Assembly of correct kinetochore architecture in Xenopus laevis egg extract requires transition of sperm DNA through interphase

Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper kinetochore assembly.     

Dissection of CENP-C–directed Centromere and Kinetochore Assembly

Eukaryotic cells ensure accurate chromosome segregation in mitosis by assembling a microtubule-binding site on each chromosome called the kinetochore that attaches to the mitotic spindle. The kinetochore is assembled specifically during mitosis on a specialized region of each chromosome called the centromere, which is constitutively bound by >15 centromere-specific proteins. These proteins, including centromere proteins A and C (CENP-A and -C), are essential for kinetochore assembly and proper chromosome segregation. How the centromere is assembled and how the centromere promotes mitotic kinetochore formation are poorly understood. We have used Xenopus egg extracts as an in vitro system to study the role of CENP-C in centromere and kinetochore assembly. We show that, unlike the histone variant CENP-A, CENP-C is not maintained at centromeres through spermatogenesis but is assembled at the sperm centromere from the egg cytoplasm. Immunodepletion of CENP-C from metaphase egg extract prevents kinetochore formation on sperm chromatin, and depleted extracts can be complemented with in vitro–translated CENP-C. Using this complementation assay, we have identified CENP-C mutants that localized to centromeres but failed to support kinetochore assembly. We find that the amino terminus of CENP-C promotes kinetochore assembly by ensuring proper targeting of the Mis12/MIND complex and CENP-K.     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindle sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G₂ nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.Key words: mitotic chromosomes, Xenopus, egg extracts, intracellular scaling, spindle, embryogenesis, cell division     

Mitotic chromosome size scaling in Xenopus

As rapid divisions without growth generate progressively smaller cells within an embryo, mitotic chromosomes must also decrease in size to permit their proper segregation, but this scaling phenomenon is poorly understood. We demonstrated previously that nuclear and spindle size scale between egg extracts of the related frog species Xenopus tropicalis and Xenopus laevis, but show here that dimensions of isolated mitotic sperm chromosomes do not differ. This is consistent with the hypothesis that chromosome scaling does not occur in early embryonic development when cell and spindles sizes are large and anaphase B segregates chromosomes long distances. To recapitulate chromosome scaling during development, we combined nuclei isolated from different stage Xenopus laevis embryos with metaphase-arrested egg extracts. Mitotic chromosomes derived from nuclei of cleaving embryos through the blastula stage were similar in size to replicated sperm chromosomes, but decreased in area approximately 50% by the neurula stage, reproducing the trend in size changes observed in fixed embryos. Allowing G2 nuclei to swell in interphase prior to mitotic condensation did not increase mitotic chromosome size, but progression through a full cell cycle in egg extract did, suggesting that epigenetic mechanisms determining chromosome size can be altered during DNA replication. Comparison of different sized mitotic chromosomes assembled in vitro provides a tractable system to elucidate underlying molecular mechanisms.     

Yeast Nkp2 is required for accurate chromosome segregation and interacts with several components of the central kinetochore

Kinetochores are macromolecular proteinaceous assemblies that are assembled on centromeres and attach chromosomes to the spindle fibres and regulate the accurate transmission of genetic material to daughter cells. Multiple protein sub-complexes within this supramolecular assembly are hierarchically assembled and contribute to the different aspects of kinetochore function. In this work we show that one of the components of the Saccharomyces cerevisiae kinetochore, Nkp2, plays an important role in ensuring accurate segregation of chromosomes. Although this protein is not conserved in higher organisms, we show that it interacts with highly conserved components of the kinetochore genetically and regulates chromosome segregation. We show that in kinetochore mutants like ctf19 and mcm21 the protein is mislocalized. Furthermore, removal of Nkp2 in these mutants restores normal levels of segregation.     

Nucleoplasmin-mediated chromatin remodelling is required for Xenopus sperm nuclei to become licensed for DNA replication

During late mitosis and early G₁, a series of proteins are assembled onto replication origins, resulting in them becoming ‘licensed’ for replication in the subsequent S phase. Four factors have so far been identified that are required for chromatin to become functionally licensed: ORC (the origin recognition complex) and Cdc6, plus the two components of the replication licensing system RLF-M and RLF-B. Here we describe the first steps of a systematic fractionation of Xenopus egg extracts to identify all the components necessary for the assembly of licensed replication origins on Xenopus sperm nuclei (the physiological DNA substrate in this system). We have purified a new activity essential for this reaction, and have shown that it is nucleoplasmin, a previously known chromatin remodelling protein. Nucleoplasmin decondenses the sperm chromatin by removing protamines, and is required at the earliest known step in origin assembly to allow ORC to bind to the DNA. Sperm nuclei can be licensed by a combination of nucleoplasmin, RLF-M and a partially purified fraction that contains ORC, Cdc6 and RLF-B. This suggests that we are likely to have identified most of the proteins required for this assembly reaction.     

Observation of nuclei reassembled from demembranated Xenopus sperm nuclei and analysis of their lamina components

A cell-free preparation obtained from extracts of activated Xenopus laevis eggs induced chromatin decondensation and nuclear formation from demembranated Xenopus sperm nuclei.Electron microscopy revealed that the reassembled nucleus had a double-layered nuclear memblane,nuclear pore complexes,and decondensed chromatin etc.Indirect immunofluorescence analysis demonstrated the presence of lamina in newly assembled nuclei.Western-blotting results showed that lamin LII was present in egg extracts and in lamina of the reassembled nuclei which were previously reported to contain only egg derived lamin LIII.     

Drosophila mars is required for organizing kinetochore microtubules during mitosis

Spindle assembly is essential for the equal distribution of genetic material to the daughter cells during mitosis. The process of spindle assembly is complicated and involves multiple levels of molecular regulation. It is generally accepted that mitotic spindles are emanated from the centrosomes and are assembled in the vicinity of chromosomes. However, the molecular mechanism involved in the spindle assembly during mitosis remains unclear. In this study, we have provided several lines of evidence to show that Drosophila Mars is required for the assembly and stabilization of kinetochore microtubules. In an immunocytochemical study, we show that Mars is mainly localized on the kinetochore microtubules during mitosis. Using RNA interference to deplete the Mars expression in Drosophila S2 cells resulted in the malformation of mitotic spindle that mainly lacked the kinetochore microtubules. The spindle defect resulted in mitotic delays by increasing the percentage of uncongressed chromosomes both in vitro and in vivo. In summary, this study has extended our previous study of Mars in cell cycle regulation and provided further evidence showing that Mars is required for the assembly of kinetochore microtubules.     

Daunomycin disrupts nuclear assembly and the coordinate initiation of DNA replication in Xenopus egg extracts

We have used Xenopus egg extracts to investigate the effects of the antitumor drug daunomycin on DNA replication in vitro. Xenopus sperm nuclei replicated nearly synchronously in our egg extracts, thereby allowing us to determine the effects of the drug on both replication initiation and elongation. Titration experiments demonstrated that daunomycin effectively inhibited replication in the extract, with 50% inhibition at a total drug concentration of 2.7 μM. However, a high concentration of daunomycin 150 μM) also inhibited nuclear envelope assembly, a prerequisite for the initiation of replication in this system. Therefore, to bypass the effects of daunomycin on nuclear envelope assembly, sperm nuclei were preassembled in extract prior to drug addition. Initiation of replication in preassembled nuclei was also inhibited by daunomycin, with 50% inhibition at a drug concentration of 3.6 μM. At low drug concentrations, where replication did occur, the synchrony of initiations within individual nuclei was lost. This drug-induced disruption of initiation events may provide important clues regarding the mechanism(s) by which these events are coordinated in eukaryotic cells. Daunomycin also inhibited replication elongation in preassembled, preinitiated nuclei. However, the concentration of drug required for 50% inhibition of elongation was nearly fourfold higher than that required for inhibition of initiation. Taken together, these data demonstrate that Xenopus egg extract can be used to investigate the effects of DNA-binding antitumor drugs on a number of interrelated cellular processes, many of which are less tractable in whole cell systems. J. Cell. Biochem. 64:476–491. © 1997 Wiley-Liss, Inc.     

pEg7, a New Xenopus Protein Required for Mitotic Chromosome Condensation in Egg Extracts

We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.     

Chromosome architecture can dictate site-specific initiation of DNA replication in 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.     

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾去膜精子染色质和卵提取物成功地进行了细胞核本外重建。当精子染色质加入卵提取物后,首先发生染色质去浓缩作用,染色质整体结构膨胀;膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜;核孔复合体以某种未知方式组装入双层膜而形成核膜结构,并逐渐完全覆盖膨大的染色质,最终形成典型的间期核结构。     

Permitted and restricted steps of human kinetochore assembly in mitotic cell extracts

Mitotic kinetochores assemble via the hierarchical recruitment of numerous cytosolic components to the centromere region of each chromosome. However, how these orderly and localized interactions are achieved without spurious macromolecular assemblies forming from soluble kinetochore components in the cell cytosol remains poorly understood. We developed assembly assays to monitor the recruitment of green fluorescent protein–tagged recombinant proteins and native proteins from human cell extracts to inner kinetochore components immobilized on microbeads. In contrast to prior work in yeast and Xenopus egg extracts, we find that human mitotic cell extracts fail to support de novo assembly of microtubule-binding subcomplexes. A subset of interactions, such as those between CENP-A–containing nucleosomes and CENP-C, are permissive under these conditions. However, the subsequent phospho-dependent binding of the Mis12 complex is less efficient, whereas recruitment of the Ndc80 complex is blocked, leading to weak microtubule-binding activity of assembled particles. Using molecular variants of the Ndc80 complex, we show that auto-inhibition of native Ndc80 complex restricts its ability to bind to the CENP-T/W complex, whereas inhibition of the Ndc80 microtubule binding is driven by a different mechanism. Together, our work reveals regulatory mechanisms that guard against the spurious formation of cytosolic microtubule-binding kinetochore particles.     

Nuclear assembly of purified Crythecodinium cohnii chromosomes in cell—free extracts of Xenopus laevis eggs

Incubation of dinoflagellate Crythecodinium cohnii chromosomes in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in chromosomes decondensation and recondensation,nuclear envelope assembly,and nuclear reconstitution.Dinoflagellate Crythecodinium cohnii is a kind of primitive eukaryote which possesses numerous permanently condensed chromosomes and discontinuous double-layered nuclear membrane throughout the cell cycle.The assembled nuclei,being surrounded by a continuous double membrane containing nuclear pores and the uniformly dispersed chromatin fibers are morphologically distinguishable from that of Dinoflagellate Crythecodinium cohnii.However,incubation of dinoflagellate Cyrthecodinium cohnii chromosomes in the extracts from dinoflagellate Crythecodinium cohnii cells does not induce nuclear reconstitution.     

Mitotic spindle assembly by two different pathways in vitro

We have used Xenopus egg extracts to study spindle morphogenesis in a cell-free system and have identified two pathways of spindle assembly in vitro using methods of fluorescent analogue cytochemistry. When demembranated sperm nuclei are added to egg extracts arrested in a mitotic state, individual nuclei direct the assembly of polarized microtubule arrays, which we term half-spindles; half-spindles then fuse pairwise to form bipolar spindles. In contrast, when sperm nuclei are added to extracts that are induced to enter interphase and arrested in the following mitosis, a single sperm nucleus can direct the assembly of a complete spindle. We find that microtubule arrays in vitro are strongly biased towards chromatin, but this does not depend on specific kinetochore-microtubule interactions. Indeed, although we have identified morphological and probably functional kinetochores in spindles assembled in vitro, kinetochores appear not to play an obligate role in the establishment of stable, bipolar microtubule arrays in either assembly pathway. Features of the two pathways suggest that spindle assembly involves a hierarchy of selective microtubule stabilization, involving both chromatin-microtubule interactions and antiparallel microtubule-microtubule interactions, and that fundamental molecular interactions are probably the same in both pathways. This in vitro reconstitution system should be useful for identifying the molecules regulating the generation of asymmetric microtubule arrays and for understanding spindle morphogenesis in general.     

Induction of nuclear envelope breakdown, chromosome condensation, and spindle formation in cell-free extracts

Incubation of demembranated sperm chromatin in cytoplasmic extracts of unfertilized Xenopus laevis eggs resulted in nuclear envelope assembly, chromosome decondensation, and sperm pronuclear formation. In contrast, egg extracts made with EGTA-containing buffers induced the sperm chromatin to form chromosomes or irregularly shaped clumps of chromatin that were incorporated into bipolar or multipolar spindles. The 150,000 g supernatants of the EGTA extracts could not alone support these changes in incubated nuclei. However, these supernatants induced not only chromosome condensation and spindle formation, but also nuclear envelope breakdown when added to sperm pronuclei or isolated Xenopus liver or brain nuclei that were incubated in extracts made without EGTA. Similar changes were induced by partially purified preparations of maturation-promoting factor. The addition of calcium chloride to extracts containing condensed chromosomes and spindles caused dissolution of the spindles, decondensation of the chromosomes, and re-formation of interphase nuclei. These results indicate that nuclear envelope breakdown, chromosome condensation, and spindle assembly, as well as the regulation of these processes by Ca2+-sensitive cytoplasmic components, can be studied in vitro using extracts of amphibian eggs.     

Core histone N-termini play an essential role in mitotic chromosome condensation

We have studied the role of core histone tails in the assembly of mitotic chromosomes using Xenopus egg extracts. Incubation of sperm nuclei in the extracts led to the formation of mitotic chromosomes, a process we found to be correlated with phosphorylation of the N-terminal tail of histone H3 at Ser10. When the extracts were supplemented with H1-depleted oligosomes, they were not able to assemble chromosomes. Selective elimination of oligosome histone tails by trypsin digestion resulted in a dramatic decrease in their ability to inhibit chromosome condensation. The chromosome assembly was also inhibited by each of the histone tails with differing efficiency. In addition, we found that nucleosomes were recruiting through the flexible histone tails some chromosome assembly factors, different from topoisomerase II and 13S condensin. These findings demonstrate that histone tails play an essential role in chromosome assembly. We also present evidence that the nucleosomes, through physical association, were able to deplete the extracts from the kinase phosphorylating histone H3 at Ser10, suggesting that this kinase could be important for chromosome condensation.     

Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components

It is widely accepted that the kinetochore is built on CENP-A–marked centromeric chromatin in a hierarchical order from inner to outer kinetochore. Recruitment of many kinetochore proteins depends on microtubule attachment status, but it remains unclear how their assembly/disassembly is orchestrated. Applying 3D structured illumination microscopy to Xenopus laevis egg extracts, here we reveal that in the absence of microtubule attachment, proteins responsible for lateral attachment and spindle checkpoint signaling expand to form micrometer-scale fibrous structures over CENP-A–free chromatin, whereas a core module responsible for end-on attachment (CENP-A, CENP-T, and Ndc80) does not. Both outer kinetochore proteins (Bub1, BubR1, Mad1, and CENP-E) and the inner kinetochore component CENP-C are integral components of the expandable module, whose assembly depends on multiple mitotic kinases (Aurora B, Mps1, and Plx1) and is suppressed by protein phosphatase 1. We propose that phospho-dependent coexpansion of CENP-C and outer kinetochore proteins promotes checkpoint signal amplification and lateral attachment, whereas their selective disassembly enables the transition to end-on attachment.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。