LAUNCHER: A single-component, light-assisted uncaging switch for endoproteolytic release

doi:10.21203/rs.3.rs-1628455/v1

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LAUNCHER: A single-component, light-assisted uncaging switch for endoproteolytic release

https://doi.org/10.21203/rs.3.rs-1628455/v1

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published 16 Nov, 2023

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Proteases can serve as molecular switches to initiate a wide spectrum of biological events. High efficiency and specificity enhance the potential of proteases as versatile elements in synthetic biological circuits. To address the unmet need for a novel protease-based toolkit with modular features, we developed a single-component photocleavable switch, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release), combining a circularly permuted potyviral protease and a blue-light-gated substrate with variable intermodular linkers. LAUNCHER enables user-controllable release of payloads that can serve in various cellular applications, such as transgene expression, multilayer-based G protein-coupled receptor (GPCR) activity sensing, and optochemogenetic manipulation. Moreover, LAUNCHER orthologs from the orthogonal potyviral protease family can be utilized in a multiple protease-based circuit without mutual interference. LAUNCHER provides a generalized method to implant optogenetic manipulations into existing synthetic biological circuits in a plug-and-play manner and adds to optogenetic toolboxes for the expansion of biomedical fields.

Diverse types of proteases, i.e., enzymes for proteolysis, have been identified in the proteomes of organisms ranging from simple microorganisms to complex plants and animals¹. Particularly, protease-dependent post-translational modification serves as a signaling cue to initiate a wide spectrum of biological events, including ectodomain shedding², apoptosis signaling³, precursor processing⁴, and transcriptional regulation⁵.

In synthetic biology, innovative bioengineering attempts have been pursued to utilize various proteases as multipurpose molecular switches such as Rewiring of Aberrant Signaling to Effector Release (RASER), Modular Extracellular Sensor Architecture (MESA) and Split-Protease-cleavable Orthogonal-CC-based (SPOC) logic^6-9. Phylogenetically distant plant viral proteases, such as tobacco etch virus protease (TEVp), are the most reasonable candidates for the proteolytic switch because they have no natural substrate in the human proteome¹⁰ and they exhibit stringent sequence specificity¹¹. TEVp is the most widely used enzyme in biomedical engineering, especially for the release of purified recombinant proteins from affinity tags¹². Furthermore, split complementation of TEVp¹³ to control its proteolytic activity through inducible protein–protein interactions enables various biological applications^7,10,14-17.

Pioneer approaches to control protease activity have been focused on chemically induced dimerization (CID) of target proteases and their substrates^9,15,18,19. Chemical complementation through CID allows reliable exogenous control and moderate “turn-on” kinetics^20,21. The complementation of split proteases occurs when chemically interacting proteins bind to each other via small-molecule drugs, such as rapamycin¹⁵. Like CID, non-invasive optogenetic heterodimer complementation allows unprecedented approaches to mediate fast signal transduction with high spatiotemporal resolution including blue-light-inducible TEV protease (BLITz)^16,17,22. However, complementation strategies consisting of separate two polypeptides, regardless of whether the input is optical or chemical, result in differential signal-to-noise ratios (SNRs) because of nonstoichiometric expression level of the individual components^23-25. Moreover, there are limits to the implementation of complex multi-component designs into existing synthetic circuits.

To overcome the above limitations, several research groups have engineered single-component proteolytic cleavable systems controlled by chemical or optical inputs. Chemically controlled systems, such as Small-Molecule-Assisted Shutoff (SMASh) and Stabilization of Polypeptide Linkages (StaPLs), are based on cis-interaction of hepatitis C virus protease (HCVp) with its inhibitor^26,27. Clinically approved drugs that inhibit HCVp offer reliable system control and moderate temporal resolution without undesired side effects. A non-protease-based optogenetic technique termed PhoCl (PhotoCleavable protein) based on violet-light-induced β-elimination reaction of a photoconvertible fluorescent protein also showed reliable photo-induced cleavage with a wide dynamic range^28,29. However, to the best of our knowledge, an optically controllable protease-based system consisting of a single component has not yet been developed.

Here, we present a novel single-component light-assisted protease-based system, termed LAUNCHER (Light-Assisted UNcaging switCH for Endoproteolytic Release). LAUNCHER enables blue-light-induced proteolytic uncaging of a user-defined payload. To this end, circularly permuted TEVp and a photo-caged substrate are connected via a size-tunable linker module. We optimized the SNR of LAUNCHER through multi-round screening and demonstrated its versatility in proof-of-concept experiments, including light-inducible transgene expression, multilayer-based G protein-coupled receptor (GPCR) activity sensing via controllable localization, and optochemical system manipulation. The protease-based photocleavable LAUNCHER provides a generalized method to implant light-induced manipulations into conventional synthetic circuits in a plug-and-play (PnP) manner.

Prototype design of a single-component photocleavable switch

To construct a protease-based photocleavable switch consisting of single-component, we generated a prototype construct integrating a membrane-tethered TEVp with its blue-light-gated substrate and transactivation domain (tTA) (Supplementary Fig. 1a, b). The blue-light-gated substrate was established by distally fusing the TEVp substrate (ENLYFQG) to the improved light-induced dimer (iLID) photosensor³⁰, as described in our previous BLITz system¹⁶. To measure the light-dependent proteolytic efficiency, we used the secreted alkaline phosphatase (SEAP) reporter gene assay in HEK293T cells³¹. Disappointingly, simply linking TEVp and its light-gated substrate in a single chain sequence did not exhibit light-dependent SEAP expression (Supplementary Fig. 1b and c upper panel). The abolishment of the caging effect could be explained by the easy accessibility and explosive cis-interactions within the single molecule. To explore new ways to modulate TEVp activity, we introduced a circularly permuted TEV protease (cpTEVp) harboring the N-terminal (amino acids (aa) 1-118) and C-terminal (aa 119-218) split fragments in the inverted order (Supplementary Fig. 1b, lower panel). Although cpTEVp exhibited lower proteolytic activity than TEVp, but it did exhibit light-dependent gene expression (Supplementary Fig. 1c, lower panel) suggesting the possibility of proteolytic activity regulation through optogenetic controls. Before further optimization of the cpTEVp switch, we tested serially deleted mutants of the light-gated substrate to confirm that the lack of caging effect was not due to the substrate peptide localization within the iLID (Supplementary Fig. 1d and e).

Development and optimization of LAUNCHER

To improve the regulation of cpTEVp activity, we focused on a flexible peptide linker connecting the split fragments of cpTEVp. We hypothesized that an appropriate linker length would render cpTEVp proteolytically active as insufficient steric accessibility of the fragments would prevent effective cis-complementation of cpTEVp (Fig. 1a). Consistent with this idea, a variant with a 25 amino-acid flexible linker (GGGGS ´5) exhibited higher proteolytic activity (19.4-fold) than a variant without the flexible linker (Fig. 1b).

We next focused on developing a size-tunable light-controlled linker. To this end, an additional iLID and nuclear export sequence (NES) were inserted between the TEVp fragments (Fig. 1c and d). We anticipated that the dual photosensors and tight tuning of the intermodular linkers would generate a differential transition of cpTEVp activity in a proximity-dependent manner (Fig. 1c). We investigated intermodular linkers of various sizes (first linker: 5, 10, 18, 23, and 28 amino acids (aa), second linker: 0, 5, 7, and 12 aa), connecting the two iLID and TEVp fragments through a four-day protocol (Fig. 1e). A two-dimensional heatmap and a summary graph displaying the cpTEVp activities of all linker-combination variants revealed that longer first linkers and shorter second linkers gave rise to higher fold changes (blue light vs. dark) (Fig. 1f and g).

For in-depth screening, we set the second linker length at 0 and tested nine first linker variants (5, 10, 18, 23, 28, 33, 38, 43, and 48 aa) (Fig. 1h). The screening results suggested a proteolytic Goldilocks zone (Fig. 1h, Supplementary Fig. 2a and b), implying that intermediate-sized first linkers (23–38 aa) may be optimal for the optogenetic regulation in our of cpTEVp configuration. Through a two-dimensional screening of first linkers of various lengths (23, 28, 33, and 38 aa) and tandem NESs (0–3), we found that the variant with the 38-aa linker and two NESs exhibited the highest light-dependent gene expression (Fig. 1i, Supplementary Fig. 2c). As additional NES insertions and aa substitution of the P1¢ substrate site may affect cpTEVp efficiency, we finally tuned the variants by either varying the first linker length or exchanging the P1¢ aa (Fig. 1j, Supplementary Fig. 2d). The best variant (first linker: 43 aa, second linker: 0 aa, two NESs, and P1¢ site: glycine) was designated as LAUNCHER 1.0 (Fig. 1j).

Characterization of LAUNCHER

To obtain integrative information of LAUNCHER, we characterized its optical induction profile in terms of the light-pulse duration, light intensity, and illumination spectrum. First, we examined blue-light-dependent SEAP expression levels over the indicated time course according to blue-light pulse duration, with a fixed duty cycle (2 s on/28 s off) (Fig. 2a and b). As expected, transgene expression levels were positively correlated with illumination duration (Fig. 2c). In our experimental setup, 6 h of illumination induced a nine-fold increase in the SNR, and 24 h of illumination resulted in a 19.0-fold increase (Fig. 2d). Similar to the findings for light-pulse duration, gene expression levels gradually increased with increasing light intensity (Supplementary Fig. 3a and b). For chromatic characterization of LAUNCHER, HEK293T cells were irradiated with light of three different wavelengths (blue: 470, green: 520, red: 630 nm). Consistent with previous findings (Fig. 1), blue light induced robust SEAP gene expression, whereas green and red induced negligible activity, demonstrating the blue-light specificity of LAUNCHER (Supplementary Fig. 3c). Finally, we tested different duty cycles of blue-light pulses with varying frequency to optimize the optical induction of LAUNCHER (Supplementary Fig. 3d left panel). We did not detect significant differences in LAUNCHER activity in the duty cycle configurations tested in this study (Supplementary Fig. 3d right panel).

Based on the above characterization results, we visualized the spatial resolution of LAUNCHER using an acryl photomask with the word “LAUNCHER” laser-cut from it (Fig. 2f). Three plasmid vectors, harboring enhanced green fluorescent protein (EGFP) as a transfection marker, the TRE-tdTomato reporter, and LAUNCHER, were transfected into HEK293T cells (Fig. 2e). The blue light entering through the photomask precisely illuminated the cellular regions corresponding to the lettering pattern of the photomask and induced selective tdTomato expression (Fig. 2f). Fluorescence cell images clearly revealed red-colored “LAUNCHER” letters (Fig. 2g), demonstrating the precise spatial induction of LAUNCHER under specific blue-light illumination.

Identification of LAUNCHER orthologs for designing multiple protease-based circuits

Establishing protease-based circuits requires multiple orthogonal proteases with high specificity because the flow of information conveyed by each individual protease must not be interfered by the other proteases. In terms of orthogonality, plant potyviruses, including TEVp, are the most suitable for application in protease-based circuits. Given the near-similar sizes and structures of potyviral proteases and their typical substrates, these proteases can be compatible with the LAUNCHER system. To identify effective LAUNCHER orthologs, we investigated nine LAUNCHER orthologs consisting of three orthogonal potyviral proteases TEVp^32,33, Soybean Mosaic Virus (SbMVp)³⁴, and Tobacco Vein Mottling Virus (TVMVp)³⁵ combined with three substrates (ENLYFQG, ESVSLQG, and ETVRFQG) (Fig. 3a–c). In line with previously reported findings^8,36-38, the three pairs with matched protease and substrate showed clear light-dependent gene expression, whereas the six mismatched pairs showed basal expression (Fig. 3d–f). A heatmap of light-dependent fold changes in expression showed strong orthogonality of the three LAUNCHER orthologs we tested (Fig. 3g). Confocal images of cells transfected with the EGFP transfection marker and the tdTomato reporter verified the stringent orthogonality of the LAUNCHER orthologs (Fig. 3h, Supplementary Fig. 4a–c). Therefore, the three LAUNCHER orthologs identified (LAUNCHER_TEVp, LAUNCHER_SbMVp, and LAUNCHER_TVMVp) can be utilized in multiple protease-based circuits without interference with the information flows of the individual proteases.

PnP of LAUNCHER to enhance the performance of protease-based circuits

We demonstrated the PnP capability of LAUNCHER in the design of custom biological circuits using our previous iTango2 system, which is a genetically encoded GPCR sensor that responds to both blue light and GPCR ligand^16,39,40. DRD2-iTango2 consists of two separate polypeptides, a plasma membrane-tethered DRD2-TevN-iLID-TEVseq-tTA and a cytosolic βarrestin2 (βArr2)-TevC. Separate expression of these two components in the iTango2 system has a potential to cause non-specific proteolytic release of the tTA via free diffusion-mediate interaction of two components. To minimize such background signals and improve the AND-gate pattern, controllable compartmentation of one component would be an effective strategy. To this end, we fused LAUNCHER and cytosolic βArr2-TEV-C and then two components were separated into different layers to stay in a ‘ready-to-go’ state (Fig. 4a, b). To identify the most effective anchoring module, we tested six different types of subcellular targeting modules (Supplementary Fig. 5a). The mitochondrial targeting sequence NTOM20⁴¹ showed the best SNR and a prominent AND-gate pattern (Supplementary Fig. 5 b–d). To demonstrate the functional superiority of LAUNCHER_TEVp-DRD2-iTango2, we directly compared its performance with that of the original DRD2-iTango2 using the SEAP assay (Fig. 4b). As expected, SEAP was detected only in the presence of blue light and the DRD2R agonist quinpirole (QU) not the DRD2R antagonist haloperidol (HA) and specially LAUNCHER_TEVp-DRD2-iTango2 showed an excellent SNR and a clear AND-gate pattern when compared with the original DRD2-iTango2 (Fig. 4c). Surprisingly, these improvements were achieved by simply inserting the LAUNCHER module, without further engineering. Finally, confocal microscopic images were acquired using the fluorescent tdTomato reporter and LAUNCHER_SbMVp ortholog (Fig. 4d). Consistent with the above results, tdTomato signal was detected only in the presence of blue light and QU not HA (Fig. 4e). Therefore, LAUNCHER and its orthologs can be easily compatible with various other conventional biological circuits in a PnP manner.

Optochemogenetic circuit fabrication using LAUNCHER

The dual control of optochemical manipulation can overcome the limitations of optical control alone, especially a leakage issue due to ambient light sources. Basically, LAUNCHER can be easily converted into an optochemogenetic system by replacing the effector module while maintaining the original structure. To this end, we used a reverse tetracycline-controlled transactivator (rtTA),⁴² which can bind to the Tet operator sequence only in the presence of tetracycline analogues such as doxycycline. When doxycycline binds to the rtTA, conformational changes occur that allow it to bind to the Tet operator sequence, inducing downstream target gene expressions. We simply created a novel variant of LAUNCHER by replacing the tTA with rtTA so that doxycycline can be used as a safeguard against undesired transgene expression (Supplementary Fig. 6a). We demonstrated selective gene expression under simultaneous stimulation with blue light and doxycycline and identified the typical AND-gate pattern of transgene expression (Supplementary Fig. 6b).

CID is a versatile tool to recruit protein–protein interaction in the presence of small ligand molecules^20,21. The rapamycin-induced FKBP12-rapamycin-binding (FRB)/FK506-binding protein (FKBP) is the most representative CID toolkit^18,43. To establish proof of concept, we attempted to fabricate a single-transcript optochemogenetic circuit by combining photocleavable LAUNCHER and FRB/FKBP. To implement a chemically induced switch via rapamycin, tTA (TetR-VP16) was first split into TetR-FRB and FKBP-VP16 and then SbMVp and its substrate sequences were inserted at the junction of the two fragments. Intramolecular self-cleavage of SbMVp generates two separate polypeptides that can rewire via rapamycin-dependent FRB/FKBP interaction, thereby acting as a chemically controllable switch. Next, we serially fused LAUNCHER with the chemically controllable tTA for optochemogenetic manipulation and tested this single-component optogenetic system in HEK293T cells (Fig. 5 a). Only doubly activated cells showed significant gene expression (Fig. 5b, 47.8, 48.1, and 19.2-fold) in compared to controls and a prominent AND-gate pattern in Fig. 5c.

To establish an optochemogenetic system with triple inputs, we fused the rapamycin controllable-split tTA into LAUNCHER-DRD2-iTango2 (Fig. 5d). The tandem AND-gate of the optochemogenetic iTango2-LAUNCHER is controlled by three types of input manipulator: light, QU, and rapamycin (Fig. 5e). We tested the tandem AND-gate LAUNCHER-DRD2-iTango2 in HEK293T cells and found that only triple-treated cells exhibited significant gene expression (Fig. 5f, 200- to 500-fold increase). These data suggest that LAUNCHER has potential value as a versatile optical switch for creating custom synthetic circuits with multimode controls.

Non-opsin optogenetics has emerged as a new biological breakthrough because of the remarkable properties that allow remote manipulation of protein–protein interactions to control cell behaviors with precise spatiotemporal resolution^44-48. In this study, we newly developed a single-component optogenetic toolkit termed LAUNCHER. Blue-light-induced uncaging of LAUNCHER leads to endoproteolytic release of customizable payloads that can serve various cellular applications.

LAUNCHER is composed of three functional parts: (1) an anchoring module, (2) a light-regulated trigger module, and (3) a payload module, as illustrated in Fig 1d. The N-terminal anchoring module can be replaced with diverse subcellular targeting sequences. Functionally, the anchoring module contributes to the spatial compartment of LAUNCHER to maximize the effectiveness of payload release. The light-induced trigger module consists of cpTEVp and a light-gated photosensor-fused substrate with size-tuned linkers. The trigger module of LAUNCHER shares the light-gated substrate with the previously reported BLITz system. However, unlike in BLITz, in which the TEVp fragments and substrate are separated from each other, in LAUNCHER, cpTEVp and the substrate are integrated as a single-chain peptide. The C-terminal payload module is an essential executor of the LAUNCHER system that drives targeted cellular behaviors.

Similar to the optically controllable LAUNCHER, chemically controllable protease-based systems have been engineered to serve in various biological applications⁹. SMASh is a representative HCVp-based technique²⁶. Clinically verified HCVp inhibitors control the proteolytic efficiency of HCVp. To control target protein stability, HCVp and its specific substrate are fused to the target protein and degron, a protein destabilization module. In the absence of an HCVp inhibitor, HCV abrogates the degron-linked module via cis-interaction, whereas in the presence of an HCVp inhibitor, the fused degron module induces proteasome-dependent degradation of the entire protein, including itself. Chemically cleavable single-component systems show robust execution in the presence exogenous drugs, but their relatively slow kinetics and poor spatial resolution are intrinsic weaknesses compared with optogenetic systems.

The first-in-class of a genetically encoded photocleavable technique consisting of single-component is PhoCl²⁸. Superficially, PhoCl and LAUNCHER have very similar positions and categories within the optogenetic taxonomy, but they differ considerably in several characteristics. In terms of mode of action, PhoCl is based on a light-induced β-elimination reaction⁴⁹ during the photoconversion of a circularly permutated fluorescent protein⁵⁰, unlike the protease-based LAUNCHER. In terms of spectral property, PhoCl is activated by violet light (~400 nm)²⁸, whereas LAUNCHER is induced by blue light (~470 nm). Although we did not investigate whether PhoCl and LAUNCHER can be simultaneously exploited, the ~70-nm spectral gap of the excitation wavelength may be sufficient to allow control through fine-tuned multiplexing.

The clear advantage of LAUNCHER over PhoCl is that the C-terminal fragment after light-induced proteolytic cleavage is shorter. The cleaved payload fragment of LAUNCHER has only one extra aa, glycine, whereas PhoCl leaves more than 12 aa residues (HYGNRVFTKYPR) after photo-induced cleavage²⁸. Because proteolytically generated extra residues can give rise to unpredictable malfunctions or structural changes⁵¹, LAUNCHER is more indicatable for applications that require near-native protein release.

Because of the structural and physical similarities among potyvirus family proteases, numerous potyvirus proteases are compatible with the LAUNCHER system while maintaining stringent substrate sequences. As published genetic information is available for more than 150 potyviruses⁵², extensive potyviral protease library screenings will lead to the discovery of functionally superior LAUNCHER orthologs. Owing to their stringent orthogonality, the technical value of LAUNCHER orthologs is maximized in multiple protease-based circuits. Recently, covalent cyclic peptides that selectively inhibit TEVp have been developed through phage display screening⁵³. These protease inhibitors can inspire the creation of an optochemogenetic system controlled by multiple inputs. Specifically, optical manipulation acts as a main master switch that simultaneously increases the activity of entire pathways, whereas chemical inhibition will be a submaster for fine-tuning to inhibit individual protease-based pathways without mutual interruption.

In conclusion, genetically encoded photocleavable LAUNCHER enables simultaneous and cell-wide release of user-selectable payload with high spatiotemporal resolution. The modular LAUNCHER system provides a generalized strategy to improve the SNR and to implement optical manipulation in conventional protease-based circuits in a PnP manner. Versatile LAUNCHER system will contribute to innovative applications in the field of biochemistry.

Methods, including statements of data availability and any associated accession codes and references, are available in the online version of the paper.

ONLINE METHODS

Plasmid vector design and cloning

All cloned plasmid DNA sequences are described in the Supplementary information. Inserts were amplified by conventional PCR using KOD Hot Start DNA Polymerase (Novagen) and purified by electrophoresis and gel extraction (HiYield Plus, RBC). The inserts and the backbone vector digested with restriction enzymes (NEB) according to the cloning scheme. The non-coding backbone sequences including CMV promoter was amplified from the Addgene #92390 plasmid vector described in a previous study^16,17,22. The digested backbone vector and inserts were ligated with T4 ligase (NEB) and the ligation mixture was transformed into DH5α competent cells (Enzynomics).

Overlap cloning was performed to rapidly clone complex vectors with multiple insert fragments. Briefly, multiple DNA fragments and a backbone vector were amplified using conventional PCR with primer sets designed to generate 15 bp overlap sequences at both ends. Using the Overlap Cloner DNA Cloning Kit (Elpisbio), multiple DNA fragments and the backbone were ligated at 37°C for 1 h.

For site-directed mutagenesis, mutations were introduced in plasmid vectors through PCR using primers containing the desired mutations. The parent plasmid was selectively digested using DpnI (NEB) (37°C for 1 h). The DpnI-treated mixtures were directly transformed into competent cells.

All plasmid sequences were confirmed by Sanger sequencing (Cosmo Genetech, Bionics). Confirmed plasmids were purified using midi prep scale kit (Macherey-Nagel). We used the SnapGene software (Insightful Science; available at snapgene.com) to electronically design and document the DNA cloning.

Cell culture, transfection, and light illumination

Human Embryonic Kidney 293 cells containing SV40 T-antigen (HEK293T) were acquired from the American Type Culture Collection and confirmed to be free from mycoplasma contamination. The cells were grown and maintained in Dulbecco’s modified Eagle’s medium (Welgene) supplemented with 10% fetal bovine serum (Gibco) and 1% streptomycin and penicillin (Gibco) in a humidified incubator at 37°C in an atmosphere of 5% CO₂. For subculture, fully grown cells were detached from culture dishes by incubating the cells with 0.25% trypsin for 1 min. The collected cells were seeded into poly-d-lysine- (Invitrogen) coated 24-well plates at a density of 1 ´ 10⁵ cells per well.

Twenty-four hours after cell seeding, the cells were transfected with the plasmid vectors using a jetOPTIMUS DNA transfection kit (Polyplus) according to the manufacturer’s instructions. In brief, 0.25 μg DNA was first diluted in 50 μL jetOPTIMUS buffer and then 0.25 μL jetOPTIMUS reagent was added into the mixture. After a 10-min incubation at 16°C, 50 μL of the precipitated solutions was carefully added into each well.

For in vitro light illumination of the cells, a 470-nm LED panel (Green Energy Star) was installed in the cell culture incubator as described previously¹⁶. To prevent overheating via direct contract with the LEDs, sample culture plates were placed atop three empty plates (total height, 6 cm). For dark condition, culture plates were tightly wrapped with aluminum foils to block ambient light sources. In our experimental setup, the light intensity measured using a power meter (PM100D, Thorlabs) was 3.0 mW/cm². Duty-cycle controls of the LED panel were established using two serially connected electronic time switches (IRT16, Hanseung).

Blue light intensity‒ and wavelength-dependent experiments were performed using a digital LED control system (TouchBright, Live Cell Instrument, maximum intensity: 3.5 mW/cm²). LED power intensity and wavelength were electronically controlled using Live Cell Instrument software.

SEAP chemiluminescence assay

Chemiluminescent reporter gene expression was quantified using the SEAP assay. Three hundred microliters of the medium from 24well cell cultured plate were transferred into fresh tubes and supernatants were collected after centrifugation at 15,800 ´g for 5 min. The cleared samples were placed on a heating block at 60°C for 1 h to inactivate the endogenous alkaline phosphatases (all except embryonic alkaline phosphatase). The inactivated samples (40 μL) were additionally incubated with L-homoarginine at 37°C for 10 min. Finally, 60 μL SEAP substrate was added to induce the chemiluminescent reaction. Spectral changes were measured using a microplate reader (Infinite F50, Tecan) every 30 s for 1 h (405 nm). Data acquisition and kinetic analysis were conducted using the Magellan software (Tecan).

Fluorescence-based imaging

Cells immobilized on coverslips were fixed with 200 μL of 4% paraformaldehyde (CURRBIO) for 30 min and then rinsed three times with PBS. For nuclear staining, the cells were incubated with 200 μL of 300 nM DAPI (Invitrogen) and then rinsed with PBS. The stained cells were mounted on a glass slide using a mounting solution (Biomeda). Confocal images were acquired using an LSM 800 confocal microscope (Zeiss) with a 40´/0.8 M27 objective lens. In the photomask experiment shown in Fig. 2, large-sized fluorescence images were acquired using a laser scanner (Typhoon FLA 9500, GE Healthcare).

Pharmacology

For chemical stimulations or inhibitions, haloperidol (Tocris), quinpirole (Tocris), rapamycin (Tocris) and doxycycline (Clontech) were used in this study. All stocks were dissolved in DMSO (Sigma-Aldrich) at 500x concentrations.

Statistics

Unpaired t-test with Welch correction was used to determine the statistical significance between SEAP results of dark and blue-light conditioned cells. One-way ANOVA with Dunnett’s multiple comparisons test was used to assess the statistical significance among experimental groups. Single, double, triple and quadruple asterisks indicate p < 0.05, p < 0.01, p < 0.005 and P < 0.001. All statistical calculations were performed with GraphPad Prism 9.

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Acknowledgments

This study was supported by National Research Foundation of Korea (NRF) grants; the Korea Government Ministry of Science and Information and Communications Technology (ICT) (NRF‐2018M3C7A1024597 and NRF-2020R1C1C1014793 to D.L.), and by the National Institutes of Health Grants DP1MH119428 (to H-B.K.). We thank all members of the laboratory for critical discussion and valuable comments.

Contributions

M.C., H-B.K., and D.L. conceived the study. M.C., S.L., S.H.B., J.Y.K., S.K.C., J.H., and Y.K. performed all experiments and collected the data. M.C., S.L., S.H.B., Y.K., K.H., Dh.L., H-B.K., and D.L. analyzed the data. M.C. and D.L. prepared all figures and schematics. M.C., S.L., S.H.B., H-B.K., and D.L. wrote the initial draft of the paper. D.L. supervised the entire study. All authors discussed and commented on the final manuscript.

Corresponding author

Dongmin Lee

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Not applicable.

Competing interests

The authors declare no competing interests.

There is NO Competing Interest.

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LAUNCHER: A single-component, light-assisted uncaging switch for endoproteolytic release

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