scholarly journals Membrane-associated lamins in Xenopus egg extracts: identification of two vesicle populations.

1993 ◽  
Vol 123 (3) ◽  
pp. 501-512 ◽  
Author(s):  
D Lourim ◽  
G Krohne
Keyword(s):  
Nuclear Envelope ◽  
Xenopus Oocyte ◽  
Mitotic Cell ◽  
Early Cleavage ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Lamin ◽  
Early Embryos ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Nuclear lamin isoforms of vertebrates can be divided into two major classes. The B-type lamins are membrane associated throughout the cell cycle, whereas A-type lamins are recovered from mitotic cell homogenates in membrane-free fractions. A feature of oogenesis in birds and mammals is the nearly exclusive presence of B-type lamins in oocyte nuclear envelopes. In contrast, oocytes and early cleavage embryos of the amphibian Xenopus laevis are believed to contain a single lamin isoform, lamin LIII, which after nuclear envelope breakdown during meiotic maturation is reported to be completely soluble. Consequently, we have reexamined the lamin complement of Xenopus oocyte nuclear envelopes, egg extracts, and early embryos. An mAb (X223) specific for the homologous B-type lamins B2 of mouse and LII of Xenopus somatic cells (Höger, T., K. Zatloukal, I. Waizenegger, and G. Krohne. 1990. Chromosoma. 99:379-390) recognized a Xenopus oocyte nuclear envelope protein biochemically distinct from lamin LIII and very similar or identical to somatic cell lamin LII. Oocyte lamin LII was detectable in nuclear envelopes of early cleavage embryos. Immunoblotting of fractionated egg extracts revealed that approximately 20-23% of lamin LII and 5-7% of lamin LIII were membrane associated. EM immunolocalization demonstrated that membrane-bound lamins LII and LIII are associated with separate vesicle populations. These findings are relevant to the interpretation of nuclear reconstitution experiments using Xenopus egg extracts.

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

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.

Lamin B receptor-mediated chromatin tethering to the nuclear envelope is detrimental to the xenopus blastula

2020 ◽  
Author(s):  
Haruka Oda ◽  
Satsuki Kato ◽  
Keita Ohsumi ◽  
Mari Iwabuchi
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Spindles ◽  
Filamentous Actin ◽  
Blastula Stage ◽  
Lamin B ◽  
Lamin B Receptor ◽  
Specific Manner ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Abstract In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear laminar architecture and nuclear morphology, and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.

scholarly journals Phosphorylation and activation of the Xenopus Cdc25 phosphatase in the absence of Cdc2 and Cdk2 kinase activity.

1995 ◽  
Vol 6 (2) ◽  
pp. 215-226 ◽  
Author(s):  
T Izumi ◽  
J L Maller
Keyword(s):  
Kinase Activity ◽  
Cdc2 Kinase ◽  
Nuclear Envelope Breakdown ◽  
Dual Specificity ◽  
M Phase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The M-phase inducer, Cdc25C, is a dual-specificity phosphatase that directly phosphorylates and activates the cyclin B/Cdc2 kinase complex, leading to initiation of mitosis. Cdc25 itself is activated at the G2/M transition by phosphorylation on serine and threonine residues. Previously, it was demonstrated that Cdc2 kinase is capable of phosphorylating and activating Cdc25, suggesting the existence of a positive feedback loop. In the present study, kinases other than Cdc2 that can phosphorylate and activate Cdc25 were investigated. Cdc25 was found to be phosphorylated and activated by cyclin A/Cdk2 and cyclin E/Cdk2 in vitro. However, in interphase Xenopus egg extracts with no detectable Cdc2 and Cdk2, treatment with the phosphatase inhibitor microcystin activated a distinct kinase that could phosphorylate and activate Cdc25. Microcystin also induced other mitotic phenomena such as chromosome condensation and nuclear envelope breakdown in extracts containing less than 5% of the mitotic level of Cdc2 kinase activity. These findings implicate a kinase other than Cdc2 and Cdk2 that may initially activate Cdc25 in vivo and suggest that this kinase may also phosphorylate M-phase substrates even in the absence of Cdc2 kinase.

scholarly journals The role of keratin filaments during nuclear envelope reassembly in Xenopus egg extracts 1

FEBS Letters ◽  
1998 ◽  
Vol 428 (1-2) ◽  
pp. 52-56 ◽  
Author(s):  
Bo Zhang ◽  
Ying Chen ◽  
Zhiyang Han ◽  
Hans Ris ◽  
Zhonghe Zhai
Keyword(s):  
Nuclear Envelope ◽  
Keratin Filaments ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

1996 ◽  
Vol 135 (5) ◽  
pp. 1207-1218 ◽  
Author(s):  
S J Lawlis ◽  
S M Keezer ◽  
J R Wu ◽  
D M Gilbert
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Cho Cells ◽  
Chromosome Condensation ◽  
G1 Phase ◽  
Unusual Site ◽  
Chromosome Architecture ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

scholarly journals Subgroup II PAK-mediated phosphorylation regulates Ran activity during mitosis

2010 ◽  
Vol 190 (5) ◽  
pp. 807-822 ◽  
Author(s):  
Guillaume Bompard ◽  
Gabriel Rabeharivelo ◽  
Marie Frank ◽  
Julien Cau ◽  
Claude Delsert ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Mitotic Progression ◽  
Subgroup Ii ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
P21 Activated Kinase ◽  
Nuclear Envelope Formation ◽  
Xenopus Egg Extracts

Ran is an essential GTPase that controls nucleocytoplasmic transport, mitosis, and nuclear envelope formation. These functions are regulated by interaction of Ran with different partners, and by formation of a Ran-GTP gradient emanating from chromatin. Here, we identify a novel level of Ran regulation. We show that Ran is a substrate for p21-activated kinase 4 (PAK4) and that its phosphorylation on serine-135 increases during mitosis. The endogenous phosphorylated Ran and active PAK4 dynamically associate with different components of the microtubule spindle during mitotic progression. A GDP-bound Ran phosphomimetic mutant cannot undergo RCC1-mediated GDP/GTP exchange and cannot induce microtubule asters in mitotic Xenopus egg extracts. Conversely, phosphorylation of GTP-bound Ran facilitates aster nucleation. Finally, phosphorylation of Ran on serine-135 impedes its binding to RCC1 and RanGAP1. Our study suggests that PAK4-mediated phosphorylation of GDP- or GTP-bound Ran regulates the assembly of Ran-dependent complexes on the mitotic spindle.

scholarly journals 23REPROGRAMMING MAMMALIAN CELLS IN XENOPUS EGG EXTRACTS: ROLE OF EMBRYONIC LAMIN B3

2004 ◽  
Vol 16 (2) ◽  
pp. 134
Author(s):  
R. Alberio ◽  
K.H.S. Campbell
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Somatic Cells ◽  
Nuclear Transplantation ◽  
Heterologous System ◽  
Fetal Fibroblasts ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The generation of animals by nuclear transplantation has demonstrated that a fully differentiated cell can be reversed into totipotency when transferred into an oocyte. Identification of oocyte specific molecules responsible for the reprogramming of somatic cells may contribute to the understanding of cell differentiation and embryo development. We have developed a heterologous system to investigate the effect of lamin B3, a major component of Xenopus laevis egg cytoplasm, on DNA replication of mammalian somatic cells. Bovine fetal fibroblasts were arrested at G1/S by incubation in aphidicolin for 18h. After permeabilization with digitonin, the cells were incubated in either (1) lamin B3 depleted, or (2) whole Xenopus egg extracts (1000 cells μL−1 extract) supplemented with an energy regenerating system for a period of 3h at 21°C. Xenopus lamin B3-depleted egg extracts were prepared by three rounds of incubation with Dynabeads coated with a mouse monoclonal lamin B3 antibody (mAbLB3). Immunodepletion was confirmed by western blotting. Purified lamin B3 was obtained by dialysis of the beads after immunodepletion, and the purified lamin B3 was used for rescue experiments. DNA replication of cells incubated in the extracts was assessed by adding 25μM Biotin-11-dUTP for 3h. After treatment cells were fixed in 70% methanol at −20°C and incubated in mAbLB3 for 30min at 37°C. This was followed by incubation in FITC-conjugated sheep anti-mouse antibody and in 5mgmL−1 Texas Red-conjugated Streptavidin for 40min at 37°C. After three hours’ incubation in egg extracts, DNA replication was detected in 60% of cells and more than 95% of cells were lamin B3 positive. In contrast, DNA replication in immunodepleted extracts was significantly lower (P≤0.01, by one-way ANOVA) than in cells incubated in whole extracts and was coincident with the few lamin B3-positive cells observed. More than 95% of cells were lamin B3-negative and did not replicate DNA. When purified lamin B3 was re-added to depleted extracts, DNA replication was detected in 60% of cells. DNA synthesis resumed in 93% of control cells 3h after release from aphidicolin into culture medium at 39°C. These experiments show that somatic nuclei, which possess a nuclear envelope with somatic variants of lamins, are able to synthesize DNA in egg extracts only when Xenopus lamin B3 is incorporated into the nuclear envelope. This heterologous system provides new information on the role of an embryonic molecule, namely Xenopus lamin B3, in the reprogramming of DNA replication of somatic cells incubated in egg environment. These results open new questions as to whether embryonic lamins also exist in mammals, and whether failure in development of cloned animals is in part due to abnormal or incomplete replacement of somatic variants of proteins with their embryonic counterparts.

scholarly journals Nup53 Is Required for Nuclear Envelope and Nuclear Pore Complex Assembly

2008 ◽  
Vol 19 (4) ◽  
pp. 1753-1762 ◽  
Author(s):  
Lisa A. Hawryluk-Gara ◽  
Melpomeni Platani ◽  
Rachel Santarella ◽  
Richard W. Wozniak ◽  
Iain W. Mattaj
Keyword(s):  
Nuclear Envelope ◽  
Critical Role ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Egg Extracts ◽  
Multiple Copies ◽  
Xenopus Egg ◽  
Pore Complexes ◽  
Xenopus Egg Extracts

Transport across the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs). These structures are composed of various subcomplexes of proteins that are each present in multiple copies and together establish the eightfold symmetry of the NPC. One evolutionarily conserved subcomplex of the NPC contains the nucleoporins Nup53 and Nup155. Using truncation analysis, we have defined regions of Nup53 that bind to neighboring nucleoporins as well as those domains that target Nup53 to the NPC in vivo. Using this information, we investigated the role of Nup53 in NE and NPC assembly using Xenopus egg extracts. We show that both events require Nup53. Importantly, the analysis of Nup53 fragments revealed that the assembly activity of Nup53 depleted extracts could be reconstituted using a region of Nup53 that binds specifically to its interacting partner Nup155. On the basis of these results, we propose that the formation of a Nup53–Nup155 complex plays a critical role in the processes of NPC and NE assembly.

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