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 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 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 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.

scholarly journals A role for nuclear lamins in nuclear envelope assembly

2001 ◽  
Vol 154 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Reynold I. Lopez-Soler ◽  
Robert D. Moir ◽  
Timothy P. Spann ◽  
Reimer Stick ◽  
Robert D. Goldman
Keyword(s):  
Nuclear Envelope ◽  
Molecular Interactions ◽  
Nuclear Membrane ◽  
Nuclear Lamina ◽  
Chromatin Decondensation ◽  
Nuclear Lamins ◽  
Nuclear Membranes ◽  
Terminal Domain ◽  
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.

scholarly journals Temporal control of nuclear envelope assembly by phosphorylation of lamin B receptor

2011 ◽  
Vol 22 (18) ◽  
pp. 3306-3317 ◽  
Author(s):  
Li-Chuan Tseng ◽  
Rey-Huei Chen
Keyword(s):  
Membrane Protein ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Chromatin Binding ◽  
Temporal Control ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Nuclear Envelope Assembly ◽  
Nuclear Membrane Protein

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Here, we use time-lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin-binding ability of LBR together with Aurora B activity contributes to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

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.

Drosophila Melanogaster Embryo Cultures: An In Vitro Teratogen Assay

1990 ◽  
Vol 18 (1_part_1) ◽  
pp. 291-300
Author(s):  
Nicole Bournias-Vardiabasis
Keyword(s):  
Stress Proteins ◽  
Embryo Cell ◽  
Drosophila Embryo ◽  
Abnormal Development ◽  
Normal Muscle ◽  
Embryo Cultures ◽  
Teratogenic Potential ◽  
Shock Proteins

An in vitro teratogen assay has been developed that uses Drosophila embryo cell cultures. The endpoints selected in assessing the teratogenic potential of any agent (physical or chemical) involves detection of interference with normal muscle and/or neuron differentiation, induction of heat stock (stress) proteins, and inhibition of normal neurotransmitter levels. Current studies involve use of reporter gene technology (protein fusions) to identify teratogenicity. Results so far suggest that the Drosophila assay is capable of accurately establishing if a particular agent tested can act as a teratogen by a variety of appropriate endpoints (morphological, biochemical, molecular). Furthermore, this assay can be used not only as a teratogen screen, but also in mechanistic studies of abnormal development, gene involvement in teratogenic resistance, and the possible role of heat shock proteins in preventing birth defects.

scholarly journals GTP hydrolysis is required for vesicle fusion during nuclear envelope assembly in vitro.

1992 ◽  
Vol 116 (2) ◽  
pp. 281-294 ◽  
Author(s):  
A L Boman ◽  
M R Delannoy ◽  
K L Wilson
Keyword(s):  
Nuclear Envelope ◽  
Light And Electron Microscopy ◽  
Vesicle Fusion ◽  
Sperm Chromatin ◽  
Fusion Pore ◽  
Soluble Factor ◽  
Gtp Hydrolysis ◽  
Nuclear Envelope Assembly ◽  
Gamma S

Nuclear envelope assembly was studied in vitro using extracts from Xenopus eggs. Nuclear-specific vesicles bound to demembranated sperm chromatin but did not fuse in the absence of cytosol. Addition of cytosol stimulated vesicle fusion, pore complex assembly, and eventual nuclear envelope growth. Vesicle binding and fusion were assayed by light and electron microscopy. Addition of ATP and GTP to bound vesicles caused limited vesicle fusion, but enclosure of the chromatin was not observed. This result suggested that nondialyzable soluble components were required for nuclear vesicle fusion. GTP gamma S and guanylyl imidodiphosphate significantly inhibited vesicle fusion but had no effect on vesicle binding to chromatin. Preincubation of membranes with 1 mM GTP gamma S or GTP did not impair vesicle binding or fusion when assayed with fresh cytosol. However, preincubation of membranes with GTP gamma S plus cytosol caused irreversible inhibition of fusion. The soluble factor mediating the inhibition by GTP gamma S, which we named GTP-dependent soluble factor (GSF), was titratable and was depleted from cytosol by incubation with excess membranes plus GTP gamma S, suggesting a stoichiometric interaction between GSF and a membrane component in the presence of GTP gamma S. In preliminary experiments, cytosol depleted of GSF remained active for fusion of chromatin-bound vesicles, suggesting that GSF may not be required for the fusion reaction itself. We propose that GTP hydrolysis is required at a step before the fusion of nuclear vesicles.

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