a-rapid-protocol-for-integrating-extrachromosomal-arrays-with-high

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the <em>C. elegans</em> Genome

Microinjecting DNA into the cytoplasm of the syncytial gonad of Caenorhabditis elegans is the main technique used to establish transgenic lines that ...

Université Claude Bernard Lyon

CNRS UMR 5534

Thyroid status is crucial in energy homeostasis, but despite extensive studies the actual mechanism by which it regulates mitochondrial respiration and ATP synthesis is still unclear. We studied oxidative phosphorylation in both intact liver cells and isolated mitochondria from in vivo models of severe not life threatening hyper- and hypothyroidism. Thyroid status correlated with cellular and mitochondrial oxygen consumption rates as well as with maximal mitochondrial ATP production. Addition of a protonophoric uncoupler, 2,4-dinitrophenol, to hepatocytes did not mimic the cellular energetic change linked to hyperthyroidism. Mitochondrial content of cytochrome oxidase, ATP synthase, phosphate and adenine nucleotide carriers were increased in hyperthyroidism and decreased in hypothyroidism as compared to controls. As a result of these complex changes, the maximal rate of ATP synthesis increased in hyperthyroidism despite a decrease in ATP/O ratio, while in hypothyroidism ATP/O ratio increased but did not compensate for the flux limitation of oxidative phosphorylation. We conclude that energy homeostasis depends on a compromise between rate and efficiency, which is mainly regulated by thyroid hormones.

Thyroid status is a key regulator of both flux and efficiency of oxidative phosphorylation in rat hepatocytes.

The permeability transition pore (PTP) is a mitochondrial inner membrane Ca2+-sensitive channel that plays a key role in different models of cell death. In a series of recent studies we have shown that the PTP is modulated by quinones, and we have identified three functional classes: (i) PTP inhibitors; (ii) PTP inducers; and (iii) PTP-inactive quinones that compete with both inhibitors and inducers. Here, we review our current understanding of pore regulation by quinones, and present the results obtained with a new series of structural variants. Based on the effects of the compounds studied so far, we confirm that minor structural changes profoundly modify the effects of quinones on the PTP. On the other hand, quinones with very different structural features may have qualitatively similar effects on the PTP. Taken together, these results support our original proposal that quinones affect the PTP through a common binding site whose occupancy modulates its open-closed transitions, possibly through secondary changes of the Ca2+-binding affinity.

Regulation of the mitochondrial permeability transition pore by ubiquinone analogs. A progress report.

The permeability transition pore (PTP) is a Ca(2+)-sensitive mitochondrial inner membrane channel involved in several models of cell death. Because the matrix concentration of PTP regulatory factors depends on matrix volume, we have investigated the role of the mitochondrial volume in PTP regulation. By incubating rat liver mitochondria in media of different osmolarity, we found that the Ca(2+) threshold required for PTP opening dramatically increased when mitochondrial volume decreased relative to the standard condition. This shrinkage-induced PTP inhibition was not related to the observed changes in protonmotive force, or pyridine nucleotide redox state and persisted when mitochondria were depleted of adenine nucleotides. On the other hand, mitochondrial volume did not affect PTP regulation when mitochondria were depleted of Mg(2+). By studying the effects of Mg(2+), cyclosporin A (CsA) and ubiquinone 0 (Ub(0)) on PTP regulation, we found that mitochondrial shrinkage increased the efficacy of Mg(2+) and Ub(0) at PTP inhibition, whereas it decreased that of CsA. The ability of mitochondrial volume to alter the activity of several PTP regulators represents a hitherto unrecognized characteristic of the pore that might lead to a new approach for its pharmacological modulation.

Effects of decreasing mitochondrial volume on the regulation of the permeability transition pore.

The fundamental contribution of the mitochondria and ER to the decision made on the cell's fate has been increasingly recognized. This progress has illuminated the need for the mechanisms these organelles use to initiate and to propagate apoptotic signals. The toolbox of the mitochondria and ER is evolutionary conserved, overlapping and complementary. Furthermore, mitochondria are often closely associated with the ER providing the conditions for a local and privileged communication between the two organelles. The present review is concerned with the spatially and temporally coordinated utilization of Bcl-2 family proteins and Ca(2+) by the mitochondria and ER to control the membrane permeabilization in the mitochondria and to regulate Ca(2+) distribution and the activity of apoptotic proteins in the ER. The apoptotic means of the mitochondria and ER will eventually come together to control the dismantling of the cell by the caspases and other enzymes.

Mitochondria and endoplasmic reticulum: the lethal interorganelle cross-talk.

The role of mitochondria in cell metabolism and survival is controlled by calcium signals that are commonly transmitted at the close associations between mitochondria and endoplasmic reticulum (ER). However, the physical linkage of the ER-mitochondria interface and its relevance for cell function remains elusive. We show by electron tomography that ER and mitochondria are adjoined by tethers that are approximately 10 nm at the smooth ER and approximately 25 nm at the rough ER. Limited proteolysis separates ER from mitochondria, whereas expression of a short "synthetic linker" (<5 nm) leads to tightening of the associations. Although normal connections are necessary and sufficient for proper propagation of ER-derived calcium signals to the mitochondria, tightened connections, synthetic or naturally observed under apoptosis-inducing conditions, make mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected dependence of cell function and survival on the maintenance of proper spacing between the ER and mitochondria.

Structural and functional features and significance of the physical linkage between ER and mitochondria.

The BH3-only BID protein (BH3-interacting domain death agonist) has a critical function in the death-receptor pathway in the liver by triggering mitochondrial outer membrane permeabilization (MOMP). Here we show that MTCH2/MIMP (mitochondrial carrier homologue 2/Met-induced mitochondrial protein), a novel truncated BID (tBID)-interacting protein, is a surface-exposed outer mitochondrial membrane protein that facilitates the recruitment of tBID to mitochondria. Knockout of MTCH2/MIMP in embryonic stem cells and in mouse embryonic fibroblasts hinders the recruitment of tBID to mitochondria, the activation of Bax/Bak, MOMP, and apoptosis. Moreover, conditional knockout of MTCH2/MIMP in the liver decreases the sensitivity of mice to Fas-induced hepatocellular apoptosis and prevents the recruitment of tBID to liver mitochondria both in vivo and in vitro. In contrast, MTCH2/MIMP deletion had no effect on apoptosis induced by other pro-apoptotic Bcl-2 family members and no detectable effect on the outer membrane lipid composition. These loss-of-function models indicate that MTCH2/MIMP has a critical function in liver apoptosis by regulating the recruitment of tBID to mitochondria.

MTCH2/MIMP is a major facilitator of tBID recruitment to mitochondria.

Prolonged opening of the mitochondrial permeability transition pore (PTP) leads to cell death. Various ubiquinone analogs have been shown to regulate PTP opening but the outcome of PTP regulation by ubiquinone analogs on cell fate has not been studied yet.

Ubiquinone analogs: a mitochondrial permeability transition pore-dependent pathway to selective cell death.

Here, we report that inactivation of the Caenorhabditis elegans dynamin-related protein DRP-1, a key component responsible for mitochondrial fission and conserved from yeast to humans, dramatically enhanced the effect of reduced insulin signaling (IIS) to extend lifespan. This represents the first report of a beneficial impact of manipulating mitochondrial dynamics on animal lifespan and suggests that mitochondrial morphology and IIS cooperate to modulate aging.

The dynamin-related protein DRP-1 and the insulin signaling pathway cooperate to modulate Caenorhabditis elegans longevity.

Recent findings indicate that perturbations of the mitochondrial electron transport chain (METC) can cause extended longevity in evolutionarily diverse organisms. To uncover the molecular basis of how altered METC increases lifespan in C. elegans, we performed an RNAi screen and revealed that three predicted transcription factors are specifically required for the extended longevity of mitochondrial mutants. In particular, we demonstrated that the nuclear homeobox protein CEH-23 uniquely mediates the longevity but not the slow development, reduced brood size, or resistance to oxidative stress associated with mitochondrial mutations. Furthermore, we showed that ceh-23 expression levels are responsive to altered METC, and enforced overexpression of ceh-23 is sufficient to extend lifespan in wild-type background. Our data point to mitochondria-to-nucleus communications to be key for longevity determination and highlight CEH-23 as a novel longevity factor capable of responding to mitochondrial perturbations. These findings provide a new paradigm for how mitochondria impact aging and age-dependent diseases.

The homeobox protein CEH-23 mediates prolonged longevity in response to impaired mitochondrial electron transport chain in C. elegans.

Duchenne muscular dystrophy (DMD) is a neuromuscular disease caused by mutations in the dystrophin gene. The subcellular mechanisms of DMD remain poorly understood and there is currently no curative treatment available. Using a Caenorhabditis elegans model for DMD as a pharmacologic and genetic tool, we found that cyclosporine A (CsA) reduces muscle degeneration at low dose and acts, at least in part, through a mitochondrial cyclophilin D, CYN-1. We thus hypothesized that CsA acts on mitochondrial permeability modulation through cyclophilin D inhibition. Mitochondrial patterns and dynamics were analyzed, which revealed dramatic mitochondrial fragmentation not only in dystrophic nematodes, but also in a zebrafish model for DMD. This abnormal mitochondrial fragmentation occurs before any obvious sign of degeneration can be detected. Moreover, we demonstrate that blocking/delaying mitochondrial fragmentation by knocking down the fission-promoting gene drp-1 reduces muscle degeneration and improves locomotion abilities of dystrophic nematodes. Further experiments revealed that cytochrome c is involved in muscle degeneration in C. elegans and seems to act, at least in part, through an interaction with the inositol trisphosphate receptor calcium channel, ITR-1. Altogether, our findings reveal that mitochondria play a key role in the early process of muscle degeneration and may be a target of choice for the design of novel therapeutics for DMD. In addition, our results provide the first indication in the nematode that (i) mitochondrial permeability transition can occur and (ii) cytochrome c can act in cell death.

Ludivine Walter

Université Claude Bernard Lyon

A Rapid Protocol for Integrating Extrachromosomal Arrays With High Transmission Rate into the C. elegans Genome