RESUMEN
Plant protoplasts provide starting material for of inducing pluripotent cell masses that are competent for tissue regeneration in vitro, analogous to animal induced pluripotent stem cells (iPSCs). Dedifferentiation is associated with large-scale chromatin reorganisation and massive transcriptome reprogramming, characterised by stochastic gene expression. How this cellular variability reflects on chromatin organisation in individual cells and what factors influence chromatin transitions during culturing are largely unknown. Here, we used high-throughput imaging and a custom supervised image analysis protocol extracting over 100 chromatin features of cultured protoplasts. The analysis revealed rapid, multiscale dynamics of chromatin patterns with a trajectory that strongly depended on nutrient availability. Decreased abundance in H1 (linker histones) is hallmark of chromatin transitions. We measured a high heterogeneity of chromatin patterns indicating intrinsic entropy as a hallmark of the initial cultures. We further measured an entropy decline over time, and an antagonistic influence by external and intrinsic factors, such as phytohormones and epigenetic modifiers, respectively. Collectively, our study benchmarks an approach to understand the variability and evolution of chromatin patterns underlying plant cell reprogramming in vitro.
Asunto(s)
Cromatina , Entropía , Células Madre Pluripotentes Inducidas , Cromatina/metabolismo , Cromatina/genética , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Protoplastos/metabolismo , Reprogramación Celular/genética , Histonas/metabolismo , Histonas/genética , Células Vegetales/metabolismo , Epigénesis GenéticaRESUMEN
Caspases are key components of apoptotic pathways. Regulation of caspases occurs at several levels, including transcription, proteolytic processing, inhibition of enzymatic function, and protein degradation. In contrast, little is known about the extent of post-transcriptional control of caspases. Here, we describe four conserved RNA-binding proteins (RBPs)-PUF-8, MEX-3, GLD-1, and CGH-1-that sequentially repress the CED-3 caspase in distinct regions of the Caenorhabditis elegans germline. We demonstrate that GLD-1 represses ced-3 mRNA translation via two binding sites in its 3' untranslated region (UTR), thereby ensuring a dual control of unwanted cell death: at the level of p53/CEP-1 and at the executioner caspase level. Moreover, we identified seven RBPs that regulate human caspase-3 expression and/or activation, including human PUF-8, GLD-1, and CGH-1 homologs PUM1, QKI, and DDX6. Given the presence of unusually long executioner caspase 3' UTRs in many metazoans, translational control of executioner caspases by RBPs might be a strategy used widely across the animal kingdom to control apoptosis.
Asunto(s)
Apoptosis/genética , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Caspasas/genética , Caspasas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Animales , Sitios de Unión , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/citología , Células HeLa , Humanos , Procesamiento Postranscripcional del ARNRESUMEN
High-content imaging using automated microscopy and computer vision allows multivariate profiling of single-cell phenotypes. Here, we present methods for the application of the CISPR-Cas9 system in large-scale, image-based, gene perturbation experiments. We show that CRISPR-Cas9-mediated gene perturbation can be achieved in human tissue culture cells in a timeframe that is compatible with image-based phenotyping. We developed a pipeline to construct a large-scale arrayed library of 2,281 sequence-verified CRISPR-Cas9 targeting plasmids and profiled this library for genes affecting cellular morphology and the subcellular localization of components of the nuclear pore complex (NPC). We conceived a machine-learning method that harnesses genetic heterogeneity to score gene perturbations and identify phenotypically perturbed cells for in-depth characterization of gene perturbation effects. This approach enables genome-scale image-based multivariate gene perturbation profiling using CRISPR-Cas9.
Asunto(s)
Sistemas CRISPR-Cas , Biblioteca de Genes , Poro Nuclear/genética , Análisis de la Célula Individual/métodos , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Aprendizaje Automático , FenotipoRESUMEN
At the onset of mitosis, cells need to break down their nuclear envelope, form a bipolar spindle and attach the chromosomes to microtubules via kinetochores. Previous studies have shown that spindle bipolarization can occur either before or after nuclear envelope breakdown. In the latter case, early kinetochore-microtubule attachments generate pushing forces that accelerate centrosome separation. However, until now, the physiological relevance of this prometaphase kinetochore pushing force was unknown. We investigated the depletion phenotype of the kinetochore protein CENP-L, which we find to be essential for the stability of kinetochore microtubules, for a homogenous poleward microtubule flux rate and for the kinetochore pushing force. Loss of this force in prometaphase not only delays centrosome separation by 5-6 minutes, it also causes massive chromosome alignment and segregation defects due to the formation of syntelic and merotelic kinetochore-microtubule attachments. By contrast, CENP-L depletion has no impact on mitotic progression in cells that have already separated their centrosomes at nuclear envelope breakdown. We propose that the kinetochore pushing force is an essential safety mechanism that favors amphitelic attachments by ensuring that spindle bipolarization occurs before the formation of the majority of kinetochore-microtubule attachments.
Asunto(s)
Centrosoma/metabolismo , Segregación Cromosómica , Cinetocoros/metabolismo , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Membrana Nuclear/metabolismo , Prometafase , Huso Acromático/metabolismoRESUMEN
The liver has the remarkable capacity to regenerate. In the clinic, regeneration is induced by portal vein embolization, which redirects portal blood flow, resulting in liver hypertrophy in locations with increased blood supply, and atrophy of embolized segments. Here, we apply single-cell and single-nucleus transcriptomics on healthy, hypertrophied, and atrophied patient-derived liver samples to explore cell states in the regenerating liver. Our data unveils pervasive upregulation of genes associated with developmental processes, cellular adhesion, and inflammation in post-portal vein embolization liver, disrupted portal-central hepatocyte zonation, and altered cell subtype composition of endothelial and immune cells. Interlineage crosstalk analysis reveals mesenchymal cells as an interaction hub between immune and endothelial cells, and highlights the importance of extracellular matrix proteins in liver regeneration. Moreover, we establish tissue-scale iterative indirect immunofluorescence imaging for high-dimensional spatial analysis of perivascular microenvironments, uncovering changes to tissue architecture in regenerating liver lobules. Altogether, our data is a rich resource revealing cellular and histological changes in human liver regeneration.
Asunto(s)
Embolización Terapéutica , Regeneración Hepática , Hígado , Vena Porta , Humanos , Regeneración Hepática/fisiología , Embolización Terapéutica/métodos , Hepatocitos/metabolismo , Análisis de la Célula Individual , Transcriptoma , Masculino , Células Endoteliales/metabolismo , Femenino , Hipertrofia , Persona de Mediana EdadRESUMEN
The formation and orientation of the mitotic spindle is a critical feature of mitosis. The morphology of the cell and the spatial distribution and composition of the cells' adhesive microenvironment all contribute to dictate the position of the spindle. However, the impact of the dimensionality of the cells' microenvironment has rarely been studied. In this study we present the use of a microwell platform, where the internal surfaces of the individual wells are coated with fibronectin, enabling the three-dimensional presentation of adhesive ligands to single cells cultured within the microwells. This platform was used to assess the effect of dimensionality and cell shape in a controlled microenvironment. Single HeLa cells cultured in circular microwells exhibited greater tilting of the mitotic spindle, in comparison to cells cultured in square microwells. This correlated with an increase in the time required to align the chromosomes at the metaphase plate due to prolonged activation of the spindle checkpoint in an actin dependent process. The comparison to 2D square patterns revealed that the dimensionality of cell adhesions alone affected both mitotic timings and spindle orientation; in particular the role of actin varied according to the dimensionality of the cells' microenvironment. Together, our data revealed that cell shape and the dimensionality of the cells' adhesive environment impacted on both the orientation of the mitotic spindle and progression through mitosis.
Asunto(s)
Forma de la Célula , Mitosis , Huso Acromático , Citoesqueleto de Actina/fisiología , Células HeLa , HumanosRESUMEN
Coordination of cell cycle events in space and time is crucial to achieve a successful cell division. Here, we demonstrate that UBXN-2, a substrate adaptor of the AAA ATPase Cdc48/p97, is required to coordinate centrosome maturation timing with mitosis. In UBXN-2-depleted Caenorhabditis elegans embryos, centrosomes recruited more AIR-1 (Aurora A), matured precociously, and alignment of the mitotic spindle with the axis of polarity was impaired. UBXN-2 and CDC-48 coimmunoprecipitated with AIR-1 and the spindle alignment defect was partially rescued by co-depleting AIR-1, indicating that UBXN-2 controls these processes via AIR-1. Similarly, depletion in human cells of the UBXN-2 orthologues p37/p47 resulted in an accumulation of Aurora A at centrosomes and a delay in centrosome separation. The latter defect was also rescued by inhibiting Aurora A. We therefore postulate that the role of this adaptor in cell cycle regulation is conserved.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Centrosoma/metabolismo , Mitosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Aurora Quinasa A , Aurora Quinasas , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Ciclo Celular , Línea Celular , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Interferencia de ARN , Proteína que Contiene ValosinaRESUMEN
Chromosome segregation in metazoans requires the alignment of sister kinetochores on the metaphase plate. During chromosome alignment, bioriented kinetochores move chromosomes by regulating the plus-end dynamics of the attached microtubules. The bundles of kinetochore-bound microtubules alternate between growth and shrinkage, leading to regular oscillations along the spindle axis. However, the molecular mechanisms that coordinate microtubule plus-end dynamics remain unknown. Here we show that centromere protein (CENP)-H, a subunit of the CENP-A nucleosome-associated and CENP-A distal complexes (CENP-A NAC/CAD), is essential for this coordination, because kinetochores lacking CENP-H establish bioriented attachments but fail to generate regular oscillations, as a result of an uncontrolled rate of microtubule plus-end turnover. These alterations lead to rapid erratic movements that disrupt metaphase plate organization. We also show that the abundance of the CENP-A NAC/CAD subunits CENP-H and CENP-I dynamically change on individual sister kinetochores in vivo, because they preferentially bind the sister kinetochore attached to growing microtubules, and that one other subunit, CENP-Q, binds microtubules in vitro. We therefore propose that CENP-A NAC/CAD is a direct regulator of kinetochore-microtubule dynamics, which physically links centromeric DNA to microtubule plus ends.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Segregación Cromosómica , Cromosomas Humanos/metabolismo , Cinetocoros/metabolismo , Metafase , Microtúbulos/metabolismo , Transducción de Señal , Autoantígenos/metabolismo , Técnicas Biosensibles , Proteínas de Ciclo Celular/genética , Proteína A Centromérica , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Células HeLa , Humanos , Metafase/genética , Microscopía Confocal , Microtúbulos/genética , Unión Proteica , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/genética , Factores de Tiempo , Transfección , Tubulina (Proteína)/metabolismoRESUMEN
Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.
Asunto(s)
Alcanos/metabolismo , Membrana Celular/enzimología , Sistema Enzimático del Citocromo P-450/metabolismo , Bacterias Grampositivas/enzimología , Oxigenasas de Función Mixta/metabolismo , Proteobacteria/enzimología , Composición de Base , Biodegradación Ambiental , Citocromo P-450 CYP4A , Sistema Enzimático del Citocromo P-450/genética , Bacterias Grampositivas/genética , Bacterias Grampositivas/metabolismo , Oxigenasas de Función Mixta/genética , Datos de Secuencia Molecular , Proteobacteria/genética , Proteobacteria/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
A number of oxygenated monoterpenes present at low concentrations in plant oils have anticarcinogenic properties. One of the most promising compounds in this respect is (-)-perillyl alcohol. Since this natural product is present only at low levels in a few plant oils, an alternative, synthetic source is desirable. Screening of 1,800 bacterial strains showed that many alkane degraders were able to specifically hydroxylate l-limonene in the 7 position to produce enantiopure (-)-perillyl alcohol. The oxygenase responsible for this was purified from the best-performing wild-type strain, Mycobacterium sp. strain HXN-1500. By using N-terminal sequence information, a 6.2-kb ApaI fragment was cloned, which encoded a cytochrome P450, a ferredoxin, and a ferredoxin reductase. The three genes were successfully coexpressed in Pseudomonas putida by using the broad-host-range vector pCom8, and the recombinant converted limonene to perillyl alcohol with a specific activity of 3 U/g (dry weight) of cells. The construct was subsequently used in a 2-liter bioreactor to produce perillyl alcohol on a scale of several grams.