scholarly journals Analysis of Bub3 spindle checkpoint function in Xenopus egg extracts

2002 ◽  
Vol 116 (4) ◽  
pp. 617-628 ◽  
Author(s):  
L. Campbell
Keyword(s):  
Spindle Checkpoint ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

scholarly journals The Nup107-160 Nucleoporin Complex Is Required for Correct Bipolar Spindle Assembly

2006 ◽  
Vol 17 (9) ◽  
pp. 3806-3818 ◽  
Author(s):  
Arturo V. Orjalo ◽  
Alexei Arnaoutov ◽  
Zhouxin Shen ◽  
Yekaterina Boyarchuk ◽  
Samantha G. Zeitlin ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Spindle Checkpoint ◽  
Spindle Assembly ◽  
Sperm Chromatin ◽  
Nuclear Pore ◽  
Checkpoint Proteins ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

The Nup107-160 complex is a critical subunit of the nuclear pore. This complex localizes to kinetochores in mitotic mammalian cells, where its function is unknown. To examine Nup107-160 complex recruitment to kinetochores, we stained human cells with antisera to four complex components. Each antibody stained not only kinetochores but also prometaphase spindle poles and proximal spindle fibers, mirroring the dual prometaphase localization of the spindle checkpoint proteins Mad1, Mad2, Bub3, and Cdc20. Indeed, expanded crescents of the Nup107-160 complex encircled unattached kinetochores, similar to the hyperaccumulation observed of dynamic outer kinetochore checkpoint proteins and motors at unattached kinetochores. In mitotic Xenopus egg extracts, the Nup107-160 complex localized throughout reconstituted spindles. When the Nup107-160 complex was depleted from extracts, the spindle checkpoint remained intact, but spindle assembly was rendered strikingly defective. Microtubule nucleation around sperm centrosomes seemed normal, but the microtubules quickly disassembled, leaving largely unattached sperm chromatin. Notably, Ran-GTP caused normal assembly of microtubule asters in depleted extracts, indicating that this defect was upstream of Ran or independent of it. We conclude that the Nup107-160 complex is dynamic in mitosis and that it promotes spindle assembly in a manner that is distinct from its functions at interphase nuclear pores.

scholarly journals Spindle Checkpoint Requires Mad1-bound and Mad1-free Mad2

2002 ◽  
Vol 13 (5) ◽  
pp. 1501-1511 ◽  
Author(s):  
Eunah Chung ◽  
Rey-Huei Chen
Keyword(s):  
Amino Acids ◽  
Mitotic Spindle ◽  
Spindle Checkpoint ◽  
Stable Complex ◽  
Anaphase Promoting Complex ◽  
Binding Region ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Active Condition ◽  
Xenopus Egg Extracts

The spindle checkpoint prevents anaphase from occurring until all chromosomes have attached properly to the mitotic spindle. The checkpoint components Mad1 and Mad2 associate with unattached kinetochores and are probably involved in triggering the checkpoint. We now demonstrate that in Xenopus egg extracts Mad1 and Mad2 form a stable complex, whereas a fraction of Mad2 molecules is not bound to Mad1. The checkpoint establishment and maintenance are lost upon titrating out free Mad2 with an excess of Mad1 or a truncated Mad1 (amino acids 326–718, Mad1C) that contains the Mad2-binding region. Mad1N (amino acids 1–445) that binds kinetochores, but not Mad2, reduces Mad1 and Mad2 at kinetochores and abolishes checkpoint maintenance. Furthermore, the association between Mad2 and Cdc20, the activator for the anaphase-promoting complex, is enhanced under checkpoint-active condition compared with that at metaphase. Immunodepletion analysis shows that the Mad1-free Mad2 protein is unable to bind Cdc20, consistent with the model that kinetochore localization of Mad2 facilitates the formation of Mad2–Cdc20 complex. This study demonstrates that the ratio between Mad1 and Mad2 is critical for maintaining a pool of Mad1-free Mad2 that is necessary for the spindle checkpoint. We propose that Mad2 may become activated and dissociated from Mad1 at kinetochores and is replenished by the pool of Mad1-free Mad2.

scholarly journals Spindle Checkpoint Protein Xmad1 Recruits Xmad2 to Unattached Kinetochores

1998 ◽  
Vol 143 (2) ◽  
pp. 283-295 ◽  
Author(s):  
Rey-Huei Chen ◽  
Andrej Shevchenko ◽  
Matthias Mann ◽  
Andrew W. Murray
Keyword(s):  
Spindle Checkpoint ◽  
Physiological Role ◽  
Yeast Saccharomyces Cerevisiae ◽  
Checkpoint Protein ◽  
Egg Extracts ◽  
Chromosome Attachment ◽  
Xenopus Egg ◽  
Spindle Microtubules ◽  
Xenopus Egg Extracts

The spindle checkpoint prevents the metaphase to anaphase transition in cells containing defects in the mitotic spindle or in chromosome attachment to the spindle. When the checkpoint protein Xmad2 is depleted from Xenopus egg extracts, adding Xmad2 to its endogenous concentration fails to restore the checkpoint, suggesting that other checkpoint component(s) were depleted from the extract through their association with Xmad2. Mass spectrometry provided peptide sequences from an 85-kD protein that coimmunoprecipitates with Xmad2 from egg extracts. This information was used to clone XMAD1, which encodes a homologue of the budding yeast (Saccharomyces cerevisiae) checkpoint protein Mad1. Xmad1 is essential for establishing and maintaining the spindle checkpoint in egg extracts. Like Xmad2, Xmad1 localizes to the nuclear envelope and the nucleus during interphase, and to those kinetochores that are not bound to spindle microtubules during mitosis. Adding an anti-Xmad1 antibody to egg extracts inactivates the checkpoint and prevents Xmad2 from localizing to unbound kinetochores. In the presence of excess Xmad2, neither chromosomes nor Xmad1 are required to activate the spindle checkpoint, suggesting that the physiological role of Xmad1 is to recruit Xmad2 to kinetochores that have not bound microtubules.

scholarly journals BubR1 is essential for kinetochore localization of other spindle checkpoint proteins and its phosphorylation requires Mad1

2002 ◽  
Vol 158 (3) ◽  
pp. 487-496 ◽  
Author(s):  
Rey-Huei Chen
Keyword(s):  
Spindle Checkpoint ◽  
Kinase Domain ◽  
Wild Type ◽  
Checkpoint Proteins ◽  
Anaphase Onset ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Spindle Microtubules ◽  
Xenopus Egg Extracts ◽  
Checkpoint Signal

The spindle checkpoint delays anaphase onset until all chromosomes have attached properly to the mitotic spindle. Checkpoint signal is generated at kinetochores that are not bound with spindle microtubules or not under tension. Unattached kinetochores associate with several checkpoint proteins, including BubR1, Bub1, Bub3, Mad1, Mad2, and CENP-E. I herein show that BubR1 is important for the spindle checkpoint in Xenopus egg extracts. The protein accumulates and becomes hyperphosphorylated at unattached kinetochores. Immunodepletion of BubR1 greatly reduces kinetochore binding of Bub1, Bub3, Mad1, Mad2, and CENP-E. Loss of BubR1 also impairs the interaction between Mad2, Bub3, and Cdc20, an anaphase activator. These defects are rescued by wild-type, kinase-dead, or a truncated BubR1 that lacks its kinase domain, indicating that the kinase activity of BubR1 is not essential for the spindle checkpoint in egg extracts. Furthermore, localization and hyperphosphorylation of BubR1 at kinetochores are dependent on Bub1 and Mad1, but not Mad2. This paper demonstrates that BubR1 plays an important role in kinetochore association of other spindle checkpoint proteins and that Mad1 facilitates BubR1 hyperphosphorylation at kinetochores.

scholarly journals Spindle Checkpoint Protein Bub1 Is Required for Kinetochore Localization of Mad1, Mad2, Bub3, and Cenp-E, Independently of Its Kinase Activity

2001 ◽  
Vol 153 (6) ◽  
pp. 1239-1250 ◽  
Author(s):  
Hilary Sharp-Baker ◽  
Rey-Huei Chen
Keyword(s):  
Mitotic Spindle ◽  
Kinase Activity ◽  
Spindle Checkpoint ◽  
Wild Type ◽  
Checkpoint Proteins ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Checkpoint Signal

The spindle checkpoint inhibits the metaphase to anaphase transition until all the chromosomes are properly attached to the mitotic spindle. We have isolated a Xenopus homologue of the spindle checkpoint component Bub1, and investigated its role in the spindle checkpoint in Xenopus egg extracts. Antibodies raised against Bub1 recognize a 150-kD phosphoprotein at both interphase and mitosis, but the molecular mass is reduced to 140 upon dephosphorylation in vitro. Bub1 is essential for the establishment and maintenance of the checkpoint and is localized to kinetochores, similar to the spindle checkpoint complex Mad1–Mad2. However, Bub1 differs from Mad1–Mad2 in that Bub1 remains on kinetochores that have attached to microtubules; the protein eventually dissociates from the kinetochore during anaphase. Immunodepletion of Bub1 abolishes the spindle checkpoint and the kinetochore binding of the checkpoint proteins Mad1, Mad2, Bub3, and CENP-E. Interestingly, reintroducing either wild-type or kinase-deficient Bub1 protein restores the checkpoint and the kinetochore localization of these proteins. Our studies demonstrate that Bub1 plays a central role in triggering the spindle checkpoint signal from the kinetochore, and that its kinase activity is not necessary for the spindle checkpoint in Xenopus egg extracts.

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.

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