Thibaud T. Renault

Bax: Addressed to kill.

The pro-apoptototic protein Bax (Bcl-2 Associated protein X) plays a central role in the mitochondria-dependent apoptotic pathway. In healthy mammalian cells, Bax is essentially cytosolic and inactive. Following a death signal, the protein is translocated to the outer mitochondrial membrane, where it promotes a permeabilization that favors the release of different apoptogenic factors, such as cytochrome c. The regulation of Bax translocation is associated to conformational changes that are under the control of different factors. The evidences showing the involvement of different Bax domains in its mitochondrial localization are presented. The interactions between Bax and its different partners are described in relation to their ability to promote (or prevent) Bax conformational changes leading to mitochondrial addressing and to the acquisition of the capacity to permeabilize the outer mitochondrial membrane.

Mitochondrial Shape Governs BAX-Induced Membrane Permeabilization and Apoptosis

Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis.

Death upon a Kiss: Mitochondrial Outer Membrane Composition and Organelle Communication Govern Sensitivity to BAK/BAX-Dependent Apoptosis

For stressed cells to induce the mitochondrial pathway of apoptosis, a cohort of pro-apoptotic BCL-2 proteins must collaborate with the outer mitochondrial membrane to permeabilize it. BAK and BAX are the two pro-apoptotic BCL-2 family members that are required for mitochondrial outer membrane permeabilization. While biochemical and structural insights of BAK/BAX function have expanded in recent years, very little is known about the role of the outer mitochondrial membrane in regulating BAK/BAX activity. In this review, we will highlight the impact of mitochondrial composition (both protein and lipid) and mitochondrial interactions with cellular organelles on BAK/BAX function and cellular commitment to apoptosis. A better understanding of how BAK/BAX and mitochondrial biology are mechanistically linked will likely reveal novel insights into homeostatic and pathological mechanisms associated with apoptosis.

Getting away with murder: how does the BCL-2 family of proteins kill with immunity?

The adult human body produces approximately one million white blood cells every second. However, only a small fraction of the cells will survive because the majority is eliminated through a genetically controlled form of cell death known as apoptosis. This review places into perspective recent studies pertaining to the BCL‐2 family of proteins as critical regulators of the development and function of the immune system, with particular attention on B cell and T cell biology. Here we discuss how elegant murine model systems have revealed the major contributions of the BCL‐2 family in establishing an effective immune system. Moreover, we highlight some key regulatory pathways that influence the expression, function, and stability of individual BCL‐2 family members, and discuss their role in immunity. From lethal mechanisms to more gentle ones, the final portion of the review discusses the nonapoptotic functions of the BCL‐2 family and how they pertain to the control of immunity.

Putting the pieces together: How is the mitochondrial pathway of apoptosis regulated in cancer and chemotherapy?

In order to solve a jigsaw puzzle, one must first have the complete picture to logically connect the pieces. However, in cancer biology, we are still gaining an understanding of all the signaling pathways that promote tumorigenesis and how these pathways can be pharmacologically manipulated by conventional and targeted therapies. Despite not having complete knowledge of the mechanisms that cause cancer, the signaling networks responsible for cancer are becoming clearer, and this information is serving as a solid foundation for the development of rationally designed therapies. One goal of chemotherapy is to induce cancer cell death through the mitochondrial pathway of apoptosis. Within this review, we present the pathways that govern the cellular decision to undergo apoptosis as three distinct, yet connected puzzle pieces: (1) How do oncogene and tumor suppressor pathways regulate apoptosis upstream of mitochondria? (2) How does the B-cell lymphoma 2 (BCL-2) family influence tumorigenesis and chemotherapeutic responses? (3) How is post-mitochondrial outer membrane permeabilization (MOMP) regulation of cell death relevant in cancer? When these pieces are united, it is possible to appreciate how cancer signaling directly impacts upon the fundamental cellular mechanisms of apoptosis and potentially reveals novel pharmacological targets within these pathways that may enhance chemotherapeutic success.

BAK/BAX activation and cytochrome c release assays using isolated mitochondria

The mitochondrial pathway of apoptosis proceeds when the outer mitochondrial membrane (OMM) is compromised by the pro-apoptotic BCL-2 family members, BAK and BAX. Once activated, BAK and BAX form proteolipid pores in the OMM leading to mitochondrial outer membrane permeabilization (MOMP), and the release of inner membrane space proteins, such as cytochrome c, which promotes caspase activation. The use of isolated mitochondria has been instrumental to understanding the key interactions necessary to engage BAK and BAX activation, MOMP, and apoptosis. Furthermore, it is possible to biochemically define the relationships between BCL-2 family function and mitochondrial physiology using isolated systems. Our laboratory uses freshly isolated mitochondria from numerous sources to better understand BCL-2 family function and requirements for BAK and BAX activation. Here, we will discuss commonly used in vitro techniques to perform MOMP and cytochrome c release assays; and provide several key methodologies to implicate BAK and BAX activity in these processes.

B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

Background: BH3 mimetics are promising chemotherapeutics, but their full mechanisms of action are underexplored. Results: BAX, BID, and BIM directly influence BH3 mimetics to pharmacologically inhibit anti-apoptotic BCL-2 proteins. Conclusion: BH3 mimetics display differential activities that are dependent upon both anti- and proapoptotic BCL-2 members function. Significance: Understanding how BH3 mimetics function within the BCL-2 family repertoire will improve the utility of these drugs. The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these “BH3 mimetics” in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins.

Bacterial flagella grow through an injection-diffusion mechanism

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell. DOI: http://dx.doi.org/10.7554/eLife.23136.001

Examining BCL-2 family function with large unilamellar vesicles.

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis(1). Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)(1). After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues(2). In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)(3-6). Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation(7,8). In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members(7,9). As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane. Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization(10). LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)(10). This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)(11). As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737

Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release.