scholarly journals Dual-color Fluorescence Cross-Correlation Spectroscopy to study Protein-Protein Interaction and Protein Dynamics in Live Cells

2021 ◽  
Author(s):  
Katherina Hemmen ◽  
Susobhan Choudhury ◽  
Mike Friedrich ◽  
Johannes Balkenhol ◽  
Felix Knote ◽  
...  
Keyword(s):  
Conformational Changes ◽  
High Reliability ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Correlation Spectroscopy ◽  
Live Cell ◽  
Fluorescence Labeling ◽  
Live Cells ◽  
Noise Levels ◽  
Dual Color

We present a protocol and workflow to perform live cell dual-color fluorescence crosscorrelation spectroscopy (FCCS) combined with Förster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represents the dynamical "fingerprint" of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known "nanoruler" to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations, and mobility constants become accessible in cellular settings. Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerable sample itself. We will show how to perform the experiments including the calibration steps. We use dual-color labeled β2-adrenergic receptor (β2AR) labeled (eGFP and SNAPtag-TAMRA). We will guide you step-by-step through the data analysis procedure using open-source software and provide templates that are easy to customize. Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis near-physiological conditions.

scholarly journals Fluorescent Probes for Live Cell Thiol Detection

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3575
Author(s):  
Shenggang Wang ◽  
Yue Huang ◽  
Xiangming Guan
Keyword(s):  
Fluorescent Probes ◽  
Biological System ◽  
High Sensitivity ◽  
Intracellular Distribution ◽  
Live Cell ◽  
Live Cells ◽  
High Demand ◽  
Non Invasive

Thiols play vital and irreplaceable roles in the biological system. Abnormality of thiol levels has been linked with various diseases and biological disorders. Thiols are known to distribute unevenly and change dynamically in the biological system. Methods that can determine thiols’ concentration and distribution in live cells are in high demand. In the last two decades, fluorescent probes have emerged as a powerful tool for achieving that goal for the simplicity, high sensitivity, and capability of visualizing the analytes in live cells in a non-invasive way. They also enable the determination of intracellular distribution and dynamitic movement of thiols in the intact native environments. This review focuses on some of the major strategies/mechanisms being used for detecting GSH, Cys/Hcy, and other thiols in live cells via fluorescent probes, and how they are applied at the cellular and subcellular levels. The sensing mechanisms (for GSH and Cys/Hcy) and bio-applications of the probes are illustrated followed by a summary of probes for selectively detecting cellular and subcellular thiols.

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

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.

scholarly journals Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

2007 ◽  
Vol 176 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Dawen Cai ◽  
Adam D. Hoppe ◽  
Joel A. Swanson ◽  
Kristen J. Verhey
Keyword(s):  
Conformational Change ◽  
Conformational Changes ◽  
Structural Organization ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Protein Activity ◽  
Kinesin Light Chain ◽  
Initial Interaction

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

scholarly journals Structural rearrangement of the intracellular domains during AMPA receptor activation

2016 ◽  
Vol 113 (27) ◽  
pp. E3950-E3959 ◽  
Author(s):  
Linda G. Zachariassen ◽  
Ljudmila Katchan ◽  
Anna G. Jensen ◽  
Darryl S. Pickering ◽  
Andrew J. R. Plested ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy ◽  
Normal Activity ◽  
Receptor Activation ◽  
Hek293 Cells ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Receptor Function ◽  
Loop Region ◽  
Intracellular Domains

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are ligand-gated ion channels that mediate the majority of fast excitatory neurotransmission in the central nervous system. Despite recent advances in structural studies of AMPARs, information about the specific conformational changes that underlie receptor function is lacking. Here, we used single and dual insertion of GFP variants at various positions in AMPAR subunits to enable measurements of conformational changes using fluorescence resonance energy transfer (FRET) in live cells. We produced dual CFP/YFP-tagged GluA2 subunit constructs that had normal activity and displayed intrareceptor FRET. We used fluorescence lifetime imaging microscopy (FLIM) in live HEK293 cells to determine distinct steady-state FRET efficiencies in the presence of different ligands, suggesting a dynamic picture of the resting state. Patch-clamp fluorometry of the double- and single-insert constructs showed that both the intracellular C-terminal domain (CTD) and the loop region between the M1 and M2 helices move during activation and the CTD is detached from the membrane. Our time-resolved measurements revealed unexpectedly complex fluorescence changes within these intracellular domains, providing clues as to how posttranslational modifications and receptor function interact.

scholarly journals Spatiotemporal imaging of small GTPases activity in live cells

2016 ◽  
Vol 113 (50) ◽  
pp. 14348-14353 ◽  
Author(s):  
Stephanie Voss ◽  
Dennis M. Krüger ◽  
Oliver Koch ◽  
Yao-Wen Wu
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Small Gtpases ◽  
Small Gtpase ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Specific Effector ◽  
Spatiotemporal Visualization ◽  
Cell Current

Ras-like small GTPases function as molecular switches and regulate diverse cellular events. To examine the dynamics of signaling requires spatiotemporal visualization of their activity in the cell. Current small GTPase sensors rely on specific effector domains that are available for only a small number of GTPases and compete for endogenous regulator/effector binding. Here, we describe versatile conformational sensors for GTPase activity (COSGAs) based on the conserved GTPase fold. Conformational changes upon GDP/GTP exchange were directly observed in solution, on beads, and in live cells by Förster resonance energy transfer (FRET). The COSGAs allow for monitoring of Rab1 and K-Ras activity in live cells using fluorescence lifetime imaging microscopy. We found that Rab1 is largely active in the cytoplasm and inactive at the Golgi, suggesting that the Golgi serves as the terminal of the Rab1 functional cycle. K-Ras displays polarized activity at the plasma membrane, with less activity at the edge of the cell and membrane ruffles.

Light up ClO−in live cells using an aza-coumarin based fluorescent probe with fast response and high sensitivity

The Analyst ◽  
2015 ◽  
Vol 140 (13) ◽  
pp. 4594-4598 ◽  
Author(s):  
Jiangli Fan ◽  
Huiying Mu ◽  
Hao Zhu ◽  
Jingyun Wang ◽  
Xiaojun Peng
Keyword(s):  
Fluorescent Probe ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
High Sensitivity ◽  
Fast Response ◽  
Live Cell ◽  
Live Cells

An aza-coumarin based fluorescent and colorimetricAC-ClOfor the ClO−determination with fast response and high sensitivity.AC-ClOwas successfully applied for the live-cell imaging of exogenous and endogenous ClO−.

scholarly journals Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response

2019 ◽  
Vol 116 (15) ◽  
pp. 7323-7332 ◽  
Author(s):  
Jieqiong Lou ◽  
Lorenzo Scipioni ◽  
Belinda K. Wright ◽  
Tara K. Bartolec ◽  
Jessie Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Resonance Energy ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
Chromatin Architecture ◽  
Damage Response

To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia–telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.

Luminescence Energy Transfer–Based Screening and Target Engagement Approaches for Chemical Biology and Drug Discovery

2021 ◽  
pp. 247255522110360
Author(s):  
Eun Jeong Cho ◽  
Kevin N. Dalby
Keyword(s):  
Energy Transfer ◽  
Drug Discovery ◽  
Early Stage ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
High Sensitivity ◽  
Live Cells ◽  
Molecular Binding ◽  
Compound Screening ◽  
Homogeneous Assay

Luminescence is characterized by the spontaneous emission of light resulting from either chemical or biological reactions. Because of their high sensitivity, reduced background interference, and applicability to numerous situations, luminescence-based assay strategies play an essential role in early-stage drug discovery. Newer developments in luminescence-based technologies have dramatically affected the ability of researchers to investigate molecular binding events. At the forefront of these developments are the nano bioluminescence resonance energy transfer (NanoBRET) and amplified luminescent proximity homogeneous assay (Alpha) technologies. These technologies have opened up numerous possibilities for analyzing the molecular biophysical properties of complexes in environments such as cell lysates. Moreover, NanoBRET enables the validation and quantitation of the interactions between therapeutic targets and small molecules in live cells, representing an essential benchmark for preclinical drug discovery. Both techniques involve proximity-based luminescence energy transfer, in which excited-state energy is transferred from a donor to an acceptor, where the efficiency of transfer depends on proximity. Both approaches can be applied to high-throughput compound screening in biological samples, with the NanoBRET assay providing opportunities for live-cell screening. Representative applications of both technologies for assessing physical interactions and associated challenges are discussed.

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