Nathalie Jourdan

Rotavirus infection induces cytoskeleton disorganization in human intestinal epithelial cells: implication of an increase in intracellular calcium concentration.

ABSTRACT Rotavirus infection is the most common cause of severe infantile gastroenteritis worldwide. In vivo, rotavirus exhibits a marked tropism for the differentiated enterocytes of the intestinal epithelium. In vitro, differentiated and undifferentiated intestinal cells can be infected. We observed that rotavirus infection of the human intestinal epithelial Caco-2 cells induces cytoskeleton alterations as a function of cell differentiation. The vimentin network disorganization detected in undifferentiated Caco-2 cells was not found in fully differentiated cells. In contrast, differentiated Caco-2 cells presented Ca2+-dependent microtubule disassembly and Ca2+-independent cytokeratin 18 rearrangement, which both require viral replication. We propose that these structural alterations could represent the first manifestations of rotavirus-infected enterocyte injury leading to functional perturbations and then to diarrhea.

Blue-light dependent reactive oxygen species formation by Arabidopsis cryptochrome may define a novel evolutionarily conserved signaling mechanism

Cryptochromes are widespread blue-light absorbing flavoproteins with important signaling roles. In plants they mediate de-etiolation, developmental and stress responses resulting from interaction with downstream signaling partners such as transcription factors and components of the proteasome. Recently, it has been shown that Arabidopsis cry1 activation by blue light also results in direct enzymatic conversion of molecular oxygen (O2 ) to reactive oxygen species (ROS) and hydrogen peroxide (H2 O2 ) in vitro. Here we explored whether direct enzymatic synthesis of ROS by Arabidopsis cry1 can play a physiological role in vivo. ROS formation resulting from cry1 expression was measured by fluorescence assay in insect cell cultures and in Arabidopsis protoplasts from cryptochrome mutant seedlings. Cell death was determined by colorimetric assay. We found that ROS formation results from cry1 activation and induces cell death in insect cell cultures. In plant protoplasts, cryptochrome activation results in rapid increase in ROS formation and cell death. We conclude that ROS formation by cryptochromes may indeed be of physiological relevance and could represent a novel paradigm for cryptochrome signaling.

Blue-light dependent ROS formation by Arabidopsis cryptochrome-2 may contribute toward its signaling role

Cryptochromes are blue-light absorbing flavoproteins with many important signaling roles in plants, including in de-etiolation, development, and stress response. They interact with downstream signaling partners such as transcription factors and components of the proteasome, and thereby alter regulation of nuclear gene expression in a light dependent manner. In a prior study, it has also been shown that Arabidopsis cry1 activation by blue light results in direct enzymatic conversion of molecular oxygen (O2) to ROS (reactive oxygen species) in vivo leading to cell death in overexpressing lines. Here we extend these observations to show that Atcry2 is translocated from the cytosol to the nucleus in response to blue light illumination, resulting in nuclear accumulation of ROS in expressing insect cell cultures. These observations suggest that ROS formation may represent a novel means of signaling by Atcry2 distinct from, and perhaps complementary to, the currently known mechanism of light-mediated conformational change.

Live-Cell Imaging Reveals Multiple Interactions between Epstein-Barr Virus Nuclear Antigen 1 and Cellular Chromatin during Interphase and Mitosis

ABSTRACT Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.

Blue-light induced biosynthesis of ROS contributes to the signaling mechanism of Arabidopsis cryptochrome

Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H202 and ROS accumulate in the plant nucleus after cryptochrome activation. They induce ROS-regulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.

Evidence of the W(O)(S2)2 core as an intermediate in the acidification of WS42−. Structural characterization of W(O)(S2)2(bpy) and W(O)(S2)2(phen)

Abstract Acidification of WS42− by HCl in MeCN in the presence of 2,2′-bipyridine (bpy) led to the discrete mononuclear compound W(O)(S2)2(bpy) (1) isolated at room temperature as crystals: monoclinic, space group C2/c, a=25.670(8), b=8.472(3), c=12.466(4) A, β=106.26(3)°, V=2603 A3, Z=8, R=0.061. W(O)(S2)2(bpy) (2) was obtained by reaction of [Ni(bpy)2]Cl2 with WS42−, followed by crystallization at −30 °C, monoclinic, space group P21/n, a=8.875(7), b=12.205(6), c=12.363(3) A, β=90.12(5)°, V=1339(1) A3, Z=4, R=0.062. Compounds 1 and 2 differ in their stackings. The alignment of the bpy ligands in 1 is parallel; in 2 the alignment is nearly perpendicular. Addition of 1,10-phenanthroline (phen) under the same conditions of acidity yielded W(O)(S2)2(phen) (3) isolated at room temperature as crystals: monoclinic, P2v a=6.798(4), b=14.807(5), c=7.048(2) A, β=92.25(3)°, V=709 A3, Z=2. The three complexes can all be described as a W-pentagonal bipyramid, with four sulfur atoms and a nitrogen atom forming the equatorial plane, the two axial positions being occupied by the remaining nitrogen atom of the bpy ligand and an oxo group. Formation of the W(O)(S2)2 core in solution is postulated through a redox process involving elemental sulfur produced in situ.

Blue-light induced accumulation of reactive oxygen species is a consequence of the Drosophila cryptochrome photocycle

Cryptochromes are evolutionarily conserved blue-light absorbing flavoproteins which participate in many important cellular processes including in entrainment of the circadian clock in plants, Drosophila and humans. Drosophila melanogaster cryptochrome (DmCry) absorbs light through a flavin (FAD) cofactor that undergoes photoreduction to the anionic radical (FAD•-) redox state both in vitro and in vivo. However, recent efforts to link this photoconversion to the initiation of a biological response have remained controversial. Here, we show by kinetic modeling of the DmCry photocycle that the fluence dependence, quantum yield, and half-life of flavin redox state interconversion are consistent with the anionic radical (FAD•-) as the signaling state in vivo. We show by fluorescence detection techniques that illumination of purified DmCry results in enzymatic conversion of molecular oxygen (O2) to reactive oxygen species (ROS). We extend these observations in living cells to demonstrate transient formation of superoxide (O2•-), and accumulation of hydrogen peroxide (H2O2) in the nucleus of insect cell cultures upon DmCry illumination. These results define the kinetic parameters of the Drosophila cryptochrome photocycle and support light-driven electron transfer to the flavin in DmCry signaling. They furthermore raise the intriguing possibility that light-dependent formation of ROS as a byproduct of the cryptochrome photocycle may contribute to its signaling role.

Sharing of mitotic pre-ribosomal particles between daughter cells.

ABSTRACT Ribosome biogenesis is a fundamental multistep process initiated by the synthesis of 90S pre-ribosomal particles in the nucleoli of higher eukaryotes. Even though synthesis of ribosomes stops during mitosis while nucleoli disappear, mitotic pre-ribosomal particles persist as observed in pre-nucleolar bodies (PNBs) during telophase. To further understand the relationship between the nucleolus and the PNBs, the presence and the fate of the mitotic pre-ribosomal particles during cell division were investigated. We demonstrate that the recently synthesized 45S precursor ribosomal RNAs (pre-rRNAs) as well as the 32S and 30S pre-rRNAs are maintained during mitosis and associated with the chromosome periphery together with pre-rRNA processing factors. Maturation of the mitotic pre-ribosomal particles, as assessed by the stability of the mitotic pre-rRNAs, is transiently arrested during mitosis by a cyclin-dependent kinase (CDK)1-cyclin-B-dependent mechanism and can be restored by CDK inhibitor treatments. At the M–G1 transition, the resumption of mitotic pre-rRNA processing in PNBs does not induce the disappearance of PNBs; this only occurs when functional nucleoli reform. Strikingly, during their maturation process, mitotic pre-rRNAs localize in reforming nucleoli. Summary: Maturation of mitotic pre-rRNAs is transiently arrested during mitosis by a CDK1–cyclin-B-dependent mechanism. Strikingly, during their maturation process, mitotic pre-rRNAs localize in reforming nucleoli.

Low-intensity electromagnetic fields induce human cryptochrome to modulate intracellular reactive oxygen species

Exposure to man-made electromagnetic fields (EMFs), which increasingly pollute our environment, have consequences for human health about which there is continuing ignorance and debate. Whereas there is considerable ongoing concern about their harmful effects, magnetic fields are at the same time being applied as therapeutic tools in regenerative medicine, oncology, orthopedics, and neurology. This paradox cannot be resolved until the cellular mechanisms underlying such effects are identified. Here, we show by biochemical and imaging experiments that exposure of mammalian cells to weak pulsed electromagnetic fields (PEMFs) stimulates rapid accumulation of reactive oxygen species (ROS), a potentially toxic metabolite with multiple roles in stress response and cellular ageing. Following exposure to PEMF, cell growth is slowed, and ROS-responsive genes are induced. These effects require the presence of cryptochrome, a putative magnetosensor that synthesizes ROS. We conclude that modulation of intracellular ROS via cryptochromes represents a general response to weak EMFs, which can account for either therapeutic or pathological effects depending on exposure. Clinically, our findings provide a rationale to optimize low field magnetic stimulation for novel therapeutic applications while warning against the possibility of harmful synergistic effects with environmental agents that further increase intracellular ROS.