Two complexes that contain histones are required for nucleosome assembly in vitro: Role of nucleoplasmin and N1 in Xenopus egg extracts

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction–recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

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Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation

The Journal of Cell Biology ◽

10.1083/jcb.124.1.19 ◽

1994 ◽

Vol 124 (1) ◽

pp. 19-31 ◽

Cited By ~ 149

Author(s):

MA Félix ◽

C Antony ◽

M Wright ◽

B Maro

Keyword(s):

Sperm Head ◽

Egg Extract ◽

Egg Extracts ◽

Pericentriolar Material ◽

Xenopus Egg ◽

Gamma Tubulin ◽

Xenopus Egg Extracts ◽

Microtubule Aster

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.

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Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.

The Journal of Cell Biology ◽

10.1083/jcb.116.6.1431 ◽

1992 ◽

Vol 116 (6) ◽

pp. 1431-1442 ◽

Cited By ~ 74

Author(s):

B Buendia ◽

G Draetta ◽

E Karsenti

Keyword(s):

Critical Concentration ◽

Cyclin A ◽

Soluble Form ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Polymer Assembly

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

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Faculty Opinions recommendation of Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1020314.231528 ◽

2004 ◽

Author(s):

Stephen Kearsey

Keyword(s):

Replication Origin ◽

Firing Time ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

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

Molecular Biology of the Cell ◽

10.1091/mbc.6.2.227 ◽

1995 ◽

Vol 6 (2) ◽

pp. 227-236 ◽

Cited By ~ 32

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.

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Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

Journal of Cell Science ◽

10.1242/jcs.108.6.2187 ◽

1995 ◽

Vol 108 (6) ◽

pp. 2187-2196 ◽

Cited By ~ 3

Author(s):

L.J. Wangh ◽

D. DeGrace ◽

J.A. Sanchez ◽

A. Gold ◽

Y. Yeghiazarians ◽

...

Keyword(s):

Cell Cycle ◽

Somatic Cell ◽

Nuclear Transplantation ◽

Optimal Time ◽

Genome Replication ◽

Cell Nuclei ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Rapid genome replication is one of the hallmarks of the frog embryonic cell cycle. We report here that complete reactivation of quiescent somatic cell nuclei in Xenopus egg extracts depends on prior restructuring of the nuclear substrate and prior preparation of cytoplasmic extract with the highest capacity to initiate and sustain DNA synthesis. Nuclei from mature erythrocytes swell, replicate their DNA efficiently, and enter mitosis in frozen/thawed extracts prepared from activated Xenopus eggs, provided the nuclei are first treated with trypsin, heparin, and an extract prepared from unactivated, meiotically arrested, eggs. Optimal replicating extracts are prepared from large batches of unfertilized eggs that are synchronously activated into the cell cycle for 28 minutes (at 20 degrees C). Because the Xenopus cell cycle progresses so rapidly, extracts prepared just a few minutes before or after this time have substantially lower DNA synthetic capacities. At the optimal time and temperature, eggs have just reached the G1/S boundary of the first cell cycle. This fact was revealed by injecting and replicating an SV40 plasmid in intact unfertilized eggs as described previously. We estimate that under optimal conditions approximately 6.14 × 10(9) base pairs of DNA/per nucleus are synthesized in 30–40 minutes, a rate that rivals that observed in the zygotic nucleus. The findings reported here are one step in our long term effort to develop a new in vitro/in vivo approach to nuclear transplantation. Nuclear transplantation in amphibian embryos has been used to establish that the genomes of many types of differentiated somatic cells are pluripotent. But very few such nuclei have ever developed into advanced tadpoles or adult frogs, probably because somatic nuclei injected directly into activated eggs fail to reactivate quickly enough to avoid being damaged during first mitosis. We have already shown that unfertilized eggs can be injected prior to activation of the first cell cycle. Future experiments will reveal whether in vitro reactivated somatic cell nuclei transplanted into such eggs reliably reach advanced stages of development.

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MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extracts

Journal of Cell Science ◽

10.1242/jcs.109.1.239 ◽

1996 ◽

Vol 109 (1) ◽

pp. 239-246 ◽

Cited By ~ 10

Author(s):

A. Abrieu ◽

T. Lorca ◽

J.C. Labbe ◽

N. Morin ◽

S. Keyse ◽

...

Keyword(s):

Map Kinase ◽

Degradation Pathway ◽

Cdc2 Kinase ◽

Vitro Assay ◽

Kinase Activation ◽

Egg Extracts ◽

Dependent Protein ◽

Xenopus Egg ◽

Xenopus Egg Extracts

Unfertilized frog eggs arrest at the second meiotic metaphase, due to cytostatic activity of the c-mos proto-oncogene (CSF). MAP kinase has been proposed to mediate CSF activity in suppressing cyclin degradation. Using an in vitro assay to generate CSF activity, and recombinant CL 100 phosphatase to inactivate MAP kinase, we confirm that the c-mos proto-oncogene blocks cyclin degradation through MAP kinase activation. We further show that for MAP kinase to suppress cyclin degradation, it must be activated before cyclin B-cdc2 kinase has effectively promoted cyclin degradation. Thus MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on. Using a constitutively active mutant of Ca2+/calmodulin dependent protein kinase II, which mediates the effects of Ca2+ at fertilization, we further show that the kinase can activate cyclin degradation in the presence of both MPF and the c-mos proto-oncogene without inactivating MAP kinase.

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

Journal of Cell Science ◽

10.1242/jcs.98.3.271 ◽

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.

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Peroxisome protein import recapitulated in Xenopus egg extracts

The Journal of Cell Biology ◽

10.1083/jcb.201901152 ◽

2019 ◽

Vol 218 (6) ◽

pp. 2021-2034 ◽

Cited By ~ 3

Author(s):

Fabian B. Romano ◽

Neil B. Blok ◽

Tom A. Rapoport

Keyword(s):

Strong Evidence ◽

Protein Import ◽

Atp Hydrolysis ◽

In Vitro Assay ◽

Vitro Assay ◽

Egg Extracts ◽

Xenopus Egg ◽

Xenopus Egg Extracts ◽

Import System

Peroxisomes import their luminal proteins from the cytosol. Most substrates contain a C-terminal Ser-Lys-Leu (SKL) sequence that is recognized by the receptor Pex5. Pex5 binds to peroxisomes via a docking complex containing Pex14, and recycles back into the cytosol following its mono-ubiquitination at a conserved Cys residue. The mechanism of peroxisome protein import remains incompletely understood. Here, we developed an in vitro import system based on Xenopus egg extracts. Import is dependent on the SKL motif in the substrate and on the presence of Pex5 and Pex14, and is sustained by ATP hydrolysis. A protein lacking an SKL sequence can be coimported, providing strong evidence for import of a folded protein. The conserved cysteine in Pex5 is not essential for import or to clear import sites for subsequent rounds of translocation. This new in vitro assay will be useful for further dissecting the mechanism of peroxisome protein import.

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2066 Functional characterization of mutant BRCA1

Journal of Clinical and Translational Science ◽

10.1017/cts.2018.77 ◽

2018 ◽

Vol 2 (S1) ◽

pp. 13-13

Author(s):

John Barrows ◽

David Long

Keyword(s):

Functional Characterization ◽

Coiled Coil ◽

Wild Type ◽

Coiled Coil Region ◽

Egg Extracts ◽

Study Population ◽

Xenopus Egg ◽

Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

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