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Mitochondria are morphologically heterogeneous within cells

Tony J. Collins^* and Martin D. Bootman

Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK

View larger version (71K): [in a new window]   Fig. 2. FRAP analysis of mitochondrial lumenal continuity. (Ai-iv) The DsRed1 fluorescence in a HeLa cell before and 10 s, 20 min and 60 min after photobleaching a small area of mitochondria in the perinuclear aggregation (denoted by white box). (Av) At 60 min post-photobleach, 1 µmol 1-1 TMRE was added to the cells for 5 min. The mitochondria that failed to recover DsRed1 fluorescence in Av were stained with TMRE, indicating that their functional integrity was not compromised during laser irradiation. Note that the laser intensity was reduced by 33% in Av compared to Ai-iv, to prevent reactive oxygen species (ROS) production and permeability transition pore (PTP) opening. See text for details. (Bi-v) The DsRed1 fluorescence in a cortical astrocyte before and 10 s, 20 min and 60 min after photobleaching the area denoted by the white box in Bi. (Bii–v) The region of the cell bounded by the broken line in A on an expanded scale. Scale bars, 5 µm.

View larger version (93K): [in a new window]   Fig. 1. Morphologically discrete mitochondria in living cells. (A) Volume render of pancreatic acini loaded with the dye TMRE (0.5 µmol 1-1, 20 min). The approximate locations of individual acinar cells are outlined in the inset image. Note that this image essentially shows the sub-plasmalemmal mitochondria around the perimeter of the individual acinar cells. (B–E) Surface render of mitochondrially targeted DsRed1-expressing cells. (B) PAE cells, (C) SH-SY5Y cells, (D) COS-7 cells and (E) cortical astrocytes. See text for details. Scale bars, 5 µm.

View larger version (60K): [in a new window]   Fig. 4. Electrical discontinuity of mitochondria, demonstrated by asynchronous depolarisation events induced by TMRE plus laser irradiation. Cells were loaded with TMRE and imaged with a moderate laser intensity (30 µW at objective). Rapid mitochondrial depolarisation events appeared as loss of fluorescence between frames (typically 2–4 s). (A) A TMRE-loaded pancreatic acinar cell. Please note that the image is a median section of a cell where the apical region would project out of the figure and the basal pole behind the figure. The locations of mitochondria that showed clear depolarisation events are mapped in (Aii). The grey areas denote TMRE-stained mitochondria that did not flicker. G, the position of the granular region, which is bounded by the perigranular mitochondria (PG). The subplasmalemmal (SPM) mitochondria can been seen around the outside of the cell. (Bi–Di) Portions of a HeLa cell (Bi), HUVEC cells (Ci) and ventricular cardiomyocyte (Di), following loading with TMRE. (Bii–Dii) The positions of electrically discrete mitochondria are depicted in the HeLa cell (Bii) and HUVEC cell (Cii). The maps of individual electrically isolated mitochondria were constructed by monitoring the locations of individual depolarisation events over time. The magenta, cyan and yellow colouration is used to indicate the positions of the electrically isolated mitochondria, and does not indicate any relationships between the organelles. (Dii) A line-scan plot derived from the region between the arrowheads in (Di), illustrating the asynchronous flickering of the majority of mitochondria. A few mitochondria do appear to depolarise synchronously (Dii, arrowheads). Scale bars, 5 µm.

View larger version (83K): [in a new window]   Fig. 3. Rapid mobility of DsRed1 within the mitochondrial matrix. A long mitochondrion in the periphery of a HeLa cell was located (A, arrowheads). A small area at one end of the mitochondrion (B, white square) was photo-bleached. (C) The fluorescence recovery was monitored for 90 s at 2 Hz. Fluorescence from a region at the photo-bleached end of the mitochondria (blue circle in 3B and corresponding blue line in D) was compared with the fluorescence from a region that was also photo-bleached but 5 µm closer to the unbleached end of the mitochondria (purple circle in 3B and corresponding purple line in D). The half-maximum fluorescence recovery (0.5 F/F0) took 5 s longer in the site 5 µm further from the unbleached end of the mitochondrion, giving an estimated linear diffusion rate for DsRed1 of approximately 1 µm s-1 along the mitochondrion. Scale bar, 5 µm.

View larger version (93K): [in a new window]   Fig. 5. Examples of atypical interconnected mitochondrial networks. (A) HUVEC cell loaded with TMRE was imaged with moderate laser intensity to trigger depolarisation of mitochondria by spontaneous PTP activation. This unusual HUVEC cell showed synchronous depolarisation across a large mitochondrial network. At 48 s (Ai) the mitochondria in the cell are all polarised, at 52 s (Aii) a large electrically continuous area was seen to depolarise, and 8 s later (Aiii) it had repolarised. After 120 s (Aiv) of imaging, this large electrically continuous mitochondrion depolarised again and did not recover during the experiment (5 min). (B) A cortical astrocyte that was found to have lumenally continuous mitochondria using FRAP. (Bii–iv) The region of the cell bounded by the dashed box in Bi on an expanded scale. The cell was imaged before (Bi,ii), approximately 10 s (Biii) and 120 s after (Biv) after bleaching a small area of the mitochondrion (the bleached region is denoted by the solid white box). During the photobleach process, the laser intensity was increased and sufficient irradiation was applied to reduce the fluorescence in the bleached area to zero. Despite this complete bleach, the fluorescence can already be seen to have partially recovered 10 s later and fully equilibrated after 120 s (Biv). Scale bar, 5 µm.