FRET analysis of cell lines expressing fluorescently tagged histones on separate nucleosomes demonstrates that variations in chromosome compaction occur during mitosis

Angus I. Lamond

Bastiaens

Belmont

Choi

Cushman

David G. Norman

David Llères

Elgin

Förster

Gerlich

Gilbert

Grewal

Grunstein

Huisinga

Huynh

John James

Kanda

Keppler

Kimura

Kruhlak

Lakowicz

Lippincott-Schwartz

Llères

Luger

Misteli

Mora-Bermúdez

Perche

Phair

Platani

Rhodes

Robinett

Sam Swift

Shaner

Shav-Tal

Strukov

Swedlow

Taddei

Telford

Treanor

Tremethick

Trinkle-Mulcahy

Turner

Tóth

Vagnarelli

van Munster

Wallrabe

Wolffe

Wouters

English

PubMed

Crossref

Llères, David

James, John

Swift, Sam

Norman, David,

Lamond, Angus,

HAL Descartes

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

We present a quantitative Forster 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.</p

Lleres, David

Norman, David G.

Lamond, Angus I.

University of Dundee Online Publications

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Quantitative analysis of chromatin compaction in living cells using FLIM-FRET

Hal-Diderot

http://discovery.dundee.ac.uk/portal/files/778663/Lleres_2009.pdf

Quantitative analysis of chromatin compaction in living cells using FLIM-FRET

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HAL Descartes

University of Dundee Online Publications

University of Dundee Online Publications

Hal-Diderot