Real-time imaging of notch activation with a luciferase complementation-based reporter

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.

Figure 1. The core Notch pathway and the various approaches for monitoring Notch activation

A. Schematic of the canonical Notch pathway: Ligand-binding leads to ectodomain shedding (at the S2 site) by ADAM, followed by intramembrane proteolysis (at S3/S4 sites) by γ-secretase. This releases the Notch intracellular domain (NICD), which translocates to the nucleus, interacts with the DNA-binding protein CSL to recruit Mastermind-like proteins (MAML) and other coactivators to activate gene expression. B. Overview of the current methods for monitoring Notch pathway activation: Left, Antibodies can specifically recognize the amino-terminus of NICD that is exposed only after γ-secretase cleavage; Middle, Overall pathway activity can be monitored by reporter proteins (luciferases or fluorescent proteins) under the control of endogenous target promoters (e.g., Hes) or multimerized CSL-binding sites (e.g., 4XCSL, TP1); Right, Regulated proteolysis of Notch-fusion proteins releases various heterologous proteins that can be monitored via transcriptional reporters or nuclear translocation. 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 allow us to interrogate different mechanistic aspects of Notch activation.

Figure 2. Development and validation of the Notch/RBPjκ LCI reporter

A. Domain organization of the Notch1 receptor and RBPjκ and their respective luciferase fragment fusions used in this study. The different mutations used for assay validation are indicated (red arrows). The full-length receptor (NotchFL) has a large extracellular domain (ECD) composed of EGF repeats and the negative regulatory region (NRR), which can be further subdivided into the Lin-Notch repeats (LNR) and the heterodimerization domain (HD). The NRR keeps the receptor in the ‘off’ state in the absence of ligand. Mature Notch receptors have been furin-processed at the S1 site within HD to generate the N^ECD and N^TMIC (Notch transmembrane and intracellular domain) fragments that are held together by interactions between the N- and C-terminal halves of HD. Following the transmembrane domain (TMD) is a large intracellular region that carries the RAM (RBPjk association module), ANK (7 ankyrin repeats) and PEST (proline/glutamic acid/serine/threonine-rich) domains. NLNG molecules cannot respond to ligand and are completely inactive unless mutations that lead to ligand-independent activation are present (e.g., cc>ss). NΔE and NICD are both constitutively active forms. RBPjκ is composed of the N-terminal domain (NTD), Beta-trefoil domain (BTD) and C-terminal domain (CTD). B. Notch-NLuc proteins were also assessed by Western analyses with the AN1 antibody, which recognizes the ANK domain. Where appropriate, NICD production was also confirmed by the α-V1744 antibody. C. The transactivation profile of the Notch-NLuc receptor variants is consistent with previous studies, with the constitutively active proteins 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. LCI can distinguish the mechanisms of action of the RAM and ANK domain mutants. Cotransfected Renilla Luc (RLuc) activities confirm equivalent transfection efficiency. E. Notch-NLuc/CLuc-RBPjκ complementation is specific: NICD-NLuc produces robust complementation with CLuc-RBPjκ but not with the CLuc alone or nls-CLuc. RAM mutations known to disrupt binding also diminish complementation.

Figure 3. LCI allows direct quantification of the modulation of NICD/RBPjk interactions in live cells

A. Wild-type (but not WFP>LAA-mutant) RAM polypeptides reduced NΔE-NLuc/CLuc-RBPjκ complementation in a dose-dependent manner. G. MAML-EGFP and dnMAML-EGFP stabilize the interactions between the Notch RAM mutants and RBPjκ. * p<0.01, ** p<0.0001.

Figure 4. Notch-NLuc/CLuc-RBPjκ complementation occurs in the nucleus

NΔE-NLuc/CLuc-RBPjκ complementation occurs in the nucleus as demonstrated by subcellular fractionation (A) and cellular bioluminescence imaging (B). This is in contrast to full-length luciferase, which partitions almost equally between the cytoplasmic and nuclear fractions (A) and can be found throughout the cell (B). Cellular bioluminescence images were processed using ImageJ to optimize the brightness and contrast and to reduce the salt-and-pepper noise in the background (i.e., despeckle).

Figure 5. Monitoring NICD/RBPjk complex stability using LCI

A. Stable lines expressing NΔE-NLuc and CLuc-RBPjκ were treated with cycloheximide (CHX) to block translation and the degradation of the NICD/RBPjκ complex was monitored by LCI in real time. The half-life of the luciferase complementation activity (t_1/2=180 min) was similar with the half-life of NICD determined using pulse-chase. Moreover, complementation activity was stabilized by the indicated proteasome inhibitors, consistent with previous studies demonstrating that NICD is ubiquitinated and degraded by the proteasome. B. Image and Western analyses of cells at 6 hr. Luciferase complementation (degradation and stabilization) correlated closely with NICD-NLuc protein (detected by α-V1744 antibody). These studies confirm that the NICD stability determines the NICD/RBPjk complex half-life. Importantly, because the Luc fusion does not alter the stability of NICD, LCI will us to accurately monitor the off-rates of pathway activation.

Figure 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 γ-secretase inhibitor 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 Westerns. 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 NΔE/RBPjκ LCI reporter cells are robust and have been validated for high throughput screening applications. Multiple 96-well plates were seeded and treated with DMSO or DAPT and assayed for bioluminescence and viability. Z’ factors obtained in these experiments were >0.5, indicating that the assay is excellent for HTS. E. Knockdown by siGL3 Luc and siNCSTN was confirmed by Western analyses. siGL3 Luc efficiently targets Notch-NLuc. siNCSTN efficiently reduced NCSTN protein as well as mature γ-secretase complexes, indicated by the reduction in Presenilin1 NTF fragments. F. Luciferase complementation activity was greatly diminished with siGL3 Luc but not with siNCSTN, suggesting that γ-secretase is not limiting in these cells. G. siNCSTN sensitizes cells to sub-inhibitory concentrations of DAPT.

Figure 7. Real-time imaging of the dynamics of ligand-independent activation by the Ca⁺⁺ chelator EGTA

A. EGTA treatment resulted in a linear time-dependent increase in bioluminescence from the NotchFL LCI reporter line (but not from the ligand-independent NICD LCI reporter line, right) that was inhibited by both BB94 and DAPT. B. The increase in bioluminescence (fold activation) correlated with the appearance and accumulation of the NICD cleavage product (see fig. S7 for additional Western analyses). C. Monitoring the activation process by LCI does not depend on downstream transcription and translation as it occurs in the presence of 1µg/ml ActinomycinD (ActD) or 10 µg/ml cycloheximide (CHX), demonstrating the real-time nature of the assay. D, E. Using the LCI reporter, we were able to perform detailed temporal and dose response analyses with EGTA. We demonstrate that complete activation occurs within a narrow EGTA concentration range.

Figure 8. Real-time imaging of the dynamics of Notch activation by ligands presented in various paradigms

A. NotchFL (but not NICD) LCI reporter cells are activated when cocultured with either CHO-Dll1 or CHO-Jag1 stable cell lines. B. The activation is diminished in the presence of DAPT, demonstrating the specificity of the response. C. Coexpression of the glycosyltransferase lunatic Fringe (LFNG) enhances activation by Dll1 but reduces responsiveness to Jag1. D. Notch activation can be regulated by the concentration of extracellular calcium. Notch receptors can be activated by ligand ECD-IgG (or -Fc) fusions that have been immobilized (E–F) or preclustered (G–J). E. Dose-dependent activation of NotchFL LCI reporter with immobilized ligands. F. Kinetics of NICD accumulation from NotchFL after DAPT removal was monitored by LCI and found to mimic kinetics of NICD accumulation from NΔE after DAPT removal. Statistical analyses were performed to assess significant differences from the DMSO control at each timepoint. G. Kinetics of Notch activation with Dll1-Fc-containing conditioned medium preclustered with different α-Fc antibody concentrations. H. The response to clustered ligand was specific to activation of full-length Notch receptors: no response is seen with the NICD reporter cells, NotchFL activation can be inhibited by DAPT and BB94 and correlates with NICD protein as assayed by Westerns (I). J. The clustered ligand-dependent activation paradigm was validated for high throughput screening applications. *p<0.001, **p<0.0001.