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

Llères, David; James, John; Swift, Sam; Norman, D.; Lamond, Angus I.

doi:10.1083/jcb.200907029

Cited by 166 publications

(208 citation statements)

References 50 publications

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“…Although our ATP depletion treatment partially inhibited transcription and had a similar effectwithActDtreatmentoflowconcentration(0.04mg/ml) (27), the ultra-structure of the nucleolus was not significantly changed (37). The results indicate that the molecular organization of the nucleolus is sensitive to cellular physiologic conditions and is then quickly reorganized at the molecular level.…”

Section: Discussionmentioning

confidence: 53%

“…The results indicate that the molecular organization of the nucleolus is sensitive to cellular physiologic conditions and is then quickly reorganized at the molecular level. Physical and chemical change of nucleolar components such as amount of molecules and electrostaticbalancemaybeaccompanied by suchnucleolar reorganization (4,25,37,43). The nucleolus needs a large amount of energy to maintain ribosomal biogenesis and the dynamic property of nucleolar organization, such as the continuous inflow and outflow of proteins.…”

Section: Discussionmentioning

confidence: 99%

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Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

Park¹,

Han²,

Sako³

et al. 2014

FASEB j.

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Organization and functions of the nucleolus is maintained by mobilities and interactions of nucleolar factors. Because the nucleolus is a densely packed structure, molecular crowding effects determined by the molecular concentrations and mobilities in the nucleolus should also be important for regulating nucleolar organization and functions. However, such molecular property of nucleolar organization is not fully understood. To understand the biophysical property of nucleolar organization, the diffusional behaviors of inert green fluorescent protein (GFP) oligomers with or without nuclear localization signals (NLSs) were analyzed under various conditions by fluorescence correlation spectroscopy. Our result demonstrates that the mobility of GFPs inside the nucleolus and the nucleoplasm can be represented by single free diffusion under normal conditions, even though the mobility in the nucleolus is considerably slower than that in the chromatin region. Moreover, the free diffusion of GFPs is found to be significantly size-and NLS-dependent only in the nucleolus. Interestingly, the mobility in the nucleolus is highly sensitive to ATP depletion, as well as actinomycin D (ActD) treatment. In contrast, the ultra-structure of the nucleolus was not significantly changed by ATP depletion but was changed by ActD treatment. These results suggest that the nucleolus behaves similarly to an open aqueous-phase medium with an increased molecular crowding effect that depends on both energy and transcription.-Park, H., Han, S.-S., Sako, Y., Pack, C.-G. Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy. FASEB J. 29, 837-848 (2015). www.fasebj.org

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Section: Discussionmentioning

confidence: 53%

Section: Discussionmentioning

confidence: 99%

Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

Park¹,

Han²,

Sako³

et al. 2014

FASEB j.

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“…These results indicate that, chromosome elongation following TSA exposure may arise as a consequence of the physical stretching induced by anaphase bridge formation most likely due to abnormal sister chromatid separation. In support of this hypothesis, FLIM-FRET analysis indicates that maximum chromatin compaction during mitosis takes place during late anaphase (Lleres et al, 2009). Thus, abnormal sister chromatid separation may interfere with this process resulting in elongation of meiotic chromosomes following TSA exposure.…”

Section: Lack Of H4k5ac Signals) As Determined By High-resolution Cytmentioning

confidence: 56%

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte

Yang¹,

Baumann²,

Viveiros³

et al. 2012

Int. J. Dev. Biol.

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Histone acetylation regulates higher-order chromatin structure and function and is critical for the control of gene expression. Histone deacetylase inhibitors (HDACi) are currently un¬der investigation as novel cancer therapeutic drugs. Here, we show that female germ cells are extremely suscep¬tible to chromatin changes induced by HDACi. Our results indicate that exposure to trichostatin A (TSA) at nanomolar levels interferes with major chromatin remodeling events in the mammalian oocyte leading to chromosome instability. High resolution analysis of chromatin structure and live-cell imaging revealed a striking euchromatin decondensation associated with histone H4 hyperacetylation following exposure to 15 nM TSA in >90% of pre-ovulatory oocytes. Dynamic changes in large-scale chromatin structure were detected after 2 h of exposure and result in the formation of misaligned chromosomes in >75% (P<0.05) of in vitro matured oocytes showing chromosome lagging as well as abnormal sister chromatid separation at anaphase I. Abnormal axial chromatid condensation during meiosis results in the formation of elongated chromosomes exhibiting hyperacetylation of histone H4 at lysine 5 and lysine 16 at interstitial chromosome segments, but not pericentric heterochro¬matin, while highly decondensed bivalents exhibit prominent histone H3 phosphorylation at centromeric domains. Notably, no changes were observed in the chromosomal localization of the condensin protein SMC4. These results indicate that HDAC activity is required for proper chromosome condensation in the mammalian oocyte and that HDACi may induce abnormal chromosome segregation by interfering with both chromosome-microtubule interactions, as well as sister chromatid separation. Thus, HDACi, proposed for cancer therapy, may disrupt the epigenetic status of female germ cells, predisposing oocytes to an¬euploidy at previously unrecognized low doses.

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“…While SIM can provide sufficient resolution to indicate chromatin territories, DNA compaction can only be inferred, e.g., from the higher intensity of a DNA label such as DAPI. As reported in previous studies, e.g., [35], FLIM FRET can be utilised to map the compaction of DNA that has been labelled with appropriate donor/acceptor fluorophores such that they come into closer proximity as the DNA folds more tightly. The increased FRET efficiency results in a lower donor fluorescence lifetime for more compacted chromatin.…”

Section: Resultsmentioning

confidence: 99%

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

et al. 2017

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Abstract:We present a new microscope integrating super-resolved imaging using structured illumination microscopy (SIM) with wide-field optically sectioned fluorescence lifetime imaging (FLIM) to provide optical mapping of molecular function and its correlation with biological nanostructure below the conventional diffraction limit. We illustrate this SIM + FLIM capability to map FRET readouts applied to the aggregation of discoidin domain receptor 1 (DDR1) in Cos 7 cells following ligand stimulation and to the compaction of DNA during the cell cycle.

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

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

Cited by 166 publications

References 50 publications

Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

Dynamic and unique nucleolar microenvironment revealed by fluorescence correlation spectroscopy

Histone hyperacetylation during meiosis interferes with large-scale chromatin remodeling, axial chromatid condensation and sister chromatid separation in the mammalian oocyte

Mapping Molecular Function to Biological Nanostructure: Combining Structured Illumination Microscopy with Fluorescence Lifetime Imaging (SIM + FLIM)

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