scholarly journals NUCLEAR MEMBRANES FROM MAMMALIAN LIVER

1970 ◽  
Vol 46 (2) ◽  
pp. 379-395 ◽  
Author(s):  
Werner W. Franke ◽  
Barbara Deumling ◽  
Baerbel Ermen ◽  
Ernst-Dieter Jarasch ◽  
Hans Kleinig
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Cytochrome B5 ◽  
Buoyant Density ◽  
Nuclear Pore ◽  
Nuclear Membranes ◽  
Dna And Rna ◽  
Mammalian Liver ◽  
Order Of Magnitude

Nuclear membranes were isolated from rat and pig liver by sonication of highly purified nuclear fractions and subsequent removal of adhering nucleoproteins in a high salt medium. The fractions were examined in the electron microscope by both negative staining and thin sectioning techniques and were found to consist of nuclear envelope fragments of widely varying sizes. Nuclear pore complex constituents still could frequently be recognized. The chemical composition of the nuclear membrane fractions was determined and compared with those of microsomal fractions prepared in parallel. For total nuclei as well as for nuclear membranes and microsomes, various enzyme activities were studied. The results indicate that a similarity exists between both fractions of cytomembranes, nuclear envelope, and endoplasmic reticulum, with respect to their RNA:protein ratio and their content of polar and nonpolar lipids. Both membranous fractions had many proteins in common including some membrane-bound enzymes. Activities in Mg-ATPase and the two examined cytochrome reductases were of the same order of magnitude. The content of cytochrome b5 as well as of P-450 was markedly lower in the nuclear membranes. The nuclear membranes were found to have a higher buoyant density and to be richer in protein. The glucose-6-phosphatase and Na-K-ATPase activities in the nuclear membrane fraction were very low. In the gel electrophoresis, in addition to many common protein bands, some characteristic ones for either microsomal or nuclear membranous material were detected. Significant small amounts of DNA and RNA were found to remain closely associated with the nuclear envelope fragments. Our findings indicate that nuclear and endoplasmic reticulum membranes which are known to be in morphological continuity have, besides a far-reaching similarity, some characteristic differences.

scholarly journals Endoplasmic reticulum-to-Golgi transitions upon herpes virus infection

F1000Research ◽  
2018 ◽  
Vol 6 ◽  
pp. 1804 ◽  
Author(s):  
Peter Wild ◽  
Andres Kaech ◽  
Elisabeth M. Schraner ◽  
Ladina Walser ◽  
Mathias Ackermann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Golgi Complex ◽  
Nuclear Membrane ◽  
Progeny Virus ◽  
Cytoplasmic Matrix ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Perinuclear Space ◽  
Hsv 1

Background: Herpesvirus capsids are assembled in the nucleus, translocated to the perinuclear space by budding, acquiring tegument and envelope, or released to the cytoplasm via impaired nuclear envelope. One model proposes that envelopment, “de-envelopment” and “re-envelopment” is essential for production of infectious virus. Glycoproteins gB/gH were reported to be essential for de-envelopment, by fusion of the “primary” envelope with the outer nuclear membrane. Yet, a high proportion of enveloped virions generated from genomes with deleted gB/gH were found in the cytoplasm and extracellular space, suggesting the existence of alternative exit routes.Methods: We investigated the relatedness between the nuclear envelope and membranes of the endoplasmic reticulum and Golgi complex, in cells infected with either herpes simplex virus 1 (HSV-1) or a Us3 deletion mutant thereof, or with bovine herpesvirus 1 (BoHV-1) by transmission and scanning electron microscopy, employing freezing technique protocols.Results:  The Golgi complex is a compact entity in a juxtanuclear position covered by a membrane on thecisface. Golgi membranes merge with membranes of the endoplasmic reticulum forming an entity with the perinuclear space. All compartments contained enveloped virions. After treatment with brefeldin A, HSV-1 virions aggregated in the perinuclear space and endoplasmic reticulum, while infectious progeny virus was still produced.Conclusions: The data suggest that virions derived by budding at nuclear membranes are intraluminally transported from the perinuclear space via Golgi -endoplasmic reticulum transitions into Golgi cisternae for packaging. Virions derived by budding at nuclear membranes are infective like Us3 deletion mutants, which  accumulate in the perinuclear space. Therefore, i) de-envelopment followed by re-envelopment is not essential for production of infective progeny virus, ii) the process taking place at the outer nuclear membrane is budding not fusion, and iii) naked capsids gain access to the cytoplasmic matrix via impaired nuclear envelope as reported earlier.

scholarly journals Induction of a Massive Endoplasmic Reticulum and Perinuclear Space Expansion by Expression of Lamin B Receptor Mutants and the Related Sterol Reductases TM7SF2 and DHCR7

2010 ◽  
Vol 21 (2) ◽  
pp. 354-368 ◽  
Author(s):  
Monika Zwerger ◽  
Thorsten Kolb ◽  
Karsten Richter ◽  
Iakowos Karakesisoglou ◽  
Harald Herrmann
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Cell Lines ◽  
Nuclear Membrane ◽  
Cultured Cells ◽  
Membrane Separation ◽  
Reductase Activity ◽  
Nuclear Pore ◽  
Lamin B ◽  
Lamin B Receptor

Lamin B receptor (LBR) is an inner nuclear membrane protein involved in tethering the nuclear lamina and the underlying chromatin to the nuclear envelope. In addition, LBR exhibits sterol reductase activity. Mutations in the LBR gene cause two different human diseases: Pelger-Huët anomaly and Greenberg skeletal dysplasia, a severe chrondrodystrophy causing embryonic death. Our study aimed at investigating the effect of five LBR disease mutants on human cultured cells. Three of the tested LBR mutants caused a massive compaction of chromatin coincidental with the formation of a large nucleus-associated vacuole (NAV) in several human cultured cell lines. Live cell imaging and electron microscopy revealed that this structure was generated by the separation of the inner and outer nuclear membrane. During NAV formation, nuclear pore complexes and components of the linker of nucleoskeleton and cytoskeleton complex were lost in areas of membrane separation. Concomitantly, a large number of smaller vacuoles formed throughout the cytoplasm. Notably, forced expression of the two structurally related sterol reductases transmembrane 7 superfamily member 2 and 7-dehydrocholesterol reductase caused, even in their wild-type form, a comparable phenotype in susceptible cell lines. Hence, LBR mutant variants and sterol reductases can severely interfere with the regular organization of the nuclear envelope and the endoplasmic reticulum.

scholarly journals Integral Membrane Proteins of the Nuclear Envelope Are Dispersed throughout the Endoplasmic Reticulum during Mitosis

1997 ◽  
Vol 137 (6) ◽  
pp. 1199-1210 ◽  
Author(s):  
Li Yang ◽  
Tinglu Guan ◽  
Larry Gerace
Keyword(s):  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Mammalian Cells ◽  
Polyclonal Antibodies ◽  
Integral Membrane Proteins ◽  
Immunogold Electron Microscopy ◽  
Nuclear Pore ◽  
Nuclear Lamins ◽  
Nuclear Membranes

We have analyzed the fate of several integral membrane proteins of the nuclear envelope during mitosis in cultured mammalian cells to determine whether nuclear membrane proteins are present in a vesicle population distinct from bulk ER membranes after mitotic nuclear envelope disassembly or are dispersed throughout the ER. Using immunofluorescence staining and confocal microscopy, we compared the localization of two inner nuclear membrane proteins (laminaassociated polypeptides 1 and 2 [LAP1 and LAP2]) and a nuclear pore membrane protein (gp210) to the distribution of bulk ER membranes, which was determined with lipid dyes (DiOC6 and R6) and polyclonal antibodies. We found that at the resolution of this technique, the three nuclear envelope markers become completely dispersed throughout ER membranes during mitosis. In agreement with these results, we detected LAP1 in most membranes containing ER markers by immunogold electron microscopy of metaphase cells. Together, these findings indicate that nuclear membranes lose their identity as a subcompartment of the ER during mitosis. We found that nuclear lamins begin to reassemble around chromosomes at the end of mitosis at the same time as LAP1 and LAP2 and propose that reassembly of the nuclear envelope at the end of mitosis involves sorting of integral membrane proteins to chromosome surfaces by binding interactions with lamins and chromatin.

scholarly journals Nuclear Envelope Proteins Modulating the Heterochromatin Formation and Functions in Fission Yeast

Cells ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 1908 ◽  
Author(s):  
Yasuhiro Hirano ◽  
Haruhiko Asakawa ◽  
Takeshi Sakuno ◽  
Tokuko Haraguchi ◽  
Yasushi Hiraoka
Keyword(s):  
Endoplasmic Reticulum ◽  
Nuclear Envelope ◽  
Fission Yeast ◽  
Histone Deacetylase ◽  
Nuclear Pore Complex ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Double Membrane ◽  
Heterochromatin Formation ◽  
Nuclear Membranes

The nuclear envelope (NE) consists of the inner and outer nuclear membranes (INM and ONM), and the nuclear pore complex (NPC), which penetrates the double membrane. ONM continues with the endoplasmic reticulum (ER). INM and NPC can interact with chromatin to regulate the genetic activities of the chromosome. Studies in the fission yeast Schizosaccharomyces pombe have contributed to understanding the molecular mechanisms underlying heterochromatin formation by the RNAi-mediated and histone deacetylase machineries. Recent studies have demonstrated that NE proteins modulate heterochromatin formation and functions through interactions with heterochromatic regions, including the pericentromeric and the sub-telomeric regions. In this review, we first introduce the molecular mechanisms underlying the heterochromatin formation and functions in fission yeast, and then summarize the NE proteins that play a role in anchoring heterochromatic regions and in modulating heterochromatin formation and functions, highlighting roles for a conserved INM protein, Lem2.

scholarly journals An ESCRT-LEM protein surveillance system is poised to directly monitor the nuclear envelope and nuclear transport system

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
David J Thaller ◽  
Matteo Allegretti ◽  
Sapan Borah ◽  
Paolo Ronchi ◽  
Martin Beck ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Surveillance System ◽  
Nuclear Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Membranes ◽  
Disease Mechanisms ◽  
Pore Complexes ◽  
Membrane Sealing

The integrity of the nuclear membranes coupled to the selective barrier of nuclear pore complexes (NPCs) are essential for the segregation of nucleoplasm and cytoplasm. Mechanical membrane disruption or perturbation to NPC assembly triggers an ESCRT-dependent surveillance system that seals nuclear pores: how these pores are sensed and sealed is ill defined. Using a budding yeast model, we show that the ESCRT Chm7 and the integral inner nuclear membrane (INM) protein Heh1 are spatially segregated by nuclear transport, with Chm7 being actively exported by Xpo1/Crm1. Thus, the exposure of the INM triggers surveillance with Heh1 locally activating Chm7. Sites of Chm7 hyperactivation show fenestrated sheets at the INM and potential membrane delivery at sites of nuclear envelope herniation. Our data suggest that perturbation to the nuclear envelope barrier would lead to local nuclear membrane remodeling to promote membrane sealing. Our findings have implications for disease mechanisms linked to NPC assembly and nuclear envelope integrity.

scholarly journals Expansion and apparent fluidity decrease of nuclear membranes induced by low Ca/Mg. Modulation of nuclear membrane lipid fluidity by the membrane-associated nuclear matrix proteins?

1978 ◽  
Vol 79 (2) ◽  
pp. 479-490 ◽  
Author(s):  
F Wunderlich ◽  
G Giese ◽  
C Bucherer
Keyword(s):  
Electron Microscopy ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Nuclear Matrix ◽  
Membrane Lipid ◽  
Ion Concentration ◽  
Nuclear Pore ◽  
Lipid Fluidity ◽  
Nuclear Membranes ◽  
Peripheral Layer

Macronuclei isolated from Tetrahymena are contracted in form (average diameter: 10.2 micron) at a final Ca/Mg (3:2)concentration of 5 mM. Lowering the ion concentration to 1 mM induces an expansion of the average nuclear diameter to 12.2 micron. Both contracted and expanded nuclei are surrounded by a largely intact nuclear envelope as revealed by thin-sectioning electron microscopy. Nuclear swelling is accompanied by an expansion of the nuclear envelope as indicated by the decrease in the frequency of nuclear pore complexes from 52.6 to 42.1 pores/micron2 determined by freeze-etch electron microscopy. Contracted nuclear membranes reveal particle-devoid areas (average size: 0.21 micron2) on 59% of their fracture faces at the optimal growth temperature of 28 degrees C. About three-fifths of the number of these smooth areas disappear upon nuclear membrane expansion. Electron spin resonance using 5-doxylstearic acid as a spin label indicates a higher lipid fluidity in contracted than in expa,ded nuclear membranes. Moreover, a thermotropic lipid clustering occurs at approximately 17 degrees C only in expanded nuclear membranes. In contrast to the nuclear membrane-bound lipids, free lipids extracted from the nuclei rigidify with increasing Ca/Mg concentrations. Our findings are compatible with the view that the peripheral layer of the fundamental nuclear protein-framework, the so-called nuclear matrix, can modulate, inter alia, the lipid distribution and fluidity, respectively, in nuclear membranes. We suggest that a contraction of the nuclear matrix's peripheral layer induces a contraction of the nuclear membranes which, in turn, leads to an isothermic lateral lipid segregation within nuclear membranes.

Nuclear envelope disassembly in mitotic extract requires functional nuclear pores and a nuclear lamina

1998 ◽  
Vol 111 (9) ◽  
pp. 1293-1303 ◽  
Author(s):  
P. Collas
Keyword(s):  
Nuclear Envelope ◽  
Sea Urchin ◽  
Nuclear Membrane ◽  
Critical Role ◽  
Nuclear Lamina ◽  
Nuclear Pore ◽  
Nuclear Pores ◽  
Lamin B ◽  
Nuclear Membranes

Using sea urchin embryonic and in-vitro-assembled nuclei incubated in sea urchin mitotic extract, I provide evidence for a requirement for functional nuclear pores and a nuclear lamina for nuclear envelope disassembly in vitro. In interphase gastrula nuclei, lamin B interacts with p56, an integral protein of inner nuclear membrane cross-reacting with antibodies to human lamin B receptor. Incubation of gastrula nuclei in mitotic cytosol containing an ATP-generating system rapidly induces hyperphosphorylation of p56 and lamin B. Subsequently, p56-lamin B interactions are weakened and the two proteins segregate into distinct nuclear envelope-derived vesicles upon disassembly of nuclear membranes and of the lamina. Nuclear disassembly is accompanied by chromatin condensation. Blocking nuclear pore function with wheat germ agglutinin or antibodies to nucleoporins prevents p56 and lamin B hyperphosphorylation, nuclear membrane breakdown and lamina solubilization. These events are not rescued by permeabilization of nuclear membranes to molecules of 150, 000 Mr with lysolecithin. In-vitro-assembled nuclei containing nuclear membranes with functional pores but no lamina do not disassemble in mitotic cytosol in spite of p56 hyperphosphorylation. Nuclear import of soluble lamin B and reformation of a lamina in interphase extract restores nuclear disassembly in mitotic cytosol. The data indicate a role for functional nuclear pores in nuclear disassembly in vitro. They show that p56 hyperphosphorylation is not sufficient for nuclear membrane disassembly in mitotic cytosol and argue that the nuclear lamina plays a critical role in nuclear disassembly at mitosis.

scholarly journals System analysis shows distinct mechanisms and common principles of nuclear envelope protein dynamics

2011 ◽  
Vol 193 (1) ◽  
pp. 109-123 ◽  
Author(s):  
Nikolaj Zuleger ◽  
David A. Kelly ◽  
A. Christine Richardson ◽  
Alastair R. W. Kerr ◽  
Martin W. Goldberg ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
System Analysis ◽  
Nuclear Pore ◽  
Binding Affinities ◽  
Inner Nuclear Membrane ◽  
Wide Range ◽  
Recovery Times

The nuclear envelope contains >100 transmembrane proteins that continuously exchange with the endoplasmic reticulum and move within the nuclear membranes. To better understand the organization and dynamics of this system, we compared the trafficking of 15 integral nuclear envelope proteins using FRAP. A surprising 30-fold range of mobilities was observed. The dynamic behavior of several of these proteins was also analyzed after depletion of ATP and/or Ran, two functions implicated in endoplasmic reticulum–inner nuclear membrane translocation. This revealed that ATP- and Ran-dependent translocation mechanisms are distinct and not used by all inner nuclear membrane proteins. The Ran-dependent mechanism requires the phenylalanine-glycine (FG)-nucleoporin Nup35, which is consistent with use of the nuclear pore complex peripheral channels. Intriguingly, the addition of FGs to membrane proteins reduces FRAP recovery times, and this also depends on Nup35. Modeling of three proteins that were unaffected by either ATP or Ran depletion indicates that the wide range in mobilities could be explained by differences in binding affinities in the inner nuclear membrane.

scholarly journals POM121 and Sun1 play a role in early steps of interphase NPC assembly

2011 ◽  
Vol 194 (1) ◽  
pp. 27-37 ◽  
Author(s):  
Jessica A. Talamas ◽  
Martin W. Hetzer
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Molecular Mechanisms ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Outer Nuclear Membrane ◽  
Nuclear Membranes ◽  
Distinct Cell ◽  
Pore Complexes

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107–160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.

scholarly journals In Vivo Dynamics of Nuclear Pore Complexes in Yeast

1997 ◽  
Vol 136 (6) ◽  
pp. 1185-1199 ◽  
Author(s):  
Mirella Bucci ◽  
Susan R. Wente
Keyword(s):  
Nuclear Envelope ◽  
Recipient Cell ◽  
Nucleocytoplasmic Transport ◽  
Yeast Cells ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Central Location ◽  
Nuclear Membranes ◽  
Mutant Strains ◽  
Pore Complexes

While much is known about the role of nuclear pore complexes (NPCs) in nucleocytoplasmic transport, the mechanism of NPC assembly into pores formed through the double lipid bilayer of the nuclear envelope is not well defined. To investigate the dynamics of NPCs, we developed a live-cell assay in the yeast Saccharomyces cerevisiae. The nucleoporin Nup49p was fused to the green fluorescent protein (GFP) of Aequorea victoria and expressed in nup49 null haploid yeast cells. When the GFP–Nup49p donor cell was mated with a recipient cell harboring only unlabeled Nup49p, the nuclei fused as a consequence of the normal mating process. By monitoring the distribution of the GFP–Nup49p, we could assess whether NPCs were able to move from the donor section of the nuclear envelope to that of the recipient nucleus. We observed that fluorescent NPCs moved and encircled the entire nucleus within 25 min after fusion. When assays were done in mutant kar1-1 strains, where nuclear fusion does not occur, GFP–Nup49p appearance in the recipient nucleus occurred at a very slow rate, presumably due to new NPC biogenesis or to exchange of GFP– Nup49p into existing recipient NPCs. Interestingly, in a number of existing mutant strains, NPCs are clustered together at permissive growth temperatures. This has been explained with two different hypotheses: by movement of NPCs through the double nuclear membranes with subsequent clustering at a central location; or, alternatively, by assembly of all NPCs at a central location (such as the spindle pole body) with NPCs in mutant cells unable to move away from this point. Using the GFP–Nup49p system with a mutant in the NPCassociated factor Gle2p that exhibits formation of NPC clusters only at 37°C, it was possible to distinguish between these two models for NPC dynamics. GFP– Nup49p-labeled NPCs, assembled at 23°C, moved into clusters when the cells were shifted to growth at 37°C. These results indicate that NPCs can move through the double nuclear membranes and, moreover, can do so to form NPC clusters in mutant strains. Such clusters may result by releasing NPCs from a nuclear tether, or by disappearance of a protein that normally prevents pore aggregation. This system represents a novel approach for identifying regulators of NPC assembly and movement in the future.

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