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.

The microtubule aster formation and its role in nuclear envelope assembly around the sperm chromatin inXenopus egg extracts

2003 ◽  
Vol 48 (18) ◽  
pp. 1912-1918
Author(s):  
Ning Yang ◽  
Zhongcai Chen ◽  
Ping Lu ◽  
Chuanmao Zhang ◽  
Zhonghe Zhai ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Sperm Chromatin ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Microtubule Aster

scholarly journals Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts.

1985 ◽  
Vol 101 (2) ◽  
pp. 518-523 ◽  
Author(s):  
M J Lohka ◽  
J L Maller
Keyword(s):  
Nuclear Envelope ◽  
Chromosome Condensation ◽  
Sperm Chromatin ◽  
Spindle Formation ◽  
Nuclear Envelope Breakdown ◽  
Multipolar Spindles ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Pronuclear Formation

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.

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 Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts

1999 ◽  
Vol 112 (14) ◽  
pp. 2453-2461 ◽  
Author(s):  
C. Zhang ◽  
M. Hughes ◽  
P.R. Clarke
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Free System ◽  
Nuclear Assembly ◽  
Microtubule Stability ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Ran Gtp ◽  
Xenopus Egg Extracts

Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.

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 Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell-free preparations from amphibian eggs.

1984 ◽  
Vol 98 (4) ◽  
pp. 1222-1230 ◽  
Author(s):  
M J Lohka ◽  
Y Masui
Keyword(s):  
Nuclear Envelope ◽  
Morphological Changes ◽  
Membrane Vesicles ◽  
Particulate Material ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Nuclear Envelope Assembly ◽  
Sperm Nuclei ◽  
Pronuclear Formation

A cell-free cytoplasmic preparation from activated Rana pipiens eggs could induce in demembranated Xenopus laevis sperm nuclei morphological changes similar to those seen during pronuclear formation in intact eggs. The condensed sperm chromatin underwent an initial rapid, but limited, dispersion. A nuclear envelope formed around the dispersed chromatin and the nuclei enlarged. The subcellular distribution of the components required for these changes was examined by separating the preparations into soluble (cytosol) and particulate fractions by centrifugation at 150,000 g for 2 h. Sperm chromatin was incubated with the cytosol or with the particulate material after it had been resuspended in either the cytosol, heat-treated (60 or 100 degrees C) cytosol or buffer. We found that the limited dispersion of chromatin occurred in each of these ooplasmic fractions, but not in the buffer alone. Nuclear envelope assembly required the presence of both untreated cytosol and particulate material. Ultrastructural examination of the sperm chromatin during incubation in the preparations showed that membrane vesicles of approximately 200 nm in diameter, found in the particulate fraction, flattened and fused together to contribute the membranous components of the nuclear envelope. The enlargement of the sperm nuclei occurred only after the nuclear envelope formed. The pronuclei formed in the cell-free preparations were able to incorporate [3H]dTTP into DNA. This incorporation was inhibited by aphidicolin, suggesting that the DNA synthesis by the pronuclei was dependent on DNA polymerase-alpha. When sperm chromatin was incubated greater than 3 h, the chromatin of the pronuclei often recondensed to form structures resembling mitotic chromosomes within the nuclear envelope. Therefore, it appeared that these ooplasmic preparations could induce, in vitro, nuclear changes resembling those seen during the first cell cycle in the zygote.

scholarly journals The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts

1995 ◽  
Vol 5 (11) ◽  
pp. 1270-1279 ◽  
Author(s):  
Mark A. Madine ◽  
Chong-Yee Khoo ◽  
Anthony D. Mills ◽  
Christine Musahl ◽  
Ronald A. Laskey
Keyword(s):  
Nuclear Envelope ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts
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