scholarly journals Ran-GTP stabilises microtubule asters and inhibits nuclear assembly in Xenopus egg extracts

1999 ◽  
Vol 112 (14) ◽  
pp. 2453-2461 ◽  
Author(s):  
C. Zhang ◽  
M. Hughes ◽  
P.R. Clarke
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Asymmetric Distribution ◽  
Nucleotide Exchange ◽  
Free System ◽  
Nuclear Assembly ◽  
Microtubule Stability ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Ran Gtp ◽  
Xenopus Egg Extracts

Ran is an abundant GTPase of the Ras superfamily that is highly conserved in eukaryotes. In interphase cells, Ran is mainly nuclear and thought to be predominantly GTP-bound, but it is also present in the cytoplasm, probably GDP-bound. This asymmetric distribution plays an important role in directing nucleocytoplasmic transport. Ran has also been implicated in cell cycle control, including the transition from mitosis to interphase when the compartmentalisation of the nucleus is established. Here, we have examined the role of Ran in this transition using a cell-free system of Xenopus egg extracts supplemented with sperm heads that provides a model for microtubule aster formation and post-M phase nuclear assembly. Ran-GTP, added as wild-type protein, a mutant defective in GTPase activity (Q69L), or generated by addition of the specific nucleotide exchange factor RCC1, stabilises large microtubule asters nucleated at the sperm centrosome, prevents the redistribution of NuMA from the aster to the nucleus and blocks chromatin decondensation. In contrast, Ran GDP does not stabilise microtubules or inhibit nuclear assembly. RanT24N and RanBP1, which oppose the generation of Ran-GTP by RCC1, arrest nuclear growth after disappearance of the aster. Ran associates with microtubule asters in egg extracts and with mitotic spindles in somatic Xenopus cells, suggesting that it may affect microtubule stability directly. These results show that Ran has a novel function in the control of microtubule stability that is clearly distinct from nucleocytoplasmic transport. The Ran GDP/GTP switch may play a role in co-ordinating changes in the structure of microtubules and the assembly of the nucleus associated with the transition from mitosis to interphase.

scholarly journals Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts

1997 ◽  
Vol 110 (24) ◽  
pp. 3019-3030 ◽  
Author(s):  
F. Nicolas ◽  
C. Zhang ◽  
M. Hughes ◽  
M. Goldberg ◽  
S. Watton ◽  
...  
Keyword(s):  
Protein Import ◽  
Binding Protein ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Assembly ◽  
Importin Alpha ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Pore Complexes ◽  
Xenopus Egg Extracts ◽  
Cycle Regulation

Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.

scholarly journals The bulk of unpolymerized actin in Xenopus egg extracts is ATP-bound.

1995 ◽  
Vol 6 (2) ◽  
pp. 227-236 ◽  
Author(s):  
J Rosenblatt ◽  
P Peluso ◽  
T J Mitchison
Keyword(s):  
Actin Polymerization ◽  
Critical Concentration ◽  
Large Fraction ◽  
Nucleotide Exchange ◽  
Chromatography Analysis ◽  
Thymosin Beta 4 ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Non-muscle cells contain 15-500 microM actin, a large fraction of which is unpolymerized. Thus, the concentration of unpolymerized actin is well above the critical concentration for polymerization in vitro (0.2 microM). This fraction of actin could be prevented from polymerization by being ADP bound (therefore less favored to polymerize) or by being ATP bound and sequestered by a protein such as thymosin beta 4, or both. We isolated the unpolymerized actin from Xenopus egg extracts using immobilized DNase 1 and assayed the bound nucleotide. High-pressure liquid chromatography analysis showed that the bulk of soluble actin is ATP bound. Analysis of actin-bound nucleotide exchange rates suggested the existence of two pools of unpolymerized actin, one of which exchanges nucleotide relatively rapidly and another that apparently does not exchange. Native gel electrophoresis of Xenopus egg extracts demonstrated that most of the soluble actin exists in complexes with other proteins, one of which might be thymosin beta 4. These results are consistent with actin polymerization being controlled by the sequestration and release of ATP-bound actin, and argue against nucleotide exchange playing a major role in regulating actin polymerization.

The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs

1991 ◽  
Vol 98 (3) ◽  
pp. 271-279
Author(s):  
J. Meier ◽  
K.H. Campbell ◽  
C.C. Ford ◽  
R. Stick ◽  
C.J. Hutchison
Keyword(s):  
Dna Replication ◽  
Membrane Structures ◽  
Chromatin Decondensation ◽  
Immunoblotting Analysis ◽  
Nuclear Assembly ◽  
Contrast Microscopy ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.

scholarly journals Barrier-to-autointegration factor

2002 ◽  
Vol 158 (3) ◽  
pp. 475-485 ◽  
Author(s):  
Miriam Segura-Totten ◽  
Amy K. Kowalski ◽  
Robert Craigie ◽  
Katherine L. Wilson
Keyword(s):  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Functional Surface ◽  
Chromatin Decondensation ◽  
Membrane Recruitment ◽  
Nuclear Assembly ◽  
Egg Extracts ◽  
Direct Binding ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain ∼12 μM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.

scholarly journals Control of microtubule dynamics and length by cyclin A- and cyclin B-dependent kinases in Xenopus egg extracts.

1992 ◽  
Vol 118 (5) ◽  
pp. 1097-1108 ◽  
Author(s):  
F Verde ◽  
M Dogterom ◽  
E Stelzer ◽  
E Karsenti ◽  
S Leibler
Keyword(s):  
Steady State ◽  
Fold Increase ◽  
Cyclin A ◽  
Microtubule Dynamics ◽  
Cyclin B ◽  
Free System ◽  
Egg Extracts ◽  
Dynamical Parameters ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

In eukaryotic cells, the onset of mitosis involves cyclin molecules which interact with proteins of the cdc2 family to produce active kinases. In vertebrate cells, cyclin A dependent kinases become active in S- and pro-phases, whereas a cyclin B-dependent kinase is mostly active in metaphase. It has recently been shown that, when added to Xenopus egg extracts, bacterially produced A- and B-type cyclins associate predominantly with the same kinase catalytic subunit, namely p34cdc2, and induce its histone H1 kinase activity with different kinetics. Here, we show that in the same cell free system, both the addition of cyclin A and cyclin B changes microtubule behavior. However, the cyclin A-dependent kinase does not induce a dramatic shortening of centrosome-nucleated microtubules whereas the cyclin B-dependent kinase does, as previously reported. Analysis of the parameters of microtubule dynamics by fluorescence video microscopy shows that the dramatic shortening induced by the cyclin B-dependent kinase is correlated with a several fold increase in catastrophe frequency, an effect not observed with the cyclin A-dependent kinase. Using a simple mathematical model, we show how the length distributions of centrosome-nucleated microtubules relate to the four parameters that describe microtubule dynamics. These four parameters define a threshold between unlimited microtubule growth and the establishment of steady-state dynamics, which implies that well defined steady-state length distributions can be produced by regulating precisely the respective values of the dynamical parameters. Moreover, the dynamical model predicts that increasing catastrophe frequency is more efficient than decreasing the rescue frequency to reduce the average steady state length of microtubules. These theoretical results are quantitatively confirmed by the experimental data.

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 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.

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