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First published online 19 February 2003
doi: 10.1242/jcs.00320

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Two phases of signalling between mitochondria during apoptosis leading to early depolarisation and delayed cytochrome c release

Department of Biochemistry and Molecular Biology, PO Box 13D, Monash University, Victoria 3800, Australia

View larger version (61K): [in a new window]   Fig. 1. Visualisation of m loss in non-irradiated mitochondria following partial irradiation of cells with filamentous and discrete punctate morphology of mitochondria. (A) Confocal imaging of two human osteosarcoma 143B cells before (0 seconds) and at end of partial irradiation (128 seconds) either treated without or with CCCP (20 µM for 60 minutes followed by 60 minutes recovery in the absence of CCCP), which induces punctate morphology. Both cells were loaded with m indicator Rh123 (10 µM) and photosensitiser CMXRos (200 nM) prior to irradiation. The area of irradiation is indicated by the dashed box. Loss of m in filamentous and punctate mitochondria is representative of that in 18 of 21 cells without CCCP and 8 of 9 cells after CCCP treatment and recovery. (B) Confocal images of a 143B cell stably expressing MtGFP before (left) and after (right) treatment with CCCP followed by recovery. Bars, 10 µm.

View larger version (17K): [in a new window]   Fig. 2. Comparative analysis of m loss in non-irradiated mitochondria and death indications in cells with either filamentous or punctate mitochondria. (A) Percentage of cells showing high m (detected by Rh123 and visualised by confocal microscopy) following various treatments. Cells were treated with CCCP (or not treated), and then allowed to recover for 60 minutes in CCCP-free medium. Cells were then loaded with Rh123 and/or CMXRos before irradiation of about half the mitochondrial area in the perceptible section of the cell. The number of cells analysed under each set of conditions is indicated above each bar. (B) Percentage of irradiated cells showing cyt c release, PS exposure, and PI uptake between 30 and 120 minutes after irradiation, either containing filamentous mitochondria in otherwise untreated cells (filled bars; n=19) or punctate mitochondria in cells treated with CCCP and allowed to recover (open bars; n=16). Cells loaded with CMXRos (200 nM) were subjected to partial irradiation, then stained with annexin V conjugated to Alexa Fluor 488 and PI, followed by visualisation with confocal microscopy before fixing and staining with anti-cytochrome c antibody. There was no significant difference between the filamentous untreated reference group and punctate CCCP-treated group in all categories (P>0.1) as assessed by chi-squared contingency test.

View larger version (92K): [in a new window]   Fig. 3. Fragmentation of mitochondria during partial irradiation with CMXRos. (A) Confocal images showing the irradiated part of a 143B cell stably expressing MtGFP (loaded with CMXRos) at various times during partial irradiation. The total duration of partial irradiation was 128 seconds. Change in mitochondrial morphology is representative of that in all 7 cells analysed. (B) Confocal images showing the irradiated part of a control cell stably expressing MtGFP that was partially irradiated without CMXRos under the same conditions as in A. Retention of filamentous morphology is representative of that in all five cells analysed. Gain levels were adjusted for images in both A and B to permit visualisation of mitochondria during intensive laser scanning, which results in some photobleaching of GFP and CMXRos. Bars, 5 µm.

View larger version (113K): [in a new window]   Fig. 4. Release of cyt c-GFP following partial irradiation. (A) Confocal images showing cells stably expressing cyt c-GFP loaded with CMXRos. One cell that had adequate mitochondrial cyt c-GFP localisation was subjected to partial irradiation, the region of irradiation indicated by the dashed box. Other adjacent cells that had diffuse cyt c-GFP localisation were not irradiated. Images were collected before irradiation, immediately after irradiation (0 minutes) and at various times after partial irradiation (25 minutes and 30 minutes). Gain levels for CMXRos signal at 25 minute and 30 minute time points were increased manually to enable visualisation of mitochondria. Note that at 30 minutes the irradiated cell had begun to detach from the growth substrate, causing it to reside in a different focal plane from that of fully adherent non-irradiated cells. Therefore, the CMXRos and cyt c-GFP fluorescence in such non-irradiated cells were not visualised when the image was captured at the particular optical Z-plane of focus of the irradiated cell. Release of cyt c-GFP is indicated by a weaker diffuse GFP fluorescence throughout the cell, the mitochondrially localised fluorescence becoming greatly reduced. This release is representative of that in 17 of 31 cells analysed up to 2.5 hours after irradiation (cf. Fig. 5). (B) Images of control cyt c-GFP expressing cells not irradiated were collected similarly to those in A. The retention of GFP fluorescence in mitochondria indicating failure to release cyt c-GFP was observed in all of 21 cells analysed. Bars, 10 µm.

View larger version (10K): [in a new window]   Fig. 5. Kinetics of cyt c-GFP release following partial irradiation and effect of zVAD-fmk. Data show temporal distribution of cyt c-GFP expressing cells that exhibit release of cyt c-GFP, monitored at half hour intervals following irradiation. Cells were loaded with CMXRos and partially irradiated in the absence of zVAD-fmk (filled bars, n=31) or in the presence of zVAD-fmk (100 µM, pre-treated for 30 minutes; open bars, n=20). Each cell was monitored every 30 minutes up to 2.5 hours after irradiation and cyt c-GFP release was scored as percentage of total cells analysed. Asterisk (*) indicates cells that did not show cyt c-GFP release by 2.5 hours after irradiation; such cells may or may not release cyt c-GFP after this time. Hash symbol (#) indicates no significant difference in the proportion of cells showing cyt c-GFP release between the untreated group and zVAD-fmk treated group (P>0.3, chi-squared contingency test); all other comparisons on inspection are not significantly different.

View larger version (54K): [in a new window]   Fig. 6. Bax is not recruited to non-irradiated mitochondria following partial irradiation. Cells were either subjected to partial irradiation (Irr) after labelling with CMXRos or treated with staurosporine (STS; 0.5 µM for 24 hours), or neither, before being fixed and stained with anti-Bax antibody. The photoirradiated cell was subjected to intensive laser scanning in its lower half (as oriented in this image; cf. Fig. 1) and fixed at 75 minutes after irradiation. Other cells partially irradiated were fixed for Bax staining at 30-150 minutes following irradiation. Bax translocation (Bax Trans) to mitochondria was visualised by confocal microscopy and scored as the percentage of the total number (n) of cells analysed in each treatment. Bars, 10 µm.

View larger version (35K): [in a new window]   Fig. 7. Effect of BAPTA and NAC on intracellular Ca2+ and ROS generation induced by partial irradiation. (A) Quantification of Fluo-3 fluorescence as a measure of intracellular Ca2+ levels in CMXRos-loaded cells (except for control cells) 30-60 minutes following partial irradiation. Prior to irradiation, CMXRos-loaded cells were either treated with BAPTA-AM (10 µM, 30 minutes) or NAC (15 mM, 30 minutes). Fluo-3 (2.5 µM) was subsequently loaded after irradiation and the fluorescence in partially irradiated cells was determined as described in Materials and Methods and expressed relative to non-irradiated cells in the same field (±standard deviation). Control cells for laser phototoxicity were neither stained with CMXRos nor treated with BAPTA and NAC prior to partial irradiation. The number of cells analysed in each group is indicated on top of each bar. Significant differences (*P<0.01; **P<0.001) between the group analysed in comparison with the group loaded with CMXRos but not treated with BAPTA or NAC were tested by student's unpaired t-test. (B) Representative images showing the increased Fluo-3 fluorescence (in this case 4.1-fold) in a partially irradiated cell compared with a non-irradiated cell. CMXRos was loaded prior to irradiation and Fluo-3 AM (2.5 µM) was loaded after irradiation. Region of irradiation is denoted by the dashed box and time after irradiation indicated in the top right. (C) Quantification of DCF fluorescence as a measure of intracellular ROS levels in CMXRos-loaded cells (except for control cells) 30-60 minutes following partial irradiation, as described in A. (D) Representative images showing increased DCF fluorescence in a partially irradiated cell (in this case 7.9-fold) collected similarly to those in B and detected by H2DCFDA (40 µM) loaded after irradiation. This partially irradiated cell shows evidence of blebbing (45 minutes). Note that in B at 30 minutes and in D at 45 minutes the gain levels of CMXRos images were increased to permit visualisation of mitochondria in partially irradiated cells. In quantitative analyses of fluorescence care was taken to ensure that only adherent cells were imaged in all cases. Thus, the various cells depicted in B and D are imaged in the same focal plane in each case. The DCF fluorescence of non-irradiated cells in D is close to the background levels. Bars, 10 µm.

View larger version (12K): [in a new window]   Fig. 8. Direct signal transmission between mitochondria during apoptosis induced by microscopic photosensitisation. Note the differential behaviour of irradiated and non-irradiated mitochondria, indicative of two phases of signalling between mitochondria. Bcl-2 not only blocks the putative Ca2+-mediated signals between these two populations of mitochondria but also suppresses cyt c release from irradiated mitochondria.