Quantitative analysis of chromatin compaction in living cells using FLIM–FRET
Open Access
- 16 November 2009
- journal article
- Published by Rockefeller University Press in The Journal of cell biology
- Vol. 187 (4), 481-496
- https://doi.org/10.1083/jcb.200907029
Abstract
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.Keywords
This publication has 49 references indexed in Scilit:
- Mitotic Chromosome Structure: Reproducibility of Folding and Symmetry between Sister ChromatidsBiophysical Journal, 2009
- Histone acetylation: truth of consequences?This paper is one of a selection of papers published in this Special Issue, entitled CSBMCB’s 51st Annual Meeting – Epigenetics and Chromatin Dynamics, and has undergone the Journal’s usual peer review process.Biochemistry and Cell Biology, 2009
- Condensin and Repo-Man–PP1 co-operate in the regulation of chromosome architecture during mitosisNature, 2006
- Microclusters of inhibitory killer immunoglobulin–like receptor signaling at natural killer cell immunological synapsesThe Journal of cell biology, 2006
- A guide to choosing fluorescent proteinsNature Methods, 2005
- A Method for the In Vitro Reconstitution of a Defined “30nm” Chromatin Fibre Containing Stoichiometric Amounts of the Linker HistoneJournal of Molecular Biology, 2005
- Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent proteinNature Biotechnology, 2004
- Dynamics of Single mRNPs in Nuclei of Living CellsScience, 2004
- The Dynamic Association of RCC1 with Chromatin Is Modulated by Ran-dependent Nuclear TransportMolecular Biology of the Cell, 2004
- Large-scale chromatin structure and functionCurrent Opinion in Cell Biology, 1999