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 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 Diazepam Accelerates GABAAR Synaptic Exchange and Alters Intracellular Trafficking

2019 ◽  
Author(s):  
Joshua M. Lorenz-Guertin ◽  
Matthew J. Bambino ◽  
Sabyasachi Das ◽  
Susan T. Weintraub ◽  
Tija C. Jacob
Keyword(s):  
Cortical Neurons ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Adult Brain ◽  
Scaffolding Protein ◽  
Gamma Aminobutyric Acid ◽  
Inhibitory Neurotransmitter

Despite 50+ years of clinical use as anxiolytics, anti-convulsants, and sedative/hypnotic agents, the mechanisms underlying benzodiazepine (BZD) tolerance are poorly understood. BZDs potentiate the actions of gamma-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the adult brain, through positive allosteric modulation of γ2 subunit containing GABA type A receptors (GABAARs). Here we define key molecular events impacting γ2 GABAAR and the inhibitory synapse gephyrin scaffold following initial sustained BZD exposure in vitro and in vivo. Using immunofluorescence and biochemical experiments, we found that cultured cortical neurons treated with the classical BZD, diazepam (DZP), presented no substantial change in surface or synaptic levels of γ2-GABAARs. In contrast, both γ2 and the postsynaptic scaffolding protein gephyrin showed diminished total protein levels following a single DZP treatment in vitro and in mouse cortical tissue. We further identified DZP treatment enhanced phosphorylation of gephyrin Ser270 and increased generation of gephyrin cleavage products. Selective immunoprecipitation of γ2 from cultured neurons revealed enhanced ubiquitination of this subunit following DZP exposure. To assess novel trafficking responses induced by DZP, we employed a γ2 subunit containing an N terminal fluorogen-activating peptide (FAP) and pH-sensitive green fluorescent protein (γ2pHFAP). Live-imaging experiments using γ2pHFAP GABAAR expressing neurons identified enhanced lysosomal targeting of surface GABAARs and increased overall accumulation in vesicular compartments in response to DZP. Using fluorescence resonance energy transfer (FRET) measurements between γ2 and γ2 subunits within a GABAAR in neurons, we identified reductions in synaptic clusters of this subpopulation of surface BZD sensitive receptor. Moreover, we found DZP simultaneously enhanced synaptic exchange of both γ2-GABAARs and gephyrin using fluorescence recovery after photobleaching (FRAP) techniques. Finally we provide the first proteomic analysis of the BZD sensitive GABAAR interactome in DZP vs. vehicle treated mice. Collectively, our results indicate DZP exposure elicits down-regulation of gephyrin scaffolding and BZD sensitive GABAAR synaptic availability via multiple dynamic trafficking processes.

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 An Improved Cerulean Fluorescent Protein with Enhanced Brightness and Reduced Reversible Photoswitching

PLoS ONE ◽  
2011 ◽  
Vol 6 (3) ◽  
pp. e17896 ◽  
Author(s):  
Michele L. Markwardt ◽  
Gert-Jan Kremers ◽  
Catherine A. Kraft ◽  
Krishanu Ray ◽  
Paula J. C. Cranfill ◽  
...  
Keyword(s):  
Quantum Yield ◽  
Fluorescence Lifetime ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Site Directed Mutagenesis ◽  
Quantum Yields

Cyan fluorescent proteins (CFPs), such as Cerulean, are widely used as donor fluorophores in Förster resonance energy transfer (FRET) experiments. Nonetheless, the most widely used variants suffer from drawbacks that include low quantum yields and unstable flurorescence. To improve the fluorescence properties of Cerulean, we used the X-ray structure to rationally target specific amino acids for optimization by site-directed mutagenesis. Optimization of residues in strands 7 and 8 of the β-barrel improved the quantum yield of Cerulean from 0.48 to 0.60. Further optimization by incorporating the wild-type T65S mutation in the chromophore improved the quantum yield to 0.87. This variant, mCerulean3, is 20% brighter and shows greatly reduced fluorescence photoswitching behavior compared to the recently described mTurquoise fluorescent protein in vitro and in living cells. The fluorescence lifetime of mCerulean3 also fits to a single exponential time constant, making mCerulean3 a suitable choice for fluorescence lifetime microscopy experiments. Furthermore, inclusion of mCerulean3 in a fusion protein with mVenus produced FRET ratios with less variance than mTurquoise-containing fusions in living cells. Thus, mCerulean3 is a bright, photostable cyan fluorescent protein which possesses several characteristics that are highly desirable for FRET experiments.

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 Far-red fluorescent tag for protein labelling

2002 ◽  
Vol 368 (1) ◽  
pp. 17-21 ◽  
Author(s):  
Arkady F. FRADKOV ◽  
Vladislav V. VERKHUSHA ◽  
Dmitry B. STAROVEROV ◽  
Maria E. BULINA ◽  
Yurii G. YANUSHEVICH ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Sea Anemones ◽  
Practical Applications ◽  
In Vivo Labelling ◽  
Covalently Linked ◽  
Green Fluorescent

Practical applications of green fluorescent protein ('GFP')-like fluorescent proteins (FPs) from species of the class Anthozoa (sea anemones, corals and sea pens) are strongly restricted owing to their oligomeric nature. Here we suggest a strategy to overcome this problem by the use of two covalently linked identical red FPs as non-oligomerizing fusion tags. We have applied this approach to the dimeric far-red fluorescent protein HcRed1 and have demonstrated superiority of the tandem tag in the in vivo labelling of fine cytoskeletal structures and tiny nucleoli. In addition, a possibility of effective fluorescence resonance energy transfer ('FRET') between enhanced yellow FP mutant ('EYFP') and tandem HcRed1 was demonstrated in a protease assay.

Programmable in vitro Co-Encapsidation of Guest Proteins Inside Protein Nanocages

2018 ◽  
Author(s):  
Noor H. Dashti ◽  
Rufika S. Abidin ◽  
Frank Sainsbury
Keyword(s):  
Self Assembly ◽  
Mammalian Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Super Resolution ◽  
Cell Engineering ◽  
Particle Analysis ◽  
Protein Cages

Bioinspired self-sorting and self-assembling systems using engineered versions of natural protein cages have been developed for biocatalysis and therapeutic delivery. The packaging and intracellular delivery of guest proteins is of particular interest for both <i>in vitro</i> and <i>in vivo</i> cell engineering. However, there is a lack of platforms in bionanotechnology that combine programmable guest protein encapsidation with efficient intracellular uptake. We report a minimal peptide anchor for <i>in vivo</i> self-sorting of cargo-linked capsomeres of the Murine polyomavirus (MPyV) major coat protein that enables controlled encapsidation of guest proteins by <i>in vitro</i> self-assembly. Using Förster resonance energy transfer (FRET) we demonstrate the flexibility in this system to support co-encapsidation of multiple proteins. Complementing these ensemble measurements with single particle analysis by super-resolution microscopy shows that the stochastic nature of co-encapsidation is an overriding principle. This has implications for the design and deployment of both native and engineered self-sorting encapsulation systems and for the assembly of infectious virions. Taking advantage of the encoded affinity for sialic acids ubiquitously displayed on the surface of mammalian cells, we demonstrate the ability of self-assembled MPyV virus-like particles to mediate efficient delivery of guest proteins to the cytosol of primary human cells. This platform for programmable co-encapsidation and efficient cytosolic delivery of complementary biomolecules therefore has enormous potential in cell engineering.

scholarly journals Biological nanoscale fluorescent probes: From structure and performance to bioimaging

2020 ◽  
Vol 39 (1) ◽  
pp. 209-221
Author(s):  
Jiafeng Wan ◽  
Xiaoyuan Zhang ◽  
Kai Zhang ◽  
Zhiqiang Su
Keyword(s):  
Fluorescent Probes ◽  
Organic Dyes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Biological Imaging ◽  
Fluorescent Materials ◽  
And Performance

Abstract In recent years, nanomaterials have attracted lots of attention from researchers due to their unique properties. Nanometer fluorescent materials, such as organic dyes, semiconductor quantum dots (QDs), metal nano-clusters (MNCs), carbon dots (CDs), etc., are widely used in biological imaging due to their high sensitivity, short response time, and excellent accuracy. Nanometer fluorescent probes can not only perform in vitro imaging of organisms but also achieve in vivo imaging. This provides medical staff with great convenience in cancer treatment. Combined with contemporary medical methods, faster and more effective treatment of cancer is achievable. This article explains the response mechanism of three-nanometer fluorescent probes: the principle of induced electron transfer (PET), the principle of fluorescence resonance energy transfer (FRET), and the principle of intramolecular charge transfer (ICT), showing the semiconductor QDs, precious MNCs, and CDs. The excellent performance of the three kinds of nano fluorescent materials in biological imaging is highlighted, and the application of these three kinds of nano fluorescent probes in targeted biological imaging is also introduced. Nanometer fluorescent materials will show their significance in the field of biomedicine.

Sign in / Sign up

Export Citation Format

Share Document