Isolation and Fractionation of Rat Brain Nuclei

Løvtrup-Rein, Huguette; McEwen, Bruce S.

doi:10.1083/jcb.30.2.405

Cited by 193 publications

(44 citation statements)

References 15 publications

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“…Electron microscopic studies of the retrograde axonal transport of NGF in sympathetic neurons have shown considerable localization of NGF in the perinuclear region but none inside the nucleus over the chromatin (20,21). Treatment of the nucleus with Triton X-100 eliminates the outer nuclear membrane (14)(15)(16)(17)(18)(19) (22,23). Cytoplasmic vesicles containing EGF with a fluorescent label were observed to form a perinuclear ring in a time-and temperature-dependent fashion (23).…”

Section: Discussionmentioning

confidence: 99%

“…RESULTS Uptake of NGF by PC12 and Its Appearance in the Nucleus. Treatment of cells with Triton X-100 results in the solubilization of membrane systems with-the exception of the nucleus, which is left with its inner membrane relatively intact (14)(15)(16)(17)(18)(19). The effects of Triton treatment on the binding of 125I-jNGF to membranes and nuclei isolated from PC12 are shown in Table 1.…”

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confidence: 99%

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Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.

Yankner¹,

Shooter²

1979

Proc. Natl. Acad. Sci. U.S.A.

170

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Cells of a continuous line of rat pheochromocytoma (PC12) were incubated with 125I-labeled P nerve growth factor (PNGF), and at given time intervals the cell nuclei were isolated by a procedure that used the detergent Triton X-100. NGF was detectable in the nucleus after 20 min and continued to accumulate in a linear fashion for several hours after the total binding to the cell had reached steady state. After 17 hr at 370C, about 60% of the NGF bound to the cell was in the nucleus. NGF was not translocated to the nucleus at 4VC. When nuclei were purified from PC12 cells and incubated with 125I-labeled j#NGF, specific binding sites were found. Binding was saturable and consistent with two sites: a high-affinity site with a Kd of 0.08 nM (+0.02 nM) and a lower-affinity site with a Kd of 9.0 nM (±2.0 nM). The receptors in the nucleus were shown to be localized to the nuclear membrane. Membrane-free chromatin did not bind NGF specifically. The translocation of NGF to the nucleus was accompanied by a commensurate decrease in the cell-surface binding capacity. In the nucleus, however, the receptor capacities of both sites were increased when PC12 cells were grown in the presence of NGF. Nerve growth factor (NGF) is a polypeptide hormone that is involved in the development and maintenance of several cell types of neural crest origin. The injection of NGF antiserum into neonatal mice results in the rapid degeneration of the sympathetic neurons of paravertebral and prevertebral ganglia. At the ultrastructural level, the first changes are observed in the nucleus; these include the disorganization of the nucleolus and the disruption of the nuclear membrane (1, 2). The most striking morphological effect of NGF is the stimulation of neurite extension. Pronounced metabolic changes are also observed; these include alterations in RNA and protein synthesis (3).The finding that NGF can be internalized in vivo by retrograde axonal transport into the cell body of the sympathetic neuron (4) prompts the consideration of an intracellular mechanism. Triton-resistant binding sites for NGF were recently demonstrated in a preparation of nuclei from the dorsal root ganglion. The sites were reported to be contained in chromatin (5).The clonal cell line of rat pheochromocytoma (PC12) responds to NGF by the extension of neuritic processes and the cessation of cell division (6, 7). In the present communication, we describe the uptake of NGF by the viable PC12 cell and the time course of its appearance in the nucleus. Receptors for NGF are found in the nucleus of PC12; they are localized exclusively to the nuclear membrane.MATERIALS AND METHODS Subfractionation of PC12. The PC12 cell line was kindly provided by Lloyd A. Greene. PC12 cells were plated on polystyrene tissue culture dishes and grown in Dulbecco's modified Eagle's medium (GIBCO) supplemented with 10% fetal calf serum and 5% horse serum. Cultures were maintained in a water-saturated atmosphere of 88% air/12% CO2. Subconfluent cultures grown either in the presence or absence...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.

Yankner¹,

Shooter²

1979

Proc. Natl. Acad. Sci. U.S.A.

170

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“…Their "nuclear suspension" contained some cytoplasmic debris, myelin fragments, and capillaries which were recovered in band 1 and band 2 separated in the subsequent density gradient centrifugation. The absence of data concerning the cytoplasmic enzyme activities in the paper of Lg~vtrup-Rein and McEwen (26) makes impossible a comparison of purity between their nuclear preparation and ours. The RNA/ DNA ratio in their nuclear suspension is of the same order as that obtained by Hadjiolov et al (5) from cat brain cortex and that obtained by us (Table II) …”

Section: Nuclear Enzyme Activitymentioning

confidence: 99%

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

Kato

Kurokawa

1967

The Journal of Cell Biology

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An aqueous method is described for the isolation of highly purified nuclei from the ccrebral cortex of adult guinea pigs. Erythrocytes were removed by a short-time perfusion of the brain, myelin fragments by a rapid mechanical method, and blood capillaries by a centrifugal sieving through densc sucrose solutions. Thc nuclear preparation retained the activity of ATP:NMN adenylyltransfcrase. Recoveries of DNA in the P4I, P4II, PL and Ps preparations werc 30, 43, 8, and 7~, respectively. Microscopy and phase contrast microscopy showed a satisfactory removal of erythrocytes, myelin fragmcnts, capillaries, and cytoplasmic clcmcnts. Biochemical purity of samplcs was vcrificd by thc absence of several cytoplasmic enzyme activities. In thc electron microscopc, the majority of nuclei showed wcll-prescrved nuclear membrancs, with nuclcar pores, and werc providcd with a finely textured nuclcoplasm. Occasional contaminants wcrc elcmcnts of endoplasmic rcticulum and of the endothelium. Assortment of nuclci on a morphological basis showed that 55-65% and 47-53% of nuclei in the P4I and P4II preparations, rcsDcctivcly, consisted of neuronal nuclei. In thc PL preparation, the population of neuronal nuclei ranged between 72 and 83 ~, while 94--99 % of the nuclei in the Ps preparation consistcd of smaller nuclei, most likely of oligodcndroglial origin.

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“…1 a). Furthermore, many reports indicate that gross morphology is unaltered, except for removal of the outer nuclear membrane during isolation of nuclei by detergent solubilization of cytomembranes (27,(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)) (see Fig. 1 b).…”

Section: Morphology Of Control and Triton X-io0 Treated Nucleimentioning

confidence: 99%

On the Attachment of the Nuclear Pore Complex

Aaronson

Blobel

1974

The Journal of Cell Biology

175

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Electron microscope examination of isolated rat liver nuclei after treatment with the detergent Triton X-100 revealed the complete removal of both the inner and outer membranes of the nuclear envelope. The envelope-denuded nuclei did not show any change in either shape or internal ultrastructure. Most strikingly, the nuclear pore complexes, which in untreated nuclei appear to be integral components of the nuclear envelope, were retained in their characteristic location at the distal ends of the channels leading through the peripheral heterochromatin.Determination of the chemical composition of detergent-treated nuclei showed that over 95% of the nuclear phospholipid was solubilized, thus corroborating the morphological absence of nuclear membranes. Furthermore, detergent treatment also solubilized approximately 10% of the nuclear protein. Analysis of the solubilized protein by polyacrylamide gel electrophoresis in the presence of SDS indicated that these proteins belong to a few specific classes which presumably represent the major polypeptides of the nuclear membranes.The total absence of the nuclear envelope on both morphological and biochemical grounds supports the idea that the nuclear pore complex does not require the membranes either for attachment to the nucleus or for maintenance of its own structural integrity. INTRODUCTIONIn eukaryotes, the nuclear pore complex is a ubiquitous organelle situated within the characteristic circular discontinuities ("pores") of the nuclear envelope (1-9). The roles which have been postulated for the pore and the complex fall primarily into two mutually compatible classes: (a) involvement in nucleo-cytoplasmic communication (9-12), and (b) organization of the interphase chromatin (13 16). Little direct evidence has been obtained to support either role, and there is only fragmentary information concerning the chemical composition of the complex (17, 18).Before attempting the isolation and chemical characterization of the pore complex, it was necessary to determine the structural relationships between the pore complex and its surrounding nuclear membranes and between the pore complex and the underlying peripheral chromatin.Nuclear pore complexes have been described in nuclear envelope fractions (19)(20)(21)(22)(23)(24)(25)(26). Their presence suggests that the bulk of the peripheral chromatin is not necessary for maintaining the gross structural integrity of the complex. However, significant amounts ofchromatin are routinely recovered 746

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Isolation and Fractionation of Rat Brain Nuclei

Cited by 193 publications

References 15 publications

Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.

Nerve growth factor in the nucleus: interaction with receptors on the nuclear membrane.

Isolation of Cell Nuclei From the Mammalian Cerebral Cortex and Their Assortment on a Morphological Basis

On the Attachment of the Nuclear Pore Complex

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