scholarly journals Transmembrane proteins are not required for early stages of nuclear envelope assembly

2006 ◽  
Vol 400 (3) ◽  
pp. 393-400 ◽  
Author(s):  
Corinne Ramos ◽  
Elvira R. Rafikova ◽  
Kamran Melikov ◽  
Leonid V. Chernomordik
Keyword(s):  
Nuclear Envelope ◽  
Transmembrane Proteins ◽  
Membrane Vesicles ◽  
Ran Gtpase ◽  
Free System ◽  
Nuclear Envelope Assembly ◽  
Cytosolic Extract ◽  
A Cell ◽  
Integral Proteins ◽  
Membrane Fusion Proteins

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.

scholarly journals Characterization of the membrane binding and fusion events during nuclear envelope assembly using purified components.

1992 ◽  
Vol 116 (2) ◽  
pp. 295-306 ◽  
Author(s):  
J Newport ◽  
W Dunphy
Keyword(s):  
Nuclear Envelope ◽  
Molecular Mechanisms ◽  
Membrane Vesicles ◽  
Sperm Chromatin ◽  
Chromosomal Dna ◽  
Free System ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg

At the end of mitosis membrane vesicles are targeted to the surface of chromatin and fuse to form a continuous nuclear envelope. To investigate the molecular mechanisms underlying these steps in nuclear envelope assembly, we have developed a defined cell-free system in which the binding and fusion steps in nuclear envelope assembly can be examined separately. We have found that extensively boiled Xenopus egg extracts efficiently promote the decondensation of demembranated Xenopus sperm chromatin. When isolated membranes are added to this decondensed chromatin a specific subfraction of membrane vesicles (approximately 70 nM in diameter) bind to the chromatin, but these vesicles do not fuse to each other. Vesicle binding is independent of ATP and insensitive to N-ethylmalamide. Quantitative analysis of these sites by EM suggests that there is at least one vesicle binding site per 100 kb of chromosomal DNA. We show by tryptic digestion that vesicle-chromatin association requires proteins on both the vesicle and on the chromatin. In addition, we show that the vesicles bound under these conditions will fuse into an intact nuclear envelope when incubated with the soluble fraction of a Xenopus egg nuclear assembly extract. With respect to vesicle fusion, we have found that vesicles prebound to chromatin will fuse to each other when ATP and GTP are present in the boiled extract. These results indicate that nuclear envelope assembly is mediated by a subset of approximately 70-nM-diam vesicles which bind to chromatin sites spaced 100 kb apart and that fusion of these vesicles is regulated by membrane-associated GTP-binding proteins.

scholarly journals Ran GTPase Cycle and Importins α and β Are Essential for Spindle Formation and Nuclear Envelope Assembly in LivingCaenorhabditis elegans Embryos

2002 ◽  
Vol 13 (12) ◽  
pp. 4355-4370 ◽  
Author(s):  
Peter Askjaer ◽  
Vincent Galy ◽  
Eva Hannak ◽  
Iain W. Mattaj
Keyword(s):  
Nuclear Envelope ◽  
Cell Function ◽  
Small Gtpase ◽  
Spindle Formation ◽  
Ran Gtpase ◽  
Early Embryos ◽  
Nuclear Envelope Assembly ◽  
Importin Α ◽  
Gtpase Cycle

The small GTPase Ran has been found to play pivotal roles in several aspects of cell function. We have investigated the role of the Ran GTPase cycle in spindle formation and nuclear envelope assembly in dividing Caenorhabditis elegans embryos in real time. We found that Ran and its cofactors RanBP2, RanGAP, and RCC1 are all essential for reformation of the nuclear envelope after cell division. Reducing the expression of any of these components of the Ran GTPase cycle by RNAi leads to strong extranuclear clustering of integral nuclear envelope proteins and nucleoporins. Ran, RanBP2, and RanGAP are also required for building a mitotic spindle, whereas astral microtubules are normal in the absence of these proteins. RCC1(RNAi) embryos have similar abnormalities in the initial phase of spindle formation but eventually recover to form a bipolar spindle. Irregular chromatin structures and chromatin bridges due to spindle failure were frequently observed in embryos where the Ran cycle was perturbed. In addition, connection between the centrosomes and the male pronucleus, and thus centrosome positioning, depends upon the Ran cycle components. Finally, we have demonstrated that both IMA-2 and IMB-1, the homologues of vertebrate importin α and β, are essential for both spindle assembly and nuclear formation in early embryos.

Three proteins required for early steps in the protein secretory pathway also affect nuclear envelope structure and cell cycle progression in fission yeast

2002 ◽  
Vol 115 (2) ◽  
pp. 421-431
Author(s):  
Anna Matynia ◽  
Sandra S. Salus ◽  
Shelley Sazer
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Ran Gtpase ◽  
A Cell ◽  
Cell Cycle Block ◽  
Er Membrane

The Ran GTPase is an essential protein that has multiple functions in eukaryotic cells. Fission yeast cells in which Ran is misregulated arrest after mitosis with condensed, unreplicated chromosomes and abnormal nuclear envelopes. The fission yeast sns mutants arrest with a similar cell cycle block and interact genetically with the Ran system. sns-A10, sns-B2 and sns-B9 have mutations in the fission yeast homologues of S. cerevisiae Sar1p, Sec31p and Sec53p, respectively, which are required for the early steps of the protein secretory pathway. The three sns mutants accumulate a normally secreted protein in the endoplasmic reticulum (ER), have an increased amount of ER membrane, and the ER/nuclear envelope lumen is dilated. Neither a post-ER block in the secretory pathway, nor ER proliferation caused by overexpression of an integral ER membrane protein, results in a cell cycle-specific defect. Therefore, the arrest seen in sns-A10, sns-B2 and sns-B9 is most likely due to nuclear envelope defects that render the cells unable to re-establish the interphase organization of the nucleus after mitosis. As a consequence, these mutants are unable to decondense their chromosomes or to initiate of the next round of DNA replication.

A cell free system to study reassembly of the nuclear envelope at the end of mitosis

Cell ◽  
1986 ◽  
Vol 44 (4) ◽  
pp. 639-652 ◽  
Author(s):  
Brian Burke ◽  
Larry Gerace
Keyword(s):  
Nuclear Envelope ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals A trypsin-sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro.

1988 ◽  
Vol 107 (1) ◽  
pp. 57-68 ◽  
Author(s):  
K L Wilson ◽  
J Newport
Keyword(s):  
Nuclear Envelope ◽  
Protein Interactions ◽  
Membrane Vesicles ◽  
Integral Membrane Protein ◽  
Electron Microscopic ◽  
Vitro Assay ◽  
Nuclear Envelope Assembly ◽  
Microscopic Studies ◽  
Nuclear Envelope Formation

The reformation of functioning organelles at the end of mitosis presents a problem in vesicle targeting. Using extracts made from Xenopus laevis frog eggs, we have studied in vitro the vesicles that reform the nuclear envelope. In the in vitro assay, nuclear envelope growth is linear with time. Furthermore, the final surface area of the nuclear envelopes formed is directly dependent upon the amount of membrane vesicles added to the assay. Egg membrane vesicles could be fractionated into two populations, only one of which was competent for nuclear envelope assembly. We found that vesicles active in nuclear envelope assembly contained markers (BiP and alpha-glucosidase II) characteristic of the endoplasmic reticulum (ER), but that the majority of ER-derived vesicles do not contribute to nuclear envelope size. This functional distinction between nuclear vesicles and ER-derived vesicles implies that nuclear vesicles are unique and possess at least one factor required for envelope assembly that is lacking in other vesicles. Consistent with this, treatment of vesicles with trypsin destroyed their ability to form a nuclear envelope; electron microscopic studies indicate that the trypsin-sensitive proteins is required for vesicles to bind to chromatin. However, the protease-sensitive component(s) is resistant to treatments that disrupt protein-protein interactions, such as high salt, EDTA, or low ionic strength solutions. We propose that an integral membrane protein, or protein tightly associated with the membrane, is critical for nuclear vesicle targeting or function.

Disassembly of the nuclear envelope of spisula oocytes in a cell-free system

1989 ◽  
Vol 131 (2) ◽  
pp. 496-504 ◽  
Author(s):  
George Dessev ◽  
Robert Palazzo ◽  
Lionel Rebhun ◽  
Robert Goldman
Keyword(s):  
Nuclear Envelope ◽  
Free System ◽  
Cell Free System ◽  
A Cell

scholarly journals Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract.

1992 ◽  
Vol 119 (1) ◽  
pp. 17-25 ◽  
Author(s):  
N Ulitzur ◽  
A Harel ◽  
N Feinstein ◽  
Y Gruenbaum
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Membrane Vesicles ◽  
Embryo Cell ◽  
Drosophila Embryo ◽  
Sperm Chromatin ◽  
Naked Dna ◽  
Nuclear Envelope Assembly

The role of the Drosophila lamin protein in nuclear envelope assembly was studied using a Drosophila in vitro assembly system that reconstitutes nuclei from added sperm chromatin or naked DNA. Upon incubation of the embryonic assembly extract with anti-Drosophila lamin antibodies, the attachment of nuclear membrane vesicles to chromatin surface and nuclear envelope formation did not occur. Lamina assembly and nuclear membrane vesicles attachment to the chromatin were inhibited only when the activity of the 75-kD lamin isoform was inhibited in both soluble and membrane-vesicles fractions. Incubation of decondensed sperm chromatin with an extract that was depleted of nuclear membranes revealed the presence of lamin molecules on the chromatin periphery. In addition, high concentrations of bacterially expressed lamin molecules added to the extract, were able to associate with the chromatin periphery, and did not inhibit nuclear envelope assembly. After nuclear reconstitution, a fraction of the lamin pool was converted into the typical 74- and 76-kD isoforms. Together, these data strongly support an essential role of the lamina in nuclear envelope assembly.

scholarly journals A lamin-independent pathway for nuclear envelope assembly.

1990 ◽  
Vol 111 (6) ◽  
pp. 2247-2259 ◽  
Author(s):  
J W Newport ◽  
K L Wilson ◽  
W G Dunphy
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Structural Integrity ◽  
Nuclear Lamina ◽  
Limited Extent ◽  
Nuclear Pores ◽  
Nuclear Assembly ◽  
Nuclear Envelope Assembly ◽  
A Cell ◽  
Nuclear Envelope Formation

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.

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