Magnesium and Calcium in Isolated Cell Nuclei

Naora, Honami; Mirsky, A. E.; Allfrey, Vincent G.

doi:10.1085/jgp.44.4.713

Cited by 58 publications

(7 citation statements)

References 34 publications

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“…Indeed, a strong case has been made that the cytoplasm does not contain free small ions at significant levels and that “there is no cytoplasmic ‘bulk’ concentration of ions and metabolites (as is often assumed in biophysical models and in the design and interpretation of in vitro experiments)” [11] . However, nuclei isolated in conventional ionic media avidly bind extra Mg 2+ and Ca 2+ from buffers to levels many-fold the endogenous levels [24] , an effect which could provide clues to resolving the paradox cited in the Introduction that chromatin from nuclei isolated in ionic media, or reconstituted in similar conditions, does not reproduce completely the properties of chromatin within the nucleus in vivo.…”

Section: Discussionmentioning

confidence: 99%

“…Secondly, chromatin isolated from nuclei prepared in conventional ionic media, or reconstituted in similar conditions, does not reproduce completely the properties of chromatin within the cell which shows constrained motion on a 100 nm-scale [19] and an irregular conformation [20] – [22] , whereas in isolated nuclei the small-scale motion of chromatin is supressed [19] and chromatin isolated from them has a regular and symmetrical conformation termed the 30 nm fibre [23] . The binding of significant amounts of Mg 2+ and Ca 2+ ions to nuclei, and possibly to chromatin, during their isolation in ionic media [24] may contribute to this discrepancy.…”

Section: Introductionmentioning

confidence: 99%

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

Hancock

Hadj-Sahraoui

2009

PLoS ONE

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Hancock

Hadj-Sahraoui

2009

PLoS ONE

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“…The remaining 24% of the total nuclear Ca content is bound to chromosomal protein and is resistant to extraction with EGTA (Naora et al, 1961). This corresponds to 2.2-11.8 pmol Caikg smooth muscle cell and represents an upper range for the nuclear contribution.…”

Section: Components Of 45ca Releasementioning

confidence: 99%

“…Approximately 76% of the nuclear Ca content is soluble and labile (Naora et al, 1961) and can diffuse through the nuclear pore complex through aqueous channels with radii of 6.5 nm. These channels are freely permeable to ions and to molecules with radii less than 2.4 nm (Reynolds and Tedeschi, 1984).…”

Section: Components Of 45ca Releasementioning

confidence: 99%

Calcium transport by sarcoplasmic reticulum of vascular smooth muscle: I. MgATP‐dependent and MgATP‐independent calcium uptake

Stout

1991

Journal Cellular Physiology

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The components of 45calcium (Ca) uptake were studied in saponin skinned rat caudal artery. The steady-state Ca content increased when the free Ca concentration was varied from 10(-8) to 10(-4) M but was reduced by azide when the free Ca concentration exceeded 3.1 microM. The azide sensitivity and low affinity for Ca were consistent with functional mitochondria. The azide-insensitive component consisted of a small bound and a larger releasable Ca fraction. After skinning in Triton X-100, approximately 4 mumol Ca/kg wet tissue remained, which represented a tightly bound but slowly exchangeable Ca pool. The Ca content was independent of the free Ca concentration and MgATP, and it was not released with A-23187 or Ca. The Ca content of the larger fraction was a higher order function of the free Ca concentration and was released with A-23187, indicating it resided within a membrane-bounded structure. Ca uptake by the releasable fraction was increased by oxalate, MgATP, phosphocreatine, temperature, phosphate, and ruthenium red and represents Ca sequestered by the sarcoplasmic reticulum (SR) with little contribution from other Ca binding or storage sites. It is described by the coefficients Umax = 96.94 mumol/kg wet tissue, K1/2 = 0.75 microM, and Hill coefficient = 1.70. The SR in this preparation regulates cytosolic Ca concentrations under physiological conditions and can accumulate Ca by MgATP-dependent and MgATP-independent process. The larger, MgATP-dependent Ca uptake is described by the coefficients Umax = 72.87 mumol/kg wet tissue, K1/2 = 0.8 microM, and Hill coefficient = 2.09 and is consistent with Ca sequestered by the Ca-transport ATPase of smooth muscle SR. The smaller, MgATP-independent uptake is described by the coefficients Umax = 24.14 mumol/kg wet tissue, K1/2 = 0.56 microM, and Hill coefficient = 1.01 and represents Ca sequestered by an unidentified mechanism or by a subpopulation of SR.

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“…This is seen especially clearly in preparations made from liver nuclei. 12 It has also been demonstrated that treatment of animal cells or nuclei with aqueous phenol under appropriate conditions separates RNA into a "low-turnover" RNA released into the aqueous phase, and a "high-turnover" RNA remaining in the gel-like interphase formed between the aqueous layer and the phenol phase.7'…”

mentioning

confidence: 99%