Perforation of the outer mitochondrial membrane triggered by BAX and facilitated by its main activator cBID is a fundamental process in cell apoptosis. Here, we employ a newly designed correlative approach based on a combination of a fluorescence cross correlation binding with a calcein permeabilization assay to understand the involvement of BAX in pore formation under oxidative stress conditions. To mimic the oxidative stress, we enriched liposomal membranes by phosphatidylcholines with truncated sn-2 acyl chains terminated by a carboxyl or aldehyde moiety. Our observations revealed that oxidative stress enhances proapoptotic conditions involving accelerated pore-opening kinetics. This enhancement is achieved through increased recruitment of BAX to the membrane and facilitation of BAX membrane insertion. Despite these effects, the fundamental mechanism of pore formation remained unchanged, suggesting an all-or-none mechanism. In line with this mechanism, we demonstrated that the minimal number of BAX molecules at the membrane necessary for pore formation remains constant regardless of BAX activation by cBID or the presence of oxidized lipids. Overall, our findings give a comprehensive picture of the molecular mechanisms underlying apoptotic pore formation and highlight the selective amplifying role of oxidized lipids in triggering formation of membrane pores.

• MeSH
• Apoptosis MeSH
• Fluoresceins chemistry   metabolism   MeSH
• Phosphatidylcholines chemistry   metabolism   MeSH
• Humans MeSH
• Liposomes chemistry   metabolism   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Oxidative Stress * MeSH
• Porosity MeSH
• bcl-2-Associated X Protein * metabolism   chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Fluoresceins MeSH
• fluorexon MeSH Browser
• Phosphatidylcholines MeSH
• Liposomes MeSH
• bcl-2-Associated X Protein * MeSH

Mitochondria represent the fundamental system for cellular energy metabolism, by not only supplying energy in the form of ATP, but also by affecting physiology and cell death via the regulation of calcium homeostasis and the activity of Bcl-2 proteins. A lot of research has recently been devoted to understanding the interplay between Bcl-2 proteins, the regulation of these interactions within the cell, and how these interactions lead to the changes in calcium homeostasis. However, the role of Bcl-2 proteins in the mediation of mitochondrial calcium homeostasis, and therefore the induction of cell death pathways, remain underestimated and are still not well understood. In this review, we first summarize our knowledge about calcium transport systems in mitochondria, which, when miss-regulated, can induce necrosis. We continue by reviewing and analyzing the functions of Bcl-2 proteins in apoptosis. Finally, we link these two regulatory mechanisms together, exploring the interactions between the mitochondrial Ca2+ transport systems and Bcl-2 proteins, both capable of inducing cell death, with the potential to determine the cell death pathway-either the apoptotic or the necrotic one.

• Keywords
• Bax, Bcl-2 proteins, MCU, VDAC, apoptosis, calcium transport, mPTP, mitochondria, necrosis,
• Publication type
• Journal Article MeSH
• Review MeSH

Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using 31P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.

• MeSH
• Apoptosis physiology   MeSH
• Calorimetry, Differential Scanning MeSH
• Escherichia coli MeSH
• Fluorescent Dyes MeSH
• Phospholipids metabolism   MeSH
• Cardiolipins metabolism   MeSH
• Humans MeSH
• Lipid Bilayers chemistry   MeSH
• Mitochondrial Membranes metabolism   MeSH
• Mitochondria metabolism   MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Oxidation-Reduction MeSH
• Oxidative Stress physiology   MeSH
• Cell Membrane Permeability MeSH
• bcl-2-Associated X Protein metabolism   MeSH
• Temperature MeSH
• Unilamellar Liposomes chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• BAX protein, human MeSH Browser
• Fluorescent Dyes MeSH
• Phospholipids MeSH
• Cardiolipins MeSH
• Lipid Bilayers MeSH
• bcl-2-Associated X Protein MeSH
• Unilamellar Liposomes MeSH