Plasma membrane permeabilization by 60- and 600-ns electric pulses is determined by the absorbed dose

We explored how the effect of plasma membrane permeabilization by nanosecond-duration electric pulses (nsEP) depends on the physical characteristics of exposure. The resting membrane resistance (R(m)) and membrane potential (MP) were measured in cultured GH3 and CHO cells by conventional whole-cell patch-clamp technique. Intact cells were exposed to a single nsEP (60 or 600 ns duration, 0-22 kV/cm), followed by patch-clamp measurements after a 2-3 min delay. Consistent with earlier findings, nsEP caused long-lasting R(m) decrease, accompanied by the loss of MP. The threshold for these effects was about 6 kV/cm for 60 ns pulses, and about 1 kV/cm for 600 ns pulses. Further analysis established that it was neither pulse duration nor the E-field amplitude per se, but the absorbed dose that determined the magnitude of the biological effect. In other words, exposure to nsEP at either pulse duration caused equal effects if the absorbed doses were equal. The threshold absorbed dose to produce plasma membrane effects in either GH3 or CHO cells at either pulse duration was found to be at or below 10 mJ/g. Despite being determined by the dose, the nsEP effect clearly is not thermal, as the maximum heating at the threshold dose is less than 0.01 degrees C. The use of the absorbed dose as a universal exposure metric may help to compare and quantify nsEP sensitivity of different cell types and of cells in different physiological conditions. The absorbed dose may also prove to be a more useful metric than the incident E-field in determining safety limits for high peak, low average power EMF emissions.     

Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis

Bax promotes cell death by permeabilizing mitochondrial outer membranes by an unresolved mechanism. However, in cells lacking the gene c-myc, membrane permeabilization by Bax is blocked by changes in the mitochondria that prevent Bax oligomerization. Drug-treated c-myc null cells and cells expressing Myc were used to map the topology of Bax in membranes prior to and after mitochondrial permeabilization. Chemical labeling of single cysteine mutants of Bax using a membrane bilayer impermeant cysteine-specific modifying agent revealed that Bax inserted both the 'pore domain' (helices alpha5-alpha6), and the tail-anchor (helix alpha9) into membranes prior to oligomerization and membrane permeabilization. Additional topology changes for Bax were not required in Myc-expressing cells to promote oligomerization and cytochrome c release. Our results suggest that unlike most pore-forming proteins, Bax membrane permeabilization results from oligomerization of transmembrane monomers rather than concerted insertion of the pore domains of a preformed oligomer.     

Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.     

Is mPTP the gatekeeper for necrosis, apoptosis, or both?

Permeabilization of the mitochondrial membranes is a crucial step in apoptosis and necrosis. This phenomenon allows the release of mitochondrial death factors, which trigger or facilitate different signaling cascades ultimately causing the execution of the cell. The mitochondrial permeability transition pore (mPTP) has long been known as one of the main regulators of mitochondria during cell death. mPTP opening can lead to matrix swelling, subsequent rupture of the outer membrane, and a nonspecific release of intermembrane space proteins into the cytosol. While mPTP was purportedly associated with early apoptosis, recent observations suggest that mitochondrial permeabilization mediated by mPTP is generally more closely linked to events of late apoptosis and necrosis. Mechanisms of mitochondrial membrane permeabilization during cell death, involving three different mitochondrial channels, have been postulated. These include the mPTP in the inner membrane, and the mitochondrial apoptosis-induced channel (MAC) and voltage-dependent anion-selective channel (VDAC) in the outer membrane. New developments on mPTP structure and function, and the involvement of mPTP, MAC, and VDAC in permeabilization of mitochondrial membranes during cell death are explored. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

Methods for the assessment of mitochondrial membrane permeabilization in apoptosis

Mitochondrial membrane permeabilization (MMP) is considered as the “point-of-no-return” in numerous models of programmed cell death. Indeed, mitochondria determine the intrinsic pathway of apoptosis, and play a major role in the extrinsic route as well. MMP affects the inner and outer mitochondrial membranes (IM and OM, respectively) to a variable degree. OM permeabilization culminates in the release of proteins that normally are confined in the mitochondrial intermembrane space (IMS), including caspase activators (e.g. cytochrome c) and caspase-independent death effectors (e.g. apoptosis-inducing factor). Partial IM permeabilization disrupts mitochondrial ion and volume homeostasis and dissipates the mitochondrial transmembrane potential (ΔΨ_m). The assessment of early mitochondrial alterations allows for the identification of cells that are committed to die but have not displayed yet the apoptotic phenotype. Several techniques to measure MMP by cytofluorometry and fluorescence microscopy have been developed. Here, we summarize the currently available methods for the detection of MMP, and provide a comparative analysis of these techniques.     

The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol

During apoptosis, an important pathway leading to caspase activation involves the release of cytochrome c from the intermembrane space of mitochondria. Using a cell-free system based on Xenopus egg extracts, we examined changes in the outer mitochondrial membrane accompanying cytochrome c efflux. The pro-apoptotic proteins, Bid and Bax, as well as factors present in Xenopus egg cytosol, each induced cytochrome c release when incubated with isolated mitochondria. These factors caused a permeabilization of the outer membrane that allowed the corelease of multiple intermembrane space proteins: cytochrome c, adenylate kinase and sulfite oxidase. The efflux process is thus nonspecific. None of the cytochrome c-releasing factors caused detectable mitochondrial swelling, arguing that matrix swelling is not required for outer membrane permeability in this system. Bid and Bax caused complete release of cytochrome c but only a limited permeabilization of the outer membrane, as measured by the accessibility of inner membrane-associated respiratory complexes III and IV to exogenously added cytochrome c. However, outer membrane permeability was strikingly increased by a macromolecular cytosolic factor, termed PEF (permeability enhancing factor). We hypothesize that PEF activity could help determine whether cells can recover from mitochondrial cytochrome c release.     

Mitochondrial membrane permeabilization: the sine qua non for cell death

Mitochondria are essential for maintaining cell life but they also play a role in regulating cell death, which occurs when their membranes become permeabilized. Mitochondria possess two distinct membrane systems including an outer membrane in close communication with the cytosol and an inner membrane involved in energy transduction. Outer membrane permeabilization is regulated by Bcl-2 family proteins, which control the release of proteins from the mitochondrial intermembrane space; these proteins then activate apoptosis. Inner membrane permeabilization is regulated by the mitochondrial permeability transition (MPT), which is activated by calcium and oxidative stress and leads to bioenergetic failure and necrosis. The purpose of this review is to discuss the biochemical mechanisms regulating mitochondrial membrane permeabilization; this is crucial to our understanding of the role of cell death in diseases such as cancer and the neurodegenerative diseases.     

Bax assembles into large ring‐like structures remodeling the mitochondrial outer membrane in apoptosis

The Bcl‐2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro‐apoptotic proteins. Yet the mechanistic details of the Bax‐induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring‐like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super‐resolution data provide direct evidence in support of large Bax‐delineated pores in the mitochondrial outer membrane as being crucial for Bax‐mediated MOMP in cells.     

Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance to TNF-induced cell killing

Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X_L overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met^mut) and Bcl-X_L-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaDTM/C) or Bcl-X_L in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X_L overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X_L appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X_L may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X_L overespression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process

During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.     

Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co²⁺). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co²⁺ (outside of the matrix). Upon induction of apoptosis, such calcein/Co²⁺-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential ( _m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co²⁺ (CC) staining can be dissociated from the _m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent _m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.     

Mitochondrial and plasma membrane potentials cause unusual accumulation and retention of rhodamine 123 by human breast adenocarcinoma-derived MCF-7 cells

Quantitative studies of MCF-7 cells (derived from human breast adenocarcinoma) and CV-1 cells (from normal African green monkey kidney epithelium), using the permeant cationic compound tetraphenylphosphonium (TPP), in conjunction with fluorescence microscopy using rhodamine 123 (Rh123), indicate that the mitochondrial and plasma membrane potentials affect both uptake and retention of these compounds. Under conditions that depolarize the plasma membrane, uptake and retention of TPP and Rh123, driven only by the mitochondrial membrane potential, is greater in MCF-7 than in CV-1. An ionophore that dissipates the mitochondrial membrane potential of MCF-7 cells causes them to resemble CV-1 cells by decreasing uptake and retention. Hyperpolarizing the mitochondrial membrane of CV-1 increases accumulation and prolongs retention; hyperpolarization of the plasma membrane further heightens this effect, causing the uptake of CV-1 cells to resemble that of MCF-7 cells even more closely. The greater uptake and retention by MCF-7 appears to be a consequence of elevated mitochondrial and plasma membrane potentials. The plasma membrane potential affects mitochondrial retention of TPP and Rh123 and its role in enhancing the effect of a difference in mitochondrial membrane potential is explained.     

Mitochondria as targets of apoptosis regulation by nitric oxide

In addition to their vital role as the cell's power stations, mitochondria exert an important function in apoptosis. In response to most if not all apoptosis inducers, mitochondrial membranes are permeabilized, leading to the release of potentially toxic proteins, mostly from the intermembrane space to the rest of the cells. Such pro-apoptotic intermembrane proteins include the caspase-independent death effector AIF, as well as cytochrome c, which can trigger the activation of caspases, once it has reached the cytosol. The mitochondrial permeabilization process can be induced by a variety of different xenobiotics, via a direct effect on mitochondrial membranes. Alternatively, mitochondrial permeabilization can be induced by endogenous second messengers, which are elicited in response to stress. The permeabilization process is controlled by the mitochondrial permeability transition pore complex (PTPC), by proteins of the Bcl-2/Bax family, as well as by lipids and metabolites. Nitric oxide (NO) is one of the second messengers that can trigger apoptosis by inducing mitochondrial membrane permeabilization. This effect may involve a direct effect on the PTPC and/or indirect effects secondary to the NO-mediated inhibition of oxidative phosphorylation. This has far-reaching implications for the pathophysiology of NO.     

Palmitic Acid Induces the Opening of a Ca2+-Dependent Pore in the Plasma Membrane of Red Blood Cells: The Possible Role of the Pore in Erythrocyte Lysis

Earlier we found that in the presence of Ca²⁺ palmitic acid (Pal) increases the nonspecific permeability of artificial (planar and liposomal) membranes and causes permeabilization of the inner mitochondrial membrane. An assumption was made that the mechanism of Pal/Ca²⁺-induced membrane permeabilization relates to the Ca²⁺-induced phase separation of Pal and can be considered as formation of fast-tightening lipid pores due to chemotropic phase transition in the lipid bilayer. In this article, we continue studying this pore. We have found that Pal plus Ca²⁺ permeabilize the plasma membrane of red blood cells in a dose-dependent manner. The same picture has been revealed for stearic acid (20 μM) but not for myristic and linoleic acids. The Pal-induced permeabilization of erythrocytic membranes can also occur in the presence of Ba²⁺ and Mn²⁺ (200 μM), but other bivalent cations (200 μM Mg²⁺, Sr²⁺, Ni²⁺, Co²⁺) are relatively ineffective. The formation of Pal/Ca²⁺-induced pores in the erythrocytic membranes has been found to result in the destruction of cells.     

Selective field effects on intracellular vacuoles and vesicle membranes with nanosecond electric pulses

Electric pulses across intact vesicles and cells can lead to transient increase in permeability of their membranes. We studied the integrity of these membranes in response to external electric pulses of high amplitude and submicrosecond duration with a primary aim of achieving selective permeabilization. These effects were examined in two separate model systems comprising of 1), a mixed population of 1,2-di-oleoyl-sn-glycero-3-phosphocholine phospholipid vesicles and in 2), single COS-7 cells, in which large endosomal membrane vacuoles were induced by stimulated endocytosis. It has been shown that large and rapidly varying external electric fields, with pulses shorter than the charging time of the outer-cell membrane, could substantially increase intracellular fields to achieve selective manipulations of intracellular organelles. The underlying principle of this earlier work is further developed and applied to the systems studied here. Under appropriate conditions, we show preferential permeabilization of one vesicle population in a mixed preparation of vesicles of similar size distribution. It is further shown that large endocytosed vacuoles in COS-7 cells can be selectively permeabilized with little effect on the integrity of outer cell membrane.     

Machine vision based stochastic analysis of cancer cell mitochondrial dysfunction induced by a BH3 domain

We have developed a versatile and rapid method for the quantitative estimation of cell death kinetics, following direct single-shot activation of the mitochondrial death pathway by a cell permeable BH3 activator peptide (D-R₈BH3^BID). This approach employs timelapse epifluorescent imaging of live cells and a machine- vision based feature extraction algorithm, to measure unidirectional stochastic transitions associated with mitochondrial inner membrane potential depolarization and/or permeability transition, at single cell resolution. This data is transformed to enable construction of a right step-wise survival function using the product limit estimator, and estimation of a median latency parameter (λ), defined for the entire imaged cell population. Estimates of λ computed for cells exhibiting two-colour fluorescence can be compared statistically using the Mantel-Hansel test. This general method has been applied to measure the kinetics and temporal ordering of BH3 domain induced mitochondrial depolarization and inner membrane permeabilization in cancer cells, and demonstrates the robustness of this technique in resolving temporally distinct intracellular events within individual cells.     

Potential-dependent membrane permeabilization and mitochondrial aggregation caused by anticancer polyarginine-KLA peptides

The anticancer activity of the polycationic peptide (KLAKLAK)₂, as a possible mitochondria-damaging agent, named KLA (l-form) or kla (d-form), has been increased by the fusion with hepta-arginine cell delivery vectors r7 and R7 (peptides r7-kla and R7-KLA, respectively), as shown in the literature. We demonstrated that 3.6 μM r7-kla or R7-KLA, but not kla, caused significant permeabilization of the inner and the outer membranes of energized rat liver mitochondria. In addition, r7-kla or R7-KLA induced mitochondrial aggregation, thus causing the inhibition of metabolic activity. Potential-dependent mechanism of permeabilization of the inner mitochondrial membrane by these peptides was also observed for the plasma membrane of red blood cells. The obtained results suggest that polyarginine cell delivery vectors of anticancer polycationic peptides not only increase their direct potential-dependent permeabilization of biological membranes, but also create the capacity to cause aggregation of mitochondria, as a new mechanism of cytotoxic action of these peptides.     

Micro-managing the executioner: pathogen targeting of mitochondria

Eukaryotic cell viability is largely regulated at the level of mitochondria, with cell death executed by endogenous proteins that act to increase the permeability of the inner and/or outer membranes of these organelles. The gastric pathogen, Helicobacter pylori, can mimic this mechanism by producing the pro-apoptotic toxin, VacA, which was recently demonstrated to (i) localize to mitochondria within epithelial cells, (ii) rapidly transport into mitochondria in vitro, and (iii) induce changes consistent with permeabilization of mitochondrial membranes by a mechanism dependent on cellular entry and toxin membrane channel activity. The targeting of mitochondrial membranes is emerging as a strategy used by pathogenic microbes to control cell viability while circumventing upstream pathways and checkpoints of cell death.     

线粒体膜完整性对细胞命运的调控

线粒体双层膜的完整性是细胞存活的关键因素,其遭到破坏后会使细胞发生凋亡、焦亡或炎症。线粒体膜的破坏包括线粒体外膜通透、线粒体内膜通透、通透性转换,三者可通过调控不同的信号通路导致不同的细胞命运。然而,这些信号通路之间存在交叉关联,使得线粒体膜对细胞命运的调控错综复杂,导致人们对其机制缺乏清晰的认识。本综述首先分析了不同程度线粒体外膜通透在细胞存活、癌变或凋亡中的作用,接着讨论了线粒体内膜通透通过引发线粒体DNA释放促进炎症发生的分子机制,然后阐述了线粒体通透性转换引发焦亡的作用机制,最后总结出线粒体膜完整性影响细胞命运决策的内在关联。深入了解线粒体膜完整性调控细胞命运的分子动力学机制,有助于为癌症和神经退行性疾病的诊疗提供思路。