Live-Cell Imaging Probes to Track Chromatin Modification Dynamics

Abstract The spatiotemporal organization of chromatin is regulated at different levels in the nucleus. Epigenetic modifications such as DNA methylation and histone modifications are involved in chromatin regulation and play fundamental roles in genome function. While the one-dimensional epigenomic landscape in many cell types has been revealed by chromatin immunoprecipitation and sequencing, the dynamic changes of chromatin modifications and their relevance to chromatin organization and genome function remain elusive. Live-cell probes to visualize chromatin and its modifications have become powerful tools to monitor dynamic chromatin regulation. Bulk chromatin can be visualized both by small fluorescent dyes and fluorescent proteins, and specific endogenous genomic loci have been detected by adapting genome-editing tools. To track chromatin modifications in living cells, various types of probes have been developed. Protein domains that bind to specific modifications weakly, such as chromodomains for histone methylation, can be repeated to create a tighter binding probe that can then be tagged with a fluorescent protein. It has also been demonstrated that antigen-binding fragments and single-chain variable fragments from modification-specific antibodies can serve as binding probes without disturbing cell division, development and, differentiation. These modification-binding modules are used in modification sensors based on fluorescence/Förster resonance energy transfer to measure the intramolecular conformation changes triggered by modifications. Other probes can be created using a bivalent binding system, such as fluorescence complementation, or luciferase chemiluminescence. Live-cell chromatin modification imaging using these probes will address dynamic chromatin regulation and will be useful for assaying and screening effective epigenome drugs in cells and organisms.

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Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽

10.3390/molecules23123105 ◽

2018 ◽

Vol 23 (12) ◽

pp. 3105 ◽

Cited By ~ 1

Author(s):

Henning Höfig ◽

Michele Cerminara ◽

Ilona Ritter ◽

Antonie Schöne ◽

Martina Pohl ◽

...

Keyword(s):

Conformational Change ◽

Single Molecule ◽

Fluorescent Dyes ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Strong Increase ◽

Single Molecule Level ◽

Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Dye selection for live cell imaging of intact siRNA

Biological Chemistry ◽

10.1515/bc-2011-256 ◽

2012 ◽

Vol 393 (1-2) ◽

pp. 23-35 ◽

Cited By ~ 10

Author(s):

Markus Hirsch ◽

Dennis Strand ◽

Mark Helm

Keyword(s):

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Dyes ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Time Lapse ◽

Live Cell ◽

High Yield ◽

Ph Variation ◽

Rnai Efficiency

Abstract Investigations into the fate of small interfering RNA (siRNA) after transfection may unravel new ways to improve RNA interference (RNAi) efficiency. Because intracellular degradation of RNA may prevent reliable observation of fluorescence-labeled siRNA, new tools for fluorescence microscopy are warranted to cover the considerable duration of the RNAi effect. Here, the characterization and application of new fluorescence resonance energy transfer (FRET) dye pairs for sensing the integrity of duplex siRNA is reported, which allows an assessment of the degradation status of an siRNA cell population by live cell imaging. A panel of high-yield fluorescent dyes has been investigated for their suitability as FRET pairs for the investigation of RNA inside the cell. Nine dyes in 13 FRET pairs were evaluated based on the performance in assays of photostability, cross-excitation, bleed-through, as well as on quantified changes of fluorescence as a consequence of, e.g., RNA strand hybridization and pH variation. The Atto488/Atto590 FRET pair has been applied to live cell imaging, and has revealed first aspects of unusual trafficking of intact siRNA. A time-lapse study showed highly dynamic movement of siRNA in large perinuclear structures. These and the resulting optimized FRET labeled siRNA are expected to have significant impact on future observations of labeled RNAs in living cells.

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Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

International Journal of Molecular Sciences ◽

10.3390/ijms20163859 ◽

2019 ◽

Vol 20 (16) ◽

pp. 3859 ◽

Cited By ~ 4

Author(s):

Michael Winkler ◽

Florian Wrensch ◽

Pascale Bosch ◽

Maike Knoth ◽

Michael Schindler ◽

...

Keyword(s):

Antiviral Activity ◽

Protein Interactions ◽

Viral Entry ◽

Cellular Localization ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

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Enhancement of correct protein folding in vivo by a non-lytic baculovirus

Biochemical Journal ◽

10.1042/bj20040007 ◽

2004 ◽

Vol 382 (2) ◽

pp. 695-702 ◽

Cited By ~ 20

Author(s):

Yu HO ◽

Huei-Ru LO ◽

Tzu-Ching LEE ◽

Carol P. Y. WU ◽

Yu-Chan CHAO

Keyword(s):

Energy Transfer ◽

Fusion Protein ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Random Mutagenesis ◽

Resonance Energy ◽

Vector System ◽

Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

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FRET Studies Between CdTe Capped by Small-Molecule Ligands and Fluorescent Protein

International Journal of Nanoscience ◽

10.1142/s0219581x14600138 ◽

2014 ◽

Vol 13 (05n06) ◽

pp. 1460013

Author(s):

Yue Zhang ◽

Dejian Zhou ◽

Junhui He

Keyword(s):

Fluorescent Protein ◽

Organic Dyes ◽

Fluorescent Proteins ◽

Semiconductor Nanocrystals ◽

Resonance Energy ◽

Emission Spectra ◽

Binding Constants ◽

Dipole Interaction ◽

Water Soluble ◽

Donor Acceptor

Water-soluble luminescent semiconductor nanocrystals also known as quantum dots (QDs) that have prominent photostability, wide absorption cross sections and tunable narrow emission, have been shown as promising probes in immunoassays. QDs are often used as donors in fluorescence resonance energy transfer (FRET) based sensors using organic dyes or fluorescent proteins as acceptors. Here, the FRET between a QD donor and fluorescent protein acceptors has been studied. The fluorescent protein (FP)mCherry appended with a hexa-histidine-tag could effectively self-assemble onto CdTe to produce small donor-acceptor distances and hence highly efficient FRET (efficiency > 80%) at relatively low FP: CdTe copy numbers (ca.1). Using the Förster dipole–dipole interaction formula, the Förster radius (R0) and respective donor-acceptor distances for the CdTe -FP FRET systems have been calculated. The binding constants (Kd) of the QD-FP systems have also been evaluated by the emission spectra.

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Dynamic measurements of mitochondrial hydrogen peroxide concentration and glutathione redox state in rat pancreatic β-cells using ratiometric fluorescent proteins: confounding effects of pH with HyPer but not roGFP1

Biochemical Journal ◽

10.1042/bj20111770 ◽

2012 ◽

Vol 441 (3) ◽

pp. 971-978 ◽

Cited By ~ 59

Author(s):

Leticia P. Roma ◽

Jessica Duprez ◽

Hilton K. Takahashi ◽

Patrick Gilon ◽

Andreas Wiederkehr ◽

...

Keyword(s):

Hydrogen Peroxide ◽

Redox State ◽

Fluorescent Dyes ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Ph Sensitivity ◽

Fluorescence Ratio ◽

Β Cells ◽

Pancreatic Β Cells ◽

Glutathione Redox

Using the ROS (reactive oxygen species)-sensitive fluorescent dyes dichlorodihydrofluorescein and dihydroethidine, previous studies yielded opposite results about the glucose regulation of oxidative stress in insulin-secreting pancreatic β-cells. In the present paper, we used the ratiometric fluorescent proteins HyPer and roGFP1 (redox-sensitive green fluorescent protein 1) targeted to mitochondria [mt-HyPer (mitochondrial HyPer)/mt-roGFP1 (mitochondrial roGFP1)] to monitor glucose-induced changes in mitochondrial hydrogen peroxide concentration and glutathione redox state in adenovirus-infected rat islet cell clusters. Because of the reported pH sensitivity of HyPer, the results were compared with those obtained with the mitochondrial pH sensors mt-AlpHi and mt-SypHer. The fluorescence ratio of the mitochondrial probes slowly decreased (mt-HyPer) or increased (mt-roGFP1) in the presence of 10 mmol/l glucose. Besides its expected sensitivity to H2O2, mt-HyPer was also highly pH sensitive. In agreement, changes in mitochondrial metabolism similarly affected mt-HyPer, mt-AlpHi and mt-SypHer fluorescence signals. In contrast, the mt-roGFP1 fluorescence ratio was only slightly affected by pH and reversibly increased when glucose was lowered from 10 to 2 mmol/l. This increase was abrogated by the catalytic antioxidant Mn(III) tetrakis (4-benzoic acid) porphyrin but not by N-acetyl-L-cysteine. In conclusion, due to its pH sensitivity, mt-HyPer is not a reliable indicator of mitochondrial H2O2 in β-cells. In contrast, the mt-roGFP1 fluorescence ratio monitors changes in β-cell mitochondrial glutathione redox state with little interference from pH changes. Our results also show that glucose acutely decreases rather than increases mitochondrial thiol oxidation in rat β-cells.

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Over the rainbow: a practical guide for fluorescent protein selection in plant FRET experiments

10.1101/734616 ◽

2019 ◽

Author(s):

Grégoire Denay ◽

Patrick Schultz ◽

Sebastian Hänsch ◽

Stefanie Weidtkamp-Peters ◽

Rüdiger Simon

Keyword(s):

Protein Interaction ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Resonance Energy ◽

Fluorescence Lifetime Imaging ◽

Excitation Spectra ◽

Expression Levels ◽

Receptor Complexes ◽

Endogenous Expression ◽

Donor And Acceptor

AbstractReceptor-like kinases (RLK) and receptor-like proteins (RLP) often interact in a combinatorial manner depending on tissue identity, membrane domains, or endo- and exogenous cues, and the same RLKs or RLPs can generate different signaling outputs depending on the composition of the receptor complexes they are involved in. Investigation of their interaction partners in a spatial and dynamic way is therefore of prime interest to understand their functions. This is however limited by the technical complexity of assessing it in endogenous conditions. A solution to close this gap is to determine protein interaction directly in the relevant tissues at endogenous expression levels using Förster resonance energy transfer (FRET). The ideal fluorophore pair for FRET must, however, fulfil specific requirements: (i) the emission and excitation spectra of the donor and acceptor, respectively, must overlap; (ii) they should not interfere with proper folding, activity, or localization of the fusion proteins; (iii) they should be sufficiently photostable in plant cells. Furthermore, the donor must yield sufficient photon counts at near-endogenous protein expression levels. Although many fluorescent proteins were reported to be suitable for FRET experiments, only a handful were already described for applications in plants. Herein, we compare a range of fluorophores, assess their usability to study RLK interactions by FRET-based fluorescence lifetime imaging (FLIM) and explore their differences in FRET efficiency. Our analysis will help to select the optimal fluorophore pair for diverse FRET applications.One-sentence summaryWe compared the performances of several different fluorescent protein pairs to study membrane protein interaction in plants with FRET.

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Generation of a Retina Reporter hiPSC Line to Label Progenitor, Ganglion, and Photoreceptor Cell Types

10.1101/658963 ◽

2019 ◽

Author(s):

Phuong T. Lam ◽

Christian Gutierrez ◽

Katia Del Rio-Tsonis ◽

Michael L. Robinson

Keyword(s):

Fluorescent Protein ◽

Ganglion Cells ◽

Fluorescent Proteins ◽

Retinal Development ◽

Retinal Disease ◽

Cell Types ◽

Retinal Cell ◽

Therapeutic Drug ◽

Retina Development ◽

Induced Pluripotent

ABSTRACTEarly in mammalian eye development, VSX2, BRN3b, and RCVRN expression marks neural retina progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist. Here, we describe a CRISPR/Cas9 gene editing strategy to generate a triple transgenic reporter hiPSC line (PGP1) that utilizes the endogenous VSX2, BRN3b, and RCVRN promoters to specifically express fluorescent proteins (Cerulean in NRPs, eGFP in RGCs and mCherry in PRs) without disrupting the function of the endogenous alleles. Retinal organoid formation from the PGP1 line demonstrated the ability of the edited cells to undergo normal retina development while exhibiting appropriate fluorescent protein expression consistent with the onset of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types. The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening.

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PIT-1 Protein Localization at Different Optical Sections in a Single Living Cell Using FRET Microscopy and Green Fluorescent Proteins

Microscopy and Microanalysis ◽

10.1017/s1431927600007558 ◽

1997 ◽

Vol 3 (S2) ◽

pp. 133-134 ◽

Cited By ~ 1

Author(s):

Ammasi Periasamy ◽

Richard N. Day

Keyword(s):

Transcription Factors ◽

Living Cell ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Protein Localization ◽

Resonance Energy ◽

Living Cells ◽

Specific Gene ◽

Green Fluorescent ◽

Prl Gene

The pituitary specific transcription factor Pit-1 is required for transcriptional activity of the prolactin (PRL) gene. The Pit-1 protein is a member of the POU homeodomain transcription factors that is expressed in several different anterior pituitary cell types, where it functions as an important determinant of pituitary-specific gene expression. The Pit-1 protein generally interacts with DNA elements in the PRL gene promoter as a dimer, and has been demonstrated to associate with other transcription factors. The objective of our research is to define the critical molecular events involved in transcriptional regulation of the PRL gene in living cells. Methods that allow monitoring of the intimate interactions between protein partners in living cells provide an unparalleled perspective on these biological processes. Using the jellyfish green fluorescent protein (GFP) as a tag, we applied the fluorescence resonance energy transfer (FRET) technique to visualize where and when the Pit-1 protein interacts in the living cell. FRET is a quantum mechanical effect that occurs between donor (D) and acceptor (A) fluorophores provided: (i) the emission energy of D is coincident with the energy required to excite A, and (ii) the distance that separating the two fluorophores is 10-100 Å. Mutant forms of GFP that fluoresce either green or blue (BFP) have excitation and emission spectra that are suitable for FRET imaging.

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