scholarly journals Multiple domains of human CLASP contribute to microtubule dynamics and organization in vitro and in Xenopus egg extracts

Cytoskeleton ◽  
2012 ◽  
Vol 69 (3) ◽  
pp. 155-165 ◽  
Author(s):  
Kieren Patel ◽  
Eva Nogales ◽  
Rebecca Heald
Keyword(s):  
Microtubule Dynamics ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Multiple Domains

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.

Efficient reactivation of Xenopus erythrocyte nuclei in Xenopus egg extracts

1995 ◽  
Vol 108 (6) ◽  
pp. 2187-2196 ◽  
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.

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

1996 ◽  
Vol 109 (1) ◽  
pp. 239-246 ◽  
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.

scholarly journals Peroxisome protein import recapitulated in Xenopus egg extracts

2019 ◽  
Vol 218 (6) ◽  
pp. 2021-2034 ◽  
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.

scholarly journals 2066 Functional characterization of mutant BRCA1

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.

scholarly journals Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

1994 ◽  
Vol 5 (2) ◽  
pp. 217-226 ◽  
Author(s):  
K E Sawin ◽  
T J Mitchison
Keyword(s):  
Basic Element ◽  
Low Light ◽  
Self Organized ◽  
Egg Extracts ◽  
Microtubule Motor ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Microtubule Motor Protein ◽  
Poleward Flux

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.

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 cdc2 kinase-induced destabilization of MAP2-coated microtubules in Xenopus egg extracts

1992 ◽  
Vol 101 (1) ◽  
pp. 69-78 ◽  
Author(s):  
S. Faruki ◽  
M. Doree ◽  
E. Karsenti
Keyword(s):  
Steady State ◽  
Bovine Brain ◽  
Microtubule Dynamics ◽  
Cdc2 Kinase ◽  
Stabilizing Effect ◽  
Egg Extracts ◽  
Phosphorylation Event ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Stabilizing Factors

During the interphase to metaphase transition, microtubules are destabilized by a cdc2 kinase-dependent phosphorylation event. This destabilization is due to a dramatic increase in the rate at which each growing microtubule starts to shrink (catastrophe rate). In principle, this could be brought about by lowering the affinity of stabilizing MAPs for the microtubule wall, by activating a factor that would actively increase the catastrophe rate or by an alteration of both parameters. Here we examine the stabilizing effect of bovine brain MAP2 on microtubules assembled in interphase Xenopus egg extracts. We show that this MAP strongly stabilizes microtubules assembled in the extracts against nocodazole-induced depolymerization. However, it does not protect them from the cdc2 kinase-induced shortening and destabilization. Moreover, the steady-state length of centrosome-nucleated microtubules in cdc2-treated extracts containing MAP2 is similar to that found in extracts lacking exogenous MAP2. We also show that although exogenous MAP2 is phosphorylated by cdc2 kinase in the extract, this is not the cause of microtubule destabilization. These results indicate that increased microtubule dynamics during mitosis is due to the activation of a factor that can function independently of the presence of active, stabilizing factors.

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.

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