scholarly journals In Vivo Biosensing Using Resonance Energy Transfer

Biosensors ◽  
2019 ◽  
Vol 9 (2) ◽  
pp. 76 ◽  
Author(s):  
Shashi Bhuckory ◽  
Joshua C. Kays ◽  
Allison M. Dennis
Keyword(s):  
Energy Transfer ◽  
Fluorescent Proteins ◽  
Low Cost ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Methods ◽  
Great Promise ◽  
Molecular Processes

Solution-phase and intracellular biosensing has substantially enhanced our understanding of molecular processes foundational to biology and pathology. Optical methods are favored because of the low cost of probes and instrumentation. While chromatographic methods are helpful, fluorescent biosensing further increases sensitivity and can be more effective in complex media. Resonance energy transfer (RET)-based sensors have been developed to use fluorescence, bioluminescence, or chemiluminescence (FRET, BRET, or CRET, respectively) as an energy donor, yielding changes in emission spectra, lifetime, or intensity in response to a molecular or environmental change. These methods hold great promise for expanding our understanding of molecular processes not just in solution and in vitro studies, but also in vivo, generating information about complex activities in a natural, organismal setting. In this review, we focus on dyes, fluorescent proteins, and nanoparticles used as energy transfer-based optical transducers in vivo in mice; there are examples of optical sensing using FRET, BRET, and in this mammalian model system. After a description of the energy transfer mechanisms and their contribution to in vivo imaging, we give a short perspective of RET-based in vivo sensors and the importance of imaging in the infrared for reduced tissue autofluorescence and improved sensitivity.

scholarly journals Förster Resonance Energy Transfer-Based Stability Assessment of PLGA Nanoparticles in Vitro and in Vivo

2019 ◽  
Vol 2 (3) ◽  
pp. 1131-1140 ◽  
Author(s):  
Edyta Swider ◽  
Sanish Maharjan ◽  
Karlijne Houkes ◽  
Nicolaas Koen van Riessen ◽  
Carl Figdor ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Plga Nanoparticles ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Stability Assessment ◽  
Förster Resonance

scholarly journals Activation Function 1 of Glucocorticoid Receptor Binds TATA-Binding Protein in Vitro and in Vivo

2006 ◽  
Vol 20 (6) ◽  
pp. 1218-1230 ◽  
Author(s):  
Alicja J. Copik ◽  
M. Scott Webb ◽  
Aaron L. Miller ◽  
Yongxin Wang ◽  
Raj Kumar ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Glucocorticoid Receptor ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Activation Function ◽  
Tata Binding Protein

Abstract The mechanism through which the glucocorticoid receptor (GR) stimulates transcription is still unclear, although it is clear that the GR affects assembly of the transcriptional machinery. The binding of the TATA-binding protein (TBP) to the TATA-box is accepted as essential in this process. It is known that the GR can interact in vitro with TBP, but the direct interaction of TBP with GR has not been previously characterized quantitatively and has not been appreciated as an important step in assembling the transcriptional complex. Herein, we demonstrate that the TBP-GR interaction is functionally significant by characterizing the association of TBP and GR in vitro by a combination of techniques and confirming the role of this interaction in vivo. Combined analysis, using native gel electrophoresis, sedimentation equilibrium, and isothermal microcalorimetry titrations, characterize the stoichiometry, affinity, and thermodynamics of the TBP-GR interaction. TBP binds recombinant GR activation function 1 (AF1) with a 1:2 stoichiometry and a dissociation constant in the nanomolar range. In vivo fluorescence resonance energy transfer experiments, using fluorescently labeled TBP and various GR constructs, transiently transfected into CV-1 cells, show GR-TBP interactions, dependent on AF1. AF1-deletion variants showed fluorescence resonance energy transfer efficiencies on the level of coexpressed cyan fluorescent protein and yellow fluorescent protein, indicating that the interaction is dependent on AF1 domain. To demonstrate the functional role of the in vivo GR-TBP interaction, increased amounts of TBP expressed in vivo stimulated expression of GR-driven reporters and endogenous genes, and the effect was also specifically dependent on AF1.

scholarly journals ERK Activity Imaging During Migration of Living Cells In Vitro and In Vivo

2019 ◽  
Vol 20 (3) ◽  
pp. 679 ◽  
Author(s):  
Eishu Hirata ◽  
Etsuko Kiyokawa
Keyword(s):  
Cancer Metastasis ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Cell Dynamics ◽  
Extracellular Signal

Extracellular signal-regulated kinase (ERK) is a major downstream factor of the EGFR-RAS-RAF signalling pathway, and thus the role of ERK in cell growth has been widely examined. The development of biosensors based on fluorescent proteins has enabled us to measure ERK activities in living cells, both after growth factor stimulation and in its absence. Long-term imaging unexpectedly revealed the oscillative activation of ERK in an epithelial sheet or a cyst in vitro. Studies using transgenic mice expressing the ERK biosensor have revealed inhomogeneous ERK activities among various cell species. In vivo Förster (or fluorescence) resonance energy transfer (FRET) imaging shed light on a novel role of ERK in cell migration. Neutrophils and epithelial cells in various organs such as intestine, skin, lung and bladder showed spatio-temporally different cell dynamics and ERK activities. Experiments using inhibitors confirmed that ERK activities are required for various pathological responses, including epithelial repair after injuries, inflammation, and niche formation of cancer metastasis. In conclusion, biosensors for ERK will be powerful and valuable tools to investigate the roles of ERK in situ.

scholarly journals Fluorescent Cell Imaging in Regenerative Medicine

2016 ◽  
Vol 7s1 ◽  
pp. BECB.S39045
Author(s):  
Etai Sapoznik ◽  
Guoguang Niu ◽  
Yu Zhou ◽  
Sean V. Murphy ◽  
Shay Soker
Keyword(s):  
Regenerative Medicine ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Dynamic Environment ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Biological Research ◽  
Protein Imaging

Fluorescent protein imaging, a promising tool in biological research, incorporates numerous applications that can be of specific use in the field of regenerative medicine. To enhance tissue regeneration efforts, scientists have been developing new ways to monitor tissue development and maturation in vitro and in vivo. To that end, new imaging tools and novel fluorescent proteins have been developed for the purpose of performing deep-tissue high-resolution imaging. These new methods, such as intravital microscopy and Förster resonance energy transfer, are providing new insights into cellular behavior, including cell migration, morphology, and phenotypic changes in a dynamic environment. Such applications, combined with multimodal imaging, significantly expand the utility of fluorescent protein imaging in research and clinical applications of regenerative medicine.

scholarly journals An endogenous green fluorescent protein–photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer

Open Biology ◽  
2014 ◽  
Vol 4 (4) ◽  
pp. 130206 ◽  
Author(s):  
Cécile Fourrage ◽  
Karl Swann ◽  
Jose Raul Gonzalez Garcia ◽  
Anthony K. Campbell ◽  
Evelyn Houliston
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Green Light ◽  
Resonance Energy ◽  
Green Fluorescent Proteins ◽  
Parallel Acquisition ◽  
Green Fluorescent

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria . It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica , we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

A selective and sensitive peptide-based fluorescent chemical DSH sensor for detection of zinc ions and application in vitro and in vivo

2019 ◽  
Vol 43 (7) ◽  
pp. 3071-3077 ◽  
Author(s):  
Lili Zhang ◽  
Jun Cao ◽  
Kai Chen ◽  
Yi Liu ◽  
Yushu Ge ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Chemical Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Zinc Ions ◽  
Design And Synthesis ◽  
Fluorescent Chemical Sensor

Here we report the design and synthesis of a peptide-based fluorescent chemical sensor (DSH sensor: Dansyl-Ser-Pro-Gly-His-Trp-Gly) for detecting Zn2+, based on fluorescence resonance energy transfer (FRET) from tryptophan (donor) to the dansyl fluorophore (acceptor).

scholarly journals Human Disease-causing Mutations Disrupt an N-C-terminal Interaction and Channel Function of Bestrophin 1

2009 ◽  
Vol 284 (24) ◽  
pp. 16473-16481 ◽  
Author(s):  
Zhiqiang Qu ◽  
Wei Cheng ◽  
Yuanyuan Cui ◽  
Yuanyuan Cui ◽  
Jie Zheng
Keyword(s):  
Plasma Membrane ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Macular Dystrophy ◽  
Fluorescence Resonance ◽  
Transfer Signal

Mutations in the human bestrophin 1 (hBest1) chloride channel cause Best vitelliform macular dystrophy. Although mutations in its transmembrane domains were found to alter biophysical properties of the channel, the mechanism for disease-causing mutations in its N and C termini remains elusive. We hypothesized that these mutations lead to channel dysfunction through disruption of an N-C-terminal interaction. Here, we present data demonstrating that hBest1 N and C termini indeed interact both in vivo and in vitro. In addition, using a spectrum-based fluorescence resonance energy transfer method, we showed that functional hBest1 channels in the plasma membrane were multimers. Disease-causing mutations in the N terminus (R19C, R25C, and K30C) and the C terminus (G299E, D301N, and D312N) caused channel dysfunction and disruption of the N-C interaction. Consistent with the functional and biochemical results, mutants D301N and D312N clearly reduced fluorescence resonance energy transfer signal, indicating that the N-C interaction was indeed perturbed. These results suggest that hBest1 functions as a multimer in the plasma membrane, and disruption of the N-C interaction by mutations leads to hBest1 channel dysfunction.

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