Damien Arnoult

Quantitation of mitochondrial dynamics by photolabeling of individual organelles shows that mitochondrial fusion is blocked during the Bax activation phase of apoptosis.

A dynamic balance of organelle fusion and fission regulates mitochondrial morphology. During apoptosis this balance is altered, leading to an extensive fragmentation of the mitochondria. Here, we describe a novel assay of mitochondrial dynamics based on confocal imaging of cells expressing a mitochondrial matrix–targeted photoactivable green fluorescent protein that enables detection and quantification of organelle fusion in living cells. Using this assay, we visualize and quantitate mitochondrial fusion rates in healthy and apoptotic cells. During apoptosis, mitochondrial fusion is blocked independently of caspase activation. The block in mitochondrial fusion occurs within the same time range as Bax coalescence on the mitochondria and outer mitochondrial membrane permeabilization, and it may be a consequence of Bax/Bak activation during apoptosis.

Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli

Mitochondrial outer membrane permeabilization by proapoptotic Bcl-2 family proteins, such as Bax, plays a crucial role in apoptosis induction. However, whether this only causes the intracytosolic release of inducers of caspase-dependent death, such as cytochrome c, or also of caspase-independent death, such as apoptosis-inducing factor (AIF) remains unknown. Here, we show that on isolated mitochondria, Bax causes the release of cytochrome c, but not of AIF, and the association of AIF with the mitochondrial inner membrane provides a simple explanation for its lack of release upon Bax-mediated outer membrane permeabilization. In cells overexpressing Bax or treated either with the Bax- or Bak-dependent proapoptotic drugs staurosporine or actinomycin D, or with hydrogen peroxide, caspase inhibitors did not affect the intracytosolic translocation of cytochrome c, but prevented that of AIF. These results provide a paradigm for mitochondria-dependent death pathways in which AIF cannot substitute for caspase executioners because its intracytosolic release occurs downstream of that of cytochrome c.

NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-κB and JNK pathways by inducing reactive oxygen species production

NOD‐like receptors (NLRs) are a family of intracellular sensors of microbial‐ or danger‐associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor‐κB (NF)‐κB‐ and type I interferon‐dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor α, Shigella infection and double‐stranded RNA, resulting in amplified NF‐κB‐dependent and JUN amino‐terminal kinases‐dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.

Mitochondria in innate immunity

Mitochondria are cellular organelles involved in host‐cell metabolic processes and the control of programmed cell death. A direct link between mitochondria and innate immune signalling was first highlighted with the identification of MAVS—a crucial adaptor for RIGI‐like receptor signalling—as a mitochondria‐anchored protein. Recently, other innate immune molecules, such as NLRX1, TRAF6, NLRP3 and IRGM have been functionally associated with mitochondria. Furthermore, mitochondrial alarmins—such as mitochondrial DNA and formyl peptides—can be released by damaged mitochondria and trigger inflammation. Therefore, mitochondria emerge as a fundamental hub for innate immune signalling.

Metalloprotease‐mediated OPA1 processing is modulated by the mitochondrial membrane potential

Background information. Human OPA1 (optic atrophy type 1) is a dynamin‐related protein of the mitochondrial IMS (intermembrane space) involved in membrane fusion and remodelling. Similarly to its yeast orthologue Mgm1p that exists in two isoforms generated by the serine protease Pcp1p/Rbd1p, OPA1 exists in various isoforms generated by alternative splicing and processing. In the present paper, we focus on protease processing of OPA1.

An N-terminal addressing sequence targets NLRX1 to the mitochondrial matrix

NLRX1 is the only member of the Nod-like receptor (NLR) family that is targeted to the mitochondria, and its overexpression induces the generation of reactive oxygen species (ROS), thus impacting on NFκB- and JNK-dependent signaling cascades. In addition, NLRX1 has been shown to interact with MAVS (also known as IPS-1, VISA and Cardif) at the mitochondrial outer membrane and to modulate antiviral responses. Here we report that NLRX1 has a functional leader sequence and fully translocates to the mitochondrial matrix via a mechanism requiring the mitochondrial inner-membrane potential, ΔΨm. Importantly, we failed to detect NLRX1 at the mitochondrial outer membrane. We also show that the leader sequence of NLRX1 is removed, which generates a mature protein lacking the first 39 amino acids through a maturation process that is common for mitochondrial-matrix proteins. Finally, we identified UQCRC2, a matrix-facing protein of the respiratory chain complex III, as an NLRX1-interacting molecule, thus providing a molecular basis for the role of NLRX1 in ROS generation. These results provide the first identification of a protein belonging to the NLR family that is targeted to the mitochondrial matrix.

A Lethal de Novo Mutation in the Middle Domain of the Dynamin-related GTPase Drp1 Impairs Higher Order Assembly and Mitochondrial Division

Mitochondria dynamically fuse and divide within cells, and the proper balance of fusion and fission is necessary for normal mitochondrial function, morphology, and distribution. Drp1 is a dynamin-related GTPase required for mitochondrial fission in mammalian cells. It harbors four distinct domains: GTP-binding, middle, insert B, and GTPase effector. A lethal mutation (A395D) within the Drp1 middle domain was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham, H. R., Koster, J., van Roermund, C. W., Mooyer, P. A., Wanders, R. J., and Leonard, J. V. (2007) N. Engl. J. Med. 356, 1736–1741). Mitochondria within patient-derived fibroblasts were markedly elongated, but the molecular mechanisms underlying these findings were not demonstrated. Because the middle domain is particularly important for the self-assembly of some dynamin superfamily proteins, we tested the hypothesis that this A395D mutation, and two other middle domain mutations (G350D, G363D) were important for Drp1 tetramerization, higher order assembly, and function. Although tetramerization appeared largely intact, each of these mutations compromised higher order assembly and assembly-dependent stimulation of Drp1 GTPase activity. Moreover, mutant Drp1 proteins exhibited impaired localization to mitochondria, indicating that this higher order assembly is important for mitochondrial recruitment, retention, or both. Overexpression of these middle domain mutants markedly inhibited mitochondrial division in cells. Thus, the Drp1 A395D lethal defect likely resulted in impaired higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria.

The role of mitochondria in cellular defense against microbial infection

Mitochondria have been long recognized for their key role in the modulation of cell death pathways. Thus, it is therefore not surprising that this organelle represents a recurrent target for pathogenic microbes, aiming to manipulate the fate of the infected host cell. More recently, mitochondria have been shown to serve as a crucial platform for innate immune signaling, as illustrated by the identification of MAVS (also known as IPS-1, VISA and Cardif), NLRX1 and STING as mitochondrial proteins. This review discusses the tight interplay between microbial infection, innate immune signaling and mitochondria.

Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice

Studying the effects of the loss of a specific cell type is a powerful approach in biology. Here we present a method based on the controlled activation of the apoptotic machinery. We expressed a modified caspase-3-containing chemical inducer of dimerization (CID)-binding sites in the livers of transgenic mice. In the absence of CID, no liver injury was detectable, underlining the absence of leakage in our system. In contrast, injection of the CID produced activation of the chimeric caspase-3, which led to a dose-dependent pure hepatocyte ablation with subsequent regeneration. This method is effective in both growing and nongrowing cells, and is therefore applicable to a wide range of cells and tissues. Moreover, because apoptosis has been described in numerous pathological circumstances, this system is useful for generating mouse models of human disorders as well as for studying the recovery or regeneration of tissues after cell loss.

NLRX1 does not inhibit MAVS-dependent antiviral signalling.

NLRX1 is a member of the Nod-like receptor family of intracellular sensors of microbial- and danger-associated molecular patterns. NLRX1 has a N-terminal mitochondrial addressing sequence that localizes the protein to the mitochondrial matrix. Recently, conflicting reports have been presented with regard to the putative implication of NLRX1 as a negative regulator of MAVS-dependent cytosolic antiviral responses. Here, we generated a new NLRX1 knockout mouse strain and observed that bone marrow-derived macrophages and murine embryonic fibroblasts from NLRX1-deficient mice displayed normal antiviral and inflammatory responses following Sendai virus infection. Importantly, wild type and NLRX1-deficient mice exhibited unaltered antiviral and inflammatory gene expression following intranasal challenge with influenza A virus or i.p. injection of Poly (I:C). Together, our results demonstrate that NLRX1 does not participate in the negative regulation of MAVS-dependent antiviral responses.