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Fixing a Photosensitizer Unlocks and Localizes Its LethalityClick to copy article linkArticle link copied!
Enzymatic cleavage simultaneously activates and localizes a photosensitizer allowing for cell ablation with high efficiency, low background, and single-cell resolution.
- Dirk TraunerDirk TraunerE-mail: [email protected]Department of Chemistry and Neuroscience Institute, New York University, New York City, New York 10002, United StatesMore by Dirk Trauner
- Johannes MorsteinJohannes MorsteinDepartment of Chemistry and Neuroscience Institute, New York University, New York City, New York 10002, United StatesMore by Johannes Morstein
The combination of genetic encoding with sophisticated photochemistry can yield powerful ways to elucidate biological function as it enables the perturbation of complex biological systems with very high temporal and spatial precision. In living organisms, a few cells, or even a single one, can play a crucial role, for instance, as progenitor cells in developing tissue or as pacemaker cells in neural networks. Therefore, the ability to control the function of a single cell or a small subset of cells in tissue is an important goal in biology. Most radically, this can be done through their controlled destruction, also known as ablation. Urano and co-workers now report a method that allows for highly effective and precise ablation of cells in vivo. (1) The ablation of cells with single-cell resolution can provide important insights into biological networks.
Cell ablation has a storied history in biology and medicine. It can be achieved through targeted expression of a lethal factor, such as diphtheria toxin, which is a comparatively slow process. (2) Irradiation with light affords much faster action but has its own challenges. Direct ablation with high-powered lasers is straightforward, but it results in the destruction of virtually every cell along the light beam and is more applicable to larger tissues. Two-photon scanning microscopes, which focus high-intensity laser light of long wavelength onto very small volumes, can be used for accurate 3D ablation of cells. However, this is technically challenging and requires expensive instrumentation. To make it effective, the cells of interest need to be identified with some labeling technique, such as genetically targeted fluorescent chromophores.
Genetic targeting can also be used to place chromophores that promote photoelectron transfer or function as photosensitizers, providing local phototoxicity, which results in ablation. Such chromophores can effectively mediate the production of reactive oxygen species through a variety of pathways. (3) Three distinct modes have emerged to produce oxidative stress in specific cells (Figure 1). The first one uses genetically encoded photoactive proteins, such as the GFP-variants KillerRed and SuperNova, which primarily produce superoxide radical anions (Figure 1A). (4) They are formed through oxidative maturation of a polypeptide and do not require the addition of an external dye. Genetically encoded chromophores, however, have the disadvantage that they cannot be quickly replenished, undergo bleaching through photoisomerism, and are generally not very efficient.
Figure 1
Figure 1. Genetic encoding of singlet oxygen producing photosensitizers: (A) KillerRed, SuperNova, and their chromophore. (B) Attachment of a photosensitizer to a genetically encoded bioconjugation tag (HaloTag). (C) Enzymatic activation of a diffusible photosensitizer. (D) Enzymatic activation of a diffusible photosensitizer with concomitant covalent attachment. The genetically encoded component is indicated in boldface.
A second approach places synthetic small-molecule photosensitizers into specific cells using genetically encoded bioconjugation motifs. These were initially developed for chromophore-assisted light inactivation (CALI) of specific proteins, but they could also be used to damage and inactivate entire cells. Early examples include biarsenicals, such as ReAsH-EDT2, which bind to tetracysteine motives. (5) Their use in biology was limited, however, due to cytotoxicity caused by nonspecific binding to endogenous proteins. More recently, genetically encoded SNAP-tags (6) and HaloTags (7) have been employed to mount photosensitizers to specific target cells. An example of such dyes, diAc-eosin-AM, is shown in Figure 1B. The alkyl chloride end of this molecule selectively reacts with the HaloTag to provide a covalent linkage. To make the molecule membrane permeable and to suppress photochemical background activity, the phenolic hydroxy groups were masked as acetates, which were presumably cleaved by esterases inside of the cell. Since this takes place more or less in every cell, and surplus reagent cannot be easily removed by washing, significant background activity can be expected, at least in vivo. In addition, all of these approaches require stoichiometric targeting of a synthetic chromophore to the genetically encoded bioconjugation motif limiting their efficiency for cell ablation.
A third mode involves activation with an enzyme that is genetically encoded in selected cells. This has the advantage that a large number of photoactive dyes (or other toxic principles) can be generated from inactive precursors. This amplification, in addition to the amplification the photosensitizer itself provides, can deliver high concentrations of singlet oxygen and effective ablation. In 2014, Urano disclosed a molecule termed HMDESeR-βGal, that could be activated by bacterial β-galactosidase (gene name: lacZ, Figure 1C). (8) This enzyme is foreign to mammalian cells but can be easily heterologously expressed. Following enzymatic hydrolysis, the probe shifts to the open xanthene form since its phenolic hydroxy group (pKa = 5.2) can be easily deprotonated at physiological pH. HMDESeR-βGal features a heavy selenium atom which promotes fast intersystem crossing and triplet oxygen sensitization following irradiation. It could be used to ablate lacZ-positive cells upon wide field irradiation with green 550 nm light. However, its resolution was limited due to the ability of the cleaved sensitizer (presumably in its closed spiro form) to diffuse into neighboring cells.
In the present paper, Urano addresses this shortcoming by adding an additional functional feature: a conditional covalent linkage (Figure 1D). Urano’s new probe, termed SPiDER-killer-βGal, is also based on a spirocyclic selenorhodol aglycone that forms a β-linked glycoside with galactose. In addition, it contains a fluoromethyl group as a “pro-electrophile”. In its glycosylated form, the probe is not colored and not photoactive as it features a spirocyclic ether, and it will not undergo covalent attachment. Enzymatic hydrolysis by β-galactosidase yields a phenol that quickly eliminates hydrogen fluoride to form an ortho-quinine methide. This highly reactive intermediate now reacts with cellular nucleophiles (e.g., lysine side chains) to form covalent adducts. Importantly, these adducts predominately reside in the open, colored, and phototoxic xanthene form of the chromophore at physiological pH. As such, enzymatic hydrolysis of SPiDER-killer-βGal simultaneously activates both its photosensitizing ability and its reactivity to nucleophiles. Hence, the phototoxic products generated by light irradiation are confined to lacZ-positive cells. The very chemistry that fixes the probe to a genetically engineered cell also unlocks its phototoxicity as it shifts the equilibrium from the closed, photochemically inactive form to the open active form.
To demonstrate the usefulness of SPiDER-killer-βGal and its advantages over existing methods, Urano and colleagues applied it to progressively more complicated biological settings. First, they demonstrated selective ablation of lacZ-positive mammalian cells through coculturing HEK293 and HEK/lacZ(+) cells. Irradiation with 550 nm light for 3 min induced selective ablation of the lacZ-positive cells. These results confirmed that SPiDER-killer-βGal allows for ablation with single-cell resolution, which was not possible with previously reported enzymatically activated small-molecular photosensitizers. Next, the new probe was used in cultured Drosophila larvae wing discs in which β-galactosidase expression was restricted to the posterior regions. This experiment enabled selective ablation of cells in this region showing that SPiDER-killer-βGal works in cultured tissue. Finally, the probe was used in vivo in Drosophila where lacZ expression was induced in a subpopulation of cells in the pupal notum using a promotor that could be induced by heat shock. A fluorescent apoptosis marker was used to detect ablation. Indeed, cells expressing lacZ were found to selectively undergo apoptosis after irradiation with 561 nm laser light demonstrating the usefulness of this new tool in vivo. This series of experiments also demonstrated compatibility of SPiDER-killer-βGal with other fluorophores such as Calcein-AM (viability marker in tissue), H2B-ECFP (expression marker), VC3Ai (apoptosis reporter), and Hoechst-33342 (nuclear stain). With its unprecedented precision and effectiveness in vivo, SPiDER-killer-βGal has clear advantages over other methods for cell ablation, and it is likely to be embraced by the biology community. However, a redesign of its synthesis, which is lengthy and contains several low-yielding steps, might be necessary to make it widely available.
Urano’s approach to the enzymatic activation and concomitant fixation of photosensitizers could prove to be fairly general. Other activating enzymes, such as engineered esterases, phosphatases, nitroreductases, or azoreductases, could be used to unleash the electrophilicity and the phototoxicity of the probe. Enzymatic activation has already been broadly explored to generate fluorophores in a genetically targeted fashion. For instance, Urano himself introduced an azoreductase to activate a rhodamine fluorophore. (9) It would be straightforward to extend some of these methods to the genetically encoded activation of photosensitizers. Nitroreductases, which convert an electron-withdrawing nitro group to an electron-donating amino group, have also been used to unleash the toxicity of prodrugs, such as metronidazole, in genetically tagged cells, resulting in cell ablation. (10) The method worked well but suffered from limited resolution due to diffusion of the toxic principle out of the target cells. As demonstrated by Urano, such limitations can be overcome with smart chemistry that enables the simultaneous activation and localization of a lethal factor.
Author Information
- Dirk Trauner - Department of Chemistry and Neuroscience Institute, New York University, New York City, New York 10002, United States;
http://orcid.org/0000-0002-6782-6056;
- Johannes Morstein - Department of Chemistry and Neuroscience Institute, New York University, New York City, New York 10002, United States;http://orcid.org/0000-0002-6940-288X
Acknowledgments
J.M. thanks the German Academic Scholarship Foundation for financial support and New York University for a MacCracken fellowship and a Margaret and Herman Sokol fellowship.
References
This article references 10 other publications.
- 1Chiba, M.; Kamiya, M.; Tsuda-Sakurai, K.; Fujisawa, Y.; Kosakamoto, H.; Kojima, R.; Miura, M.; Urano, Y. Activatable Photosensitizer for Targeted Ablation of LacZ-Positive Cells with Single-Cell Resolution. ACS Cent. Sci. 2019, in press. DOI: 10.1021/acscentsci.9b00678 .Google ScholarThere is no corresponding record for this reference.
- 2Palmiter, R. D.; Behringer, R. R.; Quaife, C. J.; Maxwell, F.; Maxwell, I. H.; Brinster, R. L. Cell Lineage Ablation in Transgenic Mice by Cell-Specific Expression of a Toxin Gene. Cell 1987, 50 (3), 435– 443, DOI: 10.1016/0092-8674(87)90497-1Google Scholar2https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXltVKhtw%253D%253D&md5=1f188bf6a118932901dc6ccf85af52d4Cell lineage ablation in transgenic mice by cell-specific expression of a toxin genePalmiter, Richard D.; Behringer, Richard R.; Quaife, Carol J.; Maxwell, Francoise; Maxwell, Ian H.; Brinster, Ralph L.Cell (Cambridge, MA, United States) (1987), 50 (3), 435-43CODEN: CELLB5; ISSN:0092-8674.A method of deleting specific cell lineages was developed that entails microinjection into fertilized eggs of a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. Microinjection of a construct in which the elastase I promoter/enhancer is fused to a gene for diphtheria toxin A polypeptide results in birth of mice lacking a normal pancreas because of expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, contg. islet and duct-like cells, was obsd. in some of the transgenic mice. This method provides a new approach for studying cell lineage relationships and for analyzing cellular interactions during development.
- 3Ogilby, P. R. Singlet Oxygen: There Is Indeed Something New under the Sun. Chem. Soc. Rev. 2010, 39 (8), 3181– 3209, DOI: 10.1039/b926014pGoogle Scholar3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXptFygs7k%253D&md5=97267464db1847dc42158b8883dae5fbSinglet oxygen: there is indeed something new under the sunOgilby, Peter R.Chemical Society Reviews (2010), 39 (8), 3181-3209CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Singlet oxygen, O2(a1Δg), the lowest excited electronic state of mol. oxygen, has been known to the scientific community for ∼80 years. It has a characteristic chem. that sets it apart from the triplet ground state of mol. oxygen, O2(X3Σ-g), and is important in fields that range from atm. chem. and materials science to biol. and medicine. For such a "mature citizen", singlet oxygen nevertheless remains at the cutting-edge of modern science. In this crit. review, recent work on singlet oxygen is summarized, focusing primarily on systems that involve light. It is clear that there is indeed still something new under the sun (243 refs.).
- 4Wojtovich, A. P.; Foster, T. H. Optogenetic control of ROS production. Redox Biol. 2014, 2, 368– 376, DOI: 10.1016/j.redox.2014.01.019Google Scholar4https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVWhtLfP&md5=c9e9aa702f2db63b7accb95096741eebOptogenetic control of ROS productionWojtovich, Andrew P.; Foster, Thomas H.Redox Biology (2014), 2 (), 368-376CODEN: RBEIB3; ISSN:2213-2317. (Elsevier B.V.)Reactive Oxygen Species (ROS) are known to cause oxidative damage to DNA, proteins and lipids. In addn., recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiol.-relevant signaling mols. However, these sensor/effector mols. are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chems. that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs) were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS prodn. This review will focus on the new frontier defined by the exptl. use of RGPs to study ROS signaling.
- 5Tour, O.; Meijer, R. M.; Zacharias, D. A.; Adams, S. R.; Tsien, R. Y. Genetically Targeted Chromophore-Assisted Light Inactivation. Nat. Biotechnol. 2003, 21 (12), 1505– 1508, DOI: 10.1038/nbt914Google Scholar5https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpt1Gltb8%253D&md5=776c01df62c057cec9840bb96210e453Genetically targeted chromophore-assisted light inactivationTour, Oded; Meijer, Rene M.; Zacharias, David A.; Adams, Stephen R.; Tsien, Roger Y.Nature Biotechnology (2003), 21 (12), 1505-1508CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochem. generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and α1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by ∼90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or α1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected mol. specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen resp. inhibit or enhance CALI.
- 6Keppler, A.; Ellenberg, J. Chromophore-Assisted Laser Inactivation of Alpha- and Gamma-Tubulin SNAP-Tag Fusion Proteins inside Living Cells. ACS Chem. Biol. 2009, 4 (2), 127– 138, DOI: 10.1021/cb800298uGoogle Scholar6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtleht7s%253D&md5=986d25efbad327aa485c62935db9a167Chromophore-Assisted Laser Inactivation of α- and γ-Tubulin SNAP-tag Fusion Proteins inside Living CellsKeppler, Antje; Ellenberg, JanACS Chemical Biology (2009), 4 (2), 127-138CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly specific tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, the authors demonstrate the effectiveness and specificity of SNAP-tag-based CALI by acute inactivation of α-tubulin and γ-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility in labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the exptl. requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.
- 7Takemoto, K.; Matsuda, T.; McDougall, M.; Klaubert, D. H.; Hasegawa, A.; Los, G. V.; Wood, K. V.; Miyawaki, A.; Nagai, T. Chromophore-Assisted Light Inactivation of HaloTag Fusion Proteins Labeled with Eosin in Living Cells. ACS Chem. Biol. 2011, 6 (5), 401– 406, DOI: 10.1021/cb100431eGoogle Scholar7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvVSnsA%253D%253D&md5=9ba99550488607a0ff05cd8c3255dc4cChromophore-Assisted Light Inactivation of HaloTag Fusion Proteins Labeled with Eosin in Living CellsTakemoto, Kiwamu; Matsuda, Tomoki; McDougall, Mark; Klaubert, Dieter H.; Hasegawa, Akira; Los, Georgyi V.; Wood, Keith V.; Miyawaki, Atsushi; Nagai, TakeharuACS Chemical Biology (2011), 6 (5), 401-406CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resoln. This technol., however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (1O2) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more prodn. of 1O2 than fluorescein and about 5-fold efficiency in CALI of β-galactosidase by using an eosin-labeled anti-β-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technol., demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomol. inactivation that is applicable to many cell biol. assays in living cells.
- 8Ichikawa, Y.; Kamiya, M.; Obata, F.; Miura, M.; Terai, T.; Komatsu, T.; Ueno, T.; Hanaoka, K.; Nagano, T.; Urano, Y. Selective Ablation of β-Galactosidase-Expressing Cells with a Rationally Designed Activatable Photosensitizer. Angew. Chem., Int. Ed. 2014, 53 (26), 6772– 6775, DOI: 10.1002/anie.201403221Google Scholar8https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXosV2hurY%253D&md5=ba8fd273346fa0b17eec515c88230c07Selective Ablation of β-Galactosidase-Expressing Cells with a Rationally Designed Activatable PhotosensitizerIchikawa, Yuki; Kamiya, Mako; Obata, Fumiaki; Miura, Masayuki; Terai, Takuya; Komatsu, Toru; Ueno, Tasuku; Hanaoka, Kenjiro; Nagano, Tetsuo; Urano, YasuteruAngewandte Chemie, International Edition (2014), 53 (26), 6772-6775CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed an activatable photosensitizer capable of specifically inducing the death of β-galactosidase-expressing cells in response to photoirradn. By using a selenium-substituted rhodol scaffold bearing β-galactoside as a targeting substituent, we designed and synthesized HMDESeR-βGal, which has a non-phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β-galactosidase efficiently converted HMDESeR-βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible-wavelength absorption and the ability to generate singlet oxygen (1O2). When HMDESeR-βGal was applied to larval Drosophila melanogaster wing disks, which express β-galactosidase only in the posterior region, photoirradn. induced cell death in the β-galactosidase-expressing region with high specificity.
- 9Shin, N.; Hanaoka, K.; Piao, W.; Miyakawa, T.; Fujisawa, T.; Takeuchi, S.; Takahashi, S.; Komatsu, T.; Ueno, T.; Terai, T. Development of an Azoreductase-Based Reporter System with Synthetic Fluorogenic Substrates. ACS Chem. Biol. 2017, 12 (2), 558– 563, DOI: 10.1021/acschembio.6b00852Google Scholar9https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlGm&md5=2bec7e7daca91444cdf152542d472da7Development of an azoreductase-based reporter system with synthetic fluorogenic substratesShin, Narae; Hanaoka, Kenjiro; Piao, Wen; Miyakawa, Takuya; Fujisawa, Tomotsumi; Takeuchi, Satoshi; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Tahara, Tahei; Tanokura, Masaru; Nagano, Tetsuo; Urano, YasuteruACS Chemical Biology (2017), 12 (2), 558-563CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biol. events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, the authors focused on azoreductase (AzoR). The authors designed and synthesized a library of azo-rhodamine derivs. as candidate fluorogenic substrates. These derivs. were non-fluorescent, probably due to ultrafast conformational change around the N:N bond after photoexcitation. The authors found that AzoR-mediated redn. of the azo bond of derivs. bearing an electron-donating group on the azobenzene moiety was followed by nonenzymic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. The authors showed that a AzoR/reporter system could detect azoreductase-expressing live cells at the single cell level.
- 10Curado, S.; Stainier, D. Y. R.; 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. 2008, 3 (6), 948– 954, DOI: 10.1038/nprot.2008.58Google Scholar10https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVemtrg%253D&md5=57fd17f5871cd47ca3443a83f203f7b1Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studiesCurado, Silvia; Stainier, Didier Y. R.; Anderson, Ryan M.Nature Protocols (2008), 3 (6), 948-954CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chem. and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, the authors provide a step-by-step protocol for a hybrid chem.-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the redn. of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.
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Abstract
Figure 1
Figure 1. Genetic encoding of singlet oxygen producing photosensitizers: (A) KillerRed, SuperNova, and their chromophore. (B) Attachment of a photosensitizer to a genetically encoded bioconjugation tag (HaloTag). (C) Enzymatic activation of a diffusible photosensitizer. (D) Enzymatic activation of a diffusible photosensitizer with concomitant covalent attachment. The genetically encoded component is indicated in boldface.
References
This article references 10 other publications.
- 1Chiba, M.; Kamiya, M.; Tsuda-Sakurai, K.; Fujisawa, Y.; Kosakamoto, H.; Kojima, R.; Miura, M.; Urano, Y. Activatable Photosensitizer for Targeted Ablation of LacZ-Positive Cells with Single-Cell Resolution. ACS Cent. Sci. 2019, in press. DOI: 10.1021/acscentsci.9b00678 .There is no corresponding record for this reference.
- 2Palmiter, R. D.; Behringer, R. R.; Quaife, C. J.; Maxwell, F.; Maxwell, I. H.; Brinster, R. L. Cell Lineage Ablation in Transgenic Mice by Cell-Specific Expression of a Toxin Gene. Cell 1987, 50 (3), 435– 443, DOI: 10.1016/0092-8674(87)90497-12https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADyaL1cXltVKhtw%253D%253D&md5=1f188bf6a118932901dc6ccf85af52d4Cell lineage ablation in transgenic mice by cell-specific expression of a toxin genePalmiter, Richard D.; Behringer, Richard R.; Quaife, Carol J.; Maxwell, Francoise; Maxwell, Ian H.; Brinster, Ralph L.Cell (Cambridge, MA, United States) (1987), 50 (3), 435-43CODEN: CELLB5; ISSN:0092-8674.A method of deleting specific cell lineages was developed that entails microinjection into fertilized eggs of a chimeric gene in which a cell-specific enhancer/promoter is used to drive the expression of a toxic gene product. Microinjection of a construct in which the elastase I promoter/enhancer is fused to a gene for diphtheria toxin A polypeptide results in birth of mice lacking a normal pancreas because of expression of the toxin in pancreatic acinar cells. A small pancreatic rudiment, contg. islet and duct-like cells, was obsd. in some of the transgenic mice. This method provides a new approach for studying cell lineage relationships and for analyzing cellular interactions during development.
- 3Ogilby, P. R. Singlet Oxygen: There Is Indeed Something New under the Sun. Chem. Soc. Rev. 2010, 39 (8), 3181– 3209, DOI: 10.1039/b926014p3https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3cXptFygs7k%253D&md5=97267464db1847dc42158b8883dae5fbSinglet oxygen: there is indeed something new under the sunOgilby, Peter R.Chemical Society Reviews (2010), 39 (8), 3181-3209CODEN: CSRVBR; ISSN:0306-0012. (Royal Society of Chemistry)A review. Singlet oxygen, O2(a1Δg), the lowest excited electronic state of mol. oxygen, has been known to the scientific community for ∼80 years. It has a characteristic chem. that sets it apart from the triplet ground state of mol. oxygen, O2(X3Σ-g), and is important in fields that range from atm. chem. and materials science to biol. and medicine. For such a "mature citizen", singlet oxygen nevertheless remains at the cutting-edge of modern science. In this crit. review, recent work on singlet oxygen is summarized, focusing primarily on systems that involve light. It is clear that there is indeed still something new under the sun (243 refs.).
- 4Wojtovich, A. P.; Foster, T. H. Optogenetic control of ROS production. Redox Biol. 2014, 2, 368– 376, DOI: 10.1016/j.redox.2014.01.0194https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXitVWhtLfP&md5=c9e9aa702f2db63b7accb95096741eebOptogenetic control of ROS productionWojtovich, Andrew P.; Foster, Thomas H.Redox Biology (2014), 2 (), 368-376CODEN: RBEIB3; ISSN:2213-2317. (Elsevier B.V.)Reactive Oxygen Species (ROS) are known to cause oxidative damage to DNA, proteins and lipids. In addn., recent evidence suggests that ROS can also initiate signaling cascades that respond to stress and modify specific redox-sensitive moieties as a regulatory mechanism. This suggests that ROS are physiol.-relevant signaling mols. However, these sensor/effector mols. are not uniformly distributed throughout the cell. Moreover, localized ROS damage may elicit site-specific compensatory measures. Thus, the impact of ROS can be likened to that of calcium, a ubiquitous second messenger, leading to the prediction that their effects are exquisitely dependent upon their location, quantity and even the timing of generation. Despite this prediction, ROS signaling is most commonly intuited through the global administration of chems. that produce ROS or by ROS quenching through global application of antioxidants. Optogenetics, which uses light to control the activity of genetically-encoded effector proteins, provides a means of circumventing this limitation. Photo-inducible genetically-encoded ROS-generating proteins (RGPs) were originally employed for their phototoxic effects and cell ablation. However, reducing irradiance and/or fluence can achieve sub-lethal levels of ROS that may mediate subtle signaling effects. Hence, transgenic expression of RGPs as fusions to native proteins gives researchers a new tool to exert spatial and temporal control over ROS prodn. This review will focus on the new frontier defined by the exptl. use of RGPs to study ROS signaling.
- 5Tour, O.; Meijer, R. M.; Zacharias, D. A.; Adams, S. R.; Tsien, R. Y. Genetically Targeted Chromophore-Assisted Light Inactivation. Nat. Biotechnol. 2003, 21 (12), 1505– 1508, DOI: 10.1038/nbt9145https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD3sXpt1Gltb8%253D&md5=776c01df62c057cec9840bb96210e453Genetically targeted chromophore-assisted light inactivationTour, Oded; Meijer, Rene M.; Zacharias, David A.; Adams, Stephen R.; Tsien, Roger Y.Nature Biotechnology (2003), 21 (12), 1505-1508CODEN: NABIF9; ISSN:1087-0156. (Nature Publishing Group)Studies of protein function would be facilitated by a general method to inactivate selected proteins in living cells noninvasively with high spatial and temporal precision. Chromophore-assisted light inactivation (CALI) uses photochem. generated, reactive oxygen species to inactivate proteins acutely, but its use has been limited by the need to microinject dye-labeled nonfunction-blocking antibodies. We now demonstrate CALI of connexin43 (Cx43) and α1C L-type calcium channels, each tagged with one or two small tetracysteine (TC) motifs that specifically bind the membrane-permeant, red biarsenical dye, ReAsH. ReAsH-based CALI is genetically targeted, requires no antibodies or microinjection, and inactivates each protein by ∼90% in <30 s of widefield illumination. Similar light doses applied to Cx43 or α1C tagged with green fluorescent protein (GFP) had negligible to slight effects with or without ReAsH exposure, showing the expected mol. specificity. ReAsH-mediated CALI acts largely via singlet oxygen because quenchers or enhancers of singlet oxygen resp. inhibit or enhance CALI.
- 6Keppler, A.; Ellenberg, J. Chromophore-Assisted Laser Inactivation of Alpha- and Gamma-Tubulin SNAP-Tag Fusion Proteins inside Living Cells. ACS Chem. Biol. 2009, 4 (2), 127– 138, DOI: 10.1021/cb800298u6https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1MXhtleht7s%253D&md5=986d25efbad327aa485c62935db9a167Chromophore-Assisted Laser Inactivation of α- and γ-Tubulin SNAP-tag Fusion Proteins inside Living CellsKeppler, Antje; Ellenberg, JanACS Chemical Biology (2009), 4 (2), 127-138CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Chromophore-assisted laser inactivation (CALI) can help to unravel localized activities of target proteins at defined times and locations within living cells. Covalent SNAP-tag labeling of fusion proteins with fluorophores such as fluorescein is a fast and highly specific tool to attach the photosensitizer to its target protein in vivo for selective inactivation of the fusion protein. Here, the authors demonstrate the effectiveness and specificity of SNAP-tag-based CALI by acute inactivation of α-tubulin and γ-tubulin SNAP-tag fusions during live imaging assays of cell division. Singlet oxygen is confirmed as the reactive oxygen species that leads to loss of fusion protein function. The major advantage of SNAP-tag CALI is the ease, reliability, and high flexibility in labeling: the genetically encoded protein tag can be covalently labeled with various dyes matching the exptl. requirements. This makes SNAP-tag CALI a very useful tool for rapid inactivation of tagged proteins in living cells.
- 7Takemoto, K.; Matsuda, T.; McDougall, M.; Klaubert, D. H.; Hasegawa, A.; Los, G. V.; Wood, K. V.; Miyawaki, A.; Nagai, T. Chromophore-Assisted Light Inactivation of HaloTag Fusion Proteins Labeled with Eosin in Living Cells. ACS Chem. Biol. 2011, 6 (5), 401– 406, DOI: 10.1021/cb100431e7https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC3MXnvVSnsA%253D%253D&md5=9ba99550488607a0ff05cd8c3255dc4cChromophore-Assisted Light Inactivation of HaloTag Fusion Proteins Labeled with Eosin in Living CellsTakemoto, Kiwamu; Matsuda, Tomoki; McDougall, Mark; Klaubert, Dieter H.; Hasegawa, Akira; Los, Georgyi V.; Wood, Keith V.; Miyawaki, Atsushi; Nagai, TakeharuACS Chemical Biology (2011), 6 (5), 401-406CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Chromophore-assisted light inactivation (CALI) is a potentially powerful tool for the acute disruption of a target protein inside living cells with high spatiotemporal resoln. This technol., however, has not been widely utilized, mainly because of the lack of an efficient chromophore as the photosensitizing agent for singlet oxygen (1O2) generation and the difficulty of covalently labeling the target protein with the chromophore. Here we choose eosin as the photosensitizing chromophore showing 11-fold more prodn. of 1O2 than fluorescein and about 5-fold efficiency in CALI of β-galactosidase by using an eosin-labeled anti-β-galactosidase antibody compared with the fluorescein-labeled one. To covalently label target protein with eosin, we synthesize a membrane-permeable eosin ligand for HaloTag technol., demonstrating easy labeling and efficient inactivation of HaloTag-fused PKC-γ and aurora B in living cells. These antibody- and HaloTag-based CALI techniques using eosin promise effective biomol. inactivation that is applicable to many cell biol. assays in living cells.
- 8Ichikawa, Y.; Kamiya, M.; Obata, F.; Miura, M.; Terai, T.; Komatsu, T.; Ueno, T.; Hanaoka, K.; Nagano, T.; Urano, Y. Selective Ablation of β-Galactosidase-Expressing Cells with a Rationally Designed Activatable Photosensitizer. Angew. Chem., Int. Ed. 2014, 53 (26), 6772– 6775, DOI: 10.1002/anie.2014032218https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2cXosV2hurY%253D&md5=ba8fd273346fa0b17eec515c88230c07Selective Ablation of β-Galactosidase-Expressing Cells with a Rationally Designed Activatable PhotosensitizerIchikawa, Yuki; Kamiya, Mako; Obata, Fumiaki; Miura, Masayuki; Terai, Takuya; Komatsu, Toru; Ueno, Tasuku; Hanaoka, Kenjiro; Nagano, Tetsuo; Urano, YasuteruAngewandte Chemie, International Edition (2014), 53 (26), 6772-6775CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA)We have developed an activatable photosensitizer capable of specifically inducing the death of β-galactosidase-expressing cells in response to photoirradn. By using a selenium-substituted rhodol scaffold bearing β-galactoside as a targeting substituent, we designed and synthesized HMDESeR-βGal, which has a non-phototoxic spirocyclic structure owing to the presence of the galactoside moiety. However, β-galactosidase efficiently converted HMDESeR-βGal into phototoxic HMDESeR, which exists predominantly in the open xanthene form. This structural change resulted in drastic recovery of visible-wavelength absorption and the ability to generate singlet oxygen (1O2). When HMDESeR-βGal was applied to larval Drosophila melanogaster wing disks, which express β-galactosidase only in the posterior region, photoirradn. induced cell death in the β-galactosidase-expressing region with high specificity.
- 9Shin, N.; Hanaoka, K.; Piao, W.; Miyakawa, T.; Fujisawa, T.; Takeuchi, S.; Takahashi, S.; Komatsu, T.; Ueno, T.; Terai, T. Development of an Azoreductase-Based Reporter System with Synthetic Fluorogenic Substrates. ACS Chem. Biol. 2017, 12 (2), 558– 563, DOI: 10.1021/acschembio.6b008529https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC2sXhtlGm&md5=2bec7e7daca91444cdf152542d472da7Development of an azoreductase-based reporter system with synthetic fluorogenic substratesShin, Narae; Hanaoka, Kenjiro; Piao, Wen; Miyakawa, Takuya; Fujisawa, Tomotsumi; Takeuchi, Satoshi; Takahashi, Shodai; Komatsu, Toru; Ueno, Tasuku; Terai, Takuya; Tahara, Tahei; Tanokura, Masaru; Nagano, Tetsuo; Urano, YasuteruACS Chemical Biology (2017), 12 (2), 558-563CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)Enzyme/substrate pairs, such as β-galactosidase with chromogenic x-gal substrate, are widely used as reporters to monitor biol. events, but there is still a requirement for new reporter systems, which may be orthogonal to existing systems. Here, the authors focused on azoreductase (AzoR). The authors designed and synthesized a library of azo-rhodamine derivs. as candidate fluorogenic substrates. These derivs. were non-fluorescent, probably due to ultrafast conformational change around the N:N bond after photoexcitation. The authors found that AzoR-mediated redn. of the azo bond of derivs. bearing an electron-donating group on the azobenzene moiety was followed by nonenzymic cleavage to afford highly fluorescent 2-methyl-rhodamine green (2-Me RG), which was well retained in cells. The authors showed that a AzoR/reporter system could detect azoreductase-expressing live cells at the single cell level.
- 10Curado, S.; Stainier, D. Y. R.; 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. 2008, 3 (6), 948– 954, DOI: 10.1038/nprot.2008.5810https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BD1cXmvVemtrg%253D&md5=57fd17f5871cd47ca3443a83f203f7b1Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studiesCurado, Silvia; Stainier, Didier Y. R.; Anderson, Ryan M.Nature Protocols (2008), 3 (6), 948-954CODEN: NPARDW; ISSN:1750-2799. (Nature Publishing Group)Ablation studies are used to elucidate cell lineage relationships, developmental roles for specific cells during embryogenesis and mechanisms of tissue regeneration. Previous chem. and genetic approaches to directed cell ablation have been hampered by poor specificity, limited efficacy, irreversibility, hypersensitivity to promoter leakiness, restriction to proliferating cells, slow inducibility or complex genetics. Here, the authors provide a step-by-step protocol for a hybrid chem.-genetic cell ablation method in zebrafish that, by combining spatial and temporal control, is cell-type specific, inducible, reversible, rapid and scaleable. Bacterial Nitroreductase (NTR) is used to catalyze the redn. of the innocuous prodrug metrodinazole (Mtz), thereby producing a cytotoxic product that induces cell death. Based on this principle, NTR is expressed in transgenic zebrafish using a tissue-specific promoter. Subsequent exposure to Mtz by adding it to the media induces cell death exclusively within NTR+ cells. This approach can be applied to regeneration studies, as removing Mtz by washing permits tissue recovery. Using this protocol, cell ablation can be achieved in 12-72 h, depending on the transgenic line used, and recovery initiates within the following 24 h.
