Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

Karpova, Tatiana S.; Baumann, Chris; He, Lin; Wu, Xiaoli; Grammer, Amrie C.; Lipsky, Peter E.; Hager, Gordon L.; McNally, James G.

doi:10.1046/j.1365-2818.2003.01100.x

Cited by 286 publications

(335 citation statements)

References 37 publications

Supporting

Mentioning

320

Contrasting

Unclassified

Order By: Relevance

“…As expected, the control reaction, in which the reducing step was omitted, did not produce any measurable signal (figure 8c). These findings were validated when FRET was analyzed through the acceptor photobleaching approach [23] (figure 9). Identical results were obtained when MIANS and FITC -two fluorophores similar to CPM and Alexa488 -were used to label respectively thiols and the antibody (data not shown).…”

Section: Resultsmentioning

confidence: 65%

A FRET-based method to study protein thiol oxidation in histological preparations

Mastroberardino

Orr

et al. 2008

Free Radical Biology and Medicine

View full text Add to dashboard Cite

Cysteine residues in proteins have important biological roles. For example, disulfides bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique -which can be used in association with standard immunological staining procedures -to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool to provide further understanding of oxidative imbalance, a phenomenon common to many diseases. Keywords fluorescence microscopy; FRET; oxidative stress; cysteine; thiols; disulfides; Parkinson's disease Thiols are the functional groups in the side chain of the amino acid, cysteine. Depending upon the oxido-reductive (redox) status of the surrounding environment, thiols exist either in the reduced form of free thiols (-SH) or oxidized to disulfides (-S-S-). Disulfides are well known to play an important structural role, contributing to the maintenance of the proper folding in proteins. More recently it was found that the formation of disulfides in proteins also exerts critical regulatory functions: vital mechanisms such as protein import, regulation of signal transduction cascades, regulation of the activity of transcription factors and proper function of the mitochondrial electron transport system rely on disulfide oxidation or reduction [1][2][3][4]. Moreover, the formation of disulfides represents an early, reversible response to oxidative *Corresponding author: mastroberardinopg@upmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Increasing interest in the study of thiols has led to the development of a variety of technical approaches to detect thiols and disulfides. In particular, several spectrophotometry-based protocols were developed to measure t...

show abstract

Section: Resultsmentioning

confidence: 65%

A FRET-based method to study protein thiol oxidation in histological preparations

Mastroberardino

Orr

et al. 2008

Free Radical Biology and Medicine

View full text Add to dashboard Cite

show abstract

“…FRET was measured in HeLa cells expressing YIC using the acceptor photobleaching method (Karpova et al, 2003). Bleaching of the YFP acceptor causes dequenching of the CFP donor, which allows a measurement of FRET efficiency (Supplementary Figure S1).…”

Section: Sorbitol Stress Causes a Defect In Nuclear Import Of A Fluormentioning

confidence: 99%

“…The bleach of the yellow fluorescent protein (YFP) was performed between images 5 and 6, such that FRET efficiency is an expression of the percent dequenching of the cyan fluorescent protein (CFP) acceptor (Karpova et al, 2003). Each time point is representative of 10 cells.…”

Section: Image Quantitationmentioning

confidence: 99%

“…If RanGTP production is reduced, import complexes formed in the cytoplasm will fail to dissociate in the nucleus, because there is insufficient RanGTP. Undissociated importin-␣/␤ complexes in the nucleus would, therefore, decrease the available importin-␤ and increase FRET.FRET was measured in HeLa cells expressing YIC using the acceptor photobleaching method (Karpova et al, 2003). Bleaching of the YFP acceptor causes dequenching of the CFP donor, which allows a measurement of FRET efficiency (Supplementary Figure S1).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Kelley

Paschal

2007

MBoC

View full text Add to dashboard Cite

The RanGTP gradient depends on nucleocytoplasmic shuttling of Ran and its nucleotide exchange in the nucleus. Here we show that hyperosmotic stress signaling induced by sorbitol disrupts the Ran protein gradient and reduces the production of RanGTP. Ran gradient disruption is rapid and is followed by early (10 -20 min) and late (30 -60 min) phases of recovery. Results from SB203580 and siRNA experiments suggest the stress kinase p38 is important for Ran gradient recovery. NTF2 and Mog1, which are transport factors that regulate the nuclear localization of Ran, showed kinetics of delocalization and recovery similar to Ran. Microinjection of a nuclear localization signal reporter protein revealed that sorbitol stress decreases the rate of nuclear import. Sorbitol stress also slowed RCC1 mobility in the nucleus, which is predicted to reduce RCC1 dissociation from chromatin and RanGTP production. This was tested using a FRET biosensor that registers nuclear RanGTP levels, which were reduced in response to sorbitol stress. Although sorbitol alters nucleotide levels, we show that inverting the GTP/GDP ratio in cells is not sufficient to disrupt the Ran gradient. Thus, the Ran system is a target of hyperosmotic stress signaling, and cells use protein localization-based mechanisms as part of a rapid stress response. INTRODUCTIONCells subjected to UV radiation or oxidative, mechanical, or aniso-osmotic stress undergo both short-and long-term adaptation. Hyperosmotic stress induces rapid dehydration that drives an increase in intracellular salt concentration and a decrease in cell volume, placing physical strain on both the cytoskeleton and the plasma membrane (Lang et al., 1998). In response to these changes, cells rapidly initiate a stress signaling cascade and attempt to restore iso-osmolarity through transport of inorganic ions and initiate an accompanying reorganization of the actin cytoskeleton (Haussinger, 1996;Di Ciano et al., 2002). Short-term recovery of cell volume is facilitated by increasing intracellular concentrations of inorganic ions; however, elevated levels of intracellular salts can be detrimental to protein structure and function (Yancey et al., 1982;Russo et al., 2003). Long-term adaptation to hyperosmotic stress is achieved through signal transduction pathways that communicate with the metabolic and transcriptional machinery, resulting in the production or accumulation of organic osmolytes that increase intracellular osmolarity without adversely affecting protein structure or function (O'Neill, 1999).One of the most thoroughly studied stress kinases is the mitogen-activated protein kinase (MAPK) p38. p38 and its yeast homologue Hog1p are activated in response to a wide variety of adverse environmental conditions. These include osmotic, UV, and mechanical stress. Cytokines, such as interleukins and tumor necrosis factor ␣ are also known to activate p38 (Ono and Han, 2000). Hog1p and p38 are both phosphorylated on a threonine, glycine, tyrosine (TGY) motif within the kinase activation loop (Thr 180 an...

show abstract

“…To the best of our knowledge, successful determination of a has not been reported for VFP-expressing cells using flow cytometry. A popular approach for the determination of FRET efficiency is based on the release of donor quenching upon photodestruction of the acceptor (20,21). Although this method has been applied to conventional fluorophores (20) and VFP-expressing cells (21), it cannot be used in flow cytometry because detection of donor fluorescence intensity before and after acceptor photobleaching is usually not feasible.…”

mentioning

confidence: 99%

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins

Nagy

Bene

Hyun³

et al. 2005

Cytometry Pt A

View full text Add to dashboard Cite

Background: The combination of fluorescence resonance energy transfer (FRET) and flow cytometry offers a statistically firm approach to study protein associations. Fusing green fluorescent protein (GFP) to a studied protein usually does not disturb the normal function of a protein, but quantitation of FRET efficiency calculated between GFP derivatives poses a problem in flow cytometry. Methods: We generated chimeras in which cyan fluorescent protein (CFP) was separated by amino acid linkers of different sizes from yellow fluorescent protein (YFP) and used them to calibrate the cell-by-cell flow cytometric FRET measurements carried out on two different duallaser flow cytometers. Then, CFP-Kip1 was coexpressed in yeast cells with YFP and cyclin-dependent kinase-2 (Cdk2) and served as a positive control for FRET measurements, and CFP-Kip1 coexpressed with a random peptide fused to YFP was the negative control. Results: We measured donor, direct, and sensitized acceptor fluorescence intensities and developed a novel

show abstract

Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser

Cited by 286 publications

References 37 publications

A FRET-based method to study protein thiol oxidation in histological preparations

A FRET-based method to study protein thiol oxidation in histological preparations

Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP

Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins

Contact Info

Product

Resources

About