Article1 April 2002free access Mitochondria are morphologically and functionally heterogeneous within cells Tony J. Collins Corresponding Author Tony J. Collins Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Michael J. Berridge Michael J. Berridge Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Peter Lipp Peter Lipp Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Martin D. Bootman Martin D. Bootman Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ UK Search for more papers by this author Tony J. Collins Corresponding Author Tony J. Collins Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Michael J. Berridge Michael J. Berridge Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Peter Lipp Peter Lipp Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Search for more papers by this author Martin D. Bootman Martin D. Bootman Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ UK Search for more papers by this author Author Information Tony J. Collins 1, Michael J. Berridge1, Peter Lipp1 and Martin D. Bootman1,2 1Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT UK 2Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:1616-1627https://doi.org/10.1093/emboj/21.7.1616 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info We investigated whether mitochondria represent morphologically continuous and functionally homogenous entities within single intact cells. Physical continuity of mitochondria was determined by three-dimensional reconstruction of fluorescence from mitochondrially targeted DsRed1 or calcein. The mitochondria of HeLa, PAEC, COS-7, HUVEC, hepatocytes, cortical astrocytes and neuronal cells all displayed heterogeneous distributions and were of varying sizes. There was a denser aggregation of mitochondria in perinuclear positions than in the cell periphery, where individual isolated mitochondria could be seen clearly. Using fluorescence-recovery after photobleaching, we observed that DsRed1 and calcein were highly mobile within the matrix of individual mitochondria, and that mitochondria within a cell were not lumenally continuous. Mitochondria were not electrically coupled, since only individual mitochondria were observed to depolarize following irradiation of TMRE-loaded cells. Functional heterogeneity of mitochondria in single cells was observed with respect to membrane potential, sequestration of hormonally evoked cytosolic calcium signals and timing of permeability transition pore opening in response to tert-butyl hydroperoxide. Our data indicate that mitochondria within individual cells are morphologically heterogeneous and unconnected, allowing them to have distinct functional properties. Introduction Renewed interest in mitochondria in recent years, sparked by the recognition of their role in processes such as apoptosis and calcium homeostasis, has reopened the debate on how many mitochondria, or populations of mitochondria, exist in a single cell. Do mitochondria exist as multiple isolated organelles, or is the mitochondrion a single network more akin to the endoplasmic reticulum (ER)? Electrical and lumenal continuity across a mitochondrial network would have important consequences for many cellular processes. Electrical continuity, for example, would tend to equalize the potential across the inner membrane of all mitochondrial regions, whether they were respiring or not. Such an electrically coupled network may allow ‘energy transmission’ across the cell from regions of low to regions of high ATP consumption (Skulachev, 2001). A lumenally continuous mitochondrial network would raise the possibility of ‘Ca2+ tunnelling’ via a non-cytoplasmic route similar to the ER (Mogami et al., 1997). Mitochondria are considered as rapid uptake, slow-release buffers of cytosolic Ca2+ (Babcock et al., 1997), with preferential access to Ca2+ at the site of its release (Rizzuto et al., 1993). Ca2+ tunnelling could result in the delivery of Ca2+ to an area of the cell distant from the site of the original signal, and with a significant delay. This would alter the spatio-temporal characteristics of the cytosolic Ca2+ response. Furthermore, Ca2+ tunnelling could promote the synchronization of increases in mitochondrial metabolism (Hajnoczky et al., 1995) or permeability transition (Ichas et al., 1994) across a cell. Mitochondria play a key role in some of the signalling pathways for apoptosis. Release of pro-apoptotic factors from the inter-membrane space is thought to occur after mitochondrial permeability transition pore (PTP) opening, matrix swelling and outer-membrane rupturing (for a review see Crompton, 1999). Paradoxically, the early stages of apoptosis can involve the depolarization of mitochondria, yet also require ATP, otherwise apoptosis turns to necrosis (Eguchi et al., 1997; Leist et al., 1997). A non-continuous mitochondrial network could allow the possibility that certain mitochondria are involved in pro-apoptotic signalling, whilst others continue to supply ATP during apoptosis. Several lines of evidence have been presented to suggest that mitochondria are both physically interconnected and functionally homogenous. By triggering mitochondrial depolarization with laser irradiation of fluorescent dye-loaded cells, large networks of electrically connected mitochondria have been demonstrated in COS-7 cells (De Giorgi et al., 2000) and cardiac myocytes (Amchenkova et al., 1988). Reconstruction of electron micrographs revealed mitochondrial networks in rat hepatocytes (Brandt et al., 1974) and a single large mitochondrion in yeast cells (Hoffman and Avers, 1973). More recently, evidence for the existence of a largely interconnected mitochondrial network in HeLa cells was presented using non-confocal, deconvolution imaging and ‘fluorescence-recovery after photobleaching’ (FRAP) (Rizzuto et al., 1998). In contrast, early electron microscopy revealed populations of mitochondria with different matrix densities within single cells (Simon et al., 1969; Ord, 1979), thought to reflect differences in metabolic states (Ord, 1979). In cardiac (Jahangir et al., 1999) and skeletal muscle (Battersby and Moyes, 1998; Lombardi et al., 2000) cells, two distinct populations of mitochondria are proposed to exist, with differing biochemical and respiratory properties. In pancreatic acinar cells, detailed spatial analysis has revealed mitochondrial populations preferentially sequestering Ca2+ from different sources, and apparently not communicating Ca2+ between one another (Park et al., 2001). In the present study, we used a combination of approaches to examine the connectivity and functional homogeneity of mitochondria in several cell types. Our analysis indicates that mitochondria exist as separate entities that vary substantially in length and cellular distribution. Furthermore, they are lumenally discontinuous. This physical compartmentalization is accompanied by a functional heterogeneity with respect to membrane potential, Ca2+ sequestration and sensitivity to PTP activation. Results Morphological heterogeneity of mitochondria HeLa cells, porcine aortic endothelial cells (PAEC), COS-7 cells, human umbilical vein endothelial cells (HUVEC), cortical astrocytes and cortical neuronal cells were transfected with DsRed1 targeted to the mitochondrial matrix (mito-DsRed1). In our hands, mito-DsRed1 has a distinct mitochondrial localization, with very little cytoplasmic signal (Collins et al., 2001) and no apparent self-aggregation of the fluorophore. For primary hepatocytes, which could not be transfected, the mitochondria were specifically visualized by loading with calcein (see Materials and methods). Deconvolved confocal images were surface-rendered to generate three-dimensional reconstructions of the mitochondria within the mito-DsRed1-expressing cells (Figure 1). Mitochondria were distributed throughout the cytosol of all cells, with a tendency to be aggregated around the nucleus. Individual, isolated mitochondria could be clearly resolved in the periphery of all cell types. In the perinuclear region, where the mitochondria were more densely packed, it was difficult to discern whether mitochondria were connected or only touching. The morphology of individual mitochondria ranged from small ‘grains’ to larger, sometimes branched, ‘threads’. Mitochondria were often observed under the nucleus, and occasionally also above it (e.g. Figure 1A). Figure 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. Download figure Download PowerPoint The three-dimensional reconstructions shown in Figure 1 were obtained with cells equilibrated at 22°C. This temperature was chosen because it dramatically slows the movement of mitochondria. This is important when taking image stacks, so that the same mitochondria do not appear in different focal planes, which could lead to a false impression of the size and connectivity of the organelles. In single confocal images, we did not see any significant effect of changing temperature from 22 to 37°C on the structure of the mitochondria (data not shown). Lumenal connectivity between mitochondria was determined using FRAP. In this process, the fluorophore is bleached in a subcellular region using brief high-intensity illumination. Any subsequent recovery of fluorescence in the bleached region occurs due to inward diffusion of unbleached fluorophore molecules. For the cell types that could be transiently transfected (HeLa, PAEC, COS-7, HUVEC, cortical astrocytes and neuronal cells), we utilized mito-DsRed1 for FRAP experiments. To examine the continuity of mitochondria in primary hepatocytes, FRAP experiments were performed using calcein-loaded cells. Since mito-DsRed1 and calcein are localized within the mitochondrial matrix, recovery of fluorescence after local photobleaching would indicate lumenal connectivity within the mitochondrial network. With all cell types examined, the brief high-intensity illumination caused bleaching of the DsRed1/calcein fluorescence in the irradiated region. Typical responses from mito-DsRed1-expressing HeLa and HUVEC cells are shown in Figure 2A, B, C. The lack of fluorescence recovery in a single calcein-loaded hepatocyte is illustrated in Figure 2D. In most cases, the area in which the fluorescence was bleached extended a few micrometres beyond the actual region illuminated. This was most probably due to the tunnelling of the fluorophores along the matrix of mitochondria that protruded outside the illumination region. Similar results were obtained using cells at 22 and 37°C (Figure 2A, B, C). Monitoring the average fluorescence intensity across the entire bleached region indicated that the fluorescence did not recover to >10% of its initial value up to 1 h after irradiation. Occasionally, there was a weak but persistent recovery of fluorescence after ∼20 min. This slow recovery of the fluorescence was more commonly observed at 37°C than at 22°C. We suggest that this recovery was not due to true FRAP within mitochondria (which is fast—see below), but rather movement of unbleached mitochondria into the bleached area or fusion between bleached and unbleached mitochondria. The FRAP procedure did not damage the mitochondria, since the organelles could be subsequently visualized by loading with a membrane-permeant mitochondrial indicator such as tetramethylrhodamine ethyl ester (TMRE) (data not shown). Figure 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. Download figure Download PowerPoint The lack of recovery of fluorescence illustrated in Figure 2 was not due to slow diffusion of DsRed1 or calcein within the mitochondrial matrix. To demonstrate this, we employed a similar FRAP protocol to that used above, except that the bleaching was performed in the periphery of cells where individual mitochondria were clearly identifiable. The high-intensity illumination was directed towards the middle of a long (∼22 μm) DsRed1-expressing mitochondrion (Figure 3Ai, arrow). This procedure caused the total loss of DsRed1 fluorescence from the irradiated area, and also decreased the DsRed1 fluorescence along the length of the mitochondrion outside the bleached area (Figure 3Aii). The DsRed1 fluorescence within the long mitochondrion was uniform before the bleach (Figure 3Ai), and had fully equilibrated within 90 s after the bleach (Figure 3Aiii). An analysis of the time-course of the fluorescence recovery revealed that this equilibration had a half-time of ∼15 s (Figure 3Bii). For the few small mitochondria that were completely encompassed within the illumination area, the fluorescence did not recover (e.g. trace a in Figure 3Bii), while those mitochondria outside the illumination region were unaffected (e.g. trace g in Figure 3Bii). The data depicted in Figure 3 were obtained from a HeLa cell, but are typical of responses from the other cell types at both 22 and 37°C (data not shown). Calcein was even more mobile than DsRed1 in the mitochondrial matrix. Equilibration of calcein fluorescence along HUVEC mitochondria at 22°C occurred within a few seconds after photobleaching (data not shown). Figure 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. Download figure Download PowerPoint The electrical continuity of mitochondria was assessed by examining the extent of irradiation-induced depolarization of the mitochondrial population. We utilized the previous observations that mitochondrial depolarization can be induced by a combination of high fluorophore concentration and laser irradiation. This is thought to be due to the generation of reactive-oxygen species (ROS), which subsequently trigger PTP opening (Amchenkova et al., 1988; Hüser et al., 1998; Hüser and Blatter, 1999; De Giorgi et al., 2000; Zorov et al., 2000; Buckman and Reynolds, 2001). TMRE is a fluorescent indicator that has been widely used to monitor mitochondrial membrane potential (Δψmit), but it can also lead to the opening of the PTP, probably via ROS production. We therefore used TMRE to both measure Δψmit and to cause spontaneous depolarizations. Cells were incubated with 1 μM TMRE for 30 min and confocal images were subsequently acquired at 0.2 Hz for periods of ∼10 min. Depolarization events were mapped frame-by-frame by following changes of TMRE fluorescence. For the HeLa cell example depicted in Figure 4Ai–v, analysis of the regions showing changes in TMRE fluorescence indicated that 85 electrically discrete mitochondria were present. In two additional HeLa cells that were analysed with the same frame-by-frame method, we observed 128 and 185 electrically discrete organelles. In keeping with the diverse morphologies of the mitochondria shown in Figure 1, the areas of TMRE fluorescence change were heterogeneous. Some of these electrically discrete mitochondria were quite long (∼45 μM) (Figure 4Avi). The Δψmit often ‘flickered’ several times before the TMRE fluorescence signal was permanently lost. The shapes of the depolarizing regions did not change from flicker to flicker, indicating that the 85 identified regions were truly discrete and the depolarization did not include different portions of an electrically connected network. Similar flickering of Δψmit was observed in all the cell types, and was present at both 22 and 37°C (data not shown). Figure 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) Depiction 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. Download figure Download PowerPoint Primary hepatocytes were consistently observed to have the shortest and least interconnected mitochondria. This was particularly evident from observations of TMRE- and laser-induced PTP flickering, as depicted in Figure 4B. The flickering of individual mitochondria occurred randomly (Figure 4Bi). Occasionally, several mitochondria depolarized simultaneously, and at other times, the same mitochondria would flicker on their own. Unlike in the other cell types, the regions of depolarization in hepatocytes were observed to be generally <5 μm (Figure 4Bix). The spontaneous depolarization of mitochondria in all cell types was dependent on a critical loading with TMRE, since with low levels of indicator spontaneous Δψmit depolarization was not observed (data not shown). Functional heterogeneity of mitochondria Δψmit. The distribution of Δψmit throughout the mitochondrial population of different cell types was monitored with the dual emission potentiometric dye tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1) (Reers et al., 1995; Salvioli et al., 1997). JC-1 accumulates preferentially in polarized mitochondria, existing as green (530 nm emission) fluorescent monomers at low membrane potentials and as orange/red (590 nm emission) fluorescent aggregates at high membrane potentials. In none of the cell types tested did we observe a homogenous red or green JC-1 fluorescence from the mitochondria. Instead, the mitochondria were either green or red, indicating a distinct heterogeneity of Δψmit (Figure 5). The proportions of green- and red-emitting mitochondria were not evenly distributed in cells. The red-fluorescing, highly energized mitochondria were proportionally more prevalent in the periphery of cells (Figure 5Ai). By randomly sampling mitochondria from the periphery and perinuclear regions of HeLa cells, it was observed that 68 ± 5% (± SEM; n = 25 cells) of peripheral mito chondrial were red fluorescing, whilst only 28 ± 4% of perinuclear mitochondria were red. To show that the JC-1 staining was specific for mitochondria, cells were co-loaded with TMRE (e.g. Figure 5Aii). JC-1 and TMRE showed the same cellular localization. In addition, the red fluorescence from both dyes disappeared upon addition of antimycin (Figure 5Aiii), indicating that the TMRE signal and the aggregation of JC-1 were dependent on respiring mitochondria. A predominance of red-fluorescing mitochondria in the periphery of the cell was also seen with JC-1-stained hepatocytes (Figure 5B), HUVECs (Figure 5Ci and ii), COS-7, PAEC and cortical astrocytes and neurons (data not shown). A similar pattern of staining was observed in cells at 22 or 37°C. Figure 5Ci depicts the JC-1 fluorescence of a HUVEC cell loaded with 1 μM JC-1 for 1 h at 22°C. Raising the temperature of the cells to 37°C and equilibrating them for a further 30 min in the continued presence of 1 μM JC-1 did not did dramatically alter the staining pattern: the green-staining mitochondria were still perinuclear, and the red-staining organelles were largely peripheral. Figure 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. Download figure Download PowerPoint TMRE partitions across the mitochondrial inner membrane in a manner proportional to Δψmit, and can also be used to monitor differences in Δψmit. We therefore examined the distribution of TMRE within cells. Although confocal imaging allows optical sectioning of cells, out-of-focus fluorescence can enter the image plane and make it appear artificially bright. The amount of out-of-focus light that enters a particular confocal plane is dependent on the intensity of fluorescence in the optical sections above and below it (Fink et al., 1998). Since the density of mitochondria is much greater around the nucleus than in the periphery (Figure 1), TMRE fluorescence from mitochondria outside a confocal plane could make the perinuclear mitochondria appear brighter than the more sparsely distributed peripheral mitochondria. To eliminate this problem and obtain a better indication of the brightness of individual mitochondria, we used image deconvolution (Fink et al., 1998). Typically, stacks of confocal images (z-stack; 0.2 μm steps) were obtained by scanning through the depth of a TMRE-loaded cell (cells were loaded with 0.1 μM TMRE for 20 min). These image stacks were deconvolved using a blind deconvolution algorithm. Since TMRE partitions between mitochondria and the cytosol in a Δψmit-dependent manner, ratios of fluorescence intensity in mitochondria and their adjacent cytosolic areas were obtained. For HeLa cells, this analysis indicated that TMRE fluorescence intensity was significantly higher (P <0.005; mean ± SEM, 33.8 ± 3.0; n = 58 mitochondria from 10 cells) than in the perinuclear region (mean ± SEM, 22.4 ± 1.8; n = 63 mitochondria from 10 cells). Similar observations were seen with the other cell types (data not shown). Differences in Δψmit should also be manifest in the rate of TMRE uptake, in that the more polarized mitochondria will load with TMRE faster than those with lower membrane potential. To locate mitochondria and compensate for differences in mitochondrial density, the increase of TMRE fluorescence was ratioed against the signal from mitochondria previously loaded with Mitotracker Green FM. Mitotracker Green FM, unlike other Mitotracker dyes, accumulates in mitochondria regardless of the Δψmit (Haugland, 1999). Cells were therefore initially stained with 1 μM Mitotracker Green FM for 20 min before adding TMRE. Dual-channel images were acquired with a Bio-Rad MRC1024 confocal microscope. It was consistently observed that the normalized TMRE accumulation in HeLa cells proceeded at a greater rate for peripheral mitochondria than in perinuclear mitochondria (mono-exponential rate constants: 0.19 ± 0.04/min for peripheral versus 0.07 ± 0.01/min for perinuclear) (Figure 6). Figure 6.Heterogeneity in Δψmit 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. Download figure Download PowerPoint The peripheral regions of cells have a higher plasma membrane surface to volume ratio than the perinuclear areas. We were concerned that the distinct rates of TMRE sequestration in peripheral and perinuclear mitochondria could simply have reflected different kinetics of TMRE accumulation in the cytosol surrounding the organelles. We therefore examined the rate of TMRE accumulation in the peripheral cytoplasm and nucleus. The time-course of TMRE fluorescence increase was identical for both regions (Figure 6, inset), suggesting that TMRE reaches these parts of the cytoplasm with the same kinetics. Ca2+ sequestration. Rapid (15 Hz) confocal imaging of histamine-stimulated rhod-2-loaded HeLa cells revealed that mitochondria in the periphery of the cells sequestered more Ca2+ than mitochondria located in the perinuclear region (Figure 7A). Although the onset of mitochondrial Ca2+ accumulation was similar for peripheral and perinuclear mitochondria, the former continued to sequester Ca2+ when their counterparts were beginning to plateau. The difference in Ca2+ sequestration was only apparent for inositol 1,4,5,triphosphate (InsP3)-evoked Ca2+ release. More slowly developing Ca2+ signals did not cause such differences in mitochondrial Ca2+ sequestration. For example, Ca2+ entry triggered by re-addition of Ca2+ to cells pre-treated with thapsigargin or cyclopiazonic acid (CPA) in Ca2+-free medium gave equivalent perinuclear and peripheral mitochondrial Ca2+ uptake (Figure 7B). Treatment with CGP37157, an inhibitor of mitochondrial sodium-Ca2+ exchange, failed to reveal any difference in Ca2+ sequestration by peripheral and perinuclear mitochondria during thapsigargin treatment or Ca2+ entry (Figure 7Biii). Figure 7.Perinuclear and peripheral
