readily detected when NICD-NLuc was coexpressed with CLuc-RBPjκ (Fig. 2, D and E), whereas nonspecific interactions between NICD-NLuc and CLuc or a nuclear-targeted CLuc (nls-CLuc) produced only low background luminescence (Fig. 2E). NotchFL-NLuc or NLNG-NLuc (both of which are inactive molecules in this paradigm) did not generate an appreciable bioluminescent signal when coexpressed with CLuc-RBPjκ, indicating that transient interactions at the endoplasmic reticulum that could occur during protein synthesis did not contribute to luciferase complementation with this amount of protein. Coexpression of CLuc-RBPjκ with RAM domain mutants (ΔRAM or WFP>LAA) did not result in a signal above background (Fig. 2, D and E), demonstrating the dependence of complementation on this high-affinity interaction domain. As anticipated, robust complementation occurred with the dominant-negative form of RBPjκ (CLuc-dnRBPjκ) (fig. S3A), confirming that unlike transcriptional reporter activation (fig. S1A), the interactions between NICD and RBPjκ do not depend on the DNA binding activity of RBPjκ (29, 30). The Notch-LCI system reported the same “signal strength” hierarchy observed with transcriptionally active constructs (Fig. 2D), with the constitutively active molecules equivalently exhibiting the highest luciferase complementation activity (that is, NΔE = NICD ≫ NΔEV1744G > NLNGCC>SS). We also verified that robust and specific complementation occurred if the Notch portion was fused to CLuc and RBPjκ was fused to NLuc (fig. S3B).

The Notch-LCI reporter system can differentiate the mechanisms underlying the effects of RAM versus ANK domain mutations on transcriptional activation. NICD molecules harboring the ANK mutations M1 and M2 failed to activate TP1-luciferase (Fig. 2C), but LCI confirmed their ability to interact with RBPjκ in intact cells (Fig. 2D). This reflects the presence of an intact RAM domain in these molecules and the negligible binding energy contributed by the ANK domain to interactions between NICD and RBPjκ relative to its importance in facilitating assembly of the NICD-RBPjκ-MAML complex (2) (fig. S4A). The ability of LCI to detect the subtle differences in stability of the NICD-RBPjκ complex with some ANK domain mutations confirms the usefulness of Notch-LCI as a sensitive cell-based reporter of protein-protein interactions. LCI also allowed us to noninvasively monitor changes in NICD-RBPjκ interactions in live cells in which we interfered with the RAM-RBPjκ interface by coexpressing RAM domain polypeptides (Fig. 3A). Soluble RAM peptides disrupt NICD-RBPjκ interactions in experiments with purified proteins in vitro and in cell lysates (30, 31). Accordingly, the RAM domain polypeptide, but not the mutant RAMWFP>LAA peptide, significantly reduced NICD-NLuc/CLuc-RBPjκ complementation (Fig. 3A).

Fig. 3

Direct quantification of the modulation of NICD-RBPjκ interactions in live cells with LCI. (A) Wild-type, but not WFP>LAA mutant, RAM polypeptides reduced NΔE-NLuc/CLuc-RBPjκ complementation in a dose-dependent manner. +, 100 ng; ++, 400 ng of RAM expression plasmid. (B) MAML-EGFP or dnMAML-EGFP stabilized the interactions between the Notch RAM mutants and RBPjκ. *P < 0.01, **P < 0.0001.

Although the Notch-LCI reporter studies performed with RAM mutants and RAM peptide competition confirm the importance of the RAM domain in mediating NICD-RBPjκ interactions, NICDΔRAM molecules can activate reporters or downstream target genes in cells with abundant amounts of Mastermind proteins (32), which act to stabilize the ANK-RBPjκ interface. The peptide fragment corresponding to amino acids 13 to 74 of MAML1, which acts as a dominant-negative MAML (dnMAML), is sufficient to stabilize the interaction between the ANK domain of NICD and RBPjκ in vitro and to prevent MAML docking and subsequent target gene expression (33) (fig. S4B). Therefore, we used our LCI reporters to examine whether dnMAML stabilized ANK-RBPjκ interactions in live cells. Coexpression of MAML or dnMAML fused to enhanced green fluorescent protein (EGFP), MAML-EGFP and dnMAML-EGFP, rescued the interaction between NICDΔRAM-NLuc and CLuc-RBPjκ (Fig. 3B). To assess the specificity of these interactions, we tested whether the stabilizing effects of dnMAML-EGFP extended to other RBPjκ partners. The viral protein EBNA2 interacts with RBPjκ to activate transcription of Epstein-Barr virus genes as well as Notch-responsive reporter genes (34) (fig. S4A). Like NICD, EBNA2 depends on a ΦWΦP motif for its interactions with RBPjκ (35), as evidenced by the reduced complementation between CLuc-RBPjκ and EBNA2WW>SR-NLuc (fig. S4C). However, EBNA2 lacks an ANK domain and is not expected to interact with MAML. Indeed, dnMAML-EGFP failed to promote EBNA2WW>SR-NLuc/CLuc-RBPjκ complementation and, as predicted, did not have a significant inhibitory effect on EBNA2-mediated transcriptional activation (fig. S4, C and D). Together, LCI accurately reported in live cells the predicted behavior of NICD-RBPjκ complex formation based on known Notch structure and biochemical analyses of functional domains.

We also showed that the Notch-LCI reporter system can monitor ternary complex formation among MAML, NICD, and RBPjκ in live cells (fig. S4E). For example, we showed that a MAML-CLuc fusion only produced robust complementation when expressed with the NΔE-NLuc fusion and not when expressed with the NΔEWFP>LAA-NLuc fusion, which cannot interact with RBPjκ (fig. S4F). This demonstrated that the LCI system accurately reported the RBPjκ-dependent interaction between MAML and NICD.

We used Notch-LCI to identify the cellular compartment in which NICD and RBPjκ interacted. Once released, NICD translocates to the nucleus, but some have reported that RBPjκ can be found both in the cytoplasm and in the nucleus (36, 37). We fractionated cells expressing either luciferase alone or NΔE-NLuc and CLuc-RBPjκ together and assayed for luciferase activities in the cytoplasmic and nuclear fractions (Fig. 4A). Whereas the luciferase-expressing cells showed almost equivalent luciferase activity in both fractions; the NΔE-NLuc/CLuc-RBPjκ–expressing cells displayed substantial luciferase activity only in the nuclear fraction. This subcellular localization was also demonstrated by cellular bioluminescence imaging (Fig. 4B) in which full-length luciferase signal was observed throughout the cell and the NΔE/RBPjκ LCI reporter signal was limited to the nucleus, confirming that luciferase complementation occurred after NICD entered the nucleus.

Fig. 4

Subcellular location of Notch-NLuc/CLuc-RBPjκ complementation. (A) Subcellular fractionation indicated that NΔE-NLuc/CLuc-RBPjκ complementation occurs in the nucleus (Nuc), whereas full-length luciferase was detected in both the cytoplasm (Cyto) and the nucleus. (B) Cellular bioluminescence imaging is consistent with the subcellular fractionation data. Cellular bioluminescence images were processed with ImageJ to optimize the brightness and contrast and to despeckle, which reduces the salt-and-pepper noise in the background.

NICD half-life unaffected by the fusion protein

The results above establish LCI as a viable alternative or complementary approach to biochemical assays for quantifying protein-protein interactions in live cells. However, these studies did not eliminate the possibility that the addition of luciferase fragments altered the half-life of Notch proteins. To assess half-life, we monitored the stability of the NICD-RBPjκ complex in real time with a HeLaTetON stable line expressing NΔE-NLuc and CLuc-RBPjκ (clonal cell line E6; this and additional lines are fully described in fig. S5). We monitored luciferase complementation activity in live NΔE-NLuc/CLuc-RBPjκ–expressing cells continuously for 6 hours in the presence or absence of the protein translation inhibitor cycloheximide (CHX) (Fig. 5A). The decay in bioluminescence indicated an NICD half-life of ~180 min, consistent with the half-life obtained by pulse-chase experiments in HeLa cells (38). As expected, inhibition of the proteasome with MG132, MG262, or epoxomicin stabilized the NICD-RBPjκ complex (Fig. 5A) and the NICD protein (Fig. 5B). The similarity between NICD half-life determined biochemically and by LCI indicated that the fusion protein did not alter the stability of the NICD-RBPjκ complexes, and confirmed that the stability of the complex was determined by the half-life of NICD. Therefore, the Notch-LCI system should enable direct and precise quantitation of both on-rates and off-rates of receptor activation and allow dynamic analyses of receptor-proximal events regulating NICD release and turnover with better temporal resolution over transcription-based reporters.

Fig. 5

Monitoring NICD-RBPjκ complex stability with LCI. (A) Stable lines expressing NΔE-NLuc and CLuc-RBPjκ were treated with CHX to block translation in the presence or absence of the indicated proteasome inhibitors, and the degradation of the NICD-RBPjκ complex was monitored by LCI in real time. The half-life based on the luciferase complementation activity is 180 min. (B) Image and Western analyses of cells at 6 hours. Luciferase complementation (degradation and stabilization) correlated with NICD-NLuc protein (detected with V1744), confirming that NICD stability determines the NICD-RBPjκ complex half-life.

Dynamics of γ-secretase–mediated cleavage of Notch and its response to γ-secretase inhibitors

To test the use of the Notch-LCI system to assess modulators of events upstream of the nuclear interactions, we monitored the dynamics of NICD release from NΔE-NLuc, a constitutively active form of Notch that is ligand-independent but γ-secretase proteolysis–dependent (Figs. 1C and 2A). We found that luciferase complementation and NICD accumulation were inhibited in a time- and dose-dependent manner by DAPT, a γ-secretase inhibitor (GSI) (Fig. 6, A and B), whereas complementation between the NICD-NLuc/CLuc-RBPjκ pair, which does not require cleavage, was unaffected (fig. S6). Furthermore, we observed an excellent correlation (R2 = 0.995) between the amount of NICD measured by Western analyses with V1744 and by luciferase complementation with RBPjκ (Fig. 6B), providing further evidence that Notch-LCI is an effective reporter for quantifying the amount of NICD. To further demonstrate the utility of Notch-LCI in monitoring the effectiveness of GSIs, we conducted dose-response analyses of a blinded set of three of these inhibitors on the NΔE-NLuc/CLuc-RBPjκ stable line. The LCI-derived IC50 (half-maximal inhibitory concentration) values correctly ordered the GSIs on the basis of efficacy and distinguished active from inactive enantiomers (fig. S6) (39).

Fig. 6

LCI as a real-time reporter of the dynamics of γ-secretase activity and inhibition in live cells. (A) NΔE/RBPjκ LCI reporter cells treated with different concentrations of the GSI DAPT exhibited a time- and dose-dependent decrease in complementation activity. (B) Complementation activity correlated well with the amount of cleavage product NICD determined by Western blots. (C) Kinetics of recovery after removal of inhibitors: All GSIs tested were confirmed to act in a reversible manner with slight differences in recovery half-times. (D) The robustness of the NΔE/RBPjκ LCI stable inducible reporter cells indicates their usefulness for HTS applications. Multiple 96-well plates were seeded, induced with doxycycline (DOX), treated with DMSO or DAPT (indicated as Compound), and assayed for bioluminescence and viability. Z′ factors obtained in these experiments were >0.5. (E) Western analyses were performed to confirm the knockdown effects of siRNAs targeting GL3 luciferase (siGL3 Luc) or nicastrin (siNCSTN). The reduction in NICD with siGL3 Luc indicates efficient targeting of Notch-NLuc. The reduction in NCSTN and the presenilin 1 N-terminal fragments (PS1 NTF) indicates effective knockdown of mature γ-secretase complexes. (F) Luciferase complementation activity was diminished with siGL3 Luc but not with siNCSTN. (G) siNCSTN sensitizes cells to subinhibitory concentrations of DAPT.

Our results showed that LCI differed from transcriptional reporter assays by enabling the kinetic analysis of GSI effects with precise temporal resolution (Fig. 6A), which permits determination of pharmacodynamic properties of GSIs in live cells. Therefore, we performed a kinetic study of different GSIs at a single effective dose with the NΔE-NLuc/CLuc-RBPjκ reporter cells. The time course for γ-secretase inhibition yielded similar half-times (t1/2s) of ~120 min for DAPT, Compound E, and L685,458 (Fig. 6C) and compared favorably with the in vivo data for inhibition of the production of amyloid β (Aβ) (40), a peptide implicated in Alzheimer’s disease that is produced by γ-secretase cleavage of the amyloid precursor protein. Maximal inhibition was observed at 6 hours and was maintained after an overnight incubation (16 hours). We next tested the kinetics of recovery after release from GSI treatment. Although the inhibition kinetics were similar, LCI differentiated recovery t1/2 values from the different GSIs: 30 min for DAPT, 90 min for Compound E, and 120 min for L685,458 (Fig. 6C). Maximal bioluminescence after recovery exceeded the bioluminescence intensity in cells treated only with dimethyl sulfoxide (DMSO), most likely due to accumulation of the NΔE substrate (20).

Precise, real-time quantitation made possible by this LCI reporter should facilitate the screening of compounds for desired properties (such as long-acting or Notch-sparing GSIs). To evaluate the HTS readiness of the Notch-LCI reporter cell lines, we performed large-scale experiments to configure the assay for HTS and to determine the Z′ factor with DAPT as the model compound. The Z′ factor is a statistical measure of assay robustness; a Z′ factor value ≥0.5 is considered an indication of an excellent assay for HTS (41). Multiple 96- and 384-well plates were seeded with NΔE-NLuc/CLuc-RBPjκ reporter cells. After 24 hours, cells were treated with DMSO alone or with different concentrations of DAPT. Cells were then assayed the next day for bioluminescence and viability (with Alamar Blue) (Fig. 6D). The assay was readily automated and highly reproducible with Z′ factors from multiple experiments in the range of 0.60 to 0.85, indicative of a robust HTS-ready cell line.

Loss-of-function genetic screens using RNA interference (RNAi) represent an approach to identifying previously unknown pathway modulators. To validate the NΔE-NLuc/CLuc-RBPjκ reporter for RNAi-based applications, we used RNAi to target the γ-secretase enzyme. γ-Secretase is a multiprotein complex composed of four proteins: the catalytic component presenilin (PS) and its three cofactors nicastrin (NCSTN), Pen2, and Aph1. We specifically targeted the protein NCSTN because it is encoded by a single gene (there are two PS homologs and two Aph1 isoforms), and it has been shown to exhibit some dosage sensitivity (42). Knockdown with NCSTN small interfering RNA (siRNA) was highly efficient; Western analyses confirmed the reduction in endogenous NCSTN protein (Fig. 6E). The amount of endogenous mature γ-secretase complexes was also reduced, as indicated by the reduction in PS1 N-terminal fragment (PS1 NTF) (Fig. 6E). This reflects the importance of NCSTN in PS stabilization and maturation of the γ-secretase complex (43, 44). Despite effective knockdown of mature γ-secretase, we failed to detect a reduction in NΔE cleavage by LCI (Fig. 6F) or in NICD production by Western analyses (Fig. 6E). This may occur if γ-secretase activity was not limiting in these cells: The few surviving γ-secretase complexes would be sufficient to cleave the substrate. To test this hypothesis, we asked whether cells treated with siNCSTN displayed a greater sensitivity to GSI. Indeed, NCSTN RNAi sensitized the NΔE-NLuc/CLuc-RBPjκ reporter cells to subinhibitory amounts of DAPT (note shift in the inhibition curve in Fig. 6G) and reduced the IC50 for DAPT fivefold (based on nonlinear regression analysis). This result is consistent with the notion that γ-secretase amounts may not be limiting for Notch cleavage in some tissues and siRNA manipulation may, on its own, be insufficient to generate a phenotype. Therefore, adding a subinhibitory amount of a pharmaceutical inhibitor can serve as a chemical genetic screening paradigm for identifying cellular activities that could modulate desired aspects of Notch signaling. To validate the robustness of this assay for high-throughput siRNA screening, we seeded multiple 96-well plates with NΔE-NLuc/CLuc-RBPjκ reporter cells and transfected them with siCONTROL or siNCSTN followed by treatment with a subinhibitory DAPT concentration (10 nM). We obtained Z′ factors of 0.50 to 0.59 in multiple experiments, indicating that the NΔE-NLuc/CLuc-RBPjκ reporter line is suitable for both small-molecule and RNAi HTS applications.

Dynamics of ligand-independent activation of NotchFL receptors

To test the sensitivity of the LCI assay for monitoring activation of NotchFL receptors, we determined the kinetics of Notch activation in response to calcium chelators in a NotchFL-NLuc/CLuc-RBPjκ stable line (line FL2, fig. S5). The NRR domain depends on calcium coordination for its structural integrity; calcium chelation disrupts the NRR structure, thereby promoting receptor proteolysis and subsequent NICD release through a mechanism that is independent of ligand (1, 2, 45, 46). With LCI, we monitored the kinetics of Notch activation in response to three calcium chelators: EDTA, BAPTA, and EGTA (Fig. 7 and fig. S7). Addition of calcium or a calcium chelator to the NICD-NLuc/CLuc-RBPjκ reporter cells led to a mild decrease in bioluminescence, which was likely due to the general effects of such treatments on various cellular processes (Fig. 7A and fig. S7A). However, despite these nonspecific effects, calcium chelator treatment of NotchFL-NLuc/CLuc-RBPjκ reporter cells resulted in an increase in photon flux within 10 min (Fig. 7A and fig. S7A), which is consistent with the appearance of dissociated NECD in the medium after EDTA treatment of NotchFL-expressing cells as has been previously reported (45). Bioluminescence increased at a fixed rate for 60 min (Fig. 7A and fig. S7A), indicating that nuclear translocation occurs rapidly, and this response correlated well with NICD accumulation detected by Western blots (Fig. 7B). Indeed, a similar linear increase was observed with a Notch-GFP nuclear translocation assay in EGTA-treated fly S2 cells (16).

Fig. 7

Real-time imaging of the dynamics of ligand-independent activation by the Ca2+ chelator EGTA and the phorbol ester PMA. (A) EGTA treatment resulted in a linear time-dependent increase in bioluminescence from the NotchFL LCI reporter line, but not from the constitutively active NICD LCI reporter line, that was inhibited by BB94 or DAPT. (B) The increase in bioluminescence (fold activation) correlated with the appearance and accumulation of the NICD cleavage product (see fig. S7B for additional Western analyses). (C) LCI reports activation in the presence of actinomycin D (ActD) (1 μg/ml) to inhibit transcription or CHX (10 μg/ml) to inhibit translation. (D) Temporal analysis of the activation of Notch by different concentrations of EGTA was performed by LCI. (E) Dose-response analyses with EGTA as detected by LCI revealed that complete activation occurred within a narrow EGTA concentration range. (F) NotchFL/RBPjκ and NLNGCC>SS/RBPjκ LCI reporters have differential sensitivity to PMA, a PKC activator that induces ADAM protease activity. Wild-type NotchFL molecules are resistant to PMA treatment. (G) In contrast, NLNGCC>SS, which contains destabilizing amino acid substitutions in the NRR domain, exhibits higher basal activity (Fig. 2, B to D) and can be stimulated by PMA with kinetics that can be monitored by LCI in real time. The specificity of this response was confirmed with DAPT and BB94.

To test for dependence on proteolytic cleavage of NotchFL, we performed the chelator treatment in the presence of an ADAM inhibitor [BB94, blocks S2 cleavage (47, 48)] or the GSI DAPT. Both abrogated luciferase complementation and NICD production (Fig. 7, A and B, and fig. S7, A and B). Together, these results confirmed that calcium chelation increased bioluminescence by triggering proteolysis of NotchFL. The chelation-induced increase in bioluminescence was observed in the presence of transcription or translation inhibitors, actinomycin D (ActD) or CHX, respectively (Fig. 7C), demonstrating the real-time nature of the Notch-LCI reporter. The ability to perform kinetic analysis with improved temporal resolution distinguishes LCI from conventional transcriptional reporter assays, which usually require hours, to assess downstream target transcriptional reporter activity. Using the NotchFL LCI reporter system, we performed detailed quantitative analyses of activation at different EGTA concentrations (Fig. 7D). We show that EGTA-dependent activation behaves as a digital switch, with Notch existing in either an inactive or a fully activated state within a narrow concentration range (5 to 20 μM; Fig. 7E).

The activating effects of calcium chelation on NotchFL reflect the importance of NRR integrity in keeping Notch in its autoinhibited state and resistant to ADAM proteolysis in the absence of ligand. Phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), activate protein kinase C (PKC), which stimulates ADAM protease activity (48). Consistent with previous studies (49, 50), PMA had no effect on wild-type NotchFL-NLuc/CLuc-RBPjκ basal luminescence because the intact NRR prevents access to ADAM proteases (Fig. 7F). Mutations that disrupt the NRR can cause T cell acute lymphoblastic leukemia (T-ALL) in humans (51) due to “leaky” Notch signaling. Consistent with this, NLNGCC>SS molecules, which carry destabilizing amino acid substitutions in the NRR domain, release a small amount of NICD protein (Fig. 2B) and exhibit a slightly increased ligand-independent basal activity in transcriptional reporter and LCI assays (Fig. 2, C and D). This basal activity should be further induced by treatment with PMA, which increases the activity of the ADAM proteases (48). We measured the kinetics of this process by monitoring NLNGCC>SS-NLuc/CLuc-RBPjκ LCI (as an indicator of NICD release) upon addition of PMA. In contrast to NotchFL, NLNGCC>SS complementation activity was stimulated by PMA (Fig. 7G). The involvement of protease cleavage in the PMA-stimulated activation was confirmed by BB94 and DAPT inhibitor treatment. Thus, the Notch-LCI assay is an effective method for performing structure-function analyses on the dynamics of NRR unfolding in live cells.

Dynamics of ligand-dependent activation of NotchFL receptors

To show that NotchFL-LCI can be used to characterize ligand-dependent activation in real time in live cells, we monitored bioluminescence in cells coexpressing NotchFL-NLuc and CLuc-RBPjκ cocultured with Chinese hamster ovary (CHO) cells expressing either the ligand Dll1 or Jag1 (CHO-Dll1 or CHO-Jag1, respectively). We observed a significant activation of the reporter upon coculturing with CHO-Dll1 or CHO-Jag1 cells (Fig. 8A), which was significantly inhibited by DAPT (Fig. 8B). Coculture with ligand-expressing cells did not affect the activity of the constitutively active NICD-NLuc/CLuc-RBPjκ reporter cells (Fig. 8A), further demonstrating the specificity of the LCI reporter activity. We also performed similar experiments with cells coexpressing the MAML-CLuc and RBPjκ-NLuc reporters and showed ligand-dependent activation of their complementation activity (fig. S8).

Fig. 8

Real-time imaging of the dynamics of Notch activation by ligands presented in various paradigms. (A) Coculture with either CHO-Dll1 or CHO-Jag1 stable cell lines activated NotchFL but did not affect the constitutively active NICD. Photon flux was normalized to the bioluminescence with CHO cell coculture. (B) DAPT reduced activation of NotchFL. (C) Coexpression of the glycosyltransferase Lunatic Fringe (LFNG) enhanced activation by Dll1 and reduced responsiveness to Jag1. (D) Increasing concentrations of extracellular calcium increased Notch activation in response to ligands presented on cocultured cells. (E) Immobilized ligands activate the NotchFL LCI reporter in a dose-dependent manner. (F) LCI analysis of the kinetics of NICD accumulation from NotchFL after DAPT removal mimicked the kinetics of NICD accumulation from NΔE after DAPT removal (Fig. 6C). Statistical analyses were performed to assess significant differences from the DMSO control at each time point. (G) LCI reported the kinetics of Notch activation with Dll1-Fc–containing conditioned medium preclustered with different concentrations of antibody against Fc. (H) The response to clustered ligand was specific to activation of full-length Notch and was blocked if cleavage was inhibited with DAPT or BB94. Clustered ligand did not alter the bioluminescence in the NICD reporter cells. (I) Western analysis indicated that NotchFL activation by clustered ligand correlated with the appearance of NICD. (J) The clustered ligand-dependent activation paradigm was validated for HTS applications. *P < 0.001, **P < 0.0001.

Because the assay detected ligand-induced activation, we assessed the effect of known modifiers of ligand-Notch interactions. The Fringe family of glycosyltransferases can modify specific EGF repeats of Notch to modulate their responsiveness to ligands in specific contexts (52). Consistent with previous studies (53), overexpression of Lunatic Fringe (LFNG) potentiated the response of the NotchFL LCI reporter to Dll1 but reduced the response to Jag1 (Fig. 8C). Notch has also been suggested to act as a sensor for changes in extracellular calcium concentrations during the establishment of the left-right axis (54). To examine the effects of extracellular calcium on receptor activation, we cocultured NotchFL LCI reporter cells with CHO-ligand stable lines under varying concentrations of CaCl2 (0 to 5.4 mM) (Fig. 8D). We observed a dose-dependent increase in fold activation by either ligand with increasing CaCl2. Thus, extracellular calcium concentration can regulate Notch signal strength.

The mammalian Notch pathway is unique in that it lacks soluble ligands. This presents challenges for biochemical studies on ligand-receptor interactions and for measuring activation rates with ligands in coculture-based systems. To overcome these limitations, other methods for presenting Notch ligands in trans have been developed. For example, ligand extracellular domains fused to immunoglobulin G (IgG) or Fc fragments can be either immobilized onto culture plates before cell seeding (55) or preclustered with antibodies against IgG or Fc and subsequently applied to cells (56). Both methods are biochemically similar to ligands presented by neighboring cells (50) and can lead to biological changes in Notch signal–receiving cells in culture and in vivo (57). Typically, in kinetic studies with immobilized ligands, the Notch-expressing cells need to be seeded onto the immobilized ligands in the presence of GSIs, and then Notch signaling is monitored after the cells are released from inhibition. In contrast, studies with the antibody-clustered ligands do not require preinhibition of Notch proteolysis; instead, Notch signaling can be activated by adding the ligands to the cells.

Studies measuring the kinetics of Notch activation by immobilized ligands using transcription-based reporters (12XCSL-GFP) determined response times to be greater than 10 hours, a reflection of transcription, translation, and GFP folding and maturation (12). Notably, because these activation rates were measured after release from DAPT inhibition, the apparent on-rate for ligand activation may not be accurate because S2-cleaved (NΔE-like) fragments would accumulate under these conditions and mimic the kinetics of NICD release from NΔE molecules after DAPT removal (Fig. 6C). We used the NotchFL LCI reporter to perform a dose-response and kinetic study with immobilized ligands. NotchFL LCI reporter cells exhibited a dose-dependent response to purified Dll1-IgG (0 to 2 μg) immobilized onto the cell culture plates, with a linear increase (R2 = 0.958) that plateaued thereafter (Fig. 8E). We observed a similar dose-dependent response to Dll1-Fc (from conditioned media) immobilized with antibodies recognizing the Fc portion (fig. S9). After establishing that we can quantify Notch activation by immobilized ligands by LCI, we tested the activation kinetics after release from DAPT. In contrast to the slower kinetics of GFP transcriptional reporters, we detected Notch activation equivalent to that of the DMSO-treated control cells within 1 hour of release from DAPT. After another 1 to 2 hours, the Notch activity in cells released from DAPT exceeded those of control (Fig. 8F), which is similar to what we observed with NΔE after DAPT release (Fig. 6C). By 24 hours, luciferase complementation reached a steady state. These data imply that therapeutic regimens involving GSIs will potentially include an acute spike in Notch activity after withdrawal from GSI, especially in tissue contexts where ligands are abundant.

Notch activation kinetics can also be measured with preclustered ligand-Fc fusion proteins, which allow the investigator to control the start time (t = 0) for ligand addition without using pathway inhibitors. We monitored NotchFL activation in real time with LCI after the addition of Dll1-Fc–containing conditioned media that had been preclustered with different amounts of antibody recognizing the Fc portion of the fusion protein (Fig. 8G). We observed a modest increase in bioluminescence in response to unclustered Dll1-Fc conditioned media, but we found that increasing concentrations of the clustering antibody concentrations increased Notch activation, reaching maximal bioluminescence 4 hours after ligand addition with the highest concentration of antibody tested. This response to clustered ligand was independent from transcription and translation (fig. S10). Clustered ligand also failed to stimulate the NICD/RBPjκ LCI reporter cells (Fig. 8H). The response to clustered ligand was blocked by protease inhibition with BB94 or DAPT in a dose-dependent manner (Fig. 8H and fig. S11, A and B) and correlated with Western analyses of NICD production (Fig. 8I).

Coupled with immobilized or clustered ligand presentation, the NotchFL-LCI reporters have expanded the use of the assay system for quantitatively monitoring signaling events upstream of nuclear interactions and γ-secretase cleavage. To illustrate this further, we conducted detailed dose-response analyses of several metalloprotease inhibitors (BB94, GM6001, and TAPI-2) in live cells by LCI (fig. S11, C and D) and found that their relative effectiveness was similar to that reported for their effects on Notch processing previously assayed by Western blots or transcriptional reporter assays (48, 58). The NotchFL-LCI system could potentially facilitate the identification and characterization of new modifiers of Notch activation by HTS. Thus, we conducted large-scale, clustered ligand-dependent LCI activation experiments and validated the assay for small-molecule screening (Z′ factors ≥0.5) (Fig. 8J).

Discussion

Our studies established LCI to be a specific, robust, flexible, and biologically relevant method for probing the mechanism and regulation of Notch activation in real time in live cells. We demonstrated that the Notch-LCI system satisfies the key validation criteria for a PCA study (18): (i) Robust signal was observed only with relevant interacting fusions (NICD and RBPjκ), but not with nonspecific proteins. (ii) Mutations or polypeptides expected to inhibit (for example, RAM domain mutations and polypeptides) or modulate (for example, ANK mutations, MAML-mediated stabilization) the protein interaction had the corresponding effects on complementation. (iii) Specific and robust complementation was also observed with luciferase fragment swapping. Moreover, the Notch-LCI system recapitulated known biochemical and regulatory profiles of NICD-RBPjκ complex formation, including subcellular location, half-life, domain contribution, dependence on ligand and proteolysis, and sensitivity to known pharmacological agents and modifiers.

The Notch-LCI reporter can be used in several different modes to address mechanistic questions regarding Notch signaling. One mode uses LCI for monitoring productive protein-protein interactions relevant to the pathway, with the capacity for performing rapid structure-function analyses in intact cells. Although most of our studies focused on NICD-RBPjκ interactions, the LCI reporter is also amenable to monitoring ternary complex formation, which we demonstrated for the RBPjκ-dependent interaction between NICD and MAML and the NICD-dependent interaction between MAML and RBPjκ. Moreover, the Notch-LCI reporter system can be adapted to probe interactions involving other NICD paralogs (Notch2, 3, and 4) and, as we demonstrated with EBNA2, to analyze other “noncanonical” interactions and address crosstalk and signal integration with other pathways in real time. The development of dual-color luciferase complementation systems (59, 60) should enable simultaneous monitoring of multiple protein interactions in the same cell.

A second mode uses LCI as an enzymatic reporter for nuclear translocation of NICD. As a protein translocation assay, optimized LCI offers advantages over other enzymatic reporters based on β-galactosidase fragment complementation (61, 62) or split intein-mediated Renilla luciferase (63). β-Galactosidase is monitored in lysates or in live cells by flow cytometry, making it less applicable as a real-time readout for protein translocation. Although Renilla luciferase activity can be imaged noninvasively, the intein-mediated complementation exhibits slower kinetic responses and is irreversible. Our analyses of the kinetics of NICD release (from NΔE and NotchFL receptors) and complementation with RBPjκ indicate that nuclear translocation occurs rapidly, with kinetics similar to those of nuclear translocation assays with Notch-GFP. The sensitivity and real-time quantitative nature of the LCI assay should facilitate analyses of the potential factors that influence the nuclear translocation process, an aspect that has not been explored extensively for the Notch pathway.

A major use for Notch-LCI is based on its ability to quantitatively report on the amount NICD-RBPjκ complexes in live cells with detailed temporal resolution. Because RBPjκ was generally unaffected by the stimuli tested, luciferase complementation highly correlated with NICD amounts and can, therefore, replace labor-intensive Western analyses while providing more quantitative information on the net amount of active complexes. Using this LCI mode, we monitored the dynamics of NICD release and accumulation under different conditions (such as those that altered protein degradation and stabilization and in response to ADAM inhibition and to γ-secretase inhibition and recovery). We compared receptor activation kinetics in response to different agonists and examined the effects of modifiers of signal strength. The ability to obtain precise temporal information can facilitate efforts in mathematically modeling pathway dynamics, especially when modifiers, target promoters, and feedback loops are added to the system.

Finally, the Notch-LCI system is a versatile tool for identifying and characterizing modulators that can target different aspects of the signal transduction pathway. We validated Notch-LCI reporter lines for HTS applications and established their usefulness in providing pharmacodynamic information and analyzing the mechanism of action of compounds that modulate the pathway. The identification of paralog-specific agents would be useful for manipulating the Notch signaling pathway in cancer, inherited diseases, stem cell differentiation, and tissue engineering for both research and potential clinical applications.

Materials and Methods

DNA constructs

To generate the various Notch1-NLuc and CLuc-RBPjκ expression plasmids, we replaced the cdc25 and 14-3-3 coding regions within the cdc25-NLuc and CLuc–14-3-3 constructs [a gift from H. Piwnica-Worms (17)], respectively, by subcloning from various established mouse Notch1 and human RBPjκ constructs (20, 22, 27). CLuc-dnRBPjκ, R178H, which is equivalent to the previously described R218H substitution in mouse RBPjκ (64), and NotchWFP>LAA-NLuc mutant constructs were generated by mutagenesis with the QuikChange kit (Stratagene) following the manufacturer’s protocols. Untagged counterparts for all Notch and RBPjκ variants were prepared by replacing the Luc coding regions with oligonucleotide linkers. For stable line generation, we created Tet-inducible pBI dual expression vectors (Clontech) carrying CLuc-RBPjκ and either NotchFL-, NΔE-, or NICD-NLuc. EBNA2-NLuc constructs were generated by subcloning from pSG5-EBNA2 and pSG5-EBNA2WW>SR expression plasmids (a gift from D. Hayward) into the NICD-NLuc plasmids. The CLuc control plasmid has been previously described (17). nls-CLuc was prepared by inserting an oligonucleotide carrying the SV40 (simian virus 40) nuclear localization signal coding sequence downstream of CLuc. SV40-GL3 luciferase (Promega) was used as a control expression plasmid; the NLuc and CLuc complementing pairs used in this study were originally derived from this luciferase (17).

To generate the RAM polypeptide expression plasmids, we amplified the RAM regions [amino acids 1748 to 1872, which are similar to RAM polypeptides described previously (30)] from wild-type and WFP>LAA mutant Notch-NLuc plasmids and subcloned the polymerase chain reaction (PCR) fragments into pCS2+ 6MT. The dnMAML (amino acids 12 to 74)–EGFP was generated by subcloning from MSCV-MAML1(12–74)-EGFP (a gift from W. Pear and J. Aster). The pcDNA3-MAML-EGFP and CLuc-MAML expression plasmids were prepared by subcloning from the MAML1 complementary DNA (cDNA) plasmid (a gift from J. Griffin).

To generate the pcDNA4-Dll1-Fc expression construct, we replaced the B7 coding region within the B7-IgG vector (65) (a gift from K. Murphy) with that of the mouse Dll1 ECD. The resulting construct encodes amino acids 1 to 535 of mouse Dll1 fused to the CH2-CH3 domain of mouse IgG1 followed by a Myc-6His (hexahistidine) tag. The corresponding control pcDNA4-Fc expression vector was also prepared by replacing B7 with the signal sequence of mouse Dll1 (amino acids 1 to 25).

The TP1-luciferase reporter construct (pGa981-6) was a gift from T. Honjo. Hes1-luciferase and Hes5-luciferase were gifts from A. Israel and R. Kageyama, respectively. CS2+ LFNG was prepared by subcloning the LFNG cDNA from pSPUTK-LFNG (a gift from S. Egan) into the CS2+ vector.

Cell lines, cell culture, and plasmid transfections

3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% bovine calf serum (BCS). OT11 (RBPjκ−/−; a gift from T. Honjo), 293T, and CHO-fN2 (a gift from S. Chiba and H. Hirai) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). HeLaTetON parental cells (Clontech) and stable line derivatives (E6, IC1, III3, and FL2 lines; see fig. S5 for details) were cultured in DMEM supplemented with 10% Tet-approved FBS. The CHO-GFP, CHO-Dll1-IRES-GFP, and CHO-Jag1-IRES-GFP stable lines have been previously described (66) and were cultured in IMDM (Iscove’s modified Dulbecco’s medium) supplemented with 10% FBS. All cell lines were maintained at 37°C in a humidified atmosphere with 5% CO2.

3T3 cells were transfected using calcium phosphate with BES (N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid)–buffered saline (pH 6.8). 293T cells were transfected with FuGENE 6 (Roche), the OT11 cells with Lipofectamine LTX (Invitrogen), and the CHO-fN2, HeLaTetON parental, and stable cell lines with Lipofectamine 2000 (Invitrogen), all according to the manufacturer’s recommended protocols.

To generate Fc or Dll1-Fc conditioned media, 293T cells were seeded in P100 dishes and transfected the next day with pcDNA4-Fc or pcDNA4-Dll1-Fc expression plasmid using FuGENE 6. After 24 hours, the cells were provided with fresh 10% FBS-Tet/DMEM without phenol red (appropriate for imaging experiments) and incubated for another 48 hours. Conditioned media were collected, filter-sterilized, and stored at 4°C. Production and secretion of the Fc and Dll1-Fc fusion proteins were confirmed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Western analyses with antibodies against Myc or His as appropriate. To precluster the Dll1-Fc ligand, we incubated conditioned medium with antibodies recognizing mouse Fc (Jackson Immunoresearch) for at least 1 hour and equilibrated the medium to 37°C before applying to reporter cells. To immobilize Dll1-Fc from conditioned medium, we initially adsorbed affinity-purified antibodies recognizing mouse Fc [Jackson Immunoresearch; diluted in phosphate-buffered saline (PBS)] onto culture plates for 2 hours, aspirated the antibody solution, and then incubated the plates with conditioned medium for another 2 hours before cell seeding.

RNAi reagents and methods

siRNAs were transfected into HeLaTetON stable lines E6, IC1, and III3 with Dharmafect3 (Dharmacon) according to the manufacturer’s protocols. All siRNAs used in this study were purchased from Dharmacon: Nontargeting siRNA (siCONTROL) was used as a negative control; an siRNA targeting GL3 luciferase (siGL3 Luc) was used for optimizing transfection conditions and as a positive control for knockdown in our reporter lines; the siGENOME SMARTpool (siNCSTN) was used to target NCSTN. Knockdown efficiency was assessed by bioluminescence assays and Western analyses.

Chemicals and reagents

Doxycycline (DOX), epoxomicin, CHX, EGTA, PMA, ActD, MG132, and human IgG (hIgG) were all purchased from Sigma. DAPT, BAPTA, MG262, GM6001, TAPI-2, and L685,458 were purchased from Calbiochem/EMD Chemicals. MRK-003 and MRK-006 were gifts from Merck. BB94 was a gift from M. Freeman. Compound E was a gift from T. Golde. Purified Dll1-IgG was a gift from I. Bernstein.

Western analyses

Cells were washed once with PBS, lysed in Laemmli buffer, and analyzed by SDS-PAGE followed by Western blotting with antibodies recognizing Notch1 [mN1A ascites (24)], activated Notch1 or NICD [cleaved Notch1 (V1744); Cell Signaling], RBPjκ (CosmoBio), β-actin (AC-15; Sigma), NCSTN (N-19; Santa Cruz Biotechnology), PS1 (H-70; Santa Cruz Biotechnology), Myc (9E10 ascites), and His (Origene).

Bioluminescence assays

Cells were seeded (and transfected, if needed) in black 96- or 24-well plates. For the HeLaTetON stable lines, cells were seeded in the presence of DOX (0.5 μg/ml) to induce reporter expression. For endpoint LCI assays, cells were imaged in PBS supplemented with 0.1% glucose, 1 mM MgCl2, 0.9 mM CaCl2, and d-luciferin (150 μg/ml; Biosynth) using an IVIS50 or IVIS100 imaging system (Caliper). The following acquisition parameters were used: exposure time, 1 to 5 min; binning, 4 or 8; no filter; f-stop, 1; field of view, 12 or 15 cm. If Renilla luciferase imaging was to be performed on the same cells, the d-luciferin–containing buffer or medium was replaced with buffer containing 400 nM coelenterazine (Biotium) and the cells were imaged with the IVIS system using the following acquisition parameters: exposure time, 1 to 2 min; binning, 4 or 8; filter <510; f-stop, 1; field of view, 12 or 15 cm.

For sequential imaging of receptor activation with Ca2+ chelators, cells were imaged in Hank’s balanced salt solution (HBSS) containing d-luciferin (150 μg/ml). An initial image was obtained (t = 0), after which an equivalent volume of HBSS/d-luciferin solution containing 2× chelator was added and images were obtained every 5 min for 1 hour. For other sequential imaging experiments (for example, those monitoring the kinetics of γ-secretase inhibition and recovery), cells were imaged in phenol red–free culture medium containing d-luciferin in an IVIS system equilibrated with 5% CO2. An initial image was also obtained before addition of an equivalent volume of d-luciferin–containing medium with 2× inhibitor or PMA. For sequential imaging of cells activated by clustered ligand, d-luciferin was directly added to the prewarmed, preclustered conditioned medium before imaging in the IVIS system equilibrated with 5% CO2.

Photon flux (photons per second) was quantified on images with regions of interest (ROIs) and analyzed with Living Image 2.6 (Caliper) and IGOR (WaveMetrics) image analysis software.

Most bioluminescence imaging experiments described herein were performed with an IVIS instrument. However, some bioluminescence assays were also performed with the Envision (1 s per well; enhanced luminescence option; Perkin Elmer) or the Biotek Synergy 2 Microplate reader (1 s per well), both of which were used for high-throughput assay development and yielded similar results and dynamic range as the IVIS, particularly for the stable lines expressing GL3 luciferase or the LCI reporters of activated forms of Notch (NICD, NΔE).

Imaging results were presented either as mean photon flux (photons per second) ± SD or normalized to the appropriate controls for a given experiment (as 1 or 100%): DMSO vehicle (for inhibitor and PMA activation studies), HBSS alone (for Ca2+ chelator activation), Fc conditioned medium or purified hIgG (for Dll1-Fc conditioned medium or purified Dll1-IgG–dependent activation, respectively), or wild-type Notch construct (for assays of mutant protein activity).

Cellular bioluminescence imaging

HeLaTetON stable lines expressing Luc alone or coexpressing NΔE-NLuc and CLuc-RBPjκ were seeded in P35 dishes. The next day, the medium was replaced with prewarmed Hepes-buffered phenol red–free DMEM containing 10% FBS and d-luciferin (150 μg/ml). The cells were then imaged with an Olympus FluoView1000 inverted microscope equipped with an Andor single-photon level, charge-coupled device image-capturing system and housed within a dark-adapted environmental chamber set at 37°C. Bioluminescence images were obtained with the 10× objective with 30-s to 1-min (Luc) or 5- to 10-min (NΔE/RBPjκ LCI) exposure times.

Transcriptional reporter assays

For reporter assays, cells were seeded in 24-well plates and were transfected the next day with TP1-, Hes1-, or Hes5-Luc (Notch-responsive reporter plasmids), CS2+ cyto–β-galactosidase (used for normalization), test untagged, NLuc- or CLuc-tagged construct, and CS2+ vector as carrier DNA (500 ng of total DNA per well; run in quadruplicate). Cells were harvested 48 hours after transfection and assayed for luciferase and β-galactosidase activities. Cells were washed once with PBS and lysed in lysis buffer [0.2% Triton X-100, 100 mM potassium phosphate buffer (pH 7.8), 1 mM dithiothreitol (DTT), and protease inhibitors] for 15 min. Luciferase activity was measured from 50 μl of lysate injected with 50 μl of 2× assay buffer [30 mM tricine (pH 7.8), 3 mM adenosine 5′-triphosphate (ATP), 15 mM MgSO4, 10 mM DTT, 0.2 mM coenzyme A, and 1 mM d-luciferin] using a Tropix TR717 luminometer. In parallel, 5 μl of lysate was used to measure β-galactosidase activity according to the Tropix Galacton Plus chemiluminescence protocols.

Nuclear fractionation

293T cells were transfected with expression constructs for full-length luciferase or a combination of NΔE-NLuc and CLuc-RBPjκ. Forty-eight hours after transfection, cells were lysed and fractionated with the Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. Nuclear and cytoplasmic fractions were then assayed for their luciferase activities (as described above for transcriptional reporter assays) and protein concentration (BCA Assay, Pierce).

LCI assay development for HTS

High-throughput, automated procedures for cell seeding, medium replacement, transfections, and cell-based assays were optimized at the Washington University Chemical Genetics Screening Core, as well as the Molecular Imaging High Throughput Core.

For pilot compound screens, NΔE-NLuc/CLuc-RBPjκ stable lines (E6) were seeded in 96-well plates at 10,000 cells per well (or in 384-well plates at 3000 cells per well) in medium containing DOX (0.5 μg/ml). After 24 hours, medium was replaced with fresh DOX-containing medium containing DMSO or DAPT (as the test compound). After another 24 hours of incubation, cells were incubated for 15 min at room temperature with d-luciferin–containing buffer and measured for bioluminescence with an Envision or Biotek Synergy 2 plate reader (1 s per well). Afterward, the cells were provided with fresh medium containing Alamar Blue [1:10 (v/v)], incubated for 1 hour at 37°C, and measured for fluorescence (excitation 544 nm, emission 590 nm; Biotek Synergy 2 or FluoOptima plate reader).

For pilot siRNA screens, NΔE-NLuc/CLuc-RBPjκ–expressing (E6) cells were seeded in 96-well plates and the next day were transfected with siRNAs (50 nM final concentration) using Dharmafect 3. Forty-eight hours later, the cells were provided with fresh medium containing DOX to induce reporter expression. Twenty-four hours later (72 hours after transfection), the cells were assayed for bioluminescence and viability as with the compound screen pilot experiments.

Statistical analyses

Mean, SD, and R2 values were calculated and plotted with Microsoft Excel. t Tests, tests for normality, and multiple comparison procedures in analysis of variance (ANOVA) were all performed with JMP software (SAS Institute). Significance was assigned where P < 0.05. Nonlinear regression analyses and curve-fitting for inhibition curves were performed with GraphPad Prism. Z′ factors were calculated according to Zhang et al. (41).

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/4/181/rs7/DC1

Fig. S1. Transcriptional activation profile of untagged and CLuc-tagged RBPjκ in RBPjκ-null (OT11) fibroblasts.

Fig. S2. Transcriptional activation profile of untagged and NLuc-tagged Notch receptor variants.

Fig. S3. Effect of DNA binding and Luc fragment domain swapping on the complementation between NΔE and RBPjκ.

Fig. S4. LCI reporting of the involvement of MAML in EBNA2-RBPjκ interactions and ternary complex formation with NICD and RBPjκ.

Fig. S5. Establishment of stable Notch/RBPjκ LCI reporter lines for pathway analyses and high-throughput screening.

Fig. S6. Determining the rank order of potency of representative γ-secretase inhibitors with the NΔE LCI reporter.

Fig. S7. Monitoring ligand-independent activation of Notch by the Ca2+ chelators BAPTA and EDTA using LCI.

Fig. S8. Monitoring the formation of the NICD-RBPjκ-MAML ternary complex after ligand-dependent activation.

Fig. S9. Activation of full-length Notch receptors with Dll1-Fc immobilized with antibodies against Fc.

Fig. S10. Monitoring NotchFL activation by clustered Dll1-Fc with LCI does not depend on downstream transcription and translation.

Fig. S11. Kinetic and dose-response analyses of S2 and S3 cleavage inhibition in NotchFL LCI reporter cells.

References and Notes

  1. Acknowledgments: We would like to thank our colleagues for providing various reagents: J. Aster, I. Bernstein, S. Chiba, S. Egan, M. Freeman, T. Golde, J. Griffin, D. Hayward, H. Hirai, T. Honjo, A. Israel, R. Kageyama, K. Murphy, W. Pear, and H. Piwnica-Worms, as well as Merck. We thank members of the Piwnica-Worms and Kopan laboratories for discussions and technical assistance, especially C. Ong for preparing the Dll1-Fc and control constructs. We thank B. Nolan (Chemical Genetics Screening Core) and J. Marasa (Molecular Imaging Center High Throughput Screening Core) for their assistance in adapting the Notch-LCI assay for automation and HTS. We also thank D. Oakley (Bakewell NeuroImaging Laboratory) for his assistance with the cellular bioluminescence imaging. We acknowledge S. Chen and M. Hass, G. Zhao, and C. Sato for their critical reading of the manuscript. Funding: Support for this work was provided by grants from the NIH: a Neuroscience Blueprint Core Grant P30 NS057105 (Washington University), R21-NS06168001 (M.X.G.I.), AG025973P50 (R.K.), CA94056 (D.P.-W.), and P50 AG005681 (J. Morris). Author contributions: M.X.G.I., D.P.-W., and R.K. conceptualized the project and designed the experiments. M.X.G.I. designed and performed most of the experiments and data analyses. S.L. performed some experiments and analyzed the data. M.F. prepared many of the constructs described herein. M.X.G.I., D.P.-W., and R.K. contributed new reagents and analytical tools. M.X.G.I. and R.K. wrote the paper, with input from all co-authors. Competing interests: M.X.G.I., D.P.-W., R.K., and Washington University may receive income based on a license of Notch-related technology by the university to Merck. Merck did not support this work. A standard academic material transfer agreement (MTA) applies for the Notch-LCI reporter expression plasmids and stable cell lines. D.P.-W. and Washington University hold a patent on the split luciferase (US 7,442,518), but no restrictions apply for academic researchers.
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