Mitochondrial flashes: From indicator characterization to in vivo imaging.
Mitochondrion is an organelle critically responsible for energy production and intracellular signaling in eukaryotic cells and its dysfunction often accompanies and contributes to human disease. Superoxide is the primary reactive oxygen species (ROS) produced in mitochondria. In vivo detection of superoxide has been a challenge in biomedical research. Here we describe the methods used to characterize a circularly permuted yellow fluorescent protein (cpYFP) as a biosensor for mitochondrial superoxide and pH dynamics. In vitro characterization reveals the high selectivity of cpYFP to superoxide over other ROS species and its dual sensitivity to pH. Confocal and two-photon imaging in conjunction with transgenic expression of the biosensor cpYFP targeted to the mitochondrial matrix detects mitochondrial flash events in living cells, perfused intact hearts, and live animals. The mitochondrial flashes are discrete and stochastic single mitochondrial events triggered by transient mitochondrial permeability transition (tMPT) and composed of a bursting superoxide signal and a transient alkalization signal. The real-time monitoring of single mitochondrial flashes provides a unique tool to study the integrated dynamism of mitochondrial respiration, ROS production, pH regulation and tMPT kinetics under diverse physiological and pathophysiological conditions.
Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA 98109, USA. Electronic address: wangwang@u.washington.edu.
Wang
W
Wang
Mitochondria and Metabolism Center, Department of Anesthesiology and Pain Medicine, University of Washington, Seattle, WA 98109, USA.
Huiliang
H
Zhang
Institute of Molecular Medicine, Peking University, Beijing 100871, China.
Heping
H
Cheng
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal / Q000379:methods; D008928:Mitochondria / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000737:chemistry / Q000302:isolation & purification*; D015398:Signal Transduction; D013481:Superoxides / Q000737:chemistry / Q000302:isolation & purification*
D001426:Bacterial Proteins; D008164:Luminescent Proteins; D017382:Reactive Oxygen Species; C054562:yellow fluorescent protein, Bacteria; D013481:Superoxides
Biosensor; In vivo imaging; Mitochondria; Mitochondrial flash; Reactive oxygen species (ROS)