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The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

Fluorescence based intracellular pH nanoprobes have been developed that overcomes the limitations imposed by shallow penetration depth of ultraviolet excitation, photostability, phototoxicity, and interference from background autofluorescence. In this study, we have constructed a Förster Resonance Energy Transfer (FRET) based pH nanoprobe using upconversion nanoparticle (UCNP) as a donor (excitation/emission @ 980/540 nm, green channel), and mOrange fluorescent protein (excitation/emission @ 548/566 nm, red channel) as acceptor. The UCNP-mOrange nanoprobe could be fluorescently imaged with 980 nm excitation, having deep penetration depth, by a fluorescence microscope on a coverslip, or uptaken in a single HeLa cell. The cellular upatake of these nanoparticles were confirmed by transmission electron microscope study. The FRET probes, with a FRET efficiency of ~20% at physiological pH of 7.0, have simultaneous self-ratiometric and ratiometric features varying linearly with local pH. The probe exhibits high accuracy, sensitivity, reversibility, and stability over a wide range of pH (3.0-8.0). The fluorescence intensity ratio from individual green, and red channels in fluorescence microscopic images could be used to estimate the pH of the intracellular compartments of HeLa cell from the pH dependent ratiometric calibration. Nigericin mediated intracellular pH (3.0, 5.0, and 7.0) could be accurately estimated from the CLSM derived FRET ratio. The pH probes demonstrate high stability and reversibility when switched between pH 3, and 8 for at least 5 cycles., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.

Genetically encoded Förster Resonance Energy Transfer (FRET)-based biosensors are powerful tools to illuminate spatiotemporal regulation of cell signaling in living cells, but the utility of the red spectrum for biosensing was limited due to a lack of bright and stable red fluorescent proteins. Here, we rationally improve the photophysical characteristics of the coral-derived fluorescent protein TagRFP-T. We show that a new single-residue mutant, super-TagRFP (stagRFP) has nearly twice the molecular brightness of TagRFP-T and negligible photoactivation. stagRFP facilitates significant improvements on multiple green-red biosensors as a FRET acceptor and is an efficient FRET donor that supports red/far-red FRET biosensing. Capitalizing on the ability of stagRFP to couple with multiple FRET partners, we develop a novel multiplex method to examine the confluence of signaling activities from three kinases simultaneously in single living cells, providing evidence for a role of Src family kinases in regulating growth factor induced Akt and ERK activities.

Detection of bioactive peptides in complex ecosystems like intestinal environment is a difficult task. In this study, we developed two new bioreporters for nisin based on Lactococcus lactis NZ9000 transformed with the vector pNZ:Nis-aFP or pNZ:Nis-mCherry, that encoded for the anaerobic fluorescent protein evoglow-Pp1 (aFP) or the fluorescent protein mCherry, respectively. The biosensors were used to study nisin A production by L. lactis INIA 650 in milk and in a colonic model. The use of L. lactis NZ9000 pNZ:Nis-aFP as a biosensor allowed the detection of nisin produced by L. lactis INIA 650 in milk, but not in the in vitro colonic model. In milk, this reporter was induced by direct addition of 10 ng/ml nisin while, in the colonic model, nisin concentrations of 50 ng/ml were necessary. However, the reporter system based on pNZ:Nis-mCherry showed a higher sensibility, detecting nisin concentrations of 1 ng/ml produced by L. lactis INIA 650 in colonic media using agar diffusion or cross streak bioassays., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Fluorescent proteins are investigated extensively as markers for the imaging of cells and tissues that are treated by gene transfection. However, limited transfection efficiency and lack of targeting restrict the clinical application of this method rooted in the challenging development of robust fluorescent proteins for in vivo bioimaging. To address this, a new type of near-infrared (NIR) fluorescent protein assemblies manufactured by genetic engineering is presented. Due to the formation of well-defined nanoparticles and spectral operation within the phototherapeutic window, the NIR protein aggregates allow stable and specific tumor imaging via simple exogenous injection. Importantly, in vivo tumor metastases are tracked and this overcomes the limitations of in vivo imaging that can only be implemented relying on the gene transfection of fluorescent proteins. Concomitantly, the efficient loading of hydrophobic drugs into the protein nanoparticles is demonstrated facilitating the therapy of tumors in a mouse model. It is believed that these theranostic NIR fluorescent protein assemblies, hence, show great potential for the in vivo detection and therapy of cancer., (© 2020 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Polycistronic expression systems in insects can be used for applications such as recombinant protein production in cells, enhanced transgenesis methods, and the development of novel pest-control strategies based on the sterile insect technique (SIT). Here we tested the performance of four picornaviral 2A self-cleaving peptides (TaV-2A, DrosCV-2A, FMDV 2A1/31 and FMDV 2A1/32) for the co-expression and differential subcellular targeting of two fluorescent marker proteins in cell lines (Anastrepha suspensa AsE01 and Drosophila melanogaster S2 cells). We found that all four 2A peptides showed comparable activity in cell lines, leading to the production of independent upstream and downstream proteins that were directed to the nucleus or membrane by a C-terminal nuclear localization signal (NLS) on the upstream protein and a poly-lysine/CAAX membrane anchor on the downstream protein. TaV-2A and DrosCV-2A were inserted into piggyBac constructs to create transgenic D. suzukii strains, confirming efficient ribosomal skipping in vivo. Interestingly, we found that the EGFP-CAAX protein was distributed homogeneously in the membrane whereas the DsRed-CAAX protein formed clumps and aggregates that induced extensive membrane blebbing. Accordingly, only flies expressing the DsRed-NLS and EGFP-CAAX proteins could be bred to homozygosity whereas expression of EGFP-NLS and DsRed-CAAX was lethal in the homozygous state. Our results therefore demonstrate that the 2A constructs and two novel targeting motifs are functional in D. suzukii, and that the combination of EGFP-NLS and DsRed-CAAX shows dosage-dependent lethality. These molecular elements could be further used to improve expression systems in insects and generate novel pest control strains., Competing Interests: Declaration of competing interest A European Patent Application 18208613.2 has been filed., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu 2+ . During Cu 2+ -induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu 2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu 2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu 2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors., Competing Interests: The authors declare that they have no conflict of interest.

Inteins are protein segments capable of joining adjacent residues via a peptide bond. In this process known as protein splicing, the intein itself is not present in the final sequence, thus achieving scarless peptide ligation. Here, we assess the splicing activity of 34 inteins (both uncharacterized and known) using a rapid split fluorescent reporter characterization platform, and establish a library of 15 mutually orthogonal split inteins for in vivo applications, 10 of which can be simultaneously used in vitro. We show that orthogonal split inteins can be coupled to multiple split transcription factors to implement complex logic circuits in living organisms, and that they can also be used for the in vitro seamless assembly of large repetitive proteins with biotechnological relevance. Our work demonstrates the versatility and vast potential of an expanded library of orthogonal split inteins for their use in the fields of synthetic biology and protein engineering.

Bacteriophytochrome photoreceptors (BphPs) containing biliverdin (BV) have great potential for the development of genetically engineered near-infrared fluorescent proteins (NIR FPs). We investigated a photoactivatable fluorescent protein PAiRFP1, was engineered through directed molecular evolution. The coexistence of both red light absorbing (Pr) and far-red light absorbing (Pfr) states in dark is essential for the photoactivation of PAiRFP1. The PCR based site-directed reverse mutagenesis, spectroscopic measurements and molecular dynamics (MD) simulations were performed on three targeted sites V386A, V480A and Y498H in PHY domain to explore their potential effects during molecular evolution of PAiRFP1. We found that these substitutions did not affect the coexistence of Pr and Pfr states but led to slight changes in the photophysical parameters. The covalent docking of biliverdin (cis and trans form) with PAiRFP1 was followed by several 100 ns MD simulations to provide some theoretical explanations for the coexistence of Pr and pfr states. The results suggested that experimentally observed coexistence of Pr and Pfr states in both PAiRFP1 and mutants were resulted from the improved stability of Pr state. The use of experimental and computational work provided useful understanding of Pr and Pfr states and the effects of these mutations on the photophysical properties of PAiRFP1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca 2+ -regulated photoproteins. These proteins bind coelenterazine and oxygen, and trap 2-hydroperoxycoelenterazine, an intermediate along the reaction pathway. The reaction is halted until Ca 2+ binding triggers the completion of the reaction. There are currently no reported experimental, atomistic descriptions of this ternary Michaelis complex. This study utilized computational techniques to develop an atomistic model of the Michaelis complex. Extensive molecular dynamics simulations were carried out to study the interactions between four tautomeric/protonation states of coelenterazine and wide-type and mutant obelin. Only minor differences in binding modes were observed across all systems. Interestingly, no basic residues were identified in the vicinity of the N7-nitrogen of coelenterazine. This observation was surprising considering that deprotonation at this position is a key mechanistic step in the proposed bioluminescent reaction. This work suggests that coelenterazine binds either as the O10H tautomer, or in the deprotonated form. Implicit ligand sampling simulations were used to identify potential O 2 binding and migration pathways within obelin. A key oxygen binding site was identified close to the coelenterazine imidazopyrazinone core. The O 2 binding free energy was observed to be dependent on the protonation state of coelenterazine. Taken together, the description of the obelin-coelenterazine-O 2 complexes established in this study provides the basis for future computational studies of the bioluminescent mechanism. © 2019 Wiley Periodicals, Inc., (© 2019 Wiley Periodicals, Inc.)

While many instances of GPCR dimerization have been reported for vertebrate receptors, invertebrate GPCR dimerization remains poorly investigated, with few invertebrate GPCRs having been shown to assemble as dimers. To date, no Drosophila GPCRs have been shown to assemble as dimers. To explore the evolutionary conservation of GPCR dimerization, we employed an acceptor-photobleaching FRET methodology to evaluate whether multiple subclasses of Drosophila GPCRs assembled as homodimers when heterologously expressed in HEK-293 T cells. We C-terminally tagged multiple Drosophila neuropeptide GPCRs that exhibited structural homology with a vertebrate GPCR family member previously shown to assemble as a dimer with CFP and YFP fluorophores and visualized these receptors through confocal microscopy. FRET responses were determined based on the increase in CFP emission intensity following YFP photobleaching for each receptor pair tested. A significant FRET response was observed for each receptor expressed as a homodimer pair, while non-significant FRET responses were displayed by both cytosolic CFP and YFP expressed alone, and a heterodimeric pair of receptors from unrelated families. These findings suggest that receptors exhibiting positive FRET responses assemble as homodimers at the plasma membrane and are the first to suggest that Drosophila GPCRs assemble as homodimeric complexes. We propose that GPCR dimerization arose early in metazoan evolution and likely plays an important and underappreciated role in the cellular signaling of all animals., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with the contents of this manuscript., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Light-sensitive Ca 2+ -regulated photoprotein berovin is responsible for the bioluminescence of the ctenophore Beroe abyssicola. It shares many properties of hydromedusan photoproteins although the degree of identity of its amino acid sequence with those of photoproteins is low. There is a hydrogen bond between C-terminal Pro and Arg situated in the N-terminal α-helix of hydromedusan photoproteins that supports a closed conformation of the internal cavity of the photoprotein molecule with bound 2-hydroperoxycoelenterazine. The C- and N-terminal hydrogen bond network is necessary to properly isolate the photoprotein active site from the solvent and consequently to provide a high quantum yield of the bioluminescence reaction. In order to find out which berovin residues perform the same function we modified the N- and C-termini of the protein by replacing or deleting various amino acid residues. The studies on berovin mutants showed that the interaction between C-terminal Tyr208 and Tyr13 localized in the first α-helix of the photoprotein is important for the stabilization and proper orientation of the oxygenated coelenterazine adduct within the internal cavity as well as for supporting the closed photoprotein conformation. We also suggest that the interplay between Tyr residues in ctenophore photoproteins occurs rather through the π-π interaction of their phenyl rings than through hydrogen bonds as in hydromedusan photoproteins.

In vivo two-photon imaging is a valuable technique for studies of viral pathogenesis and host responses to infection in vivo. In this protocol, we describe a methodology for analyzing influenza virus-infected lung in vivo by two-photon imaging microscopy. We describe the surgical procedure, how to stabilize the lung, and an approach to analyzing the data. Further, we provide a database of fluorescent dyes, antibodies, and reporter mouse lines that can be used in combination with a reporter influenza virus (Color-flu) for multicolor analysis. Setup of this model typically takes ~30 min and enables the observation of influenza virus-infected lungs for >4 h during the acute phase of the inflammation and at least 1 h in the lethal phase. This imaging system, which we termed two-photon IMPRESS (imaging pathophysiology research system), is broadly applicable to analyses of other respiratory pathogens and reveals disease progression at the cellular level in vivo.

Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signal-to-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.

We report a new series of small molecule-protein hybrid zinc sensors that combine genetic targetability with the spectroscopic profile of synthetic fluorophores. We functionalized the zinc sensor ZinPyr-1 (ZP1) with a chloroalkane linker (ZP1-12Cl) that reacts specifically with the engineered protein HaloTag. The resulting construct, ZP1-HaloTag, binds zinc ions with a threefold fluorescence enhancement. Through exploitation of the protein synthesis machinery of live cells, the HaloTag protein component was expressed, and the ZP1-HaloTag hybrid was assembled upon bath application of ZP1-12Cl. After fusion of HaloTag with targeting peptides or proteins, the resulting hybrid sensor could be directed to specific subcellular locales, including the nucleus, mitochondrial outer membrane, and endoplasmic reticulum. Furthermore, HaloTag was linked with the red fluorescent protein mCherry, permitting formation of a two-fluorophore system that provides not only targetable but also ratiometric sensing of cellular zinc. This system reversibly detects both exogenous and endogenous mobile Zn 2+ in response to reactive nitrogen species in live HeLa cells. HaloTag-based hybrid zinc sensors offer new opportunities for visualizing and quantifying biological mobile zinc at discrete subcellular compartments.

Mitochondrial Ca 2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca 2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca 2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca 2+ dynamics, which are assumed to be determined by distinct thresholds of Ca 2+ increases at each subcellular mitochondrial domain. The balance between Ca 2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca 2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca 2+ affinities are engineered to match the intramitochondrial Ca 2+ concentrations. They enable visualization of mitochondrial Ca 2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca 2+ dynamics and their functions.

Ascidians are the sister group of vertebrates and occupy a critical position in explorations of the evolution of the endocrine and nervous systems of chordates. Here, we describe the complete ventral peptidergic system in adult transgenic Ciona robusta (Ciona intestinalis Type A) which expresses the Kaede reporter gene driven by the prohormone convertase 2 (PC2) gene promoter. Numerous PC2 promoter-driven fluorescent (Kaede-positive) non-neural cells were distributed in the blood sinus located at the anterior end of the pharynx, suggesting the acquisition of a peptidergic circulatory system in Ciona. Kaede-positive ciliated columnar cells, rounded cells, and tall ciliated cells were observed in the alimentary organs, including the endostyle, pharynx, esophagus, stomach, and intestine, suggesting that digestive functions are regulated by multiple peptidergic systems. In the heart, Kaede-positive neurons were located in the ring-shaped plexus at both ends of the myocardium. Nerve fiber-like tracts ran along the raphe and appeared to be connected with the plexuses. Such unique structures suggest a role for the peptidergic system in cardiac function. Collectively, the present anatomic analysis revealed the major framework of the ventral peptidergic system of adult Ciona, which could facilitate investigations of peptidergic regulation of the pharynx, endostyle, alimentary tissues, and heart.

Members of the anthozoan green fluorescent protein (GFP) family display a diversity of photo-physical properties that can be associated with normal and damaged coral tissues. Poritid coral species often exhibit localized pink pigmentation in diseased or damaged tissues. Our spectral and histological analyses of pink-pigmented Porites lobata lesions show co-localization of bright red fluorescence with putative amoebocytes concentrating in the epidermis, suggesting an activated innate immune response. Here we report the cloning, expression, and characterization of a novel red fluorescent protein (plobRFP) from the pink-pigmented tissues associated with lesions on Porites lobata. In vitro, the recombinant plobRFP exhibits a distinct red emission signal of 614 nm (excitation maximum: 578 nm), making plobRFP the furthest red-shifted natural fluorescent protein isolated from a scleractinian coral. The recombinant protein has a high molar extinction coefficient (84,000 M -1 cm -1 ) and quantum yield (0.74), conferring a notable brightness to plobRFP. Sequence analysis suggests the distinct brightness and marked red shift may be inherent features of plobRFP's chromophore conformation. While plobRFP displays a tendency to aggregate, its high pH stability, photostability, and spectral properties make it a candidate for cell imaging applications and a potential template for engineering optimized RFPs. The association of plobRFP with a possible immune response furthers its potential use as a visual diagnostic and molecular biomarker for monitoring coral health.

Genetically encoded fluorescent protein-based kinase biosensors are a central tool for illumination of the kinome. The adaptability and versatility of biosensors have allowed for spatiotemporal observation of real-time kinase activity in living cells and organisms. In this review, we highlight various types of kinase biosensors, along with their burgeoning applications in complex biological systems. Specifically, we focus on kinase activity reporters used in neuronal systems and whole animal settings. Genetically encoded kinase biosensors are key for elucidation of the spatiotemporal regulation of protein kinases, with broader applications beyond the Petri dish., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The molecular mechanisms for the photoconversion of fluorescent proteins remain elusive owing to the challenges of monitoring chromophore structural dynamics during the light-induced processes. We implemented time-resolved electronic and stimulated Raman spectroscopies to reveal two hidden species of an engineered ancestral GFP-like protein LEA, involving semi-trapped protonated and trapped deprotonated chromophores en route to photoconversion in pH 7.9 buffer. A new dual-illumination approach was examined, using 400 and 505 nm light simultaneously to achieve faster conversion and higher color contrast. Substitution of UV irradiation with visible light benefits bioimaging, while the spectral benchmark of a trapped chromophore with characteristic ring twisting and bridge-H bending motions enables rational design of functional proteins. With the improved H-bonding network and structural motions, the photoexcited chromophore could increase the photoswitching-aided photoconversion while reducing trapped species., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Phosphoinositides constitute a critical family of lipids that regulate numerous cellular processes. Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is arguably the most important plasma membrane phosphoinositide and is involved in regulating diverse processes. It is also the precursor of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ), which is critical for growth factor signaling, as well as membrane polarization and dynamics. Studying these lipids remains challenging, because of their compartmentalized activities and location-dependent signaling profiles. Here, we introduce several new genetically encoded fluorescent biosensors, including FRET-based and dimerization-dependent fluorescent protein (ddFP)-based biosensors, that enable real-time monitoring of PIP 2 levels in live cells. In addition, we developed a red fluorescent biosensor for 3-phosphoinositides that can be co-imaged with the green PIP 2 indicator. Simultaneous visualization of the dynamics of PIP 2 and 3-phosphoinositides in the same cell shows that plasma membrane PIP 3 formation upon EGF stimulation is coupled to a decrease in the local pool of PIP 2 .

Light-inducible optogenetic systems offer precise spatiotemporal control over a myriad of biologic processes. Unfortunately, current systems are inherently limited by their dependence on external light sources for their activation. Further, the utility of laser/LED-based illumination strategies are often constrained by the need for invasive surgical procedures to deliver such devices and local heat production, photobleaching and phototoxicity that compromises cell and tissue viability. To overcome these limitations, we developed a novel BRET-activated optogenetics (BEACON) system that employs biologic light to control optogenetic tools. BEACON is driven by self-illuminating bioluminescent-fluorescent proteins that generate "spectrally tuned" biologic light via bioluminescence resonance energy transfer (BRET). Notably, BEACON robustly activates a variety of commonly used optogenetic systems in a spatially restricted fashion, and at physiologically relevant time scales, to levels that are achieved by conventional laser/LED light sources.

Since their discovery, fluorescent probes have found widespread use in biological research. Over time, multiple next-generation probes increased the fluorescence catalog by offering novel capabilities of detection that have been previously difficult or lacking with conventional probes. One of such probes is called a fluorogen-activating protein (FAP). These are bimodular sensors, composed of a single-chain antibody that exhibits high-affinity and selectivity for small-molecule fluorogens. Because fluorogens are inherently nonfluorescent unless sterically restricted, upon the formation of the noncovalent FAP-fluorogen complex the fluorogen module emits fluorescence when excited by light. More interestingly, these bimodular sensors permit improvement of their biophysical properties. For instance, the fluorescence spectra and environmental sensing capabilities of fluorogens may be altered by the method of chemical modification at the fluorogen structural level. Also, optimizations of the single-chain antibody scaffold, via amino acid substitutions at the selectivity regions, may improve the detection brightness and affinities of fluorogens; this may also improve the biophysical stability of FAPs in different cellular environments. Additionally, when utilized as biological discovery probes, FAP biosensors exhibit functional activity as genetic fusion tags with cellular proteins; this results in high fluorescent sensitivities of cell surface and intracellular targets. Also, FAPs allow the monitoring of cellular traffic of surface receptors by fluorescence methods of real-time color switching, or signal onset and offset. They find application as biological probes integrated into biomaterials, or as soluble affinity reagents for whole live animal studies. Overall, this noncovalent activation of fluorogen particles results in advanced strategies of fluorescence detection.

Cysteine plays a critical role in maintaining normal human metabolism, redox homeostasis, and immune regulation. Despite its functional versatility, cysteine metabolism in the human body is not well understood because of the lack of a robust tool for real-time measurement of cysteine at the cellular and sub-cellular level. In the present study, a genetically encoded nanosensor was developed using Cj0982 protein of Campylobacter jejuni, Enhanced Cyan Fluorescent Protein (ECFP) and Venus. The Cj0982 was sandwiched between ECFP and Venus for the construction of the nanosensor, named as Cys-FS (Cysteine-Fluorescent-Sensor). The Cys-FS is pH stable, specific to cysteine and has an affinity of 1.2 × 10 - 5  M. A range of affinity mutants were also developed with a cumulative cysteine detection range from 800 nM to 3.5 mM. The Cys-FS nanosensor was expressed in bacterial, yeast and mammalian cells, and the dynamics of cysteine level was measured in living cells using the confocal microscopy. The results showed that the Cys-FS nanosensor successfully monitored the dynamics of cysteine in both prokaryotic and eukaryotic systems without disrupting the cell. Thus, this study presents a novel nanosensor that can measure cysteine in living cells. This nanosensor is minimally invasive and non-toxic., (Copyright © 2019 Elsevier B.V. All rights reserved.)

To gain insight into factors that lead to dissociation of Bax from a complex with Hsp70 during apoptosis, we recently constructed a fluorescence resonance energy transfer (FRET) system composed of the Hsp70-YFP (YFP=yellow fluorescent protein) fusion protein and fluorescent amino acid (ANAP=6-acetyl(naphthalen-2-ylamino)-2-aminopropanoic acid)-containing Bax (Bax-ANAP), which was produced by using the genetic code expansion technique. In the current study, the FRET system was employed to elucidate how brefeldin A (an endoplasmic reticulum stress inducer), chlorpromazine and apoptozole (lysosomal membrane destabilizers), bafilomycin A1 (an inhibitor of lysosomal acidification) as well as raptinal and Az-TPP-O3 (mitochondria-targeted apoptosis inducers) affect the interaction between Bax and Hsp70. Analyses of single live cell images together with results of co-immunoprecipitation assays reveal that brefeldin A, chlorpromazine, and apoptozole promote dissociation of the Bax/Hsp70 complex through activation of the activator BH3-only protein. However, the results show that bafilomycin A1, raptinal, and Az-TPP-O3 have no influence on the interaction of Bax with Hsp70. The combined observations made in the current and previous studies demonstrate that the FRET system consisting of Bax-ANAP and Hsp70-YFP is highly useful to understand apoptotic processes associated with the Bax-Hsp70 interaction., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~10 5 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.

Monitoring of protein oligomerization has benefited greatly from Förster Resonance Energy Transfer (FRET) measurements. Although donors and acceptors are typically fluorescent molecules with different spectra, homo-FRET can occur between fluorescent molecules of the same type if the emission spectrum overlaps with the absorption spectrum. Here, we describe homo-FRET measurements by monitoring anisotropy changes in photoswitchable fluorescent proteins while photoswitching to the off state. These offer the capability to estimate anisotropy in the same specimen during homo-FRET as well as non-FRET conditions. We demonstrate photoswitching anisotropy FRET (psAFRET) with a number of test chimeras and example oligomeric complexes inside living cells. We also present an equation derived from FRET and anisotropy equations which converts anisotropy changes into a factor we call delta r FRET (drFRET). This is analogous to an energy transfer efficiency and allows experiments performed on a given homo-FRET pair to be more easily compared across different optical configurations.

We have developed probes based on the bacterial periplasmic glutamate/aspartate binding protein with either an endogenously fluorescent protein or a synthetic fluorophore as the indicator of glutamate binding for studying the kinetic mechanism of glutamate binding. iGluSnFR variants termed iGlu h , iGlu m , and iGlu l cover a broad range of K d -s (5.8 μM and 2.1 and 50 mM, respectively), and a novel fluorescently labeled indicator, Fl-GluBP, has a K d of 9.7 μM. The fluorescence response kinetics of all the probes are consistent with a two-step mechanism involving ligand binding and isomerization either of the apo or the ligand-bound binding protein. Although the previously characterized ultrafast indicators iGlu u and iGlu f had monophasic fluorescence enhancement that occurred in the rate limiting isomerization step, the sensors described here all have biphasic binding kinetics with fluorescence increases occurring both in the glutamate binding and the isomerization steps. For iGlu m and iGlu l , the data indicate prebinding conformational change followed by ligand binding. In contrast, for iGlu h and Fl-GluBP, glutamate binding is followed by isomerization. Thus, the effects of structural heterogeneity introduced by single amino acid changes around the binding site on the kinetic path of interactions with glutamate are revealed. Remarkably, glutamate binding with a diffusion-limited rate constant to iGlu h and Fl-GluBP is detected for the first time, hinting at the underlying mechanism of the supremely rapid activation of the highly homologous α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor by glutamate binding., (Copyright © 2019 Biophysical Society. All rights reserved.)

Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

A near-infrared (NIR) fluorescent nanoprobe that enables to circumvent the interference of background absorption and fluorescence in whole blood was developed for the direct sensing of blood glucose. Here, NIR fluorescent protein (iRFP) and glucose oxidase (GOx) were collectively deployed as the templates for the biomineralization of Mn2+ to prepare a NIR fluorescent nanoprobe (iRFP-GOx-MnO2 nanoparticles, iRGMs), in which the fluorescence of iRFP was effectively quenched by MnO2via energy transfer. When the iRGMs were mixed with whole blood samples, GOx can convert blood glucose into gluconic acid, as well as H2O2, which will reduce MnO2 and decompose the iRGMs. As a result, the NIR fluorescence of iRFPs was restored, providing a fluorometric assay for the direct detection of blood glucose. Owing to the high efficiency of the cascade reaction and the low background interference of the NIR fluorescence signal, accurate and rapid analysis of the glucose levels in whole blood samples was achieved using the iRGMs. Moreover, an iRGM-based paper device that only requires 5 microliters of samples was also demonstrated in the direct assay of blood glucose without any pretreatment, affording an alternative approach for the accurate monitoring of blood glucose levels.

Background: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina., Objective: To assess the properties of aeBlue chromoprotein under different physicochemical conditions., Methods: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed., Results: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography., Conclusion: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Fluorescence microscopy enables the localization of proteins to specific structures within a cell which have either been fused to a fluorescence protein or detected by immunofluorescence. Here, we describe the various procedures that can be used to prepare both the procyclic form and bloodstream form of the human pathogen Trypanosoma brucei for fluorescence microscopy. The choice of procedure to be used is determined by various parameters, including protein characteristics and the scientific question being investigated.

De novo root regeneration (DNRR) is the process in which adventitious roots are regenerated from damaged plant tissues or organs. We have developed a simple DNRR system in which adventitious roots are formed from detached leaf explants of Arabidopsis (Arabidopsis thaliana) on B5 medium without external hormones. In this chapter, we introduce the methods used to observe gene expression patterns during rooting from leaf explants. Usually, β-glucuronidase (GUS) staining is used to visualize gene expression patterns, since fluorescent proteins are difficult to observe because of the high autofluorescence in leaf explants. Here, we describe the use of the ClearSee technique with Congo red staining for deep imaging to observe fluorescent proteins. This method diminishes autofluorescence in leaf explants and preserves the stability of fluorescent proteins, thus allowing us to investigate the endogenous molecular actions guiding DNRR.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO 4 ) treatment for improved SR-CLEM.

pH is a fundamental variable in enzyme catalysis and its measurement therefore is crucial for understanding and optimizing enzyme-catalyzed reactions. Whereas measurements within homogeneous bulk liquid solution are prominently used, enzymes immobilized inside porous particles often suffer from pH gradients due to partition effects and heterogeneously catalyzed biochemical reactions. Unfortunately, the measurements of intraparticle pH are not available due to the lack of useful suitable methodologies; as a consequence the biocatalyst characterization is hampered. Here, a fully biocompatible methodology for real-time optical sensing of pH within porous materials is described. A genetically encoded ratiometric pH indicator, the superfolder yellow fluorescent protein (sYFP), is used to functionalize the internal surface of enzyme carrier supports. By using controlled, tailor-made immobilization, sYFP is homogeneously distributed within these materials, and so enables, via self-referenced imaging analysis, pH measurements in high accuracy and with useful spatiotemporal resolution. The hydrolysis of penicillin by a penicillin acylase, taking place in solution or confined to the solid surface of the porous matrix is used to show the monitoring of evolution of internal pH. Thus, pH sensing based on immobilized sYFP represents a broadly applicable technique to the study of the internally heterogeneous environment of immobilized enzymes into solid particles.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

The bionanohybrids are the junctions of at least two objects of different origin: abiotic and biotic. The abiotic part is a nanoparticle (often a fluorescent quantum dot), the biotical one may be a protein (especially fluorescent one or redox-active one), nucleic acid, carbohydrate as well as a simple organic molecule. When such a junction undergoes illumination, the energy transfer between the partners is possible. The nanoparticles, depending on their characteristics, may be donors, acceptors or mediators of the energy transfer. In most cases, the mechanism of the transfer is the Förster resonance energy transfer (FRET) or the electron transfer (ET). Here, we reviewed the newest achievements in the field with special attention paid to those bionanohybrids which allow FRET or ET. Such nanohybrids are important not only for exploration of the mechanism of the partner interaction but mainly for working out nanobiodevices for biosensing and nanotools for modern therapies.

Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions and protein folding in vivo. Conventional BRET systems have solely depended on an overlap of the energy donor and acceptor spectra. In this study, we engineered a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length Artificial Luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched between a protein pair of interest, FRB and FKBP12, and linked to a fluorescent protein as the energy acceptor. A specific ligand, rapamycin, then activates inter- and intramolecular interactions of FRB and FKBP12, which develop molecular strain in the sandwiched ALuc23 to accelerate further folding. We found that this system greatly enhanced both the total bioluminescence spectrum and the BRET signal in the far-red (FR) region. We characterized the molecular construct by studying 18 different designs categorized into four groups. The best BRET system design allowed an approximately 5-fold enhancement of the bioluminescence intensities in the FR region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR bioluminescence signals in cells and living animal models.

Genetically encoded voltage indicators (GEVIs) allow optical recording of neuronal activities with high spatial resolution. While most existing GEVIs emit in the green range, red-shifted GEVIs are highly sought after because they would enable simultaneous stimulation and recording of neuronal activities when paired with optogenetic actuators, or two-color imaging of signaling and neuronal activities when used along with GFP-based indicators. In this study, we present several improved red-shifted GEVIs based on the electrochromic Förster resonance energy transfer (eFRET) between orange/red fluorescent proteins/dyes and rhodopsin mutants. Through structure-guided mutagenesis and cell-based sensitivity screening, we identified a mutant rhodopsin with a single mutation that exhibited more than 2-fold improvement in voltage sensitivity. Notably, this mutation has been independently discovered by Pieribone et al. ( Pieribone, V. A. et al. Nat Methods 2018 , 15 ( 12 ), 1108 - 1116 ). In cultured rat hippocampal neurons, our sensors faithfully reported action potential waveforms and subthreshold activities. We also demonstrated that this mutation could enhance the sensitivity of hybrid indicators, thus providing insights for future development.

The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties., (Copyright © 2019 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from Rp BphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.

In this study, we tested the possibility of creating complexes of two proteins by fusing them with heterodimerizing helices. We used the fluorescent proteins GFP and mCHERRY expressed with a His-tag as our model system. We added heterodimer-forming sequences at the C- or N- termini of the proteins, opposite to the His-tag position. Heterodimerization was tested for both helices at the C-terminus or at the N- terminus and C-terminus. We observed complex formation with a nanomolar dissociation constant in both cases that was higher by one order of magnitude than the K d s measured for helices alone. The binding of two C-terminal helices was accompanied by an increased enthalpy change. The binding between helices could be stabilized by introducing an additional turn of the helix with cysteine, which was capable of forming disulphide bridges. Covalently linked proteins were obtained using this strategy and observed using fluorescence cross-correlation spectroscopy. Finally, we demonstrated the formation of complexes of protein dimers and quantum dots., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Fluorescent proteins have been applied in a wide variety of fields ranging from basic science to industrial applications. Apart from the naturally occurring fluorescent proteins, there is a growing interest in genetically modified variants that emit light in a specific wavelength. Genetically modifying a protein is not an easy task, especially because the exchange of one residue by other has to achieve the desired property while maintaining protein stability. To help in the choice of residue exchange, computational methods are applied to predict function and stability of proteins. In this work we have prepared a dataset composed by 109 fluorescent proteins and tested four classical supervised classification algorithms: artificial neural networks (ANNs), decision trees (DTs), support vector machines (SVMs) and random forests (RFs). This is the first time that algorithms are compared in this task. Results of comparing the algorithm's performance shows that DT, SVM and RF were significantly better than ANNs, and RF was the best method in all the scenarios. However, the interpretability of DTs is highly relevant and can provide important clues about the mechanisms involved in protein color emission. The results are promising and indicate that the use of in silico methods can greatly reduce the time and cost of the in vitro experiments., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell-based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non-invasive optical imaging at shortwave infrared region are discussed in detail., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Bioluminescence imaging is a non-invasive tool for in vivo real-time monitoring of infectious disease progression in animal models. However, no bioluminescence imaging assay has been developed to monitor Acinetobacter baumannii infections. In the current study, bioluminescent strains of A. baumannii ATCC 17978 and its isogenic ΔompA mutant were constructed by integrating the promoter of the ompA gene and the luxCDABE luciferase gene into the bacterial chromosome. In an acute murine pneumonia model, bioluminescence of the two reporter strains was clearly visible in the lungs and the bioluminescent signal increased over time. Bioluminescence was correlated with bacterial burden and histopathology in reporter strain-infected mice, suggesting that bioluminescent bacteria are useful for monitoring A. baumannii infections in animal models., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Super-resolution light microscopy (SRM) offers a unique opportunity for diffraction-unlimited imaging of biomolecular activities in living cells. To realize such potential, genetically encoded indicators were developed recently from fluorescent proteins (FPs) that exhibit phototransformation behaviors including photoactivation, photoconversion, and photoswitching, etc. Super-resolution observations of biomolecule interactions and biochemical activities have been demonstrated by exploiting the principles of bimolecular fluorescence complementation (BiFC), points accumulation for imaging nanoscale topography (PAINT), and fluorescence fluctuation increase by contact (FLINC), etc. To improve functional nanoscopy with the technology of genetically encoded indicators, it is essential to fully decipher the photo-induced chemistry of FPs and opt for innovative indicator designs that utilize not only fluorescence intensity but also multi-parametric readouts such as phototransformation kinetics. In parallel, technical improvements to both the microscopy optics and image analysis pipeline are promising avenues to increase the sensitivity and versatility of functional SRM.

We introduce a method to monitor the integrity of micellar nanocarriers using a novel fluorescent dye, IR-780-PDMS and Förster resonance energy transfer (FRET) as a readout. In addition, these dye-loaded nanocarriers can be used as a phototoxic agent in vitro. Mainly, a nanocarrier was designed, based on a previously described amphiphilic ABA-copolymer (Pip-PMOXA-PDMS-PMOXA-Pip) scaffold that incorporates the fluorescent FRET dye partners IR-780-PDMS (donor) and IR-780 (acceptor). The confirmation of FRET (that only occurs when donor and acceptor are in the required close proximity of less than ∼10 nm) in the nanocarriers is used to prove that the acceptor dye (IR-780) is still contained in its hydrophobic core. We measured such FRET signals of the nanocarriers also upon cellular uptake into HeLa cells using fluorescence-lifetime imaging microscopy (FLIM). Confocal laser scanning microscopy after incubation with nanocarriers demonstrated the intracellular uptake of the particles and their localization in an intracellular granular pattern. To demonstrate the intactness of the nanocarriers by detection of FRET we measured the fluorescence lifetime (FLIM) of the donor dye. FLIM showed that both types of lifetimes, that of the quenched donor, and that of the unquenched donor were present, in a granular pattern and homogeneously in the cytosol, respectively, indicating the presence of intracellular intact and disintegrated micellar nanocarriers. These data show that the herewith-described FRET method allows monitoring the intactness of nanocarriers while en route to the target, and also that the cargo is delivered and released within a potential target cell. In addition, near-infrared (NIR) irradiation of IR-780-loaded micellar nanocarriers leads to photocytotoxicity, which we demonstrated in in vitro experiments. Our findings open potential avenues in photodynamic therapy (PDT) of cancer.

Biliproteins have extended the spectral range of fluorescent proteins into the far-red (FR) and near-infrared (NIR) regions. These FR and NIR fluorescent proteins are suitable for the bioimaging of mammalian tissues and are indispensable for multiplex labeling. Their application, however, presents considerable challenges in increasing their brightness, while maintaining emission in FR regions and oligomerization of monomers. Two fluorescent biliprotein triads, termed BDFP1.2/1.6:3.3:1.2/1.6, are reported. In mammalian cells, these triads not only have extremely high brightness in the FR region, but also have monomeric oligomerization. The BDFP1.2 and BDFP1.6 domains covalently bind to biliverdin, which is accessible in most cells. The BDFP3.3 domain noncovalently binds phycoerythrobilin that is added externally. A new method of replacing phycoerythrobilin with proteolytically digested BDFP3.3 facilitates this labeling. BDFP3.3 has a very high fluorescence quantum yield of 66 %, with maximal absorbance at λ=608 nm and fluorescence at λ=619 nm. In BDFP1.2/1.6:3.3:1.2/1.6, the excitation energy that is absorbed in the red region by phycoerythrobilin in the BDFP3.3 domain is transferred to biliverdin in the two BDFP1.2 or BDFP1.6 domains and fluoresces at λ≈670 nm. The combination of BDFP3.3 and BDFP1.2/1.6:3.3:1.2/1.6 can realize dual-color labeling. Labeling various proteins by fusion to these new fluorescent biliproteins is demonstrated in prokaryotic and mammalian cells., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Methyl parathion hydrolase (MPH) plays an important role in degrading a range of organophosphorus compounds. In order to display MPH on the cell surface of Escherichia coli strain RosettaBlue™, the Flavin-based fluorescent protein EcFbFP was severed as an auto-anchoring matrix. With net negative charges of EcFbFP supplying the driving forces, fusion protein MPH-EcFbFP through a two-step auto-surface display process was finally verified by (a) inner membrane translocation and (b) anchoring at outer membrane. Cells with surface-displayed MPH obtained activity of 0.12 U/OD600 against substrate methyl parathion. MPH when fused with engineered EcFbFP containing 20 net negative charges exhibited fivefold higher anchoring efficiency and tenfold higher enzymatic catalytic activity of 1.10 U/OD600. The above result showed that MPH was successfully displayed on cell surface and can be used for biodegradation of methyl parathion.