Cytokine-stimulated superoxide flashes in chondrocytes <i>in situ</i>.

<p><b>A</b><b>B</b>, Time-lapse views of a typical superoxide flash <i>in situ</i> in the red boxed region of 2.5 µm×5 µm. Time course plot of the superoxide flash is shown at the bottom. Note the swelling of the mitochondrion during the flash. <b>C</b>, Activity and properties of chondrocyte superoxide flashes i<i>n vitro</i> (510 events as in Figure. 2) and <i>in situ</i> (30 events). Data are reported as the mean ± SEM values. *, <i>p</i><0.05; **, <i>p</i><0.01. <b>D</b>, Time course of the superoxide flash response to cytokine stimulation. Data are reported as the mean ± SEM values. n = 21–29 imaging planes. *, <i>p</i><0.05; **, <i>p</i><0.01 versus the respective control.</p

Imaging chondrocytes and mitochondria <i>in situ</i>.

<p><b>A</b>, Confocal or two-photon-excitation imaging of chondrocyte mitochondria in femoral head cartilage <i>in situ</i>. Insert shows an enlarged view of the femoral head. <b>B</b>, Mitochondrial distribution in intact cartilage. The femoral head was sliced lengthwise along the midcoronal panel, labeled with TMRM and imaged by a confocal microscope. Arrows mark the cartilage surface and arrowheads mark the growth plate. <b>C</b>, Two-photon excitation images of TMRM-stained mitochondria (excitation at 850 nm, red) in chondrocytes at different depths from the cartilage surface. Layer thickness: 0.77 µm, XYZ of 3D: 100*100*100 µm³ (Z axis shows depth). <b>D</b>, Profiles of averaged mitochondrial (Mito, green) and chondrocyte (Cell, blue) cross-section areas and their ratios (Mito/Cell, red) as a function of distance from the surface. Right panels shows representative images from superficial, middle and deep cartilage zones.</p

Mitochondrial superoxide flashes in cultured articular chondrocytes.

<p><b>A</b>, Colocalization of the superoxide biosensor – mitochondrial circularly permuted yellow fluorescent protein (mt-cpYFP) and tetramethylrhodamine methyl ester (TMRM) in chondrocyte mitochondria revealed by tri-wavelength excitation imaging. Chondrocytes were isolated from mt-cpYFP transgenic mice and cultured as described in the Methods. Only chondrocytes from the first passage were used for experiments. <b>B</b>, Mitochondrial superoxide flashes in articular chondrocytes. Enlarged (top panel) and pseudo-color contrast-enhanced (middle panel, scale bar shown to the bottom left) snapshots of a representative mitochondrial superoxide flash in chondrocyte are shown to the right. The lower panel shows the time course of the corresponding mt-cpYFP fluorescence intensity at 488 nm excitation. <b>C</b>, Histogram analysis of amplitude (ΔF/F₀) and full duration at half maximum (FDHM) of chondrocyte superoxide flashes. <b>D</b>, Kinetics of superoxide flashes. Note the characteristic lack of fluorescence change at 405 nm excitation. Superoxide flash coincided with transient mitochondrial depolarization (ΔΨ_m, TMRM at 543 nm excitation).</p

Enhanced superoxide flash activity and mitochondrial fragmentation during IL-1β or TNF-α challenge in cultured chondrocytes.

<p>A, Representative examples showing superoxide flash activities in basal conditions and after IL-1β or TNF-α challenges in different chondrocytes. Data are presented as an overlay of the XY view of a chondrocyte (bottom) and surface plot of all superoxide flashes combined from 100 consecutive frames obtained at 1 frame/s. The lower panel shows temporal diaries of superoxide flash incidence in nine representative cells belonging to three groups respectively, with the uppermost diary corresponding to the image shown. B, Superoxide flash frequency without or during IL-1β (10 ng/ml) or TNF-α (10 ng/ml) challenge. Data are shown as the mean ± SEM values. n = 179–576 events from 42–49 cells for each group. **, <i>p</i><0.01 versus the corresponding control group. C, Characteristics of spontaneous (control) and cytokine-stimulated (IL-1β or TNF-α) chondrocytes superoxide flashes. ΔF/F₀: amplitude. FDHM: full duration at half maximum. FAHM: full area at half maximum. Data represent the mean ± SEM values. n = 42–49 cells for each group. *, <i>p</i><0.05; **, <i>p</i><0.01 versus the corresponding control group. D, Representative chondrocyte mitochondrial morphology before (control) and 60 min after IL-1β or TNF-α treatment.</p

Remodeling of Mitochondrial Flashes in Muscular Development and Dystrophy in Zebrafish

<div><p>Mitochondrial flash (mitoflash) is a highly-conserved, universal, and physiological mitochondrial activity in isolated mitochondria, intact cells, and live organisms. Here we investigated developmental and disease-related remodeling of mitoflash activity in zebrafish skeletal muscles. In transgenic zebrafish expressing the mitoflash reporter cpYFP, <i>in vivo</i> imaging revealed that mitoflash frequency and unitary properties underwent multiphasic and muscle type-specific changes, accompanying mitochondrial morphogenesis from 2 to 14 dpf. In particular, short (S)-type mitoflashes predominated in early muscle formation, then S-, transitory (T)- and regular (R)-type mitoflashes coexisted during muscle maturation, followed by a switch to R-type mitoflashes in mature skeletal muscles. In early development of muscular dystrophy, we found accelerated S- to R-type mitoflash transition and reduced mitochondrial NAD(P)H amidst a remarkable cell-to-cell heterogeneity. This study not only unravels a profound functional and morphological remodeling of mitochondria in developing and diseased skeletal muscles, but also underscores mitoflashes as a useful reporter of mitochondrial function in milieu of live animals under physiological and pathophysiological conditions.</p></div

Mitoflash activities in red skeletal muscles of 8 dpf zebrafish embryos.

<p>(A) The same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2A</a>, except that the data were from a red skeletal muscle at 8 dpf. White contours mark mitochondria undergoing mitoflashes. Scale bar, 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.s008" target="_blank">S2 Movie</a>. (B) A representative mitoflash (marked in panel A with red-color coded contour). The same as in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2B</a>. Arrow heads mark two consecutive mitoflashes in the same mitochondria. Upper panel: time sequence of the mitoflash seen alternatively at 488 nm and 405 nm excitation and their ratio (F₄₈₈/F₄₀₅). Note that the ratiometric line plot, F₄₈₈/F₄₀₅, largely eliminated motion artifacts. (C) A family of traces showing diversified time courses of mitoflashes from red skeletal muscle at this developmental stage. The traces were arranged by the start points of mitoflash events. Photobleaching (~10%) was corrected based on global intensity decay and the dashed lines mark the basal level. Note the distinct developmental differences compared to mitoflashes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2D</a>.</p

Migrating LinuX Containers Using CRIU

<p>(A) The same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2A</a>, except that the data were from a red skeletal muscle at 8 dpf. White contours mark mitochondria undergoing mitoflashes. Scale bar, 10 μm. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.s008" target="_blank">S2 Movie</a>. (B) A representative mitoflash (marked in panel A with red-color coded contour). The same as in in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2B</a>. Arrow heads mark two consecutive mitoflashes in the same mitochondria. Upper panel: time sequence of the mitoflash seen alternatively at 488 nm and 405 nm excitation and their ratio (F₄₈₈/F₄₀₅). Note that the ratiometric line plot, F₄₈₈/F₄₀₅, largely eliminated motion artifacts. (C) A family of traces showing diversified time courses of mitoflashes from red skeletal muscle at this developmental stage. The traces were arranged by the start points of mitoflash events. Photobleaching (~10%) was corrected based on global intensity decay and the dashed lines mark the basal level. Note the distinct developmental differences compared to mitoflashes in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132567#pone.0132567.g002" target="_blank">Fig 2D</a>.</p

Mitochondrial morphogenesis in developing zebrafish skeletal muscles.

<p>(A) Top: A schematic map of Tg(<i>β-actin</i>:mt-cpYFP) reporter construct, in which mt-cpYFP was driven by the chicken <i>β</i>-actin promoter. The N-terminus of cpYFP was tagged with the COX IV mitochondrial localization signal (COX IV MTS). The transgene was flanked with Tol2 elements (Tol2) to facilitate transgenesis in zebrafish. Bottom: ubiquitous expression of mt-cpYFP in a Tg(<i>β-actin</i>:mt-cpYFP) transgenic embryo at 2 dpf. (B) Confocal images of mitochondria in white (left panels) and red (right panels) skeletal muscles of Tg(<i>β-actin</i>:mt-cpYFP) embryos from 1 to 14 dpf. Note the fibrillar mitochondria at 1 and 2 dpf, while the rod-like mitochondria at 5, 8 and 14 dpf in white skeletal muscles, whereas large brick-like at 8 dpf and smaller bead-like mitochondria at 5 and 14 dpf in red skeletal muscles. Scale bar, 10 μm. (C) Transmission electron microscopic images of mitochondria (M) in white (left panels) and red (right panels) skeletal muscle cells of Tg(<i>β-actin</i>:mt-cpYFP) transgenic embryos from 2 to 14 dpf. Red skeletal muscle cells were identified along with melanocytes. Note fewer immature cristae in mitochondria at 2 dpf but well-developed cristae in mitochondria at 5, 8 and 14 dpf in both red and white muscle fibers. Mitochondria were particularly enlarged in red muscle cells at 8 dpf. Scale bar, 1 μm.</p