scholarly journals Generation of a Transgenic Zebrafish Line for In Vivo Assessment of Hepatic Apoptosis

2021 ◽  
Vol 14 (11) ◽  
pp. 1117
Author(s):  
Aina Higuchi ◽  
Eri Wakai ◽  
Tomoko Tada ◽  
Junko Koiwa ◽  
Yuka Adachi ◽  
...  
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Caspase 3 ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Transgenic Zebrafish ◽  
Drug Induced ◽  
Drug Induced Liver Injury ◽  
In Vivo Fluorescence Imaging

Hepatic apoptosis is involved in a variety of pathophysiologic conditions in the liver, including hepatitis, steatosis, and drug-induced liver injury. The development of easy-to-perform and reliable in vivo assays would thus greatly enhance the efforts to understand liver diseases and identify associated genes and potential drugs. In this study, we developed a transgenic zebrafish line that was suitable for the assessment of caspase 3 activity in the liver by using in vivo fluorescence imaging. The larvae of transgenic zebrafish dominantly expressed Casper3GR in the liver under control of the promoter of the phosphoenolpyruvate carboxykinase 1 gene. Casper3GR is composed of two fluorescent proteins, tagGFP and tagRFP, which are connected via a peptide linker that can be cleaved by activated caspase 3. Under tagGFP excitation conditions in zebrafish that were exposed to the well-characterized hepatotoxicant isoniazid, we detected increased and decreased fluorescence associated with tagGFP and tagRFP, respectively. This result suggests that isoniazid activates caspase 3 in the zebrafish liver, which digests the linker between tagGFP and tagRFP, resulting in a reduction in the Förster resonance energy transfer to tagRFP upon tagGFP excitation. We also detected isoniazid-induced inhibition of caspase 3 activity in zebrafish that were treated with the hepatoprotectants ursodeoxycholic acid and obeticholic acid. The transgenic zebrafish that were developed in this study could be a powerful tool for identifying both hepatotoxic and hepatoprotective drugs, as well as for analyzing the effects of the genes of interest to hepatic apoptosis.

Lighting up alkaline phosphatase in drug-induced liver injury using a new chemiluminescence resonance energy transfer nanoprobe

2018 ◽  
Vol 54 (88) ◽  
pp. 12479-12482 ◽  
Author(s):  
Xueting Liu ◽  
Nannan Fan ◽  
Lijie Wu ◽  
Chuanchen Wu ◽  
Yongqing Zhou ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Energy Transfer ◽  
Liver Injury ◽  
Alkaline Phosphatase Level ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Drug Induced ◽  
Drug Induced Liver Injury

Ultra-sensitive imaging of the alkaline phosphatase levelin vivoin drug-induced liver injury with a new chemiluminescence resonance energy transfer nanoprobe.

scholarly journals FRET Microscopy in Yeast

Biosensors ◽  
2019 ◽  
Vol 9 (4) ◽  
pp. 122 ◽  
Author(s):  
Skruzny ◽  
Pohl ◽  
Abella
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Bioactive Molecules ◽  
Biophysical Processes ◽  
Microscopy Method ◽  
Fret Biosensors

Förster resonance energy transfer (FRET) microscopy is a powerful fluorescence microscopy method to study the nanoscale organization of multiprotein assemblies in vivo. Moreover, many biochemical and biophysical processes can be followed by employing sophisticated FRET biosensors directly in living cells. Here, we summarize existing FRET experiments and biosensors applied in yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, two important models of fundamental biomedical research and efficient platforms for analyses of bioactive molecules. We aim to provide a practical guide on suitable FRET techniques, fluorescent proteins, and experimental setups available for successful FRET experiments in yeasts.

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 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 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.

scholarly journals A biosensor for the direct visualization of auxin

Nature ◽  
2021 ◽  
Author(s):  
Ole Herud-Sikimić ◽  
Andre C. Stiel ◽  
Martina Kolb ◽  
Sooruban Shanmugaratnam ◽  
Kenneth W. Berendzen ◽  
...  
Keyword(s):  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Temporal Distribution ◽  
Resonance Energy ◽  
Binding Pocket ◽  
Irreversible Processes ◽  
Endogenous Auxin ◽  
In Planta ◽  
Cellular Resolution

AbstractOne of the most important regulatory small molecules in plants is indole-3-acetic acid, also known as auxin. Its dynamic redistribution has an essential role in almost every aspect of plant life, ranging from cell shape and division to organogenesis and responses to light and gravity1,2. So far, it has not been possible to directly determine the spatial and temporal distribution of auxin at a cellular resolution. Instead it is inferred from the visualization of irreversible processes that involve the endogenous auxin-response machinery3–7; however, such a system cannot detect transient changes. Here we report a genetically encoded biosensor for the quantitative in vivo visualization of auxin distribution. The sensor is based on the Escherichia coli tryptophan repressor8, the binding pocket of which is engineered to be specific to auxin. Coupling of the auxin-binding moiety with selected fluorescent proteins enables the use of a fluorescence resonance energy transfer signal as a readout. Unlike previous systems, this sensor enables direct monitoring of the rapid uptake and clearance of auxin by individual cells and within cell compartments in planta. By responding to the graded spatial distribution along the root axis and its perturbation by transport inhibitors—as well as the rapid and reversible redistribution of endogenous auxin in response to changes in gravity vectors—our sensor enables real-time monitoring of auxin concentrations at a (sub)cellular resolution and their spatial and temporal changes during the lifespan of a plant.

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