As a graduate student and short-term postdoctoral fellow at the University of Toronto I studied genetic networks that regulate cell viability in the nematode worm Caenorhabditis elegans (C. elegans) and in the single-celled eukaryotes S. cerevisiae and S. pombe, respectively. As a postdoctoral fellow, I demonstrated that the small molecule erastin inhibits the membrane cystine/glutamate transporter system xc-, depletes the cell of glutathione and activates a novel iron-dependent, oxidative cell death pathway termed ferroptosis. Currently a major goal of my lab is to understand the interaction between intracellular metabolism and cell death. Our research program integrates techniques and model systems including small molecule and proteomic screening, biochemical analysis of protein function and model organism genetics.
B.Sc., Laurentian University, Behavioral Neuroscience (2000)
Ph.D., University of Toronto, Molecular and Medical Genetics (2007)
Current Research and Scholarly Interests
My lab is interested in the relationship between cell death and metabolism. Using techniques drawn from many disciplines my laboratory is investigating how perturbation of intracellular metabolic networks can result in novel forms of cell death, such as ferroptosis. We are interested in applying this knowledge to find new ways to treat diseases characterized by insufficient (e.g. cancer) or excessive (e.g. neurodegeneration) cell death.
- Biochemistry & Molecular Biology
BIO 83 (Win)
- Frontiers in Biology
BIO 301 (Aut, Win, Spr)
Independent Studies (8)
- Advanced Research Laboratory in Experimental Biology
BIO 199 (Aut, Win, Spr)
- Directed Reading in Biology
BIO 198 (Aut, Win, Spr)
- Directed Reading in Cancer Biology
CBIO 299 (Win, Spr)
- Graduate Research
BIO 300 (Aut, Win, Spr, Sum)
- Graduate Research
CBIO 399 (Aut, Win, Spr, Sum)
- Out-of-Department Advanced Research Laboratory in Experimental Biology
BIO 199X (Win, Spr)
- Teaching in Cancer Biology
CBIO 260 (Spr)
- Teaching of Biology
BIO 290 (Win)
- Advanced Research Laboratory in Experimental Biology
- Prior Year Courses
Graduate and Fellowship Programs
Biology (School of Humanities and Sciences) (Phd Program)
GPX4 at the Crossroads of Lipid Homeostasis and Ferroptosis.
Oxygen is necessary for aerobic metabolism but can also contribute to the harmful oxidation of lipids and other macromolecules. Oxidation of cholesterol and of phospholipids containing polyunsaturated fatty acyl chains can lead to lipid peroxidation, membrane damage and cell death. Lipid hydroperoxides are key intermediates in the process of lipid peroxidation. The lipid hydroperoxidase glutathione peroxidase 4 (GPX4) converts lipid hydroperoxides to lipid alcohols, and this process prevents the iron (Fe2+ )-dependent formation of toxic lipid reactive oxygen species (ROS). Inhibition of GPX4 function leads to increased lipid ROS formation and lipid peroxidation, and can result in the induction of ferroptosis, an iron-dependent, non-apoptotic form of cell death. In this review we describe the formation of reactive lipid species, the function of GPX4 in preventing oxidative lipid damage, and the link between GPX4 dysfunction, lipid oxidation and the induction of ferroptosis. This article is protected by copyright. All rights reserved.
View details for PubMedID 30888116
Ferroptosis and Brain Injury.
Ferroptosis is a nonapoptotic form of cell death characterized by the iron-dependent accumulation of toxic lipid reactive oxygen species. Small-molecule screening and subsequent optimization have yielded potent and specific activators and inhibitors of this process. These compounds have been employed to dissect the lethal mechanism and implicate this process in pathological cell death events observed in many tissues, including the brain. Indeed, ferroptosis is emerging as an important mechanism of cell death during stroke, intracerebral hemorrhage, and other acute brain injuries, and may also play a role in certain degenerative brain disorders. Outstanding issues include the practical need to identify molecular markers of ferroptosis that can be used to detect and study this process in vivo, and the more basic problem of understanding the relationship between ferroptosis and other forms of cell death that can be triggered in the brain during injury.
View details for PubMedID 30820017
A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity.
2019; 26 (6): 1544
The tripeptide glutathione suppresses the iron-dependent, non-apoptotic cell death process of ferroptosis. How glutathione abundance is regulated in the cell and how this regulation alters ferroptosis sensitivity is poorly understood. Using genome-wide human haploid genetic screening technology coupled to fluorescence-activated cell sorting (FACS), we directly identify genes that regulate intracellular glutathione abundance and characterize their role in ferroptosis regulation. Disruption of the ATP binding cassette (ABC)-family transporter multidrug resistance protein 1 (MRP1) prevents glutathione efflux from the cell and strongly inhibits ferroptosis. High levels of MRP1 expression decrease sensitivity to certain pro-apoptotic chemotherapeutic drugs, while collaterally sensitizing to all tested pro-ferroptotic agents. By contrast, disruption of KEAP1 and NAA38, leading to the stabilization of the transcription factor NRF2, increases glutathione levels but only weakly protects from ferroptosis. This is due in part to concomitant NRF2-mediated upregulation of MRP1. These results pinpoint glutathione efflux as an unanticipated regulator of ferroptosis sensitivity.
View details for PubMedID 30726737
Exogenous Monounsaturated Fatty Acids Promote a Ferroptosis-Resistant Cell State.
Cell chemical biology
The initiation and execution of cell death can be regulated by various lipids. How the levels of environmental (exogenous) lipids impact cell death sensitivity is not well understood. We find that exogenous monounsaturated fatty acids (MUFAs) potently inhibit the non-apoptotic, iron-dependent, oxidative cell death process of ferroptosis. This protective effect is associated with the suppression of lipid reactive oxygen species (ROS) accumulation at the plasma membrane and decreased levels of phospholipids containing oxidizable polyunsaturated fatty acids. Treatment with exogenous MUFAs reduces the sensitivity of plasma membrane lipids to oxidation over several hours. This effect requires MUFA activation by acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) and is independent of lipid droplet formation. Exogenous MUFAs also protect cells from apoptotic lipotoxicity caused by the accumulation of saturated fatty acids, but in anACSL3-independent manner. Our work demonstrates that ACSL3-dependent MUFA activation promotes a ferroptosis-resistant cell state.
View details for PubMedID 30686757
The p53-p21 pathway inhibits ferroptosis during metabolic stress.
2018; 9 (37): 24572–73
View details for PubMedID 29872487
Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018
CELL DEATH AND DIFFERENTIATION
2018; 25 (3): 486–541
Over the past decade, the Nomenclature Committee on Cell Death (NCCD) has formulated guidelines for the definition and interpretation of cell death from morphological, biochemical, and functional perspectives. Since the field continues to expand and novel mechanisms that orchestrate multiple cell death pathways are unveiled, we propose an updated classification of cell death subroutines focusing on mechanistic and essential (as opposed to correlative and dispensable) aspects of the process. As we provide molecularly oriented definitions of terms including intrinsic apoptosis, extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis, necroptosis, ferroptosis, pyroptosis, parthanatos, entotic cell death, NETotic cell death, lysosome-dependent cell death, autophagy-dependent cell death, immunogenic cell death, cellular senescence, and mitotic catastrophe, we discuss the utility of neologisms that refer to highly specialized instances of these processes. The mission of the NCCD is to provide a widely accepted nomenclature on cell death in support of the continued development of the field.
View details for PubMedID 29362479
p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells
2018; 22 (3): 569–75
How cancer cells respond to nutrient deprivation remains poorly understood. In certain cancer cells, deprivation of cystine induces a non-apoptotic, iron-dependent form of cell death termed ferroptosis. Recent evidence suggests that ferroptosis sensitivity may be modulated by the stress-responsive transcription factor and canonical tumor suppressor protein p53. Using CRISPR/Cas9 genome editing, small-molecule probes, and high-resolution, time-lapse imaging, we find that stabilization of wild-type p53 delays the onset of ferroptosis in response to cystine deprivation. This delay requires the p53 transcriptional target CDKN1A (encoding p21) and is associated with both slower depletion of intracellular glutathione and a reduced accumulation of toxic lipid-reactive oxygen species (ROS). Thus, the p53-p21 axis may help cancer cells cope with metabolic stress induced by cystine deprivation by delaying the onset of non-apoptotic cell death.
View details for PubMedID 29346757
MLKL Requires the Inositol Phosphate Code to Execute Necroptosis.
2018; 70 (5): 936–48.e7
Necroptosis is an important form of lytic cell death triggered by injury and infection, but whether mixed lineage kinase domain-like (MLKL) is sufficient to execute this pathway is unknown. In a genetic selection for human cell mutants defective for MLKL-dependent necroptosis, we identified mutations in IPMK and ITPK1, which encode inositol phosphate (IP) kinases that regulate the IP code of soluble molecules. We show that IP kinases are essential for necroptosis triggered by death receptor activation, herpesvirus infection, or a pro-necrotic MLKL mutant. In IP kinase mutant cells, MLKL failed to oligomerize and localize to membranes despite proper receptor-interacting protein kinase-3 (RIPK3)-dependent phosphorylation. We demonstrate that necroptosis requires IP-specific kinase activity and that a highly phosphorylated product, but not a lowly phosphorylated precursor, potently displaces the MLKL auto-inhibitory brace region. These observations reveal control of MLKL-mediated necroptosis by a metabolite and identify a key molecular mechanism underlying regulated cell death.
View details for PubMedID 29883610
The impact of non-genetic heterogeneity on cancer cell death
CRITICAL REVIEWS IN BIOCHEMISTRY AND MOLECULAR BIOLOGY
2018; 53 (1): 99–114
The goal of cancer chemotherapy is to induce homogeneous cell death within the population of targeted cancer cells. However, no two cells are exactly alike at the molecular level, and sensitivity to drug-induced cell death, therefore, varies within a population. Genetic alterations can contribute to this variability and lead to selection for drug resistant clones. However, there is a growing appreciation for the role of non-genetic variation in producing drug-tolerant cellular states that exhibit reduced sensitivity to cell death for extended periods of time, from hours to weeks. These cellular states may result from individual variation in epigenetics, gene expression, metabolism, and other processes that impact drug mechanism of action or the execution of cell death. Such population-level non-genetic heterogeneity may contribute to treatment failure and provide a cellular "substrate" for the emergence of genetic alterations that confer frank drug resistance.
View details for PubMedID 29250983
Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease
2017; 171 (2): 273–85
Ferroptosis is a form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides to lethal levels. Emerging evidence suggests that ferroptosis represents an ancient vulnerability caused by the incorporation of polyunsaturated fatty acids into cellular membranes, and cells have developed complex systems that exploit and defend against this vulnerability in different contexts. The sensitivity to ferroptosis is tightly linked to numerous biological processes, including amino acid, iron, and polyunsaturated fatty acid metabolism, and the biosynthesis of glutathione, phospholipids, NADPH, and coenzyme Q10. Ferroptosis has been implicated in the pathological cell death associated with degenerative diseases (i.e., Alzheimer's, Huntington's, and Parkinson's diseases), carcinogenesis, stroke, intracerebral hemorrhage, traumatic brain injury, ischemia-reperfusion injury, and kidney degeneration in mammals and is also implicated in heat stress in plants. Ferroptosis may also have a tumor-suppressor function that could be harnessed for cancer therapy. This Primer reviews the mechanisms underlying ferroptosis, highlights connections to other areas of biology and medicine, and recommends tools and guidelines for studying this emerging form of regulated cell death.
View details for PubMedID 28985560
View details for PubMedCentralID PMC5685180
Ferroptosis: bug or feature?
2017; 277 (1): 150-157
Ferroptosis is an iron-dependent, oxidative form of non-apoptotic cell death. This form of cell death does not share morphological, biochemical, or genetic similarities with classic necrosis, necroptosis, parthanatos, or other forms of non-apoptotic cell death. Ferroptosis can be triggered by depleting the cell of the amino acid cysteine, or by inhibiting the phospholipid hydroperoxidase glutathione peroxidase 4 (GPX4). Why certain stimuli trigger ferroptosis instead of another form of cell death, and whether this process could be adaptive in vivo, are two major unanswered questions concerning this process. Emerging evidence and consideration of related non-apoptotic pathways suggest that ferroptosis could be an adaptive process, albeit one regulated and executed in a manner very different from apoptosis and other forms of cell death.
View details for DOI 10.1111/imr.12533
View details for Web of Science ID 000400377200011
View details for PubMedID 28462529
Heat stress induces ferroptosis-like cell death in plants.
journal of cell biology
2017; 216 (2): 463-476
In plants, regulated cell death (RCD) plays critical roles during development and is essential for plant-specific responses to abiotic and biotic stresses. Ferroptosis is an iron-dependent, oxidative, nonapoptotic form of cell death recently described in animal cells. In animal cells, this process can be triggered by depletion of glutathione (GSH) and accumulation of lipid reactive oxygen species (ROS). We investigated whether a similar process could be relevant to cell death in plants. Remarkably, heat shock (HS)-induced RCD, but not reproductive or vascular development, was found to involve a ferroptosis-like cell death process. In root cells, HS triggered an iron-dependent cell death pathway that was characterized by depletion of GSH and ascorbic acid and accumulation of cytosolic and lipid ROS. These results suggest a physiological role for this lethal pathway in response to heat stress in Arabidopsis thaliana The similarity of ferroptosis in animal cells and ferroptosis-like death in plants suggests that oxidative, iron-dependent cell death programs may be evolutionarily ancient.
View details for DOI 10.1083/jcb.201605110
View details for PubMedID 28100685
View details for PubMedCentralID PMC5294777
- Nanomedicine: An iron age for cancer therapy. Nature nanotechnology 2016; 11 (11): 921-922
Global survey of cell death mechanisms reveals metabolic regulation of ferroptosis
NATURE CHEMICAL BIOLOGY
2016; 12 (7): 497-?
Apoptosis is one type of programmed cell death. Increasingly, non-apoptotic cell death is recognized as being genetically controlled, or 'regulated'. However, the full extent and diversity of alternative cell death mechanisms remain uncharted. Here we surveyed the landscape of pharmacologically accessible cell death mechanisms. In an examination of 56 caspase-independent lethal compounds, modulatory profiling showed that 10 compounds induced three different types of regulated non-apoptotic cell death. Optimization of one of those ten resulted in the discovery of FIN56, a specific inducer of ferroptosis. Ferroptosis has been found to occur when the lipid-repair enzyme GPX4 is inhibited. FIN56 promoted degradation of GPX4. FIN56 also bound to and activated squalene synthase, an enzyme involved in isoprenoid biosynthesis, independent of GPX4 degradation. These discoveries show that dysregulation of lipid metabolism is associated with ferroptosis. This systematic approach is a means to discover and characterize novel cell death phenotypes.
View details for DOI 10.1038/NCHEMBIO.2079
View details for Web of Science ID 000378087500008
View details for PubMedID 27159577
Mechanisms of ferroptosis
CELLULAR AND MOLECULAR LIFE SCIENCES
2016; 73 (11-12): 2195-2209
Ferroptosis is a non-apoptotic form of cell death that can be triggered by small molecules or conditions that inhibit glutathione biosynthesis or the glutathione-dependent antioxidant enzyme glutathione peroxidase 4 (GPX4). This lethal process is defined by the iron-dependent accumulation of lipid reactive oxygen species and depletion of plasma membrane polyunsaturated fatty acids. Cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression may be sensitized to this process. Conversely, a number of small molecule inhibitors of ferroptosis have been identified, including ferrostatin-1 and liproxstatin-1, which can block pathological cell death events in brain, kidney and other tissues. Recent work has identified a number of genes required for ferroptosis, including those involved in lipid and amino acid metabolism. Outstanding questions include the relationship between ferroptosis and other forms of cell death, and whether activation or inhibition of ferroptosis can be exploited to achieve desirable therapeutic ends.
View details for DOI 10.1007/s00018-016-2194-1
View details for Web of Science ID 000377775800008
View details for PubMedID 27048822
Emerging roles for lipids in non-apoptotic cell death.
Cell death and differentiation
Non-apoptotic regulated cell death (RCD) is essential to maintain organismal homeostasis and may be aberrantly activated during certain pathological states. Lipids are emerging as key components of several non-apoptotic RCD pathways. For example, a direct interaction between membrane phospholipids and the pore-forming protein mixed lineage kinase domain-like (MLKL) is needed for the execution of necroptosis, while the oxidative destruction of membrane polyunsaturated fatty acids (PUFAs), following the inactivation of glutathione peroxidase 4 (GPX4), is a requisite gateway to ferroptosis. Here, we review the roles of lipids in the initiation and execution of these and other forms of non-apoptotic cell death. We also consider new technologies that are allowing for the roles of lipids and lipid metabolism in RCD to be probed in increasingly sophisticated ways. In certain cases, this new knowledge may enable the development of therapies that target lipids and lipid metabolic processes to enhance or suppress specific non-apoptotic RCD pathways.Cell Death and Differentiation advance online publication, 11 March 2016; doi:10.1038/cdd.2016.25.
View details for DOI 10.1038/cdd.2016.25
View details for PubMedID 26967968
Connectivity Homology Enables Inter-Species Network Models of Synthetic Lethality
PLOS COMPUTATIONAL BIOLOGY
2015; 11 (10)
Synthetic lethality is a genetic interaction wherein two otherwise nonessential genes cause cellular inviability when knocked out simultaneously. Drugs can mimic genetic knock-out effects; therefore, our understanding of promiscuous drugs, polypharmacology-related adverse drug reactions, and multi-drug therapies, especially cancer combination therapy, may be informed by a deeper understanding of synthetic lethality. However, the colossal experimental burden in humans necessitates in silico methods to guide the identification of synthetic lethal pairs. Here, we present SINaTRA (Species-INdependent TRAnslation), a network-based methodology that discovers genome-wide synthetic lethality in translation between species. SINaTRA uses connectivity homology, defined as biological connectivity patterns that persist across species, to identify synthetic lethal pairs. Importantly, our approach does not rely on genetic homology or structural and functional similarity, and it significantly outperforms models utilizing these data. We validate SINaTRA by predicting synthetic lethality in S. pombe using S. cerevisiae data, then identify over one million putative human synthetic lethal pairs to guide experimental approaches. We highlight the translational applications of our algorithm for drug discovery by identifying clusters of genes significantly enriched for single- and multi-drug cancer therapies.
View details for DOI 10.1371/journal.pcbi.1004506
View details for Web of Science ID 000364399700046
View details for PubMedID 26451775
Human Haploid Cell Genetics Reveals Roles for Lipid Metabolism Genes in Nonapoptotic Cell Death
ACS CHEMICAL BIOLOGY
2015; 10 (7): 1604-1609
Little is known about the regulation of nonapoptotic cell death. Using massive insertional mutagenesis of haploid KBM7 cells we identified nine genes involved in small-molecule-induced nonapoptotic cell death, including mediators of fatty acid metabolism (ACSL4) and lipid remodeling (LPCAT3) in ferroptosis. One novel compound, CIL56, triggered cell death dependent upon the rate-limiting de novo lipid synthetic enzyme ACC1. These results provide insight into the genetic regulation of cell death and highlight the central role of lipid metabolism in nonapoptotic cell death.
View details for DOI 10.1021/acschembio.5b00245
View details for Web of Science ID 000358395300004
View details for PubMedCentralID PMC4509420
Pharmacological inhibition of cystine-glutamate exchange induces endoplasmic reticulum stress and ferroptosis
Exchange of extracellular cystine for intracellular glutamate by the antiporter system xc (-) is implicated in numerous pathologies. Pharmacological agents that inhibit system xc (-) activity with high potency have long been sought, but have remained elusive. In this study, we report that the small molecule erastin is a potent, selective inhibitor of system xc (-). RNA sequencing revealed that inhibition of cystine-glutamate exchange leads to activation of an ER stress response and upregulation of CHAC1, providing a pharmacodynamic marker for system xc (-) inhibition. We also found that the clinically approved anti-cancer drug sorafenib, but not other kinase inhibitors, inhibits system xc (-) function and can trigger ER stress and ferroptosis. In an analysis of hospital records and adverse event reports, we found that patients treated with sorafenib exhibited unique metabolic and phenotypic alterations compared to patients treated with other kinase-inhibiting drugs. Finally, using a genetic approach, we identified new genes dramatically upregulated in cells resistant to ferroptosis.DOI: http://dx.doi.org/10.7554/eLife.02523.001.
View details for DOI 10.7554/eLife.02523
View details for Web of Science ID 000336253000003
View details for PubMedID 24844246
Ferrostatins Inhibit Oxidative Lipid Damage and Cell Death in Diverse Disease Models
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2014; 136 (12): 4551-4556
Ferrostatin-1 (Fer-1) inhibits ferroptosis, a form of regulated, oxidative, nonapoptotic cell death. We found that Fer-1 inhibited cell death in cellular models of Huntington's disease (HD), periventricular leukomalacia (PVL), and kidney dysfunction; Fer-1 inhibited lipid peroxidation, but not mitochondrial reactive oxygen species formation or lysosomal membrane permeability. We developed a mechanistic model to explain the activity of Fer-1, which guided the development of ferrostatins with improved properties. These studies suggest numerous therapeutic uses for ferrostatins, and that lipid peroxidation mediates diverse disease phenotypes.
View details for DOI 10.1021/ja411006a
View details for Web of Science ID 000333551800021
View details for PubMedID 24592866
- The role of iron and reactive oxygen species in cell death NATURE CHEMICAL BIOLOGY 2014; 10 (1): 9-17
Ferroptosis: An Iron-Dependent Form of Nonapoptotic Cell Death
2012; 149 (5): 1060-1072
Nonapoptotic forms of cell death may facilitate the selective elimination of some tumor cells or be activated in specific pathological states. The oncogenic RAS-selective lethal small molecule erastin triggers a unique iron-dependent form of nonapoptotic cell death that we term ferroptosis. Ferroptosis is dependent upon intracellular iron, but not other metals, and is morphologically, biochemically, and genetically distinct from apoptosis, necrosis, and autophagy. We identify the small molecule ferrostatin-1 as a potent inhibitor of ferroptosis in cancer cells and glutamate-induced cell death in organotypic rat brain slices, suggesting similarities between these two processes. Indeed, erastin, like glutamate, inhibits cystine uptake by the cystine/glutamate antiporter (system x(c)(-)), creating a void in the antioxidant defenses of the cell and ultimately leading to iron-dependent, oxidative death. Thus, activation of ferroptosis results in the nonapoptotic destruction of certain cancer cells, whereas inhibition of this process may protect organisms from neurodegeneration.
View details for DOI 10.1016/j.cell.2012.03.042
View details for Web of Science ID 000304453900012
View details for PubMedID 22632970
- DRUG DISCOVERY Engineering drug combinations NATURE CHEMICAL BIOLOGY 2010; 6 (5): 318-319
Identifying druggable disease-modifying gene products
CURRENT OPINION IN CHEMICAL BIOLOGY
2009; 13 (5-6): 549-555
Many disease genes encode proteins that are difficult to target directly using small molecule drugs. Improvements in libraries based on synthetic compounds, natural products, and other types of molecules may ultimately allow some challenging proteins to be successfully targeted; however, these developments alone are unlikely to be sufficient. A complementary strategy exploits the functional interconnectivity of intracellular networks to find druggable targets lying upstream, downstream, or in parallel to a disease-causing gene, where modulation can influence the disease process indirectly. These targets can be selected using prior knowledge of disease-associated pathways or identified using phenotypic chemical and genetic screens in model organisms and cells. These approaches should facilitate the identification of effective drug targets for many genetic disorders.
View details for DOI 10.1016/j.cbpa.2009.08.003
View details for Web of Science ID 000272984600008
View details for PubMedID 19740696
An UNC-40 pathway directs postsynaptic membrane extension in Caenorhabditis elegans
2009; 136 (6): 911-922
The postsynaptic membrane of the embryonic neuromuscular junction undergoes a dramatic expansion during later development to facilitate the depolarization of larger muscles. In C. elegans, the postsynaptic membrane resides at the termini of plasma membrane extensions called muscle arms. Membrane extension to the motor axons during larval development doubles the number of muscle arms, making them a tractable model to investigate both postsynaptic membrane expansion and guided membrane extension. To identify genes required for muscle arm extension, we performed a forward screen for mutants with fewer muscle arms. We isolated 23 mutations in 14 genes, including unc-40/Dcc, which encodes a transmembrane receptor that guides the migration of cells and extending axons in response to the secreted UNC-6/Netrin spatial cue. We discovered that UNC-40 is enriched at muscle arm termini and functions cell-autonomously to direct arm extension to the motor axons. Surprisingly, UNC-6 is dispensable for muscle arm extension, suggesting that UNC-40 relies on other spatial cues to direct arm extension. We provide the first evidence that the guanine-nucleotide exchange factor UNC-73/Trio, members of the WAVE actin-polymerization complex, and a homolog of the focal adhesion complex can function downstream of UNC-40 to direct membrane extension. Our work is the first to define a pathway for directed muscle membrane extension and illustrates that axon guidance components can play key roles in postsynaptic membrane expansion.
View details for DOI 10.1242/dev.030759
View details for Web of Science ID 000263558100005
View details for PubMedID 19211675
Systematic Mapping of Genetic Interaction Networks
ANNUAL REVIEW OF GENETICS
2009; 43: 601-625
Genetic interactions influencing a phenotype of interest can be identified systematically using libraries of genetic tools that perturb biological systems in a defined manner. Systematic screens conducted in the yeast Saccharomyces cerevisiae have identified thousands of genetic interactions and provided insight into the global structure of biological networks. Techniques enabling systematic genetic interaction mapping have been extended to other single-celled organisms, the bacteria Escherichia coli and the yeast Schizosaccharomyces pombe, opening the way to comparative investigations of interaction networks. Genetic interaction screens in Caenorhabditis elegans, Drosophila melanogaster, and mammalian models are helping to improve our understanding of metazoan-specific signaling pathways. Together, our emerging knowledge of the genetic wiring diagrams of eukaryotic and prokaryotic cells is providing a new understanding of the relationship between genotype and phenotype.
View details for DOI 10.1146/annurev.genet.39.073003.114751
View details for Web of Science ID 000273580300024
View details for PubMedID 19712041
Significant conservation of synthetic lethal genetic interaction networks between distantly related eukaryotes
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
2008; 105 (43): 16653-16658
Synthetic lethal genetic interaction networks define genes that work together to control essential functions and have been studied extensively in Saccharomyces cerevisiae using the synthetic genetic array (SGA) analysis technique (ScSGA). The extent to which synthetic lethal or other genetic interaction networks are conserved between species remains uncertain. To address this question, we compared literature-curated and experimentally derived genetic interaction networks for two distantly related yeasts, Schizosaccharomyces pombe and S. cerevisiae. We find that 23% of interactions in a novel, high-quality S. pombe literature-curated network are conserved in the existing S. cerevisiae network. Next, we developed a method, called S. pombe SGA analysis (SpSGA), enabling rapid, high-throughput isolation of genetic interactions in this species. Direct comparison by SpSGA and ScSGA of approximately 220 genes involved in DNA replication, the DNA damage response, chromatin remodeling, intracellular transport, and other processes revealed that approximately 29% of genetic interactions are common to both species, with the remainder exhibiting unique, species-specific patterns of genetic connectivity. We define a conserved yeast network (CYN) composed of 106 genes and 144 interactions and suggest that this network may help understand the shared biology of diverse eukaryotic species.
View details for DOI 10.1073/pnas.0806261105
View details for Web of Science ID 000260913500046
View details for PubMedID 18931302
Insulin-like signaling negatively regulates muscle arm extension through DAF-12 in Caenorhabditis elegans
2008; 318 (1): 153-161
The body wall muscles (BWMs) of nematodes are connected to motor axons by muscle membrane extensions called muscle arms. To better understand how muscle arm extension is regulated, we screened conserved receptor tyrosine kinases for muscle arm defects in Caenorhabditis elegans. We discovered that mutations in daf-2, which encodes the only insulin-like receptor tyrosine kinase, confer a supernumerary muscle arm (Sna) phenotype. The Sna phenotype of daf-2 mutants is suppressed by loss-of-function in the canonical downstream FOXO-family transcription factor DAF-16 in either the muscles or the intestine, demonstrating that insulin-like signaling can regulate muscle arm extension non-autonomously. Furthermore, supernumerary arm extension requires the B isoform of the down-stream DAF-12 nuclear hormone receptor, which lacks the DNA-binding domain, but retains the ligand-binding domain. daf-2 regulates many processes in C. elegans including entry into dauer, which is a diapause-like state that facilitates survival of harsh environmental conditions. We found that wild-type dauers are also Sna. Unlike other changes associated with dauer, however, the Sna phenotype of dauers persists in recovered adults. Finally, disruption of a TGF-beta pathway that regulates dauer formation in parallel to the insulin-like pathway also confers the Sna phenotype. We conclude that supernumerary muscle arms are a novel dauer-specific modification that may facilitate some aspect of dauer behavior.
View details for DOI 10.1016/j.ydbio.2008.03.019
View details for Web of Science ID 000256254200015
View details for PubMedID 18436204
FGF negatively regulates muscle membrane extension in Caenorhabditis elegans
2006; 133 (7): 1263-1275
Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.
View details for DOI 10.1242/dev.02300
View details for Web of Science ID 000236764100006
View details for PubMedID 16495308
Muscle arm development in Caenorhabditis elegans
2005; 132 (13): 3079-3092
In several types of animals, muscle cells use membrane extensions to contact motor axons during development. To better understand the process of membrane extension in muscle cells, we investigated the development of Caenorhabditis elegans muscle arms, which extend to motor axons and form the postsynaptic element of the neuromuscular junction. We found that muscle arm development is a highly regulated process: the number of muscle arms extended by each muscle, the shape of the muscle arms and the path taken by the muscle arms to reach the motor axons are largely stereotypical. We also investigated the role of several cytoskeletal components and regulators during arm development, and found that tropomyosin (LEV-11), the actin depolymerizing activity of ADF/cofilin (UNC-60B) and, surprisingly, myosin heavy chain B (UNC-54) are each required for muscle arm extension. This is the first evidence that UNC-54, which is found in thick filaments of sarcomeres, can also play a role in membrane extension. The muscle arm phenotypes produced when these genes are mutated support a 'two-phase' model that distinguishes passive muscle arm development in embryogenesis from active muscle arm extension during larval development.
View details for DOI 10.1242/dev.01883
View details for Web of Science ID 000231050700013
View details for PubMedID 15930100