RESUMO
The current utility of bioluminescence imaging is constrained by a low photon yield that limits temporal sensitivity. Here, we describe an imaging method that uses a chemiluminescent/fluorescent protein, ffLuc-cp156, which consists of a yellow variant of Aequorea GFP and firefly luciferase. We report an improvement in photon yield by over three orders of magnitude over current bioluminescent systems. We imaged cellular movement at high resolution including neuronal growth cones and microglial cell protrusions. Transgenic ffLuc-cp156 mice enabled video-rate bioluminescence imaging of freely moving animals, which may provide a reliable assay for drug distribution in behaving animals for pre-clinical studies.
Assuntos
Comportamento Animal , Células/metabolismo , Luminescência , Imagem Molecular/métodos , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Células/química , Embrião de Mamíferos/metabolismo , Células HEK293 , Humanos , Luciferases/análise , Luciferases/metabolismo , Proteínas Luminescentes/análise , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Transgênicos , Crista Neural/citologia , Crista Neural/metabolismo , Células-Tronco Neurais/metabolismoRESUMO
The structural basis for the photochromism in the fluorescent protein Dronpa is poorly understood, because the crystal structures of the bright state of the protein did not provide an answer to the mechanism of the photochromism, and structural determination of the dark state has been elusive. We performed NMR analyses of Dronpa in solution at ambient temperatures to find structural flexibility of the protein in the dark state. Light-induced changes in interactions between the chromophore and beta-barrel are responsible for switching between the two states. In the bright state, the apex of the chromophore tethers to the barrel by a hydrogen bond, and an imidazole ring protruding from the barrel stabilizes the plane of the chromophore. These interactions are disrupted by strong illumination with blue light, and the chromophore, together with a part of the beta-barrel, becomes flexible, leading to a nonradiative decay process.
Assuntos
Antozoários/química , Luz , Proteínas Luminescentes/química , Proteínas Luminescentes/efeitos da radiação , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Maleabilidade/efeitos da radiação , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , TemperaturaRESUMO
Dronpa is a green fluorescent protein homologue with a photochromic property. A green laser illumination reversibly converts Dronpa from a green-emissive bright state to a non-emissive dark state, and ultraviolet illumination converts it to the bright state. We have employed solution NMR to understand the underlying molecular mechanism of the photochromism. The detail characterization of Dronpa is hindered as it is metastable in the dark state and spontaneously converts to the bright state. To circumvent this issue, we have designed in magnet laser illumination device. By combining the device with a 150-mW argon laser at 514.5 nm, we have successfully converted and maintained Dronpa in the dark state in the NMR tube by continuous illumination during the NMR experiments. We have employed direct-detection of (13)C nuclei from the carbon skeleton of the chromophore for detailed characterization of chromophore in both states of Dronpa by using the Bruker TCI cryoprobe. The results from NMR data have provided direct evidence of the double bond formation between C(α) and C(ß) of Y63 in the chromophore, the ß-barrel structure in solution, and the ionized and protonated state of Y63 hydroxyl group in the bright and dark states, respectively. These studies have also revealed that a part of ß-barrel around the chromophore becomes polymorphic only in the dark state, which may be critical to make the fluorescence dim by increasing the contribution of non-emissive vibrational relaxation pathways.
Assuntos
Proteínas de Fluorescência Verde/química , Espectroscopia de Ressonância Magnética/métodos , Conformação Proteica , Animais , Antozoários/química , Isótopos de Carbono , Lasers , Isótopos de Nitrogênio , Processos Fotoquímicos , Tirosina/química , Raios UltravioletaRESUMO
Photoswitchable fluorophores play an essential role in super-resolution fluorescence microscopy, including techniques such as photoactivated localization microscopy (PALM). A determining factor in the precision of the images generated by PALM measurements is the photon numbers that can be detected from the fluorophores. Dronpa is a reversibly photoswitchable fluorescent protein that has been successfully used in PALM experiments. The number of photons per switching cycle that can be acquired for Dronpa depends on its off-switching rate, limiting the number of photons that can be recorded. In this study we report our discovery that the tetrameric ancestor of Dronpa, 22G, shows slower switching, and develop a mutant that displays switching kinetics between those of Dronpa and 22G. We show that the kinetics of the photoswitching are strongly related to self-association of the protein, supporting our view of dynamic flexibility as determining in the photoswitching. Similarly we find that higher-resolution PALM images can be acquired with slower-switching proteins due to their higher number of emitted photons per switching cycle.
Assuntos
Proteínas Luminescentes/química , Fótons , Actinas/química , Actinas/genética , Cinética , Proteínas Luminescentes/genética , Microscopia de Fluorescência , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
Bioluminescence is a natural light source based on luciferase catalysis of its substrate luciferin. We performed directed evolution on firefly luciferase using a red-shifted and highly deliverable luciferin analog to establish AkaBLI, an all-engineered bioluminescence in vivo imaging system. AkaBLI produced emissions in vivo that were brighter by a factor of 100 to 1000 than conventional systems, allowing noninvasive visualization of single cells deep inside freely moving animals. Single tumorigenic cells trapped in the mouse lung vasculature could be visualized. In the mouse brain, genetic labeling with neural activity sensors allowed tracking of small clusters of hippocampal neurons activated by novel environments. In a marmoset, we recorded video-rate bioluminescence from neurons in the striatum, a deep brain area, for more than 1 year. AkaBLI is therefore a bioengineered light source to spur unprecedented scientific, medical, and industrial applications.
Assuntos
Luciferases de Vaga-Lume/química , Medições Luminescentes/métodos , Neurônios/citologia , Análise de Célula Única/métodos , Animais , Benzotiazóis/química , Callithrix , Carcinogênese/química , Carcinogênese/patologia , Corpo Estriado/química , Corpo Estriado/citologia , Evolução Molecular Direcionada , Hipocampo/química , Luciferases de Vaga-Lume/genética , Pulmão/irrigação sanguínea , Camundongos , Movimento , Neurônios/química , Engenharia de Proteínas , Gravação em VídeoRESUMO
Although the consequences of Ras activation have been studied extensively in the context of oncogenesis, its regulation in physiological modes of signal transduction is not well understood. A fluorescent indicator, Raichu-Ras, was fused to the C-terminal hypervariable regions of H-Ras and K-Ras to create indicators for Ras activation within caveolae/rafts (Raichu-tH) and non-raft domains (Raichu-tK) of the plasma membrane, respectively. Raichu-tH was also found abundantly in endomembranes. To monitor Ras activation with high spatial resolution, it is imperative to observe sectioned images of the signals. We have developed a wide-field fluorescence microscope equipped with a digital micromirror device (DMD) to acquire optically sectioned images using fringe projection. This system provides reliable signals from fluorescence resonance energy transfer (FRET) between cyan and yellow mutants of green fluorescent protein. We have used this system to demonstrate that, upon stimulation with growth factors, the two indicators are activated in spatially and temporally unique patterns.
Assuntos
Cavéolas/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas ras/metabolismo , Animais , Células COS , Cavéolas/efeitos dos fármacos , Chlorocebus aethiops , Fator de Crescimento Epidérmico/farmacologia , Microdomínios da Membrana/efeitos dos fármacosRESUMO
Protein-protein interactions (PPIs) are essential components of cellular function. Current fluorescence-based technologies to measure PPIs have limited dynamic range and quantitative reproducibility. Here, we describe a genetically-encoded PPI visualization system that harnesses the dynamics of condensed liquid-phase transitions to analyze protein interactions in living cells. The fluorescent protein Azami-Green and p62-PB1 domain when fused to PPI partners triggered a rapid concatenation/oligomerization process that drove the condensation of liquid-phase droplets for real-time analysis of the interaction with unlimited dynamic range in the fluorescence signal. Proof-of-principle studies revealed novel insights on the live cell dynamics of XIAP-Smac and ERK2-dimer interactions. A photoconvertible variant allowed time-resolved optical highlighting for PPI kinetic analysis. Our system, called Fluoppi, demonstrates the unique signal amplification properties of liquid-phase condensation to detect PPIs. The findings introduce a general method for discovery of novel PPIs and modulators of established PPIs.
Assuntos
Corantes Fluorescentes/química , Mapeamento de Interação de Proteínas/métodos , Proteínas/química , Proteínas Reguladoras de Apoptose , Sítios de Ligação , Fenômenos Biofísicos , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinética , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Domínios Proteicos , Proteínas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Dual-excitation ratiometric dyes are excited alternately at two different wavelengths, but the emission is collected at a single fixed wavelength. Therefore, the pair of intensity measurements must be collected sequentially. Ratiometric-pericam is a fluorescent Ca(2+) indicator based on a chimeric fusion protein of circularly permuted green fluorescent protein and calmodulin. Upon binding to calcium, its excitation peak shifts from 415 nm to 494 nm. Ca(2+) imaging using ratiometric-pericam was thought to be inadequate to follow very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cell samples; however, we describe a technique that allows high spatial and time resolution of images acquired with ratiometric-pericam. To obtain confocal images of Ca(2+) using ratiometric-pericam, we established a system in which two laser beams (excitation 408 nm and 488 nm) are alternated on every scanning line under the control of two acousto-optic tunable filters. This system increases the rate at which ratio measurements are done to 200 Hz, and provides confocal images at 1 to 10 Hz depending on the image size. The ratio images are free from noise caused by the fluctuation of laser power, because the system is equipped with a violet laser diode (408 nm) and a diode-pumped solid-state laser (488 nm), both of which are stable. We visualized the dynamic propagation of Ca(2+) waves from the cytosol to the nucleus and changes in Ca(2+) concentrations in motile mitochondria of HeLa cells. We demonstrate that this new confocal imaging system expands the range of potential applications of ratiometric-pericam and other dual-excitation ratiometric indicators.
Assuntos
Cálcio/metabolismo , Proteínas Luminescentes/análise , Microscopia Confocal/métodos , Núcleo Celular/metabolismo , Citosol/metabolismo , Difusão , Corantes Fluorescentes/análise , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Indicadores e Reagentes/análise , Microscopia Confocal/instrumentação , Mitocôndrias/metabolismo , Células Tumorais CultivadasRESUMO
For quantitative measurements of Ca(2+) concentration ([Ca(2+)]), ratiometric dyes are preferable, because the use of such dyes allows for correction of uneven loading or partitioning of dye within the cell as well as variations in cell thickness. Although dual-excitation ratiometric dyes for measuring [Ca(2+)], such as Fura-2, Fura-Red, and ratiometric-pericam, are widely used for a variety of applications, it has been difficult to use them for monitoring very fast Ca(2+) dynamics or Ca(2+) changes in highly motile cells. To overcome this problem, we have developed three new dual-excitation ratiometry systems. (1) A system in which two laser beams are alternated on every scanning line, allowing us to obtain confocal images using dual-excitation ratiometric dyes. This system increases the rate at which ratio measurements can be made to 200 Hz and provides confocal images at 1-10 Hz depending on the image size. (2) A truly simultaneous dual-excitation ratiometry system that used linearly polarized excitation light and polarization detection, allowing us to obtain ratiometric images without any time lag. This system, however, is based on statistical features of the fluorescence polarization and is limited to samples that contain a large number of fluorophores. In addition, this method requires complicated calculations. (3) An efficient, nearly simultaneous dual-excitation ratiometry system that allows us to rapidly switch between two synchronized excitation-detection components by employing two high-power light-emitting diodes (LEDs) and two high-speed liquid crystal shutters. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity.
Assuntos
Cálcio/metabolismo , Polarização de Fluorescência/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Algoritmos , Animais , Cálcio/análise , Sinalização do Cálcio/fisiologia , Polarização de Fluorescência/instrumentação , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal/instrumentação , Microscopia de Fluorescência/instrumentaçãoRESUMO
Dual-excitation ratiometric dyes permit quantitative measurements of Ca2+ concentrations ([Ca2+]s), by minimizing the effects of several artifacts that are unrelated to changes in [Ca2+]. These dyes are excited at two different wavelengths, and the resultant fluorescence intensities are measured sequentially. Therefore, it is difficult to follow fast [Ca2+] dynamics or [Ca2+] changes in highly motile cell samples. To overcome this problem, we have developed a new dual-excitation ratiometry system that employs two high-power light-emitting diodes (LEDs), two high-speed liquid crystal shutters, and a CCD camera. The open/close operation of the two shutters is synchronized with the on/off switching of the two LEDs. This system increases the rate at which ratio measurements are made to 1 kHz, and provides ratio images at 10-100 Hz depending on the signal intensity. We demonstrate the effectiveness of this system by monitoring changes in [Ca2+] in cardiac muscle cells loaded with Fura-2.
Assuntos
Cálcio/metabolismo , Fura-2/administração & dosagem , Aumento da Imagem/instrumentação , Microscopia de Fluorescência por Excitação Multifotônica/instrumentação , Miócitos Cardíacos/metabolismo , Semicondutores , Processamento de Sinais Assistido por Computador , Animais , Células Cultivadas , Desenho de Equipamento , Análise de Falha de Equipamento , Aumento da Imagem/métodos , Iluminação , Cristais Líquidos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Miócitos Cardíacos/efeitos dos fármacos , RatosRESUMO
Highly efficient fluorescence resonance energy transfer between cyan(CFP) and yellow fluorescent proteins (YFP), the cyan- and yellow-emitting variants of the Aequorea green fluorescent protein, respectively, was achieved by tightly concatenating the two proteins. After the C-terminus of CFP and the N-terminus of YFP were truncated by 11 and 5 amino acids, respectively, the proteins were fused through a leucine-glutamate dipeptide. The resulting chimeric protein, which we called Cy11.5, exhibited a simple emission spectrum that peaked at 527 nm when the protein was excited at 436 nm. The time-resolved emission of Cy11.5 was measured using a streak camera. After excitation of Cy11.5 with a 400 nm ultrashort pulse, a fast decay of the CFP emission and a concomitant rise of the YFP emission were observed with a lifetime of 66 ps. By contrast, the emission from CFP alone showed a decay component with a lifetime of 2.9 ns. We concluded that in fully folded Cy11.5 molecules, intramolecular FRET occurred with an efficiency of 98%. Importantly, most Cy11.5 molecules were properly folded, and the protein was highly resistant to all of the tested proteases. In living cells, therefore, Cy11.5 behaved as a single fluorescent protein with a broad excitation spectrum. Moreover, Cy11.5 was used as an optical highlighter after photobleaching of YFP. When HeLa cells expressing Cy11.5 were irradiated at 514.5 nm, a 10-fold increase in the 475 nm fluorescence intensity was observed. These features make Cy11.5 useful as an optical highlighter and a new-colored fluorescent protein for multicolor imaging.
Assuntos
Proteínas de Bactérias/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Anisotropia , Proteínas de Bactérias/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinética , Lasers , Proteínas Luminescentes/metabolismo , Peptídeo Hidrolases/metabolismo , Peptídeo Hidrolases/farmacologia , Fotodegradação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
We have developed a whole-field fluorescence microscope equipped with a Digital Micromirror Device to acquire optically sectioned images by using the fringe-projection technique and the phase-shift method. This system allows free control of optical sectioning strength through computer-controlled alteration of the fringe period projected onto a sample. We have employed this system to image viable cells expressing fluorescent proteins and discussed its biological applications.
Assuntos
Cálcio/metabolismo , Células HeLa/citologia , Células HeLa/metabolismo , Aumento da Imagem/instrumentação , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Óptica e Fotônica/instrumentação , Proteínas de Ligação ao Cálcio , Proteínas de Drosophila , Desenho de Equipamento , Estudos de Viabilidade , Humanos , Aumento da Imagem/métodos , Interferometria/instrumentação , Interferometria/métodos , Miniaturização , Tomografia/instrumentação , Tomografia/métodosRESUMO
Dual-excitation ratiometric dyes permit quantitative Ca2+ measurements by minimizing the effects of several artifacts that are unrelated to changes in the concentration of free Ca2+ ([Ca2+]). These dyes are excited alternately at two different wavelengths, and the pair of intensity measurements must be collected sequentially. Therefore, it is difficult to follow very fast Ca2+ dynamics or Ca2+ changes in highly motile cell samples. Here, we present a novel but simple dual-excitation ratiometric method which overcomes this problem. By the use of our home-made illuminator, each sample is illuminated by two orthogonal linear polarized lights of different wavelengths. Fluorescence images are captured by two CCD cameras through two analyzers, whose polarization directions are at right angles. This methodology allows us to perform simultaneous measurements of any dual-excitation ratiometric dye, and we demonstrate its validity by monitoring [Ca2+] changes in rat cardiac muscle cells loaded with Fura Red.
Assuntos
Cálcio/análise , Microscopia de Fluorescência/métodos , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Polarização de Fluorescência/instrumentação , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia de Fluorescência/instrumentação , Miócitos Cardíacos/citologia , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMO
In polarized mature neurons, the asymmetrical distribution of proteins between axonal and somatodendritic plasma membrane (PM) domains may be maintained by a diffusion barrier at the axon-soma boundary. At the boundary, a complex containing membrane-associated and cytoskeletal proteins is formed, anchoring axonal membrane proteins and indirectly hindering the diffusion of other membrane proteins. We examined the latter case, i.e., secondary diffusion impedance by comparing the mobility of fluorescently labeled membrane proteins within the axon-soma and dendrite-soma boundaries. We performed fluorescence recovery after photobleaching (FRAP) experiments using mature cultured hippocampal neurons that had been labeled specifically at their PMs with fluorescent proteins (FPs). The maturation of these neurons was confirmed by immunolocalization with Ankyrin-G, which is thought to participate in the creation of the diffusion barrier at the axon-soma boundary. We developed a wide-field microscope equipped with a device (digital micromirror device) composed of 1024 x 768 binary mirrors at the field-stop, allowing free control of the illumination area and intensity. After the FPs in peripheral processes were photobleached, nonbleached FPs diffused into all the processes at equivalent speeds. These results indicate that the secondary diffusion barrier to exogenously overexpressed membrane proteins is not specific to the axon-soma boundary.
Assuntos
Axônios/metabolismo , Dendritos/metabolismo , Recuperação de Fluorescência Após Fotodegradação/métodos , Proteínas de Membrana/metabolismo , Neurônios , Animais , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Difusão , Recuperação de Fluorescência Após Fotodegradação/instrumentação , Proteínas de Membrana/genética , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The pharyngeal muscles of Caenorhabditis elegans are composed of the corpus, isthmus and terminal bulb from anterior to posterior. These components are excited in a coordinated fashion to facilitate proper feeding through pumping and peristalsis. We analysed the spatiotemporal pattern of intracellular calcium dynamics in the pharyngeal muscles during feeding. We used a new ratiometric fluorescent calcium indicator and a new optical system that allows simultaneous illumination and detection at any two wavelengths. Pumping was observed with fast, repetitive and synchronous spikes in calcium concentrations in the corpus and terminal bulb, indicative of electrical coupling throughout the muscles. The posterior isthmus, however, responded to only one out of several pumping spikes to produce broad calcium transients, leading to peristalsis, the slow and gradual motion needed for efficient swallows. The excitation-calcium coupling may be uniquely modulated in this region at the level of calcium channels on the plasma membrane.