Respective Contribution of Mitochondrial Superoxide and pH to Mt-cpYFP Flash Activity.

University of Rochester Medical Center, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2013; DOI: 10.1074/jbc.M113.455709
Source: PubMed


Superoxide flashes are transient bursts of superoxide production within the mitochondrial matrix that are detected using the superoxide sensitive biosensor, mitochondrial-targeted circularly permuted YFP (mt-cpYFP). However, due to the pH sensitivity of mt-cpYFP, flashes were suggested to reflect transient events of mitochondrial alkalinization. Here, we simultaneously monitored flashes with mt-cpYFP and mitochondrial pH with carboxy-SNARF-1. In intact cardiac myocytes and purified skeletal muscle mitochondria, robust mt-cpYFP flashes were accompanied by only a modest increase in SNARF-1 ratio (corresponding to a pH increase of <0.1) indicating that matrix alkalinization is minimal during a mt-cpYFP flash. Individual flashes were also accompanied by stepwise increases of MitoSOX signal and decreases of NADH autofluorescence, supporting the superoxide origin of mt-cpYFP flashes. Transient matrix alkalinization induced by NH4Cl only minimally influenced flash frequency and failed to alter flash amplitude. However, matrix acidification modulated superoxide flash frequency in a bimodal manner. Low concentrations of nigericin (< 100nM) that resulted in a mild dissipation of the mitochondrial pH gradient increased flash frequency, while a maximal concentration of nigericin (5 μM) collapsed the pH gradient and abolished flash activity. These results indicate that mt-cpYFP flash events reflect a burst in ETC-dependent superoxide production that is coincident with a modest increase in matrix pH. Furthermore, flash activity depends strongly on a combination of mitochondrial oxidation and pH gradient.

Download full-text


Available from: Guohua Gong, Jun 28, 2014
  • Source
    • "Ex vivo and in vivo imaging in transgenic mouse models with cardiac-specific or pan-tissue mt-cpYFP expression have allowed for detection of superoxide flashes in beating hearts under Langendorff perfusion [67] and in gastrocnemius muscle and sciatic nerve of living mice under anesthesia (Fig. 1) [70, 71]. In addition, superoxide flashes of similar characteristics are active in freshly isolated, respiratory mitochondria [83], indicating that single mitochondria contain the full machinery for the genesis of superoxide flashes. Notably, the rate of flash occurrence varies depending on species, tissue and cell types, metabolic states, the presence of stressors, and disease conditions; the amplitude and duration of the flashes, however, appear to be stereotypical at multiple levels, from isolated mitochondria, to intact or plasma membrane-permeabilized cells, to whole tissues or organs, and to living animals. "
    [Show abstract] [Hide abstract]
    ABSTRACT: 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.
    Journal of Molecular Medicine 07/2013; 91(8). DOI:10.1007/s00109-013-1067-4 · 5.11 Impact Factor
  • Source
    • "e l s e v i e r . c o m / l o c a t e / l i f e s c i e the superoxide origin of the flash (Hou et al., 2013, 2012; Huang et al., 2011; Pouvreau, 2010; Wang et al., 2008; Wei et al., 2011; Wei-Lapierre et al., 2013), a direct assay is necessary to discriminate the superoxide or pH contribution. Here we adopted an approach involving the use of multiple ROS indicators with complementary properties. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Mitochondrial flashes detected with an N- and C-terminal circularly-permuted yellow fluorescent protein (cpYFP) have been thought to represent transient and quantal bursts of superoxide production under physiological, stressful and pathophysiological conditions. However, the superoxide nature of the cpYFP-flash has been challenged, considering the pH-sensitivity of cpYFP and the distinctive regulation of the flash versus the basal production of mitochondrial reactive oxygen species (ROS). Thus, the aim of the study is to further determine the origin of mitochondrial flashes. We investigated the origin of the flashes using the widely-used pH-insensitive ROS indicators, mitoSOX, an indicator for superoxide, and 2, 7-dichlorodihydrofluorescein diacetate (DCF), an indicator for H2O2 and other oxidants. Robust, quantal, and stochastic mitochondrial flashes were detected with either mitoSOX or DCF in several cell-types and in mitochondria isolated from the heart. Both mitoSOX-flashes and DCF-flashes showed similar incidence and kinetics to those of cpYFP-flashes, and were equally sensitive to mitochondria-targeted antioxidants. Furthermore, they were markedly decreased by inhibitors or an uncoupler of the mitochondrial electron transport chain, as is the case with cpYFP-flashes. The involvement of the mitochondrial permeability transition pore in DCF-flashes was evidenced by the coincidental loss of mitochondrial membrane potential and matrix-enriched rhod-2, as well as by their sensitivity to cyclosporine A. These data indicate that all the three types of mitochondrial flashes stem from the common physiological process of bursting superoxide and ensuing H2O2 production in the matrix of single mitochondrion.
    Life sciences 06/2013; 93:178-186. DOI:10.1016/j.lfs.2013.06.012 · 2.70 Impact Factor
  • Source
    • "Transient PTP openings appear also to be at the basis of “superoxide flashes” observed with mitochondrially targeted, circularly permuted yellow fluorescent protein (mt-cpYFP) in a variety of cell types (Wang et al., 2008, 2013; Fang et al., 2011). There is an ongoing debate on whether the flashes are in fact partially or totally due to changes of matrix pH (Schwarzlander et al., 2011, 2012), although it is fair to say that the measured matrix alkalinization appears to be way too small to account for the fluorescence changes of mt-cpYFP (Wei-Lapierre et al., 2013). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The permeability transition (PT) denotes an increase of the mitochondrial inner membrane permeability to solutes with molecular masses up to about 1500 Da. It is presumed to be mediated by opening of a channel, the permeability transition pore (PTP), whose molecular nature remains a mystery. Here I briefly review the history of the PTP, discuss existing models, and present our new results indicating that reconstituted dimers of the FOF1 ATP synthase form a channel with properties identical to those of the mitochondrial megachannel (MMC), the electrophysiological equivalent of the PTP. Open questions remain, but there is now promise that the PTP can be studied by genetic methods to solve the large number of outstanding problems.
    Frontiers in Physiology 05/2013; 4(1):95. DOI:10.3389/fphys.2013.00095 · 3.53 Impact Factor
Show more