Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data

Liang, Zhen; Lou, Jieqiong; Scipioni, Lorenzo; Gratton, Enrico; Hinde, Elizabeth

doi:10.1016/j.dib.2020.105401

Cited by 16 publications

(13 citation statements)

References 12 publications

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“…These methods differ in calculation speed, minimal number of photons required for proper analysis, accuracy, complexity and type of data that can be efficiently processed. Techniques like the minimal fraction of interacting donor 34 or phasor approach 35,36,37 have the potential to perform high speed acquisitions in FRET-FLIM and still be quantitative to process large amount of data or even to reach video-rate speeds. The requirement of constructing fluorescently labelled protein that do not perturb the native functions of the proteins in cells is a major concern for scaling up the speed and the number of PPI explored.…”

Section: Discussionmentioning

confidence: 99%

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Manko

Normant

Perraud

et al. 2020

JoVE

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Section: Discussionmentioning

confidence: 99%

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Manko

Normant

Perraud

et al. 2020

JoVE

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“…FRET was quantified within each donor FLIM image by the phasor approach to lifetime analysis, where, as described in previously published papers, the donor fluorescence lifetime recorded in each pixel of a FLIM image is described by a g and s coordinate (phasor) presented in a phasor plot 27 – 29 . In pixels where donor molecules undergo FRET with acceptor molecules, the phasor coordinate is right shifted along a curved trajectory that is described by the classical definition of FRET efficiency 42 , 43 . To determine the efficiency of the FRET state, the phasor coordinates of the unquenched donor and background autofluorescence were first determined independently and then a FRET trajectory was extrapolated from this baseline.…”

Section: Methodsmentioning

confidence: 99%

Live cell dynamics of the NF-Y transcription factor

Priest

Bernardini

Lou

et al. 2021

Sci Rep

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Transcription factors (TFs) are core players in the control of gene expression, evolutionarily selected to recognise a subset of specific DNA sequences and nucleate the recruitment of the transcriptional machinery. How TFs assemble and move in the nucleus to locate and bind their DNA targets and cause a transcriptional response, remains mostly unclear. NF-Y is a highly conserved, heterotrimeric TF with important roles in both housekeeping and lineage-specific gene expression, functioning as a promoter organiser. Despite a large number of biochemical, structural and genomic studies of NF-Y, there is a lack of experiments in single living cells; therefore, basic assumptions of NF-Y biology remain unproven in vivo. Here we employ a series of dynamic fluorescence microscopy methods (FLIM-FRET, NB, RICS and FRAP) to study NF-Y dynamics and complex formation in live cells. Specifically, we provide quantitative measurement of NF-Y subunit association and diffusion kinetics in the nucleus that collectively suggest NF-Y to move and bind chromatin as a trimeric complex in vivo.

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“…1 A) microscope, which enables selective imaging of the plasma membrane of living cells, and the same dataset was analyzed with a combination of state-of-the-art fluorescence fluctuation spectroscopy (FFS) techniques. FFS techniques can be variously used to obtain important biophysical parameters such as protein size and concentration 38 – 43 , lateral diffusion coefficient 44 – 47 and diffusion modality 46 , 48 , spatiotemporal heterogeneity in FRET 49 , 50 and have also been applied in combination with super-resolution techniques 51 – 55 . In this work, we applied image-derived mean square displacement 56 , 57 (iMSD, Fig.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

Tedeschi

Scipioni

Papanikolaou

et al. 2021

Sci Rep

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Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the subunit stoichiometry and forces directing localization and diffusion of ion channel complexes in general.

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Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data

Cited by 16 publications

References 12 publications

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

Live cell dynamics of the NF-Y transcription factor

Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry

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