Towards PDT with Genetically Encoded Photosensitizer KillerRed: A Comparison of Continuous and Pulsed Laser Regimens in an Animal Tumor Model

The strong phototoxicity of the red fluorescent protein KillerRed allows it to be considered as a potential genetically encoded photosensitizer for the photodynamic therapy (PDT) of cancer. The advantages of KillerRed over chemical photosensitizers are its expression in tumor cells transduced with the appropriate gene and direct killing of cells through precise damage to any desired cell compartment. The ability of KillerRed to affect cell division and to induce cell death has already been demonstrated in cancer cell lines in vitro and HeLa tumor xenografts in vivo. However, the further development of this approach for PDT requires optimization of the method of treatment. In this study we tested the continuous wave (593 nm) and pulsed laser (584 nm, 10 Hz, 18 ns) modes to achieve an antitumor effect. The research was implemented on CT26 subcutaneous mouse tumors expressing KillerRed in fusion with histone H2B. The results showed that the pulsed mode provided a higher rate of photobleaching of KillerRed without any temperature increase on the tumor surface. PDT with the continuous wave laser was ineffective against CT26 tumors in mice, whereas the pulsed laser induced pronounced histopathological changes and inhibition of tumor growth. Therefore, we selected an effective regimen for PDT when using the genetically encoded photosensitizer KillerRed and pulsed laser irradiation.     

KillerOrange,a Genetically Encoded Photosensitizer Activated by Blue and Green Light

Genetically encoded photosensitizers, proteins that produce reactive oxygen species when illuminated with visible light, are increasingly used as optogenetic tools. Their applications range from ablation of specific cell populations to precise optical inactivation of cellular proteins. Here, we report an orange mutant of red fluorescent protein KillerRed that becomes toxic when illuminated with blue or green light. This new protein, KillerOrange, carries a tryptophan-based chromophore that is novel for photosensitizers. We show that KillerOrange can be used simultaneously and independently from KillerRed in both bacterial and mammalian cells offering chromatic orthogonality for light-activated toxicity.     

Structural basis for the phototoxicity of the fluorescent protein KillerRed

The red fluorescent protein KillerRed, engineered from the hydrozoan chromoprotein anm2CP, has been reported to induce strong cytotoxicity through the chromophore assisted light inactivation (CALI) effect. Here, we present the X-ray structures of KillerRed in its native and bleached states. A long water-filled channel is revealed, connecting the methylene bridge of the chromophore to the solvent. This channel facilitates the transit of oxygen and of reactive oxygen species (ROS) formed by reaction with the excited chromophore. The functional roles of key mutations used to produce KillerRed are discussed, strong chromophore distortions in the bleached state are revealed, and mechanisms for ROS production and self protection are proposed. The presence of a partially mature, photo-resistant, green-emitting state is characterized, which accounts for enhanced CALI by “pre-bleached” KillerRed.     

Comparative Evaluation of Genetically Encoded Voltage Indicators

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Novel Split-Luciferase-Based Genetically Encoded Biosensors for Noninvasive Visualization of Rho GTPases

Rho family GTPases are critical regulators of many important cellular processes and the dysregulation of their activities is implicated in a variety of human diseases including oncogenesis and propagation of malignancy. The traditional methods, such as “pull-down” or two-hybrid procedures, are poorly suited to dynamically evaluate the activity of Rho GTPases, especially in living mammalian cells. To provide a novel alternative approach to analyzing Rho GTPase-associated signaling pathways in vivo, we developed a series of bioluminescent biosensors based on the genetically engineered firefly luciferase. These split-luciferase-based biosensors enable non-invasive visualization and quantification of the activity of Rho GTPases in living subjects. The strategy is to reasonably split the gene of firefly luciferase protein into two inactive fragments and then respectively fuse the two fragments to Rho GTPase and the GTPase-binding domain (GBD) of the specific effector. Upon Rho GTPase interacting with the binding domain in a GTP-dependent manner, these two luciferase fragments are brought into close proximity, leading to luciferase reconstitution and photon production in the presence of the substrate. Using these bimolecular luminescence complementation (BiLC) biosensors, we successfully visualized and quantified the activities of the three best characterized Rho GTPases by measuring the luminescence in living cells. We also experimentally investigated the sensitivity of these Rho GTPase biosensors to upstream regulatory proteins and extracellular ligands without lysing cells and doing labor-intensive works. By virtue of the unique functional characteristics of bioluminescence imaging, the BiLC-based biosensors provide an enormous potential for in vivo imaging of Rho GTPase signaling pathways and high-throughput screening of therapeutic drugs targeted to Rho GTPases and (or) upstream molecules in the near future.     

Mechanistic Studies of the Genetically Encoded Fluorescent Protein Voltage Probe ArcLight

ArcLight, a genetically encoded fluorescent protein voltage probe with a large ΔF/ΔV, is a fusion between the voltage sensing domain of the Ciona instestinalis voltage sensitive phosphatase and super ecliptic pHluorin carrying a single mutation (A227D in the fluorescent protein). Without this mutation the probe produces only a very small change in fluorescence in response to voltage deflections (∼1%). The large signal afforded by this mutation allows optical detection of action potentials and sub-threshold electrical events in single-trials in vitro and in vivo. However, it is unclear how this single mutation produces a probe with such a large modulation of its fluorescence output with changes in membrane potential. In this study, we identified which residues in super ecliptic pHluorin (vs eGFP) are critical for the ArcLight response, as a similarly constructed probe based on eGFP also exhibits large response amplitude if it carries these critical residues. We found that D147 is responsible for determining the pH sensitivity of the fluorescent protein used in these probes but by itself does not result in a voltage probe with a large signal. We also provide evidence that the voltage dependent signal of ArcLight is not simply sensing environmental pH changes. A two-photon polarization microscopy study showed that ArcLight''s response to changes in membrane potential includes a reorientation of the super ecliptic pHluorin. We also explored different changes including modification of linker length, deletion of non-essential amino acids in the super ecliptic pHluorin, adding a farnesylation site, using tandem fluorescent proteins and other pH sensitive fluorescent proteins.     

Genetically Encoded FRET Biosensor Detects the Enzymatic Activity of Prostate-Specific Antigen

Prostate cancer is the most common cancer among men beyond 50 years old, and ranked the second in mortality. The level of Prostate-specific antigen (PSA) in serum has been a routine biomarker for clinical assessment of the cancer development, which is detected mostly by antibody-based immunoassays. The proteolytic activity of PSA also has important functions. Here a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) technology was developed to measure PSA activity. In vitro assay showed that the biosensor containing a substrate peptide ‘RLSSYYSGAG’ had 400% FRET change in response to 1 µg/ml PSA within 90 min, and could detect PSA activity at 25 ng/ml. PSA didn’t show enzymatic activity toward the biosensor in serum solution, likely reflecting the existence of other inhibitory factors besides Zn2+. By expressing the biosensor on cell plasma membrane, the FRET responses were significant, but couldn’t distinguish well the cultured prostate cancer cells from non-prostate cancer cells under microscopy imaging, indicating insufficient speci- ficity to PSA. The biosensor with the previously known ‘HSSKLQ’ substrate showed little response to PSA in solution. In summary, we developed a genetically encoded FRET biosensor to detect PSA activity, which may serve as a useful tool for relevant applications, such as screening PSA activation substrates or inhibitors; the purified biosensor protein can also be an alternative choice for measuring PSA activity besides currently commercialized Mu-HSSKLQ-AMC substrate from chemical synthesis.     

Understanding the Fluorescence Change in Red Genetically Encoded Calcium Ion Indicators

For over 20 years, genetically encoded Ca²⁺ indicators have illuminated dynamic Ca²⁺ signaling activity in living cells and, more recently, whole organisms. We are just now beginning to understand how they work. Various fluorescence colors of these indicators have been developed, including red. Red ones are promising because longer wavelengths of light scatter less in tissue, making it possible to image deeper. They are engineered from a red fluorescent protein that is circularly permuted and fused to a Ca²⁺-sensing domain. When Ca²⁺ binds, a conformational change in the sensing domain causes a change in fluorescence. Three factors can contribute to this fluorescence change: 1) a shift in the protonation equilibrium of the chromophore, 2) a change in fluorescence quantum yield, and 3) a change in the extinction coefficient or the two-photon cross section, depending on if it is excited with one or two photons. Here, we conduct a systematic study of the photophysical properties of a range of red Ca²⁺ indicators to determine which factors are the most important. In total, we analyzed nine indicators, including jRGECO1a, K-GECO1, jRCaMP1a, R-GECO1, R-GECO1.2, CAR-GECO1, O-GECO1, REX-GECO1, and a new variant termed jREX-GECO1. We find that these could be separated into three classes that each rely on a particular set of factors. Furthermore, in some cases, the magnitude of the change in fluorescence was larger with two-photon excitation compared to one-photon because of a change in the two-photon cross section, by up to a factor of two.     

Genetically Encoded Sensors to Elucidate Spatial Distribution of Cellular Zinc

Transition metals are essential enzyme cofactors that are required for a wide range of cellular processes. Paradoxically, whereas metal ions are essential for numerous cellular processes, they are also toxic. Therefore cells must tightly regulate metal accumulation, transport, distribution, and export. Improved tools to interrogate metal ion availability and spatial distribution within living cells would greatly advance our understanding of cellular metal homeostasis. In this work, we present genetically encoded sensors for Zn²⁺ based on the principle of fluorescence resonance energy transfer. We also develop methodology to calibrate the probes within the cellular environment. To identify both sources of and sinks for Zn²⁺, these sensors are genetically targeted to specific locations within the cell, including cytosol, plasma membrane, and mitochondria. Localized probes reveal that mitochondria contain an elevated pool of Zn²⁺ under resting conditions that can be released into the cytosol upon glutamate stimulation of hippocampal neurons. We also observed that Zn²⁺ is taken up into mitochondria following glutamate/Zn²⁺ treatment and that there is heterogeneity in both the magnitude and kinetics of the response. Our results suggest that mitochondria serve as a source of and a sink for Zn²⁺ signals under different cellular conditions.Although mammalian cells are known to concentrate transition metals, it is now well established that under resting conditions, “free” (e.g. unbound) metals are maintained at extremely low levels. Estimates of the total Zn²⁺ concentration in mammalian cells typically range from 100 to 500 μm (1); yet free Zn²⁺ concentrations are tightly buffered by proteins such as metallothionein to maintain cytosolic Zn²⁺ concentrations in the picomolar to nanomolar range (2–5). However, there is emerging evidence that this static picture is dramatically altered by different cellular conditions, such as redox perturbations caused by oxidative stress (6, 7) and cellular signals such as nitric oxide (8). Consequently, there is a pool of labile Zn²⁺ that, if mobilized by cellular signals, would result in the generation of transient Zn²⁺ signals. Recent studies suggest that these Zn²⁺ signals influence critical biological processes, such as mitochondrial function (7, 9, 10). Elucidation of the sources and dynamics of these Zn²⁺ signals would greatly advance our understanding of the interplay between metal regulation and cellular function.There has been a huge effort in the past few years to develop sensitive and selective fluorescent probes to monitor Zn²⁺ in biological systems. The majority of this work has focused on the generation of small molecule fluorescent indicators (reviewed by Que et al. (11)). Yet there are also examples of sensors based partially on Zn²⁺-binding proteins, such as carbonic anhydrase (12) and metallothionein (13), and peptide scaffolds (14). Although many of these sensors have begun to provide insight into Zn²⁺ concentrations within cells, one limitation is that it is challenging to explicitly target them to subdomains within the cell. Localized probes are necessary to generate a complete picture of cellular Zn²⁺ homeostasis in mammalian cells. For this reason, sensors that are genetically encoded (i.e. generated by translation of a nucleic acid sequence) are attractive platforms for engineering metal-specific sensors. Encoded sensors provide additional benefits such as retention of the sensor over days to weeks permitting long term imaging and the ability to systematically vary the sensor concentration to evaluate the extent to which the sensor perturbs resting Zn²⁺ concentrations.Here we present genetically encoded sensors designed with a “Zn²⁺-sensing domain” sandwiched between two fluorescent proteins. The fluorescent proteins are chosen so that they are capable of undergoing fluorescence resonance energy transfer (FRET).² Because the mechanism of FRET involves dipole-dipole coupling, it is exquisitely dependent on the distance and orientation of the fluorophores with respect to one another. Therefore, if the binding of Zn²⁺ induces a conformational change in the sensor, it will alter the energy transfer between the two fluorescent proteins. The advantage of using FRET as the optical readout is that the donor emission will decrease and the acceptor emission will increase upon Zn²⁺ binding. Hence, by taking the ratio of the acceptor to the donor emission, we can create a ratiometric sensor. These sensors are targeted to the cytosol, mitochondria, and plasma membrane by attachment of signal sequences and fusion to other proteins. These sensors reveal differences in the spatial distribution of Zn²⁺ and highlight the power and utility of localized probes.     

A Genetically Encoded Reporter for Real-Time Imaging of Cofilin-Actin Rods in Living Neurons

Filament bundles (rods) of cofilin and actin (1:1) form in neurites of stressed neurons where they inhibit synaptic function. Live-cell imaging of rod formation is hampered by the fact that overexpression of a chimera of wild type cofilin with a fluorescent protein causes formation of spontaneous and persistent rods, which is exacerbated by the photostress of imaging. The study of rod induction in living cells calls for a rod reporter that does not cause spontaneous rods. From a study in which single cofilin surface residues were mutated, we identified a mutant, cofilinR21Q, which when fused with monomeric Red Fluorescent Protein (mRFP) and expressed several fold above endogenous cofilin, does not induce spontaneous rods even during the photostress of imaging. CofilinR21Q-mRFP only incorporates into rods when they form from endogenous proteins in stressed cells. In neurons, cofilinR21Q-mRFP reports on rods formed from endogenous cofilin and induced by all modes tested thus far. Rods have a half-life of 30–60 min upon removal of the inducer. Vesicle transport in neurites is arrested upon treatments that form rods and recovers as rods disappear. CofilinR21Q-mRFP is a genetically encoded rod reporter that is useful in live cell imaging studies of induced rod formation, including rod dynamics, and kinetics of rod elimination.     

Generation and Characteristics of Genetically Encoded Fluorescent Sensors of Extracellular pH

Russian Journal of Bioorganic Chemistry - Recently, fluorescent proteins have become an indispensable tool for in vivo visualization of various processes occurring in living systems, from...     

Synthesis of Non-linear Protein Dimers through a Genetically Encoded Thiol-ene Reaction

Site-specific incorporation of bioorthogonal unnatural amino acids into proteins provides a useful tool for the installation of specific functionalities that will allow for the labeling of proteins with virtually any probe. We demonstrate the genetic encoding of a set of alkene lysines using the orthogonal PylRS/PylT_CUA pair in Escherichia coli. The installed double bond functionality was then applied in a photoinitiated thiol-ene reaction of the protein with a fluorescent thiol-bearing probe, as well as a cysteine residue of a second protein, showing the applicability of this approach in the formation of heterogeneous non-linear fused proteins.     

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate goal is to image neuronal activity in vivo, one must be able to image activity of a single cell to ensure successful in vivo preparations. This procedure will describe how to image membrane potential in a single cell to provide a foundation to eventually image in vivo. Here we describe methods for imaging GEVIs consisting of a voltage-sensing domain fused to either a single fluorescent protein (FP) or two fluorescent proteins capable of Förster resonance energy transfer (FRET) in vitro. Using an image splitter enables the projection of images created by two different wavelengths onto the same charge-coupled device (CCD) camera simultaneously. The image splitter positions a second filter cube in the light path. This second filter cube consists of a dichroic and two emission filters to separate the donor and acceptor fluorescent wavelengths depending on the FPs of the GEVI. This setup enables the simultaneous recording of both the acceptor and donor fluorescent partners while the membrane potential is manipulated via whole cell patch clamp configuration. When using a GEVI consisting of a single FP, the second filter cube can be removed allowing the mirrors in the image splitter to project a single image onto the CCD camera.     

Ultrasensitive Genetically Encoded Indicator for Hydrogen Peroxide Identifies Roles for the Oxidant in Cell Migration and Mitochondrial Function

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pHlash: A New Genetically Encoded and Ratiometric Luminescence Sensor of Intracellular pH

We report the development of a genetically encodable and ratiometic pH probe named "pHlash" that utilizes Bioluminescence Resonance Energy Transfer (BRET) rather than fluorescence excitation. The pHlash sensor-composed of a donor luciferase that is genetically fused to a Venus fluorophore-exhibits pH dependence of its spectral emission in vitro. When expressed in either yeast or mammalian cells, pHlash reports basal pH and cytosolic acidification in vivo. Its spectral ratio response is H(+) specific; neither Ca(++), Mg(++), Na(+), nor K(+) changes the spectral form of its luminescence emission. Moreover, it can be used to image pH in single cells. This is the first BRET-based sensor of H(+) ions, and it should allow the approximation of pH in cytosolic and organellar compartments in applications where current pH probes are inadequate.     

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

With the escalation of drug discovery programmes, it has become essential to visualize and monitor biological activities in healthy and pathological cells, with high spatial and temporal resolution. To this aim, the development of probes and sensors, which can report on the levels and activities of specific intracellular targets, has become essential. Together with the discovery of the Green Fluorescent Protein (GFP), and the development of GFP-based reporters, recent advances in the synthesis of small molecule fluorescent probes, and the explosion of fluorescence-based imaging technologies, the biosensor field has witnessed a dramatic expansion of fluorescence-based reporters which can be applied to complex biological samples, living cells and tissues to probe protein/protein interactions, conformational changes and posttranslational modifications. Here, we review recent developments in the field of fluorescent biosensor technology. We describe different varieties and categories of fluorescent biosensors together with an overview of the technologies commonly employed to image biosensors in cellulo and in vivo. We discuss issues and strategies related to the choice of synthetic fluorescent probes, labelling, quenching, caging and intracellular delivery of biosensors. Finally, we provide examples of some well-characterized genetically encoded FRET reporter systems, peptide and protein biosensors and describe biosensor applications in a wide variety of fields.     

A New Genetically Encoded Single-Chain Biosensor for Cdc42 Based on FRET,Useful for Live-Cell Imaging

Cdc42 is critical in a myriad of cellular morphogenic processes, requiring precisely regulated activation dynamics to affect specific cellular events. To facilitate direct observations of Cdc42 activation in live cells, we developed and validated a new biosensor of Cdc42 activation. The biosensor is genetically encoded, of single-chain design and capable of correctly localizing to membrane compartments as well as interacting with its upstream regulators including the guanine nucleotide dissociation inhibitor. We characterized this new biosensor in motile mouse embryonic fibroblasts and observed robust activation dynamics at leading edge protrusions, similar to those previously observed for endogenous Cdc42 using the organic dye-based biosensor system. We then extended our validations and observations of Cdc42 activity to macrophages, and show that this new biosensor is able to detect differential activation patterns during phagocytosis and cytokine stimulation. Furthermore, we observe for the first time, a highly transient and localized activation of Cdc42 during podosome formation in macrophages, which was previously hypothesized but never directly visualized.