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.
Highlights
Cell nuclei are commonly isolated and studied in media which have evolved from empirical observations of the conditions which maintain their ultrastructural features and functions such as transcription [1,2,3,4,5,6], or are based on the estimated concentrations of ions in the cell [5]
We show that cell nuclei which conserve their ultrastructure, internal compartmentalisation, and transcriptional activity can be stabilised by osmotic effects in solutions where an inert, volume-occupying polymer at an appropriate concentration replaces the diffusible cytoplasmic macromolecules which are dispersed upon cell lysis, and that they do not require the mM concentrations of cations which are commonly used in isolation media
Crowding agents can replace the requirement for a high ionic strength to polymerise the capsid protein CA of HIV-1 [53] and attenuate the need for Mg2+ ions for the association between 30S and 50S ribosomal subunits [49] and of K+ ions for productive folding of the chaperonin GroEL [51], and isolated chromosomes of Escherichia coli [50,52] and of eukaryotic cells are stabilised by inert polymers which reproduce their crowded environment in vivo, without the divalent cations and/or polyamines which were used earlier
Summary
Cell nuclei are commonly isolated and studied in media which have evolved from empirical observations of the conditions which maintain their ultrastructural features and functions such as transcription [1,2,3,4,5,6], or are based on the estimated concentrations of ions in the cell [5]. In these media, the volume of nuclei is conserved by including cations which screen the negative charges on chromatin and maintain its compaction [7,8]; without these cations, nuclei swell and may lyse [1,6,8,9] due to the osmotic pressure exerted by their high internal concentration of macromolecules [10]. The binding of significant amounts of Mg2+ and Ca2+ ions to nuclei, and possibly to chromatin, during their isolation in ionic media [24] may contribute to this discrepancy
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