RESUMEN
Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.
Asunto(s)
Cinetocoros , Huso Acromático , Animales , Segregación Cromosómica , Mamíferos/genética , Microtúbulos , Mitosis , Huso Acromático/genéticaRESUMEN
Microtubule attachments to kinetochores cause their deformation - a murky phenomenon known as intra-kinetochore stretching. A new study proposes that intra-kinetochore stretching is independent of microtubule-pulling forces and mediates efficient spindle assembly checkpoint silencing to prevent chromosomal instability.
Asunto(s)
Inestabilidad Cromosómica , HumanosRESUMEN
According to the prevailing 'clock' model, chromosome decondensation and nuclear envelope reformation when cells exit mitosis are byproducts of Cdk1 inactivation at the metaphase-anaphase transition, controlled by the spindle assembly checkpoint. However, mitotic exit was recently shown to be a function of chromosome separation during anaphase, assisted by a midzone Aurora B phosphorylation gradient - the 'ruler' model. Here we found that Cdk1 remains active during anaphase due to ongoing APC/CCdc20- and APC/CCdh1-mediated degradation of B-type Cyclins in Drosophila and human cells. Failure to degrade B-type Cyclins during anaphase prevented mitotic exit in a Cdk1-dependent manner. Cyclin B1-Cdk1 localized at the spindle midzone in an Aurora B-dependent manner, with incompletely separated chromosomes showing the highest Cdk1 activity. Slowing down anaphase chromosome motion delayed Cyclin B1 degradation and mitotic exit in an Aurora B-dependent manner. Thus, a crosstalk between molecular 'rulers' and 'clocks' licenses mitotic exit only after proper chromosome separation.
Asunto(s)
Anafase , Aurora Quinasa B/metabolismo , Proteína Quinasa CDC2/metabolismo , Ciclina B1/metabolismo , Proteínas de Drosophila/metabolismo , Animales , Línea Celular , Drosophila , Humanos , Proteolisis , Análisis Espacio-TemporalRESUMEN
An egg must be fertilized by a single sperm only. To prevent polyspermy, the zona pellucida, a structure that surrounds mammalian eggs, becomes impermeable upon fertilization, preventing the entry of further sperm. The structural changes in the zona upon fertilization are driven by the exocytosis of cortical granules. These translocate from the oocyte's centre to the plasma membrane during meiosis. However, very little is known about the mechanism of cortical granule translocation. Here we investigate cortical granule transport and dynamics in live mammalian oocytes by using Rab27a as a marker. We show that two separate mechanisms drive their transport: myosin Va-dependent movement along actin filaments, and an unexpected vesicle hitchhiking mechanism by which cortical granules bind to Rab11a vesicles powered by myosin Vb. Inhibiting cortical granule translocation severely impaired the block to sperm entry, suggesting that translocation defects could contribute to miscarriages that are caused by polyspermy.
Asunto(s)
Gránulos Citoplasmáticos/fisiología , Fertilización , Oocitos/fisiología , Espermatozoides/fisiología , Animales , Biomarcadores , Femenino , Fertilización In Vitro , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Masculino , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Interacciones Espermatozoide-Óvulo , Proteínas rab27 de Unión a GTP/genética , Proteínas rab27 de Unión a GTP/metabolismoRESUMEN
Histone deacetylases are important targets for cancer therapeutics, but their regulation is poorly understood. Our data show coordinated transcription of HDAC1 and HDAC2 in lung cancer cell lines, but suggest HDAC2 protein expression is cell-context specific. Through an unbiased siRNA screen we found that BRCA1-associated protein 1 (BAP1) regulates their expression, with HDAC2 reduced and HDAC1 increased in BAP1 depleted cells. BAP1 loss-of-function is increasingly reported in cancers including thoracic malignancies, with frequent mutation in malignant pleural mesothelioma. Endogenous HDAC2 directly correlates with BAP1 across a panel of lung cancer cell lines, and is downregulated in mesothelioma cell lines with genetic BAP1 inactivation. We find that BAP1 regulates HDAC2 by increasing transcript abundance, rather than opposing its ubiquitylation. Importantly, although total cellular HDAC activity is unaffected by transient depletion of HDAC2 or of BAP1 due to HDAC1 compensation, this isoenzyme imbalance sensitizes MSTO-211H cells to HDAC inhibitors. However, other established mesothelioma cell lines with low endogenous HDAC2 have adapted to become more resistant to HDAC inhibition. Our work establishes a mechanism by which BAP1 loss alters sensitivity of cancer cells to HDAC inhibitors. Assessment of BAP1 and HDAC expression may ultimately help identify patients likely to respond to HDAC inhibitors.
Asunto(s)
Histona Desacetilasa 1/biosíntesis , Inhibidores de Histona Desacetilasas/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/enzimología , Mesotelioma/tratamiento farmacológico , Mesotelioma/enzimología , Proteínas Supresoras de Tumor/deficiencia , Ubiquitina Tiolesterasa/deficiencia , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Histona Desacetilasa 2/biosíntesis , Histona Desacetilasa 2/genética , Humanos , Neoplasias Pulmonares/patología , Mesotelioma/patología , Mesotelioma Maligno , Transcripción Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Electron microscopy (EM) has dominated high-resolution cellular imaging for over 50 years, thanks to its ability to resolve on nanometer-scale intracellular structures such as the microtubules of the mitotic spindle. It is advantageous to view the cell of interest prior to processing the sample for EM. Correlative light-electron microscopy (CLEM) is a technique that allows one to visualize cells of interest by light microscopy (LM) before being transferred to EM for ultrastructural examination. Here, we describe how CLEM can be applied as an effective tool to study the spindle apparatus of mitotic cells. This approach allows transfected cells of interest, in desirable stages of mitosis, to be followed from LM to EM. CLEM has often been considered as a technically challenging and laborious technique. In this chapter, we provide step-by-step pictorial guides that allow successful CLEM to be achieved. In addition, we explain how it is possible to vary the sectioning plane, allowing spindles and microtubules to be analyzed from different angles, and the outputs that can be obtained from these methods when applied to the study of kinetochore fiber ultrastructure.
Asunto(s)
Cinetocoros/ultraestructura , Microscopía Electrónica/métodos , Microtúbulos/ultraestructura , Huso Acromático/ultraestructura , Línea Celular Tumoral , Células HeLa , Humanos , MitosisRESUMEN
Microtubule-associated proteins of the mitotic spindle are thought to be important for the initial assembly and the maintenance of spindle structure and function. However, distinguishing assembly and maintenance roles for a given protein is difficult. Most experimental methods for protein inactivation are slow and therefore affect both assembly and maintenance. Here, we have used 'knocksideways' to rapidly (â¼5 minutes) and specifically remove TACC3-ch-TOG-clathrin non-motor complexes from kinetochore fibers (K-fibers). This method allows the complex to be inactivated at defined stages of mitosis. Removal of TACC3-ch-TOG-clathrin after nuclear envelope breakdown caused severe delays in chromosome alignment. Inactivation at metaphase, following a normal prometaphase, significantly delayed progression to anaphase. In these cells, K-fiber tension was reduced and the spindle checkpoint was not satisfied. Surprisingly, there was no significant loss of K-fiber microtubules, even after prolonged removal. TACC3-ch-TOG-clathrin removal during metaphase also resulted in a decrease in spindle length and significant alteration in kinetochore dynamics. Our results indicate that TACC3-ch-TOG-clathrin complexes are important for the maintenance of spindle structure and function as well as for initial spindle assembly.
Asunto(s)
Cromosomas Humanos/metabolismo , Cinetocoros/metabolismo , Metafase/fisiología , Microtúbulos/metabolismo , Complejos Multiproteicos/metabolismo , Huso Acromático/metabolismo , Cromosomas Humanos/genética , Clatrina/genética , Clatrina/metabolismo , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/genética , Complejos Multiproteicos/genética , Huso Acromático/genéticaRESUMEN
Accurate chromosome segregation during mitosis is achieved by the kinetochore fibers (K-fibers) of the spindle apparatus. These fibers are bundles of microtubules (MTs) connected by non-motor bridges. We recently identified a TACC3/ch-TOG/clathrin complex that constitutes the shortest class of inter-MT bridge in K-fibers. TACC3 anchors the complex to MTs and this is dependent on phosphorylation by Aurora A kinase. Here we show that inhibition of Aurora A kinase using MLN8237 results in (1) loss of clathrin and TACC3 from spindles, (2) destabilization of K-fibers and (3) loss of inter-MT bridges. These results are similar to those in cells depleted of clathrin or TACC3; suggesting that TACC3/ch-TOG/clathrin bridges are the major class of bridge that is regulated by this kinase.