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. 2006 Sep 15;91(6):2326-36.
doi: 10.1529/biophysj.105.079343. Epub 2006 Jun 30.

EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells

Dipanjan Bhattacharya  1 , Aprotim Mazumder, S Annie Miriam, G V Shivashankar
Affiliations

EGFP-tagged core and linker histones diffuse via distinct mechanisms within living cells

Dipanjan Bhattacharya et al. Biophys J. .

Abstract

The effect of chromatin organization on EGFP-tagged histone protein dynamics within the cell nucleus has been probed using fluorescence correlation and recovery measurements on single living HeLa cells. Our studies reveal that free fraction of core-particle histones exist as multimers within the cell nucleus whereas the linker histones exist in monomeric forms. The multimeric state of core histones is found to be invariant across mammalian and polytene chromosomes and this is ATP dependent. In contrast, the dynamics of the linker histones exhibits two distinct diffusion timescales corresponding to its transient binding and unbinding to chromatin governed by the tail domain residues. Under conditions of chromatin condensation induced by apoptosis, the free multimeric fraction of core histones is found to become immobile, while the monomeric linker histone mobility is partially reduced. In addition, we observe differences in nuclear colocalization of linker and core particle histones. These results are validated through Brownian dynamics simulation of core and linker histone mobility. Our findings provide a framework to understand the coupling between the state of chromatin assembly and histone protein dynamics that is central to accessing regulatory sites on the genome.

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Figures

FIGURE 1
FIGURE 1
Heterogeneity in chromatin organization and its reflection on the translation diffusion of molecules within the cell nucleus. The fluorescence images of (i) EGFP-transfected and (ii) TMR-D incorporated HeLa cell nucleus. (Scale bar, 5 μm) and their associated autocorrelation function curves for EGFP (⋆), and TMR-D (•) in the HeLa cell nucleus. (Inset) Probability histogram of β-factor for TMR-D inside the cell nucleus (⋄), in the buffer (○), and in the sorted chromosome (★).
FIGURE 2
FIGURE 2
Core histone (H2B-EGFP) dynamics suggests their multimeric state. (a) Autocorrelation curves of EGFP in the cell nucleus and H2B-EGFP in the cytoplasm and in the nucleus. (Inset) Mean diffusion constants for the above cases. (b) Fluorescence images of the Drosophila salivary gland cell nucleus (polytene chromosomes) and HeLa nucleus and the associated autocorrelation curves of H2B-EGFP proteins in HeLa nucleus (★), salivary gland cell nucleus (⋄), and salivary gland cell nucleus with 600 mM NaCl (•). (Inset) Probability histograms of the diffusion correlation timescales for H2B-EGFP in HeLa nucleus and salivary gland cell nucleus and comparison of the probability histograms with H2B-EGFP diffusion in salivary gland cells in 600 mM NaCl concentration.
FIGURE 3
FIGURE 3
Linker histone shows distinct interaction timescales inside the cell nucleus. (a) Fluorescence images of H1.1-EGFP and tailless H1.1-EGFP transfected HeLa nucleus (scale bar, 5 μm) and the associated autocorrelation function curves for H1.1-EGFP (★), tailless H1.1-EGFP (○). (Inset) Fluorescence recovery curves for tailless H1.1-EGFP, H1.1-EGFP, EGFP, and core histones, H2B-EGFP and H4-EGFP. (b) Different subtypes of H1 proteins show distinct interaction timescales. Autocorrelation curves for different H1 subtypes, H1.1, H1.2, H1.4, H1.5 inside the HeLa cell nucleus. (Inset) Normalized FRAP data for different subtypes of H1 proteins, H1.1, H1.2, H1.4, and H1.5. (c) Probability histograms of different subtypes of H1 proteins H1.1-EGFP, H1.2-EGFP, H1.4-EGFP, H1.5-EGFP. (Inset to panel c (i)) Mean and standard deviations of the fractions of the noninteracting species in different H1 subtypes. (Inset to panel c (ii)) Autocorrelation function curves for H1.5-EGFP, in the cytoplasm and inside the cell nucleus.
FIGURE 4
FIGURE 4
(a) Fluorescence images of H2B-EGFP transfected HeLa nucleus before and after ATP depletion. (Scale bar, 5 μm). (i) Probability histograms of correlation timescales of H2B-EGFP diffusion inside HeLa nucleus before and after ATP depletion. The bar graphs of the mean correlation timescales are also shown in the inset. (ii) Probability histograms of H1.5-EGFP correlation timescale in milliseconds, before and after ATP depletion. (b) Time course of histone protein dynamics within cells upon induction of apoptosis. (i) Autocorrelation function curves for H2B-EGFP before and after 4 h of addition of staurosporine in HeLa cells. (Inset) Fluorescence images of H2B-EGFP transfected HeLa cells before and after addition of staurosporine. (Scale bar, 5 μm). (ii) FRAP curves for H1.1-EGFP in HeLa cells, in normal conditions and after staurosporine treatment.
FIGURE 5
FIGURE 5
Numerical simulation study of core and linker histone dynamics. (a) Autocorrelation function generated by numerical simulation for single species of 3D diffusion with confined mesh structure with varying sizes (m = 0, 10, 50). (Inset (i)) Typical single particle trajectory of 2D random walk generated within a confined mesh structure. (Inset (ii)) Mean square displacement versus time for different mesh sizes (m = 0, 11, 12, 15). (b) Autocorrelation function generated by numerical simulation for 3D diffusion with randomly distributed interacting sites (N = 0, 1500, 2000, and 3000). (Inset) Typical single particle trajectory of 2D random walk.
FIGURE 6
FIGURE 6
Schematic model of the mobility of core (i) and linker (ii) histones within the cell nucleus. Core histones (H2B-EGFP) remain in the multimeric form (red) in normal physiological condition. Upon ATP depletion the multimeric core histones (H2B-EGFP) become monomeric (green). Linker histones diffuse through the chromosomal mesh structure by diffusion with interaction (red, interacting; green, noninteracting). For tailless linker histones the motion is purely diffusive and there is no interaction.
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