Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa-Porthos axis in Drosophila.

Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD-box protein, and of two metabolic enzymes, lysine-α-ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.

Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline.

Mitochondria contain their own genomes that, unlike nuclear genomes, are inherited only in the maternal line. Owing to a high mutation rate and low levels of recombination of mitrochondrial DNA (mtDNA), special selection mechanisms exist in the female germline to prevent the accumulation of deleterious mutations1-5. However, the molecular mechanisms that underpin selection are poorly understood6. Here we visualize germline selection in Drosophila using an allele-specific fluorescent in situ-hybridization approach to distinguish wild-type from mutant mtDNA. Selection first manifests in the early stages of Drosophila oogenesis, triggered by reduction of the pro-fusion protein Mitofusin. This leads to the physical separation of mitochondrial genomes into different mitochondrial fragments, which prevents the mixing of genomes and their products and thereby reduces complementation. Once fragmented, mitochondria that contain mutant genomes are less able to produce ATP, which marks them for selection through a process that requires the mitophagy proteins Atg1 and BNIP3. A reduction in Atg1 or BNIP3 decreases the amount of wild-type mtDNA, which suggests a link between mitochondrial turnover and mtDNA replication. Fragmentation is not only necessary for selection in germline tissues, but is also sufficient to induce selection in somatic tissues in which selection is normally absent. We postulate that there is a generalizable mechanism for selection against deleterious mtDNA mutations, which may enable the development of strategies for the treatment of mtDNA disorders.

Avoiding Extinction: Recent Advances in Understanding Mechanisms of Mitochondrial DNA Purifying Selection in the Germline.

Mitochondria are unusual organelles in that they contain their own genomes, which are kept apart from the rest of the DNA in the cell. While mitochondrial DNA (mtDNA) is essential for respiration and most multicellular life, maintaining a genome outside the nucleus brings with it a number of challenges. Chief among these is preserving mtDNA genomic integrity from one generation to the next. In this review, we discuss what is known about negative (purifying) selection mechanisms that prevent deleterious mutations from accumulating in mtDNA in the germline. Throughout, we focus on the female germline, as it is the tissue through which mtDNA is inherited in most organisms and, therefore, the tissue that most profoundly shapes the genome. We discuss recent progress in uncovering the mechanisms of germline mtDNA selection, from humans to invertebrates.

Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

The role of programmed mitophagy in germline mitochondrial DNA quality control.

Mitochondrial DNA (mtDNA) is prone to the accumulation of mutations. To prevent harmful mtDNA mutations from being passed on to the next generation, the female germline, through which mtDNA is exclusively inherited, has evolved extensive mtDNA quality control. To dissect the molecular underpinnings of this process, we recently performed a large RNAi screen in Drosophila and uncovered a programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We found that PGM begins as germ cells enter meiosis, induced, at least in part, by the inhibition of the mTor (mechanistic Target of rapamycin) complex 1 (mTorC1). Interestingly, PGM requires the general macroautophagy/autophagy machinery and the mitophagy adaptor BNIP3, but not the canonical mitophagy genes Pink1 and park (parkin), even though they are critical for germline mtDNA quality control. We also identified the RNA-binding protein Atx2 as a major regulator of PGM. This work is the first to identify and implicate a programmed mitophagy event in germline mtDNA quality control, and it highlights the utility of the Drosophila ovary for studying developmentally regulated mitophagy and autophagy in vivo.

A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury.

Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

Mitochondrial DNA quality control in the female germline requires a unique programmed mitophagy.

Mitochondria have their own DNA (mtDNA), which is susceptible to the accumulation of disease-causing mutations. To prevent deleterious mutations from being inherited, the female germline has evolved a conserved quality control mechanism that remains poorly understood. Here, through a large-scale screen, we uncover a unique programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We find that PGM is developmentally triggered as germ cells enter meiosis by inhibition of the target of rapamycin complex 1 (TORC1). We identify a role for the RNA-binding protein Ataxin-2 (Atx2) in coordinating the timing of PGM with meiosis. We show that PGM requires the mitophagy receptor BNIP3, mitochondrial fission and translation factors, and members of the Atg1 complex, but not the mitophagy factors PINK1 and Parkin. Additionally, we report several factors that are critical for germline mtDNA quality control and show that pharmacological manipulation of one of these factors promotes mtDNA quality control.

Identification of S-nitrosated mitochondrial proteins by S-nitrosothiol difference in gel electrophoresis (SNO-DIGE): implications for the regulation of mitochondrial function by reversible S-nitrosation.

The S-nitrosation of mitochondrial proteins as a consequence of NO metabolism is of physiological and pathological significance. We previously developed a MitoSNO (mitochondria-targeted S-nitrosothiol) that selectively S-nitrosates mitochondrial proteins. To identify these S-nitrosated proteins, here we have developed a selective proteomic methodology, SNO-DIGE (S-nitrosothiol difference in gel electrophoresis). Protein thiols in control and MitoSNO-treated samples were blocked, then incubated with copper(II) and ascorbate to selectively reduce S-nitrosothiols. The samples were then treated with thiol-reactive Cy3 (indocarbocyanine) or Cy5 (indodicarbocyanine) fluorescent tags, mixed together and individual protein spots were resolved by 2D (two-dimensional) gel electrophoresis. Fluorescent scanning of these gels revealed S-nitrosated proteins by an increase in Cy5 red fluorescence, allowing for their identification by MS. Parallel analysis by Redox-DIGE enabled us to distinguish S-nitrosated thiol proteins from those which became oxidized due to NO metabolism. We identified 13 S-nitrosated mitochondrial proteins, and a further four that were oxidized, probably due to evanescent S-nitrosation relaxing to a reversible thiol modification. We investigated the consequences of S-nitrosation for three of the enzymes identified using SNO-DIGE (aconitase, mitochondrial aldehyde dehydrogenase and alpha-ketoglutarate dehydrogenase) and found that their activity was selectively and reversibly inhibited by S-nitrosation. We conclude that the reversible regulation of enzyme activity by S-nitrosation modifies enzymes central to mitochondrial metabolism, whereas identification and functional characterization of these novel targets provides mechanistic insight into the potential physiological and pathological roles played by this modification. More generally, the development of SNO-DIGE facilitates robust investigation of protein S-nitrosation across the proteome.

Chemical entrapment and killing of insects by bacteria.

Actinobacteria produce antibacterial and antifungal specialized metabolites. Many insects harbour actinobacteria on their bodies or in their nests and use these metabolites for protection. However, some actinobacteria produce metabolites that are toxic to insects and the evolutionary relevance of this toxicity is unknown. Here we explore chemical interactions between streptomycetes and the fruit fly Drosophila melanogaster. We find that many streptomycetes produce specialized metabolites that have potent larvicidal effects against the fly; larvae that ingest spores of these species die. The mechanism of toxicity is specific to the bacterium's chemical arsenal: cosmomycin D producing bacteria induce a cell death-like response in the larval digestive tract; avermectin producing bacteria induce paralysis. Furthermore, low concentrations of volatile terpenes like 2-methylisoborneol that are produced by streptomycetes attract fruit flies such that they preferentially deposit their eggs on contaminated food sources. The resulting larvae are killed during growth and development. The phenomenon of volatile-mediated attraction and specialized metabolite toxicity suggests that some streptomycetes pose an evolutionary risk to insects in nature.

Mitochondrial DNA Purifying Selection in Mammals and Invertebrates.

Numerous mitochondrial quality control mechanisms exist within cells, but none have been shown to effectively assess and control the quality of mitochondrial DNA (mtDNA). One reason such mechanisms have yet to be elucidated is that they do not appear to be particularly active in most somatic cells, where many studies are conducted. The female germline, the cell lineage that gives rise to eggs, appears to be an exception. In the germline, strong purifying selection pathways act to eliminate deleterious mtDNA. These pathways have apparently evolved to prevent pathogenic mtDNA mutations from accumulating over successive generations and causing a decline of species via Muller's ratchet. Despite their fundamental biological importance, the mechanisms underlying purifying selection remain poorly understood, with no genes involved in this process yet identified. In this review, we discuss recent studies exploring mechanisms of germline mtDNA purifying selection in both mammalian and invertebrate systems. We also discuss the challenges to future major advances. Understanding the molecular basis of purifying selection is not only a fundamental outstanding question in biology, but may also pave the way to controlling selection in somatic tissues, potentially leading to treatments for people suffering from mitochondrial diseases.

Phase transitioned nuclear Oskar promotes cell division of Drosophila primordial germ cells.

Germ granules are non-membranous ribonucleoprotein granules deemed the hubs for post-transcriptional gene regulation and functionally linked to germ cell fate across species. Little is known about the physical properties of germ granules and how these relate to germ cell function. Here we study two types of germ granules in the Drosophila embryo: cytoplasmic germ granules that instruct primordial germ cells (PGCs) formation and nuclear germ granules within early PGCs with unknown function. We show that cytoplasmic and nuclear germ granules are phase transitioned condensates nucleated by Oskar protein that display liquid as well as hydrogel-like properties. Focusing on nuclear granules, we find that Oskar drives their formation in heterologous cell systems. Multiple, independent Oskar protein domains synergize to promote granule phase separation. Deletion of Oskar's nuclear localization sequence specifically ablates nuclear granules in cell systems. In the embryo, nuclear germ granules promote germ cell divisions thereby increasing PGC number for the next generation.

A framework for analyzing contagion in assortative banking networks.

We introduce a probabilistic framework that represents stylized banking networks with the aim of predicting the size of contagion events. Most previous work on random financial networks assumes independent connections between banks, whereas our framework explicitly allows for (dis)assortative edge probabilities (i.e., a tendency for small banks to link to large banks). We analyze default cascades triggered by shocking the network and find that the cascade can be understood as an explicit iterated mapping on a set of edge probabilities that converges to a fixed point. We derive a cascade condition, analogous to the basic reproduction number R0 in epidemic modelling, that characterizes whether or not a single initially defaulted bank can trigger a cascade that extends to a finite fraction of the infinite network. This cascade condition is an easily computed measure of the systemic risk inherent in a given banking network topology. We use percolation theory for random networks to derive a formula for the frequency of global cascades. These analytical results are shown to provide limited quantitative agreement with Monte Carlo simulation studies of finite-sized networks. We show that edge-assortativity, the propensity of nodes to connect to similar nodes, can have a strong effect on the level of systemic risk as measured by the cascade condition. However, the effect of assortativity on systemic risk is subtle, and we propose a simple graph theoretic quantity, which we call the graph-assortativity coefficient, that can be used to assess systemic risk.

Glutathionylation of mitochondrial proteins.

Many proteins contain free thiols that can be modified by the reversible formation of mixed disulfides with low-molecular-weight thiols through a process called S-thiolation. As the majority of these modifications result from the interaction of protein thiols with the endogenous glutathione pool, protein glutathionylation is the predominant alteration. Protein glutathionylation is of significance both for defense against oxidative damage and in redox signaling. As mitochondria are at the heart of both oxidative damage and redox signaling within the cell, the glutathionylation of mitochondrial proteins is of particular importance. Here we review the mechanisms and physiological significance of the glutathionylation of mitochondrial thiol proteins.

Ultrastructural Analysis of Drosophila Ovaries by Electron Microscopy.

The Drosophila melanogaster ovary is a powerful, genetically tractable system through which one can elucidate the principles underlying cellular function and organogenesis in vivo. In order to understand the intricate process of oogenesis at the subcellular level, microscopic analysis with the highest possible resolution is required. In this chapter, we describe the preparation of ovaries for ultrastructural analysis using transmission electron microscopy and focused ion beam scanning electron microscopy. We discuss and provide protocols for chemical fixation of Drosophila ovaries that facilitate optimal imaging with particular attention paid to preserving and resolving mitochondrial membrane morphology and structure.

ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation.

The differentiation of stem cells is a tightly regulated process essential for animal development and tissue homeostasis. Through this process, attainment of new identity and function is achieved by marked changes in cellular properties. Intrinsic cellular mechanisms governing stem cell differentiation remain largely unknown, in part because systematic forward genetic approaches to the problem have not been widely used. Analysing genes required for germline stem cell differentiation in the Drosophila ovary, we find that the mitochondrial ATP synthase plays a critical role in this process. Unexpectedly, the ATP synthesizing function of this complex was not necessary for differentiation, as knockdown of other members of the oxidative phosphorylation system did not disrupt the process. Instead, the ATP synthase acted to promote the maturation of mitochondrial cristae during differentiation through dimerization and specific upregulation of the ATP synthase complex. Taken together, our results suggest that ATP synthase-dependent crista maturation is a key developmental process required for differentiation independent of oxidative phosphorylation.

Cysteine residues exposed on protein surfaces are the dominant intramitochondrial thiol and may protect against oxidative damage.

Cysteine plays a number of important roles in protecting the cell from oxidative damage through its thiol functional group. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment. In the present study, in subcellular fractions prepared from rat liver or heart, we show that the quantitatively dominant free thiols are those of cysteine residues exposed on protein surfaces and not those carried by glutathione. Within the mitochondrial matrix, the concentration of exposed protein thiols is 60-90 mm, which is approximately 26-fold higher than the glutathione concentration in that compartment. This suggests that exposed protein thiols are of greater importance than glutathione for nonenzyme catalysed reactions of thiols with reactive oxygen and nitrogen species and with electrophiles within the cell. One such antioxidant role for exposed protein thiols may be to prevent protein oxidative damage. In the present study, in mitochondrial membranes and in complex I, we show that exposed protein thiols protect against tyrosine nitration and protein dysfunction caused by peroxynitrite. Therefore, exposed protein thiols are the dominant free thiol within the cell and may play a critical role in intracellular antioxidant defences against oxidative damage.

Measuring mitochondrial protein thiol redox state.

Protein thiols are an important component of mammalian intramitochondrial antioxidant defenses owing to their selective interaction with reactive oxygen and nitrogen species (ROS and RNS). Reversible modifications of protein thiols resulting from these interactions are also an important aspect of redox signal transduction. Therefore, to assess how mitochondria respond to oxidative stress and act as nodes in redox signaling pathways, it is important to measure general changes to protein thiol redox states and also to identify the specific mitochondrial thiol proteins involved. Here we outline some of the approaches that can be used to accomplish these goals and thereby infer the multiple roles of mammalian mitochondrial protein thiols in antioxidant defense and redox signaling.

Chapter 19 Measuring redox changes to mitochondrial protein thiols with redox difference gel electrophoresis (redox-DIGE).

Low levels of reactive oxygen and nitrogen species (ROS and RNS) produced by the mitochondrial respiratory chain and ROS and RNS from other sources act as redox signals by oxidizing thiols on specific proteins. Because these thiol modifications occur on a relatively small number of proteins in the absence of bulk thiol changes, it is necessary to use sensitive methods to discover them. Recently, a number of methods have been developed to help facilitate the identification and characterization of redox-sensitive thiol proteins. In this chapter we describe one such method, redox difference gel electrophoresis (redox-DIGE), in which oxidized thiol proteins in redox-challenged samples are labeled with a thiol-reactive fluorescent tag and compared with those in control samples labeled with a different tag on the same 2-D gel. This enables the sensitive detection of redox-sensitive thiol proteins by measuring changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. With this method we have been able to identify several mitochondrial proteins whose thiol state and activity are altered by low levels of ROS from the respiratory chain, which may be an important and unexplored mode of mitochondrial redox signaling. Importantly, this method is not only applicable to studies in isolated mitochondria but can also be applied to more complicated systems such as intact cells and perhaps even whole organisms.

Complex I within oxidatively stressed bovine heart mitochondria is glutathionylated on Cys-531 and Cys-704 of the 75-kDa subunit: potential role of CYS residues in decreasing oxidative damage.

Complex I has reactive thiols on its surface that interact with the mitochondrial glutathione pool and are implicated in oxidative damage in many pathologies. However, the Cys residues and the thiol modifications involved are not known. Here we investigate complex I thiol modification within oxidatively stressed mammalian mitochondria, containing physiological levels of glutathione and glutaredoxin 2. In mitochondria incubated with the thiol oxidant diamide, complex I is only glutathionylated on the 75-kDa subunit. Of the 17 Cys residues on the 75-kDa subunit, 6 are not involved in iron-sulfur centers, making them plausible candidates for glutathionylation. Mass spectrometry of complex I from oxidatively stressed bovine heart mitochondria showed that only Cys-531 and Cys-704 were glutathionylated. The other four non-iron-sulfur center Cys residues remained as free thiols. Complex I glutathionylation also occurred in response to relatively mild oxidative stress caused by increased superoxide production from the respiratory chain. Although complex I glutathionylation within oxidatively stressed mitochondria correlated with loss of activity, it did not increase superoxide formation, and reversal of glutathionylation did not restore complex I activity. Comparison with the known structure of the 75-kDa ortholog Nqo3 from Thermus thermophilus complex I suggested that Cys-531 and Cys-704 are on the surface of mammalian complex I, exposed to the mitochondrial glutathione pool. These findings suggest that Cys-531 and Cys-704 may be important in preventing oxidative damage to complex I by reacting with free radicals and other damaging species, with subsequent glutathionylation recycling the thiyl radicals and sulfenic acids formed on the Cys residues back to free thiols.

Detection of reactive oxygen species-sensitive thiol proteins by redox difference gel electrophoresis: implications for mitochondrial redox signaling.

Reactive oxygen species (ROS) produced by the mitochondrial respiratory chain can be a redox signal, but whether they affect mitochondrial function is unclear. Here we show that low levels of ROS from the respiratory chain under physiological conditions reversibly modify the thiol redox state of mitochondrial proteins involved in fatty acid and carbohydrate metabolism. As these thiol modifications were specific and occurred without bulk thiol changes, we first had to develop a sensitive technique to identify the small number of proteins modified by endogenous ROS. In this technique, redox difference gel electrophoresis, control, and redox-challenged samples are labeled with different thiol-reactive fluorescent tags and then separated on the same two-dimensional gel, enabling the sensitive detection of thiol redox modifications by changes in the relative fluorescence of the two tags within a single protein spot, followed by protein identification by mass spectrometry. Thiol redox modification affected enzyme activity, suggesting that the reversible modification of enzyme activity by ROS from the respiratory chain may be an important and unexplored mode of mitochondrial redox signaling.