RESUMO
Faithful chromosome segregation requires biorientation, where the pair of kinetochores on the chromosome establish bipolar microtubule attachment. The integrity of the kinetochore, a macromolecular complex built on centromeric DNA, is required for biorientation, but components sufficient for biorientation remain unknown. Here, we show that tethering the outer kinetochore heterodimer NDC80-NUF2 to the surface of apolar microbeads establishes their biorientation-like state in mouse cells. NDC80-NUF2 microbeads align at the spindle equator and self-correct alignment errors. The alignment is associated with stable bipolar microtubule attachment and is independent of the outer kinetochore proteins SPC24-SPC25, KNL1, the Mis12 complex, inner kinetochore proteins, and Aurora. Larger microbeads align more rapidly, suggesting a size-dependent biorientation mechanism. This study demonstrates a biohybrid kinetochore design for synthetic biorientation of microscale particles in cells.
Assuntos
Proteínas de Ciclo Celular , Segregação de Cromossomos , Cinetocoros , Microesferas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Fuso Acromático , Animais , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas do Citoesqueleto/genética , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Fuso Acromático/metabolismoRESUMO
The centrosome of the amoebozoan model Dictyostelium discoideum provides the best-established model for an acentriolar centrosome outside the Opisthokonta. Dictyostelium exhibits an unusual centrosome cycle, in which duplication is initiated only at the G2/M transition and occurs entirely during the M phase. Little is known about the role of conserved centrosomal kinases in this process. Therefore, we have generated knock-in strains for Aurora (AurK), CDK1, cyclin B, Nek2, and Plk, replacing the endogenous genes with constructs expressing the respective green fluorescent Neon fusion proteins, driven by the endogenous promoters, and studied their behavior in living cells. Our results show that CDK1 and cyclin B arrive at the centrosome first, already during G2, followed by Plk, Nek2, and AurK. Furthermore, CDK1/cyclin B and AurK were dynamically localized at kinetochores, and AurK in addition at nucleoli. The putative roles of all four kinases in centrosome duplication, mitosis, cytokinesis, and nucleolar dynamics are discussed.
Assuntos
Proteína Quinase CDC2 , Centrossomo , Dictyostelium , Mitose , Centrossomo/metabolismo , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Dictyostelium/genética , Dictyostelium/metabolismo , Dictyostelium/enzimologia , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genética , Ciclina B/metabolismo , Ciclina B/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Cinetocoros/metabolismo , Aurora Quinases/metabolismo , Aurora Quinases/genética , Nucléolo Celular/metabolismoRESUMO
In a recent issue of Cell Reports, Bray et al. found genetic adaptation in kinetochore components and ion transporters underlying polyploid stabilization in Cochlearia. This resurrects the issue of whether nascent polyploidy in diverse organisms establish via common biological mechanisms.
Assuntos
Poliploidia , Cinetocoros/metabolismo , Animais , HumanosRESUMO
The centromere, a chromosome locus defined by the histone H3-like protein centromeric protein A (CENP-A), promotes assembly of the kinetochore to bind microtubules during cell division. Centromere maintenance requires CENP-A to be actively replenished by dedicated protein machinery in the early G1 phase of the cell cycle to compensate for its dilution after DNA replication. Cyclin-dependent kinases (CDKs) limit CENP-A deposition to once per cell cycle and function as negative regulators outside of early G1. Antithetically, Polo-like kinase 1 (PLK1) promotes CENP-A deposition in early G1, but the molecular details of this process are still unknown. We reveal here a phosphorylation network that recruits PLK1 to the deposition machinery to control a conformational switch required for licensing the CENP-A deposition reaction. Our findings clarify how PLK1 contributes to the epigenetic maintenance of centromeres.
Assuntos
Proteínas de Ciclo Celular , Proteína Centromérica A , Centrômero , Proteínas Cromossômicas não Histona , Epigênese Genética , Quinase 1 Polo-Like , Humanos , Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Fase G1 , Células HeLa , Cinetocoros/metabolismo , Fosforilação , Quinase 1 Polo-Like/genética , Quinase 1 Polo-Like/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/genéticaRESUMO
Mitosis is an important process in the cell cycle required for cells to divide. Never in mitosis (NIMA)-like kinases (NEKs) are regulators of mitotic functions in diverse organisms. Plasmodium spp., the causative agent of malaria is a divergent unicellular haploid eukaryote with some unusual features in terms of its mitotic and nuclear division cycle that presumably facilitate proliferation in varied environments. For example, during the sexual stage of male gametogenesis that occurs within the mosquito host, an atypical rapid closed endomitosis is observed. Three rounds of genome replication from 1N to 8N and successive cycles of multiple spindle formation and chromosome segregation occur within 8 min followed by karyokinesis to generate haploid gametes. Our previous Plasmodium berghei kinome screen identified 4 Nek genes, of which 2, NEK2 and NEK4, are required for meiosis. NEK1 is likely to be essential for mitosis in asexual blood stage schizogony in the vertebrate host, but its function during male gametogenesis is unknown. Here, we study NEK1 location and function, using live cell imaging, ultrastructure expansion microscopy (U-ExM), and electron microscopy, together with conditional gene knockdown and proteomic approaches. We report spatiotemporal NEK1 location in real-time, coordinated with microtubule organising centre (MTOC) dynamics during the unusual mitoses at various stages of the Plasmodium spp. life cycle. Knockdown studies reveal NEK1 to be an essential component of the MTOC in male cell differentiation, associated with rapid mitosis, spindle formation, and kinetochore attachment. These data suggest that P. berghei NEK1 kinase is an important component of MTOC organisation and essential regulator of chromosome segregation during male gamete formation.
Assuntos
Cinetocoros , Centro Organizador dos Microtúbulos , Mitose , Quinase 1 Relacionada a NIMA , Plasmodium berghei , Masculino , Cinetocoros/metabolismo , Animais , Quinase 1 Relacionada a NIMA/metabolismo , Quinase 1 Relacionada a NIMA/genética , Plasmodium berghei/fisiologia , Plasmodium berghei/genética , Plasmodium berghei/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Segregação de Cromossomos , Gametogênese , Quinases Relacionadas a NIMA/metabolismo , Quinases Relacionadas a NIMA/genéticaRESUMO
The chromosome segregation and cell division programs associated with somatic mitosis and germline meiosis display dramatic differences such as kinetochore orientation, cohesin removal, or the presence of a gap phase.1,2,3,4,5,6 These changes in chromosome segregation require alterations to the established cell division machinery.5,6 It remains unclear what aspects of kinetochore function and its regulatory control differ between the mitotic and meiotic cell divisions to rewire these core processes. Alternative RNA splicing can generate distinct protein isoforms to allow for the differential control of cell processes across cell types. However, alternative splice isoforms that differentially modulate distinct cell division programs have remained elusive. Here, we demonstrate that mammalian germ cells express an alternative mRNA splice isoform for the kinetochore component, DSN1, a subunit of the MIS12 complex that links the centromeres to spindle microtubules during chromosome segregation. This germline DSN1 isoform bypasses the requirement for Aurora kinase phosphorylation for its centromere localization due to the absence of a key regulatory region allowing DSN1 to display persistent centromere localization. Expression of the germline DSN1 isoform in somatic cells results in constitutive kinetochore localization, chromosome segregation errors, and growth defects, providing an explanation for its tight cell-type-specific expression. Reciprocally, precisely eliminating expression of the germline-specific DSN1 splice isoform in mouse models disrupts oocyte maturation and early embryonic divisions coupled with a reduction in fertility. Together, this work identifies a germline-specific splice isoform for a chromosome segregation component and implicates its role in mammalian fertility.
Assuntos
Processamento Alternativo , Segregação de Cromossomos , Desenvolvimento Embrionário , Oócitos , Isoformas de Proteínas , Animais , Oócitos/metabolismo , Camundongos , Feminino , Desenvolvimento Embrionário/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Cinetocoros/metabolismo , Células Germinativas/metabolismo , MasculinoRESUMO
The eukaryotic cell division machinery must rapidly and reproducibly duplicate and partition the cell's chromosomes in a carefully coordinated process. However, chromosome numbers vary dramatically between genomes, even on short evolutionary timescales. We sought to understand how the mitotic machinery senses and responds to karyotypic changes by using a series of budding yeast strains in which the native chromosomes have been successively fused. Using a combination of cell biological profiling, genetic engineering and experimental evolution, we show that chromosome fusions are well tolerated up until a critical point. Cells with fewer than five centromeres lack the necessary number of kinetochore-microtubule attachments needed to counter outward forces in the metaphase spindle, triggering the spindle assembly checkpoint and prolonging metaphase. Our findings demonstrate that spindle architecture is a constraining factor for karyotype evolution.
Assuntos
Cinetocoros , Saccharomyces cerevisiae , Fuso Acromático , Fuso Acromático/metabolismo , Fuso Acromático/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cinetocoros/metabolismo , Cariótipo , Cromossomos Fúngicos/genética , Mitose/genética , Evolução Molecular , Microtúbulos/metabolismo , Centrômero/genética , Centrômero/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
Assuntos
Cinetocoros , Kluyveromyces , Kluyveromyces/citologia , Cinetocoros/química , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Proteínas Associadas aos Microtúbulos/análise , Proteínas Fúngicas/análise , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Microtúbulos/metabolismo , Tomografia com Microscopia EletrônicaRESUMO
The acentrosomal spindle apparatus has kinetochore fibers organized and converged toward opposite poles; however, mechanisms underlying the organization of these microtubule fibers into an orchestrated bipolar array were largely unknown. Kinesin-14D is one of the four classes of Kinesin-14 motors that are conserved from green algae to flowering plants. In Arabidopsis thaliana, three Kinesin-14D members displayed distinct cell cycle-dependent localization patterns on spindle microtubules in mitosis. Notably, Kinesin-14D1 was enriched on the midzone microtubules of prophase and mitotic spindles and later persisted in the spindle and phragmoplast midzones. The kinesin-14d1 mutant had kinetochore fibers disengaged from each other during mitosis and exhibited hypersensitivity to the microtubule-depolymerizing herbicide oryzalin. Oryzalin-treated kinesin-14d1 mutant cells had kinetochore fibers tangled together in collapsed spindle microtubule arrays. Kinesin-14D1, unlike other Kinesin-14 motors, showed slow microtubule plus end-directed motility, and its localization and function were dependent on its motor activity and the novel malectin-like domain. Our findings revealed a Kinesin-14D1-dependent mechanism that employs interpolar microtubules to regulate the organization of kinetochore fibers for acentrosomal spindle morphogenesis.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Cinesinas , Microtúbulos , Fuso Acromático , Arabidopsis/metabolismo , Arabidopsis/genética , Cinesinas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fuso Acromático/metabolismo , Mitose , Morfogênese , Cinetocoros/metabolismo , Dinitrobenzenos/farmacologia , Sulfanilamidas/farmacologiaRESUMO
The localization of the meiotic specific regulatory molecule Moa1 to the centromere is regulated by the kinetochore protein CENP-C, and participates in the cohesion of sister chromatids in the centromere region mediated by the cohesin Rec8. To examine the interaction of these proteins, we analyzed the interactions between Moa1 and Rec8, CENP-C by yeast two-hybrid assays and identified several amino acid residues in Moa1 required for the interaction with CENP-C and Rec8. The results revealed that the interaction between Moa1 and CENP-C is crucial for the Moa1 to participate in the regulation of monopolar attachment of sister kinetochores. However, mutation at S143 and T150 of Moa1, which are required for interaction with Rec8 in the two-hybrid assay, did not show significant defects. Mutations in amino acid residues may not be sufficient to interfere with the interaction between Moa1 and Rec8 in vivo. Further research is needed to determine the interaction domain between Moa1 and Rec8. This study revealed specific amino acid sites at which Moa1 affects the meiotic homologous chromosome segregation, providing a deeper understanding of the mechanism of meiotic chromosome segregation.
Assuntos
Proteínas Cromossômicas não Histona , Meiose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Ligação Proteica , Cinetocoros/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Técnicas do Sistema de Duplo-Híbrido , Segregação de Cromossomos , Coesinas , FosfoproteínasRESUMO
Whole-genome duplication (WGD) occurs in all kingdoms and impacts speciation, domestication, and cancer outcome. However, doubled DNA management can be challenging for nascent polyploids. The study of within-species polyploidy (autopolyploidy) permits focus on this DNA management aspect, decoupling it from the confounding effects of hybridization (in allopolyploid hybrids). How is autopolyploidy tolerated, and how do young polyploids stabilize? Here, we introduce a powerful model to address this: the genus Cochlearia, which has experienced many polyploidization events. We assess meiosis and other polyploid-relevant phenotypes, generate a chromosome-scale genome, and sequence 113 individuals from 33 ploidy-contrasting populations. We detect an obvious autopolyploidy-associated selection signal at kinetochore components and ion transporters. Modeling the selected alleles, we detail evidence of the kinetochore complex mediating adaptation to polyploidy. We compare candidates in independent autopolyploids across three genera separated by 40 million years, highlighting a common function at the process and gene levels, indicating evolutionary flexibility in response to polyploidy.
Assuntos
Evolução Molecular , Genoma de Planta , Cinetocoros , Poliploidia , Cinetocoros/metabolismo , Duplicação Gênica , Adaptação Fisiológica/genética , Meiose/genéticaRESUMO
Kinetochores form the interface between chromosomes and spindle microtubules and are thus under tight control by a complex regulatory circuitry. The Aurora B kinase plays a central role within this circuitry by destabilizing improper kinetochore-microtubule attachments and relaying the attachment status to the spindle assembly checkpoint. Intriguingly, Aurora B is conserved even in kinetoplastids, a group of early-branching eukaryotes which possess a unique set of kinetochore proteins. It remains unclear how their kinetochores are regulated to ensure faithful chromosome segregation. Here, we show in Trypanosoma brucei that Aurora B activity controls the metaphase-to-anaphase transition through phosphorylation of the divergent Bub1-like protein KKT14. Depletion of KKT14 overrides the metaphase arrest resulting from Aurora B inhibition, while expression of non-phosphorylatable KKT14 delays anaphase onset. Finally, we demonstrate that re-targeting Aurora B to the outer kinetochore suffices to promote mitotic exit but causes extensive chromosome missegregation in anaphase. Our results indicate that Aurora B and KKT14 are involved in an unconventional circuitry controlling cell cycle progression in trypanosomes.
Assuntos
Anáfase , Aurora Quinase B , Segregação de Cromossomos , Cinetocoros , Proteínas de Protozoários , Trypanosoma brucei brucei , Aurora Quinase B/metabolismo , Aurora Quinase B/genética , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Trypanosoma brucei brucei/enzimologia , Cinetocoros/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , Fosforilação , Fuso Acromático/metabolismo , Fuso Acromático/genética , Microtúbulos/metabolismo , Microtúbulos/genéticaRESUMO
Spindle bipolarization, the process of a microtubule mass transforming into a bipolar spindle, is a prerequisite for accurate chromosome segregation. In contrast to mitotic cells, the process and mechanism of spindle bipolarization in human oocytes remains unclear. Using high-resolution imaging in more than 1800 human oocytes, we revealed a typical state of multipolar intermediates that form during spindle bipolarization and elucidated the mechanism underlying this process. We found that the minor poles formed in multiple kinetochore clusters contribute to the generation of multipolar intermediates. We further determined the essential roles of HAUS6, KIF11, and KIF18A in spindle bipolarization and identified mutations in these genes in infertile patients characterized by oocyte or embryo defects. These results provide insights into the physiological and pathological mechanisms of spindle bipolarization in human oocytes.
Assuntos
Segregação de Cromossomos , Cinesinas , Cinetocoros , Microtúbulos , Oócitos , Fuso Acromático , Humanos , Oócitos/metabolismo , Cinesinas/metabolismo , Cinesinas/genética , Cinetocoros/metabolismo , Fuso Acromático/metabolismo , Microtúbulos/metabolismo , Feminino , Mutação , Polos do Fuso/metabolismoRESUMO
The spindle assembly checkpoint (SAC) ensures chromosome segregation fidelity by manipulating unattached kinetochore-dependent assembly of the mitotic checkpoint complex (MCC). The MCC binds to and inhibits the anaphase promoting complex/cyclosome (APC/C) to postpone mitotic exit. However, the mechanism by which unattached kinetochores mediate MCC formation is not yet fully understood. Here, it is shown that CCDC68 is an outer kinetochore protein that preferentially localizes to unattached kinetochores. Furthermore, CCDC68 interacts with the SAC factor CDC20 to inhibit its autoubiquitination and MCC disassembly. Therefore, CCDC68 restrains APC/C activation to ensure a robust SAC and allow sufficient time for chromosome alignment, thus ensuring chromosomal stability. Hence, the study reveals that CCDC68 is required for CDC20-dependent MCC stabilization to maintain mitotic checkpoint activation.
Assuntos
Proteínas Cdc20 , Cinetocoros , Pontos de Checagem da Fase M do Ciclo Celular , Humanos , Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/genética , Proteínas Cdc20/metabolismo , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Segregação de Cromossomos/fisiologia , Células HeLa , Cinetocoros/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular/genética , Pontos de Checagem da Fase M do Ciclo Celular/fisiologia , Mitose/fisiologiaRESUMO
BACKGROUND: Aurora kinase B (Aurora-B), a member of the chromosomal passenger complex, is involved in correcting kinetochore-microtubule (KT-MT) attachment errors and regulating sister chromatid condensation and cytoplasmic division during mitosis. SUMMARY: However, few reviews have discussed its mechanism in oocyte meiosis and the differences between its role in mitosis and meiosis. Therefore, in this review, we summarize the localization, recruitment, activation, and functions of Aurora-B in mitosis and oocyte meiosis. The accurate regulation of Aurora-B is essential for ensuring accurate chromosomal segregation and correct KT-MT attachments. Aurora-B regulates the stability of KT-MT attachments by competing with cyclin-dependent kinase 1 to control the phosphorylation of the SILK and RVSF motifs on kinetochore scaffold 1 and by competing with protein phosphatase 1 to influence the phosphorylation of NDC80 which is the substrate of Aurora-B. In addition, Aurora-B regulates the spindle assembly checkpoint by promoting the recruitment and activation of mitotic arrest deficient 2. KEY MESSAGES: This review provides a theoretical foundation for elucidating the mechanism of cell division and understanding oocyte chromosomal aneuploidy.
Assuntos
Aurora Quinase B , Cinetocoros , Meiose , Microtúbulos , Mitose , Oócitos , Aurora Quinase B/metabolismo , Aurora Quinase B/genética , Cinetocoros/metabolismo , Oócitos/metabolismo , Oócitos/citologia , Humanos , Animais , Microtúbulos/metabolismo , Fosforilação , Segregação de Cromossomos , Feminino , Proteína Quinase CDC2/metabolismo , Proteína Quinase CDC2/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas do CitoesqueletoRESUMO
Chromosome segregation errors caused by centromere malfunction can lead to chromosome instability and aneuploidy. In Caenorhabditis elegans, the Argonaute protein CSR-1 is essential for proper chromosome segregation, although the specific mechanisms are not fully understood. Here, we investigated how CSR-1 regulates centromere and kinetochore function in C. elegans embryos. We found that depletion of CSR-1 results in defects in mitotic progression and chromosome positioning relative to the spindle pole. Knockdown of CSR-1 does not affect mRNA and protein levels of the centromeric histone H3 variant and CENP-A homolog HCP-3 but does increase the localization of HCP-3 and some kinetochore proteins to the mitotic chromosomes. Such elevation of HCP-3 chromatin localization depends on EGO-1, which is an upstream factor in the CSR-1 RNA interference (RNAi) pathway, and PIWI domain activity of CSR-1. Our results suggest that CSR-1 restricts the level of HCP-3 at the holocentromeres, prevents erroneous kinetochore assembly and thereby promotes accurate chromosome segregation. Our work sheds light on the role of CSR-1 in regulating deposition of HCP-3 on chromatin and centromere function in embryos.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Proteína Centromérica A , Centrômero , Segregação de Cromossomos , Cinetocoros , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteína Centromérica A/metabolismo , Proteína Centromérica A/genética , Cinetocoros/metabolismo , Centrômero/metabolismo , Proteínas Argonautas/metabolismo , Proteínas Argonautas/genética , Mitose , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Cromossômicas não Histona/genética , Interferência de RNA , Histonas/metabolismo , Histonas/genética , Cromatina/metabolismo , RNA Polimerase Dependente de RNARESUMO
In brief: Repro57 mice, bearing an Rnf212 gene mutation, exhibit infertility in both homozygous mutant males and females, revealing arrested spermatogenesis in males and investigating unclear mechanisms in females. The study highlights aneuploidy and altered kinetochore patterns in repro57 homozygous mutant oocytes, which impact later stages of embryo development. Abstract: Repro57 mice, induced with N-ethyl-N-nitrosourea and harboring a mutation in the Rnf212 gene, exhibit infertility in both homozygous mutant males and females. Rnf212 plays a crucial role in recombination and crossover designation. In male repro57 homozygous mutants, spermatocytes often degenerate during late prophase, and mature spermatozoa are absent in the seminiferous epithelium, indicating arrested spermatogenesis as the cause of infertility. Despite reports of infertility in Rnf212-knockout female mice, the specific mechanisms underlying infertility in female repro57 homozygous mutants remain elusive. This study investigates the chromosomal and kinetochore patterns of mature oocytes and their developmental potential following in vitro fertilization in female repro57 homozygous mutant mice. While all wild-type oocytes progress to metaphase II and exhibit euploidy, all repro57 homozygous mutant mouse oocytes display aneuploidy. Additionally, kinetochore distances in repro57 homozygous mutant oocytes exceed those observed in wild-type counterparts. Although no significant differences are noted in fertilization and early embryo development rates between wild-type and repro57 homozygous mutant mice, embryos derived from repro57 homozygous mutants exhibit significantly lower morula and blastocyst rates, accompanied by frequent cytokinesis failure and vacuole formation. These findings suggest that the premature segregation of sister chromatids in repro57 homozygous mutant mice adversely impacts the later stages of embryo development.
Assuntos
Desenvolvimento Embrionário , Homozigoto , Mutação , Oócitos , Animais , Feminino , Desenvolvimento Embrionário/genética , Camundongos , Masculino , Oócitos/patologia , Segregação de Cromossomos , Ubiquitina-Proteína Ligases/genética , Aneuploidia , Cinetocoros/metabolismo , Espermatogênese/genética , Camundongos Endogâmicos C57BLRESUMO
The 14-3-3 family of proteins are conserved across eukaryotes and serve myriad important regulatory functions in the cell. Homo- and hetero-dimers of these proteins mainly recognize their ligands via conserved motifs to modulate the localization and functions of those effector ligands. In most of the genetic backgrounds of Saccharomyces cerevisiae, disruption of both 14-3-3 homologs (Bmh1 and Bmh2) are either lethal or cells survive with severe growth defects, including gross chromosomal missegregation and prolonged cell cycle arrest. To elucidate their contributions to chromosome segregation, in this work, we investigated their centromere- and kinetochore-related functions of Bmh1 and Bmh2. Analysis of appropriate deletion mutants shows that Bmh isoforms have cumulative and non-shared isoform-specific contributions in maintaining the proper integrity of the kinetochore ensemble. Consequently, Bmh mutant cells exhibited perturbations in kinetochore-microtubule (KT-MT) dynamics, characterized by kinetochore declustering, mis-localization of kinetochore proteins and Mad2-mediated transient G2/M arrest. These defects also caused an asynchronous chromosome congression in bmh mutants during metaphase. In summary, this report advances the knowledge on contributions of budding yeast 14-3-3 proteins in chromosome segregation by demonstrating their roles in kinetochore integrity and chromosome congression.
Assuntos
Proteínas 14-3-3 , Segregação de Cromossomos , Cinetocoros , Mitose , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Cinetocoros/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas 14-3-3/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Microtúbulos/metabolismo , Cromossomos Fúngicos/metabolismo , Cromossomos Fúngicos/genéticaRESUMO
Centromeres are specialized chromosomal domains where the kinetochores assemble during cell division to ensure accurate transmission of the genetic information to the two daughter cells. The centromeric function is evolutionary conserved and, in most organisms, centromeres are epigenetically defined by a unique chromatin containing the histone H3 variant CENP-A. The canonical regulators of CENP-A assembly and maintenance are well-known, yet some of the molecular mechanisms regulating this complex process have only recently been unveiled. We review the most recent advances on the topic, including the emergence of new and unexpected factors that favor and regulate CENP-A assembly and/or maintenance.
Assuntos
Proteína Centromérica A , Centrômero , Proteínas Cromossômicas não Histona , Centrômero/metabolismo , Humanos , Animais , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Autoantígenos/metabolismo , Histonas/metabolismo , Cromatina/metabolismo , Cinetocoros/metabolismoRESUMO
After whole-genome duplication (WGD), tetraploid cells can undergo multipolar mitosis or pseudo-bipolar mitosis with clustered centrosomes. Kinesins play a crucial role in regulating spindle formation. However, the contribution of kinesin expression levels to the heterogeneity in centrosome clustering observed across different cell lines after WGD remains unclear. We identified two subsets of cell lines: "BP" cells efficiently cluster extra centrosomes for pseudo-bipolar mitosis, and "MP" cells primarily undergo multipolar mitosis after WGD. Diploid MP cells contained higher levels of KIF11 and KIF15 compared with BP cells and showed reduced sensitivity to centrosome clustering induced by KIF11 inhibitors. Moreover, partial inhibition of KIF11 or depletion of KIF15 converted MP cells from multipolar to bipolar mitosis after WGD. Multipolar spindle formation involved microtubules but was independent of kinetochore-microtubule attachment. Silencing KIFC1, but not KIFC3, promoted multipolar mitosis in BP cells, indicating the involvement of specific kinesin-14 family members in counteracting the forces from KIF11/KIF15 after WGD. These findings highlight the collective role of KIF11, KIF15, and KIFC1 in determining the polarity of the mitotic spindle after WGD.