Megamitochondria formation - physiology and pathology

Mitochondria undergo structural changes simultaneously with their functional changes in both physiological and pathological conditions. These structural changes of mitochondria are classified into two categories: simple swelling and the formation of megamitochondria (MG). Data have been accumulated to indicate that free radicals play a crucial role in the mechanism of the MG formation induced by various experimental conditions which are apparently various. These include ethanol-, chloramphenicol- and hydrazine-induced MG formation. Involvement of free radicals in the mechanism of MG formation is showed by the fact that MG formation is successfully suppressed by free radical scavengers such as α-tocopherol, coenzyme Q₁₀, and 4-OH-TEMPO. Detailed mechanisms and pathophysiological meanings of MG formation still remain to be investigated. However, a body of evidence strongly suggests that enormous changes in physicochemical and biochemical properties of the mitochondrial membranes during MG formation take place and these changes are favorable for membrane fusion. A recent report showed that continous exposure of cells with MG to free radicals induces apoptosis, finding which suggests that MG formation is an adaptative process to unfavorable environments at the level of intracellular organelles. Mitochondria try to decrease intracellular reactive oxygen species (ROS) levels by decreasing the consume of oxygen via MG formation. If mitochondria succeed to suppress intracellular ROS levels, MG return to normal both structurally and functionally, and they restore the ability to actively synthesize ATP. If cells are additionally exposed to excess amounts of free radicals, MG become swollen, membrane potential of mitochondria (ΔΨm) decreases, cytochrome c is released from mitochondria, leading to activation of caspases and apoptosis is induced.     

Free radical–induced megamitochondria formation and apoptosis

Pathophysiological meaning and the mechanism of the formation of megamitochondria (MG) induced under physiological and pathological conditions remain obscure. We now provide evidence suggesting that the MG formation may be a prerequisite for free radical-mediated apoptosis. MG were detected in primary cultured rat hepatocytes, rat liver cell lines RL-34 and IAR-20 and kidney cell line Cos-1 treated for 22 h with various chemicals known to generate free radicals: hydrazine, chloramphenicol, methyl-glyoxal-bis-guanylhydrazone, indomethacin, H₂O₂, and erythromycin using a fluorescent dye Mito Tracker Red CMXRos (CMXRos) for confocal laser microscopy and also by electron microscopy. Remarkable elevations of the intracellular level of reactive oxygen species (ROS), monitored by staining of cells with a fluorescent dye carboxy-H₂-DCFDA, were detected before MG were formed. Prolongation of the incubation time with various chemicals, specified above, for 36 h or longer has induced distinct structural changes of the cell, which characterize apoptosis: condensation of nuclei, the formation of apoptotic bodies, and the ladder formation. Cells treated with the chemicals for 22 h were arrested in G₁ phase, and apoptotic sub-G₁ populations then became gradually increased. The membrane potential of MG induced by chloramphenicol detected by CMXRos for flow cytometry was found to be decreased compared to that of mitochondria in control cells. Rates of the generation of H₂O₂ and O₂⁻ from MG isolated from the liver of rats treated with chloramphenicol or hydrazine were found to be lower than those of mitochondria of the liver of control animals. We suggest, based on the present results together with our previous findings, that the formation of MG may be an adaptive process at a subcellular level to unfavorable environments: when cells are exposed to excess amounts of free radicals mitochondria become enlarged decreasing the rate of oxygen consumption. Decreases in the oxygen consumption of MG may result in decreases in the rate of ROS production as shown in the present study. This will at the same time result in decreases in ATP production from MG. If cells are exposed to a large amount of free radicals beyond a certain period of time, lowered intracellular levels of ATP may result in apoptotic changes of the cell.     

Structural changes of mitochondria related to apoptosis

The original concept of apoptosis stressed the morphological changes of the nucleus, condensation with the aggregation of chromatin, and the intactness of intracellular organelles including mitochondria. However, the application of molecular biology and flow-cytometric techniques to the research field of apoptosis has led to the proposal of the apoptotic processes which emphasizes the 'swelling of mitochondria' due to the opening of the 'permeability transition pores' of the mitochondrial membranes followed by a series of events including the collapse of the transmembrane potential of mitochondria and release of cytochrome c from mitochondria into the cytosol. Enlargement of mitochondria induced by various pathological conditions are classified into two categories: the swelling and the formation of megamitochondria (MG). Recently, we have found that free radical-induced formation of MG is succeeded by apoptotic changes of the cell. If the MG formation is actually related to apoptosis, this will be a new aspect of the structural changes of mitochondria involved in apoptosis besides the simple swelling of mitochondria. First, we will discuss the 'swelling of mitochondria' which characterizes the currently accepted hypothesis on the apoptotic processes of the cell, as described above, in the light of the literature. Second, the mechanisms controlling the size and distribution of mitochondria in the cell are dealt with paying special attention to the genetic regulation and cytoskeletons. Third, we have tried to characterize the MG formation to correlate apoptotic changes of the cell. Finally, we will discuss several problems to be solved in the future which involve mitochondria in apoptotic processes of the cell.     

高糖通过线粒体途径诱导成骨细胞凋亡

线粒体途径是细胞凋亡的重要途径之一. 在特定的刺激下,例如高糖条件,可以通过caspase依赖性和非依赖性两种途径诱导多种细胞凋亡.但线粒体凋亡途径在高糖引起成骨细胞凋亡中所起的作用,目前尚不清楚.本研究证明,高糖可以通过线粒体凋亡途径诱导成骨细胞凋亡.Annexin V-FITC/PI流式细胞学检测显示,高糖可诱导MC3T3-E1细胞凋亡.Western印迹检测发现,不同浓度D-葡萄糖（11,22,33 mmol/L）可以引起线粒体中Bax蛋白表达的增加,使Bax蛋白由细胞质中易位到线粒体,激活了线粒体凋亡途径.JC-1荧光探针检测证实,高糖处理成骨细胞后,线粒体膜电位明显降低,表明线粒体途径被激活.而细胞质中的细胞色素c、凋亡诱导因子（AIF）表达增加,细胞色素c和AIF从线粒体中释放到细胞质中,释放到细胞质中的细胞色素c使caspase-3、caspase-9剪切活化,从而激活了caspase依赖性凋亡途径.因此,线粒体凋亡途径可能是高糖诱导成骨细胞凋亡过程中一个重要的途径.     

Effect of oxygen concentration on free radical-induced cytotoxicity

Free radicals induce oxidative stress in vivo, leading to various disorders and diseases. In the present study, the effect of oxygen pressure on the cytotoxicity induced by free radicals was studied. It was found that alkyl radicals markedly aggravated Jurkat cell apoptosis under low oxygen pressure and this was ascribed to a hypoxic condition caused by the consumption of oxygen by alkyl radicals giving peroxyl radicals and subsequent lipid peroxidation by a chain mechanism. The intracellular lipid hydroperoxides significantly increased at an early time point even under hypoxia. Cytochrome c was released from the mitochondria, and caspase-9 as well as caspase-3 was activated during apoptosis, indicating that cell death followed by the intrinsic, mitochondrial apoptosis pathway. Pretreatment with VAD-FMK, a caspase inhibitor, attenuated the apoptosis induced by alkyl radicals under hypoxia. Moreover, pretreatment with various antioxidants also significantly rescued the cells from apoptosis. Taken together, the results indicate that free radicals induced hypoxic conditions, which accelerated mitochondria-dependent cell apoptosis.     

Cytotoxic phospholipid oxidation products. Cell death from mitochondrial damage and the intrinsic caspase cascade

Phospholipid oxidation products accumulate in the necrotic core of atherosclerotic lesions, in apoptotic cells, and circulate in oxidized low density lipoprotein. Phospholipid oxidation generates toxic products, but little is known about which specific products are cytotoxic, their receptors, or the mechanism(s) that induces cell death. We find the most common phospholipid oxidation product of oxidized low density lipoprotein, phosphatidylcholine with esterified sn-2-azelaic acid, induced apoptosis at low micromolar concentrations. The synthetic ether phospholipid hexadecyl azelaoyl phosphatidylcholine (HAzPC) was rapidly internalized, and overexpression of PLA2g7 (PAF acetylhydrolase) that specifically hydrolyzes such oxidized phospholipids suppressed apoptosis. Internalized HAzPC associated with mitochondria, and cytochrome c, and apoptosis-inducing factor escaped from mitochondria to the cytoplasm and nucleus, respectively, in cells exposed to HAzPC. Isolated mitochondria exposed to HAzPC rapidly swelled and released cytochrome c and apoptosis-inducing factor. Other phospholipid oxidation products induced swelling, but HAzPC was the most effective and was twice as effective as its diacyl homolog. Cytoplasmic cytochrome c completes the apoptosome, and activated caspase 9 and 3 were present in cells exposed to HAzPC. Irreversible inhibition of caspase 9 blocked downstream caspase 3 activation and prevented apoptosis. Mitochondrial damage initiated this apoptotic cascade, because overexpression of Bcl-X(L), an anti-apoptotic protein localized to mitochondria, blocked cytochrome c escape and apoptosis. Thus, exogenous phospholipid oxidation products target intracellular mitochondria to activate the intrinsic apoptotic cascade.     

Partial characterization of human choriocarcinoma cell line JAR cells in regard to oxidative stress

Characterization of free radical-induced cell injury processes of placenta cells is of vital importance for clinical medicine for the maintenance of intrauterine fetal life. The present study has analyzed cell injury processes in cells of the choriocarcinoma cell line JAR treated with menadione, an anticancer drug, and H(2)O(2) in comparison to osteosarcoma 143B cells using electron microscopic and flow cytometric techniques. Flow cytometry on JAR cells exposed to 100 muM menadione and double-stained with Annexin V and propidium iodide (PI) detected apoptotic cells reaching the maximum after 4 h of incubation with a rapid decrease thereafter. Viable cells became decreased to 46% of the control after 2 h of incubation, reaching 5% after 4 h. Cells stainable with both Annexin V and PI began to increase distinctly after 2 h of incubation, reaching 55% after 4 h. Electron microscopy showed that cells stainable with both dyes specified above had condensed nuclei and swollen cytoplasm, suggesting that they were undergoing a switch of the cell death mode from apoptosis to necrosis. On the other hand, 90% of 143B cells remained intact after 4 h of menadione treatment although the intracellular levels of superoxide were always higher than those of JAR cells treated with the drug. In contrast, JAR cells were more resistant than 143B cells to H(2)O(2)-induced cytotoxicity. These results may suggest that cytotoxicity of menadione cannot be explained simply by oxygen free radicals generated from the drug. The resistance of JAR cells to oxygen free radical-induced cytotoxicity may be advantageous for intrauterine fetal life.     

Neuronal cell death caused by inhibition of intracellular cholesterol trafficking is caspase dependent and associated with activation of the mitochondrial apoptosis pathway

An elevated level of cholesterol in mitochondrial membranes of Niemann-Pick disease type C1 (NPC1) mouse brains and neural cells has been found to cause mitochondrial dysfunction. In this study, we demonstrate that inhibition of intracellular cholesterol trafficking in primary neurons by class 2 amphiphiles, which mimics the major biochemical and cellular feature of NPC1, led to not only impaired mitochondrial function but also activation of the mitochondrial apoptosis pathway. In activation of this pathway both cytochrome c and Smac/Diablo were released but apoptosis-inducing factor (AIF) was not involved. Treatment of the neurons with taurine, a caspase 9-specific inhibitor, could prevent the amphiphile-induced apoptotic cell death, suggesting that formation of apoptosome, followed by caspase 9 and caspase 3 activation, might play a critical role in the neuronal death pathway. Taken together, the mitochondria-dependent death cascade induced by blocking intracellular cholesterol trafficking was caspase dependent. The findings provide clues for both understanding the molecular basis of neurodegeneration in NPC1 disease and developing therapeutic strategies for treatment of this disorder.     

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

Nitrosylation of cytochrome c during apoptosis

Cytochrome c released from mitochondria into the cytoplasm plays a critical role in many forms of apoptosis by stimulating apoptosome formation and subsequent caspase activation. However, the mechanisms regulating cytochrome c apoptotic activity are not understood. Here we demonstrate that cytochrome c is nitrosylated on its heme iron during apoptosis. Nitrosylated cytochrome c is found predominantly in the cytoplasm in control cells. In contrast, when cytochrome c release from mitochondria is inhibited by overexpression of the anti-apoptotic proteins B cell lymphoma/leukemia (Bcl)-2 or Bcl-X(L), nitrosylated cytochrome c is found in the mitochondria. These data suggest that during apoptosis, cytochrome c is nitrosylated in mitochondria and then rapidly released into the cytoplasm in the absence of Bcl-2 or Bcl-X(L) overexpression. In vitro nitrosylation of cytochrome c increases caspase-3 activation in cell lysates. Moreover, the inhibition of intracellular cytochrome c nitrosylation is associated with a decrease in apoptosis, suggesting that cytochrome c nitrosylation is a proapoptotic modification. We conclude that nitrosylation of the heme iron of cytochrome c may be a novel mechanism of apoptosis regulation.     

And all of a sudden it's over: mitochondrial outer-membrane permeabilization in apoptosis

Identification of pro-apoptotic activities for a variety of proteins normally resident in the mitochondrial inter-membrane space has substantiated the role of mitochondria as integral to the apoptotic process. Cytochrome c is involved in apoptosome formation and caspase activation, SMAC/Diablo deregulates the inhibitor of apoptosis proteins, apoptosis-inducing factor may play a role in chromatin condensation and release of other proteins such as adenylate kinase may adversely affect cellular metabolism and contribute to the death of a cell if the downstream apoptotic pathway is blocked. It is still unclear how these proteins are released from the mitochondria. Recent advances in our knowledge of mitochondrial outer-membrane permeabilization and the consequences of this event on mitochondria will be discussed.     

Free radicals, lipid peroxidation and muscular ischemia]

The role of oxygen free radicals in ischemia and reperfusion injury of skeletal muscle has not been well defined, partly because of the relative resistance of this tissue to normothermic ischemia. Under normal conditions small quantities of oxygen free radicals are produced but they are quenched by intracellular free radical scavenging enzymes (superoxide dismutase, catalase and glutathione peroxidase) or alpha-tocopherol. The increase in malondialdehyde suggests increased lipid peroxidation initiated by free radical reactions. Lipid peroxidation is potentially a very damaging process to the organized structure and function of membranes. The results of recent studies indicate that: a) oxygen free-radicals mediates, at least in part, the increased microvascular permeability produced by reoxygenation, b) free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged ischemia. Additional evidence support the hypothesis of the interrelationship between ischemic tissue and inflammatory cells. So capillary plugging by granulocytes and oxygen free radical formation may contribute to the ischemic injury.     

Edaravone protects against MPP+ -induced cytotoxicity in rat primary cultured astrocytes via inhibition of mitochondrial apoptotic pathway

Edaravone (Eda) is a potent scavenger of hydroxyl radicals and has been demonstrated to be beneficial for patients with acute ischemic stroke. This study was set out to investigate whether Eda protect against MPP(+)-induced cytotoxicity in rat primary cultured astrocytes. The results showed that pre-treatment with Eda inhibited astrocytic apoptosis and lactate dehydrogenase release induced by MPP(+) (200 microM). Further study revealed that Eda prevented GSH depletion, down-regulated mRNA expressions of NADPH oxidase membrane subunit gp91 and membrane-translocated subunit p47, and prevented the decreases of state 3 respiration respiration and respiratory control ratio induced by MPP(+), and thereby inhibited reactive oxygen species production evoked by MPP(+). Moreover, Eda could ameliorate mitochondrial respiratory function, restrain, and prevent mitochondrial membrane potential loss induced by MPP(+). Consequently, Eda inhibited releases of cytochrome c and apoptosis-inducing factor induced by MPP(+). Taken together, these findings reveal for the first time that Eda protects against MPP(+)-induced astrocytic apoptosis via decreasing intracellular reactive oxygen species level and subsequently inhibiting mitochondrial apoptotic pathway. The antiapoptosis effects of Eda on astrocytes may provide a new perspective on neuroprotective therapy.     

活性氧、线粒体通透性转换与细胞凋亡

线粒体是真核细胞中非常重要的细胞器,细胞中的活性氧等自由基主要来源于此,线粒体膜的通透性转换(mitochondrial permeability transition,MPT)及其孔道(mitochondrialpermeability transition pore,MPTP)更是在内源性细胞凋亡中发挥了关键作用。持续性的线粒体膜通透性转换在凋亡的效应阶段起决定性作用,可介导细胞色素c等促凋亡因子从线粒体释放到胞浆中,进一步激活下游的信号通路,导致细胞不可逆地走向凋亡。瞬时性的线粒体膜通透性转换及其偶联的线粒体局部的活性氧爆发同样具有促凋亡的作用。线粒体通透性孔道的开放释放出大量活性氧,这些活性氧又能够进一步激活该孔道,以正反馈的形式进一步加剧孔道的打开,放大凋亡信号。活性氧、线粒体通透性转换与细胞凋亡之间具有密不可分的联系,本文根据已知的研究结果集中讨论了这三者的关系,并着重论述了该领域中的最新发现和成果。     

Aqueous peroxyl radical exposure to THP-1 cells causes glutathione loss followed by protein oxidation and cell death without increased caspase-3 activity

Protein oxidation within cells exposed to oxidative free radicals has been reported to occur in an uninhibited manner with both hydroxyl and peroxyl radicals. In contrast, THP-1 cells exposed to peroxyl radicals (ROO(*)) generated by thermo decomposition of the azo compound AAPH showed a distinct lag phase of at least 6 h, during which time no protein oxidation or cell death was observed. Glutathione appears to be the source of the lag phase as cellular levels were observed to rapidly decrease during this period. Removal of glutathione with buthionine sulfoxamine eliminated the lag phase. At the end of the lag phase there was a rapid loss of cellular MTT reducing activity and the appearance of large numbers of propidium iodide/annexin-V staining necrotic cells with only 10% of the cells appearing apoptotic (annexin-V staining only). Cytochrome c was released into the cytoplasm after 12 h of incubation but no increase in caspase-3 activity was found at any time points. We propose that the rapid loss of glutathione caused by the AAPH peroxyl radicals resulted in the loss of caspase activity and the initiation of protein oxidation. The lack of caspase-3 activity appears to have caused the cells to undergo necrosis in response to protein oxidation and other cellular damage.     

Impairment of the mitochondrial respiratory enzyme activity triggers sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling pathways to induce apoptosis after spinal cord injury

The mitochondrion participates in caspase-independent or caspase-dependent apoptotic pathways through the release of apoptosis-inducing factor or cytochrome c. Whether both mitochondrial apoptotic cascades are triggered in the injured spinal cord remains unknown. Here, we demonstrated that neurons, astrocytes and microglia in spinal segments proximal to a complete spinal cord transection underwent two phases of apoptotic cell death. The early phase of high-molecular weight (HMW) DNA fragmentation was associated with nuclear translocation of apoptosis-inducing factor, reduction in mitochondrial respiratory chain enzyme activity and decrease in cellular ATP concentration. The delayed phase of low-molecular weight (LMW) DNA fragmentation was accompanied by cytosolic release of cytochrome c , activation of caspases 9 and 3, and resumption of mitochondrial respiratory functions and ATP contents. Microinfusion of coenzyme Q₁₀, an electron carrier in mitochondrial respiratory chain, into the epicenter of the transected spinal cord attenuated both phases of induced apoptosis, and reversed the elicited mitochondrial dysfunction, bioenergetic failure, and activation of apoptosis-inducing factor, cytochrome c , or caspases 9 and 3. We conclude that mitochondrial dysfunction after spinal cord transection represents the initiating cellular events that trigger the sequential activation of apoptosis-inducing factor-dependent and caspase-dependent signaling cascades, leading to apoptotic cell death in the injured spinal cord.     

Cycloheximide and 4-OH-TEMPO suppress chloramphenicol-induced apoptosis in RL-34 cells via the suppression of the formation of megamitochondria

Toxic effects of chloramphenicol, an antibiotic inhibitor of mitochondrial protein synthesis, on rat liver derived RL-34 cell line were completely blocked by a combined treatment with substances endowed with direct or indirect antioxidant properties. A stable, nitroxide free radical scavenger, 4-hydroxy-2,2,6, 6-tetramethylpiperidine-1-oxyl, and a protein synthesis inhibitor, cycloheximide, suppressed in a similar manner the following manifestations of the chloramphenicol cytotoxicity: (1) Oxidative stress state as evidenced by FACS analysis of cells loaded with carboxy-dichlorodihydrofluorescein diacetate and Mito Tracker CMTH2MRos; (2) megamitochondria formation detected by staining of mitochondria with MitoTracker CMXRos under a laser confocal microscopy and electron microscopy; (3) apoptotic changes of the cell detected by the phase contrast microscopy, DNA laddering analysis and cell cycle analysis. Since increases of ROS generation in chloramphenicol-treated cells were the first sign of the chloramphenicol toxicity, we assume that oxidative stress state is a mediator of above described alternations of RL-34 cells including MG formation. Pretreatment of cells with cycloheximide or 4-hydroxy-2,2, 6,6-tetramethylpiperidine-1-oxyl, which is known to be localized into mitochondria, inhibited the megamitochondria formation and succeeding apoptotic changes of the cell. Protective effects of cycloheximide, which enhances the expression of Bcl-2 protein, may further confirm our hypothesis that the megamitochondria formation is a cellular response to an increased ROS generation and raise a possibility that antiapoptotic action of the drug is exerted via the protection of the mitochondria functions.     

Ultrastructural effects of lethal freezing on brain,muscle and Malpighian tubules from freeze-tolerant larvae of the gall fly,Eurosta solidaginis

In preparation for winter low temperatures, larvae of the gall fly, Eurosta solidaginis, accumulate the cryoprotectants glycerol, sorbitol, and trehalose. The fat body cells of these freeze-tolerant larvae can survive intracellular freezing to -80 degrees C for 48 h even though no whole larvae survive this treatment. We hypothesized that some other tissue was more susceptible to freezing and therefore may be responsible for larval death. This paper compares the ultrastructure of brain, muscle, and Malpighian tubules between non-lethally frozen and lethally frozen freeze-tolerant larvae. The nuclei of cortical brain cells from lethally frozen larvae exhibited clumped chromatin and nuclear membranes with occasional expansions or 'blebs' of the intermembranous space, while the cytoplasm contained swollen spheres of endoplasmic reticulum. In contrast, non-lethally frozen brain contained nuclei with evenly dispersed chromatin, smooth nuclear membranes and a cytoplasm free of swollen endoplasmic reticulum. Muscle tissue of lethally frozen larvae contained disrupted myofilaments surrounding the Z-line in comparison to non-lethally frozen muscle which had myofilaments extending all the way to the Z-line. Alterations of Malpighian tubule cells from lethally frozen larvae included an extracted cytoplasm with swollen and rounded mitochondria. In contrast, Malpighian tubule cells from non-lethally frozen larvae had a more concentrated cytoplasm with many rod-shaped mitochondria. Results show alterations to all three tissue types due to lethal freezing. The brain tissue contained the most observable alterations and therefore may be the most susceptible to lethal freeze damage.     

Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes

A key step in the initiation of apoptosis is the release from the mitochondrial intermembrane space of cytochrome c and other pro-apoptotic proteins such as Smac/DIABLO, Omi/HtrA2, apoptosis-inducing factor (AIF), and endonuclease G (EndoG). Discrepancies have arisen, however, as to whether all these proteins are released in different systems. Our results suggest that failure to observe cytochrome c release may be due to the use of different buffers because after permeabilization by caspase-8 cleaved human Bid (tBid), cytochrome c dissociation from mitochondria was highly dependent on ionic strength and required 50-80 mm KCl, NaCl, or LiCl. In addition, mitochondria isolated from apoptotic cells using low ionic strength buffer bound a greater proportion of endogenous cytochrome c. In contrast to cytochrome c, Smac/DIABLO and Omi/HtrA2 were released independent of ionic strength, and AIF and EndoG behaved as if they are exposed to the intermembrane space but tethered to or within the inner membrane. AIF and EndoG were also not released by active caspases, which suggests their involvement in apoptosis may be limited. In summary, whereas tBid permeabilizes the outer membrane to cytochrome c, Smac/DIABLO, and Omi/HtrA2, the release of cytochrome c during apoptosis will be underestimated unless sufficient ionic strength is maintained to overcome the electrostatic association of cytochrome c with membranes.