scholarly journals ISWI Remodeling Complexes in Xenopus Egg Extracts: Identification as Major Chromosomal Components that Are Regulated by INCENP-aurora B

2002 ◽  
Vol 13 (1) ◽  
pp. 25-39 ◽  
Author(s):  
David E. MacCallum ◽  
Ana Losada ◽  
Ryuji Kobayashi ◽  
Tatsuya Hirano
Keyword(s):  
Chromosome Condensation ◽  
Aurora B ◽  
Mitotic Chromosomes ◽  
Novel Proteins ◽  
Condensin Complex ◽  
H3 Phosphorylation ◽  
Chromatid Cohesion ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopuschromosome–associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be theXenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains theXenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.

scholarly journals Mitotic noncoding RNA processing promotes kinetochore and spindle assembly in Xenopus

2016 ◽  
Vol 214 (2) ◽  
pp. 133-141 ◽  
Author(s):  
Andrew W. Grenfell ◽  
Rebecca Heald ◽  
Magdalena Strzelecka
Keyword(s):  
Rna Processing ◽  
Rna Splicing ◽  
Transcription Initiation ◽  
Noncoding Rna ◽  
Spindle Assembly ◽  
Aurora B ◽  
Mitotic Chromosomes ◽  
Mitotic Kinase ◽  
Egg Extracts ◽  
Xenopus Egg Extracts

Transcription at the centromere of chromosomes plays an important role in kinetochore assembly in many eukaryotes, and noncoding RNAs contribute to activation of the mitotic kinase Aurora B. However, little is known about how mitotic RNA processing contributes to spindle assembly. We found that inhibition of transcription initiation or RNA splicing, but not translation, leads to spindle defects in Xenopus egg extracts. Spliceosome inhibition resulted in the accumulation of high molecular weight centromeric transcripts, concomitant with decreased recruitment of the centromere and kinetochore proteins CENP-A, CENP-C, and NDC80 to mitotic chromosomes. In addition, blocking transcript synthesis or processing during mitosis caused accumulation of MCAK, a microtubule depolymerase, on the spindle, indicating misregulation of Aurora B. These findings suggest that co-transcriptional recruitment of the RNA processing machinery to nascent mitotic transcripts is an important step in kinetochore and spindle assembly and challenge the idea that RNA processing is globally repressed during mitosis.

scholarly journals SUMO-2/3 regulates topoisomerase II in mitosis

2003 ◽  
Vol 163 (3) ◽  
pp. 477-487 ◽  
Author(s):  
Yoshiaki Azuma ◽  
Alexei Arnaoutov ◽  
Mary Dasso
Keyword(s):  
Topoisomerase Ii ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mitotic Chromosomes ◽  
Protein Inhibition ◽  
Sumo Conjugation ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Mitotic Chromatin

We have analyzed the abundance of SUMO-conjugated species during the cell cycle in Xenopus egg extracts. The predominant SUMO conjugation products associated with mitotic chromosomes arose from SUMO conjugation of topoisomerase II. Topoisomerase II was modified exclusively by SUMO-2/3 during mitosis under normal circumstances, although we observed conjugation of topoisomerase II to SUMO-1 in extracts with exogenous SUMO-1 protein. Inhibition of SUMO modification by a dominant-negative mutant of the SUMO-conjugating enzyme Ubc9 (dnUbc9) did not detectably alter topoisomerase II activity, but it did increase the amount of unmodified topoisomerase II retained on mitotic chromosomes after high salt washing. dnUbc9 did not disrupt the assembly of condensed mitotic chromosomes or block progression of extracts through mitosis, but it did block the dissociation of sister chromatids at the metaphase–anaphase transition. Together, our results suggest that SUMO conjugation is important for chromosome segregation in metazoan systems, and that mobilization of topoisomerase II from mitotic chromatin may be a key target of this modification.

scholarly journals Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts.

1993 ◽  
Vol 120 (3) ◽  
pp. 601-612 ◽  
Author(s):  
T Hirano ◽  
T J Mitchison
Keyword(s):  
Topoisomerase Ii ◽  
Mitotic Chromosome ◽  
Chromosome Morphology ◽  
Sperm Chromatin ◽  
Mitotic Chromosomes ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Topo Ii ◽  
Xenopus Egg Extracts

We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.

scholarly journals Chromosome architecture can dictate site-specific initiation of DNA replication in Xenopus egg extracts.

1996 ◽  
Vol 135 (5) ◽  
pp. 1207-1218 ◽  
Author(s):  
S J Lawlis ◽  
S M Keezer ◽  
J R Wu ◽  
D M Gilbert
Keyword(s):  
Dna Replication ◽  
Nuclear Envelope ◽  
Cho Cells ◽  
Chromosome Condensation ◽  
G1 Phase ◽  
Unusual Site ◽  
Chromosome Architecture ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

Xenopus egg extracts initiate DNA replication specifically at the dihydrofolate reductase (DHFR) origin locus with intact nuclei from late G1-phase CHO cells as a substrate, but at nonspecific sites when purified DNA is assembled by the extract into an embryonic nuclear structure. Here we show that late G1-phase CHO nuclei can be cycled through an in vitro Xenopus egg mitosis, resulting in the assembly of an embryonic nuclear envelope around G1-phase chromatin. Surprisingly, replication within these chimeric nuclei initiated at a novel specific site in the 5' region of the DHFR structural gene that does not function as an origin in cultured CHO cells. Preferential initiation at this unusual site required topoisomerase II-mediated chromosome condensation during mitosis. Nuclear envelope breakdown and reassembly in the absence of chromosome condensation resulted in nonspecific initiation. Introduction of condensed chromosomes from metaphase-arrested CHO cells directly into Xenopus egg extracts was sufficient to elicit assembly of chimeric nuclei and preferential initiation at this same site. These results demonstrate clearly that chromosome architecture can determine the sites of initiation of replication in Xenopus egg extracts, supporting the hypothesis that patterns of initiation in vertebrate cells are established by higher order features of chromosome structure.

scholarly journals The condensin complex is required for proper spindle assembly and chromosome segregation in Xenopus egg extracts

2003 ◽  
Vol 161 (6) ◽  
pp. 1041-1051 ◽  
Author(s):  
Sarah M. Wignall ◽  
Renée Deehan ◽  
Thomas J. Maresca ◽  
Rebecca Heald
Keyword(s):  
Chromosome Segregation ◽  
Spindle Assembly ◽  
Condensin Complex ◽  
Egg Extracts ◽  
Chromosomal Architecture ◽  
Xenopus Egg ◽  
Sperm Nuclei ◽  
And Function ◽  
Xenopus Egg Extracts

Chromosome condensation is required for the physical resolution and segregation of sister chromatids during cell division, but the precise role of higher order chromatin structure in mitotic chromosome functions is unclear. Here, we address the role of the major condensation machinery, the condensin complex, in spindle assembly and function in Xenopus laevis egg extracts. Immunodepletion of condensin inhibited microtubule growth and organization around chromosomes, reducing the percentage of sperm nuclei capable of forming spindles, and causing dramatic defects in anaphase chromosome segregation. Although the motor CENP-E was recruited to kinetochores pulled poleward during anaphase, the disorganized chromosome mass was not resolved. Inhibition of condensin function during anaphase also inhibited chromosome segregation, indicating its continuous requirement. Spindle assembly around DNA-coated beads in the absence of kinetochores was also impaired upon condensin inhibition. These results support an important role for condensin in establishing chromosomal architecture necessary for proper spindle assembly and chromosome segregation.

scholarly journals Aurora A Phosphorylates MCAK to Control Ran-dependent Spindle Bipolarity

2008 ◽  
Vol 19 (7) ◽  
pp. 2752-2765 ◽  
Author(s):  
Xin Zhang ◽  
Stephanie C. Ems-McClung ◽  
Claire E. Walczak
Keyword(s):  
Phosphorylation Site ◽  
Aurora B ◽  
Aurora A ◽  
Spindle Formation ◽  
Chromatin Region ◽  
Spindle Poles ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Bipolar Spindles

During mitosis, mitotic centromere-associated kinesin (MCAK) localizes to chromatin/kinetochores, a cytoplasmic pool, and spindle poles. Its localization and activity in the chromatin region are regulated by Aurora B kinase; however, how the cytoplasmic- and pole-localized MCAK are regulated is currently not clear. In this study, we used Xenopus egg extracts to form spindles in the absence of chromatin and centrosomes and found that MCAK localization and activity are tightly regulated by Aurora A. This regulation is important to focus microtubules at aster centers and to facilitate the transition from asters to bipolar spindles. In particular, we found that MCAK colocalized with NuMA and XMAP215 at the center of Ran asters where its activity is regulated by Aurora A-dependent phosphorylation of S196, which contributes to proper pole focusing. In addition, we found that MCAK localization at spindle poles was regulated through another Aurora A phosphorylation site (S719), which positively enhances bipolar spindle formation. This is the first study that clearly defines a role for MCAK at the spindle poles as well as identifies another key Aurora A substrate that contributes to spindle bipolarity.

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