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

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.

Monitoring cellular redox dynamics using newly developed BRET-based redox sensor proteins.

Reactive oxygen species are key factors that strongly affect the cellular redox state and regulate various physiological and cellular phenomena. To monitor changes in the redox state, we previously developed fluorescent redox sensors named Re-Q, the emissions of which are quenched under reduced conditions. However, such fluorescent probes are unsuitable for use in the cells of photosynthetic organisms because they require photoexcitation that may change intracellular conditions and induce autofluorescence, primarily in chlorophylls. In addition, the presence of various chromophore pigments may interfere with fluorescence-based measurements because of their strong absorbance. To overcome these problems, we adopted the bioluminescence resonance energy transfer (BRET) mechanism for the sensor and developed two BRET-based redox sensors by fusing cyan fluorescent protein-based or yellow fluorescent protein-based Re-Q with the luminescent protein Nluc. We named the resulting redox-sensitive BRET-based indicator probes "ROBINc" and "ROBINy." ROBINc is pH insensitive, which is especially vital for observation in photosynthetic organisms. By using these sensors, we successfully observed dynamic redox changes caused by an anticancer agent in HeLa cells and light/dark-dependent redox changes in the cells of photosynthetic cyanobacterium Synechocystis sp. PCC 6803. Since the newly developed sensors do not require excitation light, they should be especially useful for visualizing intracellular phenomena caused by redox changes in cells containing colored pigments.

A luminescent Nanoluc-GFP fusion protein enables readout of cellular pH in photosynthetic organisms.

pH is one of the most critical physiological parameters determining vital cellular activities, such as photosynthetic performance. Fluorescent sensor proteins capable of measuring in situ pH in animal cells have been reported. However, these proteins require an excitation laser for pH measurement that may affect photosynthetic performance and induce autofluorescence from chlorophyll. As a result, it is not possible to measure the intracellular or intraorganelle pH changes in plants. To overcome this problem, we developed a luminescent pH sensor by fusing the luminescent protein Nanoluc to a uniquely designed pH-sensitive GFP variant protein. In this system, an excitation laser is unnecessary because the fused GFP variant reports on the luminescent signal by bioluminescence resonance energy transfer from Nanoluc. The ratio of two luminescent peaks from the sensor protein was approximately linear with respect to pH in the range of 7.0 to 8.5. We designated this sensor protein as "luminescent pH indicator protein" (Luphin). We applied Luphin to the in situ pH measurement of a photosynthetic organism under fluctuating light conditions, allowing us to successfully observe the cytosolic pH changes associated with photosynthetic electron transfer in the cyanobacterium Synechocystis sp. PCC 6803. Detailed analyses of the mechanisms of the observed estimated pH changes in the cytosol in this alga suggested that the photosynthetic electron transfer is suppressed by the reduced plastoquinone pool under light conditions. These results indicate that Luphin may serve as a helpful tool to further illuminate pH-dependent processes throughout the photosynthetic organisms.

The thioredoxin (Trx) redox state sensor protein can visualize Trx activities in the light/dark response in chloroplasts.

Thiol-based redox regulation via ferredoxin-thioredoxin (Trx) reductase/Trx controls various functions in chloroplasts in response to light/dark changes. Trx is a key factor of this regulatory system, and five Trx subtypes, including 10 isoforms, have been identified as chloroplast-localized forms in Arabidopsis thaliana These subtypes display distinct target selectivity, and, consequently, they form a complicated redox regulation network in chloroplasts. In this study, we developed a FRET-based sensor protein by combining CFP, YFP, and the N-terminal region of CP12, a redox-sensitive regulatory and Trx-targeted protein in chloroplasts. This sensor protein enabled us to monitor the redox change of chloroplast thioredoxin in vivo, and we therefore designated this protein "change in redox state of Trx" (CROST). Using CP12 isoforms, we successfully prepared two types of CROST sensors that displayed different affinities for two major chloroplast Trx isoforms (f-type and m-type). These sensor proteins helped unravel the real-time redox dynamics of Trx molecules in chloroplasts during the light/dark transition.