Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2003 Nov;85(5):3350-7.
doi: 10.1016/S0006-3495(03)74754-7.

Quantitative Analysis of Spontaneous Mitochondrial Depolarizations

Affiliations
Free PMC article

Quantitative Analysis of Spontaneous Mitochondrial Depolarizations

Catherine M O'Reilly et al. Biophys J. .
Free PMC article

Abstract

Spontaneous transient depolarizations in mitochondrial membrane potential (DeltaPsi(m)), mitochondrial flickers, have been observed in isolated mitochondria and intact cells using the fluorescent probe, tetramethylrhodamine ethyl ester (TMRE). In theory, the ratio of [TMRE] in cytosol and mitochondrion allows DeltaPsi(m) to be calculated with the Nernst equation, but this has proven difficult in practice due to fluorescence quenching and binding of dye to mitochondrial membranes. We developed a new method to determine the amplitude of flickers in terms of millivolts of depolarization. TMRE fluorescence was monitored using high-speed, high-sensitivity three-dimensional imaging to track individual mitochondria in freshly dissociated smooth muscle cells. Resting mitochondrial fluorescence, an exponential function of resting DeltaPsi(m), varied among mitochondria and was approximately normally distributed. Spontaneous changes in mitochondrial fluorescence, indicating depolarizations and repolarizations in DeltaPsi(m), were observed. The depolarizations were reversible and did not result in permanent depolarization of the mitochondria. The magnitude of the flickers ranged from <10 mV to >100 mV with a mean of 17.6 +/- 1.0 mV (n = 360) and a distribution skewed to smaller values. Nearly all mitochondria flickered, and they did so independently of one another, indicating that mitochondria function as independent units in the myocytes employed here.

Figures

FIGURE 1
FIGURE 1
TMRE calibration curve. Inner axes show the TFI plotted against total number of TMRE molecules. The data were fit with a straight line (R = 0.999) having a slope of 0.93 FI/molecule of TMRE. Outer axes show mitochondrial FI plotted against [TMRE] in μM. There was no evidence of self-quenching. The dashed line shows the extrapolation of [TMRE] from the mean FI.
FIGURE 2
FIGURE 2
Mitochondrial accumulation of TMRE. A high resolution stereo-pair fluorescence image of a toad stomach smooth muscle cell loaded with TMRE. Each image is a composite consisting of a projection of a three-dimensional image stack onto a single plane. The discrete localization of the dye to the mitochondria and the arrangement of the mitochondria in alignment with the long axis of the cell are apparent. Differences in mitochondrial FI, and therefore mitochondrial ΔΨm, are clear. The scale bars are 5 μm.
FIGURE 3
FIGURE 3
Effect of FCCP upon TMRE signal. (A) Images of a myocyte before (first image) and after exposure to 1 μM FCCP. The images are at 10-s intervals. FCCP elicited an immediate decrease in FI, characteristic of mitochondrial depolarization. Scale bar is 5 μm. (B) The trace represents the FI (mean ± SE on a log scale) of 34 mitochondria before and after the application of 1 μM FCCP to the cell shown in A. The arrows above the trace indicate the time points for which images are shown in A. The last point on the trace, labeled as BG, represents the mean background FI outside the cell.
FIGURE 4
FIGURE 4
Localized transient depolarizations of ΔΨm. A and C show a selection of images taken of two cells. Each image shows one plane of a three-dimensional image stack. The arrows point to mitochondria in which changes in ΔΨm were observed during the image sequence. A decrease in FI denotes mitochondrial depolarization. Scale bars are 5 μm. B and D are a graphical representation of the FI (on a log scale) of the mitochondria indicated in A and C. The arrows above the traces indicate the time points for which images are shown. The scale bar on the right shows the change in ΔΨm (mV). Flickers vary in amplitude, frequency, time to depolarize, and duration of depolarization.
FIGURE 5
FIGURE 5
Distribution of flicker amplitudes and resting FI. (A) The frequency with which depolarizations of different amplitudes occur. The distribution is skewed to smaller values. (B) The distribution of resting FI (FI before the onset of a flicker). Resting FI varies over a wide range, suggesting that mitochondria exist at different RΔΨm. (C) Amplitudes of mitochondrial flickers plotted as a function of the resting FI. Larger flicker amplitudes are associated with brighter resting FI. The correlation, though low, r2 = 0.03, is highly significant (P ≤ 0.0005). Solid circles are those points used for determination of maximum flicker amplitude (ΔVmax) at each RΔΨm·ΔVmax are plotted against resting FI before the flicker. The slope of the line is 58.1 mV per 10-fold change in FI m pol (r2 = 0.94).

Similar articles

See all similar articles

Cited by 46 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

LinkOut - more resources

Feedback