Extending roGFP Emission via Förster-Type Resonance Energy Transfer Relay Enables Simultaneous Dual Compartment Ratiometric Redox Imaging in Live Cells
| Autor: | Jordan Snaider, Kiet Tat, Libai Huang, Keelan J. Trull, Mathew Tantama, Stevie Norcross, Sara Doan |
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| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Models Molecular Cell Survival Green Fluorescent Proteins Bioengineering Biology 010402 general chemistry medicine.disease_cause 01 natural sciences Protein Structure Secondary Article RoGFP Green fluorescent protein 03 medical and health sciences Cytosol Live cell imaging Cell Line Tumor medicine Fluorescence Resonance Energy Transfer Humans Amino Acid Sequence Instrumentation Cellular compartment Fluid Flow and Transfer Processes Process Chemistry and Technology Compartment (chemistry) 0104 chemical sciences Cell biology Mitochondria Molecular Imaging Oxidative Stress 030104 developmental biology Förster resonance energy transfer Oxidation-Reduction Oxidative stress |
| Zdroj: | ACS sensors. 2(11) |
| ISSN: | 2379-3694 |
| Popis: | Reactive oxygen species (ROS) mediate both intercellular and intraorganellar signaling, and ROS propagate oxidative stress between cellular compartments such as mitochondria and the cytosol. Each cellular compartment contains its own sources of ROS as well as antioxidant mechanisms, which contribute to dynamic fluctuations in ROS levels that occur during signaling, metabolism, and stress. However, the coupling of redox dynamics between cellular compartments has not been well studied because of the lack of available sensors to simultaneously measure more than one subcellular compartment in the same cell. Currently, the redox-sensitive green fluorescent protein, roGFP, has been used extensively to study compartment-specific redox dynamics because it provides a quantitative ratiometric readout and it is amenable to subcellular targeting as a genetically-encoded sensor. Here, we report a new family of genetically-encoded fluorescent protein sensors that extend the fluorescence emission of roGFP via Förster-type resonance energy transfer to an acceptor red fluorescent protein for dual-color live-cell microscopy. We characterize the redox and optical properties of the sensor proteins, and we demonstrate that they can be used to simultaneously measure cytosolic and mitochondrial ROS in living cells. Furthermore, we use these sensors to reveal cell-to-cell heterogeneity in redox coupling between the cytosol and mitochondria when neuroblastoma cells are exposed to reductive and metabolic stresses. |
| Databáze: | OpenAIRE |
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