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.

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

2018 ◽  
Author(s):  
Jieqiong Lou ◽  
Lorenzo Scipioni ◽  
Belinda K. Wright ◽  
Tara K. Bartolec ◽  
Jessie Zhang ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Strand Break ◽  
Double Strand Break ◽  
Image Correlation ◽  
Chromatin Dynamics ◽  
Chromatin Compaction ◽  
Chromatin Architecture ◽  
Damage Response ◽  
Nuclear Environment

AbstractTo investigate how chromatin architecture is spatiotemporally organised 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 (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.SIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

scholarly journals Streamlined histone-based fluorescence lifetime imaging microscopy reveals ATM regulation of chromatin compaction

2017 ◽  
Author(s):  
Alice Sherrard ◽  
Paul Bishop ◽  
Melanie Panagi ◽  
Maria Beatriz Villagomez ◽  
Dominic Alibhai ◽  
...  
Keyword(s):  
Dna Damage ◽  
Fluorescence Lifetime ◽  
Genotoxic Stress ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging Microscopy ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
Lifetime Imaging ◽  
Detailed Method ◽  
Chromatin Organisation

AbstractChanges in chromatin compaction are crucial during genomic responses. Thus, methods that enable such measurements are instrumental for investigating genome function. Here, we address this challenge by developing, validating, and streamlining histone-based fluorescence lifetime imaging microscopy (FLIM) that robustly detects chromatin compaction states in fixed and live cells; in 2D and 3D. We present quality-controlled and detailed method that is simpler and faster than previous approches, and uses FLIMfit open-source software. We demonstrate the versatility of our method through its combination with immunofluorescence and its implementation in immortalised cells and primary neurons. Owing to these developments, we applied this method to elucidate the function of the DNA damage response kinase, ATM, in regulating chromatin organisation after genotoxic-stress. We unravelled a role for ATM in regulating chromatin compaction independently of DNA damage. Collectively, we present an adaptable chromatin FLIM method for examining chromatin structure in cells, and establish its broader utility.

Dynamics of intracellular neonatal Fc receptor-ligand interactions in primary macrophages using biophysical fluorescence techniques

2021 ◽  
Author(s):  
Andreas Pannek ◽  
Fiona J. Houghton ◽  
Anne M. Verhagen ◽  
Steven K. Dower ◽  
Elizabeth Hinde ◽  
...  
Keyword(s):  
Fluorescent Microscopy ◽  
High Mobility ◽  
Resonance Energy ◽  
Fc Receptor ◽  
Correlation Spectroscopy ◽  
Image Correlation ◽  
Fluorescence Lifetime Imaging ◽  
Neonatal Fc Receptor ◽  
Ligand Interactions ◽  
Kinetics Of

The neonatal Fc receptor (FcRn) is responsible for the recycling of endocytosed albumin and IgG and contributes to their long plasma half-life. We recently identified a FcRn-dependent, recycling pathway from macropinosomes in macrophages (Toh et al, 2019), however, little is known about the dynamics of intracellular FcRn-ligand interactions to promote recycling. Here we demonstrate a multiplexed biophysical fluorescent microscopy approach to resolve the spatiotemporal dynamics of albumin-FcRn interactions in living bone marrow-derived macrophages (BMDMs). We used the phasor approach to fluorescence lifetime imaging microscopy (FLIM) of Förster resonance energy transfer (FRET) to detect the interaction of a FcRn-mCherry fusion protein with endocytosed Alexa Fluor 488-labelled human serum albumin (HSA-AF488) in BMDMs, and Raster Image Correlation Spectroscopy (RICS) analysis of single fluorescent-labelled albumin molecules to monitor the diffusion kinetics of internalised albumin. Our data identified a major fraction of immobile HSA-AF488 molecules in endosomal structures of human FcRn-positive mouse macrophages and an increase in FLIM- FRET following endocytosis, including detection of FRET in tubular-like structures. A non-binding mutant of albumin showed minimum FLIM-FRET and high mobility. These data reveal the kinetics of FcRn-ligand binding within endosomal structures for recruitment into transport carriers for recycling. These approaches have wide applicability for analyses of intracellular ligand-receptor interactions.

scholarly journals SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

2020 ◽  
Vol 48 (17) ◽  
pp. 10013-10014
Author(s):  
Andreas Mund ◽  
Tobias Schubert ◽  
Hannah Staege ◽  
Sarah Kinkley ◽  
Kerstin Reumann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Chromatin Compaction ◽  
Damage Response

scholarly journals Global chromatin compaction limits the strength of the DNA damage response

2007 ◽  
Vol 178 (7) ◽  
pp. 1101-1108 ◽  
Author(s):  
Matilde Murga ◽  
Isabel Jaco ◽  
Yuhong Fan ◽  
Rebeca Soria ◽  
Barbara Martinez-Pastor ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Histone Deacetylase Inhibitors ◽  
Embryonic Stem ◽  
Open Chromatin ◽  
Dna Breaks ◽  
Chromatin Compaction ◽  
Damage Response ◽  
Cell Variation ◽  
The Impact

In response to DNA damage, chromatin undergoes a global decondensation process that has been proposed to facilitate genome surveillance. However, the impact that chromatin compaction has on the DNA damage response (DDR) has not directly been tested and thus remains speculative. We apply two independent approaches (one based on murine embryonic stem cells with reduced amounts of the linker histone H1 and the second making use of histone deacetylase inhibitors) to show that the strength of the DDR is amplified in the context of “open” chromatin. H1-depleted cells are hyperresistant to DNA damage and present hypersensitive checkpoints, phenotypes that we show are explained by an increase in the amount of signaling generated at each DNA break. Furthermore, the decrease in H1 leads to a general increase in telomere length, an as of yet unrecognized role for H1 in the regulation of chromosome structure. We propose that slight differences in the epigenetic configuration might account for the cell-to-cell variation in the strength of the DDR observed when groups of cells are challenged with DNA breaks.

scholarly journals SPOC1 modulates DNA repair by regulating key determinants of chromatin compaction and DNA damage response

2012 ◽  
Vol 40 (22) ◽  
pp. 11363-11379 ◽  
Author(s):  
Andreas Mund ◽  
Tobias Schubert ◽  
Hannah Staege ◽  
Sarah Kinkley ◽  
Kerstin Reumann ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Dna Damage Response ◽  
Chromatin Compaction ◽  
Damage Response

scholarly journals Chromatin nanoscale compaction in live cells visualized by acceptor-donor ratio corrected FRET between DNA dyes

2019 ◽  
Author(s):  
Simone Pelicci ◽  
Alberto Diaspro ◽  
Luca Lanzanò
Keyword(s):  
Dna Damage ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Donor And Acceptor ◽  
Dna Dyes

AbstractChromatin nanoscale architecture in live cells can be studied by Forster Resonance Energy Transfer (FRET) between fluorescently labeled chromatin components, such as histones. A higher degree of nanoscale compaction is detected as a higher FRET level, since this corresponds to a higher degree of proximity between donor and acceptor molecules. However, in such a system the stoichiometry of the donors and acceptors engaged in the FRET process is not well defined and, in principle, FRET variations could be caused by variations in the acceptor-donor ratio rather than distance. Here we show that a FRET value independent of the acceptor-donor ratio can be obtained by Fluorescence Lifetime Imaging (FLIM) detection of FRET combined with a normalization of the FRET level to a pixel-wise estimation of the acceptor-donor ratio. We use this method to study FRET between two DNA binding dyes staining the nuclei of live cells. We show that acceptor-donor ratio corrected FRET imaging reveals variations of nanoscale compaction in different chromatin environments. As an application, we monitor the rearrangement of chromatin in response to laser-induced micro-irradiation and reveal that DNA is rapidly decompacted, at the nanoscale, in response to DNA damage induction.

scholarly journals Direct measurement of protein–protein interactions by FLIM-FRET at UV laser-induced DNA damage sites in living cells

2020 ◽  
Vol 48 (21) ◽  
pp. e122-e122
Author(s):  
Tanja Kaufmann ◽  
Sébastien Herbert ◽  
Benjamin Hackl ◽  
Johanna Maria Besold ◽  
Christopher Schramek ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cell ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy ◽  
Population Level ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Uv Laser ◽  
Protein Protein Interactions

Abstract Protein–protein interactions are essential to ensure timely and precise recruitment of chromatin remodellers and repair factors to DNA damage sites. Conventional analyses of protein–protein interactions at a population level may mask the complexity of interaction dynamics, highlighting the need for a method that enables quantification of DNA damage-dependent interactions at a single-cell level. To this end, we integrated a pulsed UV laser on a confocal fluorescence lifetime imaging (FLIM) microscope to induce localized DNA damage. To quantify protein–protein interactions in live cells, we measured Förster resonance energy transfer (FRET) between mEGFP- and mCherry-tagged proteins, based on the fluorescence lifetime reduction of the mEGFP donor protein. The UV-FLIM-FRET system offers a unique combination of real-time and single-cell quantification of DNA damage-dependent interactions, and can distinguish between direct protein–protein interactions, as opposed to those mediated by chromatin proximity. Using the UV-FLIM-FRET system, we show the dynamic changes in the interaction between poly(ADP-ribose) polymerase 1, amplified in liver cancer 1, X-ray repair cross-complementing protein 1 and tripartite motif containing 33 after DNA damage. This new set-up complements the toolset for studying DNA damage response by providing single-cell quantitative and dynamic information about protein–protein interactions at DNA damage sites.

scholarly journals Quantitative analysis of chromatin compaction in living cells using FLIM–FRET

2009 ◽  
Vol 187 (4) ◽  
pp. 481-496 ◽  
Author(s):  
David Llères ◽  
John James ◽  
Sam Swift ◽  
David G. Norman ◽  
Angus I. Lamond
Keyword(s):  
Fluorescent Protein ◽  
Trichostatin A ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Human Cell Line ◽  
Live Cells ◽  
Fluorescence Lifetime Imaging ◽  
Chromatin Compaction ◽  
Fret Efficiency ◽  
Green Fluorescent

We present a quantitative Förster resonance energy transfer (FRET)–based assay using multiphoton fluorescence lifetime imaging microscopy (FLIM) to measure chromatin compaction at the scale of nucleosomal arrays in live cells. The assay uses a human cell line coexpressing histone H2B tagged to either enhanced green fluorescent protein (FP) or mCherry FPs (HeLaH2B-2FP). FRET occurs between FP-tagged histones on separate nucleosomes and is increased when chromatin compacts. Interphase cells consistently show three populations of chromatin with low, medium, or high FRET efficiency, reflecting spatially distinct regions with different levels of chromatin compaction. Treatment with inhibitors that either increase chromatin compaction (i.e., depletion of adenosine triphosphate) or decrease chromosome compaction (trichostatin A) results in a parallel increase or decrease in the FLIM–FRET signal. In mitosis, the assay showed variation in compaction level, as reflected by different FRET efficiency populations, throughout the length of all chromosomes, increasing to a maximum in late anaphase. These data are consistent with extensive higher order folding of chromatin fibers taking place during anaphase.

scholarly journals FRET-Based Sorting of Live Cells Reveals Shifted Balance between PLK1 and CDK1 Activities During Checkpoint Recovery

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2126
Author(s):  
Lorenzo Lafranchi ◽  
Erik Müllers ◽  
Dorothea Rutishauser ◽  
Arne Lindqvist
Keyword(s):  
Dna Damage ◽  
Dna Damage Response ◽  
Cyclin B1 ◽  
Live Cells ◽  
Dependent Manner ◽  
Mitotic Entry ◽  
Protein Levels ◽  
Partial Inhibition ◽  
Damage Response ◽  
G2 Phase

Cells recovering from the G2/M DNA damage checkpoint rely more on Aurora A-PLK1 signaling than cells progressing through an unperturbed G2 phase, but the reason for this discrepancy is not known. Here, we devised a method based on a FRET reporter for PLK1 activity to sort cells in distinct populations within G2 phase. We employed mass spectroscopy to characterize changes in protein levels through an unperturbed G2 phase and validated that ATAD2 levels decrease in a proteasome-dependent manner. Comparing unperturbed cells with cells recovering from DNA damage, we note that at similar PLK1 activities, recovering cells contain higher levels of Cyclin B1 and increased phosphorylation of CDK1 targets. The increased Cyclin B1 levels are due to continuous Cyclin B1 production during a DNA damage response and are sustained until mitosis. Whereas partial inhibition of PLK1 suppresses mitotic entry more efficiently when cells recover from a checkpoint, partial inhibition of CDK1 suppresses mitotic entry more efficiently in unperturbed cells. Our findings provide a resource for proteome changes during G2 phase, show that the mitotic entry network is rewired during a DNA damage response, and suggest that the bottleneck for mitotic entry shifts from CDK1 to PLK1 after DNA damage.

Sign in / Sign up

Export Citation Format

Share Document