Control of mitochondrial motility and distribution by the calcium signal: a homeostatic circuit

Abstract

Mitochondria are dynamic organelles in cells. The control of mitochondrial motility by signaling mechanisms and the significance of rapid changes in motility remains elusive. In cardiac myoblasts, mitochondria were observed close to the microtubular array and displayed both short- and long-range movements along microtubules. By clamping cytoplasmic [Ca2+] ([Ca2+]c) at various levels, mitochondrial motility was found to be regulated by Ca2+ in the physiological range. Maximal movement was obtained at resting [Ca2+]c with complete arrest at 1-2 microM. Movement was fully recovered by returning to resting [Ca2+]c, and inhibition could be repeated with no apparent desensitization. The inositol 1,4,5-trisphosphate- or ryanodine receptor-mediated [Ca2+]c signal also induced a decrease in mitochondrial motility. This decrease followed the spatial and temporal pattern of the [Ca2+]c signal. Diminished mitochondrial motility in the region of the [Ca2+]c rise promotes recruitment of mitochondria to enhance local Ca2+ buffering and energy supply. This mechanism may provide a novel homeostatic circuit in calcium signaling.

Figure 1.

Stimulus-induced inhibition of mitochondrial motility. (A) Measurement of mitochondrial movement in H9c2 cells. Green/red overlay of two time-lapse confocal images (Δt = 10 s) of mitoYFP fluorescence in a live cell before (i) and after (iii) stimulation by 100 nM VP. (ii and iv) Processed images showing only the pixels whose values differ by more than a threshold value (+ and −) between the two time points. (v) Graph of the number of pixels that change more than the threshold value for this cell, calculated with consecutive images (Δt = 3.3 s), and normalized as the percentage of loss from the average before stimulation. (vi–x) Two cells stimulated by addition of 10 μM Iono. Graph shown is the mean of the two cells. (B) Simultaneous measurements of mitochondrial motility and [Ca²⁺]_c in an H9c2 cell expressing mitoYFP and loaded with fura2. Top row of images shows both mitoYFP fluorescence (grayscale; i scaled with higher contrast to show the structure of the mitochondria) and at each time point, the sites of mitochondrial movement calculated by subtraction of sequential images (red, positive changes; green, negative changes). Bottom row of images shows fura2 fluorescence measured using excitation of both the Ca²⁺-bound (340 nm, red) and the Ca²⁺-free form (380 nm; green). Thus, [Ca²⁺]_c elevations evoked by addition of VP (81 s) and CaCl₂ (426 s) appear as an increase in the red component. In the histogram, the decrease in mitochondrial motility (calculated as in A) and [Ca²⁺]_c (ratio of the fluorescence of the Ca²⁺ bound and Ca²⁺-free forms of fura2) are plotted in red and black, respectively. The cell was treated sequentially with 100 nM VP, 5 mM EGTA, 10 mM CaCl₂, and 5 mM EGTA. (C) Lack of change in mitochondrial motility in the absence of the VP-induced [Ca²⁺]_c rise. The cell was preincubated with 2 mM EGTA, 2 μM Tg, and 10 μM Iono in Ca²⁺-free ECM to remove extracellular Ca²⁺ and to deplete the intracellular Ca²⁺ stores before stimulation with 100 nM VP. Mitochondrial motility and [Ca²⁺]_c are plotted as in B.

Figure 2.

Relationship between [Ca ²⁺ ] _c and mitochondrial motility. (A) Stepwise increases in [Ca²⁺]_c induce stepped decreases in mitochondrial motility. MitoYFP-expressing and fura2-loaded cells were incubated in Ca²⁺-free ECM supplemented with 2 mM EGTA, 2 μM Tg, and 10 μM of Iono for 12 min. Simultaneous measurements of [Ca²⁺]_c and mitochondrial motility were performed in single cells exposed to stepped increases in extracellular CaCl₂ from 0 to 15 mM. [Ca²⁺]_c was calibrated in terms of nanomoles using in vitro calibration of fura2. (B) Dose–response relationship between [Ca²⁺]_c and mitochondrial motility. Simultaneous measurements of [Ca²⁺]_c and mitochondrial motility were performed in single cells as described for A. Mean mitochondrial motility inhibition and mean [Ca²⁺]_c were calculated for each [CaCl₂]. Mean ± SEM are marked by filled circles (n = 68 cells from 40 experiments). The IC₅₀ ≈ 400 nM, indicating that mitochondrial motility is controlled in the physiological range of [Ca²⁺]_c. (C) Mitochondrial motility (red line) and [Ca²⁺]_c (black line) were recorded in single cells stimulated by varying doses of VP (0.1–100 nM). In addition, the mean mitochondrial motility inhibition and mean [Ca²⁺]_c were calculated for each VP concentration and are plotted in B (empty circles and dashed line; n = 44 cells from 28 experiments).

Figure 3.

Spatio-temporal pattern of the calcium signal and inhibition of mitochondrial motility. (A) Calcium signal propagation to the mitochondria is not required for the inhibition of motility. Uncoupler (5 μM FCCP and 5 μg/ml Oligo; solid line) or solvent (dashed line) was added to the cells 5 min before stimulation with VP. The uncoupler causes dissipation of the ΔΨ_m in <1 min and in turn decreases the driving force of the mitochondrial Ca²⁺ uptake in H9c2 myoblasts (Szalai et al., 2000). Mitochondrial motility showed a slowly developing decrease (red solid line) compared with the control (red dash line). However uncoupler did not prevent the effect of VP on mitochondrial motility, indicating that mitochondrial Ca²⁺ uptake and ΔΨ_m were not necessary for the control of motility by the calcium signal. In another set of experiments, the cells were pretreated with uncoupler for 10 min before the recording was started. The inset shows that the residual motility was effectively inhibited by 100 nM VP. Data are the means of nine experiments with uncoupler and four experiments for the control. (B) Reversible and reproducible inhibition of mitochondrial motility by Ca²⁺. To evaluate the effect of pulsatile increases of [Ca²⁺]_c (black traces) on mitochondrial motility (red traces), the 10 μM of Iono-induced [Ca²⁺]_c elevation was recorded for 10 min (solid lines) or was reversed by addition of 5 mM EGTA and, subsequently, was reestablished by addition of 10 mM CaCl₂ (dashed lines). Data are the means of 36 cells from 22 experiments.

Figure 4.

Control of mitochondrial motility by [Ca ²⁺ ] _c oscillations and waves. Mitochondrial motility was evaluated simultaneously with [Ca²⁺]_c in cells cotransfected with constructs encoding mitoYFP and RyR1 and loaded with fura2. To promote RyR-mediated Ca²⁺ mobilization, the cells were exposed to 10 mM of caffeine (Caff). (A) In the image series, mitochondrial movements are visualized in a cell that showed [Ca²⁺]_c oscillations in response to stimulation by Caff (arrow) and in a cell that did not show a Caff-induced [Ca²⁺]_c rise (top left corner) as described for Fig. 1. Also shown is the effect of 100 nM VP that elicited a [Ca²⁺]_c signal in both cells. The time course of [Ca²⁺]_c (black trace) and mitochondrial motility (red trace) for the Caff-sensitive cell is plotted. (B) Sustained inhibition of mitochondrial motility in a cell that displayed a relatively high frequency [Ca²⁺]_c oscillation in response to stimulation by Caff. (C) Calcium waves were induced in mitoYFP-expressing cells by treatment with 75 μM thimerosal (TM) and 0.1 nM VP. The image series shows the mitoYFP fluorescence (i) and the first two calcium waves after addition of VP and TM (t = 60 s; ii–ix). The first wave begins simultaneously at the ends of the cell and converges in the center as indicated by the arrows (iv). The second wave begins at the lower end of the cell and weakens as it propagates to the top (vi–viii, arrow marks the direction of the wave propagation in vii). Measurement of [Ca²⁺]_c (x) and mitochondrial movement (ix) in three distinct regions of the cell, labeled 1–3 in panel ii, reveals spatio-temporal heterogeneity in the inhibition of movement corresponding to the local calcium concentration.

Figure 5.

Spatial relationship between MTs, MFs, and mitochondria. (A) H9c2 cells expressing tubulinGFP and mitoDsRed and incubated in the absence (i) or presence (ii) of 10 μM of an MT-stabilizing agent, taxol. (iii–v) Magnified time-lapse images of a peripheral region of the taxol-pretreated cell. Arrowheads mark the mitochondria that move substantially from the previous image. (vi–ix) Further magnified region showing a single mitochondrion sliding along an MT. (x and xi) A naive and a nocodazole-pretreated cell after permeabilization with digitonin. (B) Inhibition of mitochondrial motility and enhancement of the VP-induced mitochondrial [Ca²⁺] signal in nocodazole-pretreated cells. (top left) Mitochondrial motility and [Ca²⁺]_c in nocodazole-treated cells (solid lines, 23 cells in 10 measurements) as compared with control cells (dashed lines, 22 cells in 11 measurements). The effect of 100 nM VP is also shown. (top right) Resting [Ca²⁺]_c and the peak value of the VP-induced [Ca²⁺]_c signal in control and nocodazole-pretreated (10 μM for 25–30 min) cells. (bottom) Nuclear matrix and mitochondrial matrix [Ca²⁺] measured in cells expressing both nuclear and mitochondrial pericam using fast, ratiometric imaging (2 ratio/s). Cells were pretreated with nocodazole (10 μM for 25–30 min) or solvent (control) and stimulated by 100 nM VP. The VP-induced initial [Ca²⁺]_c signal was measured (change in nuclear pericam fluorescent ratio at the first point of the [Ca²⁺] rise, <25% of the maximal change) and the corresponding change in [Ca²⁺]_m (change in mitochondrial pericam fluorescent ratio) was calculated for each cell (mean ± SEM; n = 16). (C) MFs and mitochondria in H9c2 cells expressing actinGFP and mitoDsRed. Cells were incubated in the absence or presence of 10 μM of nocodazole.

Figure 6.

Decoding of [Ca ²⁺ ] _c signals by the mitochondrial motor machinery. The proposed mechanism for the [Ca²⁺]_c signal-dependent control of mitochondrial motility is shown.