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PubMed1984

Photoaffinity labeling of the major nucleosidetriphosphatase of rat liver nuclear envelope.

We employed the photoaffinity probe 8-azido-adenosine 5'-triphosphate (aATP) to identify the nuclear envelope (NE) nucleosidetriphosphatase activity (NTPase) implicated in control of RNA transport. The photoprobe was hydrolyzed at rates comparable to those for ATP, with a Michaelis constant of 0.225 mM. Photolabeling was dependent upon UV irradiation (300-nm max) and was not affected by quercetin. Unlabeled ATP or GTP competed with [32P]aATP in photolabeling experiments, and UTP was a less effective competitor, paralleling the substrate specificity of the NTPase. Incubation of NE with aATP led to a UV, time, and concentration dependent irreversible inactivation of NTPase. The inactivation could be blocked by ATP or GTP. Polyacrylamide gel electrophoresis and autoradiography of photolabeled NE showed selective, UV-dependent labeling of a 46-kDa protein with both [gamma-32P]aATP and [alpha-32P]aATP. This band was not labeled with [gamma-32P]ATP. Since the NE NTPase implicated in RNA transport is modulated by RNA, we examined the effects of RNA on the labeling process. Removal of RNA from the NE preparations (by RNase/DNase digestion) reduced NTPase by 30-40% and eliminated photolabeling of the 46-kDa band. Addition of yeast RNA to such preparations increased NTPase activity to control levels and selectively reinstated photolabeling of the 46-kDa band. These results suggest that the 46-kDa protein represents the major NTPase implicated in RNA transport.

Adenosine Triphosphate↗
PubMed2005

Increased nuclear envelope permeability and Pep4p-dependent degradation of nucleoporins during hydrogen peroxide-induced cell death.

The death of yeast treated with hydrogen peroxide (H(2)O(2)) shares a number of morphological and biochemical features with mammalian apoptosis. In this study, we report that the permeability of yeast nuclear envelopes (NE) increased during H(2)O(2)-induced cell death. Similar phenomena have been observed during apoptosis in mammalian tissue culture cells. Increased NE permeability in yeast was temporally correlated with an increase in the production of reactive-oxygen species (ROS). Later, after ROS levels began to decline and viability was lost, specific nuclear pore complex (NPC) proteins (nucleoporins) were degraded. Although caspases are responsible for the degradation of mammalian nucleoporins during apoptosis, the deletion of the metacaspase gene YCA1 had no effect on the stability of yeast nucleoporins. Instead, Pep4p, a vacuolar cathepsin D homolog, was responsible for the proteolysis of nucleoporins. Coincident with nucleoporin degradation, a Pep4p-EGFP reporter migrated out of the vacuole in H(2)O(2)-treated cells. We conclude that increases in ROS and NPC permeability occur relatively early during H(2)O(2)-induced cell death. Later, Pep4p migrates out of vacuoles and degrades nucleoporins after the cells are effectively dead.

Apoptosis↗
PubMed1997

[Attachment of polytene chromosomes to the nuclear envelope in ovarian pseudonurse cells in Drosophila melanogaster].

Certain aspects of the polytene chromosome attachment to the nuclear envelope (NE) of ovarian pseudonurse cells (PNC) in the mutant strains otu11 and fs(2) B of Drosophila melanogaster were studied. In contrast to salivary a local chromocenter and Rable-orientation of chromosomes were shown to be missing from the PNC nuclei. The pericentric regions were scattered in the nuclear space and were obliged to attach to the NE by specific loci of heterochromatin. These loci were assigned to the regions 20CD and 41, and the proximal regions 81 and 101 of the cytological map of secondary polytene chromosomes. Each of the loci, which was obliged to attach to the NE, had a specific morphology that was similar in the otu11 and fs(2)B strains. PNC were shown to resemble normal ovarian nurse cells of D. melanogaster in the position of the polytene chromosomes and their attachment to the NE.

Animals↗
PubMed2004

The inner nuclear membrane protein lamin B receptor forms distinct microdomains and links epigenetically marked chromatin to the nuclear envelope.

Using heterochromatin-enriched fractions, we have detected specific binding of mononucleosomes to the N-terminal domain of the inner nuclear membrane protein lamin B receptor. Mass spectrometric analysis reveals that LBR-associated particles contain complex patterns of methylated/acetylated histones and are devoid of "euchromatic" epigenetic marks. LBR binds heterochromatin as a higher oligomer and forms distinct nuclear envelope microdomains in vivo. The organization of these membrane assemblies is affected significantly in heterozygous ic (ichthyosis) mutants, resulting in a variety of structural abnormalities and nuclear defects.

Animals↗
PubMed1968

Attachment of intranuclear annulate lamellae to the nuclear envelope.

Oocytes of four species of ascidians were examined with the electron microscope. Prior to fixation, oocytes were subjected to centrifugal forces of 10-15,000 g for 5-10 min and were compared with uncentrifuged oocytes. Intranuclear annulate lamellae (IAL) are distributed uniformly around the periphery of the nucleus of the uncentrifuged oocyte. Centrifugation produces a marked flattening of the oocyte nucleus, migration of nucleoli to the centrifugal end, and often a condensation of the nucleoplasm at the centrifugal end. In contrast, the distribution of IAL is unchanged by centrifugation. Furthermore, numerous IAL profiles appear to be touching the nuclear envelope, and, in a few of these, direct continuity of the IAL with the nuclear envelope is demonstrated.

Animals↗
PubMed1999

Ultrastructural response of the nuclear envelope (NE) of C6-glioma cells to cisplatin-induced apoptosis.

The early response of the nuclear envelope of C6-glioma cells (t < or = 24 h), treated with a cytostatic dose of cisplatin in culture (5 microg/ml) included formation of slim and deep invaginations formed by either the inner or both membranes. The invaginations made of the complete NE often extended up to the enlarged nucleoli. In some of them, nucleus-like material occurred at their cytoplasmic site suggesting its enhanced nucleus-to-cytoplasm transport. Some nuclear pores in the invagination-forming cells were covered by dome-shaped "caps" protruding into the karyoplasm. The "capped" pores were absent in the cells that were initially more damaged. At 48 to 72 h, we found a small number of large and hyperlobulated cells with some small lobules containing a rarefied chromatin and focally disintegrated NE. The lamina-free remnants of the NE with a swollen perinuclear cistern were still present at 72-96 h when the population entered the execution phase of apoptotic death.

Animals↗
PubMed1994

Autoantibodies against integral membrane proteins of the nuclear envelope in patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Autoantibodies against nuclear membrane proteins have been identified in patients with primary biliary cirrhosis (PBC). The aim of the present study was to determine the incidence of these autoantibodies in patients with PBC and examine their significance. METHODS: An assay using recombinant polypeptides was designed to unequivocally detect autoantibodies against gp210 and the lamin B receptor, integral proteins of the nuclear membranes. RESULTS: Autoantibodies against gp210 were detected in 15 of 159 patients with PBC and 0 of 46 controls. Autoantibodies against lamin B receptor were detected in 2 patients with PBC and 0 controls. The presence of these autoantibodies had a sensitivity of 11% and specificity of 100% for the diagnosis of PBC. Autoantibodies against gp210 were present in 4 of 19 (21%) patients with PBC who did not have detectable antimitochondrial antibodies. Patients with PBC and gp210 autoantibodies had a higher incidence of associated arthritis. CONCLUSIONS: Autoantibodies against gp210 and the lamin B receptor are present in approximately 10% of patients with PBC. These autoantibodies are highly specific for the diagnosis of PBC and may be useful in diagnosing individuals without antimitochondrial antibodies and in identifying a subgroup of patients with an increased incidence of associated arthritis.

Autoantibodies↗
PubMed1989

In vitro translocation through the yeast nuclear envelope. Signal-dependent transport requires ATP and calcium.

Nuclei rapidly purified from yeast Saccharomyces cerevisiae using a cytochalasin B enucleation procedure are substantially free of cell wall, secretory vesicle, plasma membrane vacuolar, and cytoplasmic and mitochondrial contamination. Nuclei obtained in this manner in high yield retain transport properties comparable to nuclei in situ. An in vitro nuclear import assay system has been developed using isolated nuclei and radiolabeled proteins prepared by a coupled in vitro transcription/translation system. Both wild-type SV40 large T-antigen and nucleoplasmin are imported into isolated yeast nuclei, whereas a missense cytoplasmic mutant of the SV40 large T-antigen (Lys128----Thr) and cytoplasmic dihydrofolate reductase are not imported. Association and import of these proteins in a time- and signal-dependent manner resulted in their protection from trypsin that was tethered to agarose beads. Greater than 70% of the labeled protein harboring a karyophilic signal was imported in a reaction that could be blocked by prior treatment of nuclei with trypsin-agarose. Nuclear accumulation of SV40 large T-antigen and nucleoplasmin was unidirectional, ATP and Ca2+ dependent, and was not inhibited by a vast excess of exogenous nonnuclear competitor protein. This system provides an important new tool in combination with powerful yeast genetic methods for analysis of the mechanism and the apparatus for transport at the nuclear envelope.

Adenosine Triphosphate↗
PubMed2000

Identification of a novel signal sequence that targets transmembrane proteins to the nuclear envelope inner membrane.

Herpesvirus maturation requires translocation of glycoprotein B homologue from the endoplasmic reticulum to the inner nuclear membrane. Glycoprotein B of human cytomegalovirus was used in this context as a model protein. To identify a specific signal sequence within human cytomegalovirus glycoprotein B acting in a modular fashion, coding sequences were recombined with reporter proteins. Immunofluorescence and cell fractionation demonstrated that a short sequence element within the cytoplasmic tail of human cytomegalovirus glycoprotein B was sufficient to translocate the membrane protein CD8 to the inner nuclear membrane. This carboxyl-terminal sequence had no detectable nuclear localization signal activity for soluble beta-Galactosidase and could not be substituted by the nuclear localization signal of SV40 T antigen. For glycoprotein B of herpes simplex virus, a carboxyl-terminal element with comparable properties was found. Further experiments showed that the amino acid sequence DRLRHR of human cytomegalovirus glycoprotein B (amino acids 885-890) was sufficient for nuclear envelope translocation. Single residue mutations revealed that the arginine residues in positions 4 and 6 of the DRLRHR sequence were essential for its function. These results support the view that transmembrane protein transport to the inner nuclear membrane is controlled by a mechanism different from that of soluble proteins.

Amino Acid Sequence↗
PubMed1990

The mitosis-specific monoclonal antibody MPM-2 recognizes phosphoproteins associated with the nuclear envelope in Chlamydomonas reinhardtii cells.

The monoclonal antibody MPM-2 recognizes a family of phosphorylated proteins present in mitotic cells. In a number of organisms it stains nuclei and also cytoskeletal structures which contain or organize tubulin. In mitotic Chlamydomonas reinhardtii cells MPM-2 reacts with phosphoproteins associated with the nuclear envelope (NE). Staining of the NE region appears in preprophase, reaches a maximum intensity in metaphase/anaphase and disappears rapidly in telophase. Localized hyperphosphorylation of the anterior NE region is apparent in many cells throughout mitosis. The distribution and timing of MPM-2 labeling suggests that in Chlamydomonas MPM-2 may be interacting with lamin-like phosphoproteins.

Antibodies, Monoclonal↗
PubMed1979

Transfer of mouse nuclear envelope specific proteins to nuclei of chick erythrocytes during reactivation in heterokaryons with mouse A9 cells.

When chick erythrocyte nuclei are introduced into the cytoplasm of mouse A9 cells by cell fusion, proteins present in a fraction of the mouse nuclear envelope begin to appear in the envelope of the chick erythrocyte. The protein uptake was examined using antisera raised in chickens against the 3 major polypeptides of the nuclear pore complex-fibrous lamina fraction from rat liver nuclei. In indirect immunofluorescence studies these antisera give a strong envelope-specific staining with various mammalian but not chicken cells. Eighteen hours after cell fusion the first murine antigens can be observed in the erythrocyte nucleus. Two days after cell fusion the vast majority of the erythrocyte nuclei in cell hybrids contain some antigen and by 3 days the fluorescence of the reactivated erythrocyte nuclei reaches a level comparable to that of the mouse A9 nuclei. The rate of appearance of fluorescence in the chick nuclei depends upon the ratio of A9 cytoplasm to chick nuclei. Antigen uptake by the erythrocyte envelope is inhibited when protein synthesis is blocked suggesting that synthesis of mouse antigen, rather than a redistribution, determines the velocity or erythrocyte envelope reactivation. The early uptake of nucleospecific protein into the reactivating chick erythrocyte may not require any alteration in the nuclear envelope.

Animals↗
PubMed1994

Immunocytochemical verification of the insulin receptor's specificity in the nuclear envelope of Tetrahymena. Comparison with receptors of the plasma membrane.

The unicellular Tetrahymena possess hormone receptors in the nuclear envelope similarly to higher rank animals. These receptors bind insulin and their specificity is detectable by monoclonal antibodies developed to insulin. The hormonal (insulin) pretreatment (imprinting) of the cell did not alter the binding capacity of the nuclear membrane, demonstrated by antibody-technique. The specific binding characteristics of the plasma membrane was demonstrated and this was significantly increased following imprinting. In the nucleus of Tetrahymena presence of insulin was not detected by immunocytochemical method.

Animals↗
PubMed1962

Some dynamic aspects of the nuclear envelope.

Nuclei of frog oocytes were isolated, fixed in OsO(4) or KMnO(4), and washed. Nuclear envelopes were then dissected off, placed on grids, and air-dried for electron microscopy. Envelopes from immature oocytes at the stage of beginning yolk deposition were compared with those from mature oocytes. Envelopes from the immature stage had "pores" whose annuli contained more material and showed central globules in the center much more frequently than envelopes from mature eggs. Annuli and central globules had similar appearance and fixation properties, suggesting similar chemical composition. After fixation with KMnO(4), residual densities suggested that "pore" diaphragms are much more variable in thickness or composition in the younger stages. Envelopes of the immature oocytes had about 40 per cent more "pores" per unit area than mature envelopes. In crowding together, the "pores" tended to assume geometrical packing arrays in the young envelope, showing minimum center-to-center spacings of about 1530 A. Since the actual discontinuities in the membranes of the envelope are only about 950 A in diameter, this minimum distance of approach suggests that adjacent formations of the nuclear surface are associated with "pore" structure and perhaps set their limiting spacing distances. If this is true, then it can be deduced that "pore"-associated structures of the nuclear surface are probably circular in outline and about 1500 A in diameter. Isotopically labeled lysine was administered to intact, growing oocytes for 1 to 4 hours and the envelopes were subsequently isolated and fixed. Autoradiography of entire envelopes showed little or no incorporation of lysine into proteins, as compared with small fragments from other parts of the cell of roughly comparable mass. It was concluded that the isolated envelope, as seen in the electron micrographs, does not synthesize or turn over lysine-containing protein at a high rate.

Cell Nucleus↗
PubMed1998

Nuclear envelope remodelling during rat spermiogenesis: distribution and expression pattern of LAP2/thymopoietins.

Lamina-associated polypeptide 2 (LAP2) and the thymopoietins (TPs) are a family of proteins described in somatic cells of mammals, which are derived by alternative splicing from a single gene. For one of the members of the family (LAP2 = TPbeta) it has been shown that this integral membrane protein locates to the inner membrane of the nuclear envelope, and that it binds to chromatin and B-type lamins. In the present study, we observed that during the third phase of spermatogenesis (i.e. spermiogenesis), TP-labelling shifted progressively to one half of the nuclear periphery in round spermatids. In the elongating spermatid the signal then becomes restricted to one spot located at the posterior (centriolar) pole of the nucleus. Changes in localization are accompanied by the disappearance, first of TPgamma, and later on of LAP2/TPbeta. TPalpha is the only member of the family detectable in the mature sperm. Concomitantly, lamin B1, the only nuclear lamina protein known to be expressed in mammalian spermatids, showed a similar behaviour, i.e. shifted progressively to the centriolar pole of spermatid nuclei before it became undetectable in fully differentiated mature sperms. These results are the first demonstration that expression and localization patterns of TPs are coordinately and differentially regulated with lamins during a differentiation process.

Animals↗
PubMedDate not supplied

[Nuclear envelope of frog diplotene oocytes has telomere-binding activity].

A telomere-binding activity was found in the nuclear envelope of frog oocytes Rana temporaria by methods of binding on nitrocellulose filters and retardation. At present it is hardly possible to detect the proteins responsible for this activity. However, the results of this study allowed us to identify a specific mechanism of DNA-protein interaction, which provides the binding of the telomere to the envelope in meiotic cells.

Animals↗
PubMed1999

Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote.

In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.

Animals↗
PubMed1981

Nuclear envelope alterations accompanying thioacetamide-related enlargement of the nucleus.

Treating rats with thioacetamide results in a biphasic nuclear swelling, with an initial enlargement phase (0 to 8 hr) and a secondary phase (18 to 48 hr). The swelling coincides with and may be responsible for alterations in sedimentation properties of isolated nuclei. Nuclear swelling implies increases in nuclear surface area. Total nuclear lipid increased in proportion to the increase in nuclear-envelope (NE) surface area, suggesting that intranuclear lipid was not the source of new NE phospholipid. The increased NE was apparently not derived from mass incorporation of endoplasmic reticulum into NE, as demonstrated by monitoring enzymatic composition. Significant differences were observed in the distribution of p.o. administered [14C]stearic acid and [14C]phosphatidylcholine in lipid fractions from NE as compared with those from endoplasmic reticulum. Analysis of NE phospholipid revealed little change in composition in the heavy fraction during the first 48 hr after treatment. (An early decrease in phosphatidylserine from the light nuclear fraction may reflect the upward shift of nuclei from the heavy fraction.) A significant increase in phosphatidylserine of NE from the heavy fraction was found at 96 hr. The additional phosphatidylserine in the NE might act as an acidic polymer modifying chromatin structure. Examination of fatty acids from individual NE phospholipid fractions divulged relatively few changes related to the early or the secondary nuclear swelling phases. Major changes occurred in the neutral lipid fraction of NE which apparently served as a reservoir for 18:2, with significant decreases in 18:2 noted following both nuclear swelling phases. These changes did not occur in the neutral lipid fraction from endoplasmic reticulum obtained from the same liver preparations.

Acetamides↗
PubMed2006

Sarcoplasmic reticulum and nuclear envelope are one highly interconnected Ca2+ store throughout cardiac myocyte.

Previous ventricular myocyte studies indicated that ryanodine receptors (RyRs) are in the sarcoplasmic reticulum (SR) and are critical in excitation-contraction coupling, whereas the inositol trisphosphate (InsP(3)) receptors are separately localized on the nuclear envelope (NucEn) and involved in nuclear Ca(2+) signaling. Here, we find that both caffeine and InsP(3) receptor agonists deplete free [Ca(2+)] inside both SR and NucEn. Fluorescence recovery after photobleach (FRAP) was measured using the low-affinity Ca(2+) indicator Fluo-5N trapped inside the SR and NucEn (where its fluorescence is high because [Ca(2+)] is &1 mmol/L). After Fluo-5N photobleach in one end of the cell, FRAP occurred, accompanied by fluorescence decline in the unbleached end with similar time constants (tau&2 minutes) until fluorescence regained spatial uniformity. Notably, SR and NucEn fluorescence recovered simultaneously in the bleached end. Ca(2+) diffusion inside the SR-NucEn was also measured. SR Ca(2+)-ATPase was completely blocked but without acute SR Ca(2+) depletion. Then caffeine was applied locally to one end of the myocyte. In the caffeine-exposed end, free SR [Ca(2+)] ([Ca(2+)](SR)) declined abruptly and recovered partially (tau=20 to 30 seconds). In the noncaffeine end, [Ca(2+)](SR) gradually declined with a similar tau, until [Ca(2+)](SR) throughout the cell equalized. We conclude that the SR and NucEn lumen are extensively interconnected throughout the myocyte. Apparent intrastore diffusion coefficients of Fluo-5N and Ca(2+) were estimated (&8 microm(2) sec(-1) and 60 microm(2) sec(-1)). This rapid luminal communication may maintain homogeneously high luminal [Ca(2+)], ensuring a robust and uniform driving force for local Ca(2+) release events from either SR or NucEn.

Animals↗