Steven Zuryn

A Strategy for Direct Mapping and Identification of Mutations by Whole-Genome Sequencing

Mutant screens have proven powerful for genetic dissection of a myriad of biological processes, but subsequent identification and isolation of the causative mutations are usually complex and time consuming. We have made the process easier by establishing a novel strategy that employs whole-genome sequencing to simultaneously map and identify mutations without the need for any prior genetic mapping.

A Core Metabolic Enzyme Mediates Resistance to Phosphine Gas

Dissecting Phosphine Resistance Worldwide populations of pest insects—such as the lesser grain borer, Rhyzopertha dominica, and the rust-red flour beetle, Tribolium castaneum—have become highly resistant to the fumigant phosphine, providing a potential threat to global food security. The nematode, Caenorhabditis elegans is vulnerable to phosphine, but phosphine-resistant strains are known. Schlipalius et al. (p. 807) show that mutations in the delta-1-pyrroline-5-carboxylate dehydrogenase and dihydrolipoamide dehydrogenase (dld-1) genes both give rise to phosphine resistance in C. elegans. Phosphine resistance mutants in R. dominica, and T. castaneum also map to the dld-1 gene, which codes for a core metabolic enzyme. These mutants are, however, hypersensitive to arsenic, mimics of which might thus synergize with phosphine. Mutations in a lipoic acid metabolism enzyme confer resistance to phosphine but also result in sensitivity to arsenite. Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.

Phosphine is the most widely used fumigant for the protection of stored commodities against insect pests, especially food products such as grain. However, pest insects are developing resistance to phosphine and thereby threatening its future use. As phosphine inhibits cytochrome c oxidase (complex IV) of the mitochondrial respiratory chain and reduces the strength of the mitochondrial membrane potential (DeltaPsi(m)), we reasoned that mitochondrial uncouplers should act synergistically with phosphine. The mitochondrial uncouplers FCCP and PCP caused complete mortality in populations of both wild-type and phosphine-resistant lines of Caenorhabditis elegans simultaneously exposed to uncoupler and phosphine at concentrations that were individually nonlethal. Strong synergism was also observed with a third uncoupler DNP. We have also tested an alternative complex IV inhibitor, azide, with FCCP and found that this also caused a synergistic enhancement of toxicity in C. elegans. To investigate potential causes of the synergism, we measured DeltaPsi(m), ATP content, and oxidative damage (lipid hydroperoxides) in nematodes subjected to phosphine-FCCP treatment and found that neither an observed 50% depletion in ATP nor oxidative stress accounted for the synergistic effect. Instead, a synergistic reduction in DeltaPsi(m) was observed upon phosphine-FCCP co-treatment suggesting that this is directly responsible for the subsequent mortality. These results support the hypothesis that phosphine-induced mortality results from the in vivo disruption of normal mitochondrial activity. Furthermore, we have identified a novel pathway that can be targeted to overcome genetic resistance to phosphine.

Sequential histone-modifying activities determine the robustness of transdifferentiation

Epigenetics direct transdifferentiation To make an entire animal, many and varied cell types form and interact. Some of these differentiated cells take a U-turn and can de-differentiate or transdifferentiate to another cell fate. Although relatively rare in nature, Zuryn et al. followed such a program in the tiny roundworm Caenorhabditis elegans, where a rectal cell–to–motor neuron conversion is seen. Transcription factors with conserved roles in cell plasticity and terminal fate selection partner up with specific histone-modifying enzymes in discrete steps to specify separate sequential phases of cell identity. Science, this issue p. 826 Stepwise modifications to histones ensure efficient and reliable cell conversions in Caenorhabditis elegans. Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.

Mitochondrial dysfunction in Caenorhabditis elegans causes metabolic restructuring, but this is not linked to longevity

Lifespan in Caenorhabditis elegans, Drosophila, and mice can be extended by a decrease in mitochondrial electron transport chain (ETC) function, but the mechanism behind this extension is unknown. In the present study, we combine detailed metabolic analysis with lifespan determination following suppression of individual genes encoding respiratory complexes I-IV. We report that reduced complexes I, III, and IV activity extend lifespan but that complex II disruption does not. However, disruption to all four complexes affected metabolism in a similar manner suggesting that metabolic effects induced by ETC disruption are separable from lifespan extension. We found that suppression of ETC components induces a starvation-like metabolic response via the nuclear hormone receptor NHR-49. This includes induction of genes for mitochondrial fatty-acid β-oxidation (acs-2), the glyoxylate cycle (gei-7), gluconeogensis (PEPCK), and glycolysis (gpd-3). Interestingly, a null mutation of nhr-49 attenuated induction of these metabolic pathways, but did not affect the lifespan extension associated with decreases in complexes I, III, and IV function. Together, our results suggest that restructuring of metabolism via NHR-49 in C. elegans with mitochondrial dysfunction does not cause lifespan extension.

Deep sequencing strategies for mapping and identifying mutations from genetic screens

The development of next-generation sequencing technologies has enabled rapid and cost effective whole genome sequencing. This technology has allowed researchers to shortcut time-consuming and laborious methods used to identify nucleotide mutations in forward genetic screens in model organisms. However, causal mutations must still be mapped to a region of the genome so as to aid in their identification. This can be achieved simultaneously with deep sequencing through various methods. Here we discuss alternative deep sequencing strategies for simultaneously mapping and identifying causal mutations in Caenorhabditis elegans from mutagenesis screens. Focusing on practical considerations, such as the particular mutant phenotype obtained, this review aims to aid the reader in choosing which strategy to adopt to successfully clone their mutant.

Laser Capture Microdissection and Multiplex-Tandem PCR Analysis of Proximal Tubular Epithelial Cell Signaling in Human Kidney Disease

Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate “real time” gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.

Direct cellular reprogramming in Caenorhabditis elegans: facts, models, and promises for regenerative medicine

In vitro systems of cellular reprogramming [induced pluripotent stem (iPS) cells and direct reprogramming or transdifferentiation] are rapidly improving our repertoire of molecular techniques that can force cells in culture to change into a desired identity. However, the new frontier for regenerative medicine is in vivo cellular reprogramming, which in light of concerns about the safety of in vitro cell manipulations, is an increasingly attractive approach for regenerative medicine. Powerful in vivo approaches are currently being undertaken in the genetic model Caenorhabditis elegans. Several very distinct cell types have been induced to change or have been discovered to transform naturally, into altogether different cell types. These examples have improved our understanding of the fundamental molecular and cellular mechanisms that permit cell identity changes in live animals. In addition, the combination of a stereotyped lineage with single cell analyses allows dissection of the early and intermediate mechanisms of reprogramming, as well as their kinetics. As a result, several important concepts on in vivo cellular reprogramming have been recently developed. WIREs Dev Biol 2012, 1:138–152. doi: 10.1002/wdev.7

Alternative assembly of respiratory complex II connects energy stress to metabolic checkpoints

Cell growth and survival depend on a delicate balance between energy production and synthesis of metabolites. Here, we provide evidence that an alternative mitochondrial complex II (CII) assembly, designated as CIIlow, serves as a checkpoint for metabolite biosynthesis under bioenergetic stress, with cells suppressing their energy utilization by modulating DNA synthesis and cell cycle progression. Depletion of CIIlow leads to an imbalance in energy utilization and metabolite synthesis, as evidenced by recovery of the de novo pyrimidine pathway and unlocking cell cycle arrest from the S-phase. In vitro experiments are further corroborated by analysis of paraganglioma tissues from patients with sporadic, SDHA and SDHB mutations. These findings suggest that CIIlow is a core complex inside mitochondria that provides homeostatic control of cellular metabolism depending on the availability of energy.Mitochondrial complex II is normally composed of four subunits. Here the authors show that bioenergetic stress conditions give rise to a partially assembled variant of complex II, which shifts the anabolic pathways to less energy demanding processes.

Affinity purification of cell-specific mitochondria from whole animals resolves patterns of genetic mosaicism

Although mitochondria are ubiquitous organelles, they exhibit tissue-specific morphology, dynamics and function. Here, we describe a robust approach to isolate mitochondria from specific cells of diverse tissue systems in Caenorhabditis elegans. Cell-specific mitochondrial affinity purification (CS-MAP) yields intact and functional mitochondria with exceptional purity and sensitivity (>96% enrichment, >96% purity, and single-cell and single-animal resolution), enabling comparative analyses of protein and nucleic acid composition between organelles isolated from distinct cellular lineages. In animals harbouring a mixture of mutant and wild-type mitochondrial genomes, we use CS-MAP to reveal subtle mosaic patterns of cell-type-specific heteroplasmy across large populations of animals (>10,000 individuals). We demonstrate that the germline is more prone to propagating deleterious mitochondrial genomes than somatic lineages, which we propose is caused by enhanced mtDNA replication in this tissue.Ahier et al. describe a method to isolate intact mitochondria from specific cells in Caenorhabditis elegans and show that the germline is more prone to propagating deleterious mitochondrial genomes than somatic lineages.