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Respiratory complex I is a proton-pumping oxidoreductase key to bioenergetic metabolism. Biochemical studies have found a divide in the behavior of complex I in metazoans that aligns with the evolutionary split between Protostomia and Deuterostomia. Complex I from Deuterostomia including mammals can adopt an off-pathway “deactive” state, whereas complex I from Protostomia cannot. The presence of off-pathway states complicates the interpretation of structural results and has led to considerable mechanistic debate. Here we report the structure of mitochondrial complex I from the thoracic muscles of the model protostomianDrosophila melanogaster. We show that, althoughD. melanogastercomplex I (Dm-CI) does not deactivate the resting state ofDm-CI adopts multiple conformations. We identify a new helix-locked open state in which an N-terminal α-helix on the NDUFS4 subunit wedges between the peripheral and membrane arms. Comparison of theDm-CI structure and conformational states to those observed in bacteria, yeast and mammals provides insight into the roles of subunits across organisms, explains whyDm-CI does not deactivate and reveals incompatibilities with current mechanistic models of complex I turnover. Additionally, theDm-CI structure and novel regulatory mechanism will allow for the development of more selective pesticides for agriculture and human disease.

Because old age is associated with defects in circadian rhythm, loss of circadian regulation is thought to be pathogenic and contribute to mortality. We show instead that loss of specific circadian clock components Period (Per) and Timeless (Tim) in male Drosophila significantly extends lifespan. This lifespan extension is not mediated by canonical diet-restriction longevity pathways but is due to altered cellular respiration via increased mitochondrial uncoupling. Lifespan extension of per mutants depends on mitochondrial uncoupling in the intestine. Moreover, upregulated uncoupling protein UCP4C in intestinal stem cells and enteroblasts is sufficient to extend lifespan and preserve proliferative homeostasis in the gut with age. Consistent with inducing a metabolic state that prevents overproliferation, mitochondrial uncoupling drugs also extend lifespan and inhibit intestinal stem cell overproliferation due to aging or even tumorigenesis. These results demonstrate that circadian-regulated intestinal mitochondrial uncoupling controls longevity in Drosophila and suggest a new potential anti-aging therapeutic target., Disruption of different components of molecular circadian clocks has varying effects on health and lifespan of model organisms. Here the authors show that loss of period extends life in drosophila melanogaster.

Several subunits in the matrix domain of mitochondrial complex I (CI) have been posited to be redox sensors for CI; but how elevated levels of reactive oxygen species (ROS) impinge on CI assembly is unknown. We report that when the mitochondrial NADPH-generating enzyme – Isocitrate Dehydrogenase 2 – is genetically disrupted, ROS levels are elevated and assembly of the oxidative phosphorylation system (OXPHOS) is impaired. Mechanistically, this begins with a ROS-mediated inhibition of biosynthesis of the matrix domain of CI, which progresses to a point where ferroptotic signals are induced, the mitochondrial unfolded protein response is activated and multiple OXPHOS complexes are impaired. Disruption of other enzymes that eliminate hydrogen peroxide, but not those that eliminate the superoxide radical, recapitulates the phenotype; implicating hydrogen peroxide as the signaling molecule involved. Thus, the redox status of the mitochondrion modulates the assembly of the matrix domain of CI and ultimately that of the entire OXPHOS.

Summary Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities. For complete details on the use and execution of this protocol, please refer to Murari et al. (2020)., Graphical abstract, Highlights • Blue native polyacrylamide gel electrophoresis (BN PAGE) separates OXPHOS complexes • OXPHOS assembly in Drosophila flight muscles can be assessed with BN PAGE • OXPHOS activities can be assessed by in-gel activity assays, Drosophila flight muscles are highly enriched with mitochondria and have emerged as a powerful genetic system for studying how oxidative phosphorylation (OXPHOS) complexes are assembled. Here, we describe a series of protocols for analyzing the integrity of OXPHOS complexes in Drosophila via blue native polyacrylamide gel electrophoresis (BN PAGE). We have also included protocols for the additional steps that are typically performed after OXPHOS complexes are separated by BN PAGE, such as Coomassie staining, silver staining, and in-gel OXPHOS activities.

Impairments in mitochondrial energy metabolism have been implicated in human genetic diseases associated with mitochondrial and nuclear DNA mutations, neurodegenerative and cardiovascular disorders, diabetes, and aging. Alteration in mitochondrial complex I structure and activity has been shown to play a key role in Parkinson's disease and ischemia/reperfusion tissue injury, but significant difficulty remains in assessing the content of this enzyme complex in a given sample. The present study introduces a new method utilizing native polyacrylamide gel electrophoresis in combination with flavin fluorescence scanning to measure the absolute content of complex I, as well as α-ketoglutarate dehydrogenase complex, in any preparation. We show that complex I content is 19 ± 1 pmol/mg of protein in the brain mitochondria, whereas varies up to 10-fold in different mouse tissues. Together with the measurements of NADH-dependent specific activity, our method also allows accurate determination of complex I catalytic turnover, which was calculated as 104 min−1 for NADH:ubiquinone reductase in mouse brain mitochondrial preparations. α-ketoglutarate dehydrogenase complex content was determined to be 65 ± 5 and 123 ± 9 pmol/mg protein for mouse brain and bovine heart mitochondria, respectively. Our approach can also be extended to cultured cells, and we demonstrated that about 90 × 103 complex I molecules are present in a single human embryonic kidney 293 cell. The ability to determine complex I content should provide a valuable tool to investigate the enzyme status in samples after in vivo treatment in mutant organisms, cells in culture, or human biopsies.

SummaryMitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimer’s disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2-4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.

Summary Distinct sub-assemblies (modules) of mitochondrial complex I (CI) are assembled with the assistance of CI Assembly Factors (CIAFs) through mechanisms that are incompletely defined. Here, using genetic analyses in Drosophila, we report that when either of the CIAFs – NDUFAF3 or NDUFAF4 – is disrupted, biogenesis of the Q-, N-, and PP-b-modules of CI is impaired. This is due, at least in part, to the compromised integration of NDUFS3 and NDUFS5 into the Q-, and PP-b-modules, respectively, coupled with a destabilization of another CIAF, TIMMDC1, in assembly intermediates. Notably, forced expression of NDUFAF4 rescues the biogenesis defects in the Q-module and some aspects of the defects in the PP-b-module of CI when NDUFAF3 is disrupted. Altogether, our studies furnish new fundamental insights into the mechanism by which NDUFAF3 and NDUFAF4 regulate CI assembly and raises the possibility that certain point mutations in NDUFAF3 may be rescued by overexpression of NDUFAF4., Graphical abstract, Highlights • Disruption of NDUFAF3 and NDUFAF4 in Drosophila muscles destabilizes TIMMDC1 • NDUFAF3 and NDUFAF4 regulate biogenesis of the N, Q, and Pp modules • NDUFAF4 ameliorates some of the CI biogenesis defects in NDUFAF3 mutants, Molecular genetics; Molecular mechanism of gene regulation; Metabolic Engineering

Tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and tumor mitochondria remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign and cancerous ovarian tissue in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n=1,435) showed that gene expression of specific mitochondrial proteins in ovarian tumors is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in 51 benign and 128 high-grade epithelial ovarian tumors revealed that compared to benign tissue, tumors exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P

Tumor cells exhibit mitochondrial alterations and are also influenced by biobehavioral processes, but the intersection of biobehavioral factors and tumor mitochondria remains unexplored. Here we examined multiple biochemical and molecular markers of mitochondrial content and function in benign and cancerous ovarian tissue in parallel with exploratory analyses of biobehavioral factors. First, analysis of a publicly-available database (n=1,435) showed that gene expression of specific mitochondrial proteins in ovarian tumors is associated with survival. Quantifying multiple biochemical and molecular markers of mitochondrial content and function in 51 benign and 128 high-grade epithelial ovarian tumors revealed that compared to benign tissue, tumors exhibit 3.3-8.4-fold higher mitochondrial content and respiratory chain enzymatic activities (P

During mitochondrial complex I (CI) assembly, independently formed assembly intermediates (AIs) merge with each other to form the mature complex. Murari et al. generate a toolbox of 21 antibodies to various mitochondrial proteins and use classical Drosophila genetics and immunoblotting of AIs to define AIF’s role in CI biogenesis., An ability to comprehensively track the assembly intermediates (AIs) of complex I (CI) biogenesis in Drosophila will enable the characterization of the precise mechanism(s) by which various CI regulators modulate CI assembly. Accordingly, we generated 21 novel antibodies to various mitochondrial proteins and used this resource to characterize the mechanism by which apoptosis-inducing factor (AIF) regulates CI biogenesis by tracking the AI profile observed when AIF expression is impaired. We find that when the AIF–Mia40 translocation complex is disrupted, the part of CI that transfers electrons to ubiquinone is synthesized but fails to progress in the CI biosynthetic pathway. This is associated with a reduction in intramitochondrial accumulation of the Mia40 substrate, MIC19. Importantly, knockdown of either MIC19 or MIC60, components of the mitochondrial contact site and cristae organizing system (MICOS), fully recapitulates the AI profile observed when AIF is inhibited. Thus, AIF’s effect on CI assembly is principally due to compromised intramitochondrial transport of the MICOS complex.

Mitochondrial dysfunction has been associated with obesity and metabolic disorders. However, whether mitochondrial perturbation in a single tissue influences mitochondrial function and metabolic status of another distal tissue remains largely unknown. We analyzed the nonautonomous role of muscular mitochondrial dysfunction in Drosophila. Surprisingly, impaired muscle mitochondrial function via complex I perturbation results in simultaneous mitochondrial dysfunction in the fat body (the fly adipose tissue) and subsequent triglyceride accumulation, the major characteristic of obesity. RNA-sequencing (RNA-seq) analysis, in the context of muscle mitochondrial dysfunction, revealed that target genes of the TGF-β signaling pathway were induced in the fat body. Strikingly, expression of the TGF-β family ligand, Activin-β (Actβ), was dramatically increased in the muscles by NF-κB/Relish (Rel) signaling in response to mitochondrial perturbation, and decreasing Actβ expression in mitochondrial-perturbed muscles rescued both the fat body mitochondrial dysfunction and obesity phenotypes. Thus, perturbation of muscle mitochondrial activity regulates mitochondrial function in the fat body nonautonomously via modulation of Activin signaling.

Objective Diabetes is characterized by pancreatic β-cell dedifferentiation. Dedifferentiating β cells inappropriately metabolize lipids over carbohydrates and exhibit impaired mitochondrial oxidative phosphorylation. However, the mechanism linking the β-cell's response to an adverse metabolic environment with impaired mitochondrial function remains unclear. Methods Here we report that the oxidoreductase cytochrome b5 reductase 3 (Cyb5r3) links FoxO1 signaling to β-cell stimulus/secretion coupling by regulating mitochondrial function, reactive oxygen species generation, and nicotinamide actin dysfunction (NAD)/reduced nicotinamide actin dysfunction (NADH) ratios. Results The expression of Cyb5r3 is decreased in FoxO1-deficient β cells. Mice with β-cell-specific deletion of Cyb5r3 have impaired insulin secretion, resulting in glucose intolerance and diet-induced hyperglycemia. Cyb5r3-deficient β cells have a blunted respiratory response to glucose and display extensive mitochondrial and secretory granule abnormalities, consistent with altered differentiation. Moreover, FoxO1 is unable to maintain expression of key differentiation markers in Cyb5r3-deficient β cells, suggesting that Cyb5r3 is required for FoxO1-dependent lineage stability. Conclusions The findings highlight a pathway linking FoxO1 to mitochondrial dysfunction that can mediate β-cell failure., Highlights • Cyb5r3 is a FoxO1 target in β-cells. • Cyb5r3 ablation impairs respiration and stimulus/secretion coupling in β-cells. • β-cell-specific deletion of Cyb5r3 impairs insulin secretion and causes glucose intolerance. • Cyb5r3-deficient β-cells display mitochondrial and secretory granule abnormalities. • Cyb5r3-deficient β-cells lack key differentiation markers.

LonP1 is a mitochondrial protease that is crucial for maintaining cardiac mitochondrial homeostasis. We recently showed that LonP1 mediates cardioprotection during cardiac ischemia-reperfusion (I/R) by reducing oxidative stress and infarct size. We hypothesize that LonP1 promotes mitochondrial biogenesis during cardiac differentiation and development and protects the heart from oxidative injury by recalibrating bioenergetics. Here, we employ human induced pluripotent stem cells differentiated into cardiomyocytes (iPSC-CMs), rat neonatal ventricular myocytes (rNVMs), and mouse models with cardiac-specific LonP1 overexpression (LonTg) and heterozygous LonP1 knockout ( LONP1 +/- ). In iPSC-CMs, LonP1 is induced 4.8-fold at 39 vs. 12 days after differentiation. During the maturation of murine hearts, LonP1 is increased 4.6-fold at 3 months vs. during 1 day. Upregulation was also observed in rNVMs in which LonP1 is induced 5- fold after 5 days culturing, compared to 0 days. LonP1 induction in rNVMs is accompanied by substantial 2-fold upregulation of mitochondrial transcripts, MT-CYB and MT-COI. However, the levels of another mitochondrial matrix protease ClpP was not altered, suggesting that LonP1 specifically may play an important role during mitochondrial biogenesis and proteostasis in the heart. In vivo, LonTg protected the myocardium against ischemia-reperfusion (I/R) injury by preventing oxidative damage. In LonTg hearts, basal complex I activity was significantly (p LONP1 +/+ (0.0118 ± 0.0 mOD/min) and LONP1 +/- (0.0121 ± 0.0 mOD/min) mice (n=3 each). In addition, our preliminary native gel data of LonTg hearts shows a 40% reduction (n=1) in the levels of mitochondrial supercomplexes, compared to NTg hearts (n=4). Further studies are underway to explore the role of LonP1 during differentiation, and in response to mitochondrial cardiac stress. Together, our findings show a novel role of LonP1 in regulating cardiac function during mitochondrial biogenesis and in mitigating cardiac stress-induced injury.

NADH:ubiquinone oxidoreductase, more commonly referred to as mitochondrial complex I (CI), is the largest discrete enzyme of the oxidative phosphorylation system (OXPHOS). It is localized to the mitochondrial inner membrane. CI oxidizes NADH generated from the tricarboxylic acid cycle to NAD(+), in a series of redox reactions that culminates in the reduction of ubiquinone, and the transport of protons from the matrix across the inner membrane to the intermembrane space. The resulting proton-motive force is consumed by ATP synthase to generate ATP, or harnessed to transport ions, metabolites and proteins into the mitochondrion. CI is also a major source of reactive oxygen species. Accordingly, impaired CI function has been associated with a host of chronic metabolic and degenerative disorders such as diabetes, cardiomyopathy, Parkinson’s disease (PD) and Leigh syndrome. Studies in Drosophila have contributed to our understanding of the multiple roles of CI in bioenergetics and organismal physiology. Here, we explore and discuss some of the studies in Drosophila that have informed our understanding of this complex; and conclude with some of the open questions about CI that can be resolved by studies in Drosophila.

Cardiolipin (CL) is a mitochondrial phospholipid with a very specific and functionally important fatty acid composition, generated by tafazzin. However, in vitro tafazzin catalyzes a promiscuous acyl exchange that acquires specificity only in response to perturbations of the physical state of lipids. To identify the process that imposes acyl specificity onto CL remodeling in vivo, we analyzed a series of deletions and knockdowns in Saccharomyces cerevisiae and Drosophila melanogaster, including carriers, membrane homeostasis proteins, fission-fusion proteins, cristae-shape controlling and MICOS proteins, and the complexes I-V. Among those, only the complexes of oxidative phosphorylation (OXPHOS) affected the CL composition. Rather than any specific complex, it was the global impairment of the OXPHOS system that altered CL and at the same time shortened its half-life. The knockdown of OXPHOS expression had the same effect on CL as the knockdown of tafazzin in Drosophila flight muscles, including a change in CL composition and the accumulation of monolyso-CL. Thus, the assembly of OXPHOS complexes induces CL remodeling, which, in turn, leads to CL stabilization. We hypothesize that protein crowding in the OXPHOS system imposes packing stress on the lipid bilayer, which is relieved by CL remodeling to form tightly packed lipid-protein complexes.

Macroautophagy is required for normal cardiac function. Chaperone mediated autophagy (CMA) is distinct in that substrates are transported to lysosomes in complex with Hsc70, rather than within autophagosomes; restricted to cytoplasmic proteins; and selectively marked with a CMA-targeting motif. The role of CMA in the heart has not been assessed. We determined that CMA is induced in the heart by pressure overload with kinetics that differ markedly from those of macroautophagy in response to the same stimulus. In CMA, cargo is imported into the lysosome by LAMP2A, which is necessary, specific, and rate-limiting for this process. Accordingly, we created knockout mice lacking LAMP2A in cardiomyocytes to inactivate CMA specifically in those cells. Unexpectedly, these mice exhibited resistance to stress-induced cardiac dysfunction in both pressure overload and myocardial infarction models. Functional assessment of cardiac mitochondria from L2AKO mice and MEFs exhibited increased rates of O 2 consumption and ATP generation. Mitochondrial proteomics showed increased levels of ATP5L, a component of mitochondrial Complex V (ATP synthase). Overexpression of ATP5L phenocopied LAMP2A KO with respect to augmentation of mitochondrial function. Conversely, knockdown of ATP5L reversed increases in mitochondrial function resulting from L2AKO. We showed that ATP5L, which possesses a KFERQ motif, is a bona fide CMA substrate as it interacts with Hsc70 in a KFERQ-dependent manner; and localizes to lyososmes in a LAMP2A-dependent manner when CMA is induced. ATP5L is thought to mediate oligomerization of Complex V to enhance ATP production. In fact, we observed increases in complex V oligomerization in L2A KO mouse hearts in both basal and TAC-induced states. In summary, cardiomyocyte-specific inhibition of CMA, which renders mice resistant to stress-induced cardiac dysfunction, augments cardiac mitochondrial function through accumulation of ATP5L. Conversely, activation of CMA by cardiac stress may function as a new pathway to heart failure.

Mitochondrial CI (NADH: ubiquinone oxidoreductase) is the first and largest of the electron transport chain (ETC) complexes involved in oxidative phosphorylation, and has a molecular mass approaching 1 MDa. Mitochondrial Complex I (CI) is composed of 44 distinct subunits, but only 14 of these subunits are directly required for catalysis. A fundamental question in mitochondrial biology is to elucidate the roles of the additional 30 or so accessory subunits. In addition, CI can form different supercomplexes with other ETC complexes. It is unclear how these supercomplexes are assembled in the human heart, or any organism. We have established Drosophila flight muscles as a suitable system for studying CI assembly as well as the assembly of CI-containing supercomplexes. We show that many of the 30 accessory subunits regulate specific steps of CI assembly in vivo ; and that CI biogenesis in flight muscles proceeds via the formation of ~315-, ~370-, ~550-, and ~815 kDa CI assembly intermediates as has been reported in mammalian systems. A specific accessory subunit (dNDUFA5) is required for the formation or stabilization of the ~315 kDa assembly intermediate. Additionally, we define a specific role for the CX 9 C-containing accessory subunit (dNDUFS5); by showing that it is required for converting a transient CI assembly intermediate (an ~700 kDa assembly intermediate) into the ~815 kDa assembly intermediate. Finally, we are performing genetic screens to identify genes that specifically regulate supercomplex assembly. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover novel mechanisms of CI assembly in a living organism, and establish Drosophila as a suitable model organism for addressing questions relevant to CI biogenesis in humans.

Over the past decade, numerous reports have underscored the similarities between the metabolism of Drosophila and vertebrates, with the identification of evolutionarily conserved enzymes and analogous organs that regulate carbohydrate and lipid metabolism. It is now well established that the major metabolic, energy-sensing and endocrine signaling networks of vertebrate systems are also conserved in flies. Accordingly, studies in Drosophila are beginning to unravel how perturbed energy balance impinges on lifespan and on the ensuing diseases when energy homeostasis goes awry. Here, we highlight several emerging concepts that are at the nexus between obesity, nutrient sensing, metabolic homeostasis and aging. Specifically, we summarize the endocrine mechanisms that regulate carbohydrate and lipid metabolism, and provide an overview of the neuropeptides that regulate feeding behavior. We further describe the various efforts at modeling the effects of high-fat or -sugar diets in Drosophila and the signaling mechanisms involved in integrating organ function. Finally, we draw attention to some of the cardinal discoveries made with these disease models and how these could spur new research questions in vertebrate systems.

Summary The flight muscles of Drosophila are highly enriched with mitochondria, but the mechanism by which mitochondrial complex I (CI) is assembled in this tissue has not been described. We report the mechanism of CI biogenesis in Drosophila flight muscles and show that it proceeds via the formation of ∼315, ∼550, and ∼815 kDa CI assembly intermediates. Additionally, we define specific roles for several CI subunits in the assembly process. In particular, we show that dNDUFS5 is required for converting an ∼700 kDa transient CI assembly intermediate into the ∼815 kDa assembly intermediate. Importantly, incorporation of dNDUFS5 into CI is necessary to stabilize or promote incorporation of dNDUFA10 into the complex. Our findings highlight the potential of studies of CI biogenesis in Drosophila to uncover the mechanism of CI assembly in vivo and establish Drosophila as a suitable model organism and resource for addressing questions relevant to CI biogenesis in humans.

Many organisms have developed a robust ability to adapt and survive in the face of environmental perturbations that threaten the integrity of their genome, proteome, or metabolome. Studies in multiple model organisms have shown that, in general, when exposed to stress, cells activate a complex prosurvival signaling network that includes immune and DNA damage response genes, chaperones, antioxidant enzymes, structural proteins, metabolic enzymes, and noncoding RNAs. The manner of activation runs the gamut from transcriptional induction of genes to increased stability of transcripts to posttranslational modification of important biosynthetic proteins within the stressed tissue. Superimposed on these largely autonomous effects are nonautonomous responses in which the stressed tissue secretes peptides and other factors that stimulate tissues in different organs to embark on processes that ultimately help the organism as a whole cope with stress. This review focuses on the mechanisms by which tissues in one organ adapt to environmental challenges by regulating stress responses in tissues of different organs.

Summary Mitochondrial dysfunction is usually associated with aging. To systematically characterize the compensatory stress signaling cascades triggered in response to muscle mitochondrial perturbation, we analyzed a Drosophila model of muscle mitochondrial injury. We find that mild muscle mitochondrial distress preserves mitochondrial function, impedes the age-dependent deterioration of muscle function and architecture, and prolongs lifespan. Strikingly, this effect is mediated by at least two prolongevity compensatory signaling modules: one involving a muscle-restricted redox-dependent induction of genes that regulate the mitochondrial unfolded protein response (UPR mt ) and another involving the transcriptional induction of the Drosophila ortholog of insulin-like growth factor-binding protein 7, which systemically antagonizes insulin signaling and facilitates mitophagy. Given that several secreted IGF-binding proteins (IGFBPs) exist in mammals, our work raises the possibility that muscle mitochondrial injury in humans may similarly result in the secretion of IGFBPs, with important ramifications for diseases associated with aberrant insulin signaling.

SummaryThe binding of Hedgehog (Hh) to its receptor Patched causes derepression of Smoothened (Smo), resulting in the activation of the Hh pathway. Here, we show that Smo activation is dependent on the levels of the phospholipid phosphatidylinositol-4 phosphate (PI4P). Loss of STT4 kinase, which is required for the generation of PI4P, exhibits hh loss-of-function phenotypes, whereas loss of Sac1 phosphatase, which is required for the degradation of PI4P, results in hh gain-of-function phenotypes in multiple settings during Drosophila development. Furthermore, loss of Ptc function, which results in the activation of Hh pathway, also causes an increase in PI4P levels. Sac1 functions downstream of STT4 and Ptc in the regulation of Smo membrane localization and Hh pathway activation. Taken together, our results suggest a model in which Ptc directly or indirectly functions to suppress the accumulation of PI4P. Binding of Hh to Ptc derepresses the levels of PI4P, which, in turn, promotes Smo activation.

Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS(2). The functional significance of this difference in ROS level in the two progenitor types remains unresolved. Here we show that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. We conclude that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. Our results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response.

We conducted a screen for glossy-eye flies that fail to incorporate BrdU in the third larval instar eye disc but exhibit normal neuronal differentiation and isolated 23 complementation groups of mutants. These same phenotypes were previously seen in mutants for cytochrome c oxidase subunit Va. We have molecularly characterized six complementation groups and, surprisingly, each encodes a mitochondrial protein. Therefore, we believe our screen to be an efficient method for identifying genes with mitochondrial function.

SummaryThe precise control of the cell cycle requires regulation by many intrinsic and extrinsic factors. Whether the metabolic status of the cell exerts a direct control over cell cycle checkpoints is not well understood. We isolated a mutation, tenured (tend), in a gene encoding cytochrome oxidase subunit Va. This mutation causes a drop in intracellular ATP to levels sufficient to maintain cell survival, growth, and differentiation, but not to enable progression through the cell cycle. Analysis of this gene in vivo and in cell lines shows that a specific pathway involving AMPK and p53 is activated that causes elimination of Cyclin E, resulting in cell cycle arrest. We demonstrate that in multiple tissues the mitochondrion has a direct and specific role in enforcing a G1-S cell cycle checkpoint during periods of energy deprivation.