Centromere and Kinetochore Assembly in Xenopus laevis Egg Extract

2018 ◽  
Vol 2018 (9) ◽  
pp. pdb.prot102509 ◽  
Author(s):  
Julio C. Flores Servin ◽  
Aaron F. Straight
Keyword(s):  
Xenopus Laevis ◽  
Kinetochore Assembly ◽  
Egg Extract

Extracts from eggs and oocytes of Xenopus laevis differ in their capacities for nuclear assembly and DNA replication

1990 ◽  
Vol 97 (1) ◽  
pp. 177-184
Author(s):  
L.S. Cox ◽  
G.H. Leno
Keyword(s):  
Dna Replication ◽  
Xenopus Laevis ◽  
High Speed ◽  
De Novo ◽  
Soluble Fraction ◽  
Sperm Chromatin ◽  
Nuclear Assembly ◽  
Egg Extract ◽  
Egg Extracts ◽  
Sperm Nuclei

We describe a cell-free extract derived from the oocytes of Xenopus laevis. The oocyte extract is capable of decondensing sperm chromatin and of replicating single-stranded DNA in a semiconservative, aphidicolin-sensitive manner. In addition, oocyte extract supports the elongation phase of DNA synthesis in nuclei that have been preinitiated for replication. All of these properties are shared by previously described egg extracts. However, oocyte extracts differ from egg extracts in two important ways. First, they cannot support nuclear assembly, as visualised by phase-contrast, fluorescence and electron microscopy. Second, they do not initiate replication on chromatin or nuclei de novo. Crude low-speed supernatants can be partially fractionated into soluble and vesicular components by high-speed centrifugation. Such fractions from eggs can be functionally reconstituted, but the oocyte soluble fraction does not acquire the ability to assemble nuclei, or replicate them, even when supplemented with the egg vesicular fraction. Similarly, oocyte vesicles cannot substitute for egg vesicles on reconstitution with the egg soluble fraction. When the requirement for nuclear assembly is bypassed by using preformed, quiescent nuclei, replication is observed in egg but not oocyte extracts. However, the oocyte extract is not inhibitory for initiation of replication, as it does not prevent replication of sperm nuclei when mixed with egg extract. We suggest that the different capabilities of egg and oocyte extracts could provide the basis of an assay system for identifying factors involved in the initiation of DNA replication.

scholarly journals Poleward transport of Eg5 by dynein–dynactin in Xenopus laevis egg extract spindles

2008 ◽  
Vol 182 (4) ◽  
pp. 715-726 ◽  
Author(s):  
Marianne Uteng ◽  
Christian Hentrich ◽  
Kota Miura ◽  
Peter Bieling ◽  
Thomas Surrey
Keyword(s):  
Cell Division ◽  
Xenopus Laevis ◽  
Molecular Motors ◽  
Motor Proteins ◽  
Motor Protein ◽  
Spindle Assembly ◽  
Cross Linking ◽  
Motor Movement ◽  
Spindle Poles ◽  
Egg Extract

Molecular motors are required for spindle assembly and maintenance during cell division. How motors move and interact inside spindles is unknown. Using photoactivation and photobleaching, we measure mitotic motor movement inside a dynamic spindle. We find that dynein–dynactin transports the essential motor Eg5 toward the spindle poles in Xenopus laevis egg extract spindles, revealing a direct interplay between two motors of opposite directionality. This transport occurs throughout the spindle except at the very spindle center and at the spindle poles, where Eg5 remains stationary. The variation of Eg5 dynamics with its position in the spindle is indicative of position-dependent functions of this motor protein. Our results suggest that Eg5 drives microtubule flux by antiparallel microtubule sliding in the spindle center, whereas the dynein-dependent concentration of Eg5 outside the spindle center could contribute to parallel microtubule cross-linking. These results emphasize the importance of spatially differentiated functions of motor proteins and contribute to our understanding of spindle organization.

scholarly journals Nucleus Assembly and Import in Xenopus laevis Egg Extract

2018 ◽  
Vol 2018 (6) ◽  
pp. pdb.prot097196 ◽  
Author(s):  
Pan Chen ◽  
Daniel L. Levy
Keyword(s):  
Xenopus Laevis ◽  
Egg Extract

scholarly journals Enrichment of Aurora B kinase at the inner kinetochore controls outer kinetochore assembly

2019 ◽  
Vol 218 (10) ◽  
pp. 3237-3257 ◽  
Author(s):  
Mary Kate Bonner ◽  
Julian Haase ◽  
Jason Swinderman ◽  
Hyunmi Halas ◽  
Lisa M. Miller Jenkins ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Central Region ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Aurora B ◽  
Aurora B Kinase ◽  
Kinetochore Assembly ◽  
Egg Extracts ◽  
Chromosome Attachment

Outer kinetochore assembly enables chromosome attachment to microtubules and spindle assembly checkpoint (SAC) signaling in mitosis. Aurora B kinase controls kinetochore assembly by phosphorylating the Mis12 complex (Mis12C) subunit Dsn1. Current models propose Dsn1 phosphorylation relieves autoinhibition, allowing Mis12C binding to inner kinetochore component CENP-C. Using Xenopus laevis egg extracts and biochemical reconstitution, we found that autoinhibition of the Mis12C by Dsn1 impedes its phosphorylation by Aurora B. Our data indicate that the INCENP central region increases Dsn1 phosphorylation by enriching Aurora B at inner kinetochores, close to CENP-C. Furthermore, centromere-bound CENP-C does not exchange in mitosis, and CENP-C binding to the Mis12C dramatically increases Dsn1 phosphorylation by Aurora B. We propose that the coincidence of Aurora B and CENP-C at inner kinetochores ensures the fidelity of kinetochore assembly. We also found that the central region is required for the SAC beyond its role in kinetochore assembly, suggesting that kinetochore enrichment of Aurora B promotes the phosphorylation of other kinetochore substrates.

scholarly journals Atomic Force Microscope Imaging of Chromatin Assembled in Xenopus Laevis Egg Extract

2010 ◽  
Vol 98 (3) ◽  
pp. 474a
Author(s):  
Hongxia Fu ◽  
Benjamin Freedman ◽  
Chwee Teck Lim ◽  
Rebecca Heald ◽  
Jie Yan
Keyword(s):  
Xenopus Laevis ◽  
Atomic Force Microscope ◽  
Atomic Force ◽  
Egg Extract ◽  
Microscope Imaging

Use of dna end joining activity of a xenopus laevis egg extract for construction of deletions and expression vectors for hiv-1 tat and rev proteins

Gene ◽  
1993 ◽  
Vol 124 (2) ◽  
pp. 275-280 ◽  
Author(s):  
Heiner Schaal ◽  
Petra Pfeiffer ◽  
Michael Klein ◽  
Peter Gehrmann ◽  
Andreas Scheid
Keyword(s):  
Xenopus Laevis ◽  
Expression Vectors ◽  
End Joining ◽  
Egg Extract ◽  
Hiv 1

scholarly journals The kinesin Eg5 drives poleward microtubule flux in Xenopus laevis egg extract spindles

2004 ◽  
Vol 167 (5) ◽  
pp. 813-818 ◽  
Author(s):  
David T. Miyamoto ◽  
Zachary E. Perlman ◽  
Kendra S. Burbank ◽  
Aaron C. Groen ◽  
Timothy J. Mitchison
Keyword(s):  
Xenopus Laevis ◽  
Chromosome Segregation ◽  
Spindle Poles ◽  
Egg Extract ◽  
Constant Rate ◽  
Quantitative Image ◽  
Meiotic Spindles ◽  
Kinesin Eg5 ◽  
Poleward Flux

Although mitotic and meiotic spindles maintain a steady-state length during metaphase, their antiparallel microtubules slide toward spindle poles at a constant rate. This “poleward flux” of microtubules occurs in many organisms and may provide part of the force for chromosome segregation. We use quantitative image analysis to examine the role of the kinesin Eg5 in poleward flux in metaphase Xenopus laevis egg extract spindles. Pharmacological inhibition of Eg5 results in a dose–responsive slowing of flux, and biochemical depletion of Eg5 significantly decreases the flux rate. Our results suggest that ensembles of nonprocessive Eg5 motors drive flux in metaphase Xenopus extract spindles.

scholarly journals Atomic force microscope imaging of chromatin assembled in Xenopus laevis egg extract

Chromosoma ◽  
2011 ◽  
Vol 120 (3) ◽  
pp. 245-254 ◽  
Author(s):  
Hongxia Fu ◽  
Benjamin S. Freedman ◽  
Chwee Teck Lim ◽  
Rebecca Heald ◽  
Jie Yan
Keyword(s):  
Xenopus Laevis ◽  
Atomic Force Microscope ◽  
Atomic Force ◽  
Egg Extract ◽  
Microscope Imaging

scholarly journals Mitotic replisome disassembly depends on TRAIP ubiquitin ligase activity

2019 ◽  
Vol 2 (2) ◽  
pp. e201900390 ◽  
Author(s):  
Sara Priego Moreno ◽  
Rebecca M Jones ◽  
Divyasree Poovathumkadavil ◽  
Shaun Scaramuzza ◽  
Agnieszka Gambus
Keyword(s):  
Caenorhabditis Elegans ◽  
Xenopus Laevis ◽  
Ubiquitin Ligase ◽  
S Phase ◽  
Replication Forks ◽  
Replication Machinery ◽  
Replicative Helicase ◽  
Egg Extract ◽  
Ubiquitin Ligase Activity ◽  
Mitotic Chromatin

We have shown previously that the process of replication machinery (replisome) disassembly at the termination of DNA replication forks in the S-phase is driven through polyubiquitylation of one of the replicative helicase subunits (Mcm7) by Cul2LRR1 ubiquitin ligase. Interestingly, upon inhibition of this pathway in Caenorhabditis elegans embryos, the replisomes retained on chromatin were unloaded in the subsequent mitosis. Here, we show that this mitotic replisome disassembly pathway exists in Xenopus laevis egg extract and we determine the first elements of its regulation. The mitotic disassembly pathway depends on the formation of K6- and K63-linked ubiquitin chains on Mcm7 by TRAIP ubiquitin ligase and the activity of p97/VCP protein segregase. Unlike in lower eukaryotes, however, it does not require SUMO modifications. Importantly, we also show that this process can remove all replisomes from mitotic chromatin, including stalled ones, which indicates a wide application for this pathway over being just a “backup” for terminated replisomes. Finally, we characterise the composition of the replisome retained on chromatin until mitosis.

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