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PubMed2005

Aspects of nuclear envelope dynamics in mitotic cells.

Major features of the nuclear envelope (NE) are a pair of inner and outer nuclear membranes (INM, ONM) spanned by nuclear pore complexes. While the composition of the ONM resembles that of the endoplasmic reticulum, the INM contains a unique spectrum of proteins. Localization of INM proteins involves a mechanism of selective retention whereby integral proteins are immobilized and concentrated by virtue of interactions with nuclear components. In the case of emerin, INM localization involves interaction with A-type lamins. Interactions between membrane proteins may also play a significant role in INM localization. This conclusion stems from studies on nesprins, a family of membrane proteins that feature a large cytoplasmic domain, a single C-terminal membrane-spanning domain and a small lumenal domain. The nesprin membrane anchor and lumenal (KASH) domains are related to the Drosophila Klarsicht protein. Evidence is emerging that this KASH region interacts with other NE proteins and may influence their distributions. Overexpression of GFP-KASH causes loss of emerin and LAP2 from the NE. This is not due to global reorganization of the NE since LAP1 as well as lamins and NPCs remain unaffected. Our results suggest that interactions between NE membrane components are far more extensive and complex than current models suggest.

Animals↗
PubMed2001

Nuclear envelope disorganization in fibroblasts from lipodystrophic patients with heterozygous R482Q/W mutations in the lamin A/C gene.

Dunnigan-type familial partial lipodystrophy (FPLD), characterized by an abnormal body fat redistribution with insulin resistance, is caused by missense heterozygous mutations in A-type lamins (lamins A and C). A- and B-type lamins are ubiquitous intermediate filament proteins that polymerize at the inner face of the nuclear envelope. We have analyzed primary cultures of skin fibroblasts from three patients harboring R482Q or R482W mutations. These cells were euploid and able to cycle and divide. A subpopulation of these cells had abnormal blebbing nuclei with A-type lamins forming a peripheral meshwork, which was frequently disorganized. Inner nuclear membrane protein emerin, an A-type lamin-binding protein, strictly colocalized with this abnormal meshwork. Cells from lipodystrophic patients often had other nuclear envelope defects, mainly consisting of nuclear envelope herniations that were deficient in B-type lamins, nuclear pore complexes, lamina-associated protein 2 beta, and chromatin. The mechanical properties of nuclear envelopes were altered, as judged from the extensive deformations observed in nuclei from heat-shocked cells, and from the low stringency of extraction of their components. These structural nuclear alterations were caused by the lamins A/C mutations, as the same changes were introduced in human control fibroblasts by ectopic expression of R482W mutated lamin A.

Adult↗
PubMed2005

Relationship between RNA synthesis and the Ca2+-filled state of the nuclear envelope store.

RNA synthesis and ATP-dependent (45)Ca(2+) uptake were measured simultaneously in isolated nuclear fraction of rat liver nuclei. Maximal level of RNA synthesis was obtained under ATP-dependent (45)Ca(2+)-uptake conditions (1 microM free [Ca(2+)] and 1 mM ATP in the bathing solution). This experimental condition was defined as "stimulated nuclei" condition. ATP-dependent (45)Ca(2+) uptake was inhibited using different strategies including: (a) eliminating Ca(2+) (1 mM EGTA); (b) lowering the ATP concentration; (c) modifying nuclear envelope membranes Ca(2+) permeability (Ca(2+) ionophores); or (d) inhibiting the nuclear Ca(2+) pump (thapsigargin and 3',3'',5',5''-tetraiodophenolsulfonephthalein). Under all the above conditions, RNA synthesis was lower than in "stimulated nuclei" condition. In the presence of ionomycin, RNA synthesis was significantly higher at 500 nM free [Ca(2+)], as compared with RNA synthesis in a Ca(2+)-free medium or at 1muM free [Ca(2+)]. However, even in such condition (500 nM free [Ca(2+)]), RNA synthesis was lower than RNA synthesis obtained in "stimulated nuclei" condition. We suggest two components for the effect of Ca(2+) on RNA synthesis: (A) a direct effect of nucleoplasmic [Ca(2+)]; and (B) an effect dependent on the accumulation of Ca(2+) in the nuclear envelope store mediated by the SERCA nuclear Ca(2+) pump.

Adenosine Triphosphate↗
PubMed1978

In vitro oxidation of intrinsic sulfhydryl groups yields polymers of the two predominant polypeptides in the nuclear envelope fraction.

The two quantitatively predominant nuclear nonhistone polypeptides of the avian erythrocyte comprise a significant portion of the nuclear envelope protein. Each of these two polypeptides can be converted almost completely to homogeneous polymeric species via oxidation of intrinsic sulfhydryl groups by omicron-phenanthroline cupric ion complex. Major products are two dimeric species arising from each of the approximately 77 000-dalton pair. The larger of the two polypeptides also yields oligomers greater than dimers. These observations extend out earlier solubility and polypeptide cross-linking evidence which indicated that these polypeptides were involved in an oligomeric, proteinaceous nuclear structure. Ultramicroscopic studies from other laboratories indicate that such a structure may function as a nuclear skeleton. The architectural details of the structure now appear to be accessible at the molecular level.

Animals↗
PubMed1955

The nuclear envelope; its structure and relation to cytoplasmic membranes.

An electron microscope study of thin sections of interphase cells has revealed the following:- Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.

Animals↗
PubMed2025

Unraveling Plant Nuclear Envelope Composition Using Proximity Labeling Proteomics.

The nuclear envelope (NE) defines the eukaryotic cell and functions in a myriad of fundamental cellular processes including but not limited to signal transduction, lipid metabolism, chromatin organization, and nucleocytoplasmic transportation. Although the general structure of the NE is well-conserved across eukaryotic kingdoms, its composition and functions vary substantially between species and remain largely unknown in plants. In this chapter, we describe a proximity-labeling-based proteomic approach to profile novel NE components in the model organism Arabidopsis. This method is generally suitable for the identification of protein components in subcellular compartments or protein complexes that are poorly accessible to traditional mass spectrometry approaches and can be easily applied to other plant species. In addition to giving a step-by-step detailed description of the proximity labeling proteomics procedure in plant samples, we also provide guidelines on the appropriate use of controls and statistical analysis to achieve a highly specific selection of probed candidates.

Proteomics↗
PubMed1983

Localization of a major nuclear envelope protein by differential solubilization.

The nuclear envelope (NE) is the interface of the two major compartments of the cell. We used differential solubilization in conjunction with ultrastructural visualization to localize components of the NE in the surf clam Spisula solidissima. The high salt-resistant NE fraction can be separated into a pore complex-containing supernatant (4 M urea extract) and a membrane pellet devoid of pore complexes or pore remnants. Urea extraction of the membrane pellet reveals two major proteins with an apparent molecular weight (MWapp) of 67 000 (clam lamin) and 200 000 that are also found in the high-salt and detergent-extracted NE containing pore complexes. Urea extraction of the clam NE under reducing conditions removes the clam lamin. The 200 000 D protein remaining in the NE after removal of the pore complex is not solubilized by detergent extraction and thus can be localized on the inner nuclear part of the NE.

Animals↗
PubMed1999

The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii.

Morphological examination of the highly polarized protozoan parasite Toxoplasma gondii suggests that secretory traffic in this organism progresses from the endoplasmic reticulum to the Golgi apparatus using the nuclear envelope as an intermediate compartment. While the endoplasmic reticulum is predominantly located near the basal end of the parasite, the Golgi is invariably adjacent to the apical end of the nucleus, and the space between the Golgi and nuclear envelope is filled with numerous coatomer-coated vesicles. Staining with antiserum raised against recombinant T. gondii beta-COP confirms its association with the apical juxtanuclear region. Perturbation of protein secretion using brefeldin A, microtubule inhibitors or dithiothreitol disrupts the Golgi, causing swelling of the nuclear envelope, particularly at its basal end. Prolonged drug treatment leads to gross distention of the endoplasmic reticulum, filling the basal end of the parasite. Cloning and sequencing of the T. gondii homolog of the chaperonin protein BiP identifies the carboxy-terminal amino acid sequence HDEL as this organism's endoplasmic reticulum-retention signal. Appending the HDEL motif to a recombinant secretory protein (a chimera between the parasite's major surface protein fusion, P30, and the Green Fluorescent Protein) causes this secretory reporter to be retained intracellularly. P30-GFP-HDEL fluorescence was most intense within the nuclear envelope, particularly at the apical end. These data support a model of secretion in which protein traffic from the endoplasmic reticulum to Golgi occurs via the apical end of the nuclear envelope.

Animals↗
PubMed1995

5-Lipoxygenase is located in the euchromatin of the nucleus in resting human alveolar macrophages and translocates to the nuclear envelope upon cell activation.

5-Lipoxygenase (5-LO) and 5-lipoxygenase-activating protein (FLAP) are two key proteins involved in the synthesis of leukotrienes (LT) from arachidonic acid. Although both alveolar macrophages (AM) and peripheral blood leukocytes (PBL) produce large amounts of LT after activation, 5-LO translocates from a soluble pool to a particulate fraction upon activation of PBL, but is contained in the particulate fraction in AM irrespective of activation. We have therefore examined the subcellular localization of 5-LO in autologous human AM and PBL collected from normal donors. While immunogold electron microscopy demonstrated little 5-LO in resting PBL, resting AM exhibited abundant 5-LO epitopes in the euchromatin region of the nucleus. The presence of substantial quantities of 5-LO in the nucleus of resting AM was verified by cell fractionation and immunoblot analysis and by indirect immunofluorescence microscopy. In both AM and PBL activated by A23187, all of the observable 5-LO immunogold labeling was found associated with the nuclear envelope. In resting cells of both types, FLAP was predominantly associated with the nuclear envelope, and its localization was not affected by activation with A23187. The effects of MK-886, which binds to FLAP, were examined in ionophore-stimulated AM and PBL. Although MK-886 inhibited LT synthesis in both cell types, it failed to prevent the translocation of 5-LO to the nuclear envelope. These results indicate that the nuclear envelope is the site at which 5-LO interacts with FLAP and arachidonic acid to catalyze LT synthesis in activated AM as well as PBL, and that in resting AM the euchromatin region of the nucleus is the predominant source of the translocated enzyme. In addition, LT synthesis is a two-step process consisting of FLAP-independent translocation of 5-LO to the nuclear envelope followed by the FLAP-dependent activation of the enzyme.

Arachidonate 5-Lipoxygenase↗
PubMed1995

Two different subunits of importin cooperate to recognize nuclear localization signals and bind them to the nuclear envelope.

BACKGROUND: Selective protein import into the cell nucleus occurs in two steps: binding to the nuclear envelope, followed by energy-dependent transit through the nuclear pore complex. A 60 kD protein, importin, is essential for the first nuclear import step, and the small G protein Ran/TC4 is essential for the second. We have previously purified the 60kD importin protein (importin 60) as a single polypeptide. RESULTS: We have identified importin 90, a 90 kD second subunit that dissociates from importin 60 during affinity chromatography on nickel (II)-nitrolotriacetic acid-Sepharose, a technique that was originally used to purify importin 60. Partial amino-acid sequencing of Xenopus importin 90 allowed us to clone and sequence its human homologue; the amino-acid sequence of importin 90 is strikingly conserved between the two species. We have also identified a homologous budding yeast sequence from a database entry. Importin 90 potentiates the effects of importin 60 on nuclear protein import, indicating that the importin complex is the physiological unit responsible for import. To assess whether nuclear localization sequences are recognized by cytosolic receptor proteins, a biotin-tagged conjugate of nuclear localization signals linked to bovine serum albumin was allowed to form complexes with cytosolic proteins in Xenopus egg extracts; the complexes were then retrieved with streptavidin-agarose. The pattern of bound proteins was surprisingly simple and showed only two predominant bands: those of the importin complex. We also expressed the human homologue of importin 60, Rch1p, and found that it was able to replace its Xenopus counterpart in a functional assay. We discuss the relationship of importin 60 and importin 90 to other nuclear import factors. CONCLUSIONS: Importin consists of a 60 and a 90 kD subunit. Together, they constitute a cytosolic receptor for nuclear localization signals that enables import substrates to bind to the nuclear envelope.

Amino Acid Sequence↗
PubMed2006

Hominoid-specific SPANXA/D genes demonstrate differential expression in individuals and protein localization to a distinct nuclear envelope domain during spermatid morphogenesis.

Human sperm protein associated with the nucleus on the X chromosome consists of a five-member gene family (SPANXA1, SPANXA2, SPANXB, SPANXC and SPANXD) clustered at Xq27.1. Evolved from an ancestral SPANX-N gene family (at Xq27 and Xp11) present in all primates as well as in rats and mice, the SPANXA/D family is present only in humans, bonobos, chimpanzees and gorillas. Among hominoid-specific genes, the SPANXA/D gene family is considered to be undergoing rapid positive selection in its coding region. In this study, RT-PCR of human testis mRNA from individuals showed that, although all SPANXA/D genes are expressed in humans, differences are evident. In particular, SPANXC is expressed only in a subset of men. The SPANXa/d protein localized to the nuclear envelope of round, condensing and elongating spermatids, specifically to regions that do not underlie the developing acrosome. During spermiogenesis, the SPANXa/d-positive domain migrated into the base of the head as the redundant nuclear envelope that protrudes into the residual cytoplasm. Post-testicular modification of the SPANXa/d proteins was noted, as were PEST (proline, glutamic acid, serine, and threonine rich regions) domains. It is concluded that the duplication of the SPANX-N gene family that occurred 6-11 MYA resulted in a new gene family, SPANXA/D, that plays a role during spermiogenesis. The SPANXa/d gene products are among the few examples of X-linked nuclear proteins expressed following meiosis. Their localization to non-acrosomal domains of the nuclear envelope adjacent to regions of euchromatin and their redistribution to the redundant nuclear envelope during spermiogenesis provide a biomarker for the redundant nuclear envelope of spermatids and spermatozoa.

Acrosome↗
PubMed1991

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.

Adenosine Triphosphate↗
PubMed1997

The spindle pole body of Schizosaccharomyces pombe enters and leaves the nuclear envelope as the cell cycle proceeds.

The cycle of spindle pole body (SPB) duplication, differentiation, and segregation in Schizosaccharomyces pombe is different from that in some other yeasts. Like the centrosome of vertebrate cells, the SPB of S. pombe spends most of interphase in the cytoplasm, immediately next to the nuclear envelope. Some gamma-tubulin is localized on the SPB, suggesting that it plays a role in the organization of interphase microtubules (MTs), and serial sections demonstrate that some interphase MTs end on or very near to the SPB. gamma-Tubulin is also found on osmiophilic material that lies near the inner surface of the nuclear envelope, immediately adjacent to the SPB, even though there are no MTs in the interphase nucleus. Apparently, the MT initiation activities of gamma-tubulin in S. pombe are regulated. The SPB duplicates in the cytoplasm during late G2 phase, and the two resulting structures are connected by a darkly staining bridge until the mitotic spindle forms. As the cell enters mitosis, the nuclear envelope invaginates beside the SPB, forming a pocket of cytoplasm that accumulates dark amorphous material. The nuclear envelope then opens to form a fenestra, and the duplicated SPB settles into it. Each part of the SPB initiates intranuclear MTs, and then the two structures separate to lie in distinct fenestrae as a bipolar spindle forms. Through metaphase, the SPBs remain in their fenestrae, bound to the polar ends of spindle MTs; at about this time, a small bundle of cytoplasmic MTs forms in association with each SPB. These MTs are situated with one end near to, but not on, the SPBs, and they project into the cytoplasm at an orientation that is oblique to the simple axis. As anaphase proceeds, the nuclear fenestrae close, and the SPBs are extruded back into the cytoplasm. These observations define new fields of enquiry about the control of SPB duplication and the dynamics of the nuclear envelope.

Cell Cycle↗
PubMed2006

[Unusual nucleotide sequence of a DNA fragment isolated from nuclear envelopes of mouse hepatocytes].

Fragments of chromosomal DNA isolated from nuclear envelopes of mouse hepatocytes were previously cloned and partially characterized in our laboratory. One of the cloned fragments (EnvM4) had unusual characteristics: abundant representability in the clone library (about 50%) and homology with DNA of archebacteria. An analysis of nucleotide sequence of this fragment conducted in more detail allowed the detection of an evolutionary conserved region present in the genomes of various organisms (from bacteria to human). Other characteristics of fragment EnvM4 suggest a possible functional role of chromosomal DNA regions determining the attachment of chromosomes to the nuclear envelope.

Animals↗
PubMed1981

Nuclear envelope invaginations in hamster pyramidal cells during development and aging.

Nuclear envelope invaginations were observed in pyramidal cell nuclei of the hamster frontal cortex during development and aging. These invaginations which began to appear at 10 day did not recede at maturity as has been observed in certain other cell types, but persisted in the adult hamster and during subsequent aging. Morphometric data showed a significant increase in the number of nuclear envelope invaginations and in their length per unit of the nucleus. This increase was positively correlated with age until 500 days and is suggestive of a continued high metabolic activity that did not subside following the rapid growth phase of the pyramidal neurons.

Aging↗
PubMed1984

Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.

Animals↗
PubMed1983

Insulin receptor antiserum and plant lectins mimic the direct effects of insulin on nuclear envelope phosphorylation.

Insulin directly inhibits protein phosphorylation in isolated rat liver nuclear envelopes. In the present studies, an antiserum to insulin receptor as well as the plant lectins concanavalin A and phytohemagglutinin mimicked insulin action in isolated nuclear envelopes. These studies suggest that insulin and agents that mimic it may directly regulate nuclear functions.

Animals↗