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Journal ArticleDOI

Stable expression of FRET biosensors: A new light in cancer research

01 Apr 2012-Cancer Science (Wiley-Blackwell)-Vol. 103, Iss: 4, pp 614-619
TL;DR: The principle of FRET‐based bios Sensors is introduced, an optimized backbone of the FRET biosensors is described, techniques to express FRET BIOSensors stably in the cells are discussed, and the future perspectives of Fret biosensor in cancer research are discussed.
Abstract: The constituents of the oncogene signal transduction pathway are promising targets for anticancer drugs. Despite the wealth of available knowledge regarding their molecular properties, the spatiotemporal regulation of the signaling molecules remains elusive. Biosensors based on the principle of FRET have been developed to visualize the activities of the signaling molecules in living cells. However, difficulties in the development of sensitive FRET biosensors have prevented their widespread use in cancer research. The lack of cell lines constitutively expressing a FRET biosensor has also limited their use. In this review, we will introduce the principle of FRET-based biosensors, describe an optimized backbone of the FRET biosensors, techniques to express FRET biosensors stably in the cells, and discuss the future perspectives of FRET biosensors in cancer research.
Citations
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Journal ArticleDOI
TL;DR: Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced as discussed by the authors.
Abstract: Fluorescence microscopy provides an efficient and unique approach to study fixed and living cells because of its versatility, specificity, and high sensitivity. Fluorescence microscopes can both detect the fluorescence emitted from labeled molecules in biological samples as images or photometric data from which intensities and emission spectra can be deduced. By exploiting the characteristics of fluorescence, various techniques have been developed that enable the visualization and analysis of complex dynamic events in cells, organelles, and sub-organelle components within the biological specimen. The techniques described here are fluorescence recovery after photobleaching (FRAP), the related fluorescence loss in photobleaching (FLIP), fluorescence localization after photobleaching (FLAP), Forster or fluorescence resonance energy transfer (FRET) and the different ways how to measure FRET, such as acceptor bleaching, sensitized emission, polarization anisotropy, and fluorescence lifetime imaging microscopy (FLIM). First, a brief introduction into the mechanisms underlying fluorescence as a physical phenomenon and fluorescence, confocal, and multiphoton microscopy is given. Subsequently, these advanced microscopy techniques are introduced in more detail, with a description of how these techniques are performed, what needs to be considered, and what practical advantages they can bring to cell biological research.

429 citations

Journal ArticleDOI
14 Sep 2016-Sensors
TL;DR: An overview of methods to measure FRET changes in biological contexts is provided, the palette of FP FRET pairs developed and their relative strengths and weaknesses are discussed, and important factors to consider are noted when using FPs for FRET studies.
Abstract: Forster or fluorescence resonance energy transfer (FRET) technology and genetically encoded FRET biosensors provide a powerful tool for visualizing signaling molecules in live cells with high spatiotemporal resolution. Fluorescent proteins (FPs) are most commonly used as both donor and acceptor fluorophores in FRET biosensors, especially since FPs are genetically encodable and live-cell compatible. In this review, we will provide an overview of methods to measure FRET changes in biological contexts, discuss the palette of FP FRET pairs developed and their relative strengths and weaknesses, and note important factors to consider when using FPs for FRET studies.

323 citations


Cites background from "Stable expression of FRET biosensor..."

  • ...Lastly, stable cell lines expressing FRET biosensors are easily achievable in the presence of antibiotic pressure, which reduces cell-cell viability in FRET imaging and facilitates high-throughput drug screening [7]....

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Journal ArticleDOI
TL;DR: It is shown that stochastic ERK activity pulses regulate cell proliferation rates in a cell density-dependent manner and the involvement of serum responsive factor (SRF) transcription factors in the gene expression driven by the ERk activity pulses is suggested.

272 citations


Cites methods from "Stable expression of FRET biosensor..."

  • ...Establishment of stable cell lines To generate cell lines stably expressing FRET biosensors, we utilized a piggybac transposon system (Aoki et al., 2012)....

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  • ...Establishment of Stable Cell Lines To generate cell lines stably expressing FRET biosensors, we utilized a PiggyBac transposon system (Aoki et al., 2012)....

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  • ...We recently reported a highly sensitive ERK FRET biosensor, EKAREV, and a protocol to establish cell lines stably expressing FRET biosensors (Aoki et al., 2012)....

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Journal ArticleDOI
TL;DR: Evidence is provided that propagation waves of extracellular signal-related kinase (ERK) mitogen-activated protein kinase activation determine the direction of the collective cell migration.

186 citations

Journal ArticleDOI
TL;DR: In this paper, the authors discuss various health-monitoring devices, their ability to capture the unique state of health represented in a patient and their application in individualized diagnostics, prognosis, clinical or wellness intervention.
Abstract: Monitoring and modeling biomedical, health care and wellness data from individuals and converging data on a population scale have tremendous potential to improve understanding of the transition to the healthy state of human physiology to disease setting. Wellness monitoring devices and companion software applications capable of generating alerts and sharing data with health care providers or social networks are now available. The accessibility and clinical utility of such data for disease or wellness research are currently limited. Designing methods for streaming data capture, real-time data aggregation, machine learning, predictive analytics and visualization solutions to integrate wellness or health monitoring data elements with the electronic medical records (EMRs) maintained by health care providers permits better utilization. Integration of population-scale biomedical, health care and wellness data would help to stratify patients for active health management and to understand clinically asymptomatic patients and underlying illness trajectories. In this article, we discuss various health-monitoring devices, their ability to capture the unique state of health represented in a patient and their application in individualized diagnostics, prognosis, clinical or wellness intervention. We also discuss examples of translational bioinformatics approaches to integrating patient-generated data with existing EMRs, personal health records, patient portals and clinical data repositories. Briefly, translational bioinformatics methods, tools and resources are at the center of these advances in implementing real-time biomedical and health care analytics in the clinical setting. Furthermore, these advances are poised to play a significant role in clinical decision-making and implementation of data-driven medicine and wellness care.

153 citations


Cites background from "Stable expression of FRET biosensor..."

  • ...Biosensors [80–85] are also being used for various health applications including health monitoring....

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References
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Journal ArticleDOI
28 Aug 1997-Nature
TL;DR: New fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations are constructed and dubbed ‘cameleons’.
Abstract: Important Ca2+ signals in the cytosol and organelles are often extremely localized and hard to measure. To overcome this problem we have constructed new fluorescent indicators for Ca2+ that are genetically encoded without cofactors and are targetable to specific intracellular locations. We have dubbed these fluorescent indicators 'cameleons'. They consist of tandem fusions of a blue- or cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13, and an enhanced green- or yellow-emitting GFP. Binding of Ca2+ makes calmodulin wrap around the M13 domain, increasing the fluorescence resonance energy transfer (FRET) between the flanking GFPs. Calmodulin mutations can tune the Ca2+ affinities to measure free Ca2+ concentrations in the range 10(-8) to 10(-2) M. We have visualized free Ca2+ dynamics in the cytosol, nucleus and endoplasmic reticulum of single HeLa cells transfected with complementary DNAs encoding chimaeras bearing appropriate localization signals. Ca2+ concentration in the endoplasmic reticulum of individual cells ranged from 60 to 400 microM at rest, and 1 to 50 microM after Ca2+ mobilization. FRET is also an indicator of the reversible intermolecular association of cyan-GFP-labelled calmodulin with yellow-GFP-labelled M13. Thus FRET between GFP mutants can monitor localized Ca2+ signals and protein heterodimerization in individual live cells.

3,248 citations

Journal ArticleDOI
14 Apr 2006-Science
TL;DR: The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy.
Abstract: Advances in molecular biology, organic chemistry, and materials science have recently created several new classes of fluorescent probes for imaging in cell biology. Here we review the characteristic benefits and limitations of fluorescent probes to study proteins. The focus is on protein detection in live versus fixed cells: determination of protein expression, localization, activity state, and the possibility for combination of fluorescent light microscopy with electron microscopy. Small organic fluorescent dyes, nanocrystals ("quantum dots"), autofluorescent proteins, small genetic encoded tags that can be complexed with fluorochromes, and combinations of these probes are highlighted.

2,632 citations

Journal ArticleDOI
TL;DR: Using a multiparameter tuning model, this work describes how dimension, density, stiffness, and orientation of the extracellular matrix together with cell determinants—including cell–cell and cell–matrix adhesion, cytoskeletal polarity and stiffness, etc.—interdependently control migration mode and efficiency.
Abstract: Cell migration underlies tissue formation, maintenance, and regeneration as well as pathological conditions such as cancer invasion. Structural and molecular determinants of both tissue environment and cell behavior define whether cells migrate individually (through amoeboid or mesenchymal modes) or collectively. Using a multiparameter tuning model, we describe how dimension, density, stiffness, and orientation of the extracellular matrix together with cell determinants-including cell-cell and cell-matrix adhesion, cytoskeletal polarity and stiffness, and pericellular proteolysis-interdependently control migration mode and efficiency. Motile cells integrate variable inputs to adjust interactions among themselves and with the matrix to dictate the migration mode. The tuning model provides a matrix of parameters that control cell movement as an adaptive and interconvertible process with relevance to different physiological and pathological contexts.

1,239 citations

Journal ArticleDOI
TL;DR: Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.
Abstract: Many genetically encoded biosensors use Forster resonance energy transfer (FRET) between fluorescent proteins to report biochemical phenomena in living cells. Most commonly, the enhanced cyan fluorescent protein (ECFP) is used as the donor fluorophore, coupled with one of several yellow fluorescent protein (YFP) variants as the acceptor. ECFP is used despite several spectroscopic disadvantages, namely a low quantum yield, a low extinction coefficient and a fluorescence lifetime that is best fit by a double exponential. To improve the characteristics of ECFP for FRET measurements, we used a site-directed mutagenesis approach to overcome these disadvantages. The resulting variant, which we named Cerulean (ECFP/S72A/Y145A/H148D), has a greatly improved quantum yield, a higher extinction coefficient and a fluorescence lifetime that is best fit by a single exponential. Cerulean is 2.5-fold brighter than ECFP and replacement of ECFP with Cerulean substantially improves the signal-to-noise ratio of a FRET-based sensor for glucokinase activation.

1,154 citations

Journal ArticleDOI
TL;DR: This study optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles and generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability.
Abstract: Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca2+ indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca2+-saturated form, thereby increasing the Ca2+-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca2+ dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

1,025 citations