Francisco Javier Chichón

The Structure of Native Influenza Virion Ribonucleoproteins

Influenza Revealed Influenza virus, a single-stranded RNA virus, is responsible for substantial morbidity and mortality worldwide. The influenza ribonucleoprotein (RNP) complex, which carries out viral replication and transcription, is central to the virus life-cycle and to viral host adaptation (see the Perspective by Tao and Zheng). Structural characterization of the viral RNP has been challenging, but Moeller et al. (p. 1631, published online 22 November) and Arranz et al. (p. 1634, published online 22 November) now report the structure and assembly of this complex, using cryo-electron microscopy and negative-stain electron microscopy. The structures reveal how the viral polymerase, RNA genome, and nucleoprotein interact in the RNP providing insight into mechanisms for influenza genome replication and transcription. Electron microscopic analysis of a purified RNA-protein complex links its structure to the influenza life cycle. The influenza viruses cause annual epidemics of respiratory disease and occasional pandemics, which constitute a major public-health issue. The segmented negative-stranded RNAs are associated with the polymerase complex and nucleoprotein (NP), forming ribonucleoproteins (RNPs), which are responsible for virus transcription and replication. We describe the structure of native RNPs derived from virions. They show a double-helical conformation in which two NP strands of opposite polarity are associated with each other along the helix. Both strands are connected by a short loop at one end of the particle and interact with the polymerase complex at the other end. This structure will be relevant for unraveling the mechanisms of nuclear import of parental virus RNPs, their transcription and replication, and the encapsidation of progeny RNPs into virions.

Cryo-X-ray tomography of vaccinia virus membranes and inner compartments

Vitrified unstained purified vaccinia virus particles have been used as a test sample to evaluate the capabilities of cryo-X-ray tomography. Embedded in a thick layer of vitreous ice, the viral particles representing the mature form of the virus (MV) were visualized using full-field transmission X-ray tomography. The tomographic reconstructions reveal the viral brick-shaped characteristic structures with a size of 250x270x360nm(3). The X-ray tomograms show the presence of a clearly defined external envelope, together with an inner core surrounded by an internal envelope, including areas with clear differential density, which correlate well with those features previously described for these viral particles using electron microscopy analyses. A quantitative assessment of the resolution attained in X-ray and electron tomograms of the viral particles prepared under the same conditions yields values of 25.7 and 6.7nm half-pitch, respectively. Although the resolution of the X-ray microscope is well above the dimensions of the membranous compartments, the strong differential contrast exhibited makes it possible to precisely reveal them without any contrasting reagent within this small and complex biological sample.

Role of Helix 0 of the N-BAR Domain in Membrane Curvature Generation

A group of proteins with cell membrane remodeling properties is also able to change dramatically the morphology of liposomes in vitro, frequently inducing tubulation. For a number of these proteins, the mechanism by which this effect is exerted has been proposed to be the embedding of amphipathic helices into the lipid bilayer. For proteins presenting BAR domains, removal of an N-terminal amphipathic alpha-helix (H0-NBAR) results in much lower membrane tubulation efficiency, pointing to a fundamental role of this protein segment. Here, we studied the interaction of a peptide corresponding to H0-NBAR with model lipid membranes. H0-NBAR bound avidly to anionic liposomes but partitioned weakly to zwitterionic bilayers, suggesting an essentially electrostatic interaction with the lipid bilayer. Interestingly, it is shown that after membrane incorporation, the peptide oligomerizes as an antiparallel dimer, suggesting a potential role of H0-NBAR in the mediation of BAR domain oligomerization. Through monitoring the effect of H0-NBAR on liposome shape by cryoelectron microscopy, it is clear that membrane morphology is not radically changed. We conclude that H0-NBAR alone is not able to induce vesicle curvature, and its function must be related to the promotion of the scaffold effect provided by the concave surface of the BAR domain.

Characterization of interaction of magnetic nanoparticles with breast cancer cells

BackgroundDifferent superparamagnetic iron oxide nanoparticles have been tested for their potential use in cancer treatment, as they enter into cells with high effectiveness, do not induce cytotoxicity, and are retained for relatively long periods of time inside the cells. We have analyzed the interaction, internalization and biocompatibility of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm and negative surface charge in MCF-7 breast cancer cells.ResultsCells were incubated with dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles for different time intervals, ranging from 0.5 to 72 h. These nanoparticles showed efficient internalization and relatively slow clearance. Time-dependent uptake studies demonstrated the maximum accumulation of dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles after 24 h of incubation, and afterwards they were slowly removed from cells. Superparamagnetic iron oxide nanoparticles were internalized by energy dependent endocytosis and localized in endosomes. Transmission electron microscopy studies showed macropinocytosis uptake and clathrin-mediated internalization depending on the nanoparticles aggregate size. MCF-7 cells accumulated these nanoparticles without any significant effect on cell morphology, cytoskeleton organization, cell cycle distribution, reactive oxygen species generation and cell viability, showing a similar behavior to untreated control cells.ConclusionsAll these findings indicate that dimercaptosuccinic acid-coated superparamagnetic iron oxide nanoparticles have excellent properties in terms of efficiency and biocompatibility for application to target breast cancer cells.

Cryo X-ray nano-tomography of vaccinia virus infected cells

Abstract We have performed full-field cryo X-ray microscopy in the water window photon energy range on vaccinia virus (VACV) infected cells to produce tomographic reconstructions. PtK2 cells were infected with a GFP-expressing VACV strain and frozen by plunge fast freezing. The infected cells were selected by light fluorescence microscopy of the GFP marker and subsequently imaged in the X-ray microscope under cryogenic conditions. Tomographic tilt series of X-ray images were used to yield three-dimensional reconstructions showing different cell organelles (nuclei, mitochondria, filaments), together with other structures derived from the virus infection. Among them, it was possible to detect viral factories and two types of viral particles related to different maturation steps of VACV (immature and mature particles), which were compared to images obtained by standard electron microscopy of the same type of cells. In addition, the effect of radiation damage during X-ray tomographic acquisition was analyzed. Thin sections studied by electron microscopy revealed that the morphological features of the cells do not present noticeable changes after irradiation. Our findings show that cryo X-ray nano-tomography is a powerful tool for collecting three-dimensional structural information from frozen, unfixed, unstained whole cells with sufficient resolution to detect different virus particles exhibiting distinct maturation levels.

MISTRAL: a transmission soft X-ray microscopy beamline for cryo nano-tomography of biological samples and magnetic domains imaging.

The performance of MISTRAL is reported, the soft X-ray transmission microscopy beamline at the ALBA light source (Barcelona, Spain) which is primarily dedicated to cryo soft X-ray tomography (cryo-SXT) for three-dimensional visualization of whole unstained cells at spatial resolutions down to 30 nm (half pitch). Short acquisition times allowing for high-throughput and correlative microscopy studies have promoted cryo-SXT as an emerging cellular imaging tool for structural cell biologists bridging the gap between optical and electron microscopy. In addition, the beamline offers the possibility of imaging magnetic domains in thin magnetic films that are illustrated here with an example.

Electron tomography study of isolated human centrioles

Centrioles are components of the centrosome, which is present in most eukaryotic cells (from protozoa to mammals). They organize the microtubule skeleton during interphase and the mitotic spindle during cell division. In ciliate cells, centrioles form basal bodies that are involved in cellular motility. Despite their important roles in biology, the detailed structure of centrioles remains obscure. This work contributes to a more complete model of centriole structure. The authors used electron tomography of isolated centrosomes from the human lymphoblast KE37 to explore the details of subdistal appendages and centriole lumen organization in mother centrioles. Their results reveal that each of the nine subdistal appendages is composed of two halves (20 nm diameter each) fused in a 40 nm tip that extends 100 nm from where it anchors to microtubules. The centriole lumen is filled at the distal domain by a 45 nm periodic stack of rings. Each ring has a 30 nm diameter, is 15 nm thick, and appears to be tilted at 53° perpendicular to the centriole axis. The rings are anchored to microtubules by arms. Based on their results, the authors propose a model of the mother centriole distal structure. Microsc. Res. Tech., 2009.

Structural Changes In Cells Imaged by Soft X-ray Cryo-Tomography During Hepatitis C Virus Infection

Chronic hepatitis C virus (HCV) infection causes severe liver disease in millions of humans worldwide. Pathogenesis of HCV infection is strongly driven by a deficient immune response of the host, although intersection of different aspects of the virus life cycle with cellular homeostasis is emerging as an important player in the pathogenesis and progression of the disease. Cryo soft X-ray tomography (cryo-SXT) was performed to investigate the ultrastructural alterations induced by the interference of HCV replication with cellular homeostasis. Native, whole cell, three-dimensional (3D) maps were obtained in HCV replicon-harboring cells and in a surrogate model of HCV infection. Tomograms from HCV-replicating cells show blind-ended endoplasmic reticulum tubules with pseudospherical extrusions and marked alterations of mitochondrial morphology that correlated spatially with the presence of endoplasmic reticulum alterations, suggesting a short-range influence of the viral machinery on mitochondrial homeostasis. Both mitochondrial and endoplasmic reticulum alterations could be reverted by a combination of sofosbuvir/daclatasvir, which are clinically approved direct-acting antivirals for the treatment of chronic HCV infection. In addition to providing structural insight into cellular aspects of HCV pathogenesis, our study illustrates how cryo-SXT is a powerful 3D wide-field imaging tool for the assessment and understanding of complex cellular processes in a setting of near-native whole hydrated cells. Our results also constitute a proof of concept for the use of cryo-SXT as a platform that enables determining the potential impact of candidate compounds on the ultrastructure of the cell that may assist drug development at a preclinical level.

Single Centrosome Manipulation Reveals Its Electric Charge and Associated Dynamic Structure

The centrosome is the major microtubule-organizing center in animal cells and consists of a pair of centrioles surrounded by a pericentriolar material. We demonstrate laser manipulation of individual early Drosophila embryo centrosomes in between two microelectrodes to reveal that it is a net negatively charged organelle with a very low isoelectric region (3.1 +/- 0.1). From this single-organelle electrophoresis, we infer an effective charge smaller than or on the order of 10(3) electrons, which corresponds to a surface-charge density significantly smaller than that of microtubules. We show, however, that the charge of the centrosome has a remarkable influence over its own structure. Specifically, we investigate the hydrodynamic behavior of the centrosome by measuring its size by both Stokes law and thermal-fluctuation spectral analysis of force. We find, on the one hand, that the hydrodynamic size of the centrosome is 60% larger than its electron microscopy diameter, and on the other hand, that this physiological expansion is produced by the electric field that drains to the centrosome, a self-effect that modulates its structural behavior via environmental pH. This methodology further proves useful for studying the action of different environmental conditions, such as the presence of Ca(2+), over the thermally induced dynamic structure of the centrosome.

Membrane remodelling during vaccinia virus morphogenesis

Background information. VACV (vaccinia virus) is one of the most complex viruses, with a size exceeding 300 nm and more than 100 structural proteins. Its assembly involves sequential interactions and important rearrangements of its structural components.