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

David Lleres, John James, Sam Swift, David G. Norman, Angus I. Lamond (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    105 Citations (Scopus)
    286 Downloads (Pure)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)481-496
    Number of pages16
    JournalJournal of Cell Biology
    Volume187
    Issue number4
    DOIs
    Publication statusPublished - 16 Nov 2009

    Keywords

    • LIFETIME IMAGING MICROSCOPY
    • HISTONE ACETYLATION
    • IN-VIVO
    • FLUORESCENT PROTEINS
    • TIME-LAPSE
    • HETEROCHROMATIN
    • DYNAMICS
    • NUCLEOSOME
    • TRANSCRIPTION
    • FIBER

    Fingerprint

    Dive into the research topics of 'Quantitative analysis of chromatin compaction in living cells using FLIM-FRET'. Together they form a unique fingerprint.

    Cite this