Folia Morphol. Vol. 59, No. 2, pp. 61–75 Copyright © 2000 Via Medica Takashi Wakabayashi et al., Mitochondria and R E V I E W ARTICLE ISSN 0015–5659

Structural changes of mitochondria during free radical-induced apoptosis

Takashi Wakabayashi1, Jan H. Spodnik1,2

1Department of Cell Biology and Molecular , Nagoya University School of Medicine, Japan 2On leave of absence from the Department of Anatomy and Neurobiology, Medical University of Gdańsk, Poland

[Received 30 March 2000; Accepted 4 April 2000]

The initial proposal for apoptosis stressed nuclear change (condensation of chro- matin) and the intactness of intracellular organelles, including mitochondria, based on light and electron microscopic observations. However, data have accu- mulated to demonstrate that the opening of megachannels of mitochondrial membranes, resulting in the swelling of the organelles, notably by Ca2+ and free radicals, is the crucial step in the apoptotic processes of the cell. Application of fluorescent dyes to mitochondria, combined with , has made it possible to detect subtle changes in the structure and function of the organelles related to apoptosis. The present article overviews structural aspects of mito- chondria related to apoptosis, including the free radical-induced formation of megamitochondria.

key words: apoptosis, megachannels, free radicals, megamitochondria, fluorescent dyes, flow cytometry

INTRODUCTION excessive ATP synthesis might be disadvantageous Various cellular functions in mammalian cells are or harmful to the cell; cardiac mitochondria, which largely dependent upon ATP generated from mito- are required to produce a massive amount of ATP to chondria. Thus, changes in the structure and func- maintain the cardiac function, are rich in cristae and tion of mitochondria in physiological and patholog- contain a much larger number of cytochromes than ical conditions reflect those in the energy require- those of other tissues such as the liver; diaphragm ment of the cell: the mitochondria of various tissues mitochondria form a reticular network (“mitochon- in hibernating animals become enlarged (the forma- dria reticulare”) serving as a cable to transport mo- tion of megamitochondria (MG)), resulting in de- lecular oxygen and other substances across a large creases in the oxygen consumption by mitochondria, distance in the myocyte; adrenal cortex mitochon- which may lead to decreases in the intracellular lev- dria are characterised by the presence of “vesicular els of ATP. In non-hibernating animals in physiolog- cristae” which are common to those of steroid-pro- ical conditions, mitochondria consume a large ducing organs. When steroidogenesis is suppressed amount of oxygen to generate ATP to maintain cel- (for example, by hypophysectomy), the transition of lular activities resulting in the generation of a much the cristal membranes of mitochondria from the ve- larger amount of reactive oxygen species (ROS) than sicular configuration to the tubular one takes place. in hibernating animals. Since cells of various tissues Besides the central role described above of mito- in hibernating animals require the amount of ATP chondria as a high energy-producing machinery, data which is sufficient to maintain the basal metabo- have accumulated to demonstrate that mitochon- lism, generation of a large amount of ROS due to dria play a key role in apoptotic processes of the cell

Address for correspondence: Takashi Wakabayashi, Department of Cell Biology and , Nagoya University School of Medicine, 65, Tsurumai-cho, Showa-ku, Nagoya 466–8550, Japan, tel: +052 744 2028; fax: +052 744 2041; e-mail: [email protected]

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via the following various components localised within or to electron microscopy when isolated mitochon- the organelles: the megachannels (MGCs) dria are used in that changes in absorbance at 520 [23,35,69,71,72,86], cytochrome c, apoptosis-induc- nm (or 540 nm) reflect those of mitochondria in a ing factor (APF) [70], caspase 9 [37], caspase 3 [45] large population (Fig. 1) [81]. Mitochondria isolated and Bcl-2 family [1,6,14,21,51,52,76,80]. Pro- in a medium containing sucrose remain in the con- caspase-9 and pro-caspase-3 can reside either in the densed configuration as long as the MGCs are closed. cytosol or within mitochondria, depending on cell When the channels are opened mitochondria lose types. A small portion of the total cellular pool of impermeability to sucrose and the transition from the pro-caspase-3 enters the mitochondria [45]. When condensed to orthodox configuration takes place. cytochrome c is released from the mitochondria into Thus, the swelling of isolated mitochondria can be the cytoplasm, it binds the caspase-activating protein easily detected also by electron microscopy (Fig. 2). Apaf-1, stimulating binding of Apaf-1 to pro-caspase- However, quantitative evaluation of electron micro- 9 resulting in the activation of the caspase [40]. scopic data is inevitably associated with some diffi- According to the currently accepted hypothesis culties. When cells are used, spectrophotometric for the mechanism of the apoptotic processes of the techniques for the analysis of mitochondrial swell- cell [57,66], the following series of events take place: ing cannot be adopted since changes in absorbance 1. Opening of MGCs of the inner mitochondrial mem- at 520 nm (or 540 nm) reflect not only those ob- branes where the inner and outer membranes con- tained from mitochondria but also those from other tact notably by Ca2+ and free radicals. 2. Swelling of organelles. mitochondria. 3. Release of cytochrome c, caspase 9 Mitochondria in culture cells or in tissues remain and APF from mitochondria into the cytoplasm. 4. in the orthodox configuration since the aldehyde fix- Decreases in the membrane potential of mitochon- atives used for electron microscopy do not contain dria (DYm) 5. Activation of caspases by cytochrome sucrose. It is easy to detect mitochondria which are c resulting in proteolysis in the cytoplasmic compo- swollen distinctly by electron microscopy since the nents and degradation of nuclear DNA by APF. The mitochondrial matrix become distinctly pale and the hypothesis described here emphasises the opening cristae are pushed to the periphery in addition to of MGCs by free radicals resulting in the swelling of increases in their sizes. However, small changes in mitochondria. However, several controversial data the size of mitochondria are often overlooked and have been presented against the hypothesis in the mitochondria which are only swollen slightly are past few years, which will be discussed later. misinterpreted as “intact”. Since the routine elec- Recently, we have found that free radical-induced tron microscopy using thin-sectioned specimens formation of megamitochondria (MG) is succeeded gives us information about the structure of mito- by apoptotic changes of the cell and that apoptosis chondria on one plane of section, even a subtle is suppressed by scavengers of free radicals via the change in the size of mitochondria which are unde- suppression of the MG formation [32,33]. This may tectable or difficult to be detected on one plane of strongly suggest that MG should be regarded as section may indicate significant changes on the ba- another important structural change of mitochon- sis of their three dimensions. Thus, one of the rea- dria besides “swelling” which is intimately related sons why mitochondrial intactness during apopto- to apoptosis. sis had been emphasised for more than 20 years The present article overviews the structural chang- [5,10,15,19,34,43,48,82] before the proposal of Petit es of mitochondria during apoptotic changes of the et al. [57] and Skulachev [66] stressing the impor- cell based on the survey of the literature and on data tance of the swelling of mitochondria may be partly obtained from our laboratory. due to a misinterpretation of mitochondrial struc- tures observed on electron micrographs. This was Swelling of mitochondria as a result inevitable since only routine light and electron mi- of the opening of megachannels (MGCs) croscopic techniques were available during that pe- The opening of MGCs of mitochondria can be de- riod to detect structural changes of mitochondria in tected either by direct measurements with patch- situ. Recently, Jurgensmeier et al. [31] have shown clamp techniques [35], or by indirect measurements that Bax, a proapoptotic member of the Bcl-2 fami- using a spectrophotometer [57] or electron micros- ly, induces cytochrome c release without causing the copy. The latter indirect methods detect the swell- swelling of mitochondria. They judged the changes ing of mitochondria as a result of the opening of in the volume of mitochondria using a spectropho- MGCs. The spectrophotometric technique is superi- tometer.

62 Takashi Wakabayashi et al., Mitochondria and apoptosis

Figure 1. Detection of the swelling of mitochondria by a spectrophotometer. As a result of the opening of the megachannels. Mitochondria isolated from the livers of control rats, those of animals fed with a hydrazine-diet for 4 d or 9 d were exposed to various swelling media: potassium acetate (100 mM K-acetate, 0.1 mM EDTA, 15 mM Tris-HCl, pH 7.4, valinomycin, 0.5 mg/ml); ammonium suc- cinate (100 mM ammonium succinate, 0.1 mM EDTA, 15 mM Tris-HCl, pH 7.4, 5 mM Pi); sodium perchlorate (500 mM sodium perchlor- ate), 0.1 mM EDTA, 15 mM Tris-HCl, pH 7.4, antimycin A, 0.5 mg/ml). Mitochondria obtained from control animals and those fed with a hydrazine-diet for 4 d swell distinctly whereas those with the toxic diet for 9 d swell less distinctly indicating that the latter mitochondria are swollen already before they are exposed to swelling media.

Figure 2. Transition from the condensed to orthodox mitochondria induced by Ca. Beef heart mitochondria isolated in the condensed configuration (A) were treated with 10 mM Ca for 30 min at 30oC (B).

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Structural changes of mitochondria absence of chloramphenicol (CP) and stained with during the phase of “secondary ” CMTMRos with or without pretreatment with an ion- In vivo, apoptotic cells and apoptotic bodies are ophore, gramicidin. In control cells, the peak shifts phagocytosed by mononuclear phagocytes or sur- to the left distinctly when they are pretreated with rounding epithelial cells while culture cells undergo- gramicidin (dotted peak), indicating that DYm mea- ing apoptosis detach early on from the monolayer sured in the absence of gramicidin is influenced by and float in the culture medium. These cells escape volume changes of mitochondria. Thus, actual DYm phagocytosis by neighbouring adherent cells and is obtained by calculating the difference between proceed to undergo spontaneous degenerative the mean fluorescence intensity of the dye measured changes that result in the loss of cell membrane in- in the absence and presence of gramicidin. Howev- tegrity and swelling of intracellular organelles includ- er, the peak is much broader than that of the con- ing mitochondria. This process is called “secondary trol overlapping partly the peak obtained in the ab- necrosis” [16,34,41,60,61,82]. Cells undergoing “sec- sence of gramicidin. This indicates that DYm is de- ondary necrosis” as demonstrated in the literature creased in a large population of CP-treated cells and have condensed nuclei as evidence of apoptosis that the total volume of mitochondria per cell is dis- whereas they are swollen extremely with the rup- tinctly increased. When the total volume of mito- ture of the cell membrane and with remarkable swell- chondria per cell is increased, there are two possibil- ing of organelles including mitochondria. The cell ities: this is either due to the swelling of mitochon- demonstrated by Camilleri-Broet et al. [13] as an dria or due to the proliferation of the organelles example of secondary necrosis exhibits a prominent (increases in the mass of mitochondria). Increases in mitochondrial swelling as the authors pointed out the mass of mitochondria per cell are detected by whereas it shows a typical feature of apoptosis with the fluorescent dye Mito Tracker Green FM (FM) [75] fragmented nuclei and the aggregation of chroma- or nonyl acridine orange (NAO) [42,75]. For exam- tin. Swelling of mitochondria in this case is inter- ple, the peak intensity of FM in the cells treated with preted as a result of secondary necrosis [13]. How- CP for 22 h shifts to the right compared to that of ever, we should not misinterpret these swollen mi- those treated with CP for 12 h and it shifts further tochondria as a sign of secondary necrosis and the to the right when the CP treatment is prolonged to swelling of mitochondria in this case should be re- 36 h (Fig. 4A). In Fig. 4B, mitochondria isolated from garded as a morphological change of mitochondria control rats (A3) and those placed on a hydrazine- intrinsic to apoptosis in advanced stage as long as diet for 3 d (B3) and 7 d (C3), respectively, were ap- the cell shows a characteristic feature of apoptosis. plied to flow cytometry. Mitochondria in the liver of The cell may or may not undergo secondary necrosis animals treated with hydrazine for 3 d (B1) were with time. slightly enlarged compared to those of the control (A1). However, the peak intensity of FM in mitochon- Application of fluorescent dyes dria isolated from the former animals remained es- to the analysis of structural and functional sentially in the same position as that of control ani- changes of mitochondria in apoptosis mals. Mitochondria became extremely enlarged in Disadvantages associated with spectrophotometric the liver of animals treated with hydrazine for 7 d and electron microscopic techniques have been large- (C1), and the peak intensity of FM of mitochondria ly overcame by the application of various fluores- isolated from these animals was distinctly broadened. cent dyes combined with flow cytometry to the re- When both the mass and volume of mitochondria search field of apoptosis. For example, using culture per cell are increased we may conclude that the num- cells structural changes of mitochondria in the cell ber of mitochondria per cell has increased with or can be detected quantitatively and to some extent without the swelling of the organelles. qualitatively using Mito Tracker CMXRos [58] or Mito The rate of the generation of reactive oxygen Tracker CMTMRos [58] for confocal microscopy (Fig. species (ROS) from mitochondria can be measured

3A) combined with flow cytometry (Fig. 3B). Using with H2DCFDA [3,20,38] or carboxy-H2DCFDA [33] either dye in the presence and absence of uncou- using culture cells or isolated mitochondria. Super- plers of oxidative phosphorylation of mitochondria oxide can be specifically detected by dihydroethidi- or ionophores, the DYm and volume changes of um [44]. Overall intracellular level of ROS (mainly mitochondria per cell can be detected. In Fig. 3B, RL- generated from mitochondria) and extra-mitochon- 34 cells were cultured for 48 h in the presence and drial generations of ROS in the cell can be detected

64 Takashi Wakabayashi et al., Mitochondria and apoptosis

A

B

Figure 3. Visualisation of mitochondria and detection of their DYm and volume changes by fluorescent dyes. A) Visualisation of mitochondria by CMXRos. Cells cultured for 22 h in the absence (A) and presence of chloramphenicol (CP) (200 mg/ml) (B) were stained with CMXRos for confocal laser microscopy. B) Detection of the DYm and volume of mitochondria per cell by CMTMRos. RL-34 cells were cultured for 48 h in the presence and absence of CP and stained with CMTMRos in the presence and absence of gramicidin. (B was modified from ref. 33).

using carboxy-H2DCFDA and CM-H2TMRos, respec- presence of CP (A). Similar tendencies are obtained tively (Fig. 5) [33]. In control cells cultured for 48 h, using CM-H2TMRos both in control and CP-treated the intracellular level of ROS is slightly increased com- cells (B). Since CM-H2TMRos does not react with mi- pared to that cultured for 22 h whereas that of the tochondria unless it is oxidized (CMTMRos), these cells cultured for 48 h in the presence of CP is de- results indicate extra-mitochondrial generation of creased compared to that cultured for 22 h in the ROS. The rate of the generation of ROS from mito-

65 Folia Morphol., 2000, Vol. 59, No. 2

A

B

Figure 4. Detection of the total mass of mitochondria per cell. A) RL-34 cells were cultured for 12 h, 22 h, and 36 h, respectively, in the presence of CP, and then stained with FM for flow cytometry (modified from ref. 32). B) Mitochondria isolated from the liver of control rats (A3), those treated with hydrazine for 3 d (B3) or 7 d (C3) were stained with FM.

66 Takashi Wakabayashi et al., Mitochondria and apoptosis

A

B

C

Figure 5. Detection of overall intracellular level of ROS and the rate of mitochondrial and extra-mitochondrial generation of ROS.

A) RL-34 cell cultured for 22 h and 48 h, respectively, in the presence and absence of CP were stained with carboxy-H2DCFDA to detect intracellular level of ROS. B) Experimental conditions are the same as those shown in Fig. 5A except that cells were stained with

CM-H2TMRos to detect extra-mitochondrial generation of ROS; C) Mitochondria isolated from the cells used in Fig. 5A were stained with carboxy-H2-DCFDA to detect ROS (modified from ref. 32).

chondria isolated from the control and CP-treated has been successfully suppressed by scavengers of cells (C) show similar results to those obtained using free radicals such as 4-OH-TEMPO (Figs. 6 and 7) cells thus suggesting a possibility that increases in [32,33]. For example, mitochondria in IAR-20 cells the overall intracellular level of ROS observed in the cultured for 22 h in the presence of CP become en- cells treated with CP for 48 h are partly due to de- larged distinctly (Fig. 6B) compared to those of the creases in the mitochondrial generation of ROS. control (Fig. 6A). When the cultivation time of these cells is prolonged to 48 h, the matrix of enlarged Free radical-induced formation mitochondria becomes pale suggesting the swelling of megamitochondria is succeeded by (Fig. 6C), and apoptotic cells characterised by the apoptosis condensation of nuclear chromatin are detected (Fig. 6E). Recently, we have shown using culture cells of vari- Appearances of apoptotic cells are not accidental ous sources of tissues that free radical-induced for- since the population of apoptotic cells reaches about mation of megamitochondria (MG) is followed by 45% in cells treated with CP for 72 h (Fig. 6F). The apoptotic changes of the cell, and that the latter formation of MG and succeeding apoptotic chang-

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F

Figure 6. Induction of megamitochondria and succeeding apoptosis by CP. A–E) IAR-20 cells were cultured in the absence (A) or presence of CP for 22 h (B), 48 h (C and D) or 72 h (E). Cells in Fig. 6D were pre- treated with 4-OH-TEMPO. F. RL-34 cells were cultured for 48 h and 72 h, respectively, in the presence of CP with or without the pretreat- ment with 4-OH-TEMPO or cycloheximide (CHX). Numbers of apoptotic cells were obtained by flow cytometric analysis of propidium io- dide-stained cells. Presence of sub-G1cell population was used as an indicator of apoptosis. Data are the averages and standard error (mean±SE) of three different experiments. Values of experimental groups are statistically different from those of the control at: a (0.001

68 Takashi Wakabayashi et al., Mitochondria and apoptosis

Figure 7. Induction of MG by hydrazine in rat hepatocytes. Hepatocytes isolated from the liver of rats were cultured in the absence (A) or presence of hydrazine for 24 h (B) or 48 h (C, D) in the presence of 4-OH-TEMPO (D). the literature concerning the description of struc- than three times compared to the average sizes of tural changes of mitochondria during apoptosis [74]. control mitochondria should be designated as “MG”. When mitochondria are exposed to hypotonic me- Survey of the literature describing structural chang- dia, they increase their sizes to at most 2–3 times es of mitochondria during apoptosis discloses inap- larger than control mitochondria, and the outer mi- propriate descriptions in some cases. For example, tochondrial membrane becomes ruptured beyond Heiden et al. [78] have demonstrated an electron that point [74]. Thus, mitochondria enlarged more micrograph of mitochondria during apoptosis, which

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they have described as “swollen mitochondria”. Sim- isolated from the liver of animals treated with hy- ilarly, mitochondria enlarged extremely were de- drazine for 9 d (A). The rate of the generation of scribed as “swollen mitochondria” in several cases ROS from mitochondria isolated from the liver of the [49,54]. However, these mitochondria should be former animals is increased whereas it becomes de- designated as “MG” since the sizes of mitochondria creased in those of the latter animals (B). The DYm exceed simple swelling. in the former animals is unchanged while it is dis- Mitochondria consume more than 90% of oxy- tinctly decreased in the latter (C). We have interpret- gen taken up by the cell, and generate reactive oxy- ed these results as follows [74]: decreases in the con- gen species (ROS) via complex I, II and III linked to tent of cytochrome a+a3 result in decreases in SOD ATP synthesis [9,12,77]. Complex IV containing cy- activity of complex IV thus increase ROS generation tochrome a+a3 (cytochrome oxidase), on the other from newly formed MG. Further exposure of MG to hand, has superoxide dismutase activity [47]. When free radicals results in the opening of MGCs, namely MG are newly formed by free radicals, the membrane the swelling of MG. Swelling MG in turn causes de- potential of mitochondria (DYm) remain intact and creases in DYm and the content of cytochrome c. the rate of the generation of ROS from MG is en- These changes may enhance the rate of the genera- hanced [33]. However, a prolonged treatment of cells tion of free radicals from swollen MG. However, dis- with free radicals causes a swelling of MG resulting tinct decreases in the oxygen consumption by swol- in decreases in ROS generation as well as further len MG result in decreases in ROS generation. De- decreases in the content of cytochrome c besides creases in intracellular levels of ATP then lead to that of cytochrome a+a3 [74]. Typical example is apoptotic changes of the cell. obtained from hydrazine-induced MG (Fig. 8). The Meaning of the MG formation induced under intracellular level of ROS in hepatocytes isolated from various pathological conditions has remained ob- the liver of rats fed a hydrazine-diet for 4 d is slightly scure for a long time. However, the results described increased and becomes increased further in those above would strongly suggest the possibility that the

ABC

Figure 8. DYm and the rate of generation of ROS from “intact MG” and “swollen MG”. A) Hepatocytes were isolated from the liver of rats treated with hydrazine for 4 d when “intact MG” were induced similar to those shown in Fig. 6B, and 9 d when “swollen MG” were induced similar to those shown in Fig. 6D, respectively. They were stained with carboxy-

H2DCFDA for flow cytometry. B) Mitochondria were isolated from the livers of animals used in Fig. 8A, and stained with carboxy-H2-DCFDA for flow cytometry. C) Hepatocytes used in Fig. 8A were stained with rhodamine 123 to detect DYm (modified from ref. 74).

70 Takashi Wakabayashi et al., Mitochondria and apoptosis

MG formation is an adaptive process at an intracel- cells, the association of mitochondria with microtu- lular organelle level to unfavourable environments: bules has been emphasised [2,24,26,39,50,59]. For if decreases in the rate of the generation of ROS from example, mouse kinesin superfamily KIF1B works as swollen MG are effective enough to lower intracel- a motor for the transport of mitochondria along lular level of ROS, swollen MG may return to normal microtubules [50]. It should be stressed here that both structurally and functionally and regain the modifications of cytoskeletons result in perinuclear ability to generate enough ATP to maintain various clustering of mitochondria [17,26]. In mutants de- cellular activities. If not, the swelling of MG proceeds, fective of some genes, mitochondria aggregate intracellular levels of ATP decrease further, and cells around the nucleus: mgm [22,30,63]; drp 1 [77]; kif become apoptotic. 1B [50]; kif 5B [73]; klp 67A [55]; clue A [85]. Re- cently, we have found that microtubule-active drugs Problems to be solved concerning (MADs) such as taxol and nocodazole cause cluster- structural changes of mitochondria ing of mitochondria around the nucleus (Fig. 9). Since related to apoptosis these MADs arrest cells in the G2/M phase of the cell Data have accumulated that the size and distribu- cycle, one possibility is that mitochondria cluster tion of mitochondria in the cell are controlled by a around the nucleus just prior to the nuclear division. variety of genes detected mainly in yeast cells. Some Mitochondrial clusterings around the nucleus in- of these genes exert their actions via the connection duced by MADs are always highly condensed as with certain components of cytoskeletons. fzo (fuzzy shown in Fig. 9 whereas those in control cells are in onions) in Drosophila melanogaster encodes a trans- the orthodox configuration (Wakabayashi, unpub- membrane GTPase that becomes detectable on sper- lished observation). This may suggest that these cells matid mitochondria late in meiosis II and disappears should not be related simply to cell cycle but that soon after the fusion is complete (“Nebenkern” for- they are on the way to apoptosis. mation) [25]. Fzo1p is a mitochondrial integral mem- brane protein with GTPase domain localized to in- CONCLUDING REMARKS ner-outer membrane contact sites [25]. In yeast cells, We have discussed structural changes of mitochon- Fzo 1p is required to maintain a tubular mitochon- dria during apoptosis focussing on “the swelling of drial reticulum during mitotic growth [27]. This is mitochondria”. We have also demonstrated data to the only gene at the moment which is capable of indicate that the formation of MG is an early sign of inducing MG under physiological conditions. On the apoptosis. We emphasise that structural changes of other hand, dynamin-related GTPase, Dnm1p (yeast) mitochondria during apoptosis should be analysed is required to maintain the tubular network of mito- using various techniques to obtain correct informa- chondria. Drp1 is a human dynamin-related protein tion. Since the proposal of Petit et al. [57] for the which is essential for the tubular distribution of mi- apoptotic processes of the cell stressing the swell- tochondria in the cell [67]. mdm 10 detected both in ing of mitochondria, several arguments have been yeast cells and Podospora arseria [7,29,68] and mmm raised concerning the mechanism of the release of 1 detected in yeast cells [4] are known to regulate cytochrome c from mitochondria: Bcl-2 was thought the size of mitochondria. In both cases, MG are to block apoptosis by preventing decreases in DYm formed in mutants. Interestingly, it has been found thus preventing the release of cytochrome c from that mutants lacking both dnm 1 and fzo 1 possess mitochondria [46,53,56,84]. However, it has been a normal mitochondrial network [62]. This result shown that cytochrome c release is unaccompanied suggests as the authors have pointed out that there by changes in DYm [8,18,36]. Decreases in DYm is are balances between the fusion and fission of mi- preceded by the release of cytochrome c from mito- tochondria controlled by these genes. Whether or chondria into the cytosol [83]. Changes in the con- not this is also the case with mammalian cells re- figuration of cytochrome c instead of its release from mains to be shown. mitochondria was stressed by others [79]. The role It is also well known that distribution of mito- of free radicals as a triggering factor for apoptosis chondria in the cell is intimately related to cytoskel- was questioned [28,64,64,65]: apoptosis was ob- etons. For example, mitochondria in yeast cells are served in the cells deprived of oxygen and Bcl-2 or combined with actin microfilaments with actin-bind- Bcl-xL prevented apoptotic changes of the cells [65]. ing proteins (ABP) and move in the cell using these This may suggest that ROS is not always a triggering filaments as railroad tracks [7,11]. In mammalian factor for apoptosis and that Bcl-2 and Bcl-XL exert

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Figure 9. Perinuclear clustering of mitochondria induced by microtubule-active drugs. Human osteosarcoma-derived 143B cell were treated with taxol (A) or nocodazole (B).

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