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

Yi M, Weaver D, Hajnóczky G - J. Cell Biol. (2004)

Bottom Line: By clamping cytoplasmic [Ca2+] ([Ca2+]c) at various levels, mitochondrial motility was found to be regulated by Ca2+ in the physiological range.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.

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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.
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fig1: 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.

Mentions: As a first approach to evaluate the mitochondrial motility, two images obtained 10 s apart from each other were colored green and red, respectively, and were subsequently overlaid (Fig. 1 A, i). In the overlay, the yellow (green+red) pixels represent the mitochondria that maintained their position, whereas the green and red pixels indicate the sites of movement. One way to show only the sites of movement is to calculate the difference of the two images (F−13.3s − F−23.2s; Fig. 1 A, ii, negative values shown in green, positive values in red, respectively). The amount and distribution of green and red pixels in the difference image corresponds with that in the overlay image (Fig. 1 A, i and ii). Green and red pixels are mostly side-by-side, indicating lateral movement of the organelles, whereas the single green or red pixels are likely to reflect movement into or out of the focus plane. Similar analysis was performed with two images recorded after addition of VP. In this case, very few green and red pixels were obtained, confirming a decrease in mitochondrial mobility (Fig. 1 A, iii and iv).


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

Yi M, Weaver D, Hajnóczky G - J. Cell Biol. (2004)

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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172592&req=5

fig1: 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.
Mentions: As a first approach to evaluate the mitochondrial motility, two images obtained 10 s apart from each other were colored green and red, respectively, and were subsequently overlaid (Fig. 1 A, i). In the overlay, the yellow (green+red) pixels represent the mitochondria that maintained their position, whereas the green and red pixels indicate the sites of movement. One way to show only the sites of movement is to calculate the difference of the two images (F−13.3s − F−23.2s; Fig. 1 A, ii, negative values shown in green, positive values in red, respectively). The amount and distribution of green and red pixels in the difference image corresponds with that in the overlay image (Fig. 1 A, i and ii). Green and red pixels are mostly side-by-side, indicating lateral movement of the organelles, whereas the single green or red pixels are likely to reflect movement into or out of the focus plane. Similar analysis was performed with two images recorded after addition of VP. In this case, very few green and red pixels were obtained, confirming a decrease in mitochondrial mobility (Fig. 1 A, iii and iv).

Bottom Line: By clamping cytoplasmic [Ca2+] ([Ca2+]c) at various levels, mitochondrial motility was found to be regulated by Ca2+ in the physiological range.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.

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.

Show MeSH
Related in: MedlinePlus