RESUMEN
Various types of DNA viruses are known to elicit the formation of a large nuclear viral replication compartment and marginalization of the cell chromatin. We used three-dimensional soft x-ray tomography, confocal and electron microscopy, combined with numerical modelling of capsid diffusion to analyse the molecular organization of chromatin in herpes simplex virus 1 infection and its effect on the transport of progeny viral capsids to the nuclear envelope. Our data showed that the formation of the viral replication compartment at late infection resulted in the enrichment of heterochromatin in the nuclear periphery accompanied by the compaction of chromatin. Random walk modelling of herpes simplex virus 1-sized particles in a three-dimensional soft x-ray tomography reconstruction of an infected cell nucleus demonstrated that the peripheral, compacted chromatin restricts viral capsid diffusion, but due to interchromatin channels capsids are able to reach the nuclear envelope, the site of their nuclear egress.
Asunto(s)
Cromatina/genética , Cromatina/metabolismo , Fenómenos Fisiológicos de los Virus , Liberación del Virus , Animales , Transporte Biológico , Biomarcadores , Línea Celular , Núcleo Celular , Femenino , Histonas/metabolismo , Ratones , Microscopía ConfocalRESUMEN
The realization that nuclear distribution of DNA, RNA, and proteins differs between cell types and developmental stages suggests that nuclear organization serves regulatory functions. Understanding the logic of nuclear architecture and how it contributes to differentiation and cell fate commitment remains challenging. Here, we use soft X-ray tomography (SXT) to image chromatin organization, distribution, and biophysical properties during neurogenesis in vivo. Our analyses reveal that chromatin with similar biophysical properties forms an elaborate connected network throughout the entire nucleus. Although this interconnectivity is present in every developmental stage, differentiation proceeds with concomitant increase in chromatin compaction and re-distribution of condensed chromatin toward the nuclear core. HP1ß, but not nucleosome spacing or phasing, regulates chromatin rearrangements because it governs both the compaction of chromatin and its interactions with the nuclear envelope. Our experiments introduce SXT as a powerful imaging technology for nuclear architecture.
Asunto(s)
Cromatina/metabolismo , Neurogénesis , Tomografía por Rayos X , Animales , Diferenciación Celular , Nucléolo Celular/metabolismo , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/metabolismo , Células Epiteliales/metabolismo , Heterocromatina/metabolismo , Imagenología Tridimensional , Ratones Noqueados , Neuronas/citología , Neuronas/metabolismo , Bulbo Olfatorio/citologíaRESUMEN
Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.
Asunto(s)
Linfocitos B/virología , Cromatina/virología , Herpesvirus Humano 1/fisiología , Virión/fisiología , Animales , Linfocitos B/metabolismo , Linfocitos B/ultraestructura , Línea Celular , Cromatina/metabolismo , Cromatina/ultraestructura , Herpesvirus Humano 1/genética , Interacciones Huésped-Patógeno , Humanos , Ratones , Microscopía Confocal , Microscopía Electrónica de Transmisión , Imagen de Lapso de Tiempo/métodos , Tomografía por Rayos X , Virión/genética , Replicación Viral/genéticaRESUMEN
We report here a multipurpose dynamic-interface-based segmentation tool, suitable for segmenting planar, cylindrical, and spherical surfaces in 3D. The method is fast enough to be used conveniently even for large images. Its implementation is straightforward and can be easily realized in many environments. Its memory consumption is low, and the set of parameters is small and easy to understand. The method is based on the Edwards-Wilkinson equation, which is traditionally used to model the equilibrium fluctuations of a propagating interface under the influence of temporally and spatially varying noise. We report here an adaptation of this equation into multidimensional image segmentation, and its efficient discretization.
RESUMEN
Each class of microscope is limited to imaging specific aspects of cell structure and/or molecular organization. However, imaging the specimen by complementary microscopes and correlating the data can overcome this limitation. Whilst not a new approach, the field of correlative imaging is currently benefitting from the emergence of new microscope techniques. Here we describe the correlation of cryogenic fluorescence tomography (CFT) with soft X-ray tomography (SXT). This amalgamation of techniques integrates 3D molecular localization data (CFT) with a high-resolution, 3D cell reconstruction of the cell (SXT). Cells are imaged in both modalities in a near-native, cryopreserved state. Here we describe the current state of the art in correlative CFT-SXT, and discuss the future outlook for this method.
Asunto(s)
Imagenología Tridimensional , Microscopía Fluorescente/métodos , Tomografía por Rayos X/métodos , Levaduras/ultraestructura , Humanos , Procesamiento de Imagen Asistido por Computador , Microscopía Fluorescente/tendencias , Estadística como Asunto , Tomografía por Rayos X/tendenciasRESUMEN
Gene positioning and regulation of nuclear architecture are thought to influence gene expression. Here, we show that, in mouse olfactory neurons, silent olfactory receptor (OR) genes from different chromosomes converge in a small number of heterochromatic foci. These foci are OR exclusive and form in a cell-type-specific and differentiation-dependent manner. The aggregation of OR genes is developmentally synchronous with the downregulation of lamin b receptor (LBR) and can be reversed by ectopic expression of LBR in mature olfactory neurons. LBR-induced reorganization of nuclear architecture and disruption of OR aggregates perturbs the singularity of OR transcription and disrupts the targeting specificity of the olfactory neurons. Our observations propose spatial sequestering of heterochromatinized OR family members as a basis of monogenic and monoallelic gene expression.
Asunto(s)
Núcleo Celular/química , Neuronas Receptoras Olfatorias/metabolismo , Receptores Odorantes/genética , Animales , Núcleo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Regulación hacia Abajo , Regulación de la Expresión Génica , Heterocromatina/metabolismo , Hibridación Fluorescente in Situ , Ratones , Receptores Citoplasmáticos y Nucleares/metabolismo , Transcripción Genética , Receptor de Lamina BRESUMEN
The opportunistic pathogen Candida albicans can undergo phenotypic switching between a benign, unicellular phenotype and an invasive, multicellular form that causes candidiasis. Increasingly, strains of Candida are becoming resistant to antifungal drugs, making the treatment of candidiasis difficult, especially in immunocompromised or critically ill patients. Consequently, there is a pressing need to develop new drugs that circumvent fungal drug-resistance mechanisms. In this work we used soft X-ray tomography to image the subcellular changes that occur as a consequence of both phenotypic switching and of treating C. albicans with antifungal peptoids, a class of candidate therapeutics unaffected by drug resistance mechanisms. Peptoid treatment suppressed formation of the pathogenic hyphal phenotype and resulted in striking changes in cell and organelle morphology, most dramatically in the nucleus and nucleolus, and in the number, size, and location of lipidic bodies. In particular, peptoid treatment was seen to cause the inclusion of lipidic bodies into the nucleus.