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1.
J Biosci ; 43(4): 763-784, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30207321

RESUMO

Since the last decade, a lot of advancement has been made to understand biological processes involving complex intracellular pathways. The major challenge faced was monitoring and trafficking of metabolites in real time. Although a range of quantitative and imaging techniques have been developed so far, the discovery of green fluorescent proteins (GFPs) has revolutionized the advancement in the field of metabolomics. GFPs and their variants have enabled researchers to 'paint' a wide range of biological molecules. Fluorescence resonance energy transfer (FRET)-based genetically encoded sensors is a promising technology to decipher the real-time monitoring of the cellular events inside living cells. GFPs and their variants, due to their intrinsic fluorescence properties, are extensively being used nowadays in cell-based assays. This review focuses on structure and function of GFP and its derivatives, mechanism emission and their use in the development of FRET-based sensors for metabolites.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde/química , Proteínas Luminescentes/química , Técnicas Biossensoriais/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Humanos , Proteínas Luminescentes/genética , Metaboloma/genética
2.
Curr Opin Neurobiol ; 50: 146-153, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29501950

RESUMO

In order to understand how brain activity produces adaptive behavior we need large-scale, high-resolution recordings of neuronal activity. Fluorescent genetically encoded voltage indicators (GEVIs) offer the potential for these recordings to be performed chronically from targeted cells in a minimally invasive manner. As the number of GEVIs successfully tested for in vivo use grows, so has the number of open questions regarding the improvements that would facilitate broad adoption of this technology that surpasses mere 'proof of principle' studies. Our aim in this review is not to provide a status check of the current state of the field, as excellent publications covering this topic already exist. Here, we discuss specific questions regarding GEVI development and application that we think are crucial in achieving this goal.


Assuntos
Encéfalo/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/tendências , Proteínas Luminescentes/genética , Imagens com Corantes Sensíveis à Voltagem/tendências , Animais , Encéfalo/diagnóstico por imagem , Humanos , Proteínas Luminescentes/metabolismo , Imagens com Corantes Sensíveis à Voltagem/métodos
3.
J Neurosci ; 36(39): 9977-89, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-27683896

RESUMO

UNLABELLED: A longstanding goal in neuroscience is to understand how spatiotemporal patterns of neuronal electrical activity underlie brain function, from sensory representations to decision making. An emerging technology for monitoring electrical dynamics, voltage imaging using genetically encoded voltage indicators (GEVIs), couples the power of genetics with the advantages of light. Here, we review the properties that determine indicator performance and applicability, discussing both recent progress and technical limitations. We then consider GEVI applications, highlighting studies that have already deployed GEVIs for biological discovery. We also examine which classes of biological questions GEVIs are primed to address and which ones are beyond their current capabilities. As GEVIs are further developed, we anticipate that they will become more broadly used by the neuroscience community to eavesdrop on brain activity with unprecedented spatiotemporal resolution. SIGNIFICANCE STATEMENT: Genetically encoded voltage indicators are engineered light-emitting protein sensors that typically report neuronal voltage dynamics as changes in brightness. In this review, we systematically discuss the current state of this emerging method, considering both its advantages and limitations for imaging neural activity. We also present recent applications of this technology and discuss what is feasible now and what we anticipate will become possible with future indicator development. This review will inform neuroscientists of recent progress in the field and help potential users critically evaluate the suitability of genetically encoded voltage indicator imaging to answer their specific biological questions.


Assuntos
Potenciais de Ação/fisiologia , Transferência Ressonante de Energia de Fluorescência/tendências , Proteínas Luminescentes/genética , Potenciais da Membrana/fisiologia , Optogenética/tendências , Imagens com Corantes Sensíveis à Voltagem/tendências , Animais , Mapeamento Encefálico/métodos , Humanos , Avaliação da Tecnologia Biomédica
4.
Appl Microbiol Biotechnol ; 96(4): 895-902, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23053099

RESUMO

Förster (or fluorescence) resonance energy transfer (FRET) is a process involving the radiation-less transfer of energy from a "donor" fluorophore to an "acceptor" fluorophore. FRET technology enables the quantitative analysis of molecular dynamics in biophysics and in molecular biology, such as the monitoring of protein-protein interactions, protein-DNA interactions, and protein conformational changes. FRET-based biosensors have been utilized to monitor cellular dynamics not only in heterogeneous cellular populations, but also at the single-cell level in real time. Lately, applications of FRET-based biosensors range from basic biological to biomedical disciplines. Despite the diverse applications of FRET, FRET-based sensors still face many challenges. There is an increasing need for higher fluorescence resolution and improved specificity of FRET biosensors. Additionally, as more FRET-based technologies extend to medical diagnostics, the affordability of FRET reagents becomes a significant concern. Here, we will review current advances and limitations of FRET-based biosensor technology and discuss future FRET applications.


Assuntos
Técnicas Biossensoriais/tendências , Células/química , Metabolismo Energético , Transferência Ressonante de Energia de Fluorescência/tendências , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Células/citologia , Células/metabolismo , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Proteínas/genética , Proteínas/metabolismo
5.
Bioessays ; 34(5): 369-76, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22415767

RESUMO

New imaging methodologies in quantitative fluorescence microscopy, such as Förster resonance energy transfer (FRET), have been developed in the last few years and are beginning to be extensively applied to biological problems. FRET is employed for the detection and quantification of protein interactions, and of biochemical activities. Herein, we review the different methods to measure FRET in microscopy, and more importantly, their strengths and weaknesses. In our opinion, fluorescence lifetime imaging microscopy (FLIM) is advantageous for detecting inter-molecular interactions quantitatively, the intensity ratio approach representing a valid and straightforward option for detecting intra-molecular FRET. Promising approaches in single molecule techniques and data analysis for quantitative and fast spatio-temporal protein-protein interaction studies open new avenues for FRET in biological research.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Transferência Ressonante de Energia de Fluorescência/tendências , Proteínas de Fluorescência Verde , Microscopia de Fluorescência/tendências , Mapeamento de Interação de Proteínas/métodos
7.
Curr Pharm Biotechnol ; 12(4): 558-68, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21342101

RESUMO

Bioluminescence resonance energy transfer (BRET) assay is a comparatively new cell-based assay technology that is assuming more prominent roles in the field of studying protein-protein interactions, protein dimerization and signal transduction. In the last few years BRET related research has gained significant momentum in terms of adding versatility in the assay format as well as a variety of new applications where it has been suitably used. Beyond the scope of quantitative measurement of protein-protein interactions and protein dimerization, molecular imaging applications based on BRET assays have broaden its scope as a great tool for high-throughput screening (HTS) of pharmacologically important compounds. This article will highlight the landmarks in BRET research, with those which have significant contributions towards making it an attractive single format assay that shuttles between in vitro and in vivo measurements.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Medições Luminescentes , Animais , Bioensaio , Transferência Ressonante de Energia de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/tendências , Genes Reporter , Ensaios de Triagem em Larga Escala , Humanos , Medições Luminescentes/métodos , Medições Luminescentes/tendências , Proteínas Luminescentes/genética
8.
Lab Chip ; 10(11): 1355-64, 2010 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-20480105

RESUMO

This review focuses on the use of Förster Resonance Energy Transfer (FRET) to monitor intra- and intermolecular reactions occurring in microfluidic reactors. Microfluidic devices have recently been used for performing highly efficient and miniaturised biological assays for the analysis of biological entities such as cells, proteins and nucleic acids. Microfluidic assays are characterised by nanolitre to femtolitre reaction volumes, which necessitates the adoption of a sensitive optical detection scheme. FRET serves as a strong 'spectroscopic ruler' for elucidating the tertiary structure of biomolecules, as the efficiency of the non-radiative energy transfer is extremely sensitive to nanoscale changes in the separation between donor and acceptor markers attached to the biomolecule of interest. In this review, we will review the implementation of various microfluidic assays which employ FRET for diverse applications in the biomedical field, along with the advantages and disadvantages of the various approaches. The future prospects for development of microfluidic devices incorporating FRET detection will be discussed.


Assuntos
Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/tendências , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/tendências , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/tendências , Técnicas de Sonda Molecular/instrumentação , Técnicas de Sonda Molecular/tendências
9.
Curr Opin Biotechnol ; 21(1): 45-54, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20167470

RESUMO

The spatiotemporal patterns of ion and metabolite levels in living cells are important in understanding signal transduction and metabolite flux. Imaging approaches using genetically encoded sensors are ideal for detecting such molecule dynamics, which are hard to capture otherwise. Recent years have seen iterative improvements and evaluations of sensors, which in turn are starting to make applications in more challenging experimental settings possible. In this review, we will introduce recent progress made in the variety and properties of biosensors, and how biosensors are used for the measurement of metabolite and ion in live cells. The emerging field of applications, such as parallel imaging of two separate molecules, high-resolution transport studies and high-throughput screening using biosensors, will be discussed.


Assuntos
Técnicas Biossensoriais/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Engenharia Genética/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Metaboloma/fisiologia , Microscopia de Fluorescência/tendências , Perfilação da Expressão Gênica/métodos , Proteínas Luminescentes/análise , Técnicas de Sonda Molecular/tendências
10.
Integr Biol (Camb) ; 1(10): 565-73, 2009 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20016756

RESUMO

Since the development of green fluorescent protein (GFP) and other fluorescent proteins (FPs) with distinct colors, genetically-encoded probes and biosensors have been widely applied to visualize the molecular localization and activities in live cells. In particular, biosensors based on fluorescence resonance energy transfer (FRET) have significantly advanced our understanding of the dynamic molecular hierarchy at subcellular levels. These biosensors have also been extensively applied in recent years to study how cells perceive the mechanical environment and transmit it into intracellular molecular signals (i.e. mechanotransduction). In this review, we will first provide a brief introduction of the recent development of FPs. Different FRET biosensors based on FPs will then be described. The last part of the review will be dedicated to the introduction of examples applying FRET biosensors to visualize mechanotransduction in live cells. In summary, the integration of FRET technology and the different cutting-edge mechanical stimulation systems can provide powerful tools to allow the elucidation of the mechanisms regulating mechanobiology at cellular and molecular levels in normal and pathophysiological conditions.


Assuntos
Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Mecanotransdução Celular/fisiologia , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Desenho de Equipamento , Transferência Ressonante de Energia de Fluorescência/tendências , Humanos , Microscopia de Fluorescência/tendências
11.
J Physiol ; 587(Pt 22): 5331-5, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19752111

RESUMO

The traditional view of G protein-coupled receptor (GPCR)-mediated signalling puts the players in this signalling cascade, namely the GPCR, the G protein and its effector, as individual components in space, where the signalling specificity is obtained mainly by the interaction of the GPCR and the Galpha subunits of the G protein. A question is then raised as to how fidelity in receptor signalling is achieved, given that many systems use the same components of the G protein signalling machinery. One possible mechanism for obtaining the specific flow of the downstream signals, from the activated G protein to its specific effector target, in a timely manner, is compartmentalization, a spatial arrangement of the complex in a rather restricted space. Here we review our recent findings related to these issues, using the G protein-coupled potassium channel (GIRK) as a model effector and fluorescence-based approaches to reveal how the signalling complex is arranged and how the G protein exerts its action to activate the GIRK channel in intact cells.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/fisiologia , Ativação do Canal Iônico/fisiologia , Animais , Transferência Ressonante de Energia de Fluorescência/tendências , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos
12.
Int J Nanomedicine ; 3(2): 151-67, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18686776

RESUMO

The use of luminescent colloidal quantum dots in biological investigations has increased dramatically over the past several years due to their unique size-dependent optical properties and recent advances in biofunctionalization. In this review, we describe the methods for generating high-quality nanocrystals and report on current and potential uses of these versatile materials. Numerous examples are provided in several key areas including cell labeling, biosensing, in vivo imaging, bimodal magnetic-luminescent imaging, and diagnostics. We also explore toxicity issues surrounding these materials and speculate about the future uses of quantum dots in a clinical setting.


Assuntos
Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Aumento da Imagem/métodos , Imunoensaio/métodos , Microscopia de Fluorescência/métodos , Pontos Quânticos , Técnicas Biossensoriais/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Imunoensaio/tendências , Microscopia de Fluorescência/tendências
14.
J Biomed Opt ; 13(3): 031202, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18601526

RESUMO

We have witnessed remarkable advances over the past decade in the application of optical techniques to visualize the genetically encoded fluorescent proteins (FPs) in living systems. The imaging of the FPs inside living cells has become an essential tool for studies of cell biology and physiology. FPs are now available that span the visible spectrum from deep blue to deep red, providing a wide choice of genetically encoded fluorescent markers. Furthermore, some FPs have been identified that have unusual characteristics that make them useful reporters of the dynamic behaviors of proteins inside cells. These additions to the FP toolbox are now being used for some very innovative live-cell imaging applications. Here, we will highlight the characteristics and uses of a few of these exceptional probes. Many different optical methods can be combined with the FPs from marine organisms to provide quantitative measurements in living systems.


Assuntos
Recuperação de Fluorescência Após Fotodegradação/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Proteínas Luminescentes/análise , Microscopia de Fluorescência/tendências , Mapeamento de Interação de Proteínas/tendências , Espectrometria de Fluorescência/tendências
15.
Biotechnol J ; 3(3): 311-24, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18228541

RESUMO

The bioluminescence resonance energy transfer (BRET) method is based on resonance energy transfer between a light-emitting enzyme and a fluorescent acceptor. Since its first description in 1999, several versions of BRET have been described using different substrates and energy donor/acceptor couples. Today, BRET is considered as one of the most versatile techniques for studying the dynamics of protein-protein interactions in living cells. Various studies have applied BRET-based assays to screen new receptor ligands and inhibitors of disease-related-proteases. Inhibitors of protein-protein interactions are likely to become a new major class of therapeutic drugs, and BRET technology is expected to play an important role in the identification of such compounds. This review describes the original BRET-based methodology, more recent variants, and potential applications to drug screening.


Assuntos
Bioensaio/tendências , Desenho de Drogas , Transferência Ressonante de Energia de Fluorescência/tendências , Medições Luminescentes/tendências , Proteínas Luminescentes , Mapeamento de Interação de Proteínas/métodos
16.
Adv Biochem Eng Biotechnol ; 110: 1-24, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18219467

RESUMO

One of the key challenges of biology in the post-genomic era is to assign function to the many genes revealed by large-scale sequencing programmes, since only a small fraction of gene function can be directly inferred from the coding sequence. Identifying interactions between proteins is a substantial part in understanding their function. The main technologies for investigating protein-protein interactions and assigning functions to proteins include direct detection intermolecular interactions through protein microarray, yeast two-hybrid system, mass spectrometry fluorescent techniques to visualize protein complexes or pull-down assays, as well as technologies detecting functional interactions between genes, such as RNAi knock down or functional screening of cDNA libraries. Over recent years, considerable advances have been made in the above techniques. In this review, we discuss some recent developments and their impact on the gene function annotation.


Assuntos
Eletroforese em Gel Bidimensional/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Espectrometria de Massas/tendências , Análise Serial de Proteínas/tendências , Mapeamento de Interação de Proteínas/tendências , Proteoma/metabolismo , Técnicas do Sistema de Duplo-Híbrido/tendências , Previsões , Perfilação da Expressão Gênica/tendências
17.
Zhongguo Yi Liao Qi Xie Za Zhi ; 31(1): 39-43, 2007 Jan.
Artigo em Chinês | MEDLINE | ID: mdl-17432125

RESUMO

The theory of fluorescence resonance energy transfer (FRET) and methods of fluorescence detection in fluorescent-quantitative polymerase chain reaction (FQ-PCR) are introduced in this article. Applications of FRET in fluorescence detection of PCR are emphatically discussed, and FRET research progress and future trends are pointed out too.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/tendências , Reação em Cadeia da Polimerase/métodos
18.
Trends Biotechnol ; 24(12): 539-42, 2006 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17070948

RESUMO

Intracellular sensors for nutrients such as sugars, metabolic precursors and signaling ligands such as amino acids will help to elucidate the complex roles of these small molecules in biology. In this update, several recently developed sensors, originating from the Frommer laboratory, which combine bacterial periplasmic-binding protein-based specificity for ligand targeting with fluorescent protein-derived resonance energy transfer for signal transduction are reviewed. The insight gained from designing these sensors, along with the preliminary results gathered from their first application, serve to illustrate the impact that they can have on improving our fundamental understanding of biology.


Assuntos
Biopolímeros/química , Técnicas Biossensoriais/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes , Técnicas de Sonda Molecular , Nanoestruturas , Técnicas Biossensoriais/tendências , Transferência Ressonante de Energia de Fluorescência/tendências , Coloração e Rotulagem/métodos
20.
Sci STKE ; 2006(331): re2, 2006 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-16622184

RESUMO

The validity of experiments based on Förster resonance energy transfer (FRET), an imaging technique widely used to measure protein-protein interactions in living cells, critically depends on the accurate and precise measurement of FRET efficiency. The use of FRET standards to determine FRET efficiency, and a consideration of such factors as how the abundance of FRET acceptors and the stoichiometry of donors and acceptors in a molecular complex can affect measured FRET efficiency, will enhance the usefulness with which FRET experiments can be interpreted.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Mapeamento de Interação de Proteínas/métodos , Algoritmos , Animais , Proteínas de Bactérias/análise , Proteínas de Bactérias/química , Bibliometria , Campos Eletromagnéticos , Polarização de Fluorescência , Transferência Ressonante de Energia de Fluorescência/instrumentação , Transferência Ressonante de Energia de Fluorescência/métodos , Transferência Ressonante de Energia de Fluorescência/normas , Transferência Ressonante de Energia de Fluorescência/tendências , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Fluorometria/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/química , Humanos , Proteínas Luminescentes/análise , Proteínas Luminescentes/química , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Fotodegradação , PubMed/estatística & dados numéricos , Padrões de Referência , Projetos de Pesquisa , Técnicas do Sistema de Duplo-Híbrido
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