Balázs Enyedi

Redox State of the Endoplasmic Reticulum Is Controlled by Ero1L-alpha and Intraluminal Calcium

Formation of intra- and intermolecular disulfide bonds is an essential step in the synthesis of secretory proteins. In eukaryotic cells, this process occurs in the endoplasmic reticulum (ER) and requires an oxidative environment with the action of several chaperones and folding catalysts. During protein folding, Ero1p oxidizes protein disulfide isomerase (PDI), which then directly catalyzes the formation of disulfide bonds in folding proteins. Recent cell-free studies suggest that the terminal electron acceptor in the pathway is molecular oxygen, with the resulting formation of hydrogen peroxide (H(2)O(2)). We report for the first time the measurement of ER H(2)O(2) level in live cells. By targeting a fluorescent protein-based H(2)O(2) sensor to various intracellular compartments, we show that the ER has the highest level of H(2)O(2), and this high concentration is well confined to the lumen of the organelle. Manipulation of the Ero1-Lalpha level--either by overexpression or by siRNA-mediated inhibition--caused parallel changes in luminal H(2)O(2), proving that the activity of Ero1-Lalpha results in H(2)O(2) formation in the ER. We also found that calcium mobilization from intracellular stores induces a decrease in ER H(2)O(2) level, suggesting a complex interplay between redox and calcium signaling in the mammalian ER.

Redox Nanodomains Are Induced by and Control Calcium Signaling at the ER-Mitochondrial Interface

The ER-mitochondrial interface is central to calcium signaling, organellar dynamics, and lipid biosynthesis. The ER and mitochondrial membranes also host sources and targets of reactive oxygen species (ROS), but their local dynamics and relevance remained elusive since measurement and perturbation of ROS at the organellar interface has proven difficult. Employing drug-inducible synthetic ER-mitochondrial linkers, we overcame this problem and demonstrate that the ER-mitochondrial interface hosts a nanodomain of H2O2, which is induced by cytoplasmic [Ca(2+)] spikes and exerts a positive feedback on calcium oscillations. H2O2 nanodomains originate from the mitochondrial cristae, which are compressed upon calcium signal propagation to the mitochondria, likely due to Ca(2+)-induced K(+) and concomitant water influx to the matrix. Thus, ER-mitochondrial H2O2 nanodomains represent a component of inter-organelle communication, regulating calcium signaling and mitochondrial activities.

Tissue damage detection by osmotic surveillance

How tissue damage is detected to induce inflammatory responses is unclear. Most studies have focused on damage signals released by cell breakage and necrosis. Whether tissues use other cues in addition to cell lysis to detect that they are damaged is unknown. We find that osmolarity differences between interstitial fluid and the external environment mediate rapid leukocyte recruitment to sites of tissue damage in zebrafish by activating cytosolic phospholipase a2 (cPLA2) at injury sites. cPLA2 initiates the production of non-canonical arachidonate metabolites that mediate leukocyte chemotaxis through a 5-oxo-ETE receptor (OXE-R). Thus, tissues can detect damage through direct surveillance of barrier integrity, with cell swelling probably functioning as a pro-inflammatory intermediate in the process.

The Cell Nucleus Serves as a Mechanotransducer of Tissue Damage-Induced Inflammation

Tissue damage activates cytosolic phospholipase A2 (cPLA2), releasing arachidonic acid (AA), which is oxidized to proinflammatory eicosanoids by 5-lipoxygenase (5-LOX) on the nuclear envelope. How tissue damage is sensed to activate cPLA2 is unknown. We investigated this by live imaging in wounded zebrafish larvae, where damage of the fin tissue causes osmotic cell swelling at the wound margin and the generation of a chemotactic eicosanoid signal. Osmotic swelling of cells and their nuclei activates cPla2 by translocating it from the nucleoplasm to the nuclear envelope. Elevated cytosolic Ca(2+) was necessary but not sufficient for cPla2 translocation, and nuclear swelling was required in parallel. cPla2 translocation upon nuclear swelling was reconstituted in isolated nuclei and appears to be a simple physical process mediated by tension in the nuclear envelope. Our data suggest that the nucleus plays a mechanosensory role in inflammation by transducing cell swelling and lysis into proinflammatory eicosanoid signaling.

Spatial and Temporal Analysis of NADPH Oxidase-Generated Hydrogen Peroxide Signals by Novel Fluorescent Reporter Proteins

AIMS Hydrogen peroxide (H2O2) is an emerging signaling molecule with diverse regulatory functions. Despite its significance, the spatial and temporal organization of H2O2 signals within cells is basically unknown. Our limited knowledge about H2O2 signals is largely due to the lack of appropriate techniques for measuring intracellular H2O2. The aim of the current study was to develop novel fluorescent reporter proteins for the measurement of intracellular H2O2. RESULTS We developed two novel, fluorescence resonance energy transfer-based redox probes that undergo opposite emission ratio changes upon exposure to H2O2. We have successfully used these sensors to measure H2O2 production by NADPH oxidases (Nox). Moreover, we targeted these probes to specific cellular compartments or incorporated them into oxidase complexes to detect H2O2 at different, well-defined loci. INNOVATION Studying Nox2- and dual oxidase 1 (Duox1)-expressing cells, we provide the first analysis of how NADPH-oxidase generated H2O2 signals radiate within and between cells. CONCLUSION Our results suggest that H2O2 produced by Noxs can induce redox changes in the intracellular milieu of Nox/Duox-expressing cells while simultaneously transmitting paracrine effects to neighboring cells.

Urothelial cells produce hydrogen peroxide through the activation of Duox1

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.

Hyperspectral Microscopy of Near-Infrared Fluorescence Enables 17-Chirality Carbon Nanotube Imaging.

The intrinsic near-infrared photoluminescence (fluorescence) of single-walled carbon nanotubes exhibits unique photostability, narrow bandwidth, penetration through biological media, environmental sensitivity, and both chromatic variety and range. Biomedical applications exploiting this large family of fluorophores will require the spectral and spatial resolution of individual (n,m) nanotube species’ fluorescence and its modulation within live cells and tissues, which is not possible with current microscopy methods. We present a wide-field hyperspectral approach to spatially delineate and spectroscopically measure single nanotube fluorescence in living systems. This approach resolved up to 17 distinct (n,m) species (chiralities) with single nanotube spatial resolution in live mammalian cells, murine tissues ex vivo, and zebrafish endothelium in vivo. We anticipate that this approach will facilitate multiplexed nanotube imaging in biomedical applications while enabling deep-tissue optical penetration, and single-molecule resolution in vivo.

Osmotic surveillance mediates rapid wound closure through nucleotide release

After wounding in zebrafish, osmolarity differences between the interstitial fluid and the external environment trigger ATP release that initiates rapid wound closure through long-range activation of basal epithelial cell motility.

Mechanisms of epithelial wound detection

Efficient wound healing requires the coordinated responses of various cell types within an injured tissue. To react to the presence of a wound, cells have to first detect it. Judging from their initial biochemical and morphological responses, many cells including leukocytes, epithelial cells, and endothelial cells detect wounds from over hundreds of micrometers within seconds-to-minutes. Wound detection involves the conversion of an injury-induced homeostatic perturbation, such as cell lysis, an unconstrained epithelial edge, or permeability barrier breakdown, into a chemical or physical signal. The signal is spatially propagated through the tissue to synchronize protective responses of cells near the wound site and at a distance. This review summarizes the triggers and mechanisms of wound detection in animals.

H2O2: a chemoattractant?

H2O2 is a relatively stable, rapidly diffusing reactive oxygen species that has been recently implicated as a mediator of leukocyte recruitment to epithelial wounds and transformed cells in zebrafish. Whether H2O2 activates the innate immune response by acting as a bona fide chemoattractant, enhancing chemoattractant sensing, or triggering production of other chemoattractive ligands remains largely unclear. Here, we describe the basic experimental procedures required to study these questions. We present a detailed protocol of the zebrafish tail fin wounding assay and explain how to use it for analyzing leukocyte chemotaxis in vivo. We further outline a method for H2O2 measurement in live zebrafish larvae using the genetically encoded sensor HyPer on a wide-field and a spinning disk confocal microscope. These methods provide a basis for dissecting the role of H2O2 in leukocyte chemotaxis in a vertebrate animal.