Luciferase-LOV BRET enables versatile and specific transcriptional readout of cellular protein-protein interactions

Technologies that convert transient protein-protein interactions (PPIs) into stable expression of a reporter gene are useful for genetic selections, high-throughput screening, and multiplexing with omics technologies. We previously reported SPARK (Kim et al., 2017), a transcription factor that is activated by the coincidence of blue light and a PPI. Here, we report an improved, second-generation SPARK2 that incorporates a luciferase moiety to control the light-sensitive LOV domain. SPARK2 can be temporally gated by either external light or addition of a small-molecule luciferin, which causes luciferase to open LOV via proximity-dependent BRET. Furthermore, the nested ‘AND’ gate design of SPARK2—in which both protease recruitment to the membrane-anchored transcription factor and LOV domain opening are regulated by the PPI of interest—yields a lower-background system and improved PPI specificity. We apply SPARK2 to high-throughput screening for GPCR agonists and for the detection of trans-cellular contacts, all with versatile transcriptional readout.


Introduction
Because protein-protein interactions (PPIs) are central to every biological signaling pathway, there has been keen interest in developing ever-more specific, sensitive, and versatile reporters for detecting them in living cells. In general, existing tools to detect PPIs fall into two broad categories: real-time reporters such as those based on FRET (Truong and Ikura, 2001) or protein complementation assays (Kerppola, 2006), and transcriptional reporters such as yeast twohybrid (Miller and Stagljar, 2004) and TANGO (Barnea et al., 2008). The two categories are complementary in that the former reads out PPI dynamics, while the latter provides a stable "memory" of a previous PPI that can be used for cell-based selections or high-throughput screening. We previously reported "SPARK", for 'Specific Protein Association tool giving transcriptional Readout with rapid Kinetics', a transcriptional PPI reporter that improves upon yeast two-hybrid and TANGO designs by incorporating light-gating. As shown in Figure 1A, transcriptional activation requires both a PPI event and the presence of blue light. Not only does the light-gating dramatically reduce the background of the system, but it also enables temporal specificity because the user can sync the light window to the biological time period of interest (e.g. just before or just after addition of a drug). Instead of integrating PPI events over many hours or days, as yeast two-hybrid and TANGO do, SPARK integrates PPI events over just a 5minute user-selected time window determined by exogenous blue light delivery.
In working with first-generation SPARK (SPARK1), however, we noticed two limitations. The first is that, due to its intermolecular design, SPARK1 performance is highly expression-level dependent. As shown in Figure 1A-B, protein A-protein B interaction drives together the low-affinity tobacco etch virus protease (TEVp) and its recognition sequence (TEVcs), leading to cleavage and transcription factor (TF) release when light is also present.
When SPARK1 components are expressed at high levels, however, TEVp interaction with TEVcs becomes proximity-independent, i.e. there is significant cleavage and TF release even when A-B interaction does not occur (likely because SPARK1 component levels exceed or are close to the TEVp-TEVcs Km which is estimated to be ~450 µM (Kapust et al., 2001;Kapust et al., 2002); Figure 1C). Our practical observation is that SPARK1 performance is best when the components are expressed stably or via viral transduction. Transient transfection methods such as lipofectamine or PEI result in higher expression levels and consequently high background.
This reduction in specificity limits the robustness and therefore utility of the tool.
The second limitation is that light delivery presents a technical challenge, particularly for high-throughput screens, as many robotic platforms are not equipped to dark-cage, deliver light, and then dark-cage again multiwell plates at specified timepoints. Instead, these devices are optimized for liquid handling, i.e. delivery of compounds to multiwell plates is much more straightforward than controlled delivery of light.
To address both these challenges, we have developed second-generation SPARK (SPARK2), which incorporates a luciferase to control the LOV domain, most likely via a BRETtype mechanism (Bioluminescence Resonance Energy Transfer; Figure 1D). Luciferases are enzymes that use O2 and a small molecule "luciferin" substrate to catalyze the generation of light. First, by utilizing a luciferase, we find that it is possible to time-gate SPARK by either luciferin addition or exogenous blue light delivery. This improves the versatility of the tool and makes it more compatible with high-throughput screening platforms. Second, because we found that luciferase-LOV BRET is strongly proximity-dependent, we made LOV domain uncaging also dependent on protein A-protein B interaction by fusing the luciferase to the protease of SPARK2, rather than directly to the LOV domain ( Figure 1E). Whereas in SPARK1, only the TEVp recruitment to TEVcs was regulated by A-B interaction, now in SPARK2 the LOV domain is also regulated by A-B interaction. We find that this "double dependence" on A-B interaction makes SPARK2 a much more specific reporter of cellular PPIs, even at high expression levels ( Figure 1F). We demonstrate the utility of SPARK2 on multiple G-protein coupled receptors (GPCRs), detecting their agonist-dependent activation and subsequent arrestin recruitment with signal to noise ratios as high as 20-fold. We then extend the SPARK2 design to detection of trans-cellular interactions.

Selection of NanoLuc as the luciferase donor for LOV.
First-generation SPARK1 incorporates an evolved photosensory domain from Avena sativa (eLOV) to regulate access to the protease cleavage site (TEVcs, Figure 1B). The LOV domain contains a flavin chromophore that covalently reacts with cysteine 450 upon irradiation with 450-nm blue light. This in turn drives a large conformational change of LOV's C-terminal Jalpha helix (Harper et al., 2003), which can expose or conceal recognition sites, such as TEVcs.
A recent report describes that cell death can be mediated by the action of luciferase on the flavin-containing protein miniSOG (Proshkina et al., 2018), suggesting that luciferase-catalyzed uncaging of asLOV2 may also be possible. To explore this possibility, we considered the range of luciferases used for biological research; these include Vargula hilgendorfii (VLuc ( ). Whereas VLuc requires ATP to produce bioluminescence, limiting it to intracellular use, the others do not (Tung et al., 2016). Among the ATP-independent variants, NanoLuc has been characterized as one of the brightest-emitting luciferase variants (England et al., 2016), and its emission wavelength (450 nm) overlaps with the activation spectrum of LOV. NanoLuc's small size (19kDa) also reduces potential steric problems when fused to other proteins, and its substrate, furimazine, exhibits greater stability than the coelenterazine analogues used for RLuc, GLuc, and sbLuc (Hall et al., 2012). For these reasons, we selected NanoLuc as our luciferase donor to LOV.

NanoLuc-LOV BRET is efficient and proximity-dependent.
To determine whether NanoLuc is able to activate the LOV domain, we first used the LOVTRAP assay (Wang et al., 2016) in combination with a simple imaging-based readout. In LOVTRAP, an engineered peptide Zdk1 binds to the C-terminus of the LOV domain in the dark. Upon blue light irradiation and LOV conformational change, the Zdk1 peptide diffuses away from the LOV domain. The system is reversible, such that termination of blue light results in Zdk1 rebinding to LOV. We fused NanoLuc between β2AR and LOV (β2AR-NanoLuc-LOV) and expressed the construct in HEK293T cells together with an mCherry-Zdk1 fusion protein (Figure 2A). At baseline in the dark, we observed mCherry localization at the cell membrane, but in response to brief exposure to NanoLuc's substrate, furimazine (10µM for 1 min), or to blue light (10s), the mCherry rapidly relocalized into the cytosol (Figure 2B-C). Cytosolic mCherry fluorescence was quantified as the average fluorescence 0.5 to 1.5µm adjacent to the membrane (dashed boxes in Figure 2C). We observed comparable amounts of mCherry relocalization to cytosol between furimazine and blue light conditions (Figure 2D), suggesting that the NanoLuc-LOV activation was as efficient as exogenous blue light-LOV activation for releasing Zdk1.
To test whether NanoLuc activation of LOV is proximity-dependent, we prepared a construct in which NanoLuc was fused to the extracellular N-terminus of β2AR instead ( Figure   2E; NanoLuc-β2AR-LOV), and we compared this side-by-side to the intracellular NanoLuc fusion construct from Figure 2A (β2AR-NanoLuc-LOV). As in Figure 2B-C, the intracellular NanoLuc fusion construct released the mCherry-Zdk1 from the membrane in response to furimazine. Importantly, additional blue light did not result in further detectable release of mCherry-Zdk1 (Figure 2-figure supplement 1A-C). However, the extracellular fusion construct did not release mCherry-Zdk1 from the membrane in response to furimazine. Subsequent blue light delivered to the same cell resulted in the release of mCherry-Zdk1, indicating the LOVTRAP construct was functional (Figure 2F-H). To confirm that this difference was not due to higher expression levels of the intracellular NanoLuc versus extracellular NanoLuc fusions, we measured robust NanoLuc bioluminescence emission from both constructs (Figure 2-figure   supplement 1D). From this experiment we conclude that NanoLuc activation of LOV is proximity-dependent, as moving NanoLuc only ~5 nm away from LOV prevents its activation.

NanoLuc integration into SPARK2.
SPARK1 (Kim et al., 2017) is an "AND" logic gate transcription factor, which detects the coincidence of both a PPI and externally-delivered blue light ( Figure 1A). In this tool, protein A is fused to an evolved LOV domain (eLOV) , a protease cleavage sequence (TEVcs: ENLYFQM), and a transcription factor (e.g., Gal4). eLOV's alpha helical C-terminus cages the fused TEVcs in the dark, preventing steric access and reducing background cleavage.
The other component of SPARK1 is a low affinity protease (TEVp) fused to protein B. In order for TEVp to cut the TEVcs and release Gal4, a PPI must occur between proteins A and B, and blue light must induce a conformational change in eLOV to allow steric access to the TEVcs ( Figure 1B). Following TEVcs proteolysis, the released TF translocates to the nucleus and drives reporter gene expression.
To address the limitations of SPARK1 as outlined above, we incorporated NanoLuc to control the LOV domain within SPARK. We started with the SPARK1 constructs that have previously been used to detect the PPI between the GPCR beta-2 adrenergic receptor (β2AR) and its cytosolic effector arrestin; this interaction can be stimulated with the β2AR agonist isoetharine. We fused NanoLuc to arrestin-TEVp and co-expressed this construct in HEK293T cells along with β2AR-eLOV-TEVcs-Gal4 and the reporter gene UAS-Citrine ( Figure 3A). This "SPARK2" tool gave clear light-dependent and isoetharine-dependent Citrine expression, as we previously observed with SPARK1, showing that introduction of 19 kD NanoLuc does not impair recognition or sterics (Figure 3-figure supplement 1A-B). We then tested whether SPARK2, unlike SPARK1, could also be gated by the small-molecule luciferin, furimazine.

Figure 3B
shows that addition of isoetharine and 10 µM furimazine for 15 min (rather than external blue light) results in robust Citrine expression ~8 hours later. Minimal Citrine expression was observed in the absence of furimazine, or with furimazine but no isoetharine.
To test the generality of SPARK2's design, we also incorporated two other GPCR/arrestin pairs that previously showed light-and agonist-dependent transcription in SPARK1 (Kim et al., 2017). Figure 3B shows that SPARK2 reporters for AVPR2 (arginine vasopressin receptor 2) and DRD1 (dopamine receptor type I) both give furimazine-and agonistdependent expression of Citrine. Together, these results demonstrate that NanoLuc can indeed regulate the LOV domain of SPARK2 in place of external blue light, enabling users to toggle between luciferin-control and light-control of SPARK2 for PPI detection in living cells.
We hypothesized that the use of NanoLuc for LOV activation in a nested "AND" gate may also make SPARK2 a more specific tool for PPI detection. In SPARK1 (and in SPARK2 under light control), TEVp recruitment to TEVcs depends on protein A-protein B interaction, but LOV-TEVcs uncaging depends only on exogenous blue light. Thus, during the time period that LOV is open, even in the absence of an A-B interaction, TEVp may interact with TEVcs (if expression levels approach the TEVp-TEVcs Km). This source of background should be eliminated when using luciferin-gated SPARK2, as LOV-TEVcs uncaging is regulated by NanoLuc in a fashion that depends on A-B interaction . In this case, transient interactions between TEVp and TEVcs at   high SPARK2 expression levels may not be adequate to produce TEVcs cleavage and TF release.
To test this hypothesis, we repeated the SPARK2 experiment for β2AR/arrestin in HEK293T cells, comparing furimazine-and light-gating by microscopy and single-cell analysis ( Figure 3C and Figure 3-figure supplement 1C). The highest Citrine/HA intensity ratios were observed with external blue light and isoetharine (+light/PPI condition); however, we measured substantial background when light was delivered but isoetharine was omitted (+light/no PPI condition; Figure 3D). On the other hand, when we used luciferin (furimazine) to uncage the LOV domain instead of external blue light, we found that background Citrine signal was eliminated (no Citrine expression in the +luciferin/no PPI condition; Figure 3D). We could still observe robust Citrine/HA intensity ratios when using luciferin and isoetharine (+luciferin/PPI condition; Figure 3D). Anti-HA staining for the protease showed similar expression levels of SPARK2 components across conditions.
When directly compared to SPARK1 with light-gating, SPARK2 with nested luciferasegating also produced a higher ±PPI signal ratio (Figure 3-figure supplement 1A-B). We conclude that luciferin-gating of SPARK2 produces higher PPI-specificity than light-gating, due to the incorporation of a PPI-dependent BRET mechanism for LOV domain control that eliminates PPI-independent background. Intermolecular BRET through a nested "AND" gate is essential for improved specificity.
In SPARK2, a nested "AND" gate is achieved by fusing NanoLuc to the arrestin-TEVpmeaning that first, both the furimazine and the PPI are required for the LOV domain to be uncaged, and then both the LOV domain uncaging and the PPI are required for the TEVcs cleavage. If NanoLuc were installed in SPARK2 directly adjacent to LOV rather than on the TEVp construct, then we would not expect an improvement in specificity in the +furimazine/-PPI condition compared to the intermolecular BRET design of SPARK 2 ( Figure 3E).
Intramolecular furimazine uncaging of LOV should still occur, but as it now only requires the presence of luciferin but not A-B interaction, the TF activation would no longer have a "nested" dependence on the PPI. We tested this concept by generating a modified β2AR-NanoLuc-LOV-TEVcs-Gal4 fusion construct, which when co-expressed with arrestin-TEVp, employs only a single "AND" gate relying on furimazine and a PPI ( Figure 3E). Compared to the nested "AND" gate design of SPARK2, the single "AND" gate design resulted in high background in the +furimazine/-PPI condition, comparable to the background observed with +light/-PPI ( Figure 3F-H). Importantly with 5 minutes of blue light, the NanoLuc-LOV fusion construct produced a similar ±PPI signal ratio as light-gating with SPARK2, suggesting that there is no steric hindrance due to the fusion of NanoLuc to LOV ( Figure 3H). These data demonstrate that the nested "AND" gate via intramolecular BRET is essential for driving the improved specificity in PPI detection with SPARK2.
SPARK2 is compatible with an orthogonal luciferase as the transcriptional readout for high-throughput drug screens.
In addition to its utility for BRET, luciferase is also a convenient and scalable reporter commonly used for transcriptional assays, which we employed in our SPARK1 study (Kim et al., 2017). To check if we could continue to use luciferase as the readout, while using the luciferase NanoLuc within SPARK2, we transfected HEK293T cells with β2AR SPARK2 components in addition to the reporter UAS-Firefly luciferase (Photinus pyralis; FLuc). FLuc uses an orthogonal substrate to NanoLuc (Beetle luciferin) and also luminesces at a different wavelength (535nm instead of 450nm (Germain-Genevois et al., 2016); Figure 4A). We performed a SPARK2 assay as before, but this time measuring FLuc reporter luminescence instead of UAS-Citrine fluorescence following furimazine and agonist exposure. We observed 11.6-fold higher FLuc luminescence in cells exposed to furimazine and isoetharine for 20 min compared to cells exposed to only furimazine ( Figure 4B). As there was no FLuc luminescence detected in the control conditions, this confirms that we are detecting only the FLuc luminescence and not any cross-over NanoLuc luminescence.
We then used this multiwell plate assay configuration to again compare the specificity of SPARK2 under light versus furimazine-gating. Figure 4B shows that when SPARK2 is transiently expressed in HEK293T cells, FLuc luminescence background is significant in the +light/no PPI condition but undetectable in the +furimazine/no PPI condition (both 5 min and 20 min treatments). The consequence is that the highest ±PPI signal ratio is obtained with luciferin gating for 20 min (Figure 4B). At 5 min, the reduction in -PPI background with luciferin is offset by reduced +PPI signal, resulting in a lower ±PPI signal ratio compared to light-gating.
Overall, these results, and the single cell imaging results above, support the notion that nested luciferin-gated SPARK2 is a lower-background, higher-specificity PPI reporter alternative to SPARK1/2 with light-gating.
Given the higher-specificity of SPARK2, and the ability to perform an all-drug-gated assay, we then implemented a high-throughput compound sub-library screen to test for β2AR or DRD1 receptor activation (Figure 4C-D). In this configuration, rather than delivering timed blue light, we simply added the additional furimazine substrate along with the compounds, which makes our tool compatible with robotics-based drug-screening platforms if desired. We observed robust FLuc reporter activation for our positive control spike-in compounds isoetharine and dopamine, both delivered at 5µM. For β2AR, we detected several compounds with > 3-fold increases in FLuc reporter expression relative to a DMSO vehicle control, including known agonists such as isoproterenol, epinephrine, procaterol, cimaterol, and norepinephrine ( Figure   4E and Table 2). We could also detect weaker agonists such as the known partial β2AR agonist salbutamol and leflunomide, which drove 1.4-and 1.3-fold increases in FLuc reporter expression, respectively. Interestingly, dopamine, which is a known β2AR partial agonist, did not drive an increase in FLuc expression, supporting the notion that dopamine may act as a Gprotein-biased β2AR partial agonist (Kobilka and Deupi, 2007;Swaminath et al., 2004).
For DRD1, we detected many more compounds that drove >1.5-fold increases in FLuc reporter expression. Notably, known partial and full agonists such as fenoldopam, cabergoline, apomorphine, and pergolide drove >6-fold increases in FLuc reporter expression ( Figure 4E and Table 2). Catecholamine β2AR agonists epinephrine and norepinephrine also drove a 3and 11fold increase in FLuc expression for DRD1 activation. Surprisingly, the anti-psychotic haloperidol, which is a known DRD2 antagonist (Leysen et al., 1992), drove a 2-fold increase in FLuc expression for DRD1 activation. These screening results highlight the utility of SPARK2 for the sensitive and high-throughput detection of GPCR activation.

Proximity-dependence of NanoLuc-LOV interaction using SPARK2
Though we demonstrated using the LOVTRAP assay above that luciferase-LOV BRET is proximity-dependent, we examined proximity-dependence in the context of SPARK2 as well. To do so, we compared the β2AR-arrestin SPARK2 constructs from Figure  These results again support the notion that NanoLuc must be in close proximity (< 5 nm) to the LOV domain in order to efficiently mediate its opening.

Extending NanoLuc BRET to other molecular actuators to detect trans-cellular interactions with SPARK2.
We wondered whether the SPARK2 platform could be extended to detect trans-cellular interactions and not only intracellular PPIs. To do so, we envisioned making two modifications to SPARK2. First, for protein A, instead of a regular GPCR, we would install a light-sensitive GPCR chimera, such as "oβ2AR", previously used to enable light induction of G-protein signaling in neurons in vivo (Airan et al., 2009). Second, to activate oβ2AR across cell-cell junctions, we would utilize a second copy of the luciferase NanoLuc on the "sender" cells. With this design, addition of furimazine to cells would be expected to (1) turn on oβ2AR selectively at cell-cell junctions that are positive for NanoLuc sender and oβ2AR receiver, and (2) enable NanoLuc-arrestin-TEVp recruited to activated oβ2AR to open the LOV domain and permit cleavage and release of the TF. Reporter gene expression should then be observed specifically in "receiver" cells in contact with NanoLuc-expressing "sender" cells. to regulate their activity in vivo ("luminopsins"), luciferases have not previously been shown to turn on light-sensitive GPCR chimeras such as oβ2AR. Furthermore, proximity-dependent intermolecular luciferase regulation of retinal-binding proteins has also not been previously demonstrated.
To explore this extension of SPARK2, we started by creating an oβ2AR SPARK2 construct with NanoLuc fused directly to its N-terminus. HEK293T cells expressing this construct along with NanoLuc-arrestin-TEVp and UAS-Citrine ( Figure 5A) were incubated with the co-factor 9-cis-retinal (9cr), as HEK293T do not naturally produce 9cr. As a positive control, we delivered 15 minutes of blue light to both uncage the eLOV domain and activate oβ2AR. We observed significantly more Citrine-positive cells in the +light/+9cr condition than in the +light/-9cr condition, indicating that the oβ2AR is functional (Figure 5-figure supplement 1A-B). In a separate experimental condition, we delivered 30 minutes of furimazine and 9cr and observed significantly more Citrine-positive cells compared to control conditions ( Figure 5B-C). These results suggest that both extracellular NanoLuc-oβ2AR BRET and intracellular NanoLuc-LOV BRET are occurring in the same cells, enabling TF release to produce reporter signal.
Next we moved to test intercellular activation of oβ2AR by NanoLuc. Instead of fusing NanoLuc directly to the N-terminus of oβ2AR, we expressed NanoLuc on "Sender" cells, and oβ2AR SPARK2 components separately in "Receiver" cells, as shown in Figure 5D. For display of NanoLuc on senders, we used the pre-synaptic adhesion protein neurexin as a scaffold, and the extracellular domain of ICAM-1 (Talay et al., 2017) to extend NanoLuc further out from the cell surface. After separately transfecting the two HEK293T populations, we trypsinized the cells to lift them, and re-plated them together in the same dish at a 10:1 sender: receiver ratio ( Figure   5E).
We exposed the co-plated cells to furimazine and 9cr for 20 minutes and observed Citrine-positive cells ~8 hours later. To positively identify Sender and Receiver cells, we immunostained for HA on the Neurexin-NanoLuc Sender construct and for Flag on the oβ2AR Receiver construct ( Figure 5F). To quantify specificity, we counted the fraction of Citrinepositive Receiver cells that were contacting HA-positive Sender cells (87%; n = 43 cells from 26 FOVs). It is possible that the few Citrine+ cells not in contact with NanoLuc+ Senders were previously in contact, but the Senders were dislodged during the 8 hr incubation and washing period. To quantify Citrine expression in all Receiver cells, we calculated the ratio of Citrine fluorescence to Flag immunostain detecting the oβ2AR component. We observed a higher overall Citrine/Flag intensity ratio in the condition with furimazine and Sender NanoLuc compared to control conditions with furimazine omitted or Sender NanoLuc omitted ( Figure 5G and Figure 5-figure supplement 1C). We confirmed that in the absence of 9cr, there was no significant increase in the Citrine/Flag intensity with either blue light or furimazine (Figure 5-figure supplement 1D-E). These results provide a proof-of-concept demonstration that SPARK2 can be combined with intercellular NanoLuc-oβ2AR BRET to enable luciferin-gated, bioluminescence-mediated transcriptional readout of cell-cell contacts.

Discussion
Motivated by specificity and versatility limitations of our previously-reported SPARK1 PPI detection tool, here we developed improved, second-generation SPARK2. SPARK2 employs a novel design not previously used for PPI detection. Via a single modification of SPARK1addition of a luciferase moiety onto the protease component of SPARK-SPARK2 gains two new capabilities: the ability to toggle between light-gating and luciferin-gating for temporal specificity, and greatly reduced background leading to a more PPI-specific tool. We showed that SPARK2 could be used to detect transient arrestin recruitment to several activated GPCRs, giving stable and versatile (Citrine, FLuc) reporter gene readout many hours later. Using luciferin-gating, we used SPARK2 for high-throughput compound screening against activation of two GPCRs. We also extended the SPARK2 design to detection of cell-cell contacts, employing trans-cellular luciferase activation of a GPCR-rhodopsin chimera.
At the heart of SPARK2 is a proximity-dependent interaction between the blue lightemitting luciferase NanoLuc and the blue-light absorbing photosensory domain LOV. . It seems likely that if these tools were to also incorporate NanoLuc, this would enable users to toggle between light control and luciferin control, as we do for SPARK2. Furthermore, because we have shown that luciferase-LOV regulation is proximity-dependent, one could envision tool variants in which LOV-mediated functions (PPI induction, protein inactivation/activation, channel opening, etc.) are conditional upon PPIs or subcellular localization events designed by the user. Finally, we note that luciferase regulation is likely to extend beyond LOVs. Cryptochromes (e.g., CRY-CIBN (Kennedy et al., 2010)) also contain blue-light excitable flavin cofactors, and we showed here that luciferase can also regulate oβ2AR, a retinal-containing blue-light activated GPCR-rhodopsin chimera.
Besides enabling SPARK2 toggling between light-gating and luciferin-gating, incorporation of NanoLuc onto the protease component of SPARK substantially reduced the PPIindependent background of the system. We rationalize this by the logic diagram in Figure 1D, showing that both inputs into the committed step (TEVcs cleavage) -LOV opening and TEVp recruitmentrequire protein A-protein B interaction. This contrasts with the simpler logic diagram of SPARK1 (or in a version of SPARK where NanoLuc is directly fused to LOV), in which only TEVp recruitment depends on protein A-protein B interaction. A "double filter" for the analyte of interest (in this case, a PPI) appears to improve the specificity of a reporter for that analyte. We envision that the "double filter" or nested "AND" gate concept could also be incorporated into a range of other reporter designs, particularly those with limited dynamic range. Interestingly, there are numerous examples of natural pathways that incorporate "double filters" to increase specificity, such as tRNA synthetases that catalyze amino acid loading onto cognate tRNAs (Fersht and Dingwall, 1979). thus provide more versatile readouts, but they lack temporal-gating which can contribute to high background, and they could potentially perturb normal physiology by introducing non-natural intercellular protein-protein interactions. Trans-cellular SPARK2 has complementary features to these existing methods, although further optimization and characterization are required to make it a broadly useful tool for the study of trans-cellular interactions.  Imaging Innovations) and processed with Fiji (Schindelin et al., 2012).

Sample-size estimation and replication
No statistical methods were used to determine sample size, and instead relied on guidelines from 150 ng of P10.UAS-FLuc. ~15 hours after transfection, cells re-plated in 96-well plates were exposed to furimazine or light conditions, and ~9 hours later, UAS-FLuc reporter measurements were assayed as described above. In separate wells undergoing the same transfection and replating protocol, the total NanoLuc luminescence output was measured ~15 hours after transfection. Cells were exposed to 1.25 μL furimazine in 23.75 μL DMEM+10% FBS+20 mM HEPES in each 96-well, and luminescence was immediately analyzed at 25ºC using a 1,000ms acquisition time, the Blue filter, and linear shaking for 3 s.  Table 2) and 10µM furimazine for 1 hour (final volume in each 96-well was 100 µL). Two wells were exposed to a DMSO vehicle control, and two wells were exposed to either 5 µM isoetharine or 5 µM dopamine as positive spike-in controls. After 1 hour, the media was replaced with 100 µL of DMEM+10% FBS, and the plates were wrapped in aluminum foil and incubated in the 37°C incubator for 9 hours prior to luminescence measurements on a plate reader. FLuc measurements were performed as described above ("FLuc
For lentiviral production of each construct, a T25 flask with ~90% confluent HEK293T cells was    When NanoLuc is expressed extracellularly, we do not expect to observe NanoLuc-asLOV activation with furimazine. As a result, mCherry-Zdk1 should remain at the membrane.        Data plotted as mean±s.e.m. Figure 3C-D plotted according to their mean HA and Citrine intensities.