Huaqiang Fang

Superoxide flashes: early mitochondrial signals for oxidative stress-induced apoptosis.

Irreversible mitochondrial permeability transition and the resultant cytochrome c release signify the commitment of a cell to apoptotic death. However, the role of transient MPT (tMPT) because of flickering opening of the mitochondrial permeability transition pore remains elusive. Here we show that tMPT and the associated superoxide flashes (i.e. tMPT/superoxide flashes) constitute early mitochondrial signals during oxidative stress-induced apoptosis. Selenite (a ROS-dependent insult) but not staurosporine (a ROS-independent insult) stimulated an early and persistent increase in tMPT/superoxide flash activity prior to mitochondrial fragmentation and a global ROS rise, independently of Bax translocation and cytochrome c release. Selectively targeting tMPT/superoxide flash activity by manipulating cyclophilin D expression or scavenging mitochondrial ROS markedly impacted the progression of selenite-induced apoptosis while exerting little effect on the global ROS response. Furthermore, the tMPT/superoxide flash served as a convergence point for pro- and anti-apoptotic regulation mediated by cyclophilin D and Bcl-2 proteins. These results indicate that tMPT/superoxide flashes act as early mitochondrial signals mediating the apoptotic response during oxidative stress, and provide the first demonstration of highly efficacious local mitochondrial ROS signaling in deciding cell fate.

Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS). In intact cells, respiratory mitochondria exhibit spontaneous “superoxide flashes”, the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT). Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP, and demonstrate their coupling to whole-body glucose metabolism. Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice. In skeletal muscle, imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously. The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation, in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT. Thus, in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

Imaging ROS signaling in cells and animals

Reactive oxygen species (ROS) act as essential cellular messengers, redox regulators, and, when in excess, oxidative stressors that are widely implicated in pathologies of cancer and cardiovascular and neurodegenerative diseases. Understanding such complexity of the ROS signaling is critically hinged on the ability to visualize and quantify local, compartmental, and global ROS dynamics at high selectivity, sensitivity, and spatiotemporal resolution. The past decade has witnessed significant progress in ROS imaging at levels of intact cells, whole organs or tissues, and even live organisms. In particular, major advances include the development of novel synthetic or genetically encoded fluorescent protein-based ROS indicators, the use of protein indicator-expressing animal models, and the advent of in vivo imaging technology. Innovative ROS imaging has led to important discoveries in ROS signaling—for example, mitochondrial superoxide flashes as elemental ROS signaling events and hydrogen peroxide transients for wound healing. This review aims at providing an update of the current status in ROS imaging, while identifying areas of insufficient knowledge and highlighting emerging research directions.

Superoxide Flashes Reveal Novel Properties of Mitochondrial Reactive Oxygen Species Excitability in Cardiomyocytes

Superoxide flash represents quantal and bursting production of mitochondrial reactive oxygen species (ROS) instigated by transient opening of the mitochondrial permeability transition pore (mPTP). Given their critical role in metabolism, ischemia-reperfusion injury, and apoptosis, characterization of flash properties would be valuable to further mechanistic and physiological studies of this newly discovered mitochondrial phenomenon. Here we developed the flash detector FlashSniper based on segmentation of two-dimensional feature maps extracted from time-lapse confocal image stacks, and on the theory for correcting optical distortion of flash-amplitude histograms. Through large-scale analysis of superoxide flashes in cardiomyocytes, we demonstrated uniform mitochondrial ROS excitability among subsarcolemmal and intermyofibrillar mitochondria, and exponential distribution of intervals between consecutive flash events. Flash ignition displayed three different patterns: an abrupt rise from quiescence (44%), a rise with an exponential foot (27%), or a rise occurring after a pedestal precursor (29%), closely resembling action-potential initiation in excitable cells. However, the optical blurring-corrected amplitudes of superoxide flashes were highly variable, as were their durations, indicating stochastic automaticity of single-mitochondrion ROS excitation. Simultaneous measurement of mitochondrial membrane potential revealed that graded, rather than all-or-none, depolarization mirrored the precursor and the primary peak of the flash. We propose that superoxide flash production is a regenerative process dominated by stochastic, autonomous recruitment of a limited number of units (e.g., mPTPs) in single mitochondria.

Dendritic mitoflash as a putative signal for stabilizing long-term synaptic plasticity

Mitochondrial flashes (mitoflashes) are recently discovered excitable mitochondrial events in many cell types. Here we investigate their occurrence in the context of structural long-term potentiation (sLTP) at hippocampal synapses. At dendritic spines stimulated by electric pulses, glycine, or targeted glutamate uncaging, induction of sLTP is associated with a phasic occurrence of local, quantized mitochondrial activity in the form of one or a few mitoflashes, over a 30-min window. Low-dose nigericin or photoactivation that elicits mitoflashes stabilizes otherwise short-term spine enlargement into sLTP. Meanwhile, scavengers of reactive oxygen species suppress mitoflashes while blocking sLTP. With targeted photoactivation of mitoflashes, we further show that the stabilization of sLTP is effective within the critical 30-min time-window and a spatial extent of ~2 μm, similar to that of local diffusive reactive oxygen species. These findings indicate a potential signaling role of dendritic mitochondria in synaptic plasticity, and provide new insights into the cellular function of mitoflashes.Mitoflashes are dynamic events in mitochondria, associated with depolarization and release of reactive oxygen species, and have been associated with several cellular functions. The authors now show that in neurons, dendritic mitoflashes are involved in structural postsynaptic changes during LTP.

Exogenous nucleic acids aggregate in non-P-body cytoplasmic granules when transfected into cultured cells

To modulate gene expression in research studies or in potential clinical therapies, transfection of exogenous nucleic acids including plasmid DNA and small interference RNA (siRNA) are generally performed. However, the cellular processing and the fate of these nucleic acids remain elusive. By investigating the cellular behavior of transfected nucleic acids using confocal imaging, here we show that when siRNA was co-transfected into cultured cells with other nucleic acids, including single-stranded RNA oligonucleotides, single and double-stranded DNA oligonucleotides, as well as long double-stranded plasmid DNA, they all aggregate in the same cytoplasmic granules. Interestingly, the amount of siRNA aggregating in granules was found not to correlate with the gene silencing activity, suggesting that assembly of cytoplasmic granules triggered by siRNA transfection may be separable from the siRNA silencing event. Our results argue against the claim that the siRNA-aggregating granules are the functional site of RNA interference (RNAi). Taken together, our studies suggest that, independent of their types or forms, extraneously transfected nucleic acids are processed through a common cytoplasmic pathway and trigger the formation of a new type of cytoplasmic granules “transfection granules”.