A genome-wide CRISPR screen implicates plasma membrane asymmetry in exogenous C6-ceramide toxicity.

The bioactive sphingolipid ceramide impacts diverse cellular processes (e.g. apoptosis and cell proliferation) through its effects on membrane dynamics and intracellular signaling pathways. The dysregulation of ceramide metabolism has been implicated in cancer evasion of apoptosis and targeting ceramide metabolism has potential therapeutic benefits as a strategy to kill cancer cells and slow tumor growth. However, the mechanisms of cancer cell resistance to ceramide-mediated cell death are vastly intertwined and incompletely understood. To shed light on this mystery, we performed a genome-wide CRISPR-Cas9 screen to systematically identify regulators of cancer resistance to the soluble short chain ceramide, C6 ceramide (C6-Cer). Our results reveal a complex landscape of genetic modifiers of C6-Cer toxicity, including genes associated with ceramide and sphingolipid metabolism, vesicular trafficking, and membrane biology. Furthermore, we find that loss of the phospholipid flippase subunit TMEM30A impairs the plasma membrane trafficking of its binding partner, the P4-type ATPase ATP11B, and depletion of TMEM30A or ATP11B disrupts plasma membrane asymmetry and promotes resistance to C6-Cer toxicity. Together, our findings provide a resource of genetic modifiers of C6-Cer toxicity and reveal an unexpected role of plasma membrane asymmetry in C6-Cer induced cell death.

An integrated host-microbiome response to atrazine exposure mediates toxicity in Drosophila.

The gut microbiome produces vitamins, nutrients, and neurotransmitters, and helps to modulate the host immune system-and also plays a major role in the metabolism of many exogenous compounds, including drugs and chemical toxicants. However, the extent to which specific microbial species or communities modulate hazard upon exposure to chemicals remains largely opaque. Focusing on the effects of collateral dietary exposure to the widely used herbicide atrazine, we applied integrated omics and phenotypic screening to assess the role of the gut microbiome in modulating host resilience in Drosophila melanogaster. Transcriptional and metabolic responses to these compounds are sex-specific and depend strongly on the presence of the commensal microbiome. Sequencing the genomes of all abundant microbes in the fly gut revealed an enzymatic pathway responsible for atrazine detoxification unique to Acetobacter tropicalis. We find that Acetobacter tropicalis alone, in gnotobiotic animals, is sufficient to rescue increased atrazine toxicity to wild-type, conventionally reared levels. This work points toward the derivation of biotic strategies to improve host resilience to environmental chemical exposures, and illustrates the power of integrative omics to identify pathways responsible for adverse health outcomes.

A Tense Situation: Maintaining ER Homeostasis during Lipid Droplet Budding.

In this issue of Developmental Cell, Chorlay et al. (2019) provide evidence that asymmetric membrane surface tension determines the directionality of lipid droplet (LD) emergence. Furthermore, phospholipid synthesis "refills" the outer leaflet of the endoplasmic reticulum (ER) membrane to maintain cytosolic LD emergence and prevent disruptions to ER homeostasis.

¹9F magnetic resonance probes for live-cell detection of peroxynitrite using an oxidative decarbonylation reaction.

We report a newly discovered oxidative decarbonylation reaction of isatins that is selectively mediated by peroxynitrite (ONOO(-)) to provide anthranilic acid derivatives. We have harnessed this rapid and selective transformation to develop two reaction-based probes, 5-fluoroisatin and 6-fluoroisatin, for the low-background readout of ONOO(-) using (19)F magnetic resonance spectroscopy. 5-fluoroisatin was used to non-invasively detect ONOO(-) formation in living lung epithelial cells stimulated with interferon-γ (IFN-γ).

takeout-dependent longevity is associated with altered Juvenile Hormone signaling.

In order to understand the molecular mechanisms of longevity regulation, we recently performed a screen designed to enrich for genes common to several longevity interventions. Using this approach, we identified the Drosophila melanogaster gene takeout. takeout is upregulated in a variety of long-lived flies, and extends life span when overexpressed. Here, we investigate the mechanisms of takeout-dependent longevity. takeout overexpression specifically in the fat body is sufficient to increase fly longevity and is additive to the longevity effects of Dietary Restriction. takeout long-lived flies do not show phenotypes often associated with increased longevity, such as enhanced stress resistance or major metabolic abnormalities. However, males exhibit greatly diminished courtship behavior, leading to a reduction in fertility. Interestingly, takeout contains a binding domain for Juvenile Hormone, a fly hormone that plays a role in the regulation of developmental transitions. Importantly, the longevity and courtship phenotypes of takeout overexpressing flies are reversed by treatment with the Juvenile Hormone analog methoprene. These data suggest that takeout is a key player in the tradeoff-switch between fertility and longevity. takeout may control fertility via modulation of courtship behavior. This regulation may occur through Juvenile Hormone binding to takeout and a subsequent reduction in Juvenile Hormone signaling activity.

Development of diet-induced insulin resistance in adult Drosophila melanogaster.

The fruit fly Drosophila melanogaster is increasingly utilized as an alternative to costly rodent models to study human diseases. Fly models exist for a wide variety of human conditions, such as Alzheimer's and Parkinson's Disease, or cardiac function. Advantages of the fly system are its rapid generation time and its low cost. However, the greatest strength of the fly system are the powerful genetic tools that allow for rapid dissection of molecular disease mechanisms. Here, we describe the diet-dependent development of metabolic phenotypes in adult fruit flies. Depending on the specific type of nutrient, as well as its relative quantity in the diet, flies show weight gain and changes in the levels of storage macromolecules. Furthermore, the activity of insulin-signaling in the major metabolic organ of the fly, the fat body, decreases upon overfeeding. This decrease in insulin-signaling activity in overfed flies is moreover observed when flies are challenged with an acute food stimulus, suggesting that overfeeding leads to insulin resistance. Similar changes were observed in aging flies, with the development of the insulin resistance-like phenotype beginning at early middle ages. Taken together, these data demonstrate that imbalanced diet disrupts metabolic homeostasis in adult D. melanogaster and promotes insulin-resistant phenotypes. Therefore, the fly system may be a useful alternative tool in the investigation of molecular mechanisms of insulin resistance and the development of pharmacologic treatment options.

Dominant-negative Dmp53 extends life span through the dTOR pathway in D. melanogaster.

Expression of dominant-negative (DN) versions of the Drosophila ortholog of the tumor suppressor p53 extends fly life span in a Calorie Restriction (CR) dependent manner. DN-Dmp53 expression furthermore leads to reduction of Drosophila insulin-like peptide (dILP) 2 mRNA levels and a decrease in insulin/insulin-like growth factor-signaling activity (IIS) in the fly fat body. It is unclear by which mechanisms DN-Dmp53 extends longevity, and whether modulation of insulin-signaling activity plays a pivotal role in life span regulation by Dmp53. Here we show that life span extension due to DN-Dmp53 expression is likely due to reduction of Dmp53 activity and that decreased Dmp53 activity does not extend life span when dILP2 is concomitantly over expressed. Furthermore, extended longevity due to DN-Dmp53 expression does not further extend the life span of flies over expressing the IIS associated transcription factor dFoxO, indicating that DN-Dmp53-dependent life span extension may be related to IIS. However, reduction of dFoxO levels does not decrease DN-Dmp53-dependent longevity extension. Interestingly, when DN-Dmp53 is expressed in flies lacking the translation initiation controlling factor Thor/4E-BP, the downstream target of dTOR signaling, no increase in life span is observed. Taken together, these data suggest that Dmp53 may affect life span by differentially engaging the IIS and dTor pathways.

dSir2 and Dmp53 interact to mediate aspects of CR-dependent lifespan extension in D. melanogaster.

Calorie Restriction (CR) is a well established method of extending life span in a variety of organisms. In the fruit fly D. melanogaster, CR is mediated at least in part by activation of dSir2. In mammalian systems, one of the critical targets of Sir2 is the tumor suppressor p53. This deacetylation of p53 by Sir2 leads to inhibition of p53's transcriptional activity. We have recently shown that inhibition of Dmp53 activity in the fly brain through the use of dominant-negative (DN) constructs that inhibit DNA-binding can extend life span. This life span extension appears to be related to CR, as CR and DN-Dmp53 donot display additive effects on life span. Here we report that life span extension by DN-Dmp53 expression is highly dynamic and can be achieved even when DN-Dmp53 is expressed later in life. In addition, we demonstrate that life span extension by activation of dSir2 and DN-Dmp53 expression are not additive. Furthermore, we show that dSir2 physically interacts with Dmp53 and can deacetylate Dmp53-derived peptides. Taken together, our data demonstrate that Dmp53 is a down stream target of dSir2 enzymatic activity and mediates some aspects of the life span extending effects of CR.

Expression of dominant-negative Dmp53 in the adult fly brain inhibits insulin signaling.

In Drosophila melanogaster, p53 (Dmp53) is an important mediator of longevity. Expression of dominant-negative (DN) forms of Dmp53 in adult neurons, but not in muscle or fat body cells, extends lifespan. The lifespan of calorie-restricted flies is not further extended by simultaneously expressing DN-Dmp53 in the nervous system, indicating that a decrease in Dmp53 activity may be a part of the CR lifespan-extending pathway in flies. In this report, we show that selective expression of DN-Dmp53 in only the 14 insulin-producing cells (IPCs) in the brain extends lifespan to the same extent as expression in all neurons and this lifespan extension is not additive with CR. DN-Dmp53-dependent lifespan extension is accompanied by reduction of Drosophila insulin-like peptide 2 (dILP2) mRNA levels and reduced insulin signaling (IIS) in the fat body, which suggests that Dmp53 may affect lifespan by modulating insulin signaling in the fly.