Cargo-specific effects of hypoxia on clathrin-mediated trafficking.

Clathrin-associated trafficking is a major mechanism for intracellular communication, as well as for cells to communicate with the extracellular environment. A decreased oxygen availability termed hypoxia has been described to influence this mechanism in the past. Mostly biochemical studies were applied in these analyses, which miss spatiotemporal information. We have applied live cell microscopy and a newly developed analysis script in combination with a GFP-tagged clathrin-expressing cell line to obtain insight into the dynamics of the effect of hypoxia. Number, mobility and directionality of clathrin-coated vesicles were analysed in non-stimulated cells as well as after stimulation with epidermal growth factor (EGF) or transferrin in normoxic and hypoxic conditions. These data reveal cargo-specific effects, which would not be observable with biochemical methods or with fixed cells and add to the understanding of cell physiology in hypoxia. The stimulus-dependent consequences were also reflected in the final cellular output, i.e. decreased EGF signaling and in contrast increased iron uptake in hypoxia.

IDH3γ functions as a redox switch regulating mitochondrial energy metabolism and contractility in the heart.

Redox signaling and cardiac function are tightly linked. However, it is largely unknown which protein targets are affected by hydrogen peroxide (H2O2) in cardiomyocytes that underly impaired inotropic effects during oxidative stress. Here, we combine a chemogenetic mouse model (HyPer-DAO mice) and a redox-proteomics approach to identify redox sensitive proteins. Using the HyPer-DAO mice, we demonstrate that increased endogenous production of H2O2 in cardiomyocytes leads to a reversible impairment of cardiac contractility in vivo. Notably, we identify the γ-subunit of the TCA cycle enzyme isocitrate dehydrogenase (IDH)3 as a redox switch, linking its modification to altered mitochondrial metabolism. Using microsecond molecular dynamics simulations and experiments using cysteine-gene-edited cells reveal that IDH3γ Cys148 and 284 are critically involved in the H2O2-dependent regulation of IDH3 activity. Our findings provide an unexpected mechanism by which mitochondrial metabolism can be modulated through redox signaling processes.

In vivo dynamics of acidosis and oxidative stress in the acute phase of an ischemic stroke in a rodent model.

Ischemic cerebral stroke is one of the leading causes of death and disability in humans. However, molecular processes underlying the development of this pathology remain poorly understood. There are major gaps in our understanding of metabolic changes that occur in the brain tissue during the early stages of ischemia and reperfusion. In particular, it is generally accepted that both ischemia (I) and reperfusion (R) generate reactive oxygen species (ROS) that cause oxidative stress which is one of the main drivers of the pathology, although ROS generation during I/R was never demonstrated in vivo due to the lack of suitable methods. In the present study, we record for the first time the dynamics of intracellular pH and H2O2 during I/R in cultured neurons and during experimental stroke in rats using the latest generation of genetically encoded biosensors SypHer3s and HyPer7. We detect a buildup of powerful acidosis in the brain tissue that overlaps with the ischemic core from the first seconds of pathogenesis. At the same time, no significant H2O2 generation was found in the acute phase of ischemia/reperfusion. HyPer7 oxidation in the brain was detected only 24 h later. Comparison of in vivo experiments with studies on cultured neurons under I/R demonstrates that the dynamics of metabolic processes in these models significantly differ, suggesting that a cell culture is a poor predictor of metabolic events in vivo.

Direct optogenetic stimulation of smooth muscle cells to control gastric contractility.

Rationale: Antral peristalsis is responsible for gastric emptying. Its failure is called gastroparesis and often caused by dysfunction of enteric neurons and interstitial cells of Cajal (ICC). Current treatment options, including gastric electrical stimulation, are non-satisfying and may improve symptoms but commonly fail to restore gastric emptying. Herein, we explore direct optogenetic stimulation of smooth muscle cells (SMC) via the light-gated non-selective cation channel Channelrhodopsin2 (ChR2) to control gastric motor function. Methods: We used a transgenic mouse model expressing ChR2 in fusion with eYFP under the control of the chicken-ß-actin promoter. We performed patch clamp experiments to quantify light-induced currents in isolated SMC, Ca2+ imaging and isometric force measurements of antral smooth muscle strips as well as pressure recordings of intact stomachs to evaluate contractile responses. Light-induced propulsion of gastric contents from the isolated stomach preparation was quantified in video recordings. We furthermore tested optogenetic stimulation in a gastroparesis model induced by neuronal- and ICC-specific damage through methylene blue photo-toxicity. Results: In the stomachs, eYFP signals were restricted to SMC in which blue light (460 nm) induced inward currents typical for ChR2. These depolarizing currents led to contractions in antral smooth muscle strips that were stronger than those triggered by supramaximal electrical field stimulation and comparable to those evoked by global depolarization with high K+ concentration. In the intact stomach, panoramic illumination efficiently increased intragastric pressure achieving 239±46% (n=6) of the pressure induced by electrical field stimulation and triggered gastric transport. Within the gastroparesis model, electric field stimulation completely failed but light still efficiently generated pressure waves. Conclusions: We demonstrate direct optogenetic stimulation of SMC to control gastric contractility. This completely new approach could allow for the restoration of motility in gastroparesis in the future.

Hypoxia suppresses myofibroblast differentiation by changing RhoA activity.

Fibroblasts show a high range of phenotypic plasticity, including transdifferentiation into myofibroblasts. Myofibroblasts are responsible for generation of the contraction forces that are important for wound healing and scar formation. Overactive myofibroblasts, by contrast, are involved in abnormal scarring. Cell stretching and extracellular signals such as transforming growth factor ß can induce the myofibroblastic program, whereas microenvironmental conditions such as reduced tissue oxygenation have an inhibitory effect. We investigated the effects of hypoxia on myofibroblastic properties and linked this to RhoA activity. Hypoxia reversed the myofibroblastic phenotype of primary fibroblasts. This was accompanied by decreased αSMA (ACTA2) expression, alterations in cell contractility, actin reorganization and RhoA activity. We identified a hypoxia-inducible induction of ARHGAP29, which is critically involved in myocardin-related transcription factor-A (MRTF-A) signaling, the differentiation state of myofibroblasts and modulates RhoA activity. This novel link between hypoxia and MRTF-A signaling is likely to be important for ischemia-induced tissue remodeling and the fibrotic response.This article has an associated First Person interview with the first author of the paper.

Red fluorescent redox-sensitive biosensor Grx1-roCherry.

Redox-sensitive fluorescent proteins (roFPs) are a powerful tool for imaging intracellular redox changes. The structure of these proteins contains a pair of cysteines capable of forming a disulfide upon oxidation that affects the protein conformation and spectral characteristics. To date, a palette of such biosensors covers the spectral range from blue to red. However, most of the roFPs suffer from either poor brightness or high pH-dependency, or both. Moreover, there is no roRFP with the redox potential close to that of 2GSH/GSSG redox pair. In the present work, we describe Grx1-roCherry, the first red roFP with canonical FP topology and fluorescent excitation/emission spectra of typical RFP. Grx1-roCherry, with a midpoint redox potential of - 311 mV, is characterized by high brightness and increased pH stability (pKa 6.7). We successfully used Grx1-roCherry in combination with other biosensors in a multiparameter imaging mode to demonstrate redox changes in cells under various metabolic perturbations, including hypoxia/reoxygenation. In particular, using simultaneous expression of Grx1-roCherry and its green analog in various compartments of living cells, we demonstrated that local H2O2 production leads to compartment-specific and cell-type-specific changes in the 2GSH/GSSG ratio. Finally, we demonstrate the utility of Grx1-roCherry for in vivo redox imaging.

The Serotonin Receptor Subtype 5b Specifically Interacts with Serotonin Receptor Subtype 1A.

Previously, we described the dysregulation of serotonin (5-HT) receptor subtype 5b (5-ht5b) in a mouse model of Rett syndrome (RTT). 5-ht5b has not been extensively studied, so we set out to characterize it in more detail. Unlike common cell surface receptors, 5-ht5b displays no membrane expression, while receptor clusters are located in endosomes. This unusual subcellular localization is at least in part controlled by glycosylation of the N-terminus, with 5-ht5b possessing fewer glycosylation sites than related receptors. We analyzed whether the localization to endosomes has any functional relevance and found that 5-ht5b receptors can specifically interact with 5-HT1A receptors and retain them in endosomal compartments. This interaction reduces 5-HT1A surface expression and is mediated by interactions between the fourth and fifth trans-membrane domain (TMD). This possibly represents a mechanism by which 5-ht5b receptors regulate the activity of other 5-HT receptor.

SUMO and ubiquitin-dependent XPC exchange drives nucleotide excision repair.

XPC recognizes UV-induced DNA lesions and initiates their removal by nucleotide excision repair (NER). Damage recognition in NER is tightly controlled by ubiquitin and SUMO modifications. Recent studies have shown that the SUMO-targeted ubiquitin ligase RNF111 promotes K63-linked ubiquitylation of SUMOylated XPC after DNA damage. However, the exact regulatory function of these modifications in vivo remains elusive. Here we show that RNF111 is required for efficient repair of ultraviolet-induced DNA lesions. RNF111-mediated ubiquitylation promotes the release of XPC from damaged DNA after NER initiation, and is needed for stable incorporation of the NER endonucleases XPG and ERCC1/XPF. Our data suggest that RNF111, together with the CRL4(DDB2) ubiquitin ligase complex, is responsible for sequential XPC ubiquitylation, which regulates the recruitment and release of XPC and is crucial for efficient progression of the NER reaction, thereby providing an extra layer of quality control of NER.

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells.

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.

RNF111/Arkadia is a SUMO-targeted ubiquitin ligase that facilitates the DNA damage response.

Protein modifications by ubiquitin and small ubiquitin-like modifier (SUMO) play key roles in cellular signaling pathways. SUMO-targeted ubiquitin ligases (STUbLs) directly couple these modifications by selectively recognizing SUMOylated target proteins through SUMO-interacting motifs (SIMs), promoting their K48-linked ubiquitylation and degradation. Only a single mammalian STUbL, RNF4, has been identified. We show that human RNF111/Arkadia is a new STUbL, which used three adjacent SIMs for specific recognition of poly-SUMO2/3 chains, and used Ubc13-Mms2 as a cognate E2 enzyme to promote nonproteolytic, K63-linked ubiquitylation of SUMOylated target proteins. We demonstrate that RNF111 promoted ubiquitylation of SUMOylated XPC (xeroderma pigmentosum C) protein, a central DNA damage recognition factor in nucleotide excision repair (NER) extensively regulated by ultraviolet (UV)-induced SUMOylation and ubiquitylation. Moreover, we show that RNF111 facilitated NER by regulating the recruitment of XPC to UV-damaged DNA. Our findings establish RNF111 as a new STUbL that directly links nonproteolytic ubiquitylation and SUMOylation in the DNA damage response.