Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic

Apr 12, 2017 - ... from the Novosibirsk State University, Russian Federation, in 2009. ... Phytochrome photoreceptors absorb far-red and near-infrared...
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Near-Infrared Fluorescent Proteins, Biosensors, and Optogenetic Tools Engineered from Phytochromes Konstantin G. Chernov,†,§ Taras A. Redchuk,†,§ Evgeniya S. Omelina,†,§ and Vladislav V. Verkhusha*,†,‡ †

Department of Biochemistry and Developmental Biology, Faculty of Medicine, University of Helsinki, Helsinki 00290, Finland Department of Anatomy and Structural Biology and Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, United States

‡

ABSTRACT: Phytochrome photoreceptors absorb far-red and near-infrared (NIR) light and regulate light responses in plants, fungi, and bacteria. Their multidomain structure and autocatalytic incorporation of linear tetrapyrrole chromophores make phytochromes attractive molecular templates for the development of light-sensing probes. A subclass of bacterial phytochromes (BphPs) utilizes heme-derived biliverdin tetrapyrrole, which is ubiquitous in mammalian tissues, as a chromophore. Because biliverdin possesses the largest electron-conjugated chromophore system among linear tetrapyrroles, BphPs exhibit the most NIR-shifted spectra that reside within the NIR tissue transparency window. Here we analyze phytochrome structure and photochemistry to describe the molecular mechanisms by which they function. We then present strategies to engineer BphP-based NIR fluorescent proteins and review their properties and applications in modern imaging technologies. We next summarize designs of reporters and biosensors and describe their use in the detection of protein− protein interactions, proteolytic activities, and posttranslational modifications. Finally, we provide an overview of optogenetic tools developed from phytochromes and describe their use in light-controlled cell signaling, gene expression, and protein localization. Our review provides guidelines for the selection of NIR probes and tools for noninvasive imaging, sensing, and light-manipulation applications, specifically focusing on probes developed for use in mammalian cells and in vivo.

CONTENTS 1. Introduction 2. Structure, Function, and Photochemistry of Phytochromes 2.1. Domain Structure 2.2. Chromophore Chemistry 2.3. Light-Induced Structural Changes 3. Fluorescent Proteins 3.1. Dimeric Fluorescent Proteins 3.2. Monomeric Fluorescent Proteins 3.3. Single-Domain Monomeric Fluorescent Proteins 3.4. Photoactivatable Fluorescent Proteins 3.5. Near-Infrared Fluorescent Proteins in Advanced Imaging Technologies 3.6. Future Perspectives 4. Biosensors and Reporters 4.1. Split Protein Reporters 4.2. Protease Reporters 4.3. Biosensors 4.4. Future Perspectives 5. Optogenetic Tools 5.1. Optogenetic Tools Based on Conformational Changes 5.2. Optogenetic Tools Based on Homodimerization © 2017 American Chemical Society

5.3. Optogenetic Tools Based on Heterodimerization 5.3.1. Transcriptional Regulation by PhyBbased Optogenetic Tools 5.3.2. Control of Protein Localization by Optogenetic Tools Derived from PhyB 5.3.3. PhyB-based Optogenetic Tools Regulate Cell Signaling 5.3.4. Gene Regulation by Multiplexing Several Optogenetic Tools 5.3.5. Protein Targeting Control with RpBphP1-based Optogenetic Tool 5.3.6. Transcription Activation by Optogenetic Tool Developed from RpBphP1 5.3.7. RpBphP1-based Optogenetic Tool of Reduced Size 5.4. Future Perspectives 6. Conclusions Author Information Corresponding Author ORCID Author Contributions Notes

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6438 Received: October 11, 2016 Published: April 12, 2017

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DOI: 10.1021/acs.chemrev.6b00700 Chem. Rev. 2017, 117, 6423−6446

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Figure 1. Phytochrome structure and chromophore chemistry. (A) Light penetration depth at 480, 560, 670, and 720 nm wavelengths in muscle tissue. Adapted with permission from ref 84. Copyright 2016 Macmillan Publishers Limited. (B) Molar extinction coefficient of oxygenated hemoglobin (HbO2) and deoxygenated hemoglobin (Hb). NIR transparency window region is shown as a shaded red box. Adapted with permission from ref 128. Copyright 2015 Annual Reviews. (C) Typical domain structure of bacterial phytochromes. The PAS, GAF, and PHY domains, which form the photosensory core module (PCM), are shown in green, cyan, and magenta, respectively; the output module (OM) is shown in blue. The biliverdin (BV) chromophore is shown as red spheres. The crystal structure (PDB ID 5AKP) of the XccBphP bacterial phytochrome from Xanthomonas campestris was used to visualize the structure.12 (D) Chemical structure of BV. (E) Chemical structures of phycocyanobilin (PCB) and phytochromobilin (PΦB).

Biographies Acknowledgments Abbreviations References

Natural functions of phytochromes include transcriptional regulation of genes, which are often involved in photosynthesis, via enzymatic activity or protein−protein interactions.1 Microbial phytochromes are known to regulate pigmentation, photoprotection, redox sensing, phototaxis, the cell cycle, circadian rhythms, adjustments to the light spectrum, and virulence.2,3 Several classes of phytochromes are distinguished by their origin and structural organization, such as plant phytochromes (Phy), cyanobacterial phytochromes (Cph), bacterial phytochromes (BphP), and cyanobacteriochromes (CBCR). Here, we specifically focus on phytochromes that have been engineered into fluorescent proteins, biosensors and reporters, or optogenetic tools that were applied in eukaryotic and mammalian cells, whereas for a comprehensive description of the biological functions of phytochromes we refer readers to recent reviews.4−6

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1. INTRODUCTION Modern biology is increasingly reliant on optical technologies such as fluorescence imaging, detection, and light-induced manipulation. A major limitation in these fields is the availability of genetically encoded probes to study processes in vivo. Near-infrared (NIR) light is favorable over shorter wavelengths for use in mammalian tissues (Figure 1A) due to its low absorbance by hemoglobin (Figure 1B), melanin, and water, reduced tissue autofluorescence, and lower light scattering induced by lipids and fat. Therefore, fluorescent proteins (FPs), biosensors and reporters (BRs), and optogenetic tools (OTs) for optical imaging, readout and light control in mammals should work within a NIR tissue transparency window (∼650−900 nm). Proteins that are naturally capable of sensing light, such as photoreceptors, are found in all kingdoms of life, providing the templates for design of optical probes and controllers. Among the photoreceptor families, phytochrome photoreceptors are beneficial for the development of tools to sense light irradiation in the far-red and NIR spectral ranges, because of the nature of their light-absorbing moiety, called a chromophore. Phytochromes utilize linear tetrapyrroles, known as bilins, as chromophores. Bilins have an extended electron-conjugated system that enables them to absorb low-energy photons. Some types of bilins are ubiquitous metabolites; thus, they may be found in organisms belonging to different classes. The ability to autocatalytically incorporate a chromophore substantially simplifies the use of phytochrome-based optical probes and tools. Therefore, chromophore availability in a specific cell type or organism is crucial for probe functionality.

2. STRUCTURE, FUNCTION, AND PHOTOCHEMISTRY OF PHYTOCHROMES 2.1. Domain Structure

An analysis of the crystal structures and amino acid sequences illustrates that most plant and bacterial phytochromes share a common architecture of the photosensory core module (PCM; 55−58 kDa), typically consisting of the PAS (Per−ARNT− Sim), GAF (cGMP phosphodiesterase−adenylate cyclase− FhlA), and PHY (phytochrome-specific) domains connected by α-helical linkers7−9 (Figure 1C). Although the amino acid sequences of the PAS, GAF, and PHY domains show low sequence similarity, their structures share a common topology.8 The PAS domains are widely distributed among sensors and signal transduction proteins, usually functioning as signal receivers or interaction hubs.10,11 Typically, PAS domains are connected to another domain by α-helical linkers required for signal transduction. Of the phytochromes in the Pfam database, 48% also contain the PAS domain after the PAS-GAF-PHY 6424

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Figure 2. Structure and spectral properties of NIR FPs engineered from bacterial phytochromes. (A) Typical structure of two-domain NIR FPs engineered from BphP. The crystal structure of BphP1-FP/Cys20Ser (PDB ID 4XTQ) was used to visualize the structure.32 (B) Fluorescence excitation and emission spectra of NIR FPs with a Cys residue in either the PAS or GAF domain. Excitation spectra are shown in red and blue for biliverdin (BV) chromophore bound to PAS and GAF domain, respectively. Emission spectra are shown in magenta and cyan for BV chromophore bound to PAS and GAF domain, respectively. (C) Chemical structures of A-ring of BV and phycocyanobilin (PCB) bound to a Cys residue in PAS or GAF domain. (D) Covalent binding of BV chromophore to Cys residue in GAF domain causes a ∼40 nm blue spectral shift of NIR FPs. Symbols correspond to NIR FP oligomeric state: magenta squares designate dimers, and red triangles designate monomers.

PCM.12 These include most plant phytochromes13 and some bacterial phytochromes,2 such as those from Bradyrhizobium ORS278 (BrBphP1) and Rhodopseudomonas palustris (RpBphP1). Also, blue-light photoreceptor domains named LOV (light, oxygen, or voltage) belong to the PAS superfamily.14,15 The chromophore−protein interaction occurs in the GAF domain. A bilin-binding pocket is usually formed by a β-sheet and three α-helices. Being positioned in the GAF domain, bilin chromophore may be covalently attached to the cysteine residue in either PAS or GAF domain. Remarkably, in phytochromes with PAS-GAF-PHY architecture, N-terminal residues of PAS go through a loop formed by GAF, forming a knot-like structure, known as a figure-eight knot or lasso knot. PHY is a phytochrome-specific domain.2 The evolutionarily conserved part of the PHY domain, which extends to the bilinbinding pocket and forms a distinctive hairpin structure, is called a PHY-tongue. It serves to shield the bilin chromophore from solvents and participates in light-induced structural rearrangements.8,16 The C-terminal regions of phytochromes form an output module (OM) that is responsible for biological function. The OM in most microbial phytochromes is typically represented by histidine kinase (HisK). Other domains serving as OMs include antirepressors,17,18 enzymes involved in second-messenger signaling, such as GGDEF (diguanylate cyclase), EAL (phosphodiesterase),2,19,20 and HAMP (histidine kinase/ methyl-accepting/phosphatase) in CBCR.21 In optogenetic tool design, the modular structure of phytochromes enables researchers to replace the endogenous OMs with heterogeneous OMs for light-induced control of cellular metabolism in bacterial22 and eukaryotic cells.23,24 In this case, the signaling αhelix must be reconstructed to allow signal transduction from the PCM to the new OM. Engineering of fluorescent proteins, biosensors, and reporters from phytochromes usually includes truncation of OM and significant modification of PCM.

2.2. Chromophore Chemistry

Phytochromes incorporate the bilin chromophores (Figure 1D,E) by use of an intrinsic lyase activity. Plant and cyanobacterial phytochromes covalently attach phytochromobilin (PΦB) and phycocyanobilin (PCB) tetrapyrroles. Phycoviolobilin (PVB) serves as a light-sensing moiety in certain cyanobacteriochromes. Bacterial phytochromes incorporate a linear tetrapyrrole, biliverdin IXα (BV), which is a product of the oxidative degradation of heme by heme oxygenase (HO).7 Some phytochrome-derived fluorescent proteins can incorporate two types of bilin, for example, BV and PCB, with different binding efficiency.25 Occasionally, natural functions of phytochromes are not dependent on chromophores; in this case, phytochrome functions in an apoprotein state.26 Biliverdin IXα consists of four pyrrole rings, designated as A, B, C, and D rings, and is covalently attached to a conserved Cys amino acid residue at the N-terminus of BphP via the vinyl side chain of ring A (Figure 1D). Incorporation of BV into the BphP apoprotein likely occurs in two steps: first, BV is secured in the chromophore-binding pocket in the GAF domain, and second, a thioether bond is formed with the Cys residue at the Nterminal part of the PAS domain.27,28 PΦB in plant phytochromes is bound by a thioether linkage to a Cys residue in the GAF domain.29 The spectral properties of phytochromes depend on the position of the chromophore-binding Cys residue. For example, in BphP-derived FPs (Figure 2A), BV can bind to either the PAS or the GAF domain. The presence of the Cys residue in the PAS domain may also allosterically modulate the binding of BV to the GAF domain.30,31 Formation of the thioether bond upon binding of BV to the GAF domain induces autoisomerization of the double bond out of ring A. Consequently, reduction of the electron-conjugation system leads to ∼40 nm blue spectral shift32 (Figure 2B−D). The other autoisomerization reaction was reported to induce changes in the chromophore structure in CBCRs. In that case, PCB 6425

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Figure 3. Light-driven changes in structure and spectral properties of phytochromes. (A) Absorption spectra of bacterial phytochromes in Pr and Pfr states. (B) Reversible Z/E (Pr−Pfr) isomerization of the C15/C16 double bond in bound BV chromophore under illumination with far-red and NIR light. (C) Light-driven structural rearrangements in bacterial phytochrome. Repositioning of the PHY-tongue and the PHY domains are marked with black arrows. α-Helix and β-sheet secondary structures of the PHY-tongue in Pr and Pfr states, respectively, are indicated. Crystal structures with PDB IDs 4O0P and 4O01 were used to visualize the structure.43 (D) Domain structure of transcriptional repressor RsPpsR from Rhodobacter sphaeroides, which is the close homologue of the phytochrome binding partner RpPpsR2 from R. palustris. Multiple PAS domains are shown in shades of green. The long α-helical linkers, which are likely responsible for interaction with the photoreceptor binding partners, are shown in yellow. The crystal structure PDB ID 4HH2 was used to visualize the structure.47

pyrrole rings occurs upon illumination (Figure 3B). The resulting rotation of the D-ring induces conformational changes in the protein backbone, which are transferred from the PCM to the OM. Several mechanistic schemes for signal propagation to the OM were formulated on the basis of spectroscopic and crystallographic analyses. Studies of crystallized PCMs revealed that the α-helical spine, which is involved in the formation of phytochrome dimers, also serves as a functional linker between the PCM and the OM.8 Light-driven conformational changes in the BV chromophore in DrBphP, a bacterial phytochrome from Deinococcus radiodurans, generate rearrangement of the GAF domain and C-terminal α-helices, thus propagating a light signal to the OM and modulating its activity.39 Analysis of the structure of PhyB from Arabidopsis thaliana suggested a similar photoconversion mechanism for plant phytochromes.29 Analyses of crystal structures, single-particle electron microscopy (SPEM), and a protease sensitivity assay of DrBphP allowed further characterization of light-induced conformational changes.40−43 In the DrBphP PCM, a rotation of the D-ring causes a reorganization of hydrogen bonds in the BV chromophore binding pocket. Consequently, changes in the weak interactions of protein side chains lead to expansion of a conformational space of the protein, followed by repositioning of the PHY domains in a scissor-like manner (Figure 3C). At the same time, the PHY-tongue undergoes refolding. In the Pr state, the main part of the tongue consists of two antiparallel βstrands, whereas in the Pfr state it converts into an α-helix.40,41 Most likely, in full-length DrBphP, rotational movement in PAS−GAF domains is transduced as rotation of the OM via the PHY domain. The X-ray solution scattering data revealed that a rotational motion of HisK domains was the major light-driven structural rearrangement observed in full-length DrBphP.42

autoisomerizes to PVB after covalent attachment to the Cys residue.33,34 Slightly different Stokes shifts observed in NIR FPs are possibly determined by different properties of the chromophore electron-conjugated system, affected by chromophore planarity and presence of the hydrogen-bond chromophore conformers, and by chromophore interactions with the immediate protein environment, including rearrangement of the hydrogen bonds around the chromophore and an excited-state proton transfer.35,36 The oligomeric state of phytochromes has a strong influence on their spectral properties. Typically, oligomeric and dimeric proteins incorporate chromophore more efficiently because of a better folding of dimeric proteins than their monomeric versions. This causes a positive cooperative effect of chromophore binding to one protomer on another one in the dimer.30 Therefore, a molar extinction coefficient that depends on the amount of protein molecules with bound chromophore (holoform) is higher for dimeric and oligomeric phytochromes. 2.3. Light-Induced Structural Changes

Cyanobacterial and algal phytochromes can sense light throughout the whole visible spectrum.37,38 However, bacterial phytochromes specifically sense far-red and NIR light and therefore are good templates to develop optical probes and tools for use in mammalian tissues. Typically, BphPs exist in one of two interconvertible states, either the Pr state or the Pfr state (Figure 3A). The Pr state absorbs light at 660−700 nm, whereas the Pfr state absorbs light at 740−770 nm. In addition to the main absorption peak, known as the Q-band, all phytochromes absorb at 380−420 nm, known as the Soret band, which corresponds to individual pyrrole rings. A Z/E isomerization of the bilin chromophore around its 15/ 16 double bond in the methine bridge between the C and D 6426

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The crystal structure of the R. palustris RpBphP2 PCM showed high structural similarity to DrBphP.44 Another phytochrome with high homology to RpBphP, XccBphP from X. campestris, is the first full-length crystallized BphP12 (Figure 1C). The structure of XccBphP suggests a photoswitching mechanism similar to that described for DrBphP. The similarity of the light-driven structural rearrangements among phytochromes of different subclasses implies that the photoswitching mechanism is shared by the phytochrome family. Rearrangement of PHY domains usually leads to repositioning of the OMs in the dimer, providing the interaction needed for enzymatic activity. However, in phytochromes with no reported enzymatic activity, the light-driven rearrangements likely lead to an exposure of surfaces interacting with a partner protein. In this regard, RpBphP1 from R. palustris was reported to interact with a RpPpsR2 transcriptional repressor under NIR light.45,46 Remarkably, a RsPpsR protein from Rhodobacter sphaeroides (Figure 3D), which shares the domain structure and is homologous to RpPpsR2, consists of several PAS domains connected by helical linkers.11,47 The linker between N-PAS and PAS1 domains, known as the Q-linker, was identified in both PpsRs and may play a crucial role in the NIR light-induced RpBphP1−RpPpsR2 interaction. Structural analyses of truncated and mutated phytochromes revealed the presence of parallel, antiparallel, and butterfly-like dimers18,44 as well as monomers.48 These structures are useful for the rational design of NIR probes and tools. Importantly, BphPs have several advantages over other phytochromes for engineering of NIR probes. First, BphPs utilize the BV chromophore,7 which, in contrast to the tetrapyrrole chromophores of other phytochrome types, is ubiquitous in mammalian tissues.49 This feature renders BphP applications in mammalian cells and animals as straightforward as green fluorescent protein (GFP)-like FPs and only requires the transfection of a single gene. Second, due to the large electronically conjugated chromophore system in BV tetrapyrrole, BphPs exhibit the most NIR-shifted absorbance and fluorescence relative to other phytochromes,2 which both reside within the NIR tissue transparency window. Third, the large conformational changes observed upon BV photoisomerization make BphPs good templates for the design of photoactivatable FPs and OTs.

Figure 4. General strategy used to engineer BphP into NIR FPs. The design of permanently fluorescent NIR FPs includes truncation of BphPs to the PAS-GAF domains or to the GAF domain. Mutated PAS, GAF, and PHY domains are marked with stars. In the case of photoswitchable FPs, BphPs should contain the PAS-GAF-PHY domains, that is the whole PCM, to preserve the photoconversion properties. PCMs are subjected to random mutagenesis to increase photoactivation contrast and enhance brightness. Renilla luciferase is fused to NIR FPs to engineer chimeric NIR luciferase constructs that allow BRET from luciferase to NIR FP via the Soret band. For that, substrates that induce luciferase emission in the violet spectral range (380−450 nm) should be used.

and random mutagenesis of Cph1 that resulted in the fluorescent mutant named PR-1. Although it possessed three mutations (A47T, Y176H, and I252N) compared to parental Cph1, a single Y176H mutation in the GAF domain of Cph1 was sufficient to transform it into a fluorescent protein due to inhibition of Pr → Pfr photoconversion. This single amino acid mutant was expressed in bacteria and purified, but it required the PCB source that is not present in mammalian cells. The first FP engineered from BphP included the PAS and GAF domains of DrBphP. The FP was named IFP1.4 and was reported to be useful for whole-body liver visualization.53 However, IFP1.4 required an exogenous supply of BV or coexpression of HO, significantly limiting its use. Its successor, IFP2.0, also required a BV supply or coexpression of HO.54 Moreover, IFP2.0 tends to dimerize,55 potentially interfering with the normal functions of tagged proteins and causing their mislocalization and aggregation. Simultaneous expression of IFP2.0 and HO was required to image neurons in Drosophila larvae and brain tumors in mice. Unfortunately, this approach deeply affects the organism by changing the redox potential of the intracellular environment and generating messenger molecules.56 Probably, inefficient BV incorporation and the tendency to dimerize are intrinsic properties of DrBphP1 from which the IFPs were engineered. The first NIR FP that does not require the addition of exogenous BV was iRFP (later renamed iRFP713).57 This FP was engineered from the PAS and GAF domains of RpBphP2 by extensively screening mutants for fluorescence and efficient BV binding in mammalian cells. This approach produced iRFP713 that specifically incorporated endogenous BV and was used for whole-body imaging of organs by planar epiillumination.57 Transgenic mice that expressed iRFP713 throughout the body were completely healthy and fluorescent without the addition of exogenous BV49 (Figure 5A). Several mouse lines bearing iRFP713 cDNA between CAG promoter and SV40 polyA demonstrated variations in fluorescent intensity that could be caused by different numbers of the iRFP713 gene inserted into the genome. The fluorescent intensity also varied between different organs of the animal, probably due to the different strength of expression from CAG promoter and various amounts of biliverdin in the different

3. FLUORESCENT PROTEINS The modular structure of BphPs was exploited in the rational design of NIR FPs (Figure 4). As a rule, fluorescence is obtained from the population of molecules that are not able to undergo Pr → Pfr photoconversion. Therefore, engineering of permanently fluorescent NIR FPs involves the progressive truncation of BphPs to PAS-GAF and single GAF domains, aimed to destabilize the Pfr state and disable Pr → Pfr photoconversion. Additional stabilization of the fluorescent Pr state is achieved by introducing mutations in the immediate surroundings of the chromophore that disrupt the network of hydrogen bonds between BV and its microenvironment.50,51 Residues in the C-terminus of the GAF domain are mutated to prevent dimer formation. PHY domain is retained in photoactivatable FPs to make the photoswitching possible. 3.1. Dimeric Fluorescent Proteins

The first successful example of an engineered NIR FP used cyanobacterial phytochrome Cph1 as a template.52 The engineering involved removal of the histidine kinase domain 6427

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adenocarcinoma line that expresses both iRFP713 and Venus FPs was used to study subcutaneous and orthotopic cancer in mice.60 The combination of the two FPs allowed the visualization and quantification of tumors both in vivo and ex vivo, as well as the evaluation of anticancer treatments.60 Impaired lymphatic drainage due to metastatic spreading of cancer cells was observed in mice that were orthotopically inoculated with breast cancer cells stably expressing iRFP71361 (Figure 5C). Tracking of iRFP713-labeled cancer cells was used to visualize metastatic spread through lymphatic channels into lymph nodes.62 In the xenografted and orthotopic head and neck cancer models, iRFP713 yielded a significantly better signal-to-noise ratio than the FP tdTomato, which emits visible light.63 The combination of a NIR-labeled antitumor antibody with iRFP713 was used to detect tumor margins during surgery.64 Stem cells derived from iRFP713-expressing transgenic mice exhibit NIR fluorescence after differentiation into various types of cells and tissues. This strategy allows the noninvasive visualization of regeneration processes by use of cells stably expressing iRFP713. For example, the fate of cardiac progenitor cells transplanted in the hindlimb and ischemic heart was easily visualized in vivo.65 Recombinant Leishmania amazonensis and Leishmania infantum strains that stably express iRFP713 enable the monitoring of parasite development in living mice66,67 (Figure 5D). In the future, these strains may serve as a tool for screening antileishmanial drugs and vaccine efficiency. For development of antiviral therapies, influenza A viral strains expressing iRFP713 are useful for studies of virus replication, tropism, and pathogenesis.68 An increased concentration of BV in body fluids and excreta is an important marker in veterinary medicine. A simple highthroughput fluorescent assay based on interaction of BV with iRFP713 was developed to detect liver diseases.69 Highthroughput assays were also developed for real-time evaluation of oncogenic transformation.70,71 The advantages of these assays include better precision and speed and a much lower cost compared with standard luciferase-based assays. Gene transfer by electroporation is a promising method for antitumor therapy that has already reached the clinical evaluation phase. The iRFP713 expression vector allowed real-time evaluation of electroporation efficiency.72 Notably, conventional FPs cannot be used to label retinal cells because the excitation light causes photoreceptor bleaching and can destroy vision. iRFP713 was used to label retinal neurons to overcome this limitation. iRFP713 expression and detection do not interfere with light sensitivity, indicating that it is a new, promising tool for vision research.73 In combination with conventional FPs, iRFP713 was used as a novel fluorescent genome-editing reporter74 and for singlecell, high-speed, real-time imaging of filopodia transport.75 iRFP-labeled metastatic cells were detected in blood vessels in vivo by novel multimodal flow cytometry.76 The superior performance of iRFP713 in a wide variety of applications prompted the development of spectrally distinct dimeric NIR FPs. For this purpose, the PAS-GAF domains of RpBphP6 and iRFP713 were used as templates. Several rounds of random mutagenesis of residues located in the BV binding pocket, followed by fluorescence-activated cell sorting (FACS), resulted in mutants with blue- and red-shifted spectra. The mutants exhibiting the highest brightness in mammalian cells

Figure 5. Applications of dimeric NIR FPs. (A) Image of iRFP713 transgenic newborn mice and their wild-type, nonfluorescent littermates. Adapted with permission from ref 49. Copyright 2014 Japanese Association for Laboratory Animal Science. (B) Images of iRFP713-labeled tumor xenografts in living mice. Adapted with permission from ref 59. Copyright 2014 SPIE. (C) Impaired lymphatic drainage due to metastatic spread. Lymphatic channels are shown in green, and the iRFP713-labeled tumor is shown in red. Adapted with permission from ref 61 under the Creative Commons Attribution license. (D) Mouse foot infected with the iRFP713-expressing Leishmania strain. The fluorescent signal is shown in pseudocolor. Adapted with permission from ref 66. Copyright 2016 Elsevier. (E) Confocal microscopy with spectral detection and linear unmixing of MTLn3 cells expressing iRFP670, iRFP682, iRFP702, and iRFP720. Emission was detected from 660 to 790 nm after excitation from a 633 nm laser. Unmixed channels and overlay are shown in pseudocolor. Adapted with permission from ref 77. Copyright 2013 Macmillan Publishers Limited. (F) Flow cytometry of live HeLa cells expressing spectrally distinct iRFPs. Adapted with permission from ref 77 under the Creative Commons Attribution license. A 635 nm excitation laser and a combination of 660/20 nm and 780/60 nm emission filters were used. Ten thousand events were analyzed for each cell type.

tissues. The latter depends on the amount of heme and the balance between HO and bilirubin oxidase that shifts toward production of biliverdin upon oxidative stress. Thus, higher fluorescence intensity in the lung, pancreas, and especially liver could reflect elevated BV levels in these organs. Notably, iRFP713 was also well expressed in the primary visual cortex of living mice via a viral vector, enabling the detection of neurites at a depth of 500 μm.58 A tremendous number of animal models utilize iRFP713 to noninvasively monitor the development of diseases in vivo. Several tumor cell lines expressing iRFP713, including glioblastoma, osteosarcoma, and melanoma, were created by use of a lentiviral vector.59 iRFP713-labeled cancer cells allow the real-time monitoring of subcutaneous, deep-tissue tumors behind bone barriers 59 (Figure 5B). A human lung 6428

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Table 1. Near-Infrared Fluorescent Proteins and Their Reported Applications in Eukaryotic Cellsa NIR FP IFP1.4 iRFP713d PAiRFP1 PAiRFP2 IFP2.0f iRFP670 iRFP682 iRFP702 iRFP720 smURFP iRFP713/V256C BphP1-FP GAF-FP mIFP iBlueberry miRFP670 miRFP703 miRFP709

Ex (nm)

Em (nm)

EC (M−1cm−1)

QY (%)

684 690 659e 692e 690 643 663 673 702 642 662

708 713 703e 719e 711 670 682 702 720 670 680

92 000 98 000 67 100 63 600 98 000 114 000 90 000 93 000 96 000 180 000 94 000

7.7 6.3 4.8 4.7 8.1 12.2 11.1 8.2 6.0 18 14.5

640 635 683 644 642 674 683

669 670 704 667 670 703 709

60 000 49 800 82 000 38 000 87 400 90 900 78 400

13.0 7.3 8.4 6.9 14.0 8.6 5.4

Molecular brightness vs iRFP713 (%) Dimers 114 100 64 60 80 225 162 124 93 551 220 Monomers 126 59 74 42 198 127 69

t1/2b (s)

Brightness in HeLa cells vs iRFP713c (%)

Ref

70 960 nd nd 108 290 490 630 490 300 nd

8.0 100 25 25 7.9 119 105 61 110 2.0g 150

53 57 78 78 54 77 77 77 77 79 30

nd nd 54 954 155 394 192

nd 2.0 14 nd 72 37 30

32 25 55 80 81 81 81

Abbreviations: NIR, near-infrared; FP, fluorescent proteins; EC, extinction coefficient; QY, quantum yield; nd, not determined. Dimeric and monomeric NIR FPs are listed chronologically within each group. bt1/2 represents photostability in mammalian cells. Fluorescence decay curves obtained from transfected HeLa cells were normalized to absorbance spectra and extinction coefficients of FPs, spectrum of the lamp, and transmission of an excitation filter. cDetermined as effective NIR fluorescence in HeLa cells without exogenous BV and after normalization to fluorescence of cotransfected enhanced green fluorescent protein (EGFP). dAlso known as iRFP. eCorresponds to the photoactivated state. f Although IFP2.0 was originally reported to be a monomer,54 it was later shown to be dimer.55,82 gMeasured by us without exogenous BV 48 h after cell transfection. a

named small ultrared fluorescent protein (smURFP), exists as a 32 kDa homodimer and exhibits spectral properties similar to those of iRFP670. In contrast to iRFPs, the expression of smURFP and tandem dimeric smURFP in mammalian cell cultures required an exogenous chromophore supply, which was achieved by addition of 5-aminolevulinic acid (5-ALA, a precursor of heme) and iron(II) sulfate and coexpression of HO. This strategy is needed to reduce the accumulation of protoporphyrin IX (PpIX) that can compete with BV for binding to apoprotein and decrease brightness of the NIR FP. It occurs because PpIX does not absorb in the NIR range, unlike BV. Coexpression of HO in cells expressing smURFP supplemented with FeSO4 and 5-ALA resulted in a 7-fold increase in cell fluorescence, compared to cells grown in standard cell culture medium. The addition of BV resulted in a 4.7-fold increase in fluorescence, whereas the addition of biliverdin dimethyl ester (BVMe2) to medium resulted in an 18-fold increase in fluorescence of smURFP-expressing cells. The more hydrophobic BVMe2 probably exhibits greater membrane permeability than BV. The performance of smURFP was shown in cell and animal models. Although tumor xenografts stably expressing smURFP were visible without exogenous BV, the addition of BV or BVMe2 to excised tumors significantly increased fluorescence. smURFP was used to tag intracellular proteins, although its dimeric nature may impair their normal function and localization. With excess exogenous BV, brightness of smURFP in HeLa cells was 35% as compared to iRFP670, whereas without BV it dropped to 2% (Table 1).

were named iRFP670, iRFP682, iRFP702, and iRFP720 according to their emission maxima.77 Therefore, the molecular evolution of PAS-GAF domains of RpBphP6 and iRFP713 resulted in four novel NIR FPs with fluorescence maxima covering ∼50 nm of the NIR range (Figure 2D). Both the molecular and effective (i.e., cellular) brightness of iRFPs and lack of cytotoxicity were similar to iRFP713 (Table 1). Four types of cells labeled with different iRFPs were simultaneously detected by confocal microscopy using a spectral unmixing algorithm (Figure 5E). The different spectral properties of iRFPs allow researchers to separate several populations of cells by flow cytometry using visible red and new solid-state NIR lasers77,83 (Figure 5F). In living mice, five types of tumors were resolved by use of the spectral unmixing algorithm available in the modern imaging platforms.77 Two novel chimeric probes based on RLuc8 luciferase from Renilla reniformis fused with iRFP670 and iRFP720 NIR fluorescent proteins were created.84 They emit NIR light due to intramolecular bioluminescence resonance energy transfer (BRET) between RLuc8 and iRFPs, enabling deep-tissue imaging with 10-fold higher sensitivity, comparing to epifluorescent imaging. Recently, a new far-red FP was engineered from cyanobacterial allophycocyanine (APC) from Trichodesmium erythraeum.79 Native APC exists as a highly fluorescent hexamer containing three α + β dimers that require lyase coexpression to incorporate the PCB chromophore. The APCα subunit was subjected to several rounds of random mutagenesis to engineer the FP. Mutants were coexpressed in bacteria with HO and phycocyanobilin−ferredoxin oxidoreductase (PcyA) that provided the PCB supply. After the brightest clones were selected, the PcyA gene was removed, leaving only BV production, and the protein was mutated again. The resulting brightest clone,

3.2. Monomeric Fluorescent Proteins

Although dimeric NIR FPs are excellent tools for whole-cell labeling in cancer research, neuroscience, and parasitology, their 6429

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random mutagenesis and the brightest clones were selected. Additionally, residues in the C-terminal α-helix of the GAF domain were mutated to prevent the formation of weak dimers. The monomeric character of the resulting NIR FP variants was verified by size-exclusion chromatography, analytical ultracentrifugation, and cellular protein fusions. Ile253 in the GAF domain was substituted with a Cys residue to obtain a blueshifted variant, miRFP670.32 All miRFP proteins were superior to IFP2.0 and mIFP in terms of brightness and photostability (Table 1). Strong expression of miRFPs in preclonal cell mixtures was not cytotoxic over several cell generations. In addition, widefield microscopy of N- and C-terminal miRFP fusions was used to visualize fine cellular structures beyond the diffraction limit, using a super-resolution technique known as structured illumination microscopy (SIM) (Figure 6A). The combination

use in studies of intracellular processes is limited because they may interfere with the normal function of the tagged protein, which is particularly crucial in studies monitoring dynamic cellular processes. Moreover, the development of NIR reporters and biosensors that change their fluorescence in response to stimuli also requires the engineering of novel monomeric NIR FPs. The first reported monomeric NIR FP, IFP1.4, was later shown to form dimers55 and was essentially nonfluorescent without an exogenous BV supply. A naturally monomeric Bradyrhizobium phytochrome (BrBphP) was used as the template to generate a truly monomeric NIR FP of the IFP series. Several residues in its BV binding pocket were subjected to saturation mutagenesis, followed by DNA shuffling and random mutagenesis. The resulting brightest clone, mIFP, exhibited less brightness and photostability than iRFP71355 (Table 1). Two other previously reported monomeric FPs, WiPhy and IFP1.4rev, were not tested for their performance in mammalian cells and in vivo.50,85 A novel NIR FP was developed from wild-type RpBphP1. Engineering included truncation of the protein to the PAS-GAF domains, followed by random mutagenesis of the Asp201 and Ile202 residues in the conserved -PXSDIP- motif and screening of the library for the brightest clones. Mutations at these positions were shown to stabilize the Pr state of the chromophore and increase the fluorescence quantum yield.50,77 Several rounds of random mutagenesis and selection of the brightest clones resulted in BphP1-FP. This FP had the most blue-shifted fluorescence spectrum among the NIR FPs, with an excitation peak at 640 nm and an emission peak at 669 nm. The quantum yield of BphP1-FP was 13%, which is relatively high compared to other NIR FPs (Table 1). Interestingly, in addition to the Cys located in the PAS domain (Cys20), BphP1-FP contains a Cys in the GAF domain (Cys253). Indeed, this Cys253 was also identified in plant and cyanobacterial phytochromes, as well as in blue-shifted iRFP670 and iRFP682.32 By use of structural studies, the differences in the mode of chromophore binding between blueshifted and red-shifted variants of NIR FPs were identified. In natural BphPs and red-shifted NIR FPs, BV binds to the Cys residue in the PAS domain via the C32 carbon of the side chain of pyrrole ring A. In contrast, in BphP-FP and other blueshifted FPs, BV binds to the Cys in the GAF domain, resulting in blue-shifted chromophores that are mainly linked via the C31 carbon atoms of the side chain of pyrrole ring A32 (Figure 2C). Employing this approach, the blue-shifted version of mIFP, iBlueberry, was generated by introducing a single I251C mutation in the GAF domain proximal to the A-ring of BV80 (Table 1). As expected from the spectral difference, a combination of mIFP and iBlueberry was applied for twocolor labeling. iBlueberry was used as a fusion tag to label centrosomes in developing zebrafish embryos for the visualization of centrosome dynamics. In this case, however, coexpression of HO was required to provide an additional BV source.80 The brightest available monomeric NIR FPs, miRFP670, miRFP703, and miRFP709,81 were generated by further improving the PAS-GAF domains of RpBphP1 (Table 1). This template was chosen because, according to the RpBphP1 crystal structure, its PAS-GAF domains do not participate in dimer formation.45 The Asp201 and Ile202 residues in the GAF domain were subjected to saturation mutagenesis to engineer the miRFPs. Then the PAS-GAF domains were subjected to

Figure 6. Applications of miRFPs in super-resolution microscopy. (A) Epifluorescence wide-field (left) and super-resolution structured illumination microscopy (SIM) (right) images of a cell expressing the miRFP703−tubulin fusion protein. Adapted with permission from ref 81. Copyright 2016 Macmillan Publishers Limited. (B) Multicolor SIM image of cell coexpressing mito−TagGFP2, tubulin−mCherry, and H2B−miRFP703 fusion proteins. The NIR channel is shown on the left, and the merged image is shown on the right. Scale bars = 5 μm. Adapted with permission from ref 81. Copyright 2016 Macmillan Publishers Limited.

with green and red FPs enabled cross-talk-free multicolor SIM imaging (Figure 6B). Several reporters and biosensors based on miRFPs were created and will be described below. 3.3. Single-Domain Monomeric Fluorescent Proteins

The small size of NIR FPs is beneficial for protein tagging. RpBphP1 was truncated to the PAS-GAF domains, then 6430

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The resulting photoactivatable FPs, PAiRFP1 and PAiRFP2, had slightly different phenotypes. Half-times of dark relaxation for PAiRFP1 and PAiRFP2 were 58 and 233 min, and the photoactivation contrast for PAiRFP1 and PAiRFP2 was 9.0and 5.9-fold, respectively. Both PAiRFPs exhibited fluorescence in mammalian cells in the absence of exogenous BV, but they were significantly less bright than iRFP713. The intensity of the 660 nm light required for photoactivation of PAiRFPs was approximately 103-fold higher than the intensity used for the imaging. After subtraction of the images captured before photoactivation from the images captured after photoactivation, an approximately 20-fold increase in the S/B ratios was observed compared to conventional iRFP713. Selective photoactivation of PAiRFPs in vivo was obtained by delivering a beam of light to specific, small areas of the tumor. The light was delivered either via skin or via needle; both methods promoted the rapid and selective photoactivation of several spots in the tumor tissue. After photoactivation, fluorescence of PAiRFP1 and PAiRFP2 in tumors decreased in the darkness with half-times of 55 and 155 min, respectively.78 Tumor xenografts expressing PAiRFPs displayed significantly higher S/B ratios for in vivo imaging than iRFP713-expressing tumors.

Asp200 and Ile201 residues were subjected to saturation mutagenesis, and an additional Cys was introduced in the GAF domain to engineer a single-domain monomeric NIR FP, similar to the engineering of blue-shifted miRFPs. The mutant was truncated to the single GAF domain and the lasso knot was removed to increase protein stability. Additionally, monomerizing mutations were introduced at the C-termini, and random mutagenesis of the whole protein was performed. The brightest variants from the bacterial library were selected and beneficial mutations were combined in the single protein, named GAFFP, which contained 25 amino acid residue substitutions (15% of whole protein) and lacked the lasso knot loop. GAF-FP efficiently incorporated both BV and PCB and possessed enhanced photostability, with spectral properties similar to those of iRFP67025 (Table 1). The monomeric state of GAFFP was confirmed by size-exclusion chromatography. GAF-FP was highly tolerant to peptide insertions in the loop regions, potentially allowing circular permutations. The performance of GAF-FP was observed in HeLa cells; however, it required an exogenous BV supply. A fusion construct consisting of GAF-FP and an enhanced luciferase mutant, RLuc8, was created for deep tissue imaging. In the chimeric construct, the energy of the oxidized luciferase substrate transmits to GAF-FP via bioluminescence resonance energy transfer (BRET). This effect occurs because the RLuc8 bioluminescence emission peak overlaps with the Soret band of GAF-FP. In a phantom mouse, the GAF-FP−RLuc8 fusion enabled imaging with high signal-to-background (S/B) ratio at the depth where the green bioluminescence of RLuc8 was undetectable.25 CBCR from Acaryochloris marina binds both PCB and BV.86 The BV-bound AmCBCR displayed a red-shifted absorption spectrum compared to the PCB-bound form. Isolated GAF domains from AmCBCR were recently used to engineer NIR FPs that were relatively dim upon expression in mammalian cells in the presence of exogenous PCB.87

3.5. Near-Infrared Fluorescent Proteins in Advanced Imaging Technologies

In most applications, all available NIR FPs are imaged by planar whole-body fluorescence imaging. Although these epi-illumination imaging techniques offer simplicity and sufficient speed, they have several drawbacks. The signal from objects located at greater depth is attenuated more strongly, compared to objects located at the surface. There are several reasons for this, including tissue absorption and light scattering that leads to systematic errors in imaging quantification. Therefore, the planar images are surface-weighted due to their exceptional sensitivity to surface fluorescence that results in systematic errors in image quantification. Fluorescent molecular tomography (FMT) allows the threedimensional (3D) reconstruction of organs and tumors with submillimeter resolution at centimeter depth.88 FMT is based on the imaging of multiple source−detector pairs and 3D image reconstruction by modeling light propagation through the tissue. The simple variant of FMT is diffuse tomography (DT) that allows reconstruction of 3D organs by scanning a sample with a trans-illuminated excitation light source and acquiring multiple images. In living mice, DT was used to visualize closely located liver tissue and tumors labeled with iRFP713 and iRFP670, respectively77 (Figure 7A). The introduction of iRFPs significantly accelerated the development of advanced imaging techniques. Fluorescence time-domain imaging technology (TD) measures fluorescence decay after the delivery of short (10−11−10−10 s) laser pulses to the sample. This method neglects tissue autofluorescence, substantially increasing the detection sensitivity. By use of TD, three different tumors, each expressing a unique iRFP label, were imaged in the same animal89 (Figure 7B). Moreover, TD displayed 20-fold greater sensitivity than conventional continuous-wave imaging for the detection of metastases in deep organs. The combination of TD and X-ray computed tomography (XCT) was used for noninvasive imaging of brain tumors in mice (Figure 7B). Although phytochromes do not fluoresce upon absorption of NIR light, they generate ultrasound waves when excited with

3.4. Photoactivatable Fluorescent Proteins

Photoconversion is an inherent feature of phytochromes to undergo structural change upon illumination that is important in light signal propagation. For NIR FPs, photoconversion is beneficial because it enables differential imaging that is useful for highly autofluorescent samples and samples with low signals. There are two types of NIR FPs that undergo photoconversion. Photoactivatable FPs irreversibly convert from nonfluorescent to fluorescent state under illumination (photoactivation), whereas photoswitchable FPs reversibly convert from nonfluorescent to fluorescent state under different lights (photoswitching). Irreversible photoactivation and reversible photoswitching not only improve the S/B ratio and lead to higher resolution but also allow the spatiotemporal optical labeling and tracking of proteins, organelles, and whole cells. Therefore, photoactivatable and photoswitchable FPs are a good alternative to constitutively fluorescent FPs. Bathy BphP from Agrobacterium tumefaciens C58, AtBphP2, was used as the template for photoactivatable FPs.78 AtBphP2 was truncated to the PCM, consisting of the PAS, GAF, and PHY domains, and exhibited photoconversion properties similar to those of the full-length protein. The PCM was subjected to several rounds of random mutagenesis, and the two brightest clones with increased photoactivation contrast, enhanced brightness, and blocked Pr → Pfr photoconversion were selected. 6431

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photons in the tissues and therefore yield better resolution images than FMT. The combination of PAM and XCT positron emission tomography (PET) for imaging iRFP713expressing tumors achieved unprecedented 0.1 mm resolution in deep tissues.91 3.6. Future Perspectives

Currently, the major limitations of BphP-derived FPs are low quantum yield (