Soft X-ray tomography of phenotypic switching and the cellular response to antifungal peptoids in Candida albicans.

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 unaf…

Herpes simplex virus 1 induces egress channels through marginalized host chromatin

AbstractLytic 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, co…

Putting molecules in their place.

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…

Chromatin organization regulates viral egress dynamics.

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 s…

Quantifying Changes in Nuclear Organization in Normal vs. Cancer Cells using X-ray Tomography

Soft X-ray Tomography Reveals HSV-1-Induced Remodeling of Human B Cells.

Upon infection, viruses hijack the cell machinery and remodel host cell structures to utilize them for viral proliferation. Since viruses are about a thousand times smaller than their host cells, imaging virus-host interactions at high spatial resolution is like looking for a needle in a haystack. Scouting gross cellular changes with fluorescent microscopy is only possible for well-established viruses, where fluorescent tagging is developed. Soft X-ray tomography (SXT) offers 3D imaging of entire cells without the need for chemical fixation or labeling. Here, we use full-rotation SXT to visualize entire human B cells infected by the herpes simplex virus 1 (HSV-1). We have mapped the temporo…

Nuclear aggregation of olfactory receptor genes governs their monogenic expression.

SummaryGene 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…

Soft X-Ray Tomography Reveals Gradual Chromatin Compaction and Reorganization during Neurogenesis In Vivo

Summary - 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, differentiati…