Francisco Javier Chichón

Jose Javier Conesa, Joaquín Otón, Michele Chiappi, Jose María Carazo, Eva Pereiro, Francisco Javier Chichón, José L Carrascosa

Abstract We used soft X-ray three-dimensional imaging to quantify the mass of 
superparamagnetic iron oxide nanoparticles (SPION) within whole cells, by exploiting the 
iron oxide differential absorption contrast. Near-edge absorption soft X-ray nanotomography 
(NEASXT) combines whole-cell 3D structure determination at 50 nm resolution, with 3D 
elemental mapping and high throughput. We detected three-dimensional distribution of 
SPIONs within cells with 0.3 g/cm 3 sensitivity, sufficient for detecting the density ...

Chiappi M, Conesa JJ, Pereiro E, Sorzano CO, Rodríguez MJ, Henzler K, Schneider G, Chichón FJ, Carrascosa JL.

Here we used cryo-soft X-ray tomography (cryo-SXT) to collect comprehensive three-dimensional (3D) data to characterise the interaction of superparamagnetic iron oxide nanoparticles (SPION) with a breast cancer cell line.

We incubated MCF-7 (a human breast cancer cell line) from 0 to 24 h with SPION (15 nm average diameter, coated with dimercaptosuccinic acid), a system that has been studied previously using various microscopy and bulk techniques. This system facilitates the validation and contextualization of the new 3D data acquired using the cryo-SXT-based approach.

Aneesh Vijayan, Juan García-Arriaza, Suresh C Raman, José Javier Conesa, Francisco Javier Chichón, César Santiago, Carlos Óscar S Sorzano, José L Carrascosa, Mariano Esteban

Abstract In the HIV vaccine field, there is a need to produce highly immunogenic forms of the 
Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the 
generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by 
fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived 
from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were 
generated and the protein purified was characterized by size exclusion chromatography, ...

Andrea Sorrentino, Josep Nicolás, Ricardo Valcárcel, Francisco Javier Chichón, Marc Rosanes, Jose Avila, Andrei Tkachuk, Jeff Irwin, Salvador Ferrer, Eva Pereiro

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 ...

Ana J Pérez-Berná, Sanjin Marion, F Javier Chichón, José J Fernández, Dennis C Winkler, José L Carrascosa, Alasdair C Steven, Antonio Šiber, Carmen San Martín

Abstract Genome packing in adenovirus has long evaded precise description, since the viral 
dsDNA molecule condensed by proteins (core) lacks icosahedral order characteristic of the 
virus protein coating (capsid). We show that useful insights regarding the organization of the 
core can be inferred from the analysis of spatial distributions of the DNA and condensing 
protein units (adenosomes). These were obtained from the inspection of cryo-electron 
tomography reconstructions of individual human adenovirus particles. Our analysis shows ...

Macarena Calero, Michele Chiappi, Ana Lazaro-Carrillo, María José Rodríguez, Francisco Javier Chichón, Kieran Crosbie-Staunton, Adriele Prina-Mello, Yuri Volkov, Angeles Villanueva, José L Carrascosa

Background Different 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 
acidcoated superparamagnetic iron oxide nanoparticles with an average diameter of 15 nm 
and negative surface charge in MCF-7 breast cancer cells.

Javier M Rodríguez, Francisco J Chichón, Esther Martín-Forero, Fernando González-Camacho, José L Carrascosa, José R Castón, Daniel Luque

Abstract The infectivity of rotavirus, the main causative agent of childhood diarrhea, is 
dependent on activation of the extracellular viral particles by trypsin-like proteases in the 
host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its 
mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of 
trypsin-activated particles showed well-resolved spikes, although no density was identified 
for the spikes in uncleaved particles; these data suggested that trypsin activation triggers ...

Aránzazu Cruz-Adalia, Guillermo Ramirez-Santiago, Carmen Calabia-Linares, Mónica Torres-Torresano, Lidia Feo, Marta Galán-Díez, Elena Fernández-Ruiz, Eva Pereiro, Peter Guttmann, Michele Chiappi, Gerd Schneider, José López Carrascosa, Francisco Javier Chichón, Gloria Martínez del Hoyo, Francisco Sánchez-Madrid, Esteban Veiga

Summary Dendritic cells (DCs) phagocytose, process, and present bacterial antigens to T 
lymphocytes to trigger adaptive immunity. In vivo, bacteria can also be found inside T 
lymphocytes. However, T cells are refractory to direct bacterial infection, leaving the 
mechanisms by which bacteria invade T cells unclear. We show that T cells take up bacteria 
from infected DCs by the process of transinfection, which requires direct contact between the 
two cells and is enhanced by antigen recognition. Prior to transfer, bacteria localize to the ...

Rocío Arranz, Rocío Coloma, Francisco Javier Chichón, José Javier Conesa, José L Carrascosa, José M Valpuesta, Juan Ortín, Jaime Martín-Benito

Abstract 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 ...

Eva Pereiro, Francisco J Chichón

Abstract In the HIV vaccine field, there is a need to produce highly immunogenic forms of the 
Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the 
generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by 
fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived 
from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were 
generated and the protein purified was characterized by size exclusion chromatography, ...

Daniel Luque, José M González, Josué Gómez-Blanco, Roberto Marabini, Javier Chichón, Ignacio Mena, Iván Angulo, José L Carrascosa, Nuria Verdaguer, Benes L Trus, Juan Bárcena, José R Castón

ABSTRACT Viruses need only one or a few structural capsid proteins to build an infectious 
particle. This is possible through the extensive use of symmetry and the conformational 
polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit 
hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid 
assembly and their application in vaccine design. The RHDV capsid is based on a T= 3 
lattice containing 180 identical subunits (VP1). We determined the structure of RHDV VLP ...

Francisco Javier Chichón, Maria Josefa Rodríguez, Eva Pereiro, Michele Chiappi, Beatriz Perdiguero, Peter Guttmann, Stephan Werner, Stefan Rehbein, Gerd Schneider, Mariano Esteban, José L Carrascosa

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 ...

José L Carrascosa, Francisco Javier Chichón, Eva Pereiro, María Josefa Rodríguez, Jose Jesús Fernández, Mariano Esteban, Stefan Heim, Peter Guttmann, Gerd Schneider

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 250× 270× 360nm3. The X-ray tomograms 
show the presence of a clearly defined external envelope, together with an inner core ...

S Hormeno, B Ibarra, FJ Chichón, K Habermann, BMH Lange, JM Valpuesta, JL Carrascosa, JR Arias-Gonzalez

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 103 electrons, which corresponds to a surface- ...

Francisco Javier Chichón, María Josefa Rodríguez, Cristina Risco, Alberto Fraile‐Ramos, José Jesús Fernández, Mariano Esteban, José L Carrascosa

We have used electron tomography of sections of VACV-infected cells to follow, in 
three dimensions, the remodelling of the membrane components of the virus during 
envelope maturation. The tomograms obtained suggest that a number of independent 
'crescents' interact with each other to enclose the volume of an incomplete ellipsoid in the 
viral factory area, attaining the overall shape and size characteristic of the first immature form 
of the virus [IV (immature virus)]. The incorporation of the DNA into these forms leads to ...

Patricia de la Presa, Tatiana Rueda, María del Puerto Morales, F Javier Chichón, Rocio Arranz, Jose Maria Valpuesta, Antonio Hernando

Gold nanoparticles have been synthesized inside ethosomes, vesicles composed of 
phospholipid, ethanol, and water, which could be very efficient not only in delivery probes to 
the skin but also as diagnostic and therapeutic multimodal agents. High efficiency 
encapsulation of gold nanoparticles is achieved by a simple strategy: the nanoparticles 
synthesis occurs simultaneously with the ethosomes formation in the absence of any 
undesirable reducing agents. A three-dimensional reconstruction of a gold-embedded ...

Rana Ibrahim, Cédric Messaoudi, Francisco Javier Chichon, Claude Celati, Sergio Marco

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 ...

Fábio Fernandes, Luís MS Loura, Francisco J Chichón, Jose L Carrascosa, Alexander Fedorov, Manuel Prieto

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 α-helix (H0-NBAR) results in much 
lower membrane tubulation efficiency, pointing to a fundamental role of this protein ...