心肌细胞核Ca2+库特点及其调节的离体研究

研究核外Ca~(2+)浓度对核Ca~(2+)的影响,及细胞核Ca~(2+)摄取和释放的关系,以探讨核Ca~(2+)转运的调节机制。采用差速离心和密度梯度离心法分离纯化心肌细胞核,以Fluo-4/AM荧光指示剂负载心肌细胞核,应用激光共聚焦扫描显微镜和荧光分光光度计进行观察和测定。结果显示,分离纯化的成年大鼠心肌细胞核内自由[Ca~(2+)]随着核外[Ca~(2+)]的增加而逐渐增加,孵育液[Ca~(2+)]为1000 nmol/L达高峰,但二者增加的程度并不一致,之后随核外[Ca~(2+)]浓度的增加而呈降低趋势。ATP和100—600nmol/L的核外游离Ca~(2+),使心肌细胞核显示核被膜腔Ca~(2+)荧光,ATP和1000nmol/L的核外游离Ca~(2+)则进一步引起核浆内的Ca~(2+)荧光强度升高。荧光染色观察可见IP_3受体染色主要位于核内膜,而钙泵和ryanodine受体染色主要位于核外膜。IP_3和Ryancodine使核Ca~(2+)短暂升高1.68倍和1.93倍(P<0.001),而钙泵抑制剂Thapsigargin和IP_3受体抑制剂Heparin则分别使核Ca~(2+)降低64%和35.6%(p<0.05)。ryanodine使IP_3升高的核Ca~(2+)显著回落至正常水平以下(p<0.001)。Thapsigargin不能阻断IP_3和Ryanodine所致的核Ca~(2+)释放增加(p<0.05),但事先采用钙泵抑制剂Thapsigargin预处理心肌细胞核,则能显著的阻断IP_3和Ryanodine所致的核Ca~(2+)升高作用(Ca~(2+)释放作用)(p<0.05)。结果提示大鼠心肌细胞核可能也是细胞内的钙库之一,心肌细胞核上存在Ca~(2+)-ATPase、ryanodine受体和IP_3受体等Ca~(2+)转运系统,可能参与核Ca~(2+)摄取和释放的调节。     

心肌细胞核Ca^2＋库特点及其调节的离体研究

To investigate the regulation of Ca2+ in the isolated cardiac nuclei from rats which may illuminated the mechanism of nuclear calcium transport system. Elocity and isopyknic gradient centrifugation were employed to fractionate rat cardiac nuclei. Then fluo-4 confocal microscopy techniques was used to verify the changes of nuclear Ca2+. There are calcium-dependent Ca2+ uptake in the cardiac nuclear obtained from normal rats. The accumulation Ca2+ of cardiac nuclei in vitro from the incubating medium were not consistent with free [Ca2+] in incubating medium. The nuclear envelope was initially loaded with Ca2+ (1 mmol/L ATP and approximately 100 nmol/L Ca2+), Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope and nucleoplasm. Exposure of Ca2+ -loaded nuclei to IP3, ryanodine or ryanodine + thapsigargin, respectively, resulted in a rapid and transient elevation of nucleoplasmic Ca2+ free concentration, this effects were abolished by pretreatment of cardiac nuclei with Ca2+ -ATPase inhibitor thapsigargin. Thapsigargin and IP3 receptor antagonist heparin induced nucleoplasmic Ca2+ free concentration decrease. Fluorescence experiments indicated that both ryanodine receptors and Ca2+ -ATPase were distributed in the outer layer of nuclear envelope, and inositol 1,4,5-trisphosphate receptors mainly dispersively localized at inner layer of nuclear envelope. The present study demonstrates that nuclear calcium were regulated by free Ca2+, IP3 and ryanodine. The results suggested calcium transport system might be present in the myocardial nuclei, the myocardial nuclei might served as one of calcium pools in myocardial cell.     

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.     

Mechanism regulating nuclear calcium signaling

Although the outer nuclear membrane is continuous with the endoplasmic reticulum, it is possible to isolate nuclei both intact and free from endoplasmic reticulum contaminants. The outer and the inner nuclear membranes can be purified free from cross-contamination. Evidence in support of autonomous regulation of nuclear calcium signaling relies upon the investigations with isolated nuclei. Mechanisms for generating calcium signaling in the nucleus have been identified. Two calcium transporting systems, an ATP-dependant nuclear Ca(2+)-ATPase and an IP4-mediated inositol 1,3,4,5-tetrakisphosphate receptor, are located on the outer nuclear membrane. Thus, ATP and IP4, depending on external free calcium concentrations, are responsible for filling the nuclear envelope calcium pool. The inositol 1,4,5-trisphosphate receptor is located on the inner nuclear membrane with its ligand binding domain facing toward the nucleoplasm. Likewise, the ryanodine receptor is located on the inner nuclear membrane and its ligand cADP-ribose is generated within the nucleus. A 120 kDa protein fragment of nuclear PLC-gamma1 is stimulated in vivo by epidermal growth factor nuclear signaling coincident with the time course of nuclear membrane epidermal growth factor receptor activation. Stimulated 120 kDa protein fragment interacts with PIKE, a nuclear GTPase, and together they form a complex with PI[3]kinase serving as a module for nuclear PI[3]K stimulation. Thus, the nucleus has its own IP(3) generating system.     

Different structural systems of the nucleus are targets for SV40 large T antigen

To define the interaction of SV40 large T with different structural systems in the nuclei of SV40-transformed cells (BALB/c mKSA), we have employed an in situ cell fractionation procedure leading to the preparation of the nuclear matrix, and giving rise to defined nuclear extracts comprising soluble nuclear proteins (nucleoplasm) and the solubilized chromatin. Large T could be detected in the nucleoplasmic fraction and in the chromatin fraction, as well as in tight association with the nuclear matrix. From the nuclear matrix, large T could be solubilized by treatment with a zwitterionic detergent. Different solubility properties, differences in the amount of the cellular phosphoprotein p53 coprecipitating with large T, and different stabilities in its association with the nuclear structural systems indicate that distinct subclasses of large T were isolated from their in vivo location in SV40-transformed cells.     

Dense granular bodies: a novel nucleoplasmic structure in hibernating dormice

Dense granular bodies (DGB) are particular structural constituents observed in cell nuclei of different tissues—liver, pancreas, brown adipose tissue, adrenal cortex—of hibernating dormice. They appear as strongly electron-dense clusters of closely packed granules, with thin fibrils spreading out at their periphery. DGB always occur in the nucleoplasm, sometimes making contact with other nuclear structural constituents typical of the hibernating state, such as coiled bodies, amorphous bodies and nucleoplasmic fibrils. DGB are present only during deep hibernation and rapidly disappear upon arousal from hibernation. Cytochemical and immunocytochemical analyses showed that DGB contain ribonucleoproteins and several nucleoplasmic RNA processing factors, suggesting that DGB can represent accumulation sites of splicing factors which are provided to splicing sites when normal metabolic activity is rapidly restored during arousal.     

Immunoelectron microscope localization of snRNP,hnRNP, and ribosomal proteins in mouse spermatogenesis

We have studied the ultrastructural distribution of heterogeneous nuclear ribonucleoproteins (hnRNPs), small nuclear ribonucleoproteins (snRNPs), and ribosomal proteins during mouse spermatogenesis and spermiogenesis by means of specific antibodies and immunocytochemistry. All the above components were detectable from primary spermatocytes until the spermatid elongation phase, when the RNA synthetic activity is known to cease. Ribosomal protein (P1/P2 and L7) labeling disappeared as early as during the acrosome phase, and nucleoli were no longer labeled even during the cap phase. The nucleoplasmic structures labeled with the different anti-nucleoplasmic RNP immunoprobes corresponded, until the acrosome phase, to those previously observed as targets of the same antibodies in the nucleoplasm of somatic cell nuclei. Clusters of interchromatin granules of spermatocyte and early spermatid nuclei exhibit some labeling for hnRNP when compared with nuclei of Sertoli cells or previously analyzed liver or tissue culture cells, where these structural constituents usually remain weakly labeled or unlabeled. In spermatids in step 10, another type of nuclear granule, resembling perichromatin granules, but occurring in aggregates, can be observed. These structural constituents were labeled with antibodies recognizing nucleoplasmic snRNP antigens and therefore suggesting a non-nucleolar origin of these granules. Finally, we have observed nucleoplasmic areas of fibrogranular material, occurring only in primary spermatocytes. These components were labeled with anti-ribosomal protein antibodies but did not contain either hnRNPs or snRNPs. © 1993 Wiley-Liss, Inc.     

Presence of the inositol 1,4,5-triphosphate receptor isoforms in the nucleoplasm

Although the inositol 1,4,5-triphosphate (IP(3))-induced nuclear Ca(2+) release has been shown to play key roles in nuclear functions, the presence of IP(3) receptor (IP(3)R)/Ca(2+) channels in the nucleoplasm has not been found. Recently, the IP(3)R/Ca(2+) channels were reported to exist in the nucleoplasmic reticulum structure, an extension of the nuclear envelope. Here we investigated the potential existence of the IP(3)Rs in the nucleoplasm and found the presence of all three IP(3)R isoforms in neuroendocrine and non-neuroendocrine cells. The IP(3)Rs were widely scattered in the nucleoplasm, localizing in both the heterochromatin and euchromatin regions.     

Identification of different roles for RanGDP and RanGTP in nuclear protein import.

The importin-alpha/beta heterodimer and the GTPase Ran play key roles in nuclear protein import. Importin binds the nuclear localization signal (NLS). Translocation of the resulting import ligand complex through the nuclear pore complex (NPC) requires Ran and is terminated at the nucleoplasmic side by its disassembly. The principal GTP exchange factor for Ran is the nuclear protein RCC1, whereas the major RanGAP is cytoplasmic, predicting that nuclear Ran is mainly in the GTP form and cytoplasmic Ran is in the GDP-bound form. Here, we show that nuclear import depends on cytoplasmic RanGDP and free GTP, and that RanGDP binds to the NPC. Therefore, import might involve nucleotide exchange and GTP hydrolysis on NPC-bound Ran. RanGDP binding to the NPC is not mediated by the Ran binding sites of importin-beta, suggesting that translocation is not driven from these sites. Consistently, a mutant importin-beta deficient in Ran binding can deliver its cargo up to the nucleoplasmic side of the NPC. However, the mutant is unable to release the import substrate into the nucleoplasm. Thus, binding of nucleoplasmic RanGTP to importin-beta probably triggers termination, i.e. the dissociation of importin-alpha from importin-beta and the subsequent release of the import substrate into the nucleoplasm.     

Localization of proteasomes and proteasomal proteolysis in the mammalian interphase cell nucleus by systematic application of immunocytochemistry

Proteasomes are ATP-driven, multisubunit proteolytic machines that degrade endogenous proteins into peptides and play a crucial role in cellular events such as the cell cycle, signal transduction, maintenance of proper protein folding and gene expression. Recent evidence indicates that the ubiquitin-proteasome system is an active component of the cell nucleus. A characteristic feature of the nucleus is its organization into distinct domains that have a unique composition of macromolecules and dynamically form as a response to the requirements of nuclear function. Here, we show by systematic application of different immunocytochemical procedures and comparison with signature proteins of nuclear domains that during interphase endogenous proteasomes are localized diffusely throughout the nucleoplasm, in speckles, in nuclear bodies, and in nucleoplasmic foci. Proteasomes do not occur in the nuclear envelope region or the nucleolus, unless nucleoplasmic invaginations expand into this nuclear body. Confirmedly, proteasomal proteolysis is detected in nucleoplasmic foci, but is absent from the nuclear envelope or nucleolus. The results underpin the idea that the ubiquitin-proteasome system is not only located, but also proteolytically active in distinct nuclear domains and thus may be directly involved in gene expression, and nuclear quality control.     

Calcium microdomains in mitochondria and nucleus

Endomembranes modify the progression of the cytosolic Ca(2+) wave and contribute to generate Ca(2+) microdomains, both in the cytosol and inside the own organella. The concentration of Ca(2+) in the cytosol ([Ca(2+)](C)), the mitochondria ([Ca(2+)](M)) and the nucleus ([Ca(2+)](N)) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca(2+) from the high [Ca(2+)](C) microdomains generated during cell activation near Ca(2+) channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca(2+) signal to the bulk cytosol. This shaping of [Ca(2+)](C) signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca(2+)](M) signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca(2+)](C) signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca(2+)](N) signals could be generated by direct release of stored Ca(2+) into the nucleoplasm. Ca(2+) release could even be restricted to subnuclear domains. Putative Ca(2+) stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca(2+) into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain.     

Evidence for Ca2+- and ATP-sensitive peripheral channels in nuclear pore complexes.

In eukaryotic cells the nuclear envelope (NE) serves as a functional barrier between cytosol and nucleoplasm perforated by nuclear pore complexes (NPCs). Both active and passive transport of ions and macromolecules are thought to be mediated by the centrally located large NPC channel. However, 3-dimensional imaging of NPCs based on electron microscopy indicates the existence of additional small channels of unknown function located in the NPC periphery. By means of the recently developed nuclear hourglass technique that measures NE electrical conductance, we evaluated passive electrically driven transport through NPCs. In isolated Xenopus laevis oocyte nuclei, we varied ambient Ca2+ and ATP in the cytosolic solution and/or chelated Ca2+ in the perinuclear stores in order to assess the role of Ca2+ in regulating passive ion transport. We noticed that NE electrical conductance is large under conditions where macromolecule permeability is known to be low. In addition, atomic force microscopy applied to native NPCs detects multiple small pores in the NPC periphery consistent with channel openings. Peripheral pores were detectable only in the presence of ATP. We conclude that NPC transport of ions and macromolecules occurs through different routes. We present a model in which NE ion flux does not occur through the central NPC channel but rather through Ca2+- and ATP-activated peripheral channels of individual NPCs.     

The germinal vesicle nucleus of Xenopus laevis oocytes as a selective storage receptacle for proteins

The amorphous nucleoplasm of the germinal vesicle nucleus of Xenopus laevis oocytes has been selectively extracted under conditions which leave the nuclear formed elements morphologically intact. The nucleoplasm contains about 97% of the total nuclear proteins and on SDS- polyacrylamide gels some 68 polypeptides can be distinguished. On the basis of solubility differences, the nucleoplasmic proteins can be classified into two categories. The first consists of soluble or easily solubilized proteins which comprise about 34 polypeptides making up 87% of the nucleoplasm. A few of these proteins show electrophoretic mobilities similar to those of soluble proteins of the cytoplasm, but most are unique to the nucleus. The residual 13% of the nucleoplasmic proteins are tightly bound to a nucleoplasmic gel and can be extracted only by solubilizing the gel. The solubility characteristics of the proteinaceous gel suggest a complex held together by salt, nonpolar, hydrogen, and possibly disulfide bonding. Some 34 polypeptides can be distinguished in this gel fraction, including prominent and highly enriched polypeptides of about 115,000 and 46,000 daltons. The relatively soluble fraction of the nucleoplasm does not contain informofers and contains little or no nucleic acid. Evidence is presented that if histones are present in the germinal vesicle, they can comprise no more than about 8% of the total protein. The possibility is discussed that the unique polypeptides of the nucleoplasm may be sequestered there by selective adsorption to or in the nuclear gel.     

Nuclear proteins. II. Similarity of nonhistone proteins in nuclear sap and chromatin, and essential absence of contractile proteins from mouse liver nuclei

High resolution SDS slab gel electrophoresis has been used to examine the distribution of nonhistone proteins (NHP) in the saline-EDTA, Tris, and 0.35 M NaCl washes of isolated mouse liver nuclei. These studies led to the following conclusions: (a) all the prominent NHP which remain bound to DNA are also present in somewhat similar proportions in the saline-EDTA, Tris, and 0.35 M NaCl washes of nuclei; (b) a protein comigrating with actin is prominent in the first saline-EDTA wash of nuclei, but present as only a minor band in the subsequent washes and on washed chromatin; (c) the presence of nuclear matrix proteins in all the nuclear washes and cytosol indicates that these proteins are distributed throughout the cell; (d) a histone-binding protein (J2) analogous to the HMG1 protein of K. V. Shooter, G.H. Goodwin, and E.W. Johns (Eur J. Biochem. 47:236-270) is a prominent nucleoplasmic protein; (e) quantitation of the major NHP indicates that they are present in a range of 2.2 X 10(5)-5.2 X 10(6) copies per diploid nucleus. Most of the electrophoretically visible NHP are probably structural rather than regulatory proteins; (f) actin, myosin, tubulin, and tropomyosin, if present at all, constitute a very minor fraction of the nuclear NHP. Contractile proteins constitute a major portion of the NHP only when the chromatin is prepared from crude cell lysates instead of from purified nuclei. These studies support the conclusion that there are no clear differences between many nucleoplasmic and chromatin- bound nonhistone proteins. Except for the histones, many of the intranuclear proteins appear to be in equilibrium between DNA, HnRNA, and the nucleoplasm.     

Visualization of biphasic Ca2+ diffusion from cytosol to nucleus in contracting adult rat cardiac myocytes with an ultra-fast confocal imaging system.

In contracting cardiac myocytes, the rapid changes in cytosolic and nuclear Ca2+ make it difficult to determine whether the nuclear Ca2+ transient is caused by diffusion from the cytosol or by Ca2+ release channels on the inner nuclear membrane, or both. The propagation mechanism in the nucleoplasm also remains unknown. We have developed an ultra-fast Nipkow confocal imaging system able to acquire two-dimensional images at approximately 4 ms/full frame speed and employed it to analyze Ca2+ waves and the dynamics of the cytosolic and nuclear Ca2+ transients after electrical stimulation of cardiac myocytes. The pattern of nuclear Ca2+ upon stimulation was well described by a mathematical model of Ca2+ diffusion across the nuclear envelope. No evidence of Ca2+ release from perinuclear Ca2+ stores was obtained. The Ca2+ diffusion constant appeared to change during contraction, with essentially free diffusion of Ca2+ through nuclear pore complexes at low cytosolic Ca2+ and partially restricted diffusion at high cytosolic Ca2+. The Ca2+ in the nucleoplasm propagated by diffusion and no Ca2+ release phenomena were seen in the nucleus.     

Disruption of Nuclear Lamin Organization Alters the Distribution of Replication Factors and Inhibits DNA Synthesis

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase α, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.