RESUMEN
Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.
Asunto(s)
Imagen Óptica/métodos , Fosfotransferasas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Drosophila , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación , Fosfotransferasas/metabolismoRESUMEN
The lateral organization of proteins and lipids in the plasma membrane is fundamental to regulating a wide range of cellular processes. Compartmentalized ordered membrane domains enriched with specific lipids, often termed lipid rafts, have been shown to modulate the physicochemical and mechanical properties of membranes and to drive protein sorting. Novel methods and tools enabling the visualization, characterization, and/or manipulation of membrane compartmentalization are crucial to link the properties of the membrane with cell functions. Flipper, a commercially available fluorescent membrane tension probe, has become a reference tool for quantitative membrane tension studies in living cells. Here, we report on a so far unidentified property of Flipper, namely, its ability to photosensitize singlet oxygen (1O2) under blue light when embedded into lipid membranes. This in turn results in the production of lipid hydroperoxides that increase membrane tension and trigger phase separation. In biological membranes, the photoinduced segregated domains retain the sorting ability of intact phase-separated membranes, directing raft and nonraft proteins into ordered and disordered regions, respectively, in contrast to radical-based photo-oxidation reactions that disrupt raft protein partitioning. The dual tension reporting and photosensitizing abilities of Flipper enable simultaneous visualization and manipulation of the mechanical properties and lateral organization of membranes, providing a powerful tool to optically control lipid raft formation and to explore the interplay between membrane biophysics and cell function.
Asunto(s)
Microdominios de Membrana , Microdominios de Membrana/metabolismo , Microdominios de Membrana/química , Oxígeno Singlete/metabolismo , Oxígeno Singlete/química , Luz , Colorantes Fluorescentes/química , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/química , Procesos Fotoquímicos , Membrana Celular/metabolismo , Membrana Celular/química , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Separación de FasesRESUMEN
CRANAD-2 is a fluorogenic curcumin derivative used for near-infrared detection and imaging in vivo of amyloid aggregates, which are involved in neurodegenerative diseases. We explore the performance of CRANAD-2 in two super-resolution imaging techniques, namely stimulated emission depletion (STED) and single-molecule localization microscopy (SMLM), with markedly different fluorophore requirements. By conveniently adapting the concentration of CRANAD-2, which transiently binds to amyloid fibrils, we show that it performs well in both techniques, achieving a resolution in the range of 45-55â nm. Correlation of SMLM with atomic force microscopy (AFM) validates the resolution of fine features in the reconstructed super-resolved image. The good performance and versatility of CRANAD-2 provides a powerful tool for near-infrared nanoscopic imaging of amyloids in vitro and in vivo.
Asunto(s)
Amiloide , Curcumina , Colorantes Fluorescentes , Microscopía de Fuerza Atómica/métodos , Imagen Individual de MoléculaRESUMEN
miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.
Asunto(s)
Mononucleótido de Flavina , Oxígeno Singlete , Mononucleótido de Flavina/química , Flavoproteínas/química , Oxígeno/química , Fosfatos , Riboflavina , Oxígeno Singlete/químicaRESUMEN
A combination of time-resolved optical spectroscopy and nanoscale imaging has been used to study the complex binding to amyloids of a photocatalyst that selectively photo-oxygenates pathogenic aggregates, as well as the consequences of its irradiation. Correlative atomic force microscopy (AFM) and fluorescence microscopy reveals topography-dependent binding of the dye to model ß-lactoglobulin fibers, which may also explain the observed difference in their response to photodegradation. We provide direct evidence of the photosensitization of singlet oxygen by the photocatalyst bound to amyloid fibers by direct detection of its NIR phosphorescence. The effect of singlet oxygen at the molecular level brings about nanoscale morphological changes that can be observed with AFM at the single-fiber level. We also find differential response of two α-synuclein mutants to photodamage, which can be rationalized by the presence of amino acids susceptible to photo-oxygenation. Overall, our results help to unravel some of the complexity associated with highly heterogeneous amyloid populations and contribute to the development of improved phototherapeutic strategies for amyloid-related disorders.
Asunto(s)
Amiloide/química , Luz/efectos adversos , Microscopía de Fuerza Atómica/métodos , alfa-Sinucleína/química , Benzotiazoles/química , Lactoglobulinas/química , Microscopía Fluorescente/métodos , Especies Reactivas de Oxígeno/química , Oxígeno Singlete/química , Espectroscopía Infrarroja Corta/métodosRESUMEN
A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.
Asunto(s)
Apoptosis , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Imagen Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Animales , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Conformación Proteica en Lámina betaRESUMEN
Flavin-binding fluorescent proteins (FbFPs) are a class of fluorescent reporters that have been increasingly used as reporters in the study of cellular structures and dynamics. Flavin's intrinsic high singlet oxygen ((1)O2) quantum yield (ΦΔ = 0.51) provides a basis for the development of new FbFP mutants capable of photosensitising (1)O2 for mechanistic and therapeutic applications, as recently exemplified by the FbFP miniSOG. In the present work we report an investigation on the (1)O2 photoproduction by Pp2FbFP L30M, a novel derivative of Pseudomonas putida Pp2FbFP. Direct detection of (1)O2 through its phosphorescence at 1275 nm yielded the value ΦΔ = 0.09 ± 0.01, which is the highest (1)O2 quantum yield reported to date for any FP and is approximately 3-fold higher than the ΦΔ for miniSOG. Unlike miniSOG, transient absorption measurements revealed the existence of two independent triplet states each with a different ability to sensitise (1)O2.
Asunto(s)
Proteínas Luminiscentes/química , Oxígeno Singlete/química , Escherichia coli , Cinética , Trastornos por Fotosensibilidad , Pseudomonas putida , Análisis EspectralRESUMEN
Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.
Asunto(s)
Dinitrocresoles , Flavinas , Flavinas/metabolismo , Oxígeno/metabolismo , ProteínasRESUMEN
The Min protein system is a cell division regulator in Escherichia coli. Under normal growth conditions, MinD is associated with the membrane and undergoes pole-to-pole oscillations. The period of these oscillations has been previously proposed as a reporter for the bacterial physiological state at the single-cell level and has been used to monitor the response to sublethal challenges from antibiotics, temperature, or mechanical fatigue. Using real-time single-cell fluorescence imaging, we explore here the effect of photodynamic treatment on MinD oscillations. Irradiation of bacteria in the presence of the photosensitizer methylene blue disrupts the MinD oscillation pattern depending on its concentration. In contrast to antibiotics, which slow down the oscillation, photodynamic treatment results in an abrupt interruption, reflecting divergent physiological mechanisms leading to bacterial death. We show that MinD oscillations are sensitive to mild photodynamic effects that are overlooked by traditional methods, expanding the toolbox for mechanistic studies in antimicrobial photodynamic therapy.
Asunto(s)
Proteínas de Escherichia coli , Fotoquimioterapia , División Celular , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Fármacos FotosensibilizantesRESUMEN
We report that Thioflavin T (ThT), the reference fluorogenic probe for amyloid detection, displays photodynamic activity against bacterial biofilms. ThT recognizes key structures of the biofilm matrix, disrupting the complex architecture and efficiently inactivating bacterial cells. We also show that ThT phototherapy synergistically boosts the activity of conventional antimicrobials.
Asunto(s)
Antibacterianos/farmacología , Benzotiazoles/farmacología , Biopelículas/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Luz , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/fisiologíaRESUMEN
Singlet oxygen (1O2) is the first electronic excited state of molecular oxygen. Due to its non-radical and non-ionic character as well as its mild reactivity, 1O2 has a pivotal role in cell signaling processes at low concentration, yet it is cytotoxic at high concentrations. Quantifying the production of 1O2, particularly in biological systems, is therefore essential for understanding and controlling its effects. 1O2 can be produced by chemical and biological reactions, yet its most common method of production is by photosensitization, whereby an initially photoexcited molecule transfers its acquired electronic energy to the dioxygen molecule. The efficiency of this process is characterized by the 1O2 production quantum yield, ΦΔ, which can be determined by directly monitoring its intrinsic weak near-infrared phosphorescence or indirectly by trapping it with a suitable acceptor, a process that can be monitored by common analytical techniques. Indirect methods are thus very popular, yet they may lead to severe errors if used incorrectly. Herein we describe the common aspects of indirect methods and propose a general step-by-step procedure for the determination of ΦΔ values. In addition, we identify the key experimental conditions that need to be controlled to obtain meaningful results.
Asunto(s)
Oxígeno/química , Oxígeno Singlete/análisis , Oxígeno Singlete/química , Modelos Teóricos , Oxígeno/metabolismo , Fármacos Fotosensibilizantes/químicaRESUMEN
We report the use of the amyloid probe Thioflavin T (ThT) as a specific and exchangeable fluorophore for stimulated emission depletion (STED) super-resolution imaging of amyloid fibers. This method achieves a spatial resolution in the range of 60-70 nm, low image background and increased photostability that enables long-term STED imaging. These results expand the widespread uses of ThT and can be potentially extended to other common amyloid fluorescent probes, providing new tools for the study of amyloid diseases.
Asunto(s)
Benzotiazoles , Colorantes Fluorescentes , Amiloide , Microscopía FluorescenteRESUMEN
Mechano-bactericidal nanomaterials rely on their mechanical or physical interactions with bacteria and are promising antimicrobial strategies that overcome bacterial resistance. However, the real effect of mechanical versus chemical action on their activity is under debate. In this paper, we quantify the forces necessary to produce critical damage to the bacterial cell wall by performing simultaneous nanoindentation and fluorescence imaging of single bacterial cells. Our experimental setup allows puncturing the cell wall of an immobilized bacterium with the tip of an atomic force microscope (AFM) and following in real time the increase in the fluorescence signal from a cell membrane integrity marker. We correlate the forces exerted by the AFM tip with the fluorescence dynamics for tens of cells, and we find that forces above 20 nN are necessary to exert critical damage. Moreover, a similar experiment is performed in which bacterial viability is assessed through physiological activity, in order to gain a more complete view of the effect of mechanical forces on bacteria. Our results contribute to the quantitative understanding of the interaction between bacteria and nanomaterials.
Asunto(s)
Antibacterianos/farmacología , Viabilidad Microbiana/efectos de los fármacos , Antibacterianos/química , Bacterias/efectos de los fármacos , Bacterias/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Microscopía de Fuerza Atómica , Microscopía FluorescenteRESUMEN
miniSOG is the first flavin-binding protein that has been developed with the specific aim of serving as a genetically-encodable light-induced source of singlet oxygen (1O2). We have determined its 1.17 Å resolution structure, which has allowed us to investigate its mechanism of photosensitization using an integrated approach combining spectroscopic and structural methods. Our results provide a structural framework to explain the ability of miniSOG to produce 1O2 as a competition between oxygen- and protein quenching of its triplet state. In addition, a third excited-state decay pathway has been identified that is pivotal for the performance of miniSOG as 1O2 photosensitizer, namely the photo-induced transformation of flavin mononucleotide (FMN) into lumichrome, which increases the accessibility of oxygen to the flavin FMN chromophore and makes protein quenching less favourable. The combination of the two effects explains the increase in the 1O2 quantum yield by one order of magnitude upon exposure to blue light. Besides, we have identified several surface electron-rich residues that are progressively photo-oxidized, further contributing to facilitate the production of 1O2. Our results help reconcile the apparent poor level of 1O2 generation by miniSOG and its excellent performance in correlative light and electron microscopy experiments.
Asunto(s)
Proteínas de Arabidopsis/genética , Fármacos Fotosensibilizantes/metabolismo , Conformación Proteica , Proteínas Serina-Treonina Quinasas/genética , Oxígeno Singlete/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/ultraestructura , Fenómenos Biofísicos , Flavinas/química , Flavinas/genética , Luz , Microscopía Electrónica , Oxidación-Reducción , Oxígeno/metabolismo , Trastornos por Fotosensibilidad , Fármacos Fotosensibilizantes/química , Unión Proteica/genética , Ingeniería de Proteínas , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/ultraestructura , Oxígeno Singlete/químicaRESUMEN
Flavin-binding fluorescent proteins (FPs) are genetically encoded in vivo reporters, which are derived from microbial and plant LOV photoreceptors. In this study, we comparatively analyzed ROS formation and light-driven antimicrobial efficacy of eleven LOV-based FPs. In particular, we determined singlet oxygen (1O2) quantum yields and superoxide photosensitization activities via spectroscopic assays and performed cell toxicity experiments in E. coli. Besides miniSOG and SOPP, which have been engineered to generate 1O2, all of the other tested flavoproteins were able to produce singlet oxygen and/or hydrogen peroxide but exhibited remarkable differences in ROS selectivity and yield. Accordingly, most LOV-FPs are potent photosensitizers, which can be used for light-controlled killing of bacteria. Furthermore, the two variants Pp2FbFP and DsFbFP M49I, exhibiting preferential photosensitization of singlet oxygen or singlet oxygen and superoxide, respectively, were shown to be new tools for studying specific ROS-induced cell signaling processes. The tested LOV-FPs thus further expand the toolbox of optogenetic sensitizers usable for a broad spectrum of microbiological and biomedical applications.
Asunto(s)
Escherichia coli/metabolismo , Flavoproteínas/metabolismo , Optogenética , Fármacos Fotosensibilizantes/farmacología , Dinitrocresoles/química , Escherichia coli/patogenicidad , Escherichia coli/efectos de la radiación , Flavoproteínas/química , Luz , Trastornos por Fotosensibilidad , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Oxígeno Singlete/metabolismo , Superóxidos/metabolismoRESUMEN
Correction for 'Assessing the potential of photosensitizing flavoproteins as tags for correlative microscopy' by Alberto Rodríguez-Pulido et al., Chem. Commun., 2016, 52, 8405-8408.
RESUMEN
Photosensitizing flavoproteins have great potential as tags for correlative light and electron microscopy (CLEM). We examine the photostability of miniSOG mutants and their ability to photo-oxidize diaminobenzidine, both key aspects for CLEM. Our experiments reveal a complex relation between these parameters and the production of different reactive oxygen species.