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PubMed1992

Purification of a glucose-binding protein from rat liver nuclei. Evidence for a role in targeting of nuclear mRNP to nuclear pore complex.

A nuclear carbohydrate-binding protein with a molecular mass of 67 kDa (CBP67), which is specific for glucose residues, was purified to essential homogeneity from rat liver nuclear extracts. This protein could also be isolated from nuclear ribonucleoprotein (RNP) complexes by extraction in the presence of 0.6 M or 2 M NaCl, but it was absent in polysomal RNP complex. The binding of the purified protein, which has an isoelectric point of 7.3, to glucose-containing glycoconjugates depends on the presence of Ca2+ and Mg2+. Using closed nuclear envelope vesicles as a system to study nuclear transport of RNA, it was shown that both entrapped polysomal mRNA and nuclear RNA precursors are readily exported from the vesicles in an ATP-dependent manner. The transport was unidirectional and strongly promoted by the poly(A) segment attached to these RNAs. In contrast, nuclear RNP complexes entrapped into the vesicles together with glucose-conjugated bovine serum albumin or nucleoplasmin, or bird nest glycoprotein, were not exported into the extravesicular space. However, transport of nuclear RNP complexes could be achieved in the presence of glucose or after co-addition of a glucose-recognizing lectin from Pellina semitubulosa. In Western blots, radioiodinated CBP67 binds to an 80-kDa polypeptide both in isolated rat liver nuclear envelopes and pore-complex laminae. From these results we postulate that CBP67 may direct nuclear RNP complexes to the nuclear pore.

Animals↗
PubMedDate not supplied

[The role of lipid-nucleic acid interactions in the reconstruction in vitro of the nuclear shell with pores].

Previous studies of triple complexes DNA-phosphatidylcholine liposomes-Me2+ showed that, upon complexing, along with aggregation, a partial reversible fusion of liposomes takes place. In this case, DNA acts as a fusogen. The addition of the extract from Xenopus laevis or Drosophila melanogaster oocytes to the triple complex leads to a complete fusion of initial liposomes 100-200 nm in size and the formation of giant liposomes of up to 100-500 microns in size. Upon the addition of liposomes to the extract from Xenopus or Drosophila eggs, either fragments of the nuclear envelope with pores or structures like annulate lamellae are formed. The diameter of pores in these formations is approximately twice as small as in the native nuclear envelope, which may be associated with a lower surface tension of the lipid bilayer. This fact is in agreement with our model of the formation of nuclear pores suggested earlier.

Animals↗
PubMed1990

Nuclear architecture of human pachytene spermatocytes: quantitative analysis of associations between nucleolar and XY bivalents.

Nucleolar association and heterochromatin coalescence have both been invoked as mechanisms involved in the origin of chromosomal associations between nucleolar bivalents themselves, as well as between these bivalents and the XY pair, during meiotic prophase in human spermatocytes. However, these mechanisms do not satisfactorily explain how associating bivalents meet each other within the nuclear space. To elucidate this problem, we have characterized different types of nucleolar-nucleolar and nucleolar-XY bivalent associations, and their frequencies, in light and electron microscope serial sections of spermatocyte nuclei. In the pachytene nucleus, nucleolar bivalent associations were found to involve only one nucleolar sphere of RNP granules connected through a fibrillar center to a chromatin mass composed of two, or more, nucleolar-bivalent short arms. Structural relationships between these elements were examined using 3D computer models of various nucleolar associations. XY and nucleolar bivalents were usually located towards the nuclear periphery associated with the inner face of the nuclear envelope. Some nucleolar bivalents, whether single or associated appeared beside or over XY chromatin. When nucleolar-bivalent short arms (BK) were found over nucleolar or over XY chromatin, their telomeres were unattached to the nuclear envelope and the corresponding synaptonemal complexes were not observed. Ninety nucleoli were found in sixty pachytene nuclei. Thirty six percent of these nucleoli were bound to associated BKs and the remaining 64% to single BKs. Over 40% of individual spermatocytes showed at least one cluster of associated BKs and about 20% presented single or multiple BKs associated with the XY pair. The frequencies of random BK associations, over the total or restricted areas of the nuclear envelope, were calculated according to a probabilistic nuclear model. A correspondence was found in comparing the observed frequencies of associated BKs with those calculated on the basis of bouquet formation. Such an analysis strongly suggests that the occurrence of associations between nucleolar bivalents may arise at random within the bouquet. Thus, the architecture of the meiocyte nucleus, particularly the organization of the bouquet, may be the primary mechanism by which nucleolar bivalents meet each other and, consequently, become associated either through common nucleolus formation or by heterochromatin coalescence.

Cell Nucleolus↗
PubMed1999

Role of the membrane anchoring and cytoplasmic domains in intracellular transport and localization of viral glycoproteins.

The role of the transmembrane and the cytoplasmic regions of viral glycoproteins namely, the envelope glycoprotein gD of herpes simplex virus and the integral membrane glycoprotein E3-11.6 K of the nonenveloped adenovirus that are localized in the nuclear envelope has been studied. Chimeras of the cell surface glycoprotein G of vesicular stomatitis virus containing the transmembrane and (or) the cytoplasmic-tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K protein were examined for their intracellular transport and localization. The results show that hybrids containing the membrane anchoring and (or) the cytoplasmic tail domains of either herpes simplex virus gD or adenovirus E3-11.6 K glycoprotein were localized in the nuclear envelope as well as in the endoplasmic reticulum and the Golgi complex. These results suggest that the membrane anchoring and the cytoplasmic domains of herpes simplex virus glycoproteins gD, as well as the adenovirus integral membrane protein E3-11.6 K, were necessary for localization in the nuclear envelope and could influence retention in the endoplasmic reticulum and the Golgi complex.

Adenovirus E3 Proteins↗
PubMed1988

Perceiving mitosis in eukaryotic cells.

A sensitive method has been developed for visualizing eukaryotic cells in mitosis (M) phase. It employs Zenker's fixative, which makes the plasma membrane but not the nuclear envelope permeable to immunoglobulins. Zenker's-fixed cells are exposed to an antibody which recognizes a major constituent of chromatin. In this case the antibody is a monoclonal (MC 21) which recognizes histone H2b. Because cells in M phase do not have an intact nuclear envelope, the antibody has access to and interacts with their chromatin. The presence of a nuclear envelope in Zenker's-fixed interphase cells precludes access of the antibody to the nuclear chromatin. Consequently, this indirect immunofluorescence procedure selectively labels M-phase cells. At high enough magnification some details of the chromatin figures are revealed. MC 21 recognizes the chromatin of cells of many different species. With appropriate fixation it can be used effectively on cells in culture. With some procedural modifications it can also be used with more complex tissue systems. Detailed mitotic patterns for chick embryos up to Day 3 of development have been obtained by this method.

Animals↗
PubMed1973

Nuclear membrane fusion in fertilized Lytechinus variegatus eggs.

Fusion of apposed nuclear envelopes is frequently seen at telophase during postmitotic reorganization of the nucleus, but only rarely at other times in the cell cycle. We attempted to define an experimental system for studying changes in the nuclear envelope related to the cell cycle by varying the time of pronuclear apposition in fertilized Lytechinus variegatus eggs. This approach was based on the assumption that the period from fertilization to metaphase of the first cleavage division corresponds to the period from telophase to metaphase in the generalized cell cycle. The experimental approach used was to block the movement of the pronuclei with Colcemid and then to release this block at varying times after insemination by photochemically inactivating the Colcemid. The results show that apposed pronuclear envelopes can fuse from soon after insemination until the anticipated time of prometaphase. Fusion occurred in about 3 min as scored by light microscopy and this time did not vary significantly with the time after insemination. The potential for nuclear fusion is not restricted to pronuclei alone since diploid nuclei in binucleate cells could be fused using centrifugation in solutions of Colcemid to bring the nuclei into apposition. It is suggested that the potential for nuclear fusion is not necessarily related to the cell cycle and that modification of the nuclear envelope, possibly by association with chromatin or other fibrous material restricts nuclear fusion in most multinucleated cells.

Animals↗
PubMed2000

Pushing the envelope on lipodystrophy.

Autosomal dominant partial lipodystrophy (PLD), in which regional adipose loss is coupled with insulin resistance, is strongly associated with missense mutations in LMNA, encoding lamin A/C-a component of the nuclear envelope. This finding indicates that other proteins and functions of the nuclear envelope may have bearing on disorders of adipose tissue and insulin action.

Adipose Tissue↗
PubMed2005

LEM2 is a novel MAN1-related inner nuclear membrane protein associated with A-type lamins.

The LEM (lamina-associated polypeptide-emerin-MAN1) domain is a motif shared by a group of lamin-interacting proteins in the inner nuclear membrane (INM) and in the nucleoplasm. The LEM domain mediates binding to a DNA-crosslinking protein, barrier-to-autointegration factor (BAF). We describe a novel, ubiquitously expressed LEM domain protein, LEM2, which is structurally related to MAN1. LEM2 contains an N-terminal LEM motif, two predicted transmembrane domains and a MAN1-Src1p C-terminal (MSC) domain highly homologous to MAN1, but lacks the MAN1-specific C-terminal RNA-recognition motif. Immunofluorescence microscopy of digitonin-treated cells and subcellular fractionation identified LEM2 as a lamina-associated protein residing in the INM. LEM2 binds to the lamin C tail in vitro. Targeting of LEM2 to the nuclear envelope requires A-type lamins and is mediated by the N-terminal and transmembrane domains. Highly overexpressed LEM2 accumulates in patches at the nuclear envelope and forms membrane bridges between nuclei of adjacent cells. LEM2 structures recruit A-type lamins, emerin, MAN1 and BAF, whereas lamin B and lamin B receptor are excluded. Our data identify LEM2 as a novel A-type-lamin-associated INM protein involved in nuclear structure organization.

Animals↗
PubMed2026

Integrative chemical genetics platform identifies condensate modulators linked to neurological disorders.

Dysregulation of biomolecular condensates is implicated across multiple neurological disorders. However, approaches to systematically identify their modulators remain limited. Here, we expand the utility of MLF2 as a versatile condensate biomarker and develop CondenScreen, an integrated high-content screening and bioinformatics pipeline enabling identification of condensate modulators across chemical and genetic space. Screening 1760 bioactive compounds in a cellular DYT1 dystonia model, we validate the platform for condensate-targeted drug discovery, identifying drugs that prevent the accumulation of the MLF2 reporter into nuclear envelope condensates. In parallel, a genome-wide CRISPR/Cas9 screen correlates nuclear condensate abundance with genes implicated in microcephaly and over eight additional neurodevelopmental disorders. Machine learning and confocal imaging resolve distinct condensate phenotypes, with RNF26 deletion provoking nuclear envelope condensates that phenocopy hallmarks of torsin deficiency. Our study provides a scalable platform for identifying modulators of condensates and establishes a correlative connection between nuclear condensate accumulation and genes implicated in neurodevelopmental disorders.

Humans↗
PubMed1995

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Animals↗
PubMed2006

A-type lamin networks in light of laminopathic diseases.

Lamins are major structural components of the lamina providing mechanical support for the nuclear envelope in vertebrates. A subgroup of lamins, the A-type lamins, are only expressed in differentiated cells and serve important functions both at the nuclear envelope and in the nucleoplasm in higher order chromatin organization and gene regulation. Mutations in A-type lamins cause a variety of diseases from muscular dystrophy and lipodystrophy to systemic diseases such as premature ageing syndromes. The molecular basis of these diseases is still unknown. Here we summarize known interactions of A-type lamins with components of the nuclear envelope and the nucleoplasm and discuss their potential involvement in the etiology and molecular mechanisms of the diseases. Lamin binding partners involve chromatin proteins potentially involved in higher order chromatin organization, transcriptional regulators controlling gene expression during cell cycle progression, differentiation and senescence, and several enzymes involved in a multitude of functions.

Chromatin↗
PubMed1989

Annulate lamellae-soleplate nuclei associations in skeletal muscle fibers of rats during chronic high-dose exposure to neostigmine.

To investigate annulate lamellae (AL) with nuclear changes, ultrastructural time course studies were done of the reversible end-plate myopathy in the soleus muscles of rats exposed chronically to a high dose of the anticholinesterase drug neostigmine. At the earliest stage (2 hours) in which severe subjunctional damage involving a nuclear lesion (nuclear pyknosis) was prominent, AL profiles were undetectable. At the intermediate stage (7, 21 days), in which the subjunctional organelles tended to cluster and nuclear pyknosis often accompanied degenerative features, most AL laid near the surface of the abnormal nuclei, where there were signs of elimination or formation of pores, as well as a progression of changes leading to the loss of pores. At the late stage (56 days), in which muscle repair was nearly completed and euchromatic nuclei usually were found, there was a concomitant enhanced formation of false nuclear inclusions and of the AL profiles in these sites. Pores are packed on nuclear envelopes, which detach themselves from the nucleus and take the same profile as AL. This phenomenon can be envisioned not only in the euchromatic but also in the abnormal (pyknotic or degenerated) nuclei. Following nuclear reconstitution, the nuclear envelope folding is accelerated to induce the usual pattern of AL organization, i.e., budding from the invaginated nuclear envelope.

Animals↗
PubMed1995

Neutral sphingomyelinase: localization in rat liver nuclei and involvement in regeneration/proliferation.

We have studied the localization of neutral sphingomyelinase (N-SMas) in rat liver nuclei. The levels of neutral sphingomyelinase in regenerating liver nuclei were also assessed. We found that rat liver nuclei contain a sphingomyelinase having a pH optima of 7.2 and a kDa of 92. In intact nuclei, neutral sphingomyelinase was associated predominantly with the nuclear envelope. In regenerating/proliferating rat liver (during DNA synthesis), neutral sphingomyelinase was translocated from the nuclear envelope to the nuclear matrix. The levels of sphingomyelin in whole nuclei decreased in reverse proportion to an increase in the levels of neutral sphingomyelinase. By contrast, there was a corresponding increase in the levels of ceramide and sphingosine during cell regeneration/proliferation. Thus, endogenous nuclear neutral sphingomyelinase may play a role in the regulation of sphingomyelin levels and in relevant signal transduction reactions involving cell regeneration/proliferation. The potential significance of ceramide generation may be aimed at programmed cell death to allow the regeneration of liver mediated via target proteins such as, ceramide activated protein kinases/phospholipases or other unknown mechanisms.

Animals↗
PubMed2001

Nuclear localization of neutral sphingomyelinase 1: biochemical and immunocytochemical analyses.

To examine the intracellular localization of neutral sphingomyelinase 1 (nSMase 1), a rabbit polyclonal antibody was raised against a recombinant form of the enzyme expressed in E. coli. It has been reported that, in rat liver or in ascites hepatoma AH7974, high activity of neutral sphingomyelinase (SMase) is found at the plasma membrane, with a lesser but significant amount in nucleus and cytoplasm. The biochemical properties, dithiothreitol requirement and high salt concentration dependency, of cloned and expressed nSMase 1 resemble those of previously described nuclear neutral SMase of AH7974. The present study was therefore focused on the nuclear localization of this enzyme. Western blotting of subcellular fractions using anti-rat nSMase 1 antibody revealed most nSMase 1 to be associated with the nuclei and some with microsomes, but not with plasma membranes. Consistently, neutral SMase activity in nuclear extract was immunoprecipitated by the antibody, while that of plasma membranes was not. The results indicate that nSMase 1 mainly resides in the nucleus and may thus differ from neutral SMase in plasma membrane. On gel-filtration column chromatography of nuclear extract, the profile of neutral SMase activity corresponded well with immunoreactive protein bands on western blotting, suggesting that a large part of nuclear neutral SMase may be nSMase 1. Removal of the nuclear envelope by treatment with Triton X-100 did not significantly decrease the amount of nuclear nSMase 1, and western blotting of subnuclear fractions (i.e. nuclear envelope, chromatin, and nuclear matrix) revealed nSMase 1 signal exclusively in the nuclear matrix. Immunocytochemistry with AH7974, as well as rat fibroblast cell line 3Y1, demonstrated nSMase 1 to be localized mainly in the nucleus, with some in the cytoplasm. Moreover, immuno-electron microscopy clearly showed the signal of nSMase 1 to be more dense in the nucleus than in the cytoplasm of AH7974.

Animals↗
PubMed1997

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.

Journal Article↗
PubMed1997

High frequency of autoantibodies to insoluble cellular antigens in patients with chronic fatigue syndrome.

OBJECTIVE: To elucidate the humoral immune response in patients with chronic fatigue syndrome (CFS), by identification and characterization of autoantibodies. METHODS: Initial immunofluorescence histochemistry studies of sera using human HEp-2 cell substrate were followed by antibody class subtyping and colocalization studies with reference antibodies. Association of CFS autoantigens with insoluble cellular components was determined by in situ extraction of soluble components and subsequent immunofluorescence histochemistry studies on the extracted cell substrate. RESULTS: Of 60 CFS patients, 41 (68%) were positive for antinuclear antibodies. Localization of nuclear staining was found at the nuclear envelope (52%), in reticulated speckles (25%), in nucleoli (13%), and in dense fine speckles (5%). Twenty-eight CFS sera (47%) also had antibodies to cytoplasmic antigens. The major cytoplasmic staining pattern was of the intermediate filament type (35%). The observed nuclear envelope pattern of staining co-localized with lamina-associated polypeptide 2 (an integral nuclear membrane protein), the reticulated speckle pattern co-localized with non-small nuclear RNP splicing factor SC-35, and the intermediate filament pattern co-localized with vimentin. The intermediate filament antigen was shown to be vimentin in immunoblotting experiments using recombinant human vimentin, and one of the nuclear envelope antigens was shown previously to be lamin B1. Fifty of the 60 CFS patients (83%) had antibodies to one or another of these antigens, all of which are relatively insoluble cellular antigens, whereas a control group of patients without chronic fatigue had a significantly lower frequency of such antibodies (17%). CONCLUSION: The high frequency of autoantibodies to insoluble cellular antigens in CFS represents a unique feature which might help to distinguish CFS from other rheumatic autoimmune diseases.

Antibodies, Monoclonal↗
PubMed2005

Death of mitochondria during programmed cell death of leaf mesophyll cells.

The role of plant mitochondria in the programmed cell death (PCD) is widely discussed. However, spectrum and sequence of mitochondrial structural changes during different types of PCD in leaves are poorly described. Pea, cucumber and rye plants were grown under controlled growing conditions. A part of them were sprinkled with ethylene releaser to accelerate cell death. During yellowing the palisade parenchyma mitochondria were attracted to nuclear envelope. Mitochondrial matrix became electron translucent. Mitochondria entered vacuole by invagination of tonoplast and formed multivesicular bodies. Ethephon treatment increased the frequency of sticking of mitochondria to the nuclear envelope or chloroplasts and peroxisomes. Mitochondria divided by different mechanisms and became enclosed in Golgi and ER derived authopagic vacuoles or in the central vacuole. Several fold increase of the diameter of cristae became typical. In all cases mitochondria were attached to nuclear envelope. It can be considered as structural mechanism of promoting of PCD.

Apoptosis↗
PubMed2001

Feasibility of producing porcine nuclear transfer embryos by using G2/M-stage fetal fibroblasts as donors.

The type of donor cell most suitable for producing cloned animals is one of the topics under debate in the field of nuclear transfer. To provide useful information to answer this question, G2/M- and G0/G1-stage fetal fibroblasts were used as donor cells for nuclear transfer. In vitro-matured oocytes derived from abattoir ovaries were used as recipient cytoplasts. In both groups, nuclear envelope breakdown and premature chromosome condensation were completed within 1-2 h after donor cells were injected into the cytoplasm of oocytes. Microtubules were organized around condensed chromosomes and formed a spindle within 1-1.5 h after activation. Decondensation of chromosomes could be seen within 2-4 h after activation. Reformation of the new nuclear envelope occurred 4-6 h after activation and was followed by nuclear swelling and formation of a pronucleus-like structure (PN) 8-12 h after activation. Most (80.6%) of the reconstructed oocytes derived from G2/M cells extruded polar body-like structures (PB). However, a much lower frequency of PB (21.7%) was observed in the reconstructed oocytes derived from G0/G1 donors. A variety of PN and PB combinations were observed in reconstructed oocytes derived from G2/M-stage donors, including 1PN+0PB, 1PN+1PB, 1PN+2PB, 2PN+0PB, 2PN+1PB, 2PN+2PB, and 3PN+1PB. Chromosomes of most embryos (10/13) derived from G2/M stage were diploid. The percentage of cleavage and blastocysts and the average nuclear number of blastocysts in the G2/M and G0/G1 groups were not different. These results demonstrate that the G2/M stage can be morphologically remodeled by cytoplasm of MII oocytes in pigs. To maintain normal ploidy, the extra chromosomes derived from G2/M-stage cells could be expelled by oocytes as a second polar body. G2/M-stage fibroblast nuclei could direct reconstructed embryos to develop to the blastocyst stage.

Animals↗