RESUMEN
Numerous intra- and inter-chromosomal contacts have been mapped in eukaryotic genomes, but it remains challenging to link these 3D structures to their regulatory functions. To establish the causal relationships between chromosome conformation and genome functions, we develop a method, Chemically Induced Chromosomal Interaction (CICI), to selectively perturb the chromosome conformation at targeted loci. Using this method, long-distance chromosomal interactions can be induced dynamically between intra- or inter-chromosomal loci pairs, including the ones with very low Hi-C contact frequencies. Measurement of CICI formation time allows us to probe chromosome encounter dynamics between different loci pairs across the cell cycle. We also conduct two functional tests of CICI. We perturb the chromosome conformation near a DNA double-strand break and observe altered donor preference in homologous recombination; we force interactions between early and late-firing DNA replication origins and find no significant changes in replication timing. These results suggest that chromosome conformation plays a deterministic role in homology-directed DNA repair, but not in the establishment of replication timing. Overall, our study demonstrates that CICI is a powerful tool to study chromosome dynamics and 3D genome function.
Asunto(s)
Cromosomas/química , Cromosomas/fisiología , Genómica , Roturas del ADN de Doble Cadena , Reparación del ADN , Conformación Molecular , Origen de Réplica , LevadurasRESUMEN
To achieve spermatogenesis in vitro, one of the most challenging processes to mimic is meiosis. Meiotic problems, like incomplete synapsis of the homologous chromosomes, or impaired homologous recombination, can cause failure of crossover formation and subsequent chromosome nondisjunction, eventually leading to aneuploid sperm. These meiotic events are therefore strictly monitored by meiotic checkpoints that initiate apoptosis of aberrant spermatocytes and lead to spermatogenic arrest. However, we recently found that, in vitro derived meiotic cells proceeded to the first meiotic division (MI) stage, despite displaying incomplete chromosome synapsis, no discernible XY-body and lack of crossover formation. We therefore optimized our in vitro culture system of meiosis from male germline stem cells (mGSCs) in order to achieve full chromosome synapsis, XY-body formation and meiotic crossovers. In comparison to previous culture system, the in vitro-generated spermatocytes were transferred after meiotic initiation to a second culture dish. This dish already contained a freshly plated monolayer of proliferatively inactivated immortalized Sertoli cells supporting undifferentiated mGSCs. In this way we aimed to simulate the multiple layers of germ cell types that support spermatogenesis in vivo in the testis. We found that in this optimized culture system, although independent of the undifferentiated mGSCs, meiotic chromosome synapsis was complete and XY body appeared normal. However, meiotic recombination still occurred insufficiently and only few meiotic crossovers were formed, leading to MI-spermatocytes displaying univalent chromosomes (paired sister chromatids). Therefore, considering that meiotic checkpoints are not necessarily fully functional in vitro, meiotic crossover formation should be closely monitored when mimicking gametogenesis in vitro to prevent generation of aneuploid gametes.
Asunto(s)
Emparejamiento Cromosómico/fisiología , Cromosomas/fisiología , Meiosis/fisiología , Aneuploidia , Animales , Azoospermia/congénito , Azoospermia/fisiopatología , Diferenciación Celular/fisiología , Línea Celular , Proliferación Celular/fisiología , Masculino , Ratones , Ratones Endogámicos DBA , Células de Sertoli/fisiología , Espermatocitos/fisiología , Espermatogénesis/fisiología , Espermatozoides/fisiología , Testículo/fisiologíaRESUMEN
OBJECTIVES: Histone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood. MATERIALS AND METHODS: HDAC8-targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI-34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ-tubulin dynamics and acetylation level of α-tubulin. RESULTS: We observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ-tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI-34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α-tubulin. CONCLUSIONS: Collectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.
Asunto(s)
Histona Desacetilasas/metabolismo , Meiosis/fisiología , Oocitos/metabolismo , Huso Acromático/metabolismo , Acetilación/efectos de los fármacos , Animales , Fenómenos Biológicos/efectos de los fármacos , Cromosomas/efectos de los fármacos , Cromosomas/metabolismo , Cromosomas/fisiología , Femenino , Ácidos Hidroxámicos/farmacología , Indoles/farmacología , Meiosis/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/fisiología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Huso Acromático/efectos de los fármacos , Huso Acromático/fisiología , Porcinos , Tubulina (Proteína)/metabolismoRESUMEN
Capture Hi-C is widely used to obtain high-resolution profiles of chromosomal interactions involving, at least on one end, regions of interest such as gene promoters. Signal detection in Capture Hi-C data is challenging and cannot be adequately accomplished with tools developed for other chromosome conformation capture methods, including standard Hi-C. Capture Hi-C Analysis of Genomic Organization (CHiCAGO) is a computational pipeline developed specifically for Capture Hi-C analysis. It implements a statistical model accounting for biological and technical background components, as well as bespoke normalization and multiple testing procedures for this data type. Here we provide a step-by-step guide to the CHiCAGO workflow that is aimed at users with basic experience of the command line and R. We also describe more advanced strategies for tuning the key parameters for custom experiments and provide guidance on data preprocessing and downstream analysis using companion tools. In a typical experiment, CHiCAGO takes ~2-3 h to run, although pre- and postprocessing steps may take much longer.
Asunto(s)
Cromosomas/fisiología , Modelos Estadísticos , Programas Informáticos , Cromatina/químicaRESUMEN
In prophase of meiosis I, homologous chromosomes pair and become connected by cross-overs. Chiasmata, the connections formed by cross-overs, enable the chromosome pair, called a bivalent, to attach as a single unit to the spindle. When the meiotic spindle forms in prometaphase, most bivalents are associated with one spindle pole and then go through a series of oscillations on the spindle, attaching to and detaching from microtubules until the partners of the bivalent become bioriented-attached to microtubules from opposite sides of the spindle. The conserved kinase, Mps1, is essential for the bivalents to be pulled by microtubules across the spindle in prometaphase. Here we show that MPS1 is needed for efficient triggering of the migration of microtubule-attached kinetochores toward the poles and promotes microtubule depolymerization. Our data support the model Mps1 acts at the kinetochore to coordinate the successful attachment of a microtubule and the triggering of microtubule depolymerization to then move the chromosome.
Asunto(s)
Cromosomas/fisiología , Prometafase/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Polaridad Celular , Emparejamiento Cromosómico , Cinetocoros/fisiología , Microtúbulos/fisiología , Mutación , Prometafase/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , SaccharomycetalesRESUMEN
Polyploidy is present in many cancer types and is increasingly recognized as an important factor in promoting chromosomal instability, genome evolution, and heterogeneity in cancer cells. However, the mechanisms that trigger polyploidy in cancer cells are largely unknown. In this study, we investigated the origin of polyploidy in esophageal adenocarcinoma (EAC), a highly heterogenous cancer, using a combination of genomics and cell biology approaches in EAC cell lines, organoids, and tumors. We found the EAC cells and organoids present specific mitotic defects consistent with problems in the attachment of chromosomes to the microtubules of the mitotic spindle. Time-lapse analyses confirmed that EAC cells have problems in congressing and aligning their chromosomes, which can ultimately culminate in mitotic slippage and polyploidy. Furthermore, whole-genome sequencing, RNA-seq, and quantitative immunofluorescence analyses revealed alterations in the copy number, expression, and cellular distribution of several proteins known to be involved in the mechanics and regulation of chromosome dynamics during mitosis. Together, these results provide evidence that an imbalance in the amount of proteins implicated in the attachment of chromosomes to spindle microtubules is the molecular mechanism underlying mitotic slippage in EAC. Our findings that the likely origin of polyploidy in EAC is mitotic failure caused by problems in chromosomal attachments not only improves our understanding of cancer evolution and diversification, but may also aid in the classification and treatment of EAC and possibly other highly heterogeneous cancers.
Asunto(s)
Cromosomas/fisiología , Mitosis/fisiología , Poliploidía , Huso Acromático/metabolismo , Adenocarcinoma/genética , Línea Celular Tumoral , Segregación Cromosómica/fisiología , Neoplasias Esofágicas/genética , Humanos , MicrotúbulosRESUMEN
Oocyte production is crucial for sexual reproduction. Recent findings in zebrafish and other established model organisms emphasize that the early steps of oogenesis involve the coordination of simultaneous and tightly sequential processes across cellular compartments and between sister cells. To fully understand the mechanistic framework of these coordinated processes, cellular and morphological analysis in high temporal resolution is required. Here, we provide a protocol for four-dimensional live time-lapse analysis of cultured juvenile zebrafish ovaries. We describe how multiple-stage oocytes can be simultaneously analyzed in single ovaries, and several ovaries can be processed in single experiments. In addition, we detail adequate conditions for quantitative image acquisition. Finally, we demonstrate that using this protocol, we successfully capture rapid meiotic chromosomal movements in early prophase for the first time in zebrafish oocytes, in four dimensions and in vivo. Our protocol expands the use of the zebrafish as a model system to understand germ cell and ovarian development in postembryonic stages.
Asunto(s)
Cromosomas/fisiología , Meiosis/fisiología , Oogénesis/fisiología , Ovario/fisiología , Imagen de Lapso de Tiempo/métodos , Pez Cebra/fisiología , Animales , Femenino , Oocitos , Diferenciación Sexual/fisiologíaRESUMEN
Polar body (PB) formation is an extreme form of unequal cell division that occurs in oocytes due to the eccentric position of the small meiotic spindle near the oocyte cortex. Prior to PB formation, a chromatin-centered process causes the cortex overlying the meiotic chromosomes to become polarized. This polarized cortical subdomain marks the site where a cortical protrusion or outpocket forms at the oocyte surface creating the future PBs. Using ascidians, we observed that PB1 becomes tethered to the fertilized egg via PB2, indicating that the site of PB1 cytokinesis directed the precise site for PB2 emission. We therefore studied whether the midbody remnant left behind following PB1 emission was involved, together with the egg chromatin, in defining the precise cortical site for PB2 emission. During outpocketing of PB2 in ascidians, we discovered that a small structure around 1 µm in diameter protruded from the cortical outpocket that will form the future PB2, which we define as the "polar corps". As emission of PB2 progressed, this small polar corps became localized between PB2 and PB1 and appeared to link PB2 to PB1. We tested the hypothesis that this small polar corps on the surface of the forming PB2 outpocket was the midbody remnant from the previous round of PB1 cytokinesis. We had previously discovered that Plk1::Ven labeled midbody remnants in ascidian embryos. We therefore used Plk1::Ven to follow the dynamics of the PB1 midbody remnant during meiosis II. Plk1::Ven strongly labeled the small polar corps that formed on the surface of the cortical outpocket that created PB2. Following emission of PB2, this polar corps was rich in Plk1::Ven and linked PB2 to PB1. By labelling actin (with TRITC-Phalloidin) we also demonstrated that actin accumulates at the midbody remnant and also forms a cortical cap around the midbody remnant in meiosis II that prefigured the precise site of cortical outpocketing during PB2 emission. Phalloidin staining of actin and immunolabelling of anti-phospho aPKC during meiosis II in fertilized eggs that had PB1 removed suggested that the midbody remnant remained within the fertilized egg following emission of PB1. Dynamic imaging of microtubules labelled with Ens::3GFP, MAP7::GFP or EB3::3GFP showed that one pole of the second meiotic spindle was located near the midbody remnant while the other pole rotated away from the cortex during outpocketing. Finally, we report that failure of the second meiotic spindle to rotate can lead to the formation of two cortical outpockets at anaphase II, one above each set of chromatids. It is not known whether the midbody remnant of PB1 is involved in directing the precise location of PB2 since our data are correlative in ascidians. However, a review of the literature indicates that PB1 is tethered to the egg surface via PB2 in several species including members of the cnidarians, lophotrochozoa and echinoids, suggesting that the midbody remnant formed during PB1 emission may be involved in directing the precise site of PB2 emission throughout the invertebrates.
Asunto(s)
Meiosis/fisiología , Cuerpos Polares/fisiología , Actinas/metabolismo , Animales , Bivalvos/metabolismo , Bivalvos/fisiología , Cromatina/metabolismo , Cromatina/fisiología , Cromosomas/metabolismo , Cromosomas/fisiología , Citocinesis/fisiología , Oocitos/metabolismo , Oocitos/fisiología , Cuerpos Polares/metabolismo , Huso Acromático/metabolismo , Huso Acromático/fisiología , Urocordados/metabolismo , Urocordados/fisiología , Cigoto/metabolismo , Cigoto/fisiologíaRESUMEN
Protein Phosphatase 2A (PP2A) is a heterotrimer composed of scaffolding (A), catalytic (C), and regulatory (B) subunits. PP2A complexes with B56 subunits are targeted by Shugoshin and BUBR1 to protect centromeric cohesion and stabilise kinetochore-microtubule attachments in yeast and mouse meiosis. In Caenorhabditis elegans, the closest BUBR1 orthologue lacks the B56-interaction domain and Shugoshin is not required for meiotic segregation. Therefore, the role of PP2A in C. elegans female meiosis is unknown. We report that PP2A is essential for meiotic spindle assembly and chromosome dynamics during C. elegans female meiosis. BUB-1 is the main chromosome-targeting factor for B56 subunits during prometaphase I. BUB-1 recruits PP2A:B56 to the chromosomes via a newly identified LxxIxE motif in a phosphorylation-dependent manner, and this recruitment is important for proper chromosome congression. Our results highlight a novel mechanism for B56 recruitment, essential for recruiting a pool of PP2A involved in chromosome congression during meiosis I.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , Cromosomas/fisiología , Meiosis/fisiología , Oocitos/fisiología , Proteína Fosfatasa 2/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Segregación Cromosómica , Oocitos/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Connecting the molecular structure and function of chromatin across length and timescales remains a grand challenge to understanding and engineering cellular behaviors. Across five orders of magnitude, dynamic processes constantly reshape chromatin structures, driving spaciotemporal patterns of gene expression and cell fate. Through the interplay of structure and function, the genome operates as a highly dynamic feedback control system. Recent experimental techniques have provided increasingly detailed data that revise and augment the relatively static, hierarchical view of genomic architecture with an understanding of how dynamic processes drive organization. Here, we review how novel technologies from sequencing, imaging, and synthetic biology refine our understanding of chromatin structure and function and enable chromatin engineering. Finally, we discuss opportunities to use these tools to enhance understanding of the dynamic interrelationship of chromatin structure and function.
Asunto(s)
Ensamble y Desensamble de Cromatina/fisiología , Cromatina/fisiología , Cromosomas/genética , Cromosomas/fisiología , Expresión Génica/genética , Regulación de la Expresión Génica/genética , Genoma/genética , Genómica/métodos , Relación Estructura-Actividad , Factores de TiempoRESUMEN
Mechanisms for somatic chromosomal mosaicism (SCM) and chromosomal instability (CIN) are not completely understood. During molecular karyotyping and bioinformatic analyses of children with neurodevelopmental disorders and congenital malformations (n = 612), we observed colocalization of regular chromosomal imbalances or copy number variations (CNV) with mosaic ones (n = 47 or 7.7%). Analyzing molecular karyotyping data and pathways affected by CNV burdens, we proposed a mechanism for SCM/CIN, which had been designated as "chromohelkosis" (from the Greek words chromosome ulceration/open wound). Briefly, structural chromosomal imbalances are likely to cause local instability ("wreckage") at the breakpoints, which results either in partial/whole chromosome loss (e.g., aneuploidy) or elongation of duplicated regions. Accordingly, a function for classical/alpha satellite DNA (protection from the wreckage towards the centromere) has been hypothesized. Since SCM and CIN are ubiquitously involved in development, homeostasis and disease (e.g., prenatal development, cancer, brain diseases, aging), we have metaphorically (ironically) designate the system explaining chromohelkosis contribution to SCM/CIN as the cytogenomic "theory of everything", similar to the homonymous theory in physics inasmuch as it might explain numerous phenomena in chromosome biology. Recognizing possible empirical and theoretical weaknesses of this "theory", we nevertheless believe that studies of chromohelkosis-like processes are required to understand structural variability and flexibility of the genome.
Asunto(s)
Inestabilidad Cromosómica/genética , Cromosomas/genética , Mosaicismo/embriología , Envejecimiento/genética , Aneuploidia , Centrómero/genética , Niño , Inestabilidad Cromosómica/fisiología , Aberraciones Cromosómicas , Trastornos de los Cromosomas/genética , Cromosomas/fisiología , Anomalías Congénitas/genética , Variaciones en el Número de Copia de ADN , Diploidia , Enfermedad/genética , Femenino , Genoma/genética , Humanos , Cariotipificación/métodos , Masculino , Trastornos del Neurodesarrollo/genética , Federación de RusiaRESUMEN
Errors during meiotic resumption in oocytes can result in chromosome missegregation and infertility. Several cell cycle kinases have been linked with roles in coordinating events during meiotic resumption, including polo-like kinases (PLKs). Mammals express four kinase-proficient PLKs (PLK1-4). Previous studies assessing the role of PLK1 have relied on RNA knockdown and kinase inhibition approaches, as Plk1 null mutations are embryonically lethal. To further assess the roles of PLK1 during meiotic resumption, we developed a Plk1 conditional knockout (cKO) mouse to specifically mutate Plk1 in oocytes. Despite normal oocyte numbers and follicle maturation, Plk1 cKO mice were infertile. From analysis of meiotic resumption, Plk1 cKO oocytes underwent nuclear envelope breakdown with the same timing as control oocytes. However, Plk1 cKO oocytes failed to form compact bivalent chromosomes, and localization of cohesin and condensin were defective. Furthermore, Plk1 cKO oocytes either failed to organize α-tubulin or developed an abnormally small bipolar spindle. These abnormalities were attributed to aberrant release of the microtubule organizing center (MTOC) linker protein, C-NAP1, and the failure to recruit MTOC components and liquid-like spindle domain (LISD) factors. Ultimately, these defects result in meiosis I arrest before homologous chromosome segregation.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica/fisiología , Oocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas de Ciclo Celular/fisiología , Cromosomas/metabolismo , Cromosomas/fisiología , Femenino , Meiosis/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Centro Organizador de los Microtúbulos/metabolismo , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/metabolismo , Microtúbulos/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Huso Acromático/metabolismo , Tubulina (Proteína)/metabolismo , Quinasa Tipo Polo 1RESUMEN
Pairing of homologous chromosomes is crucial for ensuring accurate segregation of chromosomes during meiosis. Molecular mechanisms of homologous chromosome pairing in meiosis have been extensively studied in the fission yeast Schizosaccharomyces pombe. In this organism, meiosis-specific noncoding RNA transcribed from specific genes accumulates at the respective gene loci, and chromosome-associated RNA-protein complexes mediate meiotic pairing of homologous loci through phase separation. Pairing of homologous chromosomes also occurs in somatic diploid cells in certain situations. For example, somatic pairing of homologous chromosomes occurs during the early embryogenesis in diptera, and relies on the transcription-associated chromatin architecture. Earlier models also suggest that transcription factories along the chromosome mediate pairing of homologous chromosomes in plants. These studies suggest that RNA bodies formed on chromosomes mediate the pairing of homologous chromosomes. This review summarizes lessons from S. pombe to provide general insights into mechanisms of homologous chromosome pairing mediated by phase separation of chromosome-associated RNA-protein complexes.
Asunto(s)
Emparejamiento Cromosómico , Cromosomas Fúngicos/fisiología , Cromosomas/fisiología , Schizosaccharomyces/genética , Animales , Cromosomas/genética , Cromosomas Fúngicos/genética , ADN/química , ADN/genética , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Meiosis , ARN de Hongos/genética , ARN de Hongos/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Proteínas de Unión al ARN/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Homología de Secuencia de Ácido NucleicoRESUMEN
B chromosomes are extra genomic compounds found in different taxonomic groups, including plants and animals. Obtaining patterns of resolutive chromosomal bands is necessary to understand the nuclear organization, variability and nature of B chromosome chromatin and possible transcriptional regions. In this study, we analyzed 35 Astyanax scabripinnis specimens sampled from Fazenda Lavrinha, a stream in the Paraíba do Sul river basin, Brazil. Through the incorporation of the thymidine analog 5'-bromo-2'-deoxyuridine (5-BrdU) in vivo, it was possible to recognize the replicating regions of the B chromosome at the beginning of the S phase, differentially characterized in relationship to the regions of late replication. In this perspective, it is possible to suggest that the B chromosome of this species possesses a territory and the chromatin accessible for transcription, especially in the light (i.e., early replicating) bands (p1.1; p1.3; and p2.1 and q1.1, q1.3, q2.1, and q2.2). The late-replicating regions are corresponding to the blocks of constitutive heterochromatin. They show a preferential accumulation of satellite DNA As51. By the use of the fluorochrome chromomycin A3 (CMA3), it was possible to identify GC-rich chromosomal regions, corresponding to late-replicating parts of genome, confirming the revealed data by the replication banding and C-banding. In addition, the analysis by confocal microscopy in kidney cells indicates the location of a peripheral anchorage of this chromosome in the nuclear lamina, reinforcing the idea of downregulation of the associated regions.
Asunto(s)
Characidae/genética , Cromosomas/fisiología , Momento de Replicación del ADN , Riñón/fisiología , Transcripción Genética , Animales , Brasil , Cromatina/fisiología , Cromosomas/genética , Interfase , RíosRESUMEN
The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 µm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.
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Cromosomas/fisiología , Huso Acromático/fisiología , Animales , Línea Celular , Cinetocoros , Marsupiales , Tubulina (Proteína)RESUMEN
As common overexpression of Aurora A in various tumours, much attention has focused on its function in inducing cancer, and its value in cancer therapeutics, considerably less is known regarding its role in the first cleavage division of mammalian embryos. Here, we highlight an indispensable role of Aurora A during the first mitotic division progression of pig embryos just after meiosis. The expression and spatiotemporal localization of Aurora A were initially assessed in pig embryos during the first mitotic division by Western blot analysis and indirect immunofluorescent staining. Then, the potential role of Aurora A was further evaluated using a highly selective Aurora A inhibitor, MLN8054, during this mitotic progression in pig embryos. Aurora A was found to express and exhibit a specific dynamic intracellular localization pattern during the first mitotic division in pig embryos. Aurora A was diffused in the cytoplasm at the prophase stage, and then exhibited a dynamic intracellular localization which was tightly associated with the chromosome and spindle dynamics throughout subsequent mitotic phases. Inhibition of Aurora A by MLN8054 treatment led to the failure of the first cleavage, with the majority of embryos being arrested in prophase of the mitotic division. Further subcellular structure examination showed that Aurora A inhibition not only led to the failure of spindle microtubule assembly, but also resulted in severe defects in chromosome condensation, accompanied by an obvious decrease in p-TACC3(S558) expression during the prophase of the first mitosis. Together, these results illustrated that Aurora A is crucial for both spindle assembly and chromosome condensation during the first mitotic division in pig embryos, and that the regulation of Aurora A may be associated with its effects on p-TACC3(S558) expression.
Asunto(s)
Aurora Quinasa A/antagonistas & inhibidores , Benzazepinas/farmacología , Embrión de Mamíferos/efectos de los fármacos , Mitosis/efectos de los fármacos , Animales , Cromosomas/fisiología , Regulación del Desarrollo de la Expresión Génica , Huso Acromático/efectos de los fármacos , PorcinosRESUMEN
Males and females have different optimal values for some traits, such as body size. When the same genes control these traits in both sexes, selection pushes in opposite directions in males and females. Alleles at autosomal loci spend equal amounts of time in males and females, suggesting that the sexually antagonistic selective forces may approximately balance between the opposing optima. Frank and Crespi noted that alleles on the X chromosome spend twice as much time in diploid females as in haploid males. That distinction between the sexes may tend to favor X-linked genes that push more strongly toward the female optimum than the male optimum. The female bias of X-linked genes opposes the intermediate optimum of autosomal genes, potentially creating a difference between the direction of selection on traits favored by X chromosomes and autosomes. Patten has recently argued that explicit genetic assumptions about dominance and the relative magnitude of allelic effects may lead X-linked genes to favor the male rather than the female optimum, contradicting Frank and Crespi. This article combines the insights of those prior analyses into a new, more general theory. We find some parameter combinations for X-linked loci that favor a female bias and other parameter combinations that favor a male bias. We conclude that the X likely contains a mosaic pattern of loci that differ with autosomes over sexually antagonistic traits. The overall tendency for a female or male bias on the X depends on prior assumptions about the distribution of key parameters across X-linked loci. Those parameters include the dominance coefficient and the way in which ploidy influences the magnitude of allelic effects.
Asunto(s)
Cromosomas/fisiología , Conducta Sexual Animal , Animales , Cromosomas/genética , Femenino , Masculino , Modelos Genéticos , Cromosoma X/genética , Cromosoma X/fisiologíaRESUMEN
A series of well-orchestrated events help in the chromatin condensation and the formation of chromosomes. Apart from the formation of chromosomes, maintenance of their structure is important, especially for the cell division. The structural maintenance of chromosome (SMC) proteins, the non-SMC proteins and the SMC complexes are critical for the maintenance of chromosome structure. While condensins have roles for the DNA compaction, organization, and segregation, the cohesin functions in a cyclic manner through the cell cycle, as a "cohesin cycle." Specific mechanisms maintain the architecture of the centromere, the kinetochore and the telomeres which are in tandem with the cell cycle checkpoints. The presence of chromosomal territories and compactness differences through the length of the chromosomes might have implications on selective susceptibility of specific chromosomes for induced genotoxicity.
Asunto(s)
Ciclo Celular/fisiología , Cromatina/fisiología , Cromosomas/fisiología , Animales , Proteínas de Ciclo Celular/fisiología , Regulación de la Expresión Génica , HumanosRESUMEN
Aurora kinases create phosphorylation gradients within the spindle during prometaphase and anaphase, thereby locally regulating factors that promote spindle organization, chromosome condensation and movement, and cytokinesis. We show that one such factor is the kinesin KIF4A, which is present along the chromosome axes throughout mitosis and the central spindle in anaphase. These two pools of KIF4A depend on condensin I and PRC1, respectively. Previous work has shown KIF4A is activated by Aurora B at the anaphase central spindle. However, whether or not chromosome-associated KIF4A bound to condensin I is regulated by Aurora kinases remain unclear. To determine the roles of the two different pools of KIF4A, we generated specific point mutants that are unable to interact with either condensin I or PRC1 or are deficient for Aurora kinase regulation. By analyzing these mutants, we show that Aurora A phosphorylates the condensin I-dependent pool of KIF4A and thus actively promotes chromosome congression from the spindle poles to the metaphase plate.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Aurora Quinasa A/metabolismo , Segregación Cromosómica/fisiología , Cromosomas/metabolismo , Cromosomas/fisiología , Proteínas de Unión al ADN/metabolismo , Cinesinas/metabolismo , Complejos Multiproteicos/metabolismo , Anafase/fisiología , Línea Celular , Línea Celular Tumoral , Posicionamiento de Cromosoma/fisiología , Células HEK293 , Células HeLa , Humanos , Microtúbulos/metabolismo , Mitosis/fisiología , Fosforilación/fisiología , Huso Acromático/metabolismo , Huso Acromático/fisiologíaRESUMEN
Both the pericentromere and the nucleolus have unique characteristics that distinguish them amongst the rest of genome. Looping of pericentromeric DNA, due to structural maintenance of chromosome (SMC) proteins condensin and cohesin, drives its ability to maintain tension during metaphase. Similar loops are formed via condensin and cohesin in nucleolar ribosomal DNA (rDNA). Condensin and cohesin are also concentrated in transfer RNA (tRNA) genes, genes which may be located within the pericentromere as well as tethered to the nucleolus. Replication fork stalling, as well as downstream consequences such as genomic recombination, are characteristic of both the pericentromere and rDNA. Furthermore, emerging evidence suggests that the pericentromere may function as a liquid-liquid phase separated domain, similar to the nucleolus. We therefore propose that the pericentromere and nucleolus, in part due to their enrichment of SMC proteins and others, contain similar domains that drive important cellular activities such as segregation, stability, and repair.