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A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Imaging techniques are fundamental in order to understand cell organization and machinery in biological research and the related fields. Among these ...

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Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Three-dimensional (3D) structured illumination microscopy (SIM) allows imaging of fluorescently labelled cellular structures at higher resolution than ...

Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK

Beamline B24, Diamond Light Source

In Drosophila, it is thought that peptidoglycan recognition proteins (PGRPs) SA and LC structurally discriminate between bacterial peptidoglycans with lysine (Lys) or diaminopimelic (DAP) acid, respectively, thus inducing differential antimicrobial transcription response. Here, we find that accessibility to PG at the cell wall plays a central role in immunity to infection. When wall teichoic acids (WTAs) are genetically removed from S. aureus (Lys type) and Bacillus subtilis (DAP type), thus increasing accessibility, the binding of both PGRPs to either bacterium is increased. PGRP-SA and -LC double mutant flies are more susceptible to infection with both WTA-less bacteria. In addition, WTA-less bacteria grow better in PGRP-SA/-LC double mutant flies. Finally, infection with WTA-less bacteria abolishes any differential activation of downstream antimicrobial transcription. Our results indicate that accessibility to cell wall PG is a major factor in PGRP-mediated immunity and may be the cause for discrimination between classes of pathogens.

Accessibility to Peptidoglycan Is Important for the Recognition of Gram-Positive Bacteria in Drosophila.

Imaging of biological matter across resolution scales entails the challenge of preserving the direct and unambiguous correlation of subject features from the macroscopic to the microscopic level. Here, we present a correlative imaging platform developed specifically for imaging cells in 3D under cryogenic conditions by using X-rays and visible light. Rapid cryo-preservation of biological specimens is the current gold standard in sample preparation for ultrastructural analysis in X-ray imaging. However, cryogenic fluorescence localization methods are, in their majority, diffraction-limited and fail to deliver matching resolution. We addressed this technological gap by developing an integrated, user-friendly platform for 3D correlative imaging of cells in vitreous ice by using super-resolution structured illumination microscopy in conjunction with soft X-ray tomography. The power of this approach is demonstrated by studying the process of reovirus release from intracellular vesicles during the early stages of infection and identifying intracellular virus-induced structures.

3D Correlative Cryo-Structured Illumination Fluorescence and Soft X-ray Microscopy Elucidates Reovirus Intracellular Release Pathway.

The aim of the present study was to evaluate the relative mRNA expression levels of genes involved in the hypoxia inducible factor (HIF) signalling pathway in renal cell carcinoma (RCC) and to analyse their associations with clinicopathological parameters and survival outcomes. Reverse transcription-quantitative PCR was used to quantify the mRNA expression levels of HIF-1α, HIF-2α, prolyl hydroxylase (PHD)1, PHD2 and PHD3 in formalin-fixed paraffin-embedded (FFPE) tumour tissue samples from 41 patients with RCC, including 33 cases of clear cell RCC (ccRCC). FFPE samples of corresponding adjacent normal kidney tissues were used as a comparison. mRNA expression levels were analysed in regard to clinical parameters, histological type, stage, nuclear grade, cancer specific survival and overall survival. Compared with adjacent normal kidney tissue, HIF-1α levels were lower in 16/33 ccRCC samples (48.48%), while HIF-2α, PHD1 and PHD2 levels did not exhibit a specific expression pattern. By contrast, the PHD3 mRNA level was higher in 29/33 (87.87%) of the tumour samples. HIF-1α was positively associated with HIF-2α, PHD1 and PHD2. HIF-2α levels were associated with PHD1, PHD2 and PHD3, while PHD3 was strongly associated with PHD2. PHD3 mRNA levels were inversely associated with nuclear grade (P=0.015). However, in univariate analysis, PHD3 was not associated with cancer-specific or overall survival rates. The present findings suggest an important involvement of PHD3 in ccRCC, since PHD3 mRNA expression was inversely associated with nuclear grade. However, PHD3 mRNA levels did not have an independent prognostic value. Further studies are required to investigate whether PHD3 could be used as either a therapeutic target or prognostic marker.

mRNA overexpression of prolyl hydroxylase PHD3 is inversely related to nuclear grade in renal cell carcinoma.

Hypoxia inducible factors (HIF) and prolyl hydroxylase domain (PHD) enzymes play a central role in tumor progression in clear cell renal cell carcinoma (ccRCC). However, there are currently no data regarding the behavior of this pathway (HIF/PHD) in a large number of benign renal tumors, the oncocytomas. The aim of the present study was to compare the expression levels of these factors between ccRCC and oncocytoma tumors.

Decreased prolyl hydroxylase 3 mRNA expression in oncocytomas compared with clear cell renal cell carcinoma.

3D correlative microscopy methods have revolutionized biomedical research, allowing the acquisition of multidimensional information to gain an in-depth understanding of biological systems. With the advent of relevant cryo-preservation methods, correlative imaging of cryogenically preserved samples has led to nanometer resolution imaging (2-50 nm) under harsh imaging regimes such as electron and soft X-ray tomography. These methods have now been combined with conventional and super-resolution fluorescence imaging at cryogenic temperatures to augment information content from a given sample, resulting in the immediate requirement for protocols that facilitate hassle-free, unambiguous cross-correlation between microscopes. We present here sample preparation strategies and a direct comparison of different working fiducialization regimes that facilitate 3D correlation of cryo-structured illumination microscopy and cryo-soft X-ray tomography. Our protocol has been tested at two synchrotron beamlines (B24 at Diamond Light Source in the UK and BL09 Mistral at ALBA in Spain) and has led to the development of a decision aid that facilitates experimental design with the strategic use of markers based on project requirements. This protocol takes between 1.5 h and 3.5 d to complete, depending on the cell populations used (adherent cells may require several days to grow on sample carriers).

Ilias Kounatidis

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Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK

A 3D Cartographic Description of the Cell by Cryo Soft X-ray Tomography

Cryo-Structured Illumination Microscopic Data Collection from Cryogenically Preserved Cells

Beamline B24