scholarly journals Xenopus lamin B3 has a direct role in the assembly of a replication competent nucleus: evidence from cell-free egg extracts

1995 ◽  
Vol 108 (11) ◽  
pp. 3451-3461 ◽  
Author(s):  
M. Goldberg ◽  
H. Jenkins ◽  
T. Allen ◽  
W.G. Whitfield ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Nuclear Matrix ◽  
Nuclear Pore ◽  
Major Protein ◽  
Protein Species ◽  
Nuclear Pores ◽  
Thin Sections ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly

Xenopus egg extracts which assemble replication competent nuclei in vitro were depleted of lamin B3 using monoclonal antibody L6 5D5 linked to paramagnetic beads. After depletion, the extracts were still capable of assembling nuclei around demembranated sperm heads. Using field emission in lens scanning electron microscopy (FEISEM) we show that most nuclei assembled in lamin B3-depleted extracts have continuous nuclear envelopes and well formed nuclear pores. However, several consistent differences were observed. Most nuclei were small and only attained diameters which were half the size of controls. In a small number of nuclei, nuclear pore baskets, normally present on the inner aspect of the nuclear envelope, appeared on its outer surface. Finally, the assembly of nuclear pores was slower in lamin B3-depleted extracts, indicating a slower overall rate of nuclear envelope assembly. The results of FEISEM were confirmed using conventional TEM thin sections, where again the majority of nuclei assembled in lamin B3-depleted extracts had well formed double unit membranes containing a high density of nuclear pores. Since nuclear envelope assembly was mostly normal but slow in these nuclei, the lamin content of ‘depleted’ extracts was investigated. While lamin B3 was recovered efficiently from cytosolic and membrane fractions by our procedure, a second minor lamin isoform, which has characteristics similar to those of the somatic lamin B2, remained in the extract. Thus it is likely that this lamin is necessary for nuclear envelope assembly. However, while lamin B2 did not co-precipitate with lamin B3 during immunodepletion experiments, several protein species did specifically associate with lamin B3 on paramagnetic immunobeads. The major protein species associated with lamin B3 migrated with molecular masses of 102 kDa and 57 kDa, respectively, on one-dimensional polyacrylamide gels. On two-dimensional O'Farrell gels the mobility of the 102 kDa protein was identical to the mobility of a major nuclear matrix protein, indicating a specific association between lamin B3 and other nuclear matrix proteins. Nuclei assembled in lamin B3-depleted extracts did not assemble a lamina, judged by indirect immunofluorescence, and failed to initiate semi-conservative DNA replication. However, by reinoculating depleted extracts with purified lamin B3, nuclear lamina assembly and DNA replication could both be rescued. Thus it seems likely that the inability of lamin-depleted extracts to assemble a replication competent nucleus is a direct consequence of a failure to assemble a lamina.

Nuclei that lack a lamina accumulate karyophilic proteins and assemble a nuclear matrix

1993 ◽  
Vol 106 (1) ◽  
pp. 275-285 ◽  
Author(s):  
H. Jenkins ◽  
T. Holman ◽  
C. Lyon ◽  
B. Lane ◽  
R. Stick ◽  
...  
Keyword(s):  
Dna Replication ◽  
Nuclear Matrix ◽  
Magnetic Beads ◽  
Major Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Protein Components

Xenopus egg extracts, which support nuclear assembly and DNA replication in vitro, were physically depleted of lamin B3 using monoclonal antibodies linked to magnetic beads. Depleted extracts were still able to support nuclear envelope assembly around demembranated sperm heads but the resulting pronuclei lacked a lamina and were unable to initiate semiconservative DNA replication or to assemble replicases, confirming previous data. Immunoblotting analysis of isolated nuclei and nuclear matrix fractions indicated that lamin-depleted nuclei still accumulated nucleoporins and PCNA. Furthermore, the rate of PCNA uptake was identical in lamin-depleted and control nuclei. However, neither the nucleoporins nor the PCNA was associated with nuclear matrix fractions. The major protein components of sperm pronuclear matrix fractions were characterized by two-dimensional gel electrophoresis. Of these proteins only three out of 22 species, other than the lamins, were significantly reduced in lamin-depleted nuclei, indicating that these nuclei do assemble a nuclear matrix.

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent ‘envelope smoothing’ event

1997 ◽  
Vol 110 (13) ◽  
pp. 1489-1502 ◽  
Author(s):  
C. Wiese ◽  
M.W. Goldberg ◽  
T.D. Allen ◽  
K.L. Wilson
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Transmission Electron ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Attachment Proteins ◽  
Scanning Em ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying (‘rough’) vesicles, many of which bound directly to chromatin, and ‘smooth’ vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed ‘envelope smoothing’ occurred 5–15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that ‘smoothing’ required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

scholarly journals Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor

1993 ◽  
Vol 122 (5) ◽  
pp. 993-1002 ◽  
Author(s):  
JJ Blow
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Nuclear Envelope ◽  
Kinase Inhibitor ◽  
Chromatin Modification ◽  
Replication Factor ◽  
Licensing Factor ◽  
Egg Extracts ◽  
Nuclear Envelope Assembly ◽  
Xenopus Egg Extracts

Xenopus egg extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP) are unable to support the initiation of DNA replication. Nuclei assembled in 6-DMAP extracts behave as though they are in G2, and will not undergo another round of DNA replication until passage through mitosis. 6-DMAP extracts are functionally devoid of a replication factor that modifies chromatin in early G1 before nuclear envelope assembly, but which is itself incapable of crossing the nuclear envelope. This chromatin modification is capable of supporting only a single round of semiconservative replication. The behavior of this replication factor is sufficient to explain why eukaryotic DNA is replicated once and only once in each cell cycle, and conforms to the previous model of a Replication Licensing Factor. Cell cycle analysis shows that this putative Licensing Factor is inactive during metaphase, but becomes rapidly activated on exit from metaphase when it can modify chromatin before nuclear envelope assembly is complete.

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

Sequential degradation of proteins from the nuclear envelope during apoptosis

2001 ◽  
Vol 114 (20) ◽  
pp. 3643-3653 ◽  
Author(s):  
Madeleine Kihlmark ◽  
Gabriela Imreh ◽  
Einar Hallberg
Keyword(s):  
Nuclear Envelope ◽  
Apoptotic Cell ◽  
Nuclear Periphery ◽  
Condensed Chromatin ◽  
Nuclear Pore ◽  
Dependent Manner ◽  
Nuclear Pores ◽  
Double Labeling ◽  
Lamin B ◽  
Nuclear Apoptosis

We have produced new antibodies specific for the integral pore membrane protein POM121. Using these antibodies we show that during apoptosis POM121 becomes proteolytically degraded in a caspase-dependent manner. The POM121 antibodies and antibodies specific for other proteins of the nuclear envelope were used in a comparative study of nuclear apoptosis in staurosporine-treated buffalo rat liver cells. Nuclei from these cells were classified in three different stages of apoptotic progression: stage I, moderately condensed chromatin surrounded by a smooth nuclear periphery; stage II, compact patches of condensed chromatin collapsing against a smooth nuclear periphery; stage III, round compact chromatin bodies surrounded by grape-shaped nuclear periphery. We have performed double labeling immunofluorescence microscopy of individual apoptotic cells and quantitative immunoblotting analysis of total proteins from apoptotic cell cultures. The results showed that degradation of nuclear envelope marker proteins occurred in a specific order. POM121 degradation occurred surprisingly early and was initiated before nucleosomal DNA degradation could be detected using TUNEL assay and completed before clustering of the nuclear pores. POM121 was eliminated significantly more rapid compared with NUP153 (a peripheral protein located in the nucleoplasmic basket of the nuclear pore complex) and lamin B (a component of the nuclear lamina). Disappearance of NUP153 and lamin B was coincident with onset of DNA fragmentation and clustering of nuclear pores. By contrast, the peripheral NPC protein p62 was degraded much later. The results suggest that degradation of POM121 may be an important early step in propagation of nuclear apoptosis.

scholarly journals Ongoing replication forks delay nuclear envelope breakdown upon mitotic entry

2020 ◽  
pp. jbc.RA120.015142
Author(s):  
Yoshitami Hashimoto ◽  
Hirofumi Tanaka
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Dependent Manner ◽  
Repair Synthesis ◽  
Nuclear Envelope Breakdown ◽  
M Phase ◽  
Egg Extracts ◽  
Fork Stalling ◽  
Dna Repair Synthesis ◽  
Xenopus Egg Extracts

DNA replication is a major contributor to genomic instability and protection against DNA replication perturbation is essential for normal cell division. Certain types of replication stress agents, such as aphidicolin and hydroxyurea, have been shown to cause reversible replication fork stalling, wherein replisome complexes are stably maintained with competence to restart in the S-phase of the cell cycle. If these stalled forks persist into the M-phase without a replication restart, replisomes are disassembled in a p97-dependent pathway and under-replicated DNA is subjected to mitotic DNA repair synthesis. Here, using Xenopus egg extracts, we investigated the consequences that arise when stalled forks are released simultaneously with the induction of mitosis. Ara-cytidine-5’-triphosphate (Ara-CTP)-induced stalled forks were able to restart with the addition of excess dCTPduring early mitosis before the nuclear envelope breakdown (NEB). However, stalled forks could no longer restart efficiently after NEB. Although replisome complexes were finally disassembled in a p97-dependent manner during mitotic progression whether or not fork stalling was relieved, the timing of NEB was delayed with the ongoing forks, rather than the stalled forks, and the delay was dependent on Wee1/Myt1 kinase activities. Thus, ongoing DNA replication was found to be directly linked to the regulation of Wee1/Myt1 kinases to modulate cyclin-dependent kinase (CDK) activities, owing to which DNA replication and mitosis occur in a mutually exclusive and sequential manner.

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 Piecing together nuclear pore complex assembly during interphase

2009 ◽  
Vol 185 (3) ◽  
pp. 377-379 ◽  
Author(s):  
Michael Rexach
Keyword(s):  
Nuclear Envelope ◽  
Nuclear Pore Complex ◽  
Supramolecular Structures ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Assembly Process ◽  
Nuclear Pores ◽  
Complex Assembly ◽  
Cell Biol ◽  
Pore Complexes

All nucleocytoplasmic traffic of macromolecules occurs through nuclear pore complexes (NPCs), which function as stents in the nuclear envelope to keep nuclear pores open but gated. Three studies in this issue (Flemming, D., P. Sarges, P. Stelter, A. Hellwig, B. Böttcher, and E. Hurt. 2009. J. Cell Biol. 185:387–395; Makio, T., L.H. Stanton, C.-C. Lin, D.S. Goldfarb, K. Weis, and R.W. Wozniak. 2009. J. Cell Biol. 185:459–491; Onishchenko, E., L.H. Stanton, A.S. Madrid, T. Kieselbach, and K. Weis. 2009. J. Cell Biol. 185:475–491) further our understanding of the NPC assembly process by reporting what happens when the supply lines of key proteins that provide a foundation for building these marvelous supramolecular structures are disrupted.

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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