Genetically encoded biosensors Dotaz Zobrazit nápovědu
BACKGROUND AND AIM: Osmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. EXPERIMENTAL PROCEDURE: Protective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. KEY RESULTS: Quercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. CONCLUSIONS AND IMPLICATIONS: Quercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.
The strength of an excitatory synapse depends on its ability to release glutamate and on the density of postsynaptic receptors. Genetically encoded glutamate indicators (GEGIs) allow eavesdropping on synaptic transmission at the level of cleft glutamate to investigate properties of the release machinery in detail. Based on the sensor iGluSnFR, we recently developed accelerated versions of GEGIs that allow investigation of synaptic release during 100-Hz trains. Here, we describe the detailed procedures for design and characterization of fast iGluSnFR variants in vitro, transfection of pyramidal cells in organotypic hippocampal cultures, and imaging of evoked glutamate transients with two-photon laser-scanning microscopy. As the released glutamate spreads from a point source-the fusing vesicle-it is possible to localize the vesicle fusion site with a precision exceeding the optical resolution of the microscope. By using a spiral scan path, the temporal resolution can be increased to 1 kHz to capture the peak amplitude of fast iGluSnFR transients. The typical time frame for these experiments is 30 min per synapse.
- MeSH
- biosenzitivní techniky metody MeSH
- hipokampální oblast CA3 cytologie MeSH
- konfokální mikroskopie MeSH
- kultivované buňky MeSH
- kyselina glutamová analýza chemie metabolismus MeSH
- lidé MeSH
- molekulární sondy analýza chemie genetika metabolismus MeSH
- nervový přenos genetika fyziologie MeSH
- optické zobrazování MeSH
- transfekce MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Visualization of electrical activity in living cells represents an important challenge in context of basic neurophysiological studies. Here we report a new voltage sensitive fluorescent indicator which response could be detected by fluorescence monitoring in a single red channel. To the best of our knowledge, this is the first fluorescent protein-based voltage sensor which uses insertion-into-circular permutant topology to provide an efficient interaction between sensitive and reporter domains. Its fluorescent core originates from red fluorescent protein (FP) FusionRed, which has optimal spectral characteristics to be used in whole body imaging techniques. Indicators using the same domain topology could become a new perspective for the FP-based voltage sensors that are traditionally based on Förster resonance energy transfer (FRET).
- MeSH
- biosenzitivní techniky metody MeSH
- elektrofyziologické jevy MeSH
- fluorescenční barviva metabolismus MeSH
- HEK293 buňky MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- luminescentní proteiny chemie MeSH
- nádorové buněčné linie MeSH
- proteinové domény MeSH
- proteinové inženýrství metody MeSH
- rezonanční přenos fluorescenční energie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Despite the urgent need for assays to visualize insulin secretion there is to date no reliable method available for measuring insulin release from single cells. To address this need, we developed a genetically encoded reporter termed RINS1 based on proinsulin superfolder GFP (sfGFP) and mCherry fusions for monitoring insulin secretion. RINS1 expression in MIN6 β cells resulted in proper processing yielding single-labeled insulin species. Unexpectedly, glucose or drug stimulation of insulin secretion in β cells led to the preferential release of the insulin-sfGFP construct, while the mCherry-fused C-peptide remained trapped in exocytic granules. This physical separation was used to monitor glucose-stimulated insulin secretion ratiometrically by total internal reflection fluorescence microscopy in single MIN6 and primary mouse β cells. Further, RINS1 enabled parallel monitoring of pulsatile insulin release in tolbutamide-treated β cells, demonstrating the potential of RINS1 for investigations of antidiabetic drug candidates at the single-cell level.
- MeSH
- beta-buňky cytologie účinky léků metabolismus sekrece MeSH
- biosenzitivní techniky MeSH
- buněčné linie MeSH
- fluorescenční mikroskopie MeSH
- glukosa farmakologie MeSH
- hypoglykemika farmakologie MeSH
- inzulin metabolismus sekrece MeSH
- luminescentní proteiny genetika metabolismus MeSH
- myši MeSH
- rekombinantní fúzní proteiny metabolismus sekrece MeSH
- reportérové geny MeSH
- tolbutamid farmakologie MeSH
- vápník metabolismus MeSH
- zelené fluorescenční proteiny genetika metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Plant hormones are a group of naturally occurring, low-abundance organic compounds that influence physiological processes in plants. Our knowledge of the distribution profiles of phytohormones in plant organs, tissues, and cells is still incomplete, but advances in mass spectrometry have enabled significant progress in tissue- and cell-type-specific analyses of phytohormones over the last decade. Mass spectrometry is able to simultaneously identify and quantify hormones and their related substances. Biosensors, on the other hand, offer continuous monitoring; can visualize local distributions and real-time quantification; and, in the case of genetically encoded biosensors, are noninvasive. Thus, biosensors offer additional, complementary technologies for determining temporal and spatial changes in phytohormone concentrations. In this review, we focus on recent advances in mass spectrometry-based quantification, describe monitoring systems based on biosensors, and discuss validations of the various methods before looking ahead at future developments for both approaches.
