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PubMed1998

Interactions among Drosophila nuclear envelope proteins lamin, otefin, and YA.

The nuclear envelope plays many roles, including organizing nuclear structure and regulating nuclear events. Molecular associations of nuclear envelope proteins may contribute to the implementation of these functions. Lamin, otefin, and YA are the three Drosophila nuclear envelope proteins known in early embryos. We used the yeast two-hybrid system to explore the interactions between pairs of these proteins. The ubiquitous major lamina protein, lamin Dm, interacts with both otefin, a peripheral protein of the inner nuclear membrane, and YA, an essential, developmentally regulated protein of the nuclear lamina. In agreement with this interaction, lamin and otefin can be coimmunoprecipitated from the vesicle fraction of Drosophila embryos and colocalize in nuclear envelopes of Drosophila larval salivary gland nuclei. The two-hybrid system was further used to map the domains of interaction among lamin, otefin, and YA. Lamin's rod domain interacts with the complete otefin protein, with otefin's hydrophilic NH2-terminal domain, and with two different fragments derived from this domain. Analogous probing of the interaction between lamin and YA showed that the lamin rod and tail plus part of its head domain are needed for interaction with full-length YA in the two-hybrid system. YA's COOH-terminal region is necessary and sufficient for interaction with lamin. Our results suggest that interactions with lamin might mediate or stabilize the localization of otefin and YA in the nuclear lamina. They also suggest that the need for both otefin and lamin in mediating association of vesicles with chromatin might reflect the function of a protein complex that includes these two proteins.

Animals↗
PubMed2000

The nuclear envelope, muscular dystrophy and gene expression.

Lamins and other nuclear envelope proteins organize nuclear architecture through structural attachments that vary dynamically during the cell cycle and cell differentiation. Genetic studies have now shown that people with mutations in either lamins A/C or emerin, a nuclear membrane protein, develop Emery-Dreifuss muscular dystrophy. A mouse model for this rare disease has been created by knocking out the gene that encodes lamin A/C. This article discusses these and other recent results in the wider context of nuclear envelope function, as a framework for thinking about the possible ways in which defects in nuclear envelope proteins can lead to disease.

Animals↗
PubMed1989

Interleukin-3 and bryostatin 1 mediate rapid nuclear envelope protein phosphorylation in growth factor-dependent FDC-P1 hematopoietic cells. A possible role for nuclear protein kinase C.

Interleukin-3 (IL-3) is a lymphokine which stimulates the proliferation of normal and transformed multilineage hematopoietic cells. Recently we reported that bryostatin 1, a macrocyclic lactone and potent activator of protein kinase C, could stimulate normal multipotential hematopoietic progenitor cells in vitro in the absence of added polypeptide growth factors. We have now used the murine IL-3-dependent cell line FDC-P1, derived from normal murine marrow cells, to examine the early biochemical events associated with stimulation of hematopoietic cells. We find that both IL-3 and bryostatin 1 are mitogenic and stimulate the growth of FDC-P1 cells. Cells grown for extended periods in the presence of bryostatin 1 (1 nM) alone retain IL-3 responsiveness, indicating that bryostatin 1 does not induce an IL-3-independent state. Protein phosphorylation studies in cells treated with either IL-3 or bryostatin 1 indicate that both stimulators can mediate the rapid (within 5 min) serine-specific phosphorylation of several nuclear envelope polypeptides, including lamin B. Both IL-3- and bryostatin 1-mediated nuclear envelope phosphorylation is dose-dependent, occurring at concentrations which are mitogenic to FDC-P1 cells. The extent of nuclear envelope phosphorylation mediated by IL-3 and bryostatin 1 correlates with the mitogenic response. Furthermore, both mitogens mediate the rapid immunologic translocation of protein kinase C to the nuclear envelope where phosphorylation occurs. These data indicate that the early mitogenic signal(s) generated by IL-3 and bryostatin 1 may converge at the level of the nuclear envelope, perhaps through a protein kinase C-like activity which mediates phosphorylation of specific nuclear envelope polypeptides such as lamin B.

Animals↗
PubMed2004

Nucleocytoplasmic transport and nuclear envelope integrity in the fission yeast Schizosaccharomyces pombe.

The nuclear envelope is essential for compartmentalizing the nucleus from the cytoplasm in all eukaryotic cells. There is a tremendous flux of both RNA and proteins across the nuclear envelope, which is intact throughout the entire cell cycle of yeasts but breaks down during mitosis of animal cells. Transport across the nuclear envelope requires the recognition of cargo molecules by receptors, docking at the nuclear pore, transit through the nuclear pore, and then dissociation of the cargo from the receptor. This process depends on the RanGTPase system, transport receptors, and the nuclear pore complex. We provide an overview of the nuclear transport process, with particular emphasis on the fission yeast Schizosaccharomyces pombe, including strategies for predicting and experimentally verifying the signals that determine the sub-cellular localization of a protein of interest. We also describe a variety of reagents and experimental strategies, including the use of mutants and chemical inhibitors, to study nuclear protein import, nuclear protein export, nucleocytoplasmic protein shuttling, and mRNA export in fission yeast. The RanGTPase and its regulators also play an essential transport independent role in nuclear envelope re-assembly after mitosis in animal cells and in the maintenance of nuclear envelope integrity at mitosis in S. pombe. Several experimental strategies and reagents for studying nuclear size, nuclear shape, the localization of nuclear pores, and the integrity of the nuclear envelope in living fission yeast cells are described.

Active Transport, Cell Nucleus↗
PubMed1998

Velcro in the nuclear envelope: LBR and LAPs.

The nuclear envelope is crucial for the functional organization of the nucleus. Lamin B receptor (LBR) and several lamina-associated proteins (LAPs), residing in the inner membrane, provide attachment sites for chromatin and the nuclear lamina. LAPs and LAP-related proteins are members of a growing family of proteins, whose genes are expressed in a tissue and development specific manner, opening the opportunity for a complex regulation of membrane-chromatin and membrane-lamina interactions. Post-translational modifications of LBR and LAPs are likely to modulate their binding to lamins and chromatin, interactions that need to be dynamic to accommodate nuclear growth in interphase and nuclear envelope disassembly in mitosis. Accumulation of proteins in the inner nuclear membrane is believed to depend on their retention mediated by the interaction with nuclear components such as chromatin and lamins.

Laminin↗
PubMed1984

Localization of nucleoside triphosphatase activity to the inner nuclear envelope and associated heterochromatin.

Nuclear-envelope nucleoside triphosphatase activity (NTPase), an enzymatic activity thought to participate in RNA transport, was localized in rat liver in situ after brief perfusion with 3% paraformaldehyde. Reaction product was distributed along the nucleoplasmic side of the nuclear envelope (NE) in heterochromatin, was only occasionally found at nuclear pores, and nuclear deposition was selectively blocked by inhibitors of NE NTPase activity. Our results suggest that NTPases, which are active in the NE and which participate in RNA transport, are not specifically associated with nuclear-pore complexes.

Animals↗
PubMed1999

In vitro nuclear assembly with affinity-purified nuclear envelope precursor vesicle fractions, PV1 and PV2.

Nuclear envelope precursor vesicles were affinity purified from a Xenopus egg extract by a chromatin binding method. Vesicles bound to chromatin at 4 degrees C were dissociated with a high salt buffer and further fractionated into nuclear envelope precursor vesicle fractions 1 (PV1) and 2 (PV2) by differential centrifugation. PV1 contained larger vesicles. When chromatin was incubated in a Xenopus egg cytosol fraction supplemented with PV1, vesicles bound to chromatin, fused with each other, formed a bilayered nuclear envelope, and assembled into spherical small nuclei. However, the thus assembled nuclei did not grow to the normal size. Nuclear pore complexes were not found on the thus assembled nuclei. On the other hand, PV2 contained smaller vesicles. PV2 vesicles bound to chromatin, fused little with each other in the Xenopus egg cytosol fraction, and no nuclei were assembled. When PV1 supplemented with PV2 was used for the nuclear assembly reaction, the assembled nuclei grew to the normal size. Nuclear pore complexes existed in the thus assembled nuclear envelopes. These results suggested that 1) two vesicle populations, PV1 and PV2, are necessary for the assembly of normal sized nuclei, 2) PV1 contains a chromatin targeting molecule(s) and membrane fusion machinery, 3) PV2 contains a chromatin targeting molecule(s) and a molecule(s) necessary for nuclear pore complex assembly, and 4) PV1 has the ability to assemble a nuclear membrane, and PV2 is necessary for the assembly of nuclear pore complexes and for nuclei to grow to the normal size. An in vitro nuclear assembly system constituted with affinity-purified vesicle fractions, PV1 and PV2, was established.

Animals↗
PubMed1989

The anchorosome, a special chromatin granule for the anchorage of the interphase chromosome to the nuclear envelope.

Peripheral chromatin granules bound to the nuclear envelope of rat liver nuclei have been further investigated. Judging by the results of Staphylococcal nuclease digestion of nuclei and electron microscopical observations, the peripheral granules have nucleosomal organization. As shown by ultraviolet radiation DNA-protein cross-linkage, the histone-like proteins present in the peripheral chromatin instead of histone H1 (Fais et al., 1982) are in close contact with DNA. The peripheral chromatin contains a DNA firmly bound to the lamina. This DNA, resistant to extraction in high salt, heparin and SDS, is protected against a DNase attack since, as shown by DNA electrophoresis data, high molecular weight molecules (up to 20 kbas) are still present in the lamina residue. However, the high molecular weight DNA disappeared if the nuclear envelope fraction was again DNase-digested after high salt treatment. Altogether, the data of the previous (Fais et al., 1982; Prusov et al., 1980: Prusov et al., 1982) and the present investigations demonstrate that the peripheral chromatin granules are endowed with properties which distinguish them from the bulk chromatin and account for the chromosome bond to the nuclear envelope during interphase. This is why we suggest the term "anchorosome" for the peripheral protein granule attached to the nuclear envelope.

Animals↗
PubMedDate not supplied

[Features of interactions of p68 anchorosomal protein with the nuclear envelope in the cell cycle].

Chromatin associated with the nuclear envelope appears in the interphase nuclei as a layer of anchorosomes, granules 20-25 nm in diameter. The fraction of chromatin directly associated with the nuclear envelope is resistant to decondensation, shows a low level of DNA methylation, and contains specific acid-soluble proteins. However, mechanisms underlying the interaction of chromatin with the nuclear envelope are not fully understood. Specifically, it is not known whether anchorosomes are permanent structures or if they undergo reversible disassembly during mitosis, when contacts between chromatin and the nuclear envelope are destroyed. We obtained immune serum recognizing a 68 kDa protein from the nuclear envelopes fraction and studied the localization of this protein in interphase and mitotic cells. We show that this protein present in the NE/anchorosomal fraction does not remain bound with chromosomes during mitosis. It dissociates from chromosomes at the beginning of the prophase and then can be identified again at the periphery of the newly forming nuclei in the telophase.

3T3 Cells↗
PubMed1990

Selective digestion of nuclear envelopes from Xenopus oocyte germinal vesicles: possible structural role for the nuclear lamina.

We have used enzymic digestion as a structural probe to investigate components of the nuclear envelope of germinal vesicles from Xenopus oocytes. Previous studies have shown that these envelopes are composed of a double membrane in which nuclear pore complexes are embedded. The nuclear pore complexes are linked to a fibrous lamina that underlies the nucleoplasmic face of the envelope. The pores are also linked by pore-connecting fibrils that attach near their cytoplasmic face. Xenopus oocyte nuclear envelopes were remarkably resistant to extraction with salt solutions and, even after treatment with 1 M NaCl or 3 M MgCl2, pores, lamina and pore-connecting fibrils remained intact. However, mild proteolysis with trypsin selectively removed the lamina fibres from Triton-extracted nuclear envelopes to leave only the pore complexes and connecting fibrils. This observation confirmed that the pore-connecting fibrils were different from the lamina fibres and were probably constructed from different proteins. Trypsin digestion followed by Triton treatment resulted in the complete disintegration of the nuclear envelope, providing direct evidence for a structural role for the lamina in maintaining envelope integrity. Digestion with ribonuclease did not produce any marked change in the structure of Triton-extracted nuclear envelopes, indicating that probably neither the pore-connecting fibrils nor the cytoplasmic granules on the pore complexes contained a substantial proportion of RNA that was vital for their structural integrity.

Animals↗
PubMed1995

Integral membrane proteins associated with the nuclear lamina are novel autoimmune antigens of the nuclear envelope.

We have analyzed sera from 55 patients, most with rheumatic diseases, that all react with the nuclear envelope of human cells in immunofluorescence microscopy. The molecular targets of these autoantibodies were characterized by immunoblot analysis of fractions derived from rat liver nuclear envelopes. While numerous sera were found to react with previously characterized autoimmune antigens of the nuclear envelope including nuclear lamins and the pore complex glycoprotein gp210, a substantial number of the sera were found to recognize relatively minor integral membrane proteins of the nuclear envelope associated with the nuclear lamina (LAP 1A and LAP 2), which have not been previously identified as autoantigens. Autoantibodies to LAP 1A and LAP 2 are present in 9 and 29% of the patient sera, respectively. Only autoantibodies to lamins A/C were encountered more frequently (in 31% of the sera) than autoantibodies to LAP 2, suggesting that LAP 2 may be among one of the most prominent autoantigens of the nuclear envelope in rheumatic disease patients. Since recent studies have suggested that LAP 1A and LAP 2 may be involved in attaching lamins and chromosomes to the nuclear envelope, these findings could promoted understanding of nuclear envelope functions as well as autoimmunity.

Autoantibodies↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗
PubMed2001

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Animals↗
PubMed1993

Immunoelectron microscopic localization of herpes simplex virus glycoprotein gB in the nuclear envelope of infected cells.

Herpesvirus, such as herpes simplex type 1 (HSV-1) acquire their envelope by budding through a modified inner membrane of the nuclear envelope which forms thick and dense patches at the site of budding. This suggests that some of the viral envelope glycoproteins must be transported to the nuclear envelope in order to be incorporated into the virus. In an effort to establish the localization of the HSV-1 glycoprotein gB-1 in the nuclear envelope of HSV-1 infected cells directly, we have studied the distribution of the glycoprotein gB-1 by immunoelectron microscopy using a polyclonal anti gB-1 antibody. A specific accumulation of gB-1 in the nuclear envelope, which was five times more labeled than the plasma membrane was observed. The glycoprotein gB-1 was localized in both the outer and the inner membrane of the nuclear envelope. The labeling over the nuclear envelope was distributed evenly and no preferential concentration of gB-1 around or within the patches where the virus buds was detected. The nucleocapsids were found to be labeled only when they become associated with the nuclear envelope indicating that gB-1 is incorporated into the virus at this site.

Animals↗
PubMed1992

Regulation of nuclear envelope precursor functions during cell division.

Previously, we have shown that nuclear envelope assembly in cell-free extracts of Xenopus eggs requires two distinct vesicle-containing fractions, called Nuclear Envelope Precursor Fractions A and B (NEP-A and NEP-B). These fractions are characterized further in this paper and the manner in which they are regulated during metaphase is examined. Antisera against the NEP-B fraction recognized several proteins common to NEP-B and Xenopus oocyte or liver nuclei, but not to NEP-A or cytosol. A known glycoprotein component of the nuclear pore complex, p62, also co-fractionated with NEP-B, whereas the Xenopus egg lamin LIII did not. Together, these results provide further evidence that the NEP-B fraction contains precursors of the nuclear envelope. The regulation of NEP-A and -B function during metaphase, when the nuclear envelope is disassembled, was examined by treating each fraction with metaphase cytosol or purified protein kinase preparations isolated from metaphase-arrested eggs. Treatment of NEP-B with metaphase cytosol, under conditions where proteins are irreversibly phosphorylated, inhibited the subsequent assembly of the nuclear envelope by preventing the binding of NEP-B to chromatin. In contrast, similar treatment of NEP-A did not affect its ability to form nuclear envelopes. The changes in NEP-B during metaphase did not appear to be regulated directly by either p34cdc2/cyclin B, S6 kinase II or MAP kinase.

Animals↗
PubMed1983

Phosphorylation of rat liver nuclear envelopes. I. Characterization of in vitro protein phosphorylation.

Nuclear envelopes were prepared from purified rat liver nuclei by lysis with heparin, digestion with deoxyribonuclease I (DNase I), or sonication. The envelopes were fractionated by centrifugation on sucrose density gradients and analyzed for protein kinase activity using endogenous and exogenous protein substrates and [gamma-32 P]ATP. The protein kinase activity toward endogenous proteins was markedly affected by the method used to isolate the envelopes, with sonication producing a preparation with very low activity. At least 12 phosphoproteins in nuclear envelopes isolated by the heparin or DNase I method were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A 32P-labeled material migrating with an apparent Mr = 3000 was extracted with chloroform:methanol:HCl and was identified as a mixture of phospholipids. Total 32P incorporation into nuclear envelopes peaked at 5 min of incubation, followed by a decrease in labeled products. This decrease was due to both phosphoprotein phosphatase activity and degradation of the lipid products. The highest protein kinase activity toward endogenous proteins was expressed with [gamma-32P]ATP in the presence of MgCl2; however, some phosphorylation also occurred with MnCl2, CoCl2, NiCl2, and [gamma-32P]GTP in the presence of MgCl2. Nuclear envelope protein phosphorylation was unaffected by cyclic nucleotides and calmodulin, slightly inhibited by CaCl2, MnCl2, CoCl2, disulfides, and sulfhydryl alkylating agents, and strongly inhibited by LaCl3 and phosphatidylglycerol. Nuclear porelamina complexes isolated from phosphorylated envelopes contained phosphoproteins of 7, 20, 51, 59, and 70 kDa. Incubation of pore-lamina complexes isolated from unlabeled envelopes with [gamma-32P]ATP resulted in 32P incorporation into the 20-, 51-, and 50-kDa proteins.

Adenosine Triphosphatases↗
PubMed2004

Bidirectional increase in permeability of nuclear envelope upon poliovirus infection and accompanying alterations of nuclear pores.

Poliovirus and some other picornaviruses trigger relocation of certain nuclear proteins into the cytoplasm. Here, by using a protein changing its fluorescence color with time and containing a nuclear localization signal (NLS), we demonstrate that the poliovirus-triggered relocation is largely due to the exit of presynthesized nuclear protein into the cytoplasm. The leakiness of the nuclear envelope was also documented by the inability of nuclei from digitonin-permeabilized, virus-infected (but not mock-infected) cells to retain an NLS-containing derivative of green fluorescent protein (GFP). The cytoplasm-to-nucleus traffic was also facilitated during infection, as evidenced by experiments with GAPDH (glyceraldehyde-3-phosphate dehydrogenase), cyclin B1, and an NLS-lacking derivative of GFP, which are predominantly cytoplasmic in uninfected cells. Electron microscopy demonstrated that a bar-like barrier structure in the channel of the nuclear pores, seen in uninfected cells, was missing in the infected cells, giving the impression of fully open pores. Transient expression of poliovirus 2A protease also resulted in relocation of the nuclear proteins. Lysates from poliovirus-infected or 2A-expressing cells induced efflux of 3xEGFP-NLS from the nuclei of permeabilized uninfected cells. This activity was inhibited by the elastase inhibitors elastatinal and N-(methoxysuccinyl)-L-alanyl-L-alanyl-L-prolyl-L-valine chloromethylketone (drugs known also to be inhibitors of poliovirus protease 2A), a caspase inhibitor zVAD(OMe), fmk, and some other protease inhibitors. These data suggest that 2A elicited nuclear efflux, possibly in cooperation with a zVAD(OMe).fmk-sensitive protease. However, poliovirus infection facilitated nuclear protein efflux also in cells deficient in caspase-3 and caspase-9, suggesting that the efflux may occur without the involvement of these enzymes. The biological relevance of nucleocytoplasmic traffic alterations in infected cells is discussed.

Active Transport, Cell Nucleus↗
PubMed2006

Nuclear envelope localization of human UNC84A does not require nuclear lamins.

The SUN proteins are a conserved family of proteins in eukaryotes. Human UNC84A (Sun1) is a homolog of Caenorhabditis elegans UNC-84, a protein involved in nuclear anchorage and migration. We have analyzed targeting of UNC84A to the nuclear envelope (NE) and show that the N-terminal 300 amino acids are crucial for efficient NE localization of UNC84A whereas the conserved C-terminal SUN domain is not required. Furthermore, we demonstrate by combining RNA interference with immunofluorescence and fluorescence recovery after photobleaching analysis that localization and anchoring of UNC84A is not dependent on the lamin proteins, in contrast to what had been observed for C. elegans UNC-84.

Amino Acid Sequence↗