Articles
A genetically targetable near-infrared photosensitizer
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Jianjun He1, Yi Wang2, Maria A Missinato3, Ezenwa Onuoha3, Lydia A Perkins2, Simon C Watkins4, Claudette M St Croix4, Michael Tsang3 & Marcel P Bruchez1,2,5 Upon illumination, photosensitizer molecules produce reactive oxygen species that can be used for functional manipulation of living cells, including protein inactivation, targeted-damage introduction and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. We describe a genetically encoded fluorogen-activating protein (FAP) that binds a heavy atom−substituted fluorogenic dye, forming an ‘on-demand’ activated photosensitizer that produces singlet oxygen and fluorescence when activated with near-infrared light. This targeted and activated photosensitizer (TAPs) approach enables protein inactivation, targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs provides a new spectral range for photosensitizer proteins that could be useful for imaging, manipulation and cellular ablation deep within living organisms.
Light provides precise spatiotemporal control of biological pro cesses when combined with suitable genetic constructs or chemical reagents1–3. Photosensitizer dyes and proteins exploit absorbed light to create short-lived reactive oxygen species (ROS) that can mediate biological effects at the target site4,5. Traditional photosen sitizers such as methylene blue have no selectivity, and off-target phototoxicity produced during light exposure limits the applica tions6,7. Photosensitizers with improved efficiency of ROS gen eration, photostability and near-infrared (NIR) light absorption enhance optical tissue penetration and allow real-time fluorescence visualization8. More recently, genetically targeted photosensitiz ers, such as FlAsH and ReAsH, KillerRed and MiniSOG9–12, have been developed to improve targeting and specificity in living cells. The genetic fusion approach allows the photosensitizing protein to produce ROS at the target, selectively inactivating it through chromophore-assisted light inactivation (CALI) of directly linked proteins. These photosensitizer proteins require a very high light dose to reach effective inactivation or cell killing, and the spectral properties of these sensitizers overlap with those of
biological chromophores, resulting in some ROS generation even in the absence of the photosensitizer proteins. Photosensitizers with far-red and/or NIR light excitation wavelengths (>620 nm) are required for deep tissue applications and to avoid photosensi tization by endogenous chromophores, yet no genetically targeted photosensitizers are available in this spectral range. Activation of a photosensitizer at a target site improves specifi city by minimizing the damage to nontargeted tissues13. Current activatable photosensitizers either are responsive to local envi ronmental changes, such as changes in pH, or contain a quench ing group that is cleaved, increasing photosensitizer activity14–16. Although activation increases the ROS production by 10–50-fold, these tools still show some off-target effects from nonspecifically localized materials. Thus, selectively targeting and activating a photosensitizer remains an important goal. To target and activate an ROS-generating photosensitizer, we exploited a genetically targetable and efficient FAP17. FluorogenFAP complexes have been adapted to a number of applications such as single-molecule imaging18, physiological pH measure ments19 and protein detection as recombinant affinity probes 20. FAPdL5** (previously MBIC5; referred to hereinafter as dL5**) is a 25-kDa binder for malachite green (MG) derivatives that func tions throughout living cells with thousands-fold fluorescence activation and a low-picomolar dissociation constant17,21. We reasoned that suppression of the nonradiative relaxation, which enhances fluorescence in MG fluorogens, could be exploited to modify other photochemical properties, in particular intersystem crossing, when combined with chemically tailored fluorogens22. We prepared an iodine-substituted MG analog (Supplementary Note 1) with low free fluorescence and ROS generation that binds to dL5**, producing an NIR light–excitable fluorescent complex with high singlet oxygen (1O2) quantum yield (Fig. 1a). We used the dye-protein complex to photo-inactivate fused proteins; to photosensitize cells expressing dL5** at the plasma membrane, cytosol, mitochondrial matrix or nucleus in culture; and to ablate cardiac cells in living larval and adult zebrafish expressing dL5** in the cytoplasm. The free dye showed no photosensitization in cells or zebrafish, indicating that the iodinated MG analog is a potent, NIR light–excitable FAP-TAPs.
1Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. 2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
Pennsylvania, USA. 3Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 4Center for Biologic Imaging, Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 5Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. Correspondence should be addressed to M.P.B. ([email protected]). Received 19 August 2015; accepted 4 December 2015; published online 25 january 2016; doi:10.1038/nmeth.3735
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RESULTS MG-2I–dL5** as a 1O2-specific photosensitizer Heavy-atom substitutions at some sites on the MG chromo phore, which are expected to increase intersystem crossing23,24, retained high-affinity dL5** binding and fluorescent properties (Supplementary Fig. 1). A di-iodinated derivative of the cellpermeable MG-ester fluorogen (MG-2I) showed dramatically increased photogenerated 1O2 when bound to dL5** (FAP-TAPs). The iodination redshifted the major absorption band, moving the excitation maximum of the complex into the NIR light range (666 nm; Supplementary Table 1 and Fig. 1b). We evaluated the generation of 1O2 via bleaching of anthracene-9,10-dipropionic acid (ADPA), a commonly used 1O2 scavenger25. Relative to aluminum phthalo cyanine tetrasulfonate (AlPcS4)26, the FAP-TAPs 1O2 quantum yield (Φ∆) was 0.13 (Fig. 1c), whereas generation of 1O2 from the MG-ester–dL5** or free TAPs dye was not detectable under the same conditions. ADPA bleaching by FAP-TAPs, evaluated using filters for various fluorescent proteins (Supplementary Fig. 2a,b), revealed little FAP-TAPs activation under YFP and RFP excitation, suggesting that these fluorescent proteins are optimal for use with FAP-TAPs. In deuterated PBS buffer, the bleaching rate of ADPA by FAP-TAPs was enhanced. As D2O is known to increase the lifetime of 1O2 (4 µs to 52 µs) (ref. 27) while having little effect on other ROS, the increased ADPA bleaching is consistent with 1O production (Supplementary Fig. 3a). Diaminobenzidine was 2 photo-oxidized to produce brown precipitate in vitro, which is potentially useful for correlative light and electron microscopy (Supplementary Fig. 3b). When we compared ADPA bleaching in deuterated PBS between MG-2I and FAP-TAPs (Supplementary Fig. 3c), we noted that the 1O2 generation of the MG-2I dye increased up to 450-fold upon binding to dL5**. This ensures that free dye is nonphotosensitizing, a critical difference with respect to other dye-targeting or photosensitizer-activating approaches. CALI of the PLC δ1 Plekstrin homology (PH) domain To assess the utility of FAP-TAPs for targeted protein inactiva tion, we compared membrane release of EGFP-PH-KillerRed and
FAP-TAPs mediated cellular photoablation We evaluated light-induced cytotoxicity of FAP-TAPs on HEK cells expressing a cell surface−anchored FAP (TM-dL5**). We also incubated a mixed population of TM-dL5**−expressing HEK cells and wild-type HEK cells with 400 nM dye for 30 min and then illuminated it for 1 min with a continuous laser source (40× objective, 640-nm excitation, 0.76 W cm−2). Only fluorescently labeled TM-dL5**−expressing HEK cells treated with MG-2I and light were stained dead within 30 min using a live/dead cell viabil ity kit, whereas the wild-type HEK cells in the illumination field remained alive and metabolically active (Fig. 3a). The labeled cells began to lose cell morphology with swelling and blebbing within a very short period after illumination (Supplementary Video 1). MG-ester bound to TM-dL5**−expressing HEK cells,
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EGFP-PH-dL5** fusion proteins upon suitable illumination in HEK293 (HEK) cells9. When the membrane-bound PH domain from PLC δ1 is inactivated by CALI, it is released to the cyto plasm, increasing the ratio of cytoplasmic to membrane EGFP. After 5 min of illumination with a 560-nm laser (60× objective, 2.03 W cm−2), the cytoplasm-to-membrane signal ratio increased by 37% under KillerRed-mediated CALI (Fig. 2), similar to pre viously reported observations. The fluorescence of KillerRed was bleached (>75%) after 1 min of illumination. In contrast, FAP-TAPs illumination resulted in a 33% increase in ratio after 10 s of 640-nm laser illumination (60× objective, 2.07 W cm−2). Further illumination of FAP-TAPs induced no additional change in EGFP ratio, but noticeable morphology changes and minor photobleach ing were observed. We evaluated the potential collateral dam age by coexpressing EGFP-PH with PH-KillerRed or PH-dL5**. Both KillerRed and FAP-TAPs induced similar inactivation of EGFP-PH in proportion to the amount of target inactivation, indicating that the FAP-TAPs are spatially restricted similarly to KillerRed under CALI conditions. The high efficacy and specifi city of FAP-TAPs−mediated protein inactivation could facilitate well-controlled experiments based on acute protein inactivation, when proximal collateral damage can be properly controlled.
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Figure 1 | Characterization of ROS generation by FAP-TAPs. (a) Mechanism of ROS generation by FAP-TAPs. 0 IC, internal conversion by molecule’s free rotation; ISC, intersystem crossing. (b) Normalized excitation (dashed lines) and emission (solid lines) spectra of MG-ester and MG-2I binding to dL5**, where 500 nM 0 20 40 60 Time (s) fluorogen was complexed with 3 µM dL5** and the fluorescence intensity was individually normalized to the peak maxima. (c) 1O2 generation by MG-2I–dL5** assessed on the basis of ADPA bleaching, where bleaching of ADPA fluorescence was monitored at 374-nm excitation and 402-nm emission as a function of the duration of 669-nm light exposure. AlPcS 4 was used as standard for the 1O2 generation (Φ∆ = 0.34). Optically matched solutions of MG-2I–dL5** and AlPcS4 at 669 nm were used. n = 4; data are shown as mean ± s.e.m.; in instances of very small s.e.m., error bars overlap points and may not be visible. | ADVANCE ONLINE PUBLICATION | nature methods
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Figure 2 | FAP-TAPs mediated light-induced protein inactivation of the PLC δ1 PH domain. (a) Outline of the experimental design. We constructed EGFPPH-KillerRed or EGFP-PH-dL5** triple fusion protein to evaluate the effectiveness of specific protein inactivation from KillerRed and FAP-TAPs (top). EGFP-PH was coexpressed with PH-KillerRed or PH-dL5** to allow estimation of the collateral damage from KillerRed and FAP-TAPs (bottom). (b) Change in EGFP cytoplasm-to-membrane ratio after illumination of MG-2I with EGFP-PH-dL5** and of MG-ester with EGFP-PH-dL5** and EGFP-PH-KillerRed (solid lines). Dashed lines represent the corresponding collateral damage from coexpressed proteins. n = 8; mean ± s.e.m. (c) Representative EGFP fluorescence images from each condition, with imaging at the indicated intervals. Scale bar, 5 µm. Illumination conditions were as follows: KillerRed, 560-nm laser, 60× objective, 2.03 W cm−2; MG-ester–dL5** and MG-2I–dL5**, 640-nm laser, 60× objective, 2.07 W cm −2.
MG-2I−treated wild-type HEK cells and nontargeted FAP-TAPs added to the medium showed no apparent cytotoxicity or photo toxicity upon illumination (Fig. 3a), suggesting that close contact of FAP-TAPs on target cells was required for effective delivery of ROS owing to the very short radius of action of 1O2. These results established the dual requirements of targeted FAP expression and TAPs binding for efficient photosensitization. We observed lim ited self-bleaching ( 0.05, one-way analysis of variance (ANOVA) using MG-2I–only group as a control) suppression of phototoxicity in cells incubated with high concentrations of catalase (peroxide quencher; 1,000 U ml−1) or superoxide dismutase (superoxide quencher; 500 U ml−1)34. Results from these quenching experi ments along with the in vitro ADPA bleaching assay indicate that 1O generated from FAP-TAPs was the primary ROS mediating 2 the observed phototoxic responses. 1O is known to react with nearby molecules to produce 2 secondary peroxide species as initial products, which then create other reactive species and lead to cytotoxicity 35. Thus chemical sensors can be used to identify the cascade generation of ROS after photosensitization. We used dihydroethidium (DHE), which reacts with superoxide (O2−) to form a DNA-intercalating nature methods | ADVANCE ONLINE PUBLICATION |
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fluorescent species (2-hydroxyethidium, 490-nm excitation and 567-nm emission), to detect ROS in HEK cells expressing FAP-TAPs in the nucleus36. We observed significant (P < 0.0001, one-way ANOVA) ROS-dependent activation of DHE in the nucleus only when MG-2I with FAP and light were present, and not in identical but unilluminated cells (P > 0.05) or FAP-expressing cells labeled and illuminated in the presence of MG-ester dye (P > 0.05) (Supplementary Fig. 8). FAP-TAPs mediated cardiac ablation in larval zebrafish In vivo cellular ablation has been used to study development and regeneration37. Zebrafish (Danio rerio) is a vertebrate model organ ism that is optically transparent, is easily genetically manipulated and shows conserved developmental processes with respect to other vertebrates38,39. Lineage ablation can be accomplished in zebrafish by tissue-specific expression of a bacterial nitroreductase and sub sequent treatment for ~12−24 h with metronidazole, a prodrug that is reduced to a DNA-synthesis inhibitor in nitroreductasepositive cells, causing cell death40. As zebrafish develop rapidly, 12−24 h of treatment limits the temporal resolution of lineage ablation studies. Because cultured cells are ablated rapidly, we evaluated rapid cellular ablation in living larval zebrafish. We produced transgenic zebrafish lines that expressed a cytoplasmic dL5**-mCerulean3 (dL5**-mCer3) tandem protein under control of the heart-specific myosin light chain 7 (myl7, also known as cmlc2) promoter, Tg(myl7:MBIC5-mCer3). We confirmed expression of dL5**-mCer3 by fluorescence imaging of mCer3 and MG-ester–dL5** or MG-2I–dL5** complexes, which were restricted to the beating heart (Supplementary Fig. 9a and Supplementary Video 3). To test the effectiveness of FAP-TAPs, we treated embryos at 48 h after fertilization with 500 nM dye (MG-2I or MG-ester) for 3 h and then subjected them to 12 min of laser illumination (659 nm, 242 mW cm−2). Immediately after illu mination, Tg(myl7:MBIC5-mCer3)pt22 (herein referred to as Tgpt22) larvae treated with MG-2I showed no sign of heartbeat or blood circulation, whereas MG-ester−treated Tgpt22 larvae and wild-type larvae treated with MG-2I were normal (Supplementary Video 4). | ADVANCE ONLINE PUBLICATION | nature methods
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Figure 3 | Phototoxicity of FAP-TAPs in HEK cells expressing surfacetargeted FAP. (a) Images of cells that were labeled with 400 nM of the indicated dye for 30 min before illumination (without removal of the unbound dye), taken before laser illumination (top; merge of c640 (fluorogen-FAP, red) and differential interference contrast (DIC)). Live/dead cell viability was assayed 30 min after illumination (bottom; merge of c488 (live cells; cyan), c560 (dead cells; yellow) and DIC). Scale bar, 10 µm (applies to all images in panel). (b) Viability of cocultured TM-dL5**−expressing HEK cells (TM-dL5** HEK) and wild-type HEK cells (WT HEK) incubated with MG-2I and subjected to 0, 30, 60 and 120 s of illumination (640 nm, 40× objective, 0.76 W cm−2). n = 8, one-way ANOVA. Tukey post-hoc tests were performed with multiple comparisons; data are shown as mean and s.e.m. ****P < 0.0001; ns, not significant. (c) Viability of TM-dL5**−expressing cells labeled with MG-2I under variable light intensity (blue) and variable illumination duration (red, 0.089 W cm −2 light intensity with 2, 5, 7, 10, 12 and 15 min of illumination), MG-ester with TM-dL5**−expressing HEK cells (dotted black), and MG-2I with wild-type HEK cells (dashed-dotted green). Cells were illuminated using an LED light box. Cells were stained with propidium iodide (dead) and Hoechst (total); more than 300 cells were counted for each data point (n = 4, mean and s.e.m.; in instances of very small s.e.m., error bars overlap points and may not be visible).
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At 24 h post-illumination (h.p.i.), we found that illuminated MG-2I−treated Tgpt22 larvae exhibited cardiac edema and lower mCer3 fluorescence (Fig. 4a). At 96 h.p.i., these larvae had smaller eyes and larger yolk than larvae in other groups did (Supplementary Fig. 9b). At 96 h.p.i., we detected no mCer3+ cells in illuminated MG-2I−treated Tgpt22 larvae, indicating that acute loss of myl7 lineage cells was not renewed later in development. We used deoxynucleotidyl transferase−mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay with anti-GFP staining to assess on-target and off-target cell death in wholemount Tgpt22 larvae at 24 h.p.i. (Supplementary Fig. 10a). In MG-2I–treated larvae exposed to 12 min of laser illumination, we saw fewer mCer3+ cells and more TUNEL+mCer3+ cells, without any increase in TUNEL+mCer3− cell death compared with that in MG-ester–treated larvae exposed to 12 min of laser illumination (Supplementary Fig. 10b,c). The fraction of TUNEL+mCer+ cells in MG-2I–treated larvae was greater than that in MG-ester–treated larvae, both of which were exposed to 12 min of laser illumination (31% versus 4.4%, P < 0.01) (Supplementary Fig. 10d). A shorter illumination time (MG-2I–treated larvae exposed to 4 min of illu mination) resulted in decreased specific cytotoxicity, confirming the light-dose dependence of the FAP-TAPs ablation. Cardiac photoablation in adult zebrafish demonstrated the utility of FAP-TAPs in deep tissue. Tg(myl7:MBIC5-mCer3)pt23 (herein referred to as Tgpt23) zebrafish uniformly express mCer3dL5** in the heart at the adult stage (Supplementary Fig. 11a). We retro-orbitally injected zebrafish with dye and illuminated them for a total of 30 min using the LED light box (2.5 W cm−2 illu mination from below the fish) (Supplementary Fig. 11b). At 3 d post-illumination (d.p.i.), we assessed cell death using TUNEL staining of hearts, livers and intestines. We observed increased cell death only in the hearts of transgenic zebrafish injected with MG-2I, compared to all the control groups (Fig. 4b) and nontar geted tissues (Supplementary Fig. 11c), demonstrating effective and specific ablation in deep tissue. To confirm cardiomyocyte ablation, we visualized intact cardiac muscle at 5 d.p.i. with Acid Fuchsin Orange G. Hearts from the Tgpt23 fish treated with MG-2I
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showed damaged cardiac structure and decreased ventricular car diac muscle compared to the controls (Fig. 4c). We observed no structural defects in livers or intestines (Supplementary Fig. 11d). Damaged cardiac muscle can regenerate through proliferation of pre-existing cardiomyocytes41–43. At 5 d.p.i., only illuminated trans genic zebrafish injected with MG-2I had significantly increased numbers of proliferating cardiomyocytes compared to similarly treated wild-type zebrafish (P < 0.001, MG-2I–treated Tgpt23 versus MG-2I–treated wild-type; P > 0.05 MG-ester–treated Tgpt23 versus MG-ester–treated wild-type) (Fig. 4d), showing that the ablation inflicted sufficient damage to induce a regenerative response. DISCUSSION We have demonstrated a two-component targeted and activated photosensitizing approach that enables specific NIR light− mediated protein inactivation and phototoxicity in FAP-expressing cells and living transgenic organisms. The TAPs dye (MG-2I) and FAP (dL5**) had no photosensitizing effects until they associated to form the FAP-TAPs. Photosensitization is primed by the on-demand addition of TAPs dye to the FAP-expressing cells rather than removal of quenching groups by cellular processes. This method allows temporally, spatially and subcellularly con trolled, target-specific generation of 1O2. The flexible genetic targeting strategy of the FAP along with efficient activation of ROS generation makes FAP-TAPs an immediately useful tool for targeted cellular ablation and subcellular protein inactivation, and
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Figure 4 | FAP-TAPs induced photo-ablation of cardiac function. (a) Merge of DIC and mCer3 fluorescence (cyan) showing phenotype development from 0 h.p.i. to 96 h.p.i. of larval zebrafish (n = 20 for each group): Tg(myl7: MBIC5-mCer3)pt22 (Tgpt22) larvae treated with MG-ester (MG-ester/Tgpt22), Tgpt22 larvae treated with MG-2I (MG-2I/Tgpt22) and wild-type larvae treated with MG-2I (MG-2I/WT). In the MG-2I/Tgpt22 group, larvae developed visible defects: large cardiac edema, small eyes and collapsed, nonfunctional heart chambers. In both control groups, development proceeded normally. Scale bar, 1,000 µm (applies to all images in panel). (b) Images of FAP-TAPs– photoinduced cardiac damage in adult zebrafish hearts extracted at 3 d.p.i., subjected to TUNEL and stained with DAPI. Scale bar, 100 µm (applies to all images in panel). (c) Acid Fuchsin Orange G (AFOG) staining of hearts at 5 d.p.i. showing the damaged cardiac structure in transgenic fish injected with MG-2I, as marked by reduced myocardium (brown labeling). Scale bar, 100 µm (applies to all images in panel). (d) Mef2c (cardiomyocyte) and PCNA (proliferation) staining at 5 d.p.i. showing enhanced cardiomyocyte (CM) proliferation (yellow arrowheads) in transgenic fish injected with MG-2I. Scale bar, 100 µm. n = 9 for all groups; one-way ANOVA with Tukey post-hoc tests was performed with multiple comparisons of the mean for each group. ***P < 0.001, ****P < 0.0001; ns, not significant. Data are shown as mean ± s.e.m. Each individual point represents the average from four heart sections per zebrafish.
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it could potentially be multiplexed with other photosensitizers (for example, KillerRed and MiniSOG). The fact that the same encoded FAP (dL5**) can be used to bind a nonphotosensitiz ing dye (MG-ester) allows many studies that rely on imaging to be extended to photosensitization only when the researcher uses the TAPs dye. This alternative labeling strategy can facilitate the selection of stable cells and transgenic animals usable for imag ing, photoablation or photosensitization studies, depending on the dye and light dose used in the study. Methods Methods and any associated references are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments This work was supported in part with funds from the US National Institutes of Health (NIH), Technology Centers for Networks and Pathways program (grant U54GM103529 to M.P.B., S.C.W., J.H. and Y.W.), NIH grant R01EB017268 (to J.H., Y.W., M.A.M., E.O., C.M.S., M.T. and M.P.B.) and NIH grant R21ES025606 (to M.P.B. and S.C.W.). We thank E. Kelley and C.J. Bakkenist for helpful discussion and guidance on establishing ROS involved in cellular toxicity; G. Daskivich for help in establishing zebrafish lines; and E. Drill, C.T. Wallace and M.A. Ross for help in larval zebrafish TUNEL imaging. AUTHOR CONTRIBUTIONS J.H. designed and performed experiments, analyzed data and wrote the paper. Y.W. provided cell culture, performed experiments and wrote the paper.
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Articles E.O., M.A.M. and M.T. developed and provided zebrafish lines, designed and performed experiments and analyzed data. L.A.P. provided reagents. S.C.W. and C.M.C. performed experiments and analyzed data. M.P.B. designed experiments, analyzed data and wrote the paper. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper.
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21. Szent-Gyorgyi, C. et al. Malachite green mediates homodimerization of antibody V–L domains to form a fluorescent ternary complex with singular symmetric interfaces. J. Mol. Biol. 425, 4595–4613 (2013). 22. Koziar, J.C. & Cowan, D.O. Photochemical heavy-atom effects. Acc. Chem. Res. 11, 334–341 (1978). 23. Gandin, E., Lion, Y. & Van de Vorst, A. Quantum yield of singlet oxygen production by xanthene derivatives. Photochem. Photobiol. 37, 271–278 (1983). 24. Yogo, T., Urano, Y., Ishitsuka, Y., Maniwa, F. & Nagano, T. Highly efficient and photostable photosensitizer based on BODIPY chromophore. J. Am. Chem. Soc. 127, 12162–12163 (2005). 25. Lindig, B.A., Rodgers, M.A.J. & Schaap, A.P. Determination of the Lifetime of Singlet Oxygen in D2O Using 9,10-Anthracenedipropionic Acid, a Water-Soluble Probe. J. Am. Chem. Soc. 102, 5590–5593 (1980). 26. Davila, J. & Harriman, A. Photosensitized oxidation of biomaterials and related model compounds. Photochem. Photobiol. 50, 29–35 (1989). 27. Ogilby, P.R. Singlet oxygen: there is indeed something new under the sun. Chem. Soc. Rev. 39, 3181–3209 (2010). 28. Shibuya, T. & Tsujimoto, Y. Deleterious effects of mitochondrial ROS generated by KillerRed photodynamic action in human cell lines and C. elegans. J. Photochem. Photobiol. B 117, 1–12 (2012). 29. Qi, Y.B., Garren, E.J., Shu, X., Tsien, R.Y. & Jin, Y. Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG. Proc. Natl. Acad. Sci. USA 109, 7499–7504 (2012). 30. Williams, D.C. et al. Rapid and permanent neuronal inactivation in vivo via subcellular generation of reactive oxygen with the use of KillerRed. Cell Rep. 5, 553–563 (2013). 31. Patterson, M.S., Wilson, B.C. & Graff, R. In vivo tests of the concept of photodynamic threshold dose in normal rat-liver photosensitized by aluminum chlorosulfonated phthalocyanine. Photochem. Photobiol. 51, 343–349 (1990). 32. Schäfer, M. et al. Systematic study of parameters influencing the action of Rose Bengal with visible light on bacterial cells: comparison between the biological effect and singlet-oxygen production. Photochem. Photobiol. 71, 514–523 (2000). 33. Kuimova, M.K., Yahioglu, G. & Ogilby, P.R. Singlet oxygen in a cell: spatially dependent lifetimes and quenching rate constants. J. Am. Chem. Soc. 131, 332–340 (2009). 34. Tilly, J.L. & Tilly, K.I. Inhibitors of oxidative stress mimic the ability of follicle-stimulating-hormone to suppress apoptosis in cultured rat ovarian follicles. Endocrinology 136, 242–252 (1995). 35. Simon, H.U., Haj-Yehia, A. & Levi-Schaffer, F. Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 5, 415–418 (2000). 36. Zhao, H. et al. Superoxide reacts with hydroethidine but forms a fluorescent product that is distinctly different from ethidium: potential implications in intracellular fluorescence detection of superoxide. Free Radic. Biol. Med. 34, 1359–1368 (2003). 37. Fuchs, Y. & Steller, H. Programmed cell death in animal development and disease. Cell 147, 742–758 (2011). 38. Dodd, A., Curtis, P.M., Williams, L.C. & Love, D.A. Zebrafish: bridging the gap between development and disease. Hum. Mol. Genet. 9, 2443–2449 (2000). 39. Lieschke, G.J. & Currie, P.D. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet. 8, 353–367 (2007). 40. Curado, S., Stainier, D.Y. & Anderson, R.M. Nitroreductase-mediated cell/ tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies. Nat. Protoc. 3, 948–954 (2008). 41. Poss, K.D., Wilson, L.G. & Keating, M.T. Heart regeneration in zebrafish. Science 298, 2188–2190 (2002). 42. Kikuchi, K. et al. Primary contribution to zebrafish heart regeneration by gata4(+) cardiomyocytes. Nature 464, 601–605 (2010). 43. Jopling, C. et al. Zebrafish heart regeneration occurs by cardiomyocyte dedifferentiation and proliferation. Nature 464, 606–609 (2010).
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ONLINE METHODS Materials. We purchased 4,4-dimethylaniline and 3,5-diiodo-4hydroxybenzaldehyde from VWR International; anthracene-9, 10-dipropionic acid disodium salt (ADPA), catalasepolyethyl ene glycol and SODpolyethylene glycol from Sigma-Aldrich; tetrasulfonated aluminum phthalocyanine (AlPcS4) from Frontier Scientific; and propidium iodide (P3566), Hoechst dye 33342, a live/dead cell viability/cytotoxicity kit (L-3224) and dihydroethid ium (hydroethidine) (D11347) from Invitrogen. 1H NMR spec troscopy and 13C NMR spectroscopy data were recorded with a Bruker Avance at 300 MHz and 500 MHz. Mass spectra were obtained with a Thermo-Fisher LCQ ESI/APCI ion trap mass spectrometer. Final products were purified with silica, neutral alumina and reverse-phase chromatography; purity was tested by ultrahigh-performance liquid chromatography using a diode array absorbance detector. Raw absorbance values of free dyes and dye-FAP complex were measured on a PerkinElmer Lambda45 spectrophotometer. Fluorogenic enhancement was measured in 96-well microplates on a Tecan Infinite plate spectrometer. Corrected emission spectra were acquired on a Quantamaster monochromator fluorimeter (Photon Technology International). Live larval zebrafish imaging was done with an EVOS FL manual microscope. Cell culture. HEK 293 cells (Sigma-Aldrich) were cultured in DMEM (Thermo Fisher) supplemented with 10% FBS (FisherBrand). To construct cell lines expressing FAP or KillerRed in different cellular compartments, we transfected HEK cells with pcDNA plasmid using Lipofectamine 2000 (Invitrogen). The transfected cells were selected by G418 for 2 weeks and then sorted into single clones for stable cell line generation. In pcDNA, sequence encoding dL5** was cloned downstream of the Kozak sequence for cytosolic expression, upstream of the sequence encoding PDGFR-derived transmembrane domain for membrane surface localization, downstream of a nuclear localization sig nal for nuclear targeting and downstream of a COXIV/COXVIII signal for mitochondrial labeling as previously reported44. We routinely monitored for mycoplasma contamination in cell culture by fluorescent Hoechst 33258 stain. HEK 293 cells were selected for this study because they are widely used in many biology-related studies as a model cell line. Cloning and purification. The Escherichia coli bacterial strain MACH1-T1 (Invitrogen) was used as the host for cloning. FAP dL5** was cloned into pET21 vector with sequence encoding a 6× His tag and sequence encoding an HRV protease site at the 5′ end as previously reported44. Determination of singlet-oxygen quantum yield. PBS (pH 7.4) buffer containing optically matched samples (AlPcS 4, MG-2I, MG-2I–dL5** and MG-ester–dL5**) and 0.1 mM ADPA were illuminated by a 669-nm light LED light box. The fluorescence intensity of ADPA was monitored with an excitation of 374 nm and an emission of 402 nm using a Tecan Infinite M1000 plate spectrometer. The normalized fluorescence intensity was plot ted against exposure time at 669 nm, and Φ∆ was then calculated from the slopes using equation (1) (ref. 25). Here kr is the chemi cal quenching of singlet oxygen by ADPA and kd is the constant of deactivation of singlet oxygen via the solvent; both values are doi:10.1038/nmeth.3735
constant regardless of the photosensitizer being used.[A] is the ADPA concentration, and Iabs is the absorbance at excitation wavelength. −
d[A] k = I absΦ ∆ r [A] dt kd
(1)
Determination of quantum yields. We determined quantum yields by comparing integrated emission spectra of fluorogenFAP complexes to those of reference dyes. Corrected emission spectra were taken on a Quantamaster monochromator fluorim eter (Photon Technology International). Cy5 was used as standard for MG-2I–dL5** complex. The emission spectra (620−820 nm) of a set of five concentrations were integrated and plotted against absorbance at 600 nm, and ΦF was then calculated from the slopes using equation (2). 2 Grad X hX Φ X = Φ ST 2 GradST hST
(2)
where ΦX is the sample quantum yield, ΦST is the standard quantum yield, GradX is the sample slope, GradST is the standard slope, ηX is the refraction index of the sample solvent, and ηST is the refraction index of the standard solvent. Photoconversion of 3,3′-diaminobenzidine (DAB). 100-µl sam ples (AlPcS4, MG-2I–dL5**, MG-ester–dL5** and MG-2I) in pH 7.4 sodium cacodylate buffer with the same optical density at 669 nm were mixed with 100 µl of 1 mg/ml DAB in pH 7.4 sodium cacodylate buffer for 5 min. Then all samples were transferred to a 96-well plate and subjected to 2 h of illumination using the LED light box. Pictures were taken at 0 min, 10 min, 30 min, 60 min and 120 min. Determination of dissociation constant. 5 nM of purified dL5** was precomplexed with a series of dye dilutions, and the fluores cence intensity was measured. The fluorescence of dye without protein at each concentration was also obtained and subtracted as background. On the basis of the plateau intensity in the binding curve calculated from a hyperbolic binding model, we normalized the results to the concentration of fluorogen-FAP complex. Using these scaled data, we calculated the dissociation constant from a one-site binding-ligand depletion model in GraphPad Prism 6. Microscopy. Cells were seeded onto 35-mm glass-bottom dishes (MatTek) one day before imaging and maintained overnight at 37 °C in OptiMEM (Invitrogen) with 10% FBS. Cell images were acquired on an Andor Revolution XD system with a spinning disk and processed with ImageJ. Imaging specifications were as follows: c405, excitation (ex) 405 nm, emission (em) 435/25 nm; c488, ex 488 nm, em 515/25 nm; c560, ex 560 nm, em 585/25 nm; c640, ex 640 nm, em 670/25 nm. Differential interference contrast imaging conditions were optimized for image contrast and kept the same for each experiment. Dead/live cell viability assay. After illumination by a laser source from an Andor confocal microscope, cell medium containing the dye was replaced with 2 µM calcein AM and 4 µM EthD-1 in PBS (pH 7.4) buffer and allowed to stain for 30 min at room temperature. nature methods
Then we conducted the two-color fluorescence cell viability test with the cells using the same microscope (40× objective; for calcein AM, 7.5% laser power; for EthD-1, 560-nm excitation, 610/20-nm emission filter, 35% laser power).
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Light-induced cytotoxicity experiment and cell death count. Cells were incubated with MG-ester, MG-2I or fluorogen-FAP complex for 30 min before illumination. A laser and a light box were used as illumination sources, and different light doses were achieved with different light intensities (using 10 cm × 10 cm neutral density filters placed over the sample in the light box or by varying the laser power on the microscope; power was measured at the sample for each condition) or illumination times. For counting of dead cells, the medium was replaced with PBS containing 2 µM propidium iodide (PI) and 8 µM Hoechst after illumination. Allowing 30 min of staining, we calculated the ratio of cell death from the count of cells stained by PI (dead cells) versus those stained by Hoechst (total cells). Detection of ROS in nucleus by dihydroethidium. HEK cells with nuclear-targeted dL5** were incubated with 400 nM of dyes and then exposed to 2 µM dihydroethidium (diluted from freshly prepared ethanol stock solution) for 10 min. Then cells were illuminated for 2 min (60× objective, 640 nm, 2.43 W cm−2). In the presence of superoxide, DHE is oxidized to 2-hydroethid ium (EOH), which binds to DNA and becomes fluorescent. The generation of EOH was monitored with an excitation of 488 nm and emission of 535/25 nm immediately after photosensitizing illumination. Discussion of fold of activation of singlet-oxygen generation. To estimate the fold of activation of singlet-oxygen generation, we made a concentration series of free MG-2I (in dPBS) and MG2I–dL5** (in 1:9 H2O:dPBS) and plotted the absorbance at 669 nm against the ADPA bleaching. However, in the same absorb ance range, MG-2I–dL5** is much faster in bleaching ADPA than MG-2I, so we used different light exposure times for different samples (60-s exposure time for MG-2I and 10-s exposure time for MG-2I–dL5**). Assuming the bleaching rate does not change within 60 s for MG-2I, we estimated the change in singlet-oxygen generation as >~100-fold on the basis of the ratio of the slopes, scaled by a factor of 6 to account for the sixfold longer exposure of the MG-2I. Owing to the change of absorption maximum upon binding, the OD666 of an equimolar solution of MG-2I dye is 4.5-fold lower than that of an MG-2I–dL5** complex (0.022 versus 0.097). Taken together, the overall activation for singletoxygen production upon binding is given by equation (3), where Rε and RΦ∆ are the ratio of extinction coefficients at the excitation maximum for the complex and the ratio of the singlet-oxygen quantum yield, respectively. R∆ = Re RΦ ∆
(3)
Generation of transgenic zebrafish (Tg(myl7:MBIC5-mCer3)). The zebrafish experiments were performed according to a proto col approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh that conforms to NIH guidelines. MBIC5-mCer3 was cloned into the pISceI vector that contained the Myl7 promoter. 20 pg of pI-SceI-Myl7:MBIC5-mCer3 nature methods
plasmid DNA was injected into the one-cell embryo with I-Sce 1 restriction enzyme as described45. These F0 injected embryos were raised to adulthood and incrossed to identify transgenic founders. We identified three founder lines that expressed mCer3 at 28 h after fertilization in the beating cardiac tube. Tg(myl7:MBIC5-mCer3)pt22 and Tg(myl7:MBIC5-mCer3)pt23 were used in this study. In the embryo ablation studies, cardiac mCer3+ embryos at 28 h post-fertilization were removed from their chorion and treated with 500 nM MG-ester or MG-2I at 48 h post-fertilization for 3 h. The embryos were then illuminated for 12 min using a 659-nm laser (242 mW cm−2). Zebrafish maintenance and retro-orbital injections. The zebrafish experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh that conforms to NIH guidelines. Adult (6−18 months old) wild-type AB* and trans genic Tg(myl7:MBIC5-mCer3)pt23 zebrafish were anesthetized for 3 min in Tricaine (0.168 mg/l of MS222; Sigma) and retro-orbitally injected with 3 µl of 12 µM MG-ester (0.06 mg/kg) or MG-2I (0.09 mg/kg) solution dissolved in filtered 1× PBS. Retro-orbital injection was performed as previously described46. We returned the fish to water and stimulated them to breathe by vigorously squirting water over their gills with a pipette. 20−30 min after retro-orbital injection, five fish were placed in a 100-ml beaker containing 50 ml of fish water and illuminated with a custombuilt light box emitting at 669 nm for 30 min. The fish water in the beaker was replaced with fresh water every 10 min. The fish were then returned to the water system and monitored daily. Fish were killed at a determined time point after the illumination, and their hearts, livers and intestines were extracted and fixed in 4% paraformaldehyde for histochemical analysis. Immunohistochemistry. Larval zebrafish were fixed 24 h after illumination in 4% paraformaldehyde overnight. Whole-mount apoptosis was measured via TUNEL assay (Invitrogen, C10617) according to the manufacturer’s instructions. mCerulean3 was labeled by a primary rabbit anti-GFP antibody (Abcam, ab6556); the secondary antibody in use was donkey anti-rabbit IgG H&L (Alexa Fluor 555) (Abcam, ab150074). Fixed hearts, livers and intestines were cryopreserved in 30% sucrose in PBS before immersion in embedding medium (Leica). Fourteen-micrometer cryosections were stained with Acid Fuchsin Orange G (AFOG) as previously described47. Images were captured with a Leica MZ16 microscope and a Q Imaging Retiga 1300 camera. Apoptosis was measured via TUNEL assay (Millipore; S7165) according to the manufacturer’s instructions. Primary antibodies used for immu nostaining were anti-Mef2c (Santa Cruz Biotechnology; sc-313) (1:500) and anti-PCNA (Sigma; P8825) (1:1,000). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG peroxidase conjugate (Thermo Fisher; A-11008) (1:1,000) and Alexa Fluor 594 goat anti-mouse IgG (H + L) (Thermo Fisher; A-11036) (1:1,000). Slides were mounted with Vectashield mount ing medium with DAPI (Vector Laboratories; H-1200). Images were taken with a Zeiss LSM 700 confocal microscope. For each experiment, two to six sections were analyzed for each heart, liver and intestine. The cardiomyocyte proliferation index was calculated as the percentage of the number of Mef2c+PCNA+ cells divided by the number of total Mef2c+ cells. We measured the doi:10.1038/nmeth.3735
mean ± s.e.m. For one-way ANOVA, Tukey post-hoc tests were performed with multiple comparison of the mean for each group. P < 0.05 was considered significant.
Statistical analysis. Sample sizes were based on practical consid erations (for example, the number of fish that can fit in a single 100-ml beaker or the number of samples required to fill a 96-well plate) to allow effective observations and to avoid unnecessary waste of resources. No systematic randomization or blinding was used in sample allocation or analysis. For animal studies, transgenic fish were assigned to experimental groups randomly. Samples were excluded only for technical failures (i.e., perva sive nonspecific staining). Statistical significance was analyzed by one-way ANOVA or unpaired t-test, and data are shown as
44. Telmer, C.A. et al. Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins. ACS Chem. Biol. 10, 1239–1246 (2015). 45. Molina, G.A., Watkins, S.C. & Tsang, M. Generation of FGF reporter transgenic zebrafish and their utility in chemical screens. BMC Dev. Biol. 7, 62 (2007). 46. Pugach, E.K., Li, P., White, R. & Zon, L. Retro-orbital injection in adult zebrafish. J. Vis. Exp. 34, 1645 (2009). 47. Missinato, M.A., Tobita, K., Romano, N., Carroll, J.A. & Tsang, M. Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration. Cardiovasc. Res. 107, 487–498 (2015).
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myofiber area by imaging four sections for each heart stained with AFOG and quantifying the total ventricular area and the cardiac tissue using ImageJ software (NIH).
doi:10.1038/nmeth.3735
nature methods
A genetically targetable near-infrared photosensitizer
© 2016 Nature America, Inc. All rights reserved.
Jianjun He1, Yi Wang2, Maria A Missinato3, Ezenwa Onuoha3, Lydia A Perkins2, Simon C Watkins4, Claudette M St Croix4, Michael Tsang3 & Marcel P Bruchez1,2,5 Upon illumination, photosensitizer molecules produce reactive oxygen species that can be used for functional manipulation of living cells, including protein inactivation, targeted-damage introduction and cellular ablation. Photosensitizers used to date have been either exogenous, resulting in delivery and removal challenges, or genetically encoded proteins that form or bind a native photosensitizing molecule, resulting in a constitutively active photosensitizer inside the cell. We describe a genetically encoded fluorogen-activating protein (FAP) that binds a heavy atom−substituted fluorogenic dye, forming an ‘on-demand’ activated photosensitizer that produces singlet oxygen and fluorescence when activated with near-infrared light. This targeted and activated photosensitizer (TAPs) approach enables protein inactivation, targeted cell killing and rapid targeted lineage ablation in living larval and adult zebrafish. The near-infrared excitation and emission of this FAP-TAPs provides a new spectral range for photosensitizer proteins that could be useful for imaging, manipulation and cellular ablation deep within living organisms.
Light provides precise spatiotemporal control of biological pro cesses when combined with suitable genetic constructs or chemical reagents1–3. Photosensitizer dyes and proteins exploit absorbed light to create short-lived reactive oxygen species (ROS) that can mediate biological effects at the target site4,5. Traditional photosen sitizers such as methylene blue have no selectivity, and off-target phototoxicity produced during light exposure limits the applica tions6,7. Photosensitizers with improved efficiency of ROS gen eration, photostability and near-infrared (NIR) light absorption enhance optical tissue penetration and allow real-time fluorescence visualization8. More recently, genetically targeted photosensitiz ers, such as FlAsH and ReAsH, KillerRed and MiniSOG9–12, have been developed to improve targeting and specificity in living cells. The genetic fusion approach allows the photosensitizing protein to produce ROS at the target, selectively inactivating it through chromophore-assisted light inactivation (CALI) of directly linked proteins. These photosensitizer proteins require a very high light dose to reach effective inactivation or cell killing, and the spectral properties of these sensitizers overlap with those of
biological chromophores, resulting in some ROS generation even in the absence of the photosensitizer proteins. Photosensitizers with far-red and/or NIR light excitation wavelengths (>620 nm) are required for deep tissue applications and to avoid photosensi tization by endogenous chromophores, yet no genetically targeted photosensitizers are available in this spectral range. Activation of a photosensitizer at a target site improves specifi city by minimizing the damage to nontargeted tissues13. Current activatable photosensitizers either are responsive to local envi ronmental changes, such as changes in pH, or contain a quench ing group that is cleaved, increasing photosensitizer activity14–16. Although activation increases the ROS production by 10–50-fold, these tools still show some off-target effects from nonspecifically localized materials. Thus, selectively targeting and activating a photosensitizer remains an important goal. To target and activate an ROS-generating photosensitizer, we exploited a genetically targetable and efficient FAP17. FluorogenFAP complexes have been adapted to a number of applications such as single-molecule imaging18, physiological pH measure ments19 and protein detection as recombinant affinity probes 20. FAPdL5** (previously MBIC5; referred to hereinafter as dL5**) is a 25-kDa binder for malachite green (MG) derivatives that func tions throughout living cells with thousands-fold fluorescence activation and a low-picomolar dissociation constant17,21. We reasoned that suppression of the nonradiative relaxation, which enhances fluorescence in MG fluorogens, could be exploited to modify other photochemical properties, in particular intersystem crossing, when combined with chemically tailored fluorogens22. We prepared an iodine-substituted MG analog (Supplementary Note 1) with low free fluorescence and ROS generation that binds to dL5**, producing an NIR light–excitable fluorescent complex with high singlet oxygen (1O2) quantum yield (Fig. 1a). We used the dye-protein complex to photo-inactivate fused proteins; to photosensitize cells expressing dL5** at the plasma membrane, cytosol, mitochondrial matrix or nucleus in culture; and to ablate cardiac cells in living larval and adult zebrafish expressing dL5** in the cytoplasm. The free dye showed no photosensitization in cells or zebrafish, indicating that the iodinated MG analog is a potent, NIR light–excitable FAP-TAPs.
1Department of Chemistry, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. 2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
Pennsylvania, USA. 3Department of Developmental Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA. 4Center for Biologic Imaging, Department of Cell Biology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 5Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA. Correspondence should be addressed to M.P.B. ([email protected]). Received 19 August 2015; accepted 4 December 2015; published online 25 january 2016; doi:10.1038/nmeth.3735
nature methods | ADVANCE ONLINE PUBLICATION |
RESULTS MG-2I–dL5** as a 1O2-specific photosensitizer Heavy-atom substitutions at some sites on the MG chromo phore, which are expected to increase intersystem crossing23,24, retained high-affinity dL5** binding and fluorescent properties (Supplementary Fig. 1). A di-iodinated derivative of the cellpermeable MG-ester fluorogen (MG-2I) showed dramatically increased photogenerated 1O2 when bound to dL5** (FAP-TAPs). The iodination redshifted the major absorption band, moving the excitation maximum of the complex into the NIR light range (666 nm; Supplementary Table 1 and Fig. 1b). We evaluated the generation of 1O2 via bleaching of anthracene-9,10-dipropionic acid (ADPA), a commonly used 1O2 scavenger25. Relative to aluminum phthalo cyanine tetrasulfonate (AlPcS4)26, the FAP-TAPs 1O2 quantum yield (Φ∆) was 0.13 (Fig. 1c), whereas generation of 1O2 from the MG-ester–dL5** or free TAPs dye was not detectable under the same conditions. ADPA bleaching by FAP-TAPs, evaluated using filters for various fluorescent proteins (Supplementary Fig. 2a,b), revealed little FAP-TAPs activation under YFP and RFP excitation, suggesting that these fluorescent proteins are optimal for use with FAP-TAPs. In deuterated PBS buffer, the bleaching rate of ADPA by FAP-TAPs was enhanced. As D2O is known to increase the lifetime of 1O2 (4 µs to 52 µs) (ref. 27) while having little effect on other ROS, the increased ADPA bleaching is consistent with 1O production (Supplementary Fig. 3a). Diaminobenzidine was 2 photo-oxidized to produce brown precipitate in vitro, which is potentially useful for correlative light and electron microscopy (Supplementary Fig. 3b). When we compared ADPA bleaching in deuterated PBS between MG-2I and FAP-TAPs (Supplementary Fig. 3c), we noted that the 1O2 generation of the MG-2I dye increased up to 450-fold upon binding to dL5**. This ensures that free dye is nonphotosensitizing, a critical difference with respect to other dye-targeting or photosensitizer-activating approaches. CALI of the PLC δ1 Plekstrin homology (PH) domain To assess the utility of FAP-TAPs for targeted protein inactiva tion, we compared membrane release of EGFP-PH-KillerRed and
FAP-TAPs mediated cellular photoablation We evaluated light-induced cytotoxicity of FAP-TAPs on HEK cells expressing a cell surface−anchored FAP (TM-dL5**). We also incubated a mixed population of TM-dL5**−expressing HEK cells and wild-type HEK cells with 400 nM dye for 30 min and then illuminated it for 1 min with a continuous laser source (40× objective, 640-nm excitation, 0.76 W cm−2). Only fluorescently labeled TM-dL5**−expressing HEK cells treated with MG-2I and light were stained dead within 30 min using a live/dead cell viabil ity kit, whereas the wild-type HEK cells in the illumination field remained alive and metabolically active (Fig. 3a). The labeled cells began to lose cell morphology with swelling and blebbing within a very short period after illumination (Supplementary Video 1). MG-ester bound to TM-dL5**−expressing HEK cells,
b
+
In vivo imaging
S1
S1
606 nm
lC
633 nm S0
S0 Heavy-atom effect
Protein trafficking
+
CALI 1
+ S1 628 nm
S1
FAP
666 nm
lC
S0
S0 TAPs
pH, ion sensor
Fluorescence
FAP-TAPs
ISC
T1
O2
Cell killing ROS signaling
Photosensitization O2
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EGFP-PH-dL5** fusion proteins upon suitable illumination in HEK293 (HEK) cells9. When the membrane-bound PH domain from PLC δ1 is inactivated by CALI, it is released to the cyto plasm, increasing the ratio of cytoplasmic to membrane EGFP. After 5 min of illumination with a 560-nm laser (60× objective, 2.03 W cm−2), the cytoplasm-to-membrane signal ratio increased by 37% under KillerRed-mediated CALI (Fig. 2), similar to pre viously reported observations. The fluorescence of KillerRed was bleached (>75%) after 1 min of illumination. In contrast, FAP-TAPs illumination resulted in a 33% increase in ratio after 10 s of 640-nm laser illumination (60× objective, 2.07 W cm−2). Further illumination of FAP-TAPs induced no additional change in EGFP ratio, but noticeable morphology changes and minor photobleach ing were observed. We evaluated the potential collateral dam age by coexpressing EGFP-PH with PH-KillerRed or PH-dL5**. Both KillerRed and FAP-TAPs induced similar inactivation of EGFP-PH in proportion to the amount of target inactivation, indicating that the FAP-TAPs are spatially restricted similarly to KillerRed under CALI conditions. The high efficacy and specifi city of FAP-TAPs−mediated protein inactivation could facilitate well-controlled experiments based on acute protein inactivation, when proximal collateral damage can be properly controlled.
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Figure 1 | Characterization of ROS generation by FAP-TAPs. (a) Mechanism of ROS generation by FAP-TAPs. 0 IC, internal conversion by molecule’s free rotation; ISC, intersystem crossing. (b) Normalized excitation (dashed lines) and emission (solid lines) spectra of MG-ester and MG-2I binding to dL5**, where 500 nM 0 20 40 60 Time (s) fluorogen was complexed with 3 µM dL5** and the fluorescence intensity was individually normalized to the peak maxima. (c) 1O2 generation by MG-2I–dL5** assessed on the basis of ADPA bleaching, where bleaching of ADPA fluorescence was monitored at 374-nm excitation and 402-nm emission as a function of the duration of 669-nm light exposure. AlPcS 4 was used as standard for the 1O2 generation (Φ∆ = 0.34). Optically matched solutions of MG-2I–dL5** and AlPcS4 at 669 nm were used. n = 4; data are shown as mean ± s.e.m.; in instances of very small s.e.m., error bars overlap points and may not be visible. | ADVANCE ONLINE PUBLICATION | nature methods
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Figure 2 | FAP-TAPs mediated light-induced protein inactivation of the PLC δ1 PH domain. (a) Outline of the experimental design. We constructed EGFPPH-KillerRed or EGFP-PH-dL5** triple fusion protein to evaluate the effectiveness of specific protein inactivation from KillerRed and FAP-TAPs (top). EGFP-PH was coexpressed with PH-KillerRed or PH-dL5** to allow estimation of the collateral damage from KillerRed and FAP-TAPs (bottom). (b) Change in EGFP cytoplasm-to-membrane ratio after illumination of MG-2I with EGFP-PH-dL5** and of MG-ester with EGFP-PH-dL5** and EGFP-PH-KillerRed (solid lines). Dashed lines represent the corresponding collateral damage from coexpressed proteins. n = 8; mean ± s.e.m. (c) Representative EGFP fluorescence images from each condition, with imaging at the indicated intervals. Scale bar, 5 µm. Illumination conditions were as follows: KillerRed, 560-nm laser, 60× objective, 2.03 W cm−2; MG-ester–dL5** and MG-2I–dL5**, 640-nm laser, 60× objective, 2.07 W cm −2.
MG-2I−treated wild-type HEK cells and nontargeted FAP-TAPs added to the medium showed no apparent cytotoxicity or photo toxicity upon illumination (Fig. 3a), suggesting that close contact of FAP-TAPs on target cells was required for effective delivery of ROS owing to the very short radius of action of 1O2. These results established the dual requirements of targeted FAP expression and TAPs binding for efficient photosensitization. We observed lim ited self-bleaching ( 0.05, one-way analysis of variance (ANOVA) using MG-2I–only group as a control) suppression of phototoxicity in cells incubated with high concentrations of catalase (peroxide quencher; 1,000 U ml−1) or superoxide dismutase (superoxide quencher; 500 U ml−1)34. Results from these quenching experi ments along with the in vitro ADPA bleaching assay indicate that 1O generated from FAP-TAPs was the primary ROS mediating 2 the observed phototoxic responses. 1O is known to react with nearby molecules to produce 2 secondary peroxide species as initial products, which then create other reactive species and lead to cytotoxicity 35. Thus chemical sensors can be used to identify the cascade generation of ROS after photosensitization. We used dihydroethidium (DHE), which reacts with superoxide (O2−) to form a DNA-intercalating nature methods | ADVANCE ONLINE PUBLICATION |
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fluorescent species (2-hydroxyethidium, 490-nm excitation and 567-nm emission), to detect ROS in HEK cells expressing FAP-TAPs in the nucleus36. We observed significant (P < 0.0001, one-way ANOVA) ROS-dependent activation of DHE in the nucleus only when MG-2I with FAP and light were present, and not in identical but unilluminated cells (P > 0.05) or FAP-expressing cells labeled and illuminated in the presence of MG-ester dye (P > 0.05) (Supplementary Fig. 8). FAP-TAPs mediated cardiac ablation in larval zebrafish In vivo cellular ablation has been used to study development and regeneration37. Zebrafish (Danio rerio) is a vertebrate model organ ism that is optically transparent, is easily genetically manipulated and shows conserved developmental processes with respect to other vertebrates38,39. Lineage ablation can be accomplished in zebrafish by tissue-specific expression of a bacterial nitroreductase and sub sequent treatment for ~12−24 h with metronidazole, a prodrug that is reduced to a DNA-synthesis inhibitor in nitroreductasepositive cells, causing cell death40. As zebrafish develop rapidly, 12−24 h of treatment limits the temporal resolution of lineage ablation studies. Because cultured cells are ablated rapidly, we evaluated rapid cellular ablation in living larval zebrafish. We produced transgenic zebrafish lines that expressed a cytoplasmic dL5**-mCerulean3 (dL5**-mCer3) tandem protein under control of the heart-specific myosin light chain 7 (myl7, also known as cmlc2) promoter, Tg(myl7:MBIC5-mCer3). We confirmed expression of dL5**-mCer3 by fluorescence imaging of mCer3 and MG-ester–dL5** or MG-2I–dL5** complexes, which were restricted to the beating heart (Supplementary Fig. 9a and Supplementary Video 3). To test the effectiveness of FAP-TAPs, we treated embryos at 48 h after fertilization with 500 nM dye (MG-2I or MG-ester) for 3 h and then subjected them to 12 min of laser illumination (659 nm, 242 mW cm−2). Immediately after illu mination, Tg(myl7:MBIC5-mCer3)pt22 (herein referred to as Tgpt22) larvae treated with MG-2I showed no sign of heartbeat or blood circulation, whereas MG-ester−treated Tgpt22 larvae and wild-type larvae treated with MG-2I were normal (Supplementary Video 4). | ADVANCE ONLINE PUBLICATION | nature methods
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Figure 3 | Phototoxicity of FAP-TAPs in HEK cells expressing surfacetargeted FAP. (a) Images of cells that were labeled with 400 nM of the indicated dye for 30 min before illumination (without removal of the unbound dye), taken before laser illumination (top; merge of c640 (fluorogen-FAP, red) and differential interference contrast (DIC)). Live/dead cell viability was assayed 30 min after illumination (bottom; merge of c488 (live cells; cyan), c560 (dead cells; yellow) and DIC). Scale bar, 10 µm (applies to all images in panel). (b) Viability of cocultured TM-dL5**−expressing HEK cells (TM-dL5** HEK) and wild-type HEK cells (WT HEK) incubated with MG-2I and subjected to 0, 30, 60 and 120 s of illumination (640 nm, 40× objective, 0.76 W cm−2). n = 8, one-way ANOVA. Tukey post-hoc tests were performed with multiple comparisons; data are shown as mean and s.e.m. ****P < 0.0001; ns, not significant. (c) Viability of TM-dL5**−expressing cells labeled with MG-2I under variable light intensity (blue) and variable illumination duration (red, 0.089 W cm −2 light intensity with 2, 5, 7, 10, 12 and 15 min of illumination), MG-ester with TM-dL5**−expressing HEK cells (dotted black), and MG-2I with wild-type HEK cells (dashed-dotted green). Cells were illuminated using an LED light box. Cells were stained with propidium iodide (dead) and Hoechst (total); more than 300 cells were counted for each data point (n = 4, mean and s.e.m.; in instances of very small s.e.m., error bars overlap points and may not be visible).
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At 24 h post-illumination (h.p.i.), we found that illuminated MG-2I−treated Tgpt22 larvae exhibited cardiac edema and lower mCer3 fluorescence (Fig. 4a). At 96 h.p.i., these larvae had smaller eyes and larger yolk than larvae in other groups did (Supplementary Fig. 9b). At 96 h.p.i., we detected no mCer3+ cells in illuminated MG-2I−treated Tgpt22 larvae, indicating that acute loss of myl7 lineage cells was not renewed later in development. We used deoxynucleotidyl transferase−mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assay with anti-GFP staining to assess on-target and off-target cell death in wholemount Tgpt22 larvae at 24 h.p.i. (Supplementary Fig. 10a). In MG-2I–treated larvae exposed to 12 min of laser illumination, we saw fewer mCer3+ cells and more TUNEL+mCer3+ cells, without any increase in TUNEL+mCer3− cell death compared with that in MG-ester–treated larvae exposed to 12 min of laser illumination (Supplementary Fig. 10b,c). The fraction of TUNEL+mCer+ cells in MG-2I–treated larvae was greater than that in MG-ester–treated larvae, both of which were exposed to 12 min of laser illumination (31% versus 4.4%, P < 0.01) (Supplementary Fig. 10d). A shorter illumination time (MG-2I–treated larvae exposed to 4 min of illu mination) resulted in decreased specific cytotoxicity, confirming the light-dose dependence of the FAP-TAPs ablation. Cardiac photoablation in adult zebrafish demonstrated the utility of FAP-TAPs in deep tissue. Tg(myl7:MBIC5-mCer3)pt23 (herein referred to as Tgpt23) zebrafish uniformly express mCer3dL5** in the heart at the adult stage (Supplementary Fig. 11a). We retro-orbitally injected zebrafish with dye and illuminated them for a total of 30 min using the LED light box (2.5 W cm−2 illu mination from below the fish) (Supplementary Fig. 11b). At 3 d post-illumination (d.p.i.), we assessed cell death using TUNEL staining of hearts, livers and intestines. We observed increased cell death only in the hearts of transgenic zebrafish injected with MG-2I, compared to all the control groups (Fig. 4b) and nontar geted tissues (Supplementary Fig. 11c), demonstrating effective and specific ablation in deep tissue. To confirm cardiomyocyte ablation, we visualized intact cardiac muscle at 5 d.p.i. with Acid Fuchsin Orange G. Hearts from the Tgpt23 fish treated with MG-2I
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showed damaged cardiac structure and decreased ventricular car diac muscle compared to the controls (Fig. 4c). We observed no structural defects in livers or intestines (Supplementary Fig. 11d). Damaged cardiac muscle can regenerate through proliferation of pre-existing cardiomyocytes41–43. At 5 d.p.i., only illuminated trans genic zebrafish injected with MG-2I had significantly increased numbers of proliferating cardiomyocytes compared to similarly treated wild-type zebrafish (P < 0.001, MG-2I–treated Tgpt23 versus MG-2I–treated wild-type; P > 0.05 MG-ester–treated Tgpt23 versus MG-ester–treated wild-type) (Fig. 4d), showing that the ablation inflicted sufficient damage to induce a regenerative response. DISCUSSION We have demonstrated a two-component targeted and activated photosensitizing approach that enables specific NIR light− mediated protein inactivation and phototoxicity in FAP-expressing cells and living transgenic organisms. The TAPs dye (MG-2I) and FAP (dL5**) had no photosensitizing effects until they associated to form the FAP-TAPs. Photosensitization is primed by the on-demand addition of TAPs dye to the FAP-expressing cells rather than removal of quenching groups by cellular processes. This method allows temporally, spatially and subcellularly con trolled, target-specific generation of 1O2. The flexible genetic targeting strategy of the FAP along with efficient activation of ROS generation makes FAP-TAPs an immediately useful tool for targeted cellular ablation and subcellular protein inactivation, and
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Figure 4 | FAP-TAPs induced photo-ablation of cardiac function. (a) Merge of DIC and mCer3 fluorescence (cyan) showing phenotype development from 0 h.p.i. to 96 h.p.i. of larval zebrafish (n = 20 for each group): Tg(myl7: MBIC5-mCer3)pt22 (Tgpt22) larvae treated with MG-ester (MG-ester/Tgpt22), Tgpt22 larvae treated with MG-2I (MG-2I/Tgpt22) and wild-type larvae treated with MG-2I (MG-2I/WT). In the MG-2I/Tgpt22 group, larvae developed visible defects: large cardiac edema, small eyes and collapsed, nonfunctional heart chambers. In both control groups, development proceeded normally. Scale bar, 1,000 µm (applies to all images in panel). (b) Images of FAP-TAPs– photoinduced cardiac damage in adult zebrafish hearts extracted at 3 d.p.i., subjected to TUNEL and stained with DAPI. Scale bar, 100 µm (applies to all images in panel). (c) Acid Fuchsin Orange G (AFOG) staining of hearts at 5 d.p.i. showing the damaged cardiac structure in transgenic fish injected with MG-2I, as marked by reduced myocardium (brown labeling). Scale bar, 100 µm (applies to all images in panel). (d) Mef2c (cardiomyocyte) and PCNA (proliferation) staining at 5 d.p.i. showing enhanced cardiomyocyte (CM) proliferation (yellow arrowheads) in transgenic fish injected with MG-2I. Scale bar, 100 µm. n = 9 for all groups; one-way ANOVA with Tukey post-hoc tests was performed with multiple comparisons of the mean for each group. ***P < 0.001, ****P < 0.0001; ns, not significant. Data are shown as mean ± s.e.m. Each individual point represents the average from four heart sections per zebrafish.
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it could potentially be multiplexed with other photosensitizers (for example, KillerRed and MiniSOG). The fact that the same encoded FAP (dL5**) can be used to bind a nonphotosensitiz ing dye (MG-ester) allows many studies that rely on imaging to be extended to photosensitization only when the researcher uses the TAPs dye. This alternative labeling strategy can facilitate the selection of stable cells and transgenic animals usable for imag ing, photoablation or photosensitization studies, depending on the dye and light dose used in the study. Methods Methods and any associated references are available in the online version of the paper. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. Acknowledgments This work was supported in part with funds from the US National Institutes of Health (NIH), Technology Centers for Networks and Pathways program (grant U54GM103529 to M.P.B., S.C.W., J.H. and Y.W.), NIH grant R01EB017268 (to J.H., Y.W., M.A.M., E.O., C.M.S., M.T. and M.P.B.) and NIH grant R21ES025606 (to M.P.B. and S.C.W.). We thank E. Kelley and C.J. Bakkenist for helpful discussion and guidance on establishing ROS involved in cellular toxicity; G. Daskivich for help in establishing zebrafish lines; and E. Drill, C.T. Wallace and M.A. Ross for help in larval zebrafish TUNEL imaging. AUTHOR CONTRIBUTIONS J.H. designed and performed experiments, analyzed data and wrote the paper. Y.W. provided cell culture, performed experiments and wrote the paper.
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Articles E.O., M.A.M. and M.T. developed and provided zebrafish lines, designed and performed experiments and analyzed data. L.A.P. provided reagents. S.C.W. and C.M.C. performed experiments and analyzed data. M.P.B. designed experiments, analyzed data and wrote the paper. COMPETING FINANCIAL INTERESTS The authors declare competing financial interests: details are available in the online version of the paper.
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ONLINE METHODS Materials. We purchased 4,4-dimethylaniline and 3,5-diiodo-4hydroxybenzaldehyde from VWR International; anthracene-9, 10-dipropionic acid disodium salt (ADPA), catalasepolyethyl ene glycol and SODpolyethylene glycol from Sigma-Aldrich; tetrasulfonated aluminum phthalocyanine (AlPcS4) from Frontier Scientific; and propidium iodide (P3566), Hoechst dye 33342, a live/dead cell viability/cytotoxicity kit (L-3224) and dihydroethid ium (hydroethidine) (D11347) from Invitrogen. 1H NMR spec troscopy and 13C NMR spectroscopy data were recorded with a Bruker Avance at 300 MHz and 500 MHz. Mass spectra were obtained with a Thermo-Fisher LCQ ESI/APCI ion trap mass spectrometer. Final products were purified with silica, neutral alumina and reverse-phase chromatography; purity was tested by ultrahigh-performance liquid chromatography using a diode array absorbance detector. Raw absorbance values of free dyes and dye-FAP complex were measured on a PerkinElmer Lambda45 spectrophotometer. Fluorogenic enhancement was measured in 96-well microplates on a Tecan Infinite plate spectrometer. Corrected emission spectra were acquired on a Quantamaster monochromator fluorimeter (Photon Technology International). Live larval zebrafish imaging was done with an EVOS FL manual microscope. Cell culture. HEK 293 cells (Sigma-Aldrich) were cultured in DMEM (Thermo Fisher) supplemented with 10% FBS (FisherBrand). To construct cell lines expressing FAP or KillerRed in different cellular compartments, we transfected HEK cells with pcDNA plasmid using Lipofectamine 2000 (Invitrogen). The transfected cells were selected by G418 for 2 weeks and then sorted into single clones for stable cell line generation. In pcDNA, sequence encoding dL5** was cloned downstream of the Kozak sequence for cytosolic expression, upstream of the sequence encoding PDGFR-derived transmembrane domain for membrane surface localization, downstream of a nuclear localization sig nal for nuclear targeting and downstream of a COXIV/COXVIII signal for mitochondrial labeling as previously reported44. We routinely monitored for mycoplasma contamination in cell culture by fluorescent Hoechst 33258 stain. HEK 293 cells were selected for this study because they are widely used in many biology-related studies as a model cell line. Cloning and purification. The Escherichia coli bacterial strain MACH1-T1 (Invitrogen) was used as the host for cloning. FAP dL5** was cloned into pET21 vector with sequence encoding a 6× His tag and sequence encoding an HRV protease site at the 5′ end as previously reported44. Determination of singlet-oxygen quantum yield. PBS (pH 7.4) buffer containing optically matched samples (AlPcS 4, MG-2I, MG-2I–dL5** and MG-ester–dL5**) and 0.1 mM ADPA were illuminated by a 669-nm light LED light box. The fluorescence intensity of ADPA was monitored with an excitation of 374 nm and an emission of 402 nm using a Tecan Infinite M1000 plate spectrometer. The normalized fluorescence intensity was plot ted against exposure time at 669 nm, and Φ∆ was then calculated from the slopes using equation (1) (ref. 25). Here kr is the chemi cal quenching of singlet oxygen by ADPA and kd is the constant of deactivation of singlet oxygen via the solvent; both values are doi:10.1038/nmeth.3735
constant regardless of the photosensitizer being used.[A] is the ADPA concentration, and Iabs is the absorbance at excitation wavelength. −
d[A] k = I absΦ ∆ r [A] dt kd
(1)
Determination of quantum yields. We determined quantum yields by comparing integrated emission spectra of fluorogenFAP complexes to those of reference dyes. Corrected emission spectra were taken on a Quantamaster monochromator fluorim eter (Photon Technology International). Cy5 was used as standard for MG-2I–dL5** complex. The emission spectra (620−820 nm) of a set of five concentrations were integrated and plotted against absorbance at 600 nm, and ΦF was then calculated from the slopes using equation (2). 2 Grad X hX Φ X = Φ ST 2 GradST hST
(2)
where ΦX is the sample quantum yield, ΦST is the standard quantum yield, GradX is the sample slope, GradST is the standard slope, ηX is the refraction index of the sample solvent, and ηST is the refraction index of the standard solvent. Photoconversion of 3,3′-diaminobenzidine (DAB). 100-µl sam ples (AlPcS4, MG-2I–dL5**, MG-ester–dL5** and MG-2I) in pH 7.4 sodium cacodylate buffer with the same optical density at 669 nm were mixed with 100 µl of 1 mg/ml DAB in pH 7.4 sodium cacodylate buffer for 5 min. Then all samples were transferred to a 96-well plate and subjected to 2 h of illumination using the LED light box. Pictures were taken at 0 min, 10 min, 30 min, 60 min and 120 min. Determination of dissociation constant. 5 nM of purified dL5** was precomplexed with a series of dye dilutions, and the fluores cence intensity was measured. The fluorescence of dye without protein at each concentration was also obtained and subtracted as background. On the basis of the plateau intensity in the binding curve calculated from a hyperbolic binding model, we normalized the results to the concentration of fluorogen-FAP complex. Using these scaled data, we calculated the dissociation constant from a one-site binding-ligand depletion model in GraphPad Prism 6. Microscopy. Cells were seeded onto 35-mm glass-bottom dishes (MatTek) one day before imaging and maintained overnight at 37 °C in OptiMEM (Invitrogen) with 10% FBS. Cell images were acquired on an Andor Revolution XD system with a spinning disk and processed with ImageJ. Imaging specifications were as follows: c405, excitation (ex) 405 nm, emission (em) 435/25 nm; c488, ex 488 nm, em 515/25 nm; c560, ex 560 nm, em 585/25 nm; c640, ex 640 nm, em 670/25 nm. Differential interference contrast imaging conditions were optimized for image contrast and kept the same for each experiment. Dead/live cell viability assay. After illumination by a laser source from an Andor confocal microscope, cell medium containing the dye was replaced with 2 µM calcein AM and 4 µM EthD-1 in PBS (pH 7.4) buffer and allowed to stain for 30 min at room temperature. nature methods
Then we conducted the two-color fluorescence cell viability test with the cells using the same microscope (40× objective; for calcein AM, 7.5% laser power; for EthD-1, 560-nm excitation, 610/20-nm emission filter, 35% laser power).
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Light-induced cytotoxicity experiment and cell death count. Cells were incubated with MG-ester, MG-2I or fluorogen-FAP complex for 30 min before illumination. A laser and a light box were used as illumination sources, and different light doses were achieved with different light intensities (using 10 cm × 10 cm neutral density filters placed over the sample in the light box or by varying the laser power on the microscope; power was measured at the sample for each condition) or illumination times. For counting of dead cells, the medium was replaced with PBS containing 2 µM propidium iodide (PI) and 8 µM Hoechst after illumination. Allowing 30 min of staining, we calculated the ratio of cell death from the count of cells stained by PI (dead cells) versus those stained by Hoechst (total cells). Detection of ROS in nucleus by dihydroethidium. HEK cells with nuclear-targeted dL5** were incubated with 400 nM of dyes and then exposed to 2 µM dihydroethidium (diluted from freshly prepared ethanol stock solution) for 10 min. Then cells were illuminated for 2 min (60× objective, 640 nm, 2.43 W cm−2). In the presence of superoxide, DHE is oxidized to 2-hydroethid ium (EOH), which binds to DNA and becomes fluorescent. The generation of EOH was monitored with an excitation of 488 nm and emission of 535/25 nm immediately after photosensitizing illumination. Discussion of fold of activation of singlet-oxygen generation. To estimate the fold of activation of singlet-oxygen generation, we made a concentration series of free MG-2I (in dPBS) and MG2I–dL5** (in 1:9 H2O:dPBS) and plotted the absorbance at 669 nm against the ADPA bleaching. However, in the same absorb ance range, MG-2I–dL5** is much faster in bleaching ADPA than MG-2I, so we used different light exposure times for different samples (60-s exposure time for MG-2I and 10-s exposure time for MG-2I–dL5**). Assuming the bleaching rate does not change within 60 s for MG-2I, we estimated the change in singlet-oxygen generation as >~100-fold on the basis of the ratio of the slopes, scaled by a factor of 6 to account for the sixfold longer exposure of the MG-2I. Owing to the change of absorption maximum upon binding, the OD666 of an equimolar solution of MG-2I dye is 4.5-fold lower than that of an MG-2I–dL5** complex (0.022 versus 0.097). Taken together, the overall activation for singletoxygen production upon binding is given by equation (3), where Rε and RΦ∆ are the ratio of extinction coefficients at the excitation maximum for the complex and the ratio of the singlet-oxygen quantum yield, respectively. R∆ = Re RΦ ∆
(3)
Generation of transgenic zebrafish (Tg(myl7:MBIC5-mCer3)). The zebrafish experiments were performed according to a proto col approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh that conforms to NIH guidelines. MBIC5-mCer3 was cloned into the pISceI vector that contained the Myl7 promoter. 20 pg of pI-SceI-Myl7:MBIC5-mCer3 nature methods
plasmid DNA was injected into the one-cell embryo with I-Sce 1 restriction enzyme as described45. These F0 injected embryos were raised to adulthood and incrossed to identify transgenic founders. We identified three founder lines that expressed mCer3 at 28 h after fertilization in the beating cardiac tube. Tg(myl7:MBIC5-mCer3)pt22 and Tg(myl7:MBIC5-mCer3)pt23 were used in this study. In the embryo ablation studies, cardiac mCer3+ embryos at 28 h post-fertilization were removed from their chorion and treated with 500 nM MG-ester or MG-2I at 48 h post-fertilization for 3 h. The embryos were then illuminated for 12 min using a 659-nm laser (242 mW cm−2). Zebrafish maintenance and retro-orbital injections. The zebrafish experiments were performed according to a protocol approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pittsburgh that conforms to NIH guidelines. Adult (6−18 months old) wild-type AB* and trans genic Tg(myl7:MBIC5-mCer3)pt23 zebrafish were anesthetized for 3 min in Tricaine (0.168 mg/l of MS222; Sigma) and retro-orbitally injected with 3 µl of 12 µM MG-ester (0.06 mg/kg) or MG-2I (0.09 mg/kg) solution dissolved in filtered 1× PBS. Retro-orbital injection was performed as previously described46. We returned the fish to water and stimulated them to breathe by vigorously squirting water over their gills with a pipette. 20−30 min after retro-orbital injection, five fish were placed in a 100-ml beaker containing 50 ml of fish water and illuminated with a custombuilt light box emitting at 669 nm for 30 min. The fish water in the beaker was replaced with fresh water every 10 min. The fish were then returned to the water system and monitored daily. Fish were killed at a determined time point after the illumination, and their hearts, livers and intestines were extracted and fixed in 4% paraformaldehyde for histochemical analysis. Immunohistochemistry. Larval zebrafish were fixed 24 h after illumination in 4% paraformaldehyde overnight. Whole-mount apoptosis was measured via TUNEL assay (Invitrogen, C10617) according to the manufacturer’s instructions. mCerulean3 was labeled by a primary rabbit anti-GFP antibody (Abcam, ab6556); the secondary antibody in use was donkey anti-rabbit IgG H&L (Alexa Fluor 555) (Abcam, ab150074). Fixed hearts, livers and intestines were cryopreserved in 30% sucrose in PBS before immersion in embedding medium (Leica). Fourteen-micrometer cryosections were stained with Acid Fuchsin Orange G (AFOG) as previously described47. Images were captured with a Leica MZ16 microscope and a Q Imaging Retiga 1300 camera. Apoptosis was measured via TUNEL assay (Millipore; S7165) according to the manufacturer’s instructions. Primary antibodies used for immu nostaining were anti-Mef2c (Santa Cruz Biotechnology; sc-313) (1:500) and anti-PCNA (Sigma; P8825) (1:1,000). Secondary antibodies used were Alexa Fluor 488 goat anti-rabbit IgG peroxidase conjugate (Thermo Fisher; A-11008) (1:1,000) and Alexa Fluor 594 goat anti-mouse IgG (H + L) (Thermo Fisher; A-11036) (1:1,000). Slides were mounted with Vectashield mount ing medium with DAPI (Vector Laboratories; H-1200). Images were taken with a Zeiss LSM 700 confocal microscope. For each experiment, two to six sections were analyzed for each heart, liver and intestine. The cardiomyocyte proliferation index was calculated as the percentage of the number of Mef2c+PCNA+ cells divided by the number of total Mef2c+ cells. We measured the doi:10.1038/nmeth.3735
mean ± s.e.m. For one-way ANOVA, Tukey post-hoc tests were performed with multiple comparison of the mean for each group. P < 0.05 was considered significant.
Statistical analysis. Sample sizes were based on practical consid erations (for example, the number of fish that can fit in a single 100-ml beaker or the number of samples required to fill a 96-well plate) to allow effective observations and to avoid unnecessary waste of resources. No systematic randomization or blinding was used in sample allocation or analysis. For animal studies, transgenic fish were assigned to experimental groups randomly. Samples were excluded only for technical failures (i.e., perva sive nonspecific staining). Statistical significance was analyzed by one-way ANOVA or unpaired t-test, and data are shown as
44. Telmer, C.A. et al. Rapid, specific, no-wash, far-red fluorogen activation in subcellular compartments by targeted fluorogen activating proteins. ACS Chem. Biol. 10, 1239–1246 (2015). 45. Molina, G.A., Watkins, S.C. & Tsang, M. Generation of FGF reporter transgenic zebrafish and their utility in chemical screens. BMC Dev. Biol. 7, 62 (2007). 46. Pugach, E.K., Li, P., White, R. & Zon, L. Retro-orbital injection in adult zebrafish. J. Vis. Exp. 34, 1645 (2009). 47. Missinato, M.A., Tobita, K., Romano, N., Carroll, J.A. & Tsang, M. Extracellular component hyaluronic acid and its receptor Hmmr are required for epicardial EMT during heart regeneration. Cardiovasc. Res. 107, 487–498 (2015).
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myofiber area by imaging four sections for each heart stained with AFOG and quantifying the total ventricular area and the cardiac tissue using ImageJ software (NIH).
doi:10.1038/nmeth.3735
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