Isolation of Cell Nuclei Using Inert Macromolecules to Mimic the Crowded Cytoplasm

Abstract

Cell nuclei are commonly isolated and studied in media which include millimolar concentrations of cations, which conserve the nuclear volume by screening the negative charges on chromatin and maintaining its compaction. However, two factors question if these ionic conditions correctly reproduce the environment of nuclei in vivo: the small-scale motion and conformation of chromatin in vivo are not reproduced in isolated nuclei, and experiments and theory suggest that small ions in the cytoplasm are not free in the soluble phase but are predominantly bound to macromolecules. We studied the possible role in maintaining the structure and functions of nuclei in vivo of a further but frequently overlooked property of the cytoplasm, the crowding or osmotic effects caused by diffusible macromolecules whose concentration, measured in several studies, is in the range of 130 mg/ml. Nuclei which conserved their volume in the cell and their ultrastructure seen by electron microscopy were released from K562 cells in media containing the inert polymer 70 kDa Ficoll (50% w/v) or 70 kDa dextran (35% w/v) to replace the diffusible cytoplasmic molecules which were dispersed on cell lysis with digitonin, with 100 µM K-Hepes buffer as the only source of ions. Immunofluorescence labelling and experiments using cells expressing GFP-fusion proteins showed that internal compartments (nucleoli, PML and coiled bodies, foci of RNA polymerase II) were conserved in these nuclei, and nascent RNA transcripts could be elongated. Our observations are consistent with the hypothesis that crowding by diffusible cytoplasmic macromolecules is a crucial but overlooked factor which supports the nucleus in vivo by equilibrating the opposing osmotic pressure cause by the high concentration of macromolecules in the nucleus, and suggest that crowded media provide more physiological conditions to study nuclear structure and functions. They may also help to resolve the long-standing paradox that the small-scale motion and irregular conformation of chromatin seen in vivo are not reproduced in nuclei isolated in conventional ionic media.

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“…In addition, this observation suggests that there are changes in the elasticity of the nucleus (Hancock and Hadj-Sahraoui, 2009), which likely dictates the F-actin cytoskeleton organization to achieve cell stability. The idea of a mechano-directed pathway between the nucleus and cytoplasm may explain the signaling between Runx2 and the F-actin cytoskeleton organization.…”

Structures and functions in the crowded nucleus: new biophysical insights

“…These [20]). (B) Discrete territories of the 24 chromosomes in a human fibroblast nucleus labeled with different fluorochrome combinations (reproduced from [21]).…”

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.…”

“…38 Isolating nuclei using solutions containing uncharged inert polymers is able to mimic such a crowding effect and preserve physiological nuclear structure and function. 39 Given that Ficoll polymers have been used to retain transcription factor GFRI in yeast nucleus during fractionation, 40 we included different amount of Ficoll in digitonin extraction solution to test whether the increased crowding effect can prevent SIRT1 leakage. As indicated in Figure 2D, addition of high concentration of Ficoll effectively retained GFP-SIRT1 in the nuclear fraction.…”