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 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 Mammalian nuclei become licensed for DNA replication during late telophase

2002 ◽  
Vol 115 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Daniela S. Dimitrova ◽  
Tatyana A. Prokhorova ◽  
J. Julian Blow ◽  
Ivan T. Todorov ◽  
David M. Gilbert
Keyword(s):  
Cho Cells ◽  
Chinese Hamster ◽  
Significant Degree ◽  
Initiation Of Replication ◽  
Egg Extracts ◽  
Late Telophase ◽  
Xenopus Egg ◽  
Mcm Proteins ◽  
Nuclear Envelope Formation ◽  
Xenopus Egg Extracts

Mcm 2-7 are essential replication proteins that bind to chromatin in mammalian nuclei during late telophase. Here, we have investigated the relationship between Mcm binding, licensing of chromatin for replication, and specification of the dihydrofolate reductase (DHFR) replication origin. Approximately 20% of total Mcm3 protein was bound to chromatin in Chinese hamster ovary (CHO) cells during telophase, while an additional 25% bound gradually and cumulatively throughout G1-phase. To investigate the functional significance of this binding, nuclei prepared from CHO cells synchronized at various times after metaphase were introduced into Xenopus egg extracts, which were either immunodepleted of Mcm proteins or supplemented with geminin, an inhibitor of the Mcm-loading protein Cdt1. Within 1 hour after metaphase, coincident with completion of nuclear envelope formation, CHO nuclei were fully competent to replicate in both of these licensing-defective extracts. However, sites of initiation of replication in each of these extracts were found to be dispersed throughout the DHFR locus within nuclei isolated between 1 to 5 hours after metaphase, but became focused to the DHFR origin within nuclei isolated after 5 hours post-metaphase. Importantly, introduction of permeabilized post-ODP, but not pre-ODP, CHO nuclei into licensing-deficient Xenopus egg extracts resulted in the preservation of a significant degree of DHFR origin specificity, implying that the previously documented lack of specific origin selection in permeabilized nuclei is at least partially due to the licensing of new initiation sites by proteins in the Xenopus egg extracts. We conclude that the functional association of Mcm proteins with chromatin (i.e. replication licensing) in CHO cells takes place during telophase, several hours prior to the specification of replication origins at the DHFR locus.

scholarly journals A requirement for MAP kinase in the assembly and maintenance of the mitotic spindle

2003 ◽  
Vol 161 (6) ◽  
pp. 1021-1028 ◽  
Author(s):  
Melinda M. Horne ◽  
Thomas M. Guadagno
Keyword(s):  
Map Kinase ◽  
Mitotic Spindle ◽  
Kinase Inhibitor ◽  
Map Kinase Phosphatase ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Multiple Spindle ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Bipolar Spindles

Circumstantial evidence has suggested the possibility of microtubule-associated protein (MAP) kinase's involvement in spindle regulation. To test this directly, we asked whether MAP kinase was required for spindle assembly in Xenopus egg extracts. Either the inhibition or the depletion of endogenous p42 MAP kinase resulted in defective spindle structures resembling asters or half-spindles. Likewise, an increase in the length and polymerization of microtubules was measured in aster assays suggesting a role for MAP kinase in regulating microtubule dynamics. Consistent with this, treatment of extracts with either a specific MAP kinase kinase inhibitor or a MAP kinase phosphatase resulted in the rapid disassembly of bipolar spindles into large asters. Finally, we report that mitotic progression in the absence of MAP kinase signaling led to multiple spindle abnormalities in NIH 3T3 cells. We therefore propose that MAP kinase is a key regulator of the mitotic spindle.

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.

Targeting of Ran: variation on a common theme?

2001 ◽  
Vol 114 (18) ◽  
pp. 3233-3241 ◽  
Author(s):  
Markus Künzler ◽  
Ed Hurt
Keyword(s):  
Nuclear Envelope ◽  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Common Theme ◽  
Spindle Formation ◽  
Ran Gtpase ◽  
Cellular Processes ◽  
Important Target ◽  
Transport Receptors ◽  
Nuclear Envelope Formation

The Ran GTPase plays a key role in nucleocytoplasmic transport. In its GTP-bound form, it directly interacts with members of the importin β family of nuclear transport receptors and modulates their association with cargo. Work in cell-free higher-eukaryote systems has demonstrated additional roles for Ran in spindle and nuclear envelope formation during mitosis. However, until recently, no Ran-target proteins in these cellular processes were known. Several groups have now identified importin β as one important target of Ran during mitotic spindle formation. This finding suggests that Ran uses the same effectors to regulate different cellular processes.

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 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.

Initiation of DNA synthesis by nuclei from scrape-ruptured quiescent mammalian cells in high-speed supernatants of Xenopus egg extracts

1994 ◽  
Vol 107 (11) ◽  
pp. 3045-3053 ◽  
Author(s):  
M. Hola ◽  
S. Castleden ◽  
M. Howard ◽  
R.F. Brooks
Keyword(s):  
Nuclear Envelope ◽  
Dna Synthesis ◽  
Mammalian Cells ◽  
High Speed ◽  
Nuclear Assembly ◽  
Naked Dna ◽  
Egg Extracts ◽  
Eukaryotic Dna ◽  
Nuclear Envelope Formation ◽  
Xenopus Egg Extracts

Demembranated sperm heads, detergent-isolated somatic nuclei and even naked DNA are efficiently replicated in cytoplasmic extracts of activated amphibian eggs, but only after nuclear assembly and the formation of an intact nuclear envelope. DNA synthesis has not previously been shown to be initiated in high-speed (200,000 g) supernatants of egg cytoplasm because they are depleted of the vesicular material required to support nuclear envelope formation. Here we show that mammalian nuclei prepared by scrape-rupture are able to initiate DNA replication in such high-speed supernatants. These nuclei begin DNA synthesis asynchronously. This asynchrony cannot be attributed to differences in the time taken for nuclear assembly. Instead, we suggest that the asynchrony reflects intrinsic differences between nuclei and that these differences are a major cause of cell cycle variability. Our demonstration of initiation in high-speed supernatants now enables the initiation of eukaryotic DNA synthesis to be studied independently of nuclear assembly.

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