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 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.

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

Metaphase protein phosphorylation in Xenopus laevis eggs

1987 ◽  
Vol 7 (2) ◽  
pp. 760-768
Author(s):  
M J Lohka ◽  
J L Kyes ◽  
J L Maller
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Protein Phosphorylation ◽  
Tyrosine Kinases ◽  
Chromosome Condensation ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Nuclear Events ◽  
Gamma S

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.

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 Targeting of membranes to sea urchin sperm chromatin is mediated by a lamin B receptor-like integral membrane protein.

1996 ◽  
Vol 135 (6) ◽  
pp. 1715-1725 ◽  
Author(s):  
P Collas ◽  
J C Courvalin ◽  
D Poccia
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Sea Urchin ◽  
Binding Capacity ◽  
Integral Membrane Protein ◽  
Sperm Chromatin ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Pronuclear Formation

We have identified an integral membrane protein of sea urchin gametes with an apparent molecular mass of 56 kD that cross-reacts with an antibody against the nucleoplasmic NH2-terminal domain of human lamin B receptor (LBR). In mature sperm, p56 is located at the tip and base of the nucleus from where it is removed by egg cytosol in vitro. In the egg, p56 is present in a subset of cytoplasmic membranes (MV2 beta) which contributes the bulk of the nuclear envelope during male pronuclear formation. p56-containing vesicles are required for nuclear envelope assembly and have a chromatin-binding capacity that is mediated by p56. Lamin B is not present in these vesicles and is imported into the nucleus from a soluble pool at a later stage of pronuclear formation. Lamin B incorporation and addition of new membranes are necessary for pronuclear swelling and nuclear envelope growth. We suggest that p56 is a sea urchin LBR homologue that targets membranes to chromatin and later anchors the membrane to the lamina.

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 Metaphase protein phosphorylation in Xenopus laevis eggs.

1987 ◽  
Vol 7 (2) ◽  
pp. 760-768 ◽  
Author(s):  
M J Lohka ◽  
J L Kyes ◽  
J L Maller
Keyword(s):  
Xenopus Laevis ◽  
Nuclear Envelope ◽  
Protein Phosphorylation ◽  
Tyrosine Kinases ◽  
Chromosome Condensation ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Nuclear Events ◽  
Gamma S

Cytoplasmic extracts of metaphase (M-phase)-arrested Xenopus laevis eggs support nuclear envelope breakdown and chromosome condensation in vitro. Induction of nuclear breakdown is inhibited by AMPP(NH)P, a nonhydrolyzable ATP analog, but not by ATP or gamma-S-ATP, a hydrolyzable ATP analog, suggesting that protein phosphorylation may be required for M-phase nuclear events in vitro. By addition of [gamma-32P]ATP, we have identified in cytoplasmic extracts and in intact eggs at least six phosphoproteins that are present during M-phase but absent in G1/S-phase. These phosphoproteins also appear in response to partially purified preparations of maturation-promoting factor. A subset of these proteins are thiophosphorylated by gamma-S-ATP under conditions that promote nuclear envelope breakdown and chromosome condensation. Each of these proteins is phosphorylated on serine and threonine, and one, a 42-kilodalton protein, is also phosphorylated on tyrosine both in extracts and in intact eggs. These results indicate that activation of protein kinases accounts for at least part of the increased phosphorylation in M-phase and that both protein-serine-threonine kinases and protein-tyrosine kinases may play a role in controlling M-phase nuclear behavior.

scholarly journals An in vitro nuclear disassembly system reveals a role for the RanGTPase system and microtubule-dependent steps in nuclear envelope breakdown

2007 ◽  
Vol 178 (4) ◽  
pp. 595-610 ◽  
Author(s):  
Petra Mühlhäusser ◽  
Ulrike Kutay
Keyword(s):  
Nuclear Envelope ◽  
Fluorescent Protein ◽  
Dynamic Rupture ◽  
Vitro Assay ◽  
Nuclear Envelope Breakdown ◽  
Egg Extracts ◽  
Guanosine Triphosphatase ◽  
Green Fluorescent

During prophase, vertebrate cells disassemble their nuclear envelope (NE) in the process of NE breakdown (NEBD). We have established an in vitro assay that uses mitotic Xenopus laevis egg extracts and semipermeabilized somatic cells bearing a green fluorescent protein–tagged NE marker to study the molecular requirements underlying the dynamic changes of the NE during NEBD by live microscopy. We applied our in vitro system to analyze the role of the Ran guanosine triphosphatase (GTPase) system in NEBD. Our study shows that high levels of RanGTP affect the dynamics of late steps of NEBD in vitro. Also, inhibition of RanGTP production by RanT24N blocks the dynamic rupture of nuclei, suggesting that the local generation of RanGTP around chromatin may serve as a spatial cue in NEBD. Furthermore, the microtubule-depolymerizing drug nocodazole interferes with late steps of nuclear disassembly in vitro. High resolution live cell imaging reveals that microtubules are involved in the completion of NEBD in vivo by facilitating the efficient removal of membranes from chromatin.

scholarly journals The cdc25 homologue twine is required for only some aspects of the entry into meiosis in Drosophila

1993 ◽  
Vol 106 (4) ◽  
pp. 1035-1044 ◽  
Author(s):  
H. White-Cooper ◽  
L. Alphey ◽  
D.M. Glover
Keyword(s):  
Nuclear Envelope ◽  
Chromosome Condensation ◽  
Wild Type ◽  
Spindle Formation ◽  
Nuclear Envelope Breakdown ◽  
Spindle Poles ◽  
Meiotic Spindles ◽  
Metaphase I

The twineHB5 mutation prevents spindle formation during the entry into meiosis in Drosophila males, but chromosome condensation and nuclear envelope breakdown both still occur. This suggests the possibility that this particular cdc25 homologue is required to activate a p34cdc2 kinase required for only some of the events of this G2-M transition. In contrast, meiotic spindles do form in twineHB5 females, although these appear abnormal. However, the female meiotic divisions do not arrest at metaphase I as in wild type, but continue repeatedly, leading to gross non-disjunction. Small chromatin masses, corresponding in size to the fourth chromosomes, often segregate properly to the spindle poles. These can persist into the embryos derived from twineHB5 females, where they appear to participate in mitotic divisions on thin spindles. In addition, these embryos contain a small number of large chromatin masses that are not associated with spindles.

Sign in / Sign up

Export Citation Format

Share Document