Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells

Abstract

Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca²⁺ are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling.

Keywords: Ca(2+) sparks; Carcinoma cells; Membrane potential; Mitochondria; ROS flashes.

Fig. 1

Representative imaging of the changes in mitochondrial membrane potential visualized by TMRM (red) and mitoROS generation detected by DCF (green).MitoROS generation and ROS flashes occur simultaneously with mitochondrial membrane depolarization in HT-29 cells during photoactivated oxidative stress. Full co-localization of ROS production (DCF) and mitochondria (TMRM) in individual mitochondria (M) in HT-29 cells can be seen at the initial steps of time series (see also merge images, bottom panel). In contrast, the continuous increase in DCF signal was also a characteristic of extra-mitochondrial (cytosolic) regions of the cells (C). Notably, a high level of the heterogeneity of mitoROS (DCF signals) was observed. Scale bar, 15 μm.

Fig. 2

Dynamics of the changes in ROS and membrane potential (TMRM/DCF fluorescence) during photoactivated oxidative stress. Heterogeneous rates of ROS generation can be seen in distinct mitochondria (mROIs) in HT-29 (A, C) and HRT-18 (B, D) cells. Initiation times of ROS flashes varied between 300 and 700 s. Acquisition time intervals: 20 s. Notably, mitoROS flashes (DCF signal sparks in mROIs) always occur simultaneously with a sharp drop in TMRM signal (ΔΨm collapse, probably due to MPTP opening) (arrows). Different graphics color indicates distinct individual mitochondria. C, D: This drop was synchronized with TMRM release from mitochondria detectable as a signal increase in near-extra-mitochondrial regions (cROIs). Similarly, ROS release from mitochondria can be seen as a decrease in the DCF signal in mROI and an increase in cROI.

Fig. 3

Mitochondrial and near-extra-mitochondrial ROS communications trigger ROS flashes. In individual mitochondria, despite a very different (more than 5-fold difference) initiation time, the mitoROS flash and collapse of membrane potential occur at (or are triggered by) the time when the same level of extra-mitoROS (see DCF fluorescence signals in cROI, dashed line) is reached. Different graphics color indicates distinct individual mitochondria. Notably, in distinct individual mitochondria, ROS bursts occur at very different times but after reaching the same level of extra-mitoROS. A: - HT-29 cells, B: - HRT-18 cells.

Fig. 4

Effects of antioxidants, MnSOD overexpression and MPTP blocker cyclosporin A during photoactivated oxidative stress. A: Effects of antioxidants, MnSOD overexpression and cyclosporin A on ROS production. B: Effects of antioxidants, MnSOD overexpression and cyclosporin A on mitochondrial depolarization (probably due to MPTP opening). ^*P < 0.05; ^**P < 0.01; N = 3. C: Notably, averaged (bulk) cellular ROS level negatively correlated with the percentage of normally polarized mitochondria. As normally polarized mitochondria we considered mitochondria with more than 90% fluorescence intensity of TMRM comparing with control cells. Laser irradiation time: 400–500 s.

Fig. 5

Mitochondria and ROS communications. ROS-induced ROS release (RIRR).A: Representative images, showing wave propagation of mitoROS flashes (green DCF fluorescence) and depolarization (red TMRM fluorescence) between neighboring mitochondria (arrows) within a single cell. Scale bar, 10 μm. B: Quantification of TMRM and DCF fluorescence signal dynamics in mitochondria #1, 2, 3 and 4 presented in Fig. 6A. C: Direction of wave propagation (schematic sequence of events, shown by red arrows) presented in Fig. 6A from mitochondrion #1 to #4. D: Distance versus initiation time for ROS flashes and ΔΨm collapses. Wave propagation velocity: 0.28 μm/s.

Fig. 6

Representative images of ΔΨm oscillations in HT-29 cells monitored by TMRM, demonstrating reversible mitochondrial depolarization-repolarization. Oscillating mitochondria #1, 2 and 3 are indicated by arrows and show a periodical decrease and increase in the TMRM fluorescence signal, whereas mitochondrion #4 (asterisk) from the same cell demonstrates a relatively stable TMRM signal and therefore membrane potential. Scale bar, 5 μm.

Fig. 7

MitoROS and Ca²⁺ imaging. A: Representative simultaneous imaging of the mitoROS (DCF fluorescence, green) dynamics and mitochondrial calcium visualized with Rhod-2 (red fluorescence) in HT-29 cells. Scale bar, 20 μm.B: Higher magnification of images 0 and 100 s. Scale bar, 20 μm.

Fig. 8

Simultaneous increase in mitoROS and mitoCa²⁺.A: Heterogeneous mitochondria. Averaged initial and final calcium levels are shown by dashed lines. B: Homogeneous increase in mitochondria. MitoROS flash (flash duration: ~150 s) occurs after reaching a certain mitoCa²⁺ level (dashed line). C: Heterogeneous initiation time of ROS flashes and concomitant calcium sparks in distinct individual mitochondria in the same cell. Cells were loaded with DCF-DA and Rhod-2-AM (see Methods). Different graphics color indicates distinct individual mitochondria.

Fig. 9

Oscillations of mitoROS and mitoCa²⁺. Reversibility of ROS flashes and Ca²⁺ sparks in mitochondria. A: Representative confocal time series imaging of mitoROS and mitoCa²⁺ oscillations (arrows) in cells double-loaded with DCF and Rhod-2. Scale bar, 20 μm. B, C: Quantification of the DCF and Rhod-2 fluorescence dynamics, demonstrating repetitive, oscillating ROS flashes and calcium sparks in single mitochondria.

Fig. 10

Tentative scheme representing possible complex interactions between ROS, calcium and MPTP opening/depolarization.