Liposome-nucleus interactions. Flow cytometric study on the role of the nuclear surface

The water-soluble probe carboxyfluorescein (CF), contained in the internal aqueous phase of liposomes, was used to investigate the interaction of phospholipid vesicles with isolated nuclei. Ultrastructural analysis indicated that adherent liposomes coated the nuclear surface, and fluorescence microscopy showed that they contained quenching concentrations of the dye. Flow cytometry revealed that the transfer of the entrapped dye from the adhering liposomes to nuclei was blocked by chilling at 0 degrees C. Chase experiments demonstrated that the most reliable mechanism of dye transfer involved fusion phenomena between the liposomal and the nuclear membranes. After the release of the fluorophore into the nucleus, empty liposomes could withdraw the intranuclear soluble fraction of the dye.     

Cell age-related differences in the interaction of a potential-sensitive fluorescent dye with nuclear envelopes of Acetabularia mediterranea

Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC₆(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC₆(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.     

Glucocorticoid receptors in cytosol and nuclear extract of human leukocytes

Cortisol binding by cytosol and 0.4 M KCl extract of the nuclear fraction of human leukocytes were studied by gel chromatography and ion exchange filtration on DEAE cellulose. The cytoplasmic cortisol binding protein has a molecular weight 95 000 and the soluble nuclear binding protein 50 000. The absence of the uptake of radioactive cortisol by isolated nuclei and the apparent requirement of the cytosol for glucocorticoid specific binding in nuclear receptor sites was observed. The association constant characterising the binding of cortisol to cytosol was KA = 3.5 . 10(9) l/mol.     

Progesterone-binding components of chick oviduct. VIII. Receptor activation and hormone-dependent binding to purified nuclei.

A cell-free system prepared from the estrogen-primed chick oviduct was developed and used to study the uptake of cytoplasmic progesterone-receptor complex by isolated nuclei. The receptor and purified nuclei were shown to be stable at 25 degrees, but not at 37 degrees. Thus, nuclear incubations were routinely performed at 25 degrees. Such incubations revealed greater nuclear uptake of the cytoplasmic hormone-receptor complex as compared to control incubations performed at 0 degrees. The uptake process showed a quantitative preference for oviduct nuclei. No net uptake occurred during 0 degrees incubations when the nuclei were preincubated in the absence of cytoplasmic components at 25 degrees. In contrast, the temperature requirement was partially removed by preincubation of the hormone-receptor complex at 25 degrees prior to incubation with nuclei at 0 degrees. Nuclear uptake was not accompanied by measurable alterations in the sedimentation properties of the progesterone receptor. The activation and nuclear uptake of receptor was clearly dependent upon prior binding of steroid hormone to the receptor indicating that the active nuclear form of the receptor could not be generated in the absence of the hormone. Receptor precipitation with ammonium sulfate also partially removed the temperature requirement for nuclear binding. In contrast to temperature activation, ammonium sulfate precipitation activated the receptor in the absence of hormone. It thus seemed likely that temperature and salt activation of receptor occurred via different mechanisms. Although we were able to destroy up to 60% of the nuclear DNA content by treatment with DNase prior to nuclear incubation, some 80 to 85% of the receptor-binding capacity was still present in the treated nuclei. Thus, chick progesterone receptors apparently bind to a relatively DNase-resistant portion of the oviduct genome. The properties of this system indicate its value for further investigation into the initial events of progesterone action in the chick oviduct.     

6-Iodoacetamidofluorescein labelling to assess the state of sulphhydril groups after thermal stabilization of isolated nuclei

Summary Isolated nuclei and nuclear matrices, prepared from mouse erythroleukaemia cells, were reacted with the sulphhydryl-specific dye 6-iodoacetamidofluorescein. To determine whether in vitro formation of disulphide bonds might play a role in the nuclear matrix stabilization triggered by exposure of isolated nuclei to the physiological temperature of 37°C, a variety of techniques were employed to assess the state of cysteinyl residues after such an incubation. Both flow cytometry and confocal microscopy quantitative analysis did not reveal major differences in the fluorescence intensity of nuclei incubated at 37°C in comparison with those maintained at 0°C. Confocal scanning laser microscopy revealed that 6-iodoacetamidofluorescein labelled a fibrogranular network in isolated nuclei. The fluorescent pattern of the network was not affected by a 37°C exposure of nuclei. However, such a network was not detectable in isolated nuclear matrices, thus suggesting a possible protein re-arrangement during matrix preparation. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of fluorescent-labelled nuclear proteins showed no difference between heat-exposed and control samples. We conclude that oxidation of cysteinyl residues is not a major factor leading to the stabilization of nuclei incubated at 37°C.     

Binding of the glucocorticoid receptor to the rat liver nuclear matrix. The role of disulfide bond formation

The nuclear matrix is a putative skeletal structure which has been implicated in many nuclear functions. To assess a possible role of the nuclear matrix in glucocorticoid action, purified rat liver nuclei containing glucocorticoid-receptor complexes were treated with DNase I +/- RNase A followed by 1.6 M NaCl, thus yielding salt-extractable and salt-resistant (nuclear matrix) fractions. The subnuclear distribution of hormone-receptor complexes was determined by following the fate of unmetabolized radiolabel after injection of labeled triamcinolone acetonide into adrenalectomized animals and subjecting various subfractions to immunoblotting using a monoclonal antibody which recognizes the glucocorticoid receptor. Both techniques indicated that 50-70% of the total nuclear hormone-receptor complexes were recovered in the nuclear matrix fraction. Previous results (Kaufmann, S. H., and Shaper, J. H. (1984) Exp. Cell Res. 155, 477-495) suggest that a variety of nuclear polypeptides become nuclease- and salt-resistant as a result of the formation of intermolecular disulfide bonds. The following evidence suggests that disulfide bonds mediate the association between the glucocorticoid receptor and the nuclear matrix. When nuclei were isolated in the absence of sulfhydryl-blocking and -cross-linking reagents, sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions revealed that the receptor was present as a high molecular weight disulfide-cross-linked complex. When nuclei were isolated in the presence of the irreversible sulfhydryl-blocking reagent iodoacetamide, the disulfide bonds which cross-linked the receptor into high molecular weight complexes were absent; and 85-100% of the hormone-receptor complexes were salt-extractable. When nuclei (isolated in the absence of iodoacetamide) were treated with the sulfhydryl-cross-linking reagent sodium tetrathionate, greater than 95% of the nuclear hormone-receptor complexes became resistant to extraction with nucleases and 1.6 M NaCl. The implications of these results for other matrix-associated nuclear functions are discussed.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

On the attachment of the nuclear pore complex

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.     

Dynamic inorganic ion-protein interactions in structural organization of DNA of living cell nuclei.

Staining polarization optical techniques showed differences in the structural organization of DNA of chromatin in interphase nuclei and in mitotic chromosomes. The DNA was non-birefringent in intact interphase cell nuclei, but birefringent in chromosomes and in isolated nuclei incubated in a physiological electrolyte solution. The birefringence of DNA appears to be related to an unfolding of DNA filaments induced by free cations and to the oriented binding of dye molecules to DNA phosphates. We propose that the actual concentration of free cations inside the living cell nuclei is regulated by a dynamic interaction between nuclear proteins and ions.     

The nuclear matrix continues DNA synthesis at in vivo replicational forks

Alkaline cesium chloride gradient analysis of in vivo [3H]bromodeoxyuridine-labeled and in vitro [alpha-32P]dCTP-labeled DNA was used to determine whether in vitro DNA synthesis in regenerating rat liver nuclei and nuclear matrices continued from sites of replication initiated in vivo. At least 70 and 50% of the products of total nuclear and matrix-bound in vitro DNA synthesis, respectively, were continuations of in vivo initiated replicational forks. The relationship of the in vitro DNA synthetic sites in total nuclei versus the nuclear matrix was examined by using [3H]bromodeoxyuridine triphosphate to density label in vitro synthesized DNA in isolated nuclei and [alpha-32P]dCTP to label DNA synthesized in isolated nuclear matrix. A minimum of about 40% of matrix-bound DNA synthesis continued from sites being used in vitro by isolated nuclei. Furthermore, nuclear matrices prepared from in vitro labeled nuclei were 5-fold enriched in DNA synthesized by the nuclei and were several-fold enriched, compared to total nuclear DNA, in a particularly high density labeled population of DNA molecules.     

Transfer of liposome encapsulated carboxyfluorescein to isolated nuclei

The interaction between phosphatidylcholine vesicles and isolated rat liver nuclei has been examined by studying the uptake of the fluorophore carboxyfluorescein. The kinetics of transfer of the dye, analyzed by flow cytofluorimetry with a Fluorescence Activated Cell Sorter (FACS IV), indicate an efficient delivery to the nucleoplasm. The results reflect a liposome-nuclear membrane interaction which may contribute to the processes which underlie our previously described morphological and functional changes in isolated nuclei treated with phospholipids.     

RNA-containing nuclear binding sites for glucocorticoid-receptor complexes

RNase A treatment of HeLa cell nuclei causes a time- and concentration-dependent release of dexamethasone-receptor complexes. If nuclei are incubated in the absence of enzyme, only 60% of RNase-releasable complexes can be detected. Sucrose density gradient analysis of nuclear extracts shows that receptor complexes released by RNase treatment sediment at 3.6 S, whereas complexes obtained from untreated nuclei sediment between 7 and 3.6 S. Our results show that a fraction of dexamethasone-receptor complexes retained by HeLa cell nuclei is located in binding sites involving RNA.     

Binding of a 23 kD endonuclease to the rat liver nuclear matrix

In a previous paper we have described a 23 kD nuclear endonuclease (p23) that was mostly found to exist in a state of association with the isolated rat hepatocyte nuclear matrix. To investigate the nature of this interaction, the nuclear matrix was prepared using different procedures and examined for the presence/absence of the enzyme by activity gel analysis. Treatment of isolated nuclei with sodium tetrathionate (NaTT), a sulfhydryl-cross-linking agent, led to the complete recovery of p23 in the nuclear matrix, whereas incubation of nuclei with dithiothreitol (DTT), a sulfhydryl-reducing agent, led to its complete solubilization and resulting absence from the nuclear matrix. Exposure of the isolated nuclear matrix to DTT in high-ionic strength buffer, a procedure that promotes the solubilization of the internal nuclear matrix, caused the nearly complete solubilization of p23. It was concluded that disulfide bonds play an essential role in the association of p23 with the nuclear matrix and that p23 is mostly localized in the nuclear matrix interior.     

Light microscopic demonstration of myoid material in nuclei.

During the development of configurational staining methods for proteins of the myosin-fibrin group, nuclei showed staining properties similar to those of myofibrils. This dye binding could be attributed to nuclear alpha-helical proteins. More recent chemical and electron microscopic studies demonstrated actomyosins in nuclei of various species. Possible roles of nuclear actomyosin in chromosome movements and condensation and in cell proliferation have been suggested. It seems therefore permissible to assume that the tannic acid-phosphomolybdic acid (TP)-Levanol Fast Cyanine 5RN method and similar technics visualize myosin in nuclei. Comparative studies of actomyosins from various sites indicated significant chemical an histochemical differences. It is therefore suggested that, in analogy to the different classes of collagens, there may be several subgroups of myosin which differ in their physico-chemical properties and sensitivity to fixation procedures and pathological conditions.     

Specific binding of phorbol esters to nuclei of human promyelocytic leukemia cells

In this report, we demonstrate that HL-60 nuclei isolated in calcium but not EGTA containing buffers specifically bind PE and express approximately 37,000 receptor sites/nucleus. Nuclear phorbol ester binding is lost by isolation in the absence of calcium, but can be repleted by the addition of partially purified protein kinase C and calcium. When HL-60 cells are treated with bryostatin 1, a compound which activates protein kinase C in a similar fashion to phorbol esters but does not induce differentiation of HL-60 cells, and nuclei are isolated in the presence of EGTA, these nuclei continue to bind phorbol esters. These experiments suggest that HL-60 nuclei bind PE in vitro, and that compounds that activate protein kinase C may increase nuclear binding of PE in situ.     

Interrelationship between nuclear histone binding and cell proliferation

1. Binding of non-enzymatically [methyl-14C]-labeled histone H3 to nuclei isolated from young and old rat livers, regenerating rat liver, and tumor cells has been investigated. 2. Scatchard plot analysis indicated that various cell types had different binding capacity and different dissociation constant (Kd). 3. Nuclei isolated from younger rats had fewer binding sites and lower Kd (or higher Ka) values for [methyl-14C]H3 than those from older rats. 4. Fewer binding sites and lower Kd values were also observed with nuclei isolated from the maximally regenerating liver (24 hr after partial hepatectomy) and the fast-growing ascites tumor and Novikoff hepatomas. 5. These results strongly suggest that the number of binding sites and affinity of histone H3 for nuclei appears to be correlated with the degree of cell proliferation. 6. Fractionation of the [methyl-14C]H3 bound nuclei into nuclear membrane and nucleoplasm demonstrates that approx. 94% of radioactivity is associated with the former in which less than 6% of DNA is found, whereas 94% of total DNA is found in nucleoplasm. 7. This suggests that the binding of [methyl-14C]H3 to nuclei is independent of DNA present in each fraction.     

Staining kinetics in single cells

Summary Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.     

Staining kinetics in single cells. Part II. Diffusion processes inside the cell

Applying hydrodynamic conditions, which certify a negligible influence of convective diffusion, the time-dependent uptake of thionin in lymphocytes, monkey kidney cells, and their separated nuclei was measured spectroscopically. Using fixed cell material the dye transport inside the cell is not hindered due to plasma membrane and cytoplasm. The staining rate depends on the dye concentration, the pretreatment of the cell, and on the electrolyte concentration of the dye solution. The mechanism of dye migration inside the cell is in accordance with a porous matrix model. The diffusion process takes place inside the pores and channels filled with liquid and is modified by adsorption of dye molecules on the walls of the pores. A dynamic reversible equilibrium exists between migrating dye molecules and the binding sites on the pore walls described by the Freundlich adsorption isotherm. The proposed model explains the observed order of reaction of the staining kinetics.