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

ABSTRACT: 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.

SUBMITTER: Sugiura K

PROVIDER: S-EPMC7354920 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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

Sugiura Kazunori K Mihara Shoko S Fu Nae N Hisabori Toru T

Proceedings of the National Academy of Sciences of the United States of America 20200623 27

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 w ...[more]

PMID: 32576684

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Project description:The performance of supramolecular nanocarriers as drug delivery systems depends on their stability in the complex and dynamic biological media. After administration, nanocarriers are challenged by physiological barriers such as shear stress and proteins present in blood, endothelial wall, extracellular matrix, and eventually cancer cell membrane. While early disassembly will result in a premature drug release, extreme stability of the nanocarriers can lead to poor drug release and low efficiency. Therefore, comprehensive understanding of the stability and assembly state of supramolecular carriers in each stage of delivery is the key factor for the rational design of these systems. One of the main challenges is that current 2D in vitro models do not provide exhaustive information, as they fail to recapitulate the 3D tumor microenvironment. This deficiency in the 2D model complexity is the main reason for the differences observed in vivo when testing the performance of supramolecular nanocarriers. Herein, we present a real-time monitoring study of self-assembled micelles stability and extravasation, combining spectral confocal microscopy and a microfluidic cancer-on-a-chip. The combination of advanced imaging and a reliable 3D model allows tracking of micelle disassembly by following the spectral properties of the amphiphiles in space and time during the crucial steps of drug delivery. The spectrally active micelles were introduced under flow and their position and conformation continuously followed by spectral imaging during the crossing of barriers, revealing the interplay between carrier structure, micellar stability, and extravasation. Integrating the ability of the micelles to change their fluorescent properties when disassembled, spectral confocal imaging and 3D microfluidic tumor blood vessel-on-a-chip resulted in the establishment of a robust testing platform suitable for real-time imaging and evaluation of supramolecular drug delivery carrier's stability.

Dataset Information

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

Publications

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

Similar Datasets

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