scholarly journals Real-time monitoring of the in vivo redox state transition using the ratiometric redox state sensor protein FROG/B

2020 ◽  
Vol 117 (27) ◽  
pp. 16019-16026 ◽  
Author(s):  
Kazunori Sugiura ◽  
Shoko Mihara ◽  
Nae Fu ◽  
Toru Hisabori
Keyword(s):  
Redox State ◽  
Fluorescent Protein ◽  
Fluorescent Sensor ◽  
Blue Emission ◽  
Redox Potentials ◽  
Excitation Peak ◽  
Redox Sensor ◽  
Sensor Protein ◽  
Sensor Proteins ◽  
Intracellular Redox

The intracellular redox state is one of the key factors regulating various physiological phenomena in the cell. Monitoring this state is therefore important for understanding physiological homeostasis in cells. Various fluorescent sensor proteins have already been developed to monitor intracellular redox state. We also developed fluorescent redox sensor proteins named Oba-Q and Re-Q, the emissions of which are quenched under oxidized and reduced conditions, respectively. Although these sensors were useful to visualize the redox changes in the cell over time, they have the weakness that their emission signals are directly influenced by their in situ expression levels. To overcome this problem, we developed a redox sensor protein with a single excitation peak and dual variable emission peaks. This sensor protein shows green emission under oxidized conditions and blue emission under reduced conditions. We therefore named this sensor FROG/B, fluorescent protein with redox-dependent change in green/blue. By using this sensor, we successfully measured the changes in intracellular redox potentials in cyanobacterial cells quantitatively caused by light/dark transition just by calculating the ratio of emission between green and blue signals.

scholarly journals Redox sensor proteins for highly sensitive direct imaging of intracellular redox state

2015 ◽  
Vol 457 (3) ◽  
pp. 242-248 ◽  
Author(s):  
Kazunori Sugiura ◽  
Takeharu Nagai ◽  
Masahiro Nakano ◽  
Hiroshi Ichinose ◽  
Takakazu Nakabayashi ◽  
...  
Keyword(s):  
Redox State ◽  
Direct Imaging ◽  
Redox Sensor ◽  
Highly Sensitive ◽  
Sensor Proteins ◽  
Intracellular Redox

scholarly journals An Epithelial Ca2+-Sensor Protein is an Alternative to Calmodulin to Compose Functional KCNQ1 Channels

2015 ◽  
Vol 36 (5) ◽  
pp. 1847-1861 ◽  
Author(s):  
Atsushi Inanobe ◽  
Chizuru Tsuzuki ◽  
Yoshihisa Kurachi
Keyword(s):  
Fluorescent Protein ◽  
Size Exclusion ◽  
Cellular Functions ◽  
Kcnq Channels ◽  
C Terminus ◽  
Sensor Protein ◽  
Voltage Dependent ◽  
Exclusion Chromatography ◽  
Green Fluorescent ◽  
Sensor Proteins

Background/Aims: KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+-sensor protein, calmodulin (CaM), to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. Methods: We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL) and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. Results: When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. Conclusion: Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels.

Monitoring cellular redox state under hypoxia using a fluorescent sensor based on eel fluorescent protein

2018 ◽  
Vol 120 ◽  
pp. 255-265 ◽  
Author(s):  
Hanyang Hu ◽  
Aoxue Wang ◽  
Li Huang ◽  
Yejun Zou ◽  
Yanfang Gu ◽  
...  
Keyword(s):  
Redox State ◽  
Fluorescent Protein ◽  
Fluorescent Sensor ◽  
Cellular Redox ◽  
Cellular Redox State

scholarly journals How Bacterial Redox Sensors Transmit Redox Signals via Structural Changes

Antioxidants ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 502
Author(s):  
In-Gyun Lee ◽  
Bong-Jin Lee
Keyword(s):  
Structural Biology ◽  
Redox State ◽  
Structural Changes ◽  
Normal Growth ◽  
Chemical Information ◽  
Cellular Redox ◽  
Downstream Signaling ◽  
Redox Sensor ◽  
Protective Strategies ◽  
Sensor Proteins

Bacteria, like humans, face diverse kinds of stress during life. Oxidative stress, which is produced by cellular metabolism and environmental factors, can significantly damage cellular macromolecules, ultimately negatively affecting the normal growth of the cell. Therefore, bacteria have evolved a number of protective strategies to defend themselves and respond to imposed stress by changing the expression pattern of genes whose products are required to convert harmful oxidants into harmless products. Structural biology combined with biochemical studies has revealed the mechanisms by which various bacterial redox sensor proteins recognize the cellular redox state and transform chemical information into structural signals to regulate downstream signaling pathways.

scholarly journals ATP compartmentation in plastids and cytosol ofArabidopsis thalianarevealed by fluorescent protein sensing

2018 ◽  
Vol 115 (45) ◽  
pp. E10778-E10787 ◽  
Author(s):  
Chia Pao Voon ◽  
Xiaoqian Guan ◽  
Yuzhe Sun ◽  
Abira Sahu ◽  
May Ngor Chan ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Fluorescent Protein ◽  
Higher Plants ◽  
Electron Flow ◽  
Cyclic Electron Flow ◽  
Linear Electron ◽  
Protein Sensing ◽  
Atp Production ◽  
Respiratory Inhibitors ◽  
Sensor Protein

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin–Benson–Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in livingArabidopsis thalianaseedlings, we found that MgATP2−concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) inArabidopsismesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

scholarly journals Resolving diurnal dynamics of the chloroplastic glutathione redox state in Arabidopsis reveals its photosynthetically-derived oxidation

2021 ◽  
Author(s):  
Zechariah Haber ◽  
Nardy Lampl ◽  
Andreas J Meyer ◽  
Einat Zelinger ◽  
Matanel Hipsch ◽  
...  
Keyword(s):  
Redox State ◽  
Fluorescent Protein ◽  
Operating Efficiency ◽  
High Temporal Resolution ◽  
Fluctuating Light ◽  
Gsh Metabolism ◽  
Glutathione Cycle ◽  
Green Fluorescent ◽  
Glutathione Redox

Abstract Plants are subjected to fluctuations in light intensity, and this causes unbalanced photosynthetic electron fluxes and overproduction of reactive oxygen species (ROS). Electrons needed for ROS detoxification are drawn, at least partially, from the cellular glutathione (GSH) pool via the ascorbate-glutathione cycle. Here, we explore the dynamics of the chloroplastic glutathione redox potential (chl-EGSH) using high-temporal-resolution monitoring of Arabidopsis (Arabidopsis thaliana) lines expressing the reduction-oxidation sensitive green fluorescent protein 2 (roGFP2in chloroplasts. This was carried out over several days, under dynamic environmental conditions and in correlation with PSII operating efficiency. Peaks in chl-EGSH oxidation during dark-to-light and light-to-dark transitions were observed. Increasing light intensities triggered a binary oxidation response, with a threshold around the light saturating point, suggesting two regulated oxidative states of the chl-EGSH. These patterns were not affected in npq1 plants, which are impaired in nonphotochemical quenching. Oscillations between the two oxidation states were observed under fluctuating light in WT and npq1 plants, but not in pgr5 plants, suggesting a role for PSI photoinhibition in regulating the chl-EGSH dynamics. Remarkably, pgr5 plants showed an increase in chl-EGSH oxidation during the nights following light stresses, linking daytime photoinhibition and nighttime GSH metabolism. This work provides a systematic view of the dynamics of the in vivo chloroplastic glutathione redox state during varying light conditions.

Intracellular Redox State and Stimulation of Gluconeogenesis by Glucagon and Norepinephrine in the Perfused Rat Liver

1980 ◽  
Vol 87 (1) ◽  
pp. 153-166 ◽  
Author(s):  
Tsukasa SUGANO ◽  
Masakazu SHIOTA ◽  
Takashi TANAKA ◽  
Yhoichi MIYAMAE ◽  
Masakazu SHIMADA ◽  
...  
Keyword(s):  
Rat Liver ◽  
Redox State ◽  
Perfused Rat Liver ◽  
Stimulation Of ◽  
Intracellular Redox
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