RESUMEN
Advances in light-sheet and confocal microscopy now allow imaging of cleared large biological tissue samples and enable the 3D appreciation of cell and protein localization in their native organ environment. However, the sample preparations for such imaging are often onerous, and their capability for antigen detection is limited. Here, we describe FLASH (fast light-microscopic analysis of antibody-stained whole organs), a simple, rapid, fully customizable technique for molecular phenotyping of intact tissue volumes. FLASH utilizes non-degradative epitope recovery and membrane solubilization to enable the detection of a multitude of membranous, cytoplasmic and nuclear antigens in whole mouse organs and embryos, human biopsies, organoids and Drosophila. Retrieval and immunolabeling of epithelial markers, an obstacle for previous clearing techniques, can be achieved with FLASH. Upon volumetric imaging, FLASH-processed samples preserve their architecture and integrity and can be paraffin-embedded for subsequent histopathological analysis. The technique can be performed by scientists trained in light microscopy and yields results in <1 week.
Asunto(s)
Antígenos/análisis , Técnica del Anticuerpo Fluorescente/métodos , Imagenología Tridimensional/métodos , Microscopía Confocal/métodos , Animales , Drosophila , Epítopos/análisis , Femenino , Humanos , Riñón/ultraestructura , Aparato Lagrimal/ultraestructura , Hígado/ultraestructura , Pulmón/ultraestructura , Masculino , Glándulas Mamarias Humanas/ultraestructura , Ratones , Organoides/ultraestructura , Páncreas/ultraestructura , Estómago/ultraestructuraRESUMEN
Determining the site of cell cleavage is crucial for cytokinesis and involves precise activation of the RhoGEF ECT2. A new study demonstrates how a non-canonical interaction of ECT2 with centralspindlin underlies cytokinesis in animal cells, solving a mechanistic conundrum.
Asunto(s)
Citocinesis , Fosfopéptidos , Animales , División Celular , Células HeLa , Humanos , Proteínas Proto-Oncogénicas/metabolismo , Proteína de Unión al GTP rhoARESUMEN
Cohesin is essential for genome folding and inheritance. In somatic cells, these functions are both mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is expressed but neither required nor sufficient for cohesion, and its function remains unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Here, we show that Wapl is required for accurate meiosis I chromosome segregation, predominantly releases Scc1-cohesin from chromosomes, and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromosome organization in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in somatic cells, does not increase loop size. We conclude that distinct cohesin complexes generate loops and cohesion in oocytes and propose that the same principle applies to all cell types and species.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Proteínas de Unión al ADN/metabolismo , Oocitos/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , CohesinasRESUMEN
In mammalian cell lines, the endosomal sorting complex required for transport (ESCRT)-III mediates abscission, the process that physically separates daughter cells and completes cell division. Cep55 protein is regarded as the master regulator of abscission, because it recruits ESCRT-III to the midbody (MB), the site of abscission. However, the importance of this mechanism in a mammalian organism has never been tested. Here we show that Cep55 is dispensable for mouse embryonic development and adult tissue homeostasis. Cep55-knockout offspring show microcephaly and primary neural progenitors require Cep55 and ESCRT for survival and abscission. However, Cep55 is dispensable for cell division in embryonic or adult tissues. In vitro, division of primary fibroblasts occurs without Cep55 and ESCRT-III at the midbody and is not affected by ESCRT depletion. Our work defines Cep55 as an abscission regulator only in specific tissue contexts and necessitates the re-evaluation of an alternative ESCRT-independent cell division mechanism.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Citocinesis , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Animales , Animales Recién Nacidos , Apoptosis , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Células Cultivadas , Corteza Cerebral/anomalías , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Genotipo , Riñón/anomalías , Ratones Endogámicos C57BL , Ratones Noqueados , Microcefalia/patología , MitosisRESUMEN
Mammalian genomes are spatially organized by CCCTC-binding factor (CTCF) and cohesin into chromatin loops and topologically associated domains, which have important roles in gene regulation and recombination. By binding to specific sequences, CTCF defines contact points for cohesin-mediated long-range chromosomal cis-interactions. Cohesin is also present at these sites, but has been proposed to be loaded onto DNA elsewhere and to extrude chromatin loops until it encounters CTCF bound to DNA. How cohesin is recruited to CTCF sites, according to this or other models, is unknown. Here we show that the distribution of cohesin in the mouse genome depends on transcription, CTCF and the cohesin release factor Wings apart-like (Wapl). In CTCF-depleted fibroblasts, cohesin cannot be properly recruited to CTCF sites but instead accumulates at transcription start sites of active genes, where the cohesin-loading complex is located. In the absence of both CTCF and Wapl, cohesin accumulates in up to 70 kilobase-long regions at 3'-ends of active genes, in particular if these converge on each other. Changing gene expression modulates the position of these 'cohesin islands'. These findings indicate that transcription can relocate mammalian cohesin over long distances on DNA, as previously reported for yeast cohesin, that this translocation contributes to positioning cohesin at CTCF sites, and that active genes can be freed from cohesin either by transcription-mediated translocation or by Wapl-mediated release.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas de los Mamíferos/metabolismo , Genoma/genética , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética/genética , Animales , Sitios de Unión , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/genética , Células Cultivadas , Proteoglicanos Tipo Condroitín Sulfato/deficiencia , Proteoglicanos Tipo Condroitín Sulfato/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/deficiencia , Proteínas Cromosómicas no Histona/genética , Cromosomas de los Mamíferos/genética , ADN/genética , ADN/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Masculino , Ratones , Transporte de Proteínas , Proteínas/genética , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Sitio de Iniciación de la Transcripción , CohesinasRESUMEN
Mammalian genomes contain several billion base pairs of DNA that are packaged in chromatin fibres. At selected gene loci, cohesin complexes have been proposed to arrange these fibres into higher-order structures, but how important this function is for determining overall chromosome architecture and how the process is regulated are not well understood. Using conditional mutagenesis in the mouse, here we show that depletion of the cohesin-associated protein Wapl stably locks cohesin on DNA, leads to clustering of cohesin in axial structures, and causes chromatin condensation in interphase chromosomes. These findings reveal that the stability of cohesin-DNA interactions is an important determinant of chromatin structure, and indicate that cohesin has an architectural role in interphase chromosome territories. Furthermore, we show that regulation of cohesin-DNA interactions by Wapl is important for embryonic development, expression of genes such as c-myc (also known as Myc), and cell cycle progression. In mitosis, Wapl-mediated release of cohesin from DNA is essential for proper chromosome segregation and protects cohesin from cleavage by the protease separase, thus enabling mitotic exit in the presence of functional cohesin complexes.
Asunto(s)
Cromatina/química , Cromatina/metabolismo , Segregación Cromosómica , Proteínas/metabolismo , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cromatina/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Cromosomas de los Mamíferos/química , Cromosomas de los Mamíferos/genética , Cromosomas de los Mamíferos/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Endopeptidasas/metabolismo , Regulación de la Expresión Génica/genética , Genes myc/genética , Interfase , Ratones , Mitosis , Profase , Proteínas/genética , Separasa , CohesinasRESUMEN
Cohesin is a chromosome-associated multisubunit protein complex that is highly conserved in eukaryotes and has close homologs in bacteria. Cohesin mediates cohesion between replicated sister chromatids and is therefore essential for chromosome segregation in dividing cells. Cohesin is also required for efficient repair of damaged DNA and has important functions in regulating gene expression in both proliferating and post-mitotic cells. Here we discuss how cohesin associates with DNA, how these interactions are controlled during the cell cycle; how binding of cohesin to DNA may mediate sister chromatid cohesion, DNA repair, and gene regulation; and how defects in these processes can lead to human disease.
Asunto(s)
Proteínas de Ciclo Celular/fisiología , Cromátides/fisiología , Proteínas Cromosómicas no Histona/fisiología , Segregación Cromosómica/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , CohesinasRESUMEN
The GTPase RAN has an established role in spindle assembly and in mitotic progression, although not all mechanisms are fully understood in somatic cells. Here, we have downregulated RAN-binding protein 1 (RANBP1), a RAN partner that has highest abundance in G2 and mitosis, in human cells. RANBP1-depleted cells underwent prolonged prometaphase delay often followed by apoptosis. Cells that remained viable assembled morphologically normal spindles; these spindles, however, were hyperstable and failed to recruit cyclin B1 or to restrict the localization of HURP (DLG7), a microtubule-stabilizing factor, to plus-ends. RANBP1 depletion did not increase the frequency of unattached chromosomes; however, RANBP1-depleted cells frequently showed lagging chromosomes in anaphase, suggesting that merotelic attachments form and are not efficiently resolved. These data indicate that RANBP1 activity is required for the proper localization of specific factors that regulate microtubule function; loss of this activity contributes to the generation of aneuploidy in a microtubule-dependent manner.