PMC

Fig. 1. Surface-rendered three-dimensional reconstructions of cells expressing mito-DsRed1. (A–E) Typical three-dimensional reconstructions of mito-DsRed1 fluorescence in the following cell types: (A) HeLa, (B) COS-7, (C) cortical astrocyte, (D) cortical neuron and (E) HUVEC. (F) A reconstruction of mitochondrial calcein fluorescence in a hepatocyte. The dashed circles indicate the position of nuclei. Scale bars, 5 µm.

Fig. 6. Heterogeneity in Δψ_mit reported by TMRE. The figure illustrates TMRE loading of peripheral (thick black line) and perinuclear (thin grey line) mitochondria pre-loaded with Mitotracker Green FM (1 µM, 25 min). The arrowhead indicates addition of TMRE (0.1 µM). TMRE loading is expressed as a ratio of TMRE fluorescence:Mitotracker Green FM fluorescence. The data in this figure were obtained by monitoring TMRE sequestration in six cells. For each of these cells, TMRE uptake was analysed in 10 randomly chosen perinuclear and peripheral mitochondria. The graph therefore represents the averaged response of 60 mitochondria for both the peripheral and perinuclear traces. The inset image shows the increase in TMRE fluorescence in the nucleus and peripheral cytoplasm.

Fig. 2. Assessment of lumenal continuity between mitochondria using FRAP. A HeLa cell (Ai–iii and Bi–iii) and a HUVEC cell (Ci–iii) expressing mito-DsRed1, and a primary hepatocyte loaded with calcein (Di–iii) were imaged before (Ai–Di), ∼10 s after (Aii–Dii) and 20 min after (Aiii–Diii) photobleaching a subcellular region of each cell type. The white boxes in (Aii–Dii) denote the bleached regions in each cell. The experiments depicted in (A) and (D) were performed at 22°C, and those in (B) and (C) were at 37°C. Scale bars, 5 µm. The results shown in this figure are typical of every trial (n >5) for each cell type and temperature.

Fig. 9. Asynchronous PTP activation within a single cell. Location of mito-DsRed1 (Ai) and calcein (Aii) fluorescence in the same cell. (Aiii) Difference image [i.e. (Aii) subtracted from (Ai)], indicating that mito-DsRed1 and calcein co-localized to the same organelles, except in a few small spherical vesicles [e.g. spot marked by arrowhead in panel (Aiii)]. (Bi) Another single calcein-loaded HeLa cell. The circled mitochondria in (Bi) indicate the locations of organelles from which the fluorescence in (Bii) was recorded. PTP activation was initiated by addition of 100 µM tBuOOH [arrowhead in (Bii)]. The drop in fluorescence indicates PTP opening. The traces are expressed as normalized fluorescence calculated as (F – F_min)/(F_max – F_min), except for those mitochondria that did not undergo PTP, which are expressed as F/F_max (i.e. where there was no F_min). The numbered mitochondria in (Bi) are those that did not show PTP activation during the experiment. The fluorescence of calcein in these mitochondria is depicted by the correspondingly numbered traces in (Bii). Scale bar, 5 µm.

Fig. 8. Association between ER and mitochondria in the periphery and perinuclear regions of a cell. (A) Maximum z-projection of a HeLa cell expressing ER-targeted EGFP (shown as green) and mito-DsRed1 (shown as red). The nucleus is out of view to the left of the image. (B) Yellow regions denote where ER and mitochondria co-localize within the resolution of the deconvolved, confocal image. The regions bounded by white boxes in (A) are shown on expanded scales in (Ci) (perinuclear region) and (Di) (peripheral region). The same expanded regions are shown as surface-rendered versions in (Cii) and (Dii), respectively, to show that the perinuclear mitochondria are embedded in ER, whilst the peripheral mitochondria are more remote from the ER. The images are presented as if the cell is viewed from above. The view from underneath the cell shows a similar pattern. Scale bars, 5 µm.

Fig. 5. Heterogeneity in membrane potential revealed by JC-1. (Ai) HeLa cell mitochondria after incubation with JC-1, illustrating the heterogeneity in mitochondria membrane potential within a single cell. To demonstrate co-localization of TMRE with JC-1, the same cells were subsequently co-loaded with TMRE (0.1 µM, 20 min). As illustrated in (Aii), the same organelles are stained. The mitochondria within the cells were then depolarized by addition of antimycin (10 µM) plus oligomycin (2 µM). (B) The peripheral location of red staining mitochondria in a hepatocyte stained with JC-1 at 22°C. (Ci and ii) A section of the same JC-1-loaded HUVEC cell at 22 and 37°C. The HUVEC cell was incubated in JC-1 at 22°C for 1 h before the image in (Ci) was obtained. The cell was then slowly warmed to 37°C (∼10 min) and then incubated for a further 30 min in the continual presence of JC-1. Scale bar, 5 µm.

Fig. 3. DsRed1 is rapidly diffusible in the mitochondrial matrix. (Ai) A portion of a HeLa cell expressing mito-DsRed1. Both long (denoted by an arrow) and short discrete mitochondria can be seen in this region. The region bounded by the white box in (Aii) was photobleached using a 5 s illumination as described in Materials and methods. The bleached region encompassed the middle portion of the long mitochondria and completely surrounded several smaller mitochondria. The images in (Aii) and (Aiii) show the gradual recovery of fluorescence in the long mitochondrion, and were obtained at times corresponding to 0.5 and 90 s after the photobleach. (B) A quantitation of the fluorescence recovery within the long mitochondrion. The fluorescence was monitored in the regions denoted in (Bi). Areas a, b, c and d were within the bleached zone, but e, f and g were not. The traces in (Bii) illustrate the intensity of mito-DsRed1 fluorescence in these regions before and after the photobleach. Note that regions a and g were not part of the long mitochondrion. Scale bar, 5 µm. The data presented show a typical response observed in more than five cells.

Fig. 7. Perinuclear and peripheral mitochondria sequester agonist- induced Ca²⁺ signals to different extents. (A) HeLa cells loaded with rhod-2 were treated with 100 µM histamine (arrowhead) to trigger Ca²⁺ release from the ER. The resultant cytosolic Ca²⁺ change [thin dashed line in (A)] was monitored by recording rhod-2 fluorescence in the nucleus as described previously (). In addition, Ca²⁺ increases in the perinuclear mitochondria (thin grey line) and peripheral mitochondria (thick black line) were followed. (B) Depiction of the similarity of Ca²⁺ sequestration by perinuclear and peripheral mitochondria during Ca²⁺ entry. Prior to the traces shown in (Bi–iii), the ER Ca²⁺ pool was depleted by pre-treatment of cells with 2 µM thapsigargin in 4 mM EGTA or 50 µM CPA in 4 mM EGTA. Ca²⁺ entry was initiated by substituting the EGTA in the extracellular medium with 1.8 mM CaCl₂ (arrowheads). The sodium/Ca²⁺ exchanger was inhibited for the response shown in (Biii) by the addition of 10 µM CGP37157 for 5 min before the replacement of EGTA with CaCl₂. For the responses in (A), where the histamine-stimulated Ca²⁺ signals were rapid, the cells were imaged at 15 Hz. The slower Ca²⁺ entry signals in (B) were imaged at 0.5 Hz. Traces represent the mean of 10 cells, with >10 individual mitochondria or small clusters of mitochondria being analysed in each cell.

Fig. 4. Induced depolarization events reveal multiple, electrically discrete mitochondria. (Ai–v) A TMRE-loaded HeLa cell showing random depolarization of discrete mitochondria. The depolarization events were assessed using a frame-by-frame subtraction. This generated a series of images in which the positions of depolarized mitochondria appear from a dark background. These events were pseudocoloured red and superimposed on the original image of the mitochondria as shown. The experiment was actually recorded for 10 min, but only representative frames are shown. The times at which the images were captured relative to the start of the recording are shown. (Avi) Map of individual electrically isolated mitochondria. This was constructed by monitoring the locations of individual depolarization events over time. The red, green and yellow colouration is used to indicate the locations of the electrically isolated mitochondria. The colours do not indicate any relationships between mitochondria. The arrowhead in (Avi) indicates a single 45 µm mitochondria seen to depolarize in (Ai). Scale bar, 5 µm. (B) Depolarizations of mitochondria within a TMRE-loaded primary hepatocyte imaged at 2 Hz. (Bi) The change in fluorescence of individual mitochondria (the locations at which the mitochondrial fluorescence was monitored are depicted by black dots in the inset cell image) plotted over 8 min of laser irradiation. Note that depolarization events only start after a significant period of imaging (∼90 s), confirming that the PTP ‘flickering’ is a laser- and dye-induced phenomenon. (Bii) De piction of the image of the TMRE-loaded cell. The region bounded by the dashed box is shown on an expanded scale in (Biii–viii), and the depolarization events are superimposed with red colouration. (Bix) Map of individual electrically isolated mitochondria constructed by monitoring the locations of individual depolarization events over time. The red, green, cyan and yellow colouration is used to indicate the locations of the electrically isolated mitochondria, and does not indicate any relationships between mitochondria. Scale bar, 5 µm.