RESUMO
Ferroptosis is a type of programmed cell death distinct from apoptosis and necroptosis that plays an essential role in pathophysiological conditions such as neurodegenerative diseases and tumorigenesis. Massive lipid oxidation in an iron-dependent manner is a hallmark of ferroptosis.This modality of cell death is also characterized by perturbation of several metabolic pathways, predominantly fatty acid metabolism, thiol metabolism, iron homeostasis and the mevalonate pathway. We aimed to acquire data from different timepoints of ferroptotic death in order to get information about the primary and delayed phases of the ferroptotic response. For this purpose, we used model Pfa1 cells, which are 4-OH-TAM-inducible Gpx4-/- mouse immortalized fibroblasts [1]. GPX4 is one of the main intracellular ferroptosis regulators and inhibiting it is a classic approach to induce ferroptosis. Measuring protein fold changes at different ferroptotic stages and in nontreated Pfa1 cells could give useful information on the activation of genes involved in ferroptosis and non-genomic protein regulation during ferroptotic progression. Bottom-up proteomic data were acquired from samples obtained 24 and 48 hours after genetic induction of ferroptosis. Chromato-mass spectra were registered in DDA mode and are suitable for further label-free quantification. These data might be a valuable proteome basis for further investigation of ferroptosis and complement other available omics.
RESUMO
We demonstrate label-free imaging of genetically induced hepatocellular carcinoma (HCC) in a murine model provided by two- and three-photon fluorescence microscopy of endogenous fluorophores excited at the central wavelengths of 790, 980 and 1250 nm and reinforced by second and third harmonic generation microscopy. We show, that autofluorescence imaging presents abundant information about cell arrangement and lipid accumulation in hepatocytes and hepatic stellate cells (HSCs), harmonics generation microscopy provides a versatile tool for fibrogenesis and steatosis study. Multimodal images may be performed by a single ultrafast laser source at 1250 nm falling in tissue transparency window. Various grades of HCC are examined revealing fibrosis, steatosis, liver cell dysplasia, activation of HSCs and hepatocyte necrosis, that shows a great ability of multimodal label-free microscopy to intravital visualization of liver pathology development.
Assuntos
Carcinoma Hepatocelular , Fígado Gorduroso , Neoplasias Hepáticas , Camundongos , Animais , Carcinoma Hepatocelular/diagnóstico por imagem , Neoplasias Hepáticas/diagnóstico por imagem , Hepatócitos , Células Estreladas do Fígado/patologia , Microscopia/métodosRESUMO
Reactive oxygen species (ROS) are highly reactive products of the cell metabolism derived from oxygen molecules, and their abundant level is observed in many diseases, particularly tumors, such as hepatocellular carcinoma (HCC). In vivo imaging of ROS is a necessary tool in preclinical research to evaluate the efficacy of drugs with antioxidant activity and for diagnosis and monitoring of diseases. However, most known sensors cannot be used for in vivo experiments due to low stability in the blood and rapid elimination from the body. In this work, we focused on the development of an effective delivery system of fluorescent probes for intravital ROS visualization using the HCC model. We have synthesized various lipid nanoparticles (LNPs) loaded with ROS-inducible hydrocyanine pro-fluorescent dye or plasmid DNA (pDNA) with genetically encoded protein sensors of hydrogen peroxide (HyPer7). LNP with an average diameter of 110 ± 12 nm, characterized by increased stability and pDNA loading efficiency (64 ± 7%), demonstrated preferable accumulation in the liver compared to 170 nm LNPs. We evaluated cytotoxicity and demonstrated the efficacy of hydrocyanine-5 and HyPer7 formulated in LNP for ROS visualization in mouse hepatocytes (AML12 cells) and in the mouse xenograft model of HCC. Our results demonstrate that obtained LNP could be a valuable tool in preclinical research for visualization ROS in liver diseases.
RESUMO
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.
RESUMO
Zyxin is a cytoskeletal LIM-domain protein that regulates actin cytoskeleton assembly and gene expression. In the present work, we find that zyxin downregulation in Xenopus laevis embryos reduces the expression of numerous genes that regulate cell differentiation, but it enhances that of several genes responsible for embryonic stem cell status, specifically klf4, pou5f3.1, pou5f3.2, pou5f3.3, and vent2.1/2. For pou5f3 family genes (mammalian POU5F1/OCT4 homologs), we show that this effect is the result of mRNA stabilization due to complex formation with the Y-box protein Ybx1. When bound to Ybx1, zyxin interferes with the formation of these complexes, thereby stimulating pou5f3 mRNA degradation. In addition, in zebrafish embryos and human HEK293 cells, zyxin downregulation increases mRNA levels of the pluripotency genes KLF4, NANOG, and POU5F1/OCT4. Our findings indicate that zyxin may play a role as a switch among morphogenetic cell movement, differentiation, and embryonic stem cell status.
Assuntos
Células-Tronco Embrionárias/metabolismo , Zixina/metabolismo , Zixina/fisiologia , Animais , Padronização Corporal/genética , Diferenciação Celular/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Células-Tronco Embrionárias/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Células HEK293 , Humanos , Fator 4 Semelhante a Kruppel , Morfogênese , Placa Neural/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Xenopus laevis/metabolismo , Peixe-Zebra/metabolismoRESUMO
Hydrogen peroxide (H2O2) is a key redox intermediate generated within cells. Existing probes for H2O2 have not solved the problem of detection of the ultra-low concentrations of the oxidant: these reporters are not sensitive enough, or pH-dependent, or insufficiently bright, or not functional in mammalian cells, or have poor dynamic range. Here we present HyPer7, the first bright, pH-stable, ultrafast, and ultrasensitive ratiometric H2O2 probe. HyPer7 is fully functional in mammalian cells and in other higher eukaryotes. The probe consists of a circularly permuted GFP integrated into the ultrasensitive OxyR domain from Neisseria meningitidis. Using HyPer7, we were able to uncover the details of H2O2 diffusion from the mitochondrial matrix, to find a functional output of H2O2 gradients in polarized cells, and to prove the existence of H2O2 gradients in wounded tissue in vivo. Overall, HyPer7 is a probe of choice for real-time H2O2 imaging in various biological contexts.
Assuntos
Movimento Celular , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Oxidantes/metabolismo , Animais , Transporte Biológico , Extensões da Superfície Celular/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Células HeLa , Humanos , Imageamento Tridimensional , Larva/metabolismo , Membranas Mitocondriais/metabolismo , Peixe-ZebraRESUMO
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 -â¯311â¯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.
Assuntos
Técnicas Biossensoriais , Glutarredoxinas/genética , Proteínas Luminescentes/genética , Oxirredução , Proteínas Recombinantes de Fusão , Animais , Expressão Gênica , Genes Reporter , Glutarredoxinas/metabolismo , Glutationa/metabolismo , Dissulfeto de Glutationa/metabolismo , Glicólise , Células HEK293 , Células HeLa , Humanos , Hipóxia/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Estresse Oxidativo , Peixe-ZebraRESUMO
Thermogenetics is a promising innovative neurostimulation technique, which enables robust activation of neurons using thermosensitive transient receptor potential (TRP) cation channels. Broader application of this approach in neuroscience is, however, hindered by a limited variety of suitable ion channels, and by low spatial and temporal resolution of neuronal activation when TRP channels are activated by ambient temperature variations or chemical agonists. Here, we demonstrate rapid, robust and reproducible repeated activation of snake TRPA1 channels heterologously expressed in non-neuronal cells, mouse neurons and zebrafish neurons in vivo by infrared (IR) laser radiation. A fibre-optic probe that integrates a nitrogen-vacancy (NV) diamond quantum sensor with optical and microwave waveguide delivery enables thermometry with single-cell resolution, allowing neurons to be activated by exceptionally mild heating, thus preventing the damaging effects of excessive heat. The neuronal responses to the activation by IR laser radiation are fully characterized using Ca2+ imaging and electrophysiology, providing, for the first time, a complete framework for a thermogenetic manipulation of individual neurons using IR light.