Quantitative Fluorescence Resonance Energy Transfer Analysis on the Direct Interaction of Activation-2b with Histone H3/Switch-3B Protein in Arabidopsis Mesophyll Protoplasts

2021 ◽  
Author(s):  
Lu Gao ◽  
Fangrui Lin ◽  
Danlu Han ◽  
Jieming Jiang ◽  
Chengwei Yang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Histone H3 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Mesophyll Protoplasts ◽  
Fluorescence Resonance ◽  
Quantitative Fluorescence

scholarly journals Identification of Small Molecule Inhibitors of the Mitotic Kinase Haspin by High-Throughput Screening Using a Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assay

2008 ◽  
Vol 13 (10) ◽  
pp. 1025-1034 ◽  
Author(s):  
Debasis Patnaik ◽  
Jun Xian ◽  
Marcie A. Glicksman ◽  
Gregory D. Cuny ◽  
Ross L. Stein ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
High Throughput ◽  
Histone H3 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Kinase Assay ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Fluorescence Resonance

Haspin/Gsg2 is a kinase that phosphorylates histone H3 at Thr-3 (H3T3ph) during mitosis. Its depletion by RNA interference results in failure of chromosome alignment and a block in mitosis. Haspin, therefore, is a novel target for development of antimitotic agents. We report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) kinase assay for haspin. Histone H3 peptide was used as a substrate, and a europium-labeled H3T3ph phosphospecific monoclonal antibody was used to detect phosphorylation. A library of 137632 small molecules was screened at Km concentrations of ATP and peptide to allow identification of diverse inhibitor types. Reconfirmation of hits and IC 50 determinations were carried out with the TR-FRET assay and by a radiometric assay using recombinant histone H3 as the substrate. A preliminary assessment of specificity was made by testing inhibition of 2 unrelated kinases. EC 50 values in cells were determined using a cell-based ELISA of H3T3ph. Five compounds were selected as leads based on potency and chemical structure considerations. These leads form the basis for the development of specific inhibitors of haspin that will have clear utility in basic research and possible use as starting points for development of antimitotic anticancer therapeutics. ( Journal of Biomolecular Screening 2008:1025-1034)

Fluorescence resonance energy transfer (FRET) microscopy in living cells as a novel tool for the study of cytokine action

2005 ◽  
Vol 72 (S1) ◽  
pp. 14-19 ◽  
Author(s):  
Arieh Gertler ◽  
Eva Biener ◽  
Krishnan V. Ramanujan ◽  
Jean Djiane ◽  
Brian Herman
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Receptor Internalization ◽  
Placental Lactogen ◽  
Hormonal Stimulation ◽  
Intact Cells ◽  
Fluorescence Resonance

Fluorescence resonance energy transfer (FRET) microscopy was used to study interactions between proteins in intact cells. We showed that growth hormone (GH) causes transient homodimerization of GH receptors tagged with yellow or cyan fluorescent proteins. The peak of FRET signaling occurred 2 to 4 min after hormonal stimulation and was followed by a decrease in FRET signal. Repeating those experiments in cells pretreated with the inhibitor of internalization methyl-β-cyclodextrin, or in potassium-depleted cells showed no difference in the kinetics of FRET signaling as compared with the non-treated cells, indicating that the decrease in FRET signal does not result from receptor internalization by the pathways inhibited by methyl-β-cyclodextrin or potassium depleted but might occur by other pathways of internalization. Using a similar methodology, we also demonstrated that ovine placental lactogen (oPL) causes transient heterodimerization of GH and prolactin (PRL) receptors 2·5 to 3 min after oPL application. On the other hand, oGH or oPRL had no effect at all, further substantiating the finding the oPL, which lacks a specific receptor, acts in homologous systems by heterodimerization of GH and PRL receptors. We also demonstrated that both PRL and leptin (LEP) are capable of transactivation of the oncogenic receptors erbB2 and erbB3. Upon PRL or LEP stimulation of HEK-293T cells transfected with LEP or PRL receptors and erbB2 or erbB3, erbB proteins are first phosphorylated and then activate MAPK (erk1/erk2). However, the FRET experiments failed to document any evidence of a direct interaction between erbB2 and the PRL or LEP receptors, suggesting that erbB activation probably occurs via activated JAK2, translocated from the respective receptors to erbB2.

scholarly journals Oligomeric Size of the M2Muscarinic Receptor in Live Cells as Determined by Quantitative Fluorescence Resonance Energy Transfer

2010 ◽  
Vol 285 (22) ◽  
pp. 16723-16738 ◽  
Author(s):  
Luca F. Pisterzi ◽  
David B. Jansma ◽  
John Georgiou ◽  
Michael J. Woodside ◽  
Judy Tai-Chieh Chou ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Resonance ◽  
Quantitative Fluorescence

Fluorescence resonance energy transfer as a method for dissecting in vivo mechanisms of transcriptional activation

2006 ◽  
Vol 73 ◽  
pp. 217-224 ◽  
Author(s):  
Sara K. Evans ◽  
David P. Aiello ◽  
Michael R. Green
Keyword(s):  
Energy Transfer ◽  
Rna Polymerase Ii ◽  
Fluorescence Resonance Energy Transfer ◽  
Transcriptional Activation ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Direct Interaction ◽  
Fluorescence Resonance

The first step in transcriptional activation of protein-coding genes involves the assembly on the promoter of a large PIC (pre-initiation complex) comprising RNA polymerase II and a suite of general transcription factors. Transcription is greatly enhanced by the action of promoter-specific activator proteins (activators) that function, at least in part, by increasing PIC formation. Activator-mediated stimulation of PIC assembly is thought to result from a direct interaction between the activator and one or more components of the transcription machinery, termed the ‘target’. The unambiguous identification of direct, physiologically relevant in vivo targets of activators has been a considerable challenge in the transcription field. The major obstacle has been the lack appropriate experimental methods to measure direct interactions with activators in vivo. The development of spectral variants of green fluorescent protein has made it possible to perform FRET (fluorescence resonance energy transfer) analysis in living cells, thereby allowing the detection of direct protein–protein interactions in vivo. Here we discuss how FRET can be used to identify activator targets and to dissect in vivo mechanisms of transcriptional activation.

scholarly journals Highly-efficient quantitative fluorescence resonance energy transfer measurements based on deep learning

2020 ◽  
Vol 13 (06) ◽  
pp. 2050021
Author(s):  
Lin Ge ◽  
Fei Liu ◽  
Jianwen Luo
Keyword(s):  
Energy Transfer ◽  
Deep Learning ◽  
Fluorescence Resonance Energy Transfer ◽  
High Efficiency ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Optical Diffraction ◽  
Fluorescence Resonance ◽  
Quantitative Fluorescence ◽  
Image Analyses

Intensity-based quantitative fluorescence resonance energy transfer (FRET) is a technique to measure the distance of molecules in scale of a few nanometers which is far beyond optical diffraction limit. This widely used technique needs complicated experimental process and manual image analyses to obtain precise results, which take a long time and restrict the application of quantitative FRET especially in living cells. In this paper, a simplified and automatic quantitative FRET (saqFRET) method with high efficiency is presented. In saqFRET, photoactivatable acceptor PA-mCherry and optimized excitation wavelength of donor enhanced green fluorescent protein (EGFP) are used to simplify FRET crosstalk elimination. Traditional manual image analyses are time consuming when the dataset is large. The proposed automatic image analyses based on deep learning can analyze 100 samples within 30[Formula: see text]s and demonstrate the same precision as manual image analyses.

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