scholarly journals The Xenopus TACC Homologue, Maskin, Functions in Mitotic Spindle Assembly

2005 ◽  
Vol 16 (6) ◽  
pp. 2836-2847 ◽  
Author(s):  
Lori L. O'Brien ◽  
Alison J. Albee ◽  
Lingling Liu ◽  
Wei Tao ◽  
Pawel Dobrzyn ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Coiled Coil ◽  
Spindle Assembly ◽  
Cyclin B1 ◽  
Time Points ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Mitotic Spindle Assembly ◽  
Xenopus Egg Extracts

Maskin is the Xenopus homolog of the transforming acidic coiled coil (TACC)-family of microtubule and centrosome-interacting proteins. Members of this family share a ∼200 amino acid coiled coil motif at their C-termini, but have only limited homology outside of this domain. In all species examined thus far, perturbations of TACC proteins lead to disruptions of cell cycle progression and/or embryonic lethality. In Drosophila, Caenorhabditis elegans, and humans, these disruptions have been attributed to mitotic spindle assembly defects, and the TACC proteins in these organisms are thought to function as structural components of the spindle. In contrast, cell division failure in early Xenopus embryo blastomeres has been attributed to a role of maskin in regulating the translation of, among others, cyclin B1 mRNA. In this study, we show that maskin, like other TACC proteins, plays a direct role in mitotic spindle assembly in Xenopus egg extracts and that this role is independent of cyclin B. Maskin immunodepletion and add-back experiments demonstrate that maskin, or a maskin-associated activity, is required for two distinct steps during spindle assembly in Xenopus egg extracts that can be distinguished by their response to “rescue” experiments. Defects in the “early” step, manifested by greatly reduced aster size during early time points in maskin-depleted extracts, can be rescued by readdition of purified full-length maskin. Moreover, defects in this step can also be rescued by addition of only the TACC-domain of maskin. In contrast, defects in the “late” step during spindle assembly, manifested by abnormal spindles at later time points, cannot be rescued by readdition of maskin. We show that maskin interacts with a number of proteins in egg extracts, including XMAP215, a known modulator of microtubule dynamics, and CPEB, a protein that is involved in translational regulation of important cell cycle regulators. Maskin depletion from egg extracts results in compromised microtubule asters and spindles and the mislocalization of XMAP215, but CPEB localization is unaffected. Together, these data suggest that in addition to its previously reported role as a translational regulator, maskin is also important for mitotic spindle assembly.

scholarly journals Mitotic Spindle Assembly around RCC1-Coated Beads in Xenopus Egg Extracts

PLoS Biology ◽  
2011 ◽  
Vol 9 (12) ◽  
pp. e1001225 ◽  
Author(s):  
David Halpin ◽  
Petr Kalab ◽  
Jay Wang ◽  
Karsten Weis ◽  
Rebecca Heald
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Mitotic Spindle Assembly ◽  
Xenopus Egg Extracts

Chapter 20 The Use of Xenopus Egg Extracts to Study Mitotic Spindle Assembly and Function in Vitro

1998 ◽  
pp. 385-412 ◽  
Author(s):  
Arshad Desai ◽  
Andrew Murray ◽  
Timothy J. Mitchison ◽  
Claire E. Walczak
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Mitotic Spindle Assembly ◽  
And Function ◽  
Xenopus Egg Extracts

scholarly journals Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis

2013 ◽  
Vol 24 (22) ◽  
pp. 3522-3533 ◽  
Author(s):  
Shusheng Wang ◽  
Stephanie A. Ketcham ◽  
Arne Schön ◽  
Benjamin Goodman ◽  
Yueju Wang ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
Spindle Poles ◽  
Interesting Possibility ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end–directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein–dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.

scholarly journals Multiple mechanisms determine ER network morphology during the cell cycle in Xenopus egg extracts

2013 ◽  
Vol 203 (5) ◽  
pp. 801-814 ◽  
Author(s):  
Songyu Wang ◽  
Fabian B. Romano ◽  
Christine M. Field ◽  
Tim J. Mitchison ◽  
Tom A. Rapoport
Keyword(s):  
Cell Cycle ◽  
Adaptor Protein ◽  
Sensitive Factor ◽  
Protein Receptors ◽  
Egg Extracts ◽  
Organizing Center ◽  
Xenopus Egg ◽  
Guanosine Triphosphatase ◽  
Xenopus Egg Extracts ◽  
Network Morphology

In metazoans the endoplasmic reticulum (ER) changes during the cell cycle, with the nuclear envelope (NE) disassembling and reassembling during mitosis and the peripheral ER undergoing extensive remodeling. Here we address how ER morphology is generated during the cell cycle using crude and fractionated Xenopus laevis egg extracts. We show that in interphase the ER is concentrated at the microtubule (MT)-organizing center by dynein and is spread by outward extension of ER tubules through their association with plus ends of growing MTs. Fusion of membranes into an ER network is dependent on the guanosine triphosphatase atlastin (ATL). NE assembly requires fusion by both ATL and ER-soluble N-ethyl-maleimide–sensitive factor adaptor protein receptors. In mitotic extracts, the ER converts into a network of sheets connected by ER tubules and loses most of its interactions with MTs. Together, these results indicate that fusion of ER membranes by ATL and interaction of ER with growing MT ends and dynein cooperate to generate distinct ER morphologies during the cell cycle.

scholarly journals Characterization of the TPX2 Domains Involved in Microtubule Nucleation and Spindle Assembly in Xenopus Egg Extracts

2004 ◽  
Vol 15 (12) ◽  
pp. 5318-5328 ◽  
Author(s):  
Stéphane Brunet ◽  
Teresa Sardon ◽  
Timo Zimmerman ◽  
Torsten Wittmann ◽  
Rainer Pepperkok ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Nucleation ◽  
Terminal Domain ◽  
Large N ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Domain Functional ◽  
Xenopus Egg Extracts

TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.

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 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 Cell Cycle Regulation of the Replication Licensing System: Involvement of a Cdk-dependent Inhibitor

1997 ◽  
Vol 136 (1) ◽  
pp. 125-135 ◽  
Author(s):  
Hiro M. Mahbubani ◽  
James P.J. Chong ◽  
Stephane Chevalier ◽  
Pia Thömmes ◽  
J. Julian Blow
Keyword(s):  
Cell Cycle ◽  
Single Cell ◽  
Kinase Inhibitor ◽  
Cyclin B ◽  
Initiation Factor ◽  
Chromosomal Dna ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Cycle Regulation

The replication licensing factor (RLF) is an essential initiation factor that is involved in preventing re-replication of chromosomal DNA in a single cell cycle. In Xenopus egg extracts, it can be separated into two components: RLF-M, a complex of MCM/P1 polypeptides, and RLF-B, which is currently unpurified. In this paper we investigate variations in RLF activity throughout the cell cycle. Total RLF activity is low in metaphase, due to a lack of RLF-B activity and the presence of an RLF inhibitor. RLF-B is rapidly activated on exit from metaphase, and then declines during interphase. The RLF inhibitor present in metaphase extracts is dependent on the activity of cyclin-dependent kinases (Cdks). Affinity depletion of Cdks from metaphase extracts removed the RLF inhibitor, while Cdc2/cyclin B directly inhibited RLF activity. In metaphase extracts treated with the protein kinase inhibitor 6-dimethylaminopurine (6-DMAP), both cyclin B and the RLF inhibitor were stabilized although the extracts morphologically entered interphase. These results are consistent with studies in other organisms that invoke a key role for Cdks in preventing re-replication of DNA in a single cell cycle.

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