Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Guanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with Fluorescence Lifetime Imaging Microscopy (FLIM) can identify G4 within nuclei of live and fixed cells. We present a new FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4 and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4 in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

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Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy

Nature Communications ◽

10.1038/s41467-020-20414-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peter A. Summers ◽

Benjamin W. Lewis ◽

Jorge Gonzalez-Garcia ◽

Rosa M. Porreca ◽

Aaron H. M. Lim ◽

...

Keyword(s):

Fluorescence Lifetime ◽

Mammalian Cells ◽

Live Cells ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Dna Dynamics ◽

Drug Candidates ◽

Lifetime Imaging ◽

Wide Range

AbstractGuanine rich regions of oligonucleotides fold into quadruple-stranded structures called G-quadruplexes (G4s). Increasing evidence suggests that these G4 structures form in vivo and play a crucial role in cellular processes. However, their direct observation in live cells remains a challenge. Here we demonstrate that a fluorescent probe (DAOTA-M2) in conjunction with fluorescence lifetime imaging microscopy (FLIM) can identify G4s within nuclei of live and fixed cells. We present a FLIM-based cellular assay to study the interaction of non-fluorescent small molecules with G4s and apply it to a wide range of drug candidates. We also demonstrate that DAOTA-M2 can be used to study G4 stability in live cells. Reduction of FancJ and RTEL1 expression in mammalian cells increases the DAOTA-M2 lifetime and therefore suggests an increased number of G4s in these cells, implying that FancJ and RTEL1 play a role in resolving G4 structures in cellulo.

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Faculty Opinions recommendation of Visualising G-quadruplex DNA dynamics in live cells by fluorescence lifetime imaging microscopy.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.739335370.793581858 ◽

2021 ◽

Author(s):

Richard Willson

Keyword(s):

Fluorescence Lifetime ◽

Live Cells ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Dna Dynamics ◽

Quadruplex Dna ◽

Lifetime Imaging ◽

G Quadruplex

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

10.1101/191890 ◽

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.

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Coarse-grained modeling of mitochondrial metabolism enables subcellular flux inference from fluorescence lifetime imaging microscopy

10.1101/2020.11.20.392225 ◽

2020 ◽

Author(s):

Xingbo Yang ◽

Daniel J. Needleman

Keyword(s):

Fluorescence Lifetime ◽

Metabolic Flux ◽

Living Cells ◽

Coarse Grained ◽

Fluorescence Lifetime Imaging Microscopy ◽

Fluorescence Lifetime Imaging ◽

Lifetime Imaging ◽

Metabolic Fluxes ◽

Subcellular Resolution

AbstractMitochondria are central to metabolism and their dysfunctions are associated with many diseases1–9. Metabolic flux, the rate of turnover of molecules through a metabolic pathway, is one of the most important quantities in metabolism, but it remains a challenge to measure spatiotemporal variations in mitochondrial metabolic fluxes in living cells. Fluorescence lifetime imaging microscopy (FLIM) of NADH is a label-free technique that is widely used to characterize the metabolic state of mitochondria in vivo10–18. However, the utility of this technique has been limited by the inability to relate FLIM measurement to the underlying metabolic activities in mitochondria. Here we show that, if properly interpreted, FLIM of NADH can be used to quantitatively measure the flux through a major mitochondrial metabolic pathway, the electron transport chain (ETC), in vivo with subcellular resolution. This result is based on the use of a coarse-grained NADH redox model, which we test in mouse oocytes subject to a wide variety of perturbations by comparing predicted fluxes to direct biochemical measurements and by self-consistency criterion. Using this method, we discovered a subcellular spatial gradient of mitochondrial metabolic flux in mouse oocytes. We showed that this subcellular variation in mitochondrial flux correlates with a corresponding subcellular variation in mitochondrial membrane potential. The developed model, and the resulting procedure for analyzing FLIM of NADH, are valid under nearly all circumstances of biological interest. Thus, this approach is a general procedure to measure metabolic fluxes dynamically in living cells, with subcellular resolution.

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