Ligand-Mediated Assembly and Real-Time Cellular Dynamics of Estrogen Receptor α-Coactivator Complexes in Living Cells

ABSTRACT Studies with live cells demonstrate that agonist and antagonist rapidly (within minutes) modulate the subnuclear dynamics of estrogen receptor α (ER) and steroid receptor coactivator 1 (SRC-1). A functional cyan fluorescent protein (CFP)-taggedlac repressor-ER chimera (CFP-LacER) was used in live cells to discretely immobilize ER on stably integratedlac operator arrays to study recruitment of yellow fluorescent protein (YFP)-steroid receptor coactivators (YFP–SRC-1 and YFP-CREB binding protein [CBP]). In the absence of ligand, YFP–SRC-1 is found dispersed throughout the nucleoplasm, with a surprisingly high accumulation on the CFP-LacER arrays. Agonist addition results in the rapid (within minutes) recruitment of nucleoplasmic YFP–SRC-1, while antagonist additions diminish YFP–SRC-1–CFP-LacER associations. Less ligand-independent colocalization is observed with CFP-LacER and YFP-CBP, but agonist-induced recruitment occurs within minutes. The agonist-induced recruitment of coactivators requires helix 12 and critical residues in the ER–SRC-1 interaction surface, but not the F, AF-1, or DNA binding domains. Fluorescence recovery after photobleaching indicates that YFP–SRC-1, YFP-CBP, and CFP-LacER complexes undergo rapid (within seconds) molecular exchange even in the presence of an agonist. Taken together, these data suggest a dynamic view of receptor-coregulator interactions that is now amenable to real-time study in living cells.

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Ligand-Independent Interactions of p160/Steroid Receptor Coactivators and CREB-Binding Protein (CBP) with Estrogen Receptor-α: Regulation by Phosphorylation Sites in the A/B Region Depends on Other Receptor Domains

Molecular Endocrinology ◽

10.1210/me.2001-0316 ◽

2003 ◽

Vol 17 (7) ◽

pp. 1296-1314 ◽

Cited By ~ 98

Author(s):

Martin Dutertre ◽

Carolyn L. Smith

Keyword(s):

Estrogen Receptor ◽

Binding Protein ◽

Steroid Receptor ◽

Estrogen Receptor Α ◽

Phosphorylation Sites ◽

Creb Binding Protein ◽

Steroid Receptor Coactivators

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Detection of Mitochondrial Caspase Activity in Real Time In Situ in Live Cells

Microscopy and Microanalysis ◽

10.1017/s1431927604040401 ◽

2004 ◽

Vol 10 (4) ◽

pp. 442-448 ◽

Cited By ~ 6

Author(s):

Yingpei Zhang ◽

Catherine Haskins ◽

Marisa Lopez-Cruzan ◽

Jianhua Zhang ◽

Victoria E. Centonze ◽

...

Keyword(s):

Real Time ◽

Caspase 3 ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Subcellular Location ◽

Live Cells ◽

Excitation Peak ◽

Caspase Substrate

Apoptosis plays an important role in many physiological and pathological processes. The initiation and execution of the cell death program requires activation of multiple caspases in a stringently temporal order. Here we describe a method that allows real-time observation of caspase activation in situ in live cells based on fluorescent resonance energy transfer (FRET) measurement using the prism and reflector imaging spectroscopy system (PARISS). When a fusion protein consisting of CFP connected to YFP via an intervening caspase substrate that has been targeted to a specific subcellular location is excited with a light source whose wavelength matches the cyan fluorescent protein (CFP) excitation peak, the energy absorbed by the CFP fluorophore is not emitted as fluorescence. Instead, the excitation energy is absorbed by the nearby yellow fluorescent protein (YFP) fluorophore that is covalently linked to CFP through a short peptide containing the caspase substrate. Cleavage of the linker peptide by caspases results in loss of FRET due to the separation of CFP and YFP fluorophores. Using a mitochondrially targeted CFP–caspase 3 substrate–YFP construct (mC3Y), we demonstrate for the first time that there is caspase-3-like activity in the mitochondrial matrix of some cells at very late stage of apoptosis.

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Subcellular and Subnuclear Distributions of Estrogen Receptor α in Living Cells Using Green Fluorescent Protein and Immunohistochemistry

ACTA HISTOCHEMICA ET CYTOCHEMICA ◽

10.1267/ahc.34.413 ◽

2001 ◽

Vol 34 (6) ◽

pp. 413-422 ◽

Cited By ~ 1

Author(s):

Maki Yoshida ◽

Mayumi Nishi ◽

Zenro Kizaki ◽

Tadashi Sawada ◽

Mitsuhiro Kawata

Keyword(s):

Estrogen Receptor ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Estrogen Receptor Α ◽

Living Cells ◽

Green Fluorescent

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Imaging Analysis of Subcellular Correlation of Androgen Receptor and Estrogen Receptor α in Single Living Cells Using Green Fluorescent Protein Color Variants

Molecular Endocrinology ◽

10.1210/me.2002-0262 ◽

2004 ◽

Vol 18 (1) ◽

pp. 26-42 ◽

Cited By ~ 24

Author(s):

Ikuo Ochiai ◽

Ken-ichi Matsuda ◽

Mayumi Nishi ◽

Hitoshi Ozawa ◽

Mitsuhiro Kawata

Keyword(s):

Estrogen Receptor ◽

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Estrogen Receptor Α ◽

Living Cells ◽

Imaging Analysis ◽

Green Fluorescent ◽

Single Living Cells ◽

Single Living ◽

Color Variants

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Real-time imaging of steroid hormone receptor and steroid receptor coactivator-1 in living cells with color variants of green fluorescent protein

Neuroscience Research ◽

10.1016/s0168-0102(00)81227-4 ◽

2000 ◽

Vol 38 ◽

pp. S63

Author(s):

M Nishi

Keyword(s):

Green Fluorescent Protein ◽

Real Time ◽

Hormone Receptor ◽

Fluorescent Protein ◽

Steroid Hormone ◽

Living Cells ◽

Steroid Hormone Receptor ◽

Steroid Receptor Coactivator ◽

Green Fluorescent ◽

Color Variants

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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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Construction of a Nanosensor for Non-Invasive Imaging of Hydrogen Peroxide Levels in Living Cells

Biology ◽

10.3390/biology9120430 ◽

2020 ◽

Vol 9 (12) ◽

pp. 430

Author(s):

Amreen ◽

Hayssam M. Ali ◽

Mohammad Ahmad ◽

Mohamed Z. M. Salem ◽

Altaf Ahmad

Keyword(s):

Hydrogen Peroxide ◽

Real Time ◽

Mammalian Cells ◽

Dynamic Range ◽

Resonance Energy ◽

Living Cells ◽

Stress Conditions ◽

Live Cells ◽

Cell Physiology ◽

Broad Dynamic Range

Hydrogen peroxide (H2O2) serves fundamental regulatory functions in metabolism beyond the role as damage signal. During stress conditions, the level of H2O2 increases in the cells and causes oxidative stress, which interferes with normal cell growth in plants and animals. The H2O2 also acts as a central signaling molecule and regulates numerous pathways in living cells. To better understand the generation of H2O2 in environmental responses and its role in cellular signaling, there is a need to study the flux of H2O2 at high spatio–temporal resolution in a real-time fashion. Herein, we developed a genetically encoded Fluorescence Resonance Energy Transfer (FRET)-based nanosensor (FLIP-H2O2) by sandwiching the regulatory domain (RD) of OxyR between two fluorescent moieties, namely ECFP and mVenus. This nanosensor was pH stable, highly selective to H2O2, and showed insensitivity to other oxidants like superoxide anions, nitric oxide, and peroxynitrite. The FLIP-H2O2 demonstrated a broad dynamic range and having a binding affinity (Kd) of 247 µM. Expression of sensor protein in living bacterial, yeast, and mammalian cells showed the localization of the sensor in the cytosol. The flux of H2O2 was measured in these live cells using the FLIP-H2O2 under stress conditions or by externally providing the ligand. Time-dependent FRET-ratio changes were recorded, which correspond to the presence of H2O2. Using this sensor, real-time information of the H2O2 level can be obtained non-invasively. Thus, this nanosensor would help to understand the adverse effect of H2O2 on cell physiology and its role in redox signaling.

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