The Connection between Chromatin Motion on the 100 nm Length Scale and Core Histone Dynamics in Live XTC-2 Cells and Isolated Nuclei

Abstract

The diffusive motion of DNA-containing chromatin in live cells and isolated nuclei is investigated using a two-photon standing wave fluorescence photobleaching experiment with 100 nm spatial resolution. The chromatin is labeled using the minor groove binding dye Hoechst 33342. In live cells, the mean diffusion rate is 5 x 10(-4) micro m2/s, with considerable cell-to-cell variation. This diffusion is highly constrained and cannot be observed in a standard, single beam fluorescence recovery after photobleaching experiment. To determine the chemical origin of the diffusion, we study motion in isolated nuclei and vary the strength of the histone-DNA interactions by changing the ionic strength and using chemical and photocross-linking experiments. At higher NaCl concentrations, we see increased chromatin diffusion as the histone-DNA interaction is weakened due to ionic screening, whereas photocross-linking the core histones to the DNA results in a complete absence of diffusive motion. These trends are consistent with the 100 nm scale motion being correlated with the interactions of histone proteins with the DNA. If chromatin diffusion is connected to the nucleosomal dynamics on much smaller length scales, this may provide a way to assay biochemical activity in vivo based on larger scale macromolecular dynamics observed via fluorescence microscopy.

In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow

“…An image of the Hoechst 33342 stain was also acquired before and after each measurement to check for DNA movement (in those files displaying no cell movement). As reported by others (23,24), no detectable motion of the DNA was ever observed. The autocorrelation function (ACF) carpet obtained was then analyzed and pair correlation functions derived (mathematical derivation of the pCF is reported in SI Materials and Methods).…”

Macromolecular crowding effect is critical for maintaining SIRT1's nuclear localization in cancer cells

“…48 In contrast, nuclei isolated in inert polymers containing solution maintain their intact ultrastructure and transcription activity, 39 indicating that cytoplasmic macromolecular crowding effect is critical to maintain a more physiological condition of nuclei. Consistently, using inert polymers to mimic cytoplasmic crowding effect successfully preserved SIRT1 in the nucleus during fractionation, as it did previously for GRFI which leaked out similarly during isolation of nuclei.…”

Beyond the diffraction limit: far-field fluorescence imaging with ultrahigh resolution

“…SWFM has been applied to study the diffusive motion of DNA-containing chromatin in live cells and isolated nuclei. 10 This study centred on imaging processes involving histone proteins and DNA which occurred on 100 nm length scales, something not possible using diffraction limited fluorescence microscopy. At the same time as studies using SWFM were being performed the first set of studies applying 4Pi to biosystems were also being undertaken.…”

“…Davis and Bardeen used two-photon SWFM to study the diffusive motion of DNA-containing chromatin in live cells and isolated nuclei. 10 The diffusion processes studied centred on understanding the interactions of histone proteins with DNA which occurred on 100 nm length scales which was accessible when imaging using two-photon SWFM. These studies demonstrated that the ultrahigh image resolution achievable using SWFM enabled better understanding of fundamental questions related to structural biology and also to protein biology.…”