Mechanism for the selection of nuclear polypeptides in Xenopus oocytes

The function of the nuclear envelope in regulating the cellular distribution of proteins was studied by experimentally altering nuclear permeability and determing the effect of the procedure on the incorporation of exogenous and endogenous polypeptides into the nucleoplasm. Using fine glass needles, nuclear envelopes were disrupted by puncturing oocytes in that region of the animal pole occupied by the germinal vesicle. This resulted in a highly significant increase in the nuclear uptake of cytoplasmically injected [125I]-bovine serum albumin ([125I]BSA), deomonstrating that the envelopes had lost their capacity to act as effective barriers to the diffusion of macromolecules. Endogenous proteins were labeled by incubating oocytes in L-[3H]lecuine. After appropriate intervals, nuclei were isolated from punctured and control cells and analyzed for tritiated polypeptides. Both total precipitable counts and the proportion of label in different size classes of polypeptides were compared. The results showed that puncturing the oocytes had no apparent quantitative or qualitative effects on the uptake of endogenous polypeptides by the nuclei. It can be concluded that the accumulation of specific nuclear proteins is not controlled by the envelope but rather by selective binding within the nucleoplasm.     

The function of the nuclear envelope in nuclear protein accumulation

The mechanism by which proteins accumulate in the cell nucleus is not yet known. Two alternative mechanisms are discussed here: (a) selective unidirectional entry of karyophilic proteins through the nuclear pores, and (b) free diffusion of all proteins through the nuclear pores and specific binding of nuclear proteins to nondiffusible components of the nucleoplasm. We present experiments designed to distinguish between these alternatives. After mechanical injury of the Xenopus oocyte nuclear envelope, nuclear proteins were detected in the cytoplasm by immunohistochemical methods. In a second approach, nuclei from X. borealis oocytes were isolated under oil, the nuclear envelopes were removed, and the pure nucleoplasm was injected into the vegetal pole of X. laevis oocytes. With immunohistochemical methods, it was found that each of five nuclear proteins rapidly diffuses out of the injected nucleoplasm into the surrounding cytoplasm. The subsequent transport and accumulation in the intact host nucleus could be shown for the nuclear protein N1 with the aid of a species-specific mAb that reacts only with X. borealis N1. Purified and iodinated nucleoplasmin was injected into the cytoplasm of Xenopus oocytes and its uptake into the nucleus was studied by biochemical methods.     

The localization of the nuclear protein pool in Xenopus oocytes

It has been demonstrated previously that nuclear proteins in Xenopus oocytes are synthesized in the cytoplasm and maintained in a cellular pool. The present study was performed to determine if any portion of this pool is associated specifically with the nuclear envelope. This was accomplished by first micro-injecting oocytes with [³H]leucine; at various times after injection, nuclear envelope and nucleoplasmic fractions were run on SDS-polyacrylamide gels. In this way labeled polypeptides available in the envelope fraction could be compared to polypeptides which were subsequently incorporated into the nucleoplasm. No evidence was obtained that the nuclear protein pool is associated with the envelope.     

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.     

Identification of specific polypeptides of the nuclear envelope by iodination of mouse liver nuclei.

A sensitive technique is described for the rapid identification of nuclear-envelope proteins. Mouse liver nuclei (purified on sucrose gradients) were iodinated with Na125I by the immobilized water-insoluble reagent Iodogen. Iodinated nuclei were digested with RNAase A and DNAase I and then salt-extracted to obtain labelled nuclear envelopes. Nuclear envelopes were characterized by morphological and biochemical criteria and by SDS/polyacrylamide-gel electrophoresis. In all, 13 polypeptides of molecular masses 145, 115, 98, 85, 75, 70, 65, 54, 50, 45, 40, 38 and 36 kDa were identified in the labelled nuclear envelopes. The labelled polypeptides were localized to the nuclear envelope by extraction of the envelope with Triton X-100 and different concentrations of salt. Iodination of intact nuclei was shown to be specific for the nuclear envelope by the absence of labelling of histones and cytoplasmic contaminants.     

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.     

In vivo analysis of Cajal body movement, separation, and joining in live human cells

Cajal bodies (also known as coiled bodies) are subnuclear organelles that contain specific nuclear antigens, including splicing small nuclear ribonucleoproteins (snRNPs) and a subset of nucleolar proteins. Cajal bodies are localized in the nucleoplasm and are often found at the nucleolar periphery. We have constructed a stable HeLa cell line, HeLa(GFP-coilin), that expresses the Cajal body marker protein, p80 coilin, fused to the green fluorescent protein (GFP-coilin). The localization pattern and biochemical properties of the GFP-coilin fusion protein are identical to the endogenous p80 coilin. Time-lapse recordings on 63 nuclei of HeLa(GFP-coilin) cells showed that all Cajal bodies move within the nucleoplasm. Movements included translocations through the nucleoplasm, joining of bodies to form larger structures, and separation of smaller bodies from larger Cajal bodies. Also, we observed Cajal bodies moving to and from nucleoli. The data suggest that there may be at least two classes of Cajal bodies that differ in their size, antigen composition, and dynamic behavior. The smaller size class shows more frequent and faster rates of movement, up to 0.9 microm/min. The GFP-coilin protein is dynamically associated with Cajal bodies as shown by changes in their fluorescence intensity over time. This study reveals an unexpectedly high level of movement and interactions of nuclear bodies in human cells and suggests that these movements may be driven, at least in part, by regulated mechanisms.     

Nuclear ribonucleoprotein complexes of amphibian liver. II. Changes in the protein moiety during development.

Ribonucleoprotein complexes composed of small molecular weight nuclear RNA (4--9 S) and proteins were isolated from hepatic nuclei of Rana catesbeiana (bullfrog) and the protein moiety of this nuclear ribonucleoprotein complex compared during different stages of development. SDS-polyacrylamide gel analysis of premetamorphic tadpoles and adult frog nuclear ribonucleoprotein complexes revealed that while the protein profiles of these two particles were very similar polypeptides of 47,000, 70,000, and 11,000 molecular weight were present in significantly higher concentrations in the frog ribonucleoprotein complexes. Comparison of the chromatin proteins isolated from these two developmental stages demonstrated that these three polypeptides of frog ribonucleoprotein were not contaminants from chromatin. Since these three polypeptides could not be preferentially extracted from the frog ribonucleoprotein complex by 0.5 M KCl or 1 M urea, it was unlikely that these polypeptides were bound nonspecifically to the ribonucleoprotein particle. Polypeptide analysis of the nuclear ribonucleoprotein complexes isolated from tadpoles immersed in the thyroid hormone L-thyroxine revealed an increase in two polypeptides of 37,000 and 45,000 molecular weight during metamorphosis. The absence of reduced amount of these two polypeptides in either the premetamorphic tadpole or adult frog demonstrated that their presence in Rana catesbeiana nuclear ribonucleoprotein was transient during development and specifically associated with tadpole metamorphosis. We conclude from these experiments that the nuclear ribonucleoprotein complex is a dynamic structure during Rana catesbeiana development and that specific changes in its protein composition are associated with discrete stages of amphibian development.     

Identification of nuclear envelope proteins and glycoproteins which co-isolate with the nuclear protein matrix

Nuclear envelopes and nuclear matrices were isolated from rat liver nuclei. Although differences in polypeptide composition of the structures are evident on SDS gel electrophoresis, they have an almost identical distribution of concanavalin A-binding glycoproteins. These matrix-associated concanavalin A-binding glycoproteins derive entirely from the nuclear envelope and are recovered almost quantitatively in the matrix. They constitute easily identifiable markers for nuclear envelope association with matrix or other nuclear subfractions. Surface labelling of nuclei with 125I using solid-phase lactoperoxidase further confirmed that a large number of envelope-associated nuclear surface proteins co-isolate with the matrix. Protein kinase activity, as well as endogenous substrates for the kinase(s) are shown to be the same in both envelopes and matrix. Envelope-derived proteins and glycoproteins may comprise a substantial proportion of total matrix protein.     

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.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [gamma-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [gamma-32P]GTP, low levels of [gamma-32P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.     

Late nonstructural 100,000- and 33,000-dalton proteins of adenovirus type 2. I. Subcellular localization during the course of infection.

We analyzed the subcellular locations of the late adenovirus type 2 nonstructural 100,000-dalton (100K) and 33K proteins in adenovirus type 2-infected HeLa cells both by biochemical cell fractionation and by immunofluorescence microscopy, using specific antisera against purified sodium dodecyl sulfate-denatured 100K and 33K polypeptides. Both methods showed that the 100K protein was present in the cytoplasm as well as in the nuclei of infected cells and that it accumulated in the nuclei during the course of infection. Phosphorylated 100K protein also was found both in the cytoplasm and in nuclei. However, the nuclear 100K protein pool was phosphorylated to a higher degree than the cytoplasmic pool. In all experiments the 33K protein, which also is a phosphoprotein, was present exclusively in the nuclei of infected cells. The 100K and 33K proteins were associated with different nuclear substructures; this was demonstrated serologically by an analysis of infected cells in which double color immunofluorescence microscopy was used. In these experiments antibodies against the 100K protein decorated different nuclear structures than antibodies against the 33K protein.     

Nonlamin components of the lamina: a paucity of proteins.

Current models of nuclear organization propose that nuclear functions are modulated in part by reversible tethering of chromatin loops to structural elements of the nucleoplasm and the nuclear envelope. Lamins are the best-characterized proteins of the lamina portion of the nuclear envelope and are involved in binding chromatin to the inner nuclear membrane. However, they are not a universal feature of eukaryotic nuclei and do not account fully for the putative functions of the lamina in all organisms. It is possible that nonlamin components of the lamina may substitute for lamins in organisms from which they are absent and modify the properties of lamins during development and the cell cycle. We review the properties of the relatively small number of such components that have been reported, including the young arrest (fs(1)Ya) protein of Drosophila, statin, circumferin, and the MAN antigens. The experimental evidence indicates they are a diverse group of proteins, and that at least some have the potential to modulate the interactions of chromatin, lamins, and the nuclear membranes.     

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.     

Spindle formation in Aspergillus is coupled to tubulin movement into the nucleus

In many important organisms, including many algae and most fungi, the nuclear envelope does not disassemble during mitosis. This fact raises the possibility that mitotic onset and/or exit might be regulated, in part, by movement of important mitotic proteins into and out of the nucleoplasm. We have used two methods to determine whether tubulin levels in the nucleoplasm are regulated in the fungus Aspergillus nidulans. First, we have used benomyl to disassemble microtubules and create a pool of free tubulin that can be readily observed by immunofluorescence. We find that tubulin is substantially excluded from interphase nuclei, but is present in mitotic nuclei. Second, we have observed a green fluorescent protein/alpha-tubulin fusion in living cells by time-lapse spinning-disk confocal microscopy. We find that tubulin is excluded from interphase nuclei, enters the nucleus seconds before the mitotic spindle begins to form, and is removed from the nucleoplasm during the M-to-G1 transition. Our data indicate that regulation of intranuclear tubulin levels plays an important, perhaps essential, role in the control of mitotic spindle formation in A. nidulans. They suggest that regulation of protein movement into the nucleoplasm may be important for regulating mitotic onset in organisms with intranuclear mitosis.     

The effect of carboxyl-terminal deletions on the nuclear transport rate of rat hsc70.

Rat brain hsc70 is a constitutively expressed member of the 70-kDa family of heat shock proteins that is capable of bidirectional transport across the nuclear envelope when microinjected into Xenopus oocytes [1]. The objective of this study was to identify domains involved in its bidirectional transport. Limited proteolytic digestion with chymotrypsin generated three major truncated proteins of approximately 67.5, 59.5, and 56.5 kDa. Reactivity with NH2-terminal-specific antibodies showed that carboxyl-terminal fragments were removed. Nuclear uptake studies were performed by microinjecting 125I-labeled proteins into the cytoplasm and determining their subsequent nucleocytoplasmic distribution. The accumulation rates, while faster than bovine serum albumin controls, were inversely related to the size of the truncated proteins and greatly reduced compared to undigested hsc70. Nuclear efflux was assayed by microinjecting labeled proteins directly into oocyte nuclei. The relative efflux rates of the truncated polypeptides were less than the undigested protein, and, as observed for uptake, were inversely related to size. These results indicate that the carboxyl-terminal domain of hsc70 is involved in its bidirectional exchange.     

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。