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The TWK-26 potassium channel governs nutrient absorption in the C. elegans intestine
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Abstract
Ion channels are necessary for proper water and nutrient absorption in the intestine, which supports cellular metabolism and organismal growth. While a role for Na+ co-transporters and pumps in intestinal nutrient absorption is well defined, how individual K+ uniporters function to maintain ion homeostasis is poorly understood. Using Caenorhabditis elegans , we show that a gain-of-function mutation in twk-26 , which encodes a two-pore domain K+ ion channel orthologous to human KCNK3, facilitates nutrient absorption and suppresses the metabolic and developmental defects displayed by impaired intestinal MAP Kinase (MAPK) signaling. Mutations in drl-1 and flr-4 , which encode two components of this MAPK pathway, cause severe growth defects, reduced lipid storage, and a dramatic increase in autophagic lysosomes, which mirror dietary restriction phenotypes. Additionally, these MAPK mutants display structural defects of the intestine and an impaired defecation motor program. We find that activation of TWK-26 reverses the dietary restriction-like state of the MAPK mutants by restoring intestinal nutrient absorption without correcting the intestinal bloating or defecation defects. This study provides unique insight into the mechanisms by which intestinal K+ ion channels support intestinal metabolic homeostasis.
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Calcineurin is a highly conserved calcium/calmodulin-dependent serine/threonine protein phosphatase with diverse functions. Inhibition of calcineurin is known to enhance the lifespan of Caenorhabditis elegans through multiple signaling pathways. Aiming to study the role of calcineurin in regulating innate immunity, we discover that calcineurin is required for the rhythmic defecation motor program (DMP) in C. elegans . Calcineurin inhibition leads to defects in the DMP, resulting in intestinal bloating, rapid colonization of the gut by bacteria, and increased susceptibility to bacterial infection. We demonstrate that intestinal bloating caused by calcineurin inhibition mimics the effects of calorie restriction, resulting in enhanced lifespan. The TFEB ortholog, HLH-30, is required for lifespan extension mediated by calcineurin inhibition. Finally, we show that the nuclear hormone receptor, NHR-8, is upregulated by calcineurin inhibition and is necessary for the increased lifespan. Our studies uncover a role for calcineurin in the C. elegans DMP and provide a new mechanism for calcineurin inhibition-mediated longevity extension.
The popularity of the ancient, probiotic-rich beverage Kombucha Tea (KT) has surged in part due to its purported health benefits, which include protection against metabolic diseases; however, these claims have not been rigorously tested and the mechanisms underlying host response to the probiotics in KT are unknown. Here, we establish a reproducible method to maintain C. elegans on a diet exclusively consisting of Kombucha Tea-associated microbes (KTM), which mirrors the microbial community found in the fermenting culture. KT microbes robustly colonize the gut of KTM-fed animals and confer normal development and fecundity. Intriguingly, animals consuming KTMs display a marked reduction in total lipid stores and lipid droplet size. We find that the reduced fat accumulation phenotype is not due to impaired nutrient absorption, but rather it is sustained by a programed metabolic response in the intestine of the host. KTM consumption triggers widespread transcriptional changes within core lipid metabolism pathways, including upregulation of a suite of lysosomal lipase genes that are induced during lipophagy. The elevated lysosomal lipase activity, coupled with a decrease in lipid droplet biogenesis, is partially required for the reduction in host lipid content. We propose that KTM consumption stimulates a fasting-like response in the C. elegans intestine by rewiring transcriptional programs to promote lipid utilization. Our results provide mechanistic insight into how the probiotics in Kombucha Tea reshape host metabolism and how this popular beverage may impact human metabolism.
Animals integrate developmental and nutritional signals before committing crucial resources to growth and reproduction; however, the pathways that perceive and respond to these inputs remain poorly understood. Here, we demonstrate that DRL-1 and FLR-4, which share similarity with mammalian mitogen-activated protein kinases, maintain lipid homeostasis in the C. elegans intestine. DRL-1 and FLR-4 function in a protein complex at the plasma membrane to promote development, as mutations in drl-1 or flr-4 confer slow growth, small body size, and impaired lipid homeostasis. To identify factors that oppose DRL-1/FLR-4, we performed a forward genetic screen for suppressors of the drl-1 mutant phenotypes and identified mutations in flr-2 and fshr-1, which encode the orthologues of follicle stimulating hormone and its putative G protein–coupled receptor, respectively. In the absence of DRL-1/FLR-4, neuronal FLR-2 acts through intestinal FSHR-1 and protein kinase A signaling to restrict growth. Furthermore, we show that opposing signaling through DRL-1 and FLR-2 coordinates TIR-1 oligomerization, which modulates downstream p38/PMK-1 activity, lipid homeostasis, and development. Finally, we identify a surprising noncanonical role for the developmental transcription factor PHA-4/FOXA in the intestine where it restricts growth in response to impaired DRL-1 signaling. Our work uncovers a complex multi-tissue signaling network that converges on p38 signaling to maintain homeostasis during development.
mTORC1 senses nutrients and growth factors and phosphorylates downstream targets, including the transcription factor TFEB, to coordinate metabolic supply and demand. These functions position mTORC1 as a central controller of cellular homeostasis, but the behavior of this system in individual cells has not been well characterized. Here, we provide measurements necessary to refine quantitative models for mTORC1 as a metabolic controller. We developed a series of fluorescent protein-TFEB fusions and a multiplexed immunofluorescence approach to investigate how combinations of stimuli jointly regulate mTORC1 signaling at the single-cell level. Live imaging of individual MCF10A cells confirmed that mTORC1-TFEB signaling responds continuously to individual, sequential, or simultaneous treatment with amino acids and the growth factor insulin. Under physiologically relevant concentrations of amino acids, we observe correlated fluctuations in TFEB, AMPK, and AKT signaling that indicate continuous activity adjustments to nutrient availability. Using partial least squares regression modeling, we show that these continuous gradations are connected to protein synthesis rate via a distributed network of mTORC1 effectors, providing quantitative support for the qualitative model of mTORC1 as a homeostatic controller and clarifying its functional behavior within individual cells.
Fat is stored or mobilized according to food availability. Malfunction of the mechanisms that ensure this coordination underlie metabolic diseases in humans. In mammals, lysosomal and autophagic function is required for normal fat storage and mobilization in the presence or absence of food. Autophagy is tightly linked to nutrients. However, if and how lysosomal lipolysis is coupled to nutritional status remains to be determined. Here we identify MXL-3 and HLH-30 as transcriptional switches coupling lysosomal lipolysis and autophagy to nutrient availability and controlling fat storage and ageing in Caenorhabditis elegans. Transcriptional coupling of lysosomal lipolysis and autophagy to nutrients is also observed in mammals. Thus, MXL-3 and HLH-30 orchestrate an adaptive and conserved cellular response to nutritional status and regulate lifespan. The importance of lipolysis to general metabolism became apparent when it was discovered that fat can only be mobilized in its hydrolysed form 1. Classically, lipolysis has been associated with cytosolic or ER-associated neutral lipases. However, cytosolic lipids are also catabolized through autophagy-mediated lipolysis, also termed lipophagy 2. Both lysosomal acid lipases 3 (LALs) and Atg15 (ref. 4), an autophagy protein with predicted triglyceride-lipase activity, have been proposed to catabolize lipid-droplet fat stores through lipophagy. Although lysosomal inhibitors induce increased levels of lipid-droplet fats, lysosomal inhibition impairs the function of the endocytic pathway and autophagy, making this approach insufficient to define which lipases actually break down lipids through lipophagy. Similar to general macroautophagy, lipophagy is activated by fasting. However, it is unknown if the assembly of autophagosomes is the only regulated step or if the lipases breaking down lipids in the autophagolysosome would be separately regulated by changes in nutrient availability. If the lipases were independently regulated, then the molecular players that link their activity to nutrient availability would remain to be uncovered. Finally, it has not been established if lipophagy is confined to mammals or if it is an ancient mechanism of energy homeostasis. Here we present the lysosomal lipases LIPL-1 and LIPL-3 as key enzymes breaking down lipid-droplet fats through lipophagy in C. elegans. In addition, we show that the transcription factors MXL-3 and HLH-30 link nutrient availability to lysosomal lipolysis, uncovering two fat regulators. We also show that lysosomal lipolysis and autophagy are similarly linked to nutrient availability in mammalian cells in culture. Finally, we show that lysosomal lipolysis and its regulators MXL-3 and HLH-30 influence C. elegans ageing. RESULTS lipl-1, 2, 3, 4 and 5 respond to fasting A list of 84 conserved C. elegans genes likely to be regulated by nutritional status was generated through comparative analyses of the transcriptional response to starvation of C. elegans 5 , Drosophila melanogaster 6 , and mice 7 (Supplementary Tables S1 and S2). We tested these genes for differential expression in 6 h-fasted versus well-fed fertile adult worms. The most upregulated genes of the set belonged to a family of predicted triglyceride lipases. lipl-1, 2, 3, 4 and 5 were upregulated 44-, 12-, 25-, 7-and 2-fold, respectively (Fig. 1). lipl-6, 7 and 8 showed no change in expression (Fig. 1). Transcriptional reporters for lipl-1, 2 and 3 are observed in the intestine of fasted, but not well-fed, larvae and adults (Supplementary Fig. S1). The lipl-4 P::GFP (green fluorescent protein) fusion gene is expressed in the intestine of starvation-induced dauer larvae, and in the pharynx of both well-fed and fasted animals (Supplementary Fig. S1). lipl-5 P::GFP is expressed in the intestine 8. LIPL-1 and LIPL-3 are lysosomal lipases controlling lipid-droplet fats The lipl genes encode for uncharacterized triglyceride lipases with extensive sequence similarity to human lysosomal acid lipase (BLAST scores 9e-78 and 3e-75 for lipl-1 and lipl-3, respectively). The primary sequence of the fasting-responsive LIPL proteins contains predicted NATURE CELL BIOLOGY ADVANCE ONLINE PUBLICATION 1
The DEG/ENaC family of ion channels was defined based on the sequence similarity between degenerins (DEG) from the nematode Caenorhabditis elegans and subunits of the mammalian epithelial sodium channel (ENaC), and also includes a diverse array of non‐voltage‐gated cation channels from across animal phyla, including the mammalian acid‐sensing ion channels (ASICs) and Drosophila pickpockets. ENaCs and ASICs have wide ranging medical importance; for example, ENaCs play an important role in respiratory and renal function, and ASICs in ischaemia and inflammatory pain, as well as being implicated in memory and learning. Electrophysiological approaches, both in vitro and in vivo, have played an essential role in establishing the physiological properties of this diverse family, identifying an array of modulators and implicating them in an extensive range of cellular functions, including mechanosensation, acid sensation and synaptic modulation. Likewise, genetic studies in both invertebrates and vertebrates have played an important role in linking our understanding of channel properties to function at the cellular and whole animal/behavioural level. Drawing together genetic and physiological evidence is essential to furthering our understanding of the precise cellular roles of DEG/ENaC channels, with the diversity among family members allowing comparative physiological studies to dissect the molecular basis of these diverse functions. image
Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its link with sleep apnea remains poorly understood. Here we describe a new developmental disorder with associated sleep apnea (developmental delay with sleep apnea, or DDSA) caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the ‘X-gate’, a gating motif that controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein-coupled receptor pathways. However, despite their defective X-gating, these mutant channels can still be inhibited by a range of known TASK channel inhibitors. These results not only highlight an important new role for TASK-1 K+ channels and their link with sleep apnea but also identify possible therapeutic strategies. Heterozygous de novo gain-of-function mutations in KCNK3, which encodes the two-pore-domain K+ channel TASK-1, cause a channelopathy characterized by developmental delay with sleep apnea.
Despite its physiological and pathological importance, the mechanical relationship between glucose uptake in the intestine and intestinal flows is unclear. In the intestine of the nematode Caenorhabditis elegans, the defecation motor program (DMP) causes reciprocating intestinal flows. Although the DMP is frequently activated in the intestines, its physiological function is unknown. We evaluated the mechanical signature of enhanced glucose uptake by the DMP in worms. Glucose uptake tended to increase with increasing flow velocity during the DMP because of mechanical mixing and transport. However, the increase in input energy required for the DMP was low compared with the calorie intake. The findings suggest that animals with gastrointestinal motility exploit the reciprocating intestinal flows caused by peristalsis to promote nutrient absorption by intestinal cells.
Biological clocks are fundamental to an organism’s health, controlling periodicity of behaviour and metabolism. Here, we identify two acid-sensing ion channels, with very different proton sensing properties, and describe their role in an ultradian clock, the defecation motor program (DMP) of the nematode Caenorhabditis elegans . An ACD-5-containing channel, on the apical membrane of the intestinal epithelium, is essential for maintenance of luminal acidity, and thus the rhythmic oscillations in lumen pH. In contrast, the second channel, composed of FLR-1, ACD-3 and/or DEL-5, located on the basolateral membrane, controls the intracellular Ca ²⁺ wave and forms a core component of the master oscillator that controls timing and rhythmicity of the DMP. flr-1 and acd-3/del-5 mutants show severe developmental and metabolic defects. We thus directly link the proton-sensing properties of these channels to their physiological roles in pH regulation and Ca ²⁺ signalling, the generation of an ultradian oscillator, and its metabolic consequences.
Intracellular signaling regulators can be concentrated into membrane-free, higher-ordered protein assemblies to initiate protective responses during stress - a process known as phase transition. Here, we show that a phase transition of the Caenorhabditis elegans Toll/interleukin-1 receptor domain protein (TIR-1), an NAD ⁺ glycohydrolase homologous to mammalian sterile alpha and TIR motif-containing 1 (SARM1), underlies p38 PMK-1 immune pathway activation in C. elegans intestinal epithelial cells. Through visualization of fluorescently labeled TIR-1/SARM1 protein, we demonstrate that physiologic stresses, both pathogen and non-pathogen, induce multimerization of TIR-1/SARM1 into visible puncta within intestinal epithelial cells. In vitro enzyme kinetic analyses revealed that, like mammalian SARM1, the NAD ⁺ glycohydrolase activity of C. elegans TIR-1 is dramatically potentiated by protein oligomerization and a phase transition. Accordingly, C. elegans with genetic mutations that specifically block either multimerization or the NAD ⁺ glycohydrolase activity of TIR-1/SARM1 fail to induce p38 PMK phosphorylation, are unable to increase immune effector expression, and are dramatically susceptible to bacterial infection. Finally, we demonstrate that a loss-of-function mutation in nhr-8 , which alters cholesterol metabolism and is used to study conditions of sterol deficiency, causes TIR-1/SARM1 to oligomerize into puncta in intestinal epithelial cells. Cholesterol scarcity increases p38 PMK-1 phosphorylation, primes immune effector induction in a manner that requires TIR-1/SARM1 oligomerization and its intrinsic NAD ⁺ glycohydrolase activity, and reduces pathogen accumulation in the intestine during a subsequent infection. These data reveal a new adaptive response that allows a metazoan host to anticipate pathogen threats during cholesterol deprivation, a time of relative susceptibility to infection. Thus, a phase transition of TIR-1/SARM1 as a prerequisite for its NAD ⁺ glycohydrolase activity is strongly conserved across millions of years of evolution and is essential for diverse physiological processes in multiple cell types.
Injured axons must regenerate to restore nervous system function, and regeneration is regulated in part by external factors from non-neuronal tissues. Many of these extrinsic factors act in the immediate cellular environment of the axon to promote or restrict regeneration, but the existence of long-distance signals regulating axon regeneration has not been clear. Here we show that the Rab GTPase rab-27 inhibits regeneration of GABAergic motor neurons in C. elegans through activity in the intestine. Re-expression of RAB-27, but not the closely related RAB-3, in the intestine of rab-27 mutant animals is sufficient to rescue normal regeneration. Several additional components of an intestinal neuropeptide secretion pathway also inhibit axon regeneration, including NPDC1/cab-1, SNAP25/aex-4, KPC3/aex-5, and the neuropeptide NLP-40, and re-expression of these genes in the intestine of mutant animals is sufficient to restore normal regeneration success. Additionally, NPDC1/cab-1 and SNAP25/aex-4 genetically interact with rab-27 in the context of axon regeneration inhibition. Together these data indicate that RAB-27-dependent neuropeptide secretion from the intestine inhibits axon regeneration, and point to distal tissues as potent extrinsic regulators of regeneration.
The ability of organisms to sense nutrient availability and tailor their metabolic states to withstand nutrient deficiency is critical for survival. To identify previously unknown regulators that couple nutrient deficiency to body fat utilization, we performed a cherry-picked RNAi screen in C. elegans and found that the transcription factor HLH-11 regulates lipid metabolism in response to food availability. In well-fed worms, HLH-11 suppresses transcription of lipid catabolism genes. Upon fasting, the HLH-11 protein level is reduced through lysosome- and proteasome-mediated degradation, thus alleviating the inhibitory effect of HLH-11, activating the transcription of lipid catabolism genes, and utilizing fat. Additionally, lipid profiling revealed that reduction in the HLH-11 protein level remodels the lipid landscape in C. elegans. Moreover, TFAP4, the mammalian homolog of HLH-11, plays an evolutionarily conserved role in regulating lipid metabolism in response to starvation. Thus, TFAP4 may represent a potential therapeutic target for lipid storage disorders.
The metabolic state of an organism instructs gene expression modalities, leading to changes in complex life history traits, such as longevity. Dietary restriction (DR), which positively affects health and life span across species, leads to metabolic reprogramming that enhances utilisation of fatty acids for energy generation. One direct consequence of this metabolic shift is the upregulation of cytoprotective (CyTP) genes categorized in the Gene Ontology (GO) term of "Xenobiotic Detoxification Program" (XDP). How an organism senses metabolic changes during nutritional stress to alter gene expression programs is less known. Here, using a genetic model of DR, we show that the levels of polyunsaturated fatty acids (PUFAs), especially linoleic acid (LA) and eicosapentaenoic acid (EPA), are increased following DR and these PUFAs are able to activate the CyTP genes. This activation of CyTP genes is mediated by the conserved p38 mitogen-activated protein kinase (p38-MAPK) pathway. Consequently, genes of the PUFA biosynthesis and p38-MAPK pathway are required for multiple paradigms of DR-mediated longevity, suggesting conservation of mechanism. Thus, our study shows that PUFAs and p38-MAPK pathway function downstream of DR to help communicate the metabolic state of an organism to regulate expression of CyTP genes, ensuring extended life span.
CRISPR genome editing has revolutionized genetics in many organisms. In the nematode Caenorhabditis elegans one injection into each of the two gonad arms of an adult hermaphrodite exposes hundreds of meiotic germ cells to editing mixtures, permitting the recovery of multiple indels or small precision edits from each successfully injected animal. Unfortunately, particularly for long insertions, editing efficiencies can vary widely, necessitating multiple injections, and often requiring co-selection strategies. Here we show that melting double stranded DNA (dsDNA) donor molecules prior to injection increases the frequency of precise homology-directed repair (HDR) by several fold for longer edits. We describe troubleshooting strategies that enable consistently high editing efficiencies resulting, for example, in up to 100 independent GFP knock-ins from a single injected animal. These efficiencies make C. elegans by far the easiest metazoan to genome edit, removing barriers to the use and adoption of this facile system as a model for understanding animal biology.
TWIK-related acid-sensitive potassium (TASK) channels—members of the two pore domain potassium (K2P) channel family—are found in neurons1, cardiomyocytes2–4 and vascular smooth muscle cells5, where they are involved in the regulation of heart rate6, pulmonary artery tone5,7, sleep/wake cycles8 and responses to volatile anaesthetics8–11. K2P channels regulate the resting membrane potential, providing background K+ currents controlled by numerous physiological stimuli12–15. Unlike other K2P channels, TASK channels are able to bind inhibitors with high affinity, exceptional selectivity and very slow compound washout rates. As such, these channels are attractive drug targets, and TASK-1 inhibitors are currently in clinical trials for obstructive sleep apnoea and atrial fibrillation16. In general, potassium channels have an intramembrane vestibule with a selectivity filter situated above and a gate with four parallel helices located below; however, the K2P channels studied so far all lack a lower gate. Here we present the X-ray crystal structure of TASK-1, and show that it contains a lower gate—which we designate as an ‘X-gate’—created by interaction of the two crossed C-terminal M4 transmembrane helices at the vestibule entrance. This structure is formed by six residues (243VLRFMT248) that are essential for responses to volatile anaesthetics10, neurotransmitters13 and G-protein-coupled receptors13. Mutations within the X-gate and the surrounding regions markedly affect both the channel-open probability and the activation of the channel by anaesthetics. Structures of TASK-1 bound to two high-affinity inhibitors show that both compounds bind below the selectivity filter and are trapped in the vestibule by the X-gate, which explains their exceptionally low washout rates. The presence of the X-gate in TASK channels explains many aspects of their physiological and pharmacological behaviour, which will be beneficial for the future development and optimization of TASK modulators for the treatment of heart, lung and sleep disorders. The X-ray crystal structure of the potassium channel TASK-1 reveals the presence of an X-gate, which traps small-molecule inhibitors in the intramembrane vestibule and explains their low washout rates from the channel.
In their seminal papers Hanahan and Weinberg described oncogenic processes a normal cell undergoes to be transformed into a cancer cell. The functions of ion channels in the gastrointestinal (GI) tract influence a variety of cellular processes, many of which overlap with these hallmarks of cancer. In this review we focus on the roles of the calcium (Ca2+), sodium (Na+), potassium (K+), chloride (Cl-) and zinc (Zn2+) transporters in GI cancer, with a special emphasis on the roles of the KCNQ1 K+ channel and CFTR Cl- channel in colorectal cancer (CRC). Ca2+ is a ubiquitous second messenger, serving as a signaling molecule for a variety of cellular processes such as control of the cell cycle, apoptosis, and migration. Various members of the TRP superfamily, including TRPM8, TRPM7, TRPM6 and TRPM2, have been implicated in GI cancers, especially through overexpression in pancreatic adenocarcinomas and down-regulation in colon cancer. Voltage-gated sodium channels (VGSCs) are classically associated with the initiation and conduction of action potentials in electrically excitable cells such as neurons and muscle cells. The VGSC NaV1.5 is abundantly expressed in human colorectal CRC cell lines as well as being highly expressed in primary CRC samples. Studies have demonstrated that conductance through NaV1.5 contributes significantly to CRC cell invasiveness and cancer progression. Zn2+ transporters of the ZIP/SLC39A and ZnT/SLC30A families are dysregulated in all major GI organ cancers, in particular, ZIP4 up-regulation in pancreatic cancer (PC). More than 70 K+ channel genes, clustered in four families, are found expressed in the GI tract, where they regulate a range of cellular processes, including gastrin secretion in the stomach and anion secretion and fluid balance in the intestinal tract. Several distinct types of K+ channels are found dysregulated in the GI tract. Notable are hERG1 upregulation in PC, gastric cancer (GC) and CRC, leading to enhanced cancer angiogenesis and invasion, and KCNQ1 down-regulation in CRC, where KCNQ1 expression is associated with enhanced disease-free survival in stage II, III, and IV disease. Cl- channels are critical for a range of cellular and tissue processes in the GI tract, especially fluid balance in the colon. Most notable is CFTR, whose deficiency leads to mucus blockage, microbial dysbiosis and inflammation in the intestinal tract. CFTR is a tumor suppressor in several GI cancers. Cystic fibrosis patients are at a significant risk for CRC and low levels of CFTR expression are associated with poor overall disease-free survival in sporadic CRC. Two other classes of chloride channels that are dysregulated in GI cancers are the chloride intracellular channels (CLIC1, 3 & 4) and the chloride channel accessory proteins (CLCA1,2,4). CLIC1 & 4 are upregulated in PC, GC, gallbladder cancer, and CRC, while the CLCA proteins have been reported to be down-regulated in CRC. In summary, it is clear, from the diverse influences of ion channels, that their aberrant expression and/or activity can contribute to malignant transformation and tumor progression. Further, because ion channels are often localized to the plasma membrane and subject to multiple layers of regulation, they represent promising clinical targets for therapeutic intervention including the repurposing of current drugs.
Diet profoundly affects metabolism and incidences of age-related diseases. Animals adapt their physiology to different food-types, modulating complex life-history traits like aging. The molecular mechanisms linking adaptive capacity to diet with aging are less known. We identify FLR-4 kinase as a novel modulator of aging in C. elegans, depending on bacterial diet. FLR-4 functions to prevent differential activation of the p38MAPK pathway in response to diverse food-types, thereby maintaining normal life span. In a kinase-dead flr-4 mutant, E. coli HT115 (K12 strain), but not the standard diet OP50 (B strain), is able to activate p38MAPK, elevate expression of cytoprotective genes through the nuclear hormone receptor NHR-8 and enhance life span. Interestingly, flr-4 and dietary restriction utilize similar pathways for longevity assurance, suggesting cross-talks between cellular modules that respond to diet quality and quantity. Together, our study discovers a new C. elegans gene-diet pair that controls the plasticity of aging.
Autophagy has been linked to longevity in many species, but the underlying mechanisms are unclear. Using a GFP-tagged and a new tandem-tagged Atg8/LGG-1 reporter, we quantified autophagic vesicles and performed autophagic flux assays in multiple tissues of wild-type Caenorhabditis elegans and long-lived daf-2/insulin/IGF-1 and glp-1/Notch mutants throughout adulthood. Our data are consistent with an age-related decline in autophagic activity in the intestine, body-wall muscle, pharynx, and neurons of wild-type animals. In contrast, daf-2 and glp-1 mutants displayed unique age-and tissue-specific changes in autophagic activity, indicating that the two longevity paradigms have distinct effects on autophagy during aging. Although autophagy appeared active in the intestine of both long-lived mutants, inhibition of intestinal autophagy significantly abrogated lifespan extension only in glp-1 mutants. Collectively, our data suggest that autophagic activity normally decreases with age in C. elegans, whereas daf-2 and glp-1 long-lived mutants regulate autophagy in distinct spatiotemporal-specific manners to extend lifespan.
Animals integrate metabolic, developmental, and environmental information before committing key resources to reproduction. In Caenorhabditis elegans, adult animals transport fat from intestinal cells to the germline to promote reproduction. Weidentified a microRNA (miRNA)-regulated developmental timing pathway that functions in the hypodermis to nonautonomously coordinate the mobilization of intestinal fat stores to the germline upon initiation of adulthood. This developmental timing pathway, which is controlled by the lin-4 and let-7 miRNAs, engages mTOR signaling in the intestine. The intestinal signaling component is specific to mTORC2 and functions in parallel to the insulin pathway to modulate the activity of the serum/glucocorticoid-regulated kinase (SGK-1). Surprisingly, SGK-1 functions independently of DAF-16/FoxO; instead, SGK-1 promotes the cytoplasmic localization of the PQM-1 transcription factor, which antagonizes intestinal fat mobilization at the transcriptional level when localized to the nucleus. These results revealed that a non-cell-autonomous developmental input regulates intestinal fat metabolism by engaging mTORC2 signaling to promote the intertissue transport of fat reserves from the soma to the germline.
Aims/hypothesis:
Type 2 diabetes is closely associated with pathological lipid accumulation in the liver, which is suggested to actively contribute to the development of insulin resistance. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of liver steatosis, whole-body glucose tolerance and insulin sensitivity in a mouse model system. The aim of this study was to assess the role of STK25 in the control of lipid metabolism in human liver.
Methods:
Intracellular fat deposition, lipid metabolism and insulin sensitivity were studied in immortalised human hepatocytes (IHHs) and HepG2 hepatocellular carcinoma cells in which STK25 was overexpressed or knocked down by small interfering RNA. The association between STK25 mRNA expression in human liver biopsies and hepatic fat content was analysed.
Results:
Overexpression of STK25 in IHH and HepG2 cells enhanced lipid deposition by suppressing β-oxidation and triacylglycerol (TAG) secretion, while increasing lipid synthesis. Conversely, knockdown of STK25 attenuated lipid accumulation by stimulating β-oxidation and TAG secretion, while inhibiting lipid synthesis. Furthermore, TAG hydrolase activity was repressed in hepatocytes overexpressing STK25 and reciprocally increased in cells with STK25 knockdown. Insulin sensitivity was reduced in STK25-overexpressing cells and enhanced in STK25-deficient hepatocytes. We also found a statistically significant positive correlation between STK25 mRNA expression in human liver biopsies and hepatic fat content.
Conclusions/interpretation:
Our data suggest that STK25 regulates lipid partitioning in human liver cells by controlling TAG synthesis as well as lipolytic activity and thereby NEFA release from lipid droplets for β-oxidation and TAG secretion. Our findings highlight STK25 as a potential drug target for the prevention and treatment of type 2 diabetes.
The molecular mechanisms by which dietary fatty acids are absorbed by the intestine, and the way in which the process is regulated are poorly understood. In a genetic screen for mutations affecting fat accumulation in the intestine of Caenorhabditis elegans, nematode worms, we have isolated mutations in the aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase known to be required for maturation of certain neuropeptides, and for a discrete step in an ultradian rhythmic phenomenon called the defecation motor program. We demonstrate that aex-5 mutants have markedly lower steady-state levels of fat in the intestine, and that this defect is associated with a significant reduction in the rate at which labeled fatty acid derivatives are taken up from the intestinal lumen. Other mutations affecting the defecation motor program also affect steady-state levels of triglycerides, suggesting that the program is required per se for the proper accumulation of neutral lipids. Our results suggest that an important function of the defecation motor program in C. elegans is to promote the uptake of an important class of dietary nutrients. They also imply that modulation of the program might be one way in which worms adjust nutrient uptake in response to altered metabolic status.
TOPCONS (http://topcons.net/) is a widely used web server for consensus prediction of membrane protein topology. We hereby present a major update to the server, with some substantial improvements, including the following: (i) TOPCONS can now efficiently separate signal peptides from transmembrane regions. (ii) The server can now differentiate more successfully between globular and membrane proteins. (iii) The server now is even slightly faster, although a much larger database is used to generate the multiple sequence alignments. For most proteins, the final prediction is produced in a matter of seconds. (iv) The user-friendly interface is retained, with the additional feature of submitting batch files and accessing the server programmatically using standard interfaces, making it thus ideal for proteome-wide analyses. Indicatively, the user can now scan the entire human proteome in a few days. (v) For proteins with homology to a known 3D structure, the homology-inferred topology is also displayed. (vi) Finally, the combination of methods currently implemented achieves an overall increase in performance by 4% as compared to the currently available best-scoring methods and TOPCONS is the only method that can identify signal peptides and still maintain a state-of-the-art performance in topology predictions.
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
K+ channels participate in many biological functions from ion homeostasis to generation and modulation of the electrical membrane potential. They are involved in a large variety of diseases. In the human genome, 15 genes code for K+ channels with two pore-domains (K2P). These channels form dimers of pore-forming subunits that produce background conductances finely regulated by a range of natural and chemical effectors, including signaling lipids, temperature, pressure, pH, antidepressants and volatile anesthetics. Since the cloning of TWIK1, the prototypical member of this family, a lot of work has been carried out about their structure and biology. These studies are still in progress, but data gathered so far show that K2P channels are central players in many processes including ion homeostasis, hormone secretion, cell development and excitability. A growing number of studies underline their implication in physiopathological mechanisms such as vascular and pulmonary hypertension, cardiac arrhythmias, nociception, neuroprotection and depression. This review gives a synthetic view of the most noticeable features of these channels.This article is protected by copyright. All rights reserved
Although dietary restriction (DR) is known to extend lifespan across species, from yeast to mammals, the signalling events downstream of food/nutrient perception are not well understood. In Caenorhabditis elegans, DR is typically attained either by using the eat-2 mutants that have reduced pharyngeal pumping leading to lower food intake or by feeding diluted bacterial food to the worms. In this study, we show that knocking down a mammalian MEKK3-like kinase gene, mekk-3 in C. elegans, initiates a process similar to DR without compromising food intake. This DR-like state results in upregulation of beta-oxidation genes through the nuclear hormone receptor NHR-49, a HNF-4 homolog, resulting in depletion of stored fat. This metabolic shift leads to low levels of reactive oxygen species (ROS), potent oxidizing agents that damage macromolecules. Increased beta-oxidation, in turn, induces the phase I and II xenobiotic detoxification genes, through PHA-4/FOXA, NHR-8 and aryl hydrocarbon receptor AHR-1, possibly to purge lipophilic endotoxins generated during fatty acid catabolism. The coupling of a metabolic shift with endotoxin detoxification results in extreme longevity following mekk-3 knock-down. Thus, MEKK-3 may function as an important nutrient sensor and signalling component within the organism that controls metabolism. Knocking down mekk-3 may signal an imminent nutrient crisis that results in initiation of a DR-like state, even when food is plentiful.
Autophagy is a cellular recycling process that has an important anti-aging role, but the underlying molecular mechanism is not well understood. The mammalian transcription factor EB (TFEB) was recently shown to regulate multiple genes in the autophagy process. Here we show that the predicted TFEB orthologue HLH-30 regulates autophagy in Caenorhabditis elegans and, in addition, has a key role in lifespan determination. We demonstrate that hlh-30 is essential for the extended lifespan of Caenorhabditis elegans in six mechanistically distinct longevity models, and overexpression of HLH-30 extends lifespan. Nuclear localization of HLH-30 is increased in all six Caenorhabditis elegans models and, notably, nuclear TFEB levels are augmented in the livers of mice subjected to dietary restriction, a known longevity-extending regimen. Collectively, our results demonstrate a conserved role for HLH-30 and TFEB in autophagy, and possibly longevity, and identify HLH-30 as a uniquely important transcription factor for lifespan modulation in Caenorhabditis elegans.
The lysosomal–autophagic pathway is activated by starvation and plays an important role in both cellular clearance and lipid catabolism. However, the transcriptional regulation of this pathway in response to metabolic cues is uncharacterized. Here we show that the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, is induced by starvation through an autoregulatory feedback loop and exerts a global transcriptional control on lipid catabolism via Ppargc1α and Ppar1α. Thus, during starvation a transcriptional mechanism links the autophagic pathway to cellular energy metabolism. The conservation of this mechanism in Caenorhabditis elegans suggests a fundamental role for TFEB in the evolution of the adaptive response to food deprivation. Viral delivery of TFEB to the liver prevented weight gain and metabolic syndrome in both diet-induced and genetic mouse models of obesity, suggesting a new therapeutic strategy for disorders of lipid metabolism.
Whole genome sequencing (WGS) allows researchers to pinpoint genetic differences between individuals and significantly shortcuts the costly and time-consuming part of forward genetic analysis in model organism systems. Currently, the most effort-intensive part of WGS is the bioinformatic analysis of the relatively short reads generated by second generation sequencing platforms. We describe here a novel, easily accessible and cloud-based pipeline, called CloudMap, which greatly simplifies the analysis of mutant genome sequences. Available on the Galaxy web platform, CloudMap requires no software installation when run on the cloud, but it can also be run locally or via Amazon's Elastic Compute Cloud (EC2) service. CloudMap uses a series of pre-defined workflows to pinpoint sequence variations in animal genomes, such as those of pre-mutagenized and mutagenized Caenorhabditis elegans strains. In combination with a variant-based mapping procedure, CloudMap allows users to sharply define genetic map intervals graphically and to retrieve very short lists of candidate variants with a few simple clicks. Automated workflows and extensive video user guides are available to detail the individual analysis steps performed (http://usegalaxy.org/cloudmap). We demonstrate the utility of CloudMap for WGS analysis of C. elegans and Arabidopsis genomes and describe how other organisms (e.g. Zebrafish, Drosophila) can easily be accommodated by this software platform. To accommodate rapid analysis of many mutants from large scale genetic screens, CloudMap contains an in silico complementation testing tool which allows users to rapidly identify instances where multiple alleles of the same gene are present in the mutant collection. Lastly, we describe the application of a novel mapping/WGS method ("Variant Discovery Mapping") that does not rely on a defined polymorphic mapping strain and we integrate the application of this method into CloudMap. CloudMap tools and documentation are continually updated at http://usegalaxy.org/cloudmap.
Fiji is a distribution of the popular open-source software ImageJ focused on biological-image analysis. Fiji uses modern software engineering practices to combine powerful software libraries with a broad range of scripting languages to enable rapid prototyping of image-processing algorithms. Fiji facilitates the transformation of new algorithms into ImageJ plugins that can be shared with end users through an integrated update system. We propose Fiji as a platform for productive collaboration between computer science and biology research communities.
Lysosomes are the major cellular site for clearance of defective organelles and digestion of internalized material. Demand on lysosomal capacity can vary greatly, and lysosomal function must be adjusted to maintain cellular homeostasis. Here, we identified an interaction between the lysosome-localized mechanistic target of rapamycin complex 1 (mTORC1) and the transcription factor TFEB (transcription factor EB), which promotes lysosome biogenesis. When lysosomal activity was adequate, mTOR-dependent phosphorylation of TFEB on Ser(211) triggered the binding of 14-3-3 proteins to TFEB, resulting in retention of the transcription factor in the cytoplasm. Inhibition of lysosomal function reduced the mTOR-dependent phosphorylation of TFEB, resulting in diminished interactions between TFEB and 14-3-3 proteins and the translocation of TFEB into the nucleus, where it could stimulate genes involved in lysosomal biogenesis. These results identify TFEB as a target of mTOR and suggest a mechanism for matching the transcriptional regulation of genes encoding proteins of autophagosomes and lysosomes to cellular need. The closely related transcription factors MITF (microphthalmia transcription factor) and TFE3 (transcription factor E3) also localized to lysosomes and accumulated in the nucleus when lysosome function was inhibited, thus broadening the range of physiological contexts under which this regulatory mechanism may prove important.
Diarrhoea in ulcerative colitis (UC) mainly reflects impaired colonic Na(+) and water absorption. Colonocyte membrane potential, an important determinant of electrogenic Na(+) absorption, is reduced in UC. Colonocyte potential is principally determined by basolateral IK (KCa3.1) channel activity. To determine whether reduced Na(+) absorption in UC might be associated with decreased IK channel expression and activity, we used molecular and patch clamp recording techniques to evaluate IK channels in colon from control patients and patients with active UC. In control patients, immunolabelling revealed basolateral IK channels distributed uniformly along the surface-crypt axis, with substantially decreased immunolabelling in patients with active UC, although IK mRNA levels measured by quantitative PCR were similar in both groups. Patch clamp analysis indicated that cell conductance was dominated by basolateral IK channels in control patients, but channel abundance and overall activity were reduced by 53% (p = 0.03) and 61% (p = 0.04), respectively, in patients with active UC. These changes resulted in a 75% (p = 0.003) decrease in the estimated basolateral membrane K(+) conductance in UC patients compared with controls. Levels of IK channel immunolabelling and activity in UC patients in clinical remission were similar to those in control patients. We conclude that a substantial decrease in basolateral IK channel expression and activity in active UC most likely explains the epithelial cell depolarization observed in this disease, and decreases the electrical driving force for electrogenic Na(+) transport, thereby impairing Na(+) absorption (and as a consequence, Cl(-) and water absorption) across the inflamed mucosa.
Whole-genome sequencing (WGS) is becoming a fast and cost-effective method to pinpoint molecular lesions in mutagenized genetic model systems, such as Caenorhabditis elegans. As mutagenized strains contain a significant mutational load, it is often still necessary to map mutations to a chromosomal interval to elucidate which of the WGS-identified sequence variants is the phenotype-causing one. We describe here our experience in setting up and testing a simple strategy that incorporates a rapid SNP-based mapping step into the WGS procedure. In this strategy, a mutant retrieved from a genetic screen is crossed with a polymorphic C. elegans strain, individual F2 progeny from this cross is selected for the mutant phenotype, the progeny of these F2 animals are pooled and then whole-genome-sequenced. The density of polymorphic SNP markers is decreased in the region of the phenotype-causing sequence variant and therefore enables its identification in the WGS data. As a proof of principle, we use this strategy to identify the molecular lesion in a mutant strain that produces an excess of dopaminergic neurons. We find that the molecular lesion resides in the Pax-6/Eyeless ortholog vab-3. The strategy described here will further reduce the time between mutant isolation and identification of the molecular lesion.
The genetically tractable model host Caenorhabditis elegans provides a valuable tool to dissect host-microbe interactions in vivo. Pseudomonas aeruginosa and Staphylococcus aureus utilize virulence factors involved in human disease to infect and kill C. elegans. Despite much progress, virtually nothing is known regarding the cytopathology of infection and the proximate causes of nematode death. Using light and electron microscopy, we found that P. aeruginosa infection entails intestinal distention, accumulation of an unidentified extracellular matrix and P. aeruginosa-synthesized outer membrane vesicles in the gut lumen and on the apical surface of intestinal cells, the appearance of abnormal autophagosomes inside intestinal cells, and P. aeruginosa intracellular invasion of C. elegans. Importantly, heat-killed P. aeruginosa fails to elicit a significant host response, suggesting that the C. elegans response to P. aeruginosa is activated either by heat-labile signals or pathogen-induced damage. In contrast, S. aureus infection causes enterocyte effacement, intestinal epithelium destruction, and complete degradation of internal organs. S. aureus activates a strong transcriptional response in C. elegans intestinal epithelial cells, which aids host survival during infection and shares elements with human innate responses. The C. elegans genes induced in response to S. aureus are mostly distinct from those induced by P. aeruginosa. In contrast to P. aeruginosa, heat-killed S. aureus activates a similar response as live S. aureus, which appears to be independent of the single C. elegans Toll-Like Receptor (TLR) protein. These data suggest that the host response to S. aureus is possibly mediated by pathogen-associated molecular patterns (PAMPs). Because our data suggest that neither the P. aeruginosa nor the S. aureus-triggered response requires canonical TLR signaling, they imply the existence of unidentified mechanisms for pathogen detection in C. elegans, with potentially conserved roles also in mammals.
Genetic conservation allows ancient features of fat storage endocrine pathways to be explored in C. elegans. Multiple studies have used Nile red or BODIPY-labeled fatty acids to identify regulators of fat mass. When mixed with their food, E. coli bacteria, Nile red, and BODIPY-labeled fatty acids stain multiple spherical cellular structures in the C. elegans major fat storage organ, the intestine. However, here we demonstrate that, in the conditions previously reported, the lysosome-related organelles stained by Nile red and BODIPY-labeled fatty acids are not the C. elegans major fat storage compartment. We show that the major fat stores are contained in a distinct cellular compartment that is not stained by Nile red. Using biochemical assays, we validate oil red O staining as a method to assess major fat stores in C. elegans, allowing for efficient and accurate genetic and functional genomic screens for genes that control fat accumulation at the organismal level.
In Caenorhabditis elegans, oscillations of intestinal pH contribute to the rhythmic defecation behavior, but the acid-base transport mechanisms that facilitate proton movement are not well understood. Here, we demonstrate that VHA-6, an intestine-specific a-subunit of the H(+)-K(+)-ATPase complex (V-ATPase), resides in the apical membrane of the intestinal epithelial cells and is required for luminal acidification. Disruption of the vha-6 gene led to early developmental arrest; the arrest phenotype could be complemented by expression of a fluorescently labeled vha-6 transgene. To study the contribution of vha-6 to pH homeostasis in larval worms, we used a partial reduction of function through postembryonic single-generation RNA interference. We demonstrate that the inability to fully acidify the intestinal lumen coincides with a defect in pH recovery of the intestinal epithelial cells, suggesting that VHA-6 is essential for proton pumping following defecation. Moreover, intestinal dipeptide accumulation and fat storage are compromised by the loss of VHA-6, suggesting that luminal acidification promotes nutrient uptake in worms, as well as in mammals. Since acidified intracellular vesicles and autofluorescent storage granules are indistinguishable between the vha-6 mutant and controls, it is likely that the nutrient-restricted phenotype is due to a loss of plasma membrane V-ATPase activity specifically. These data establish a simple genetic model for proton pump-driven acidification. Since defecation occurs at 45-s intervals in worms, this model represents an opportunity to study acute regulation of V-ATPase activity on a short time scale and may be useful in the study of alternative treatments for acid-peptic disorders.
Amino acid absorption in the form of di- and tripeptides is mediated by the intestinal proton-coupled peptide transporter PEPT-1 (formally OPT-2) in Caenorhabditits elegans. Transporter-deficient animals (pept-1(lg601)) show impaired growth, slowed postembryonal development and major changes in amino acid status.
Here we demonstrate that abolished intestinal peptide transport also leads to major metabolic alterations that culminate in a two fold increase in total body fat content. Feeding of C. elegans with [U-(13)C]-labelled E. coli revealed a decreased de novo synthesis of long-chain fatty acids in pept-1(lg601) and reduced levels of polyunsaturated fatty acids. mRNA profiling revealed increased transcript levels of enzymes/transporters needed for peroxisomal beta-oxidation and decreased levels for those required for fatty acid synthesis, elongation and desaturation. As a prime and most fundamental process that may account for the increased fat content in pept-1(lg601) we identified a highly accelerated absorption of free fatty acids from the bacterial food in the intestine.
The influx of free fatty acids into intestinal epithelial cells is strongly dependent on alterations in intracellular pH which is regulated by the interplay of PEPT-1 and the sodium-proton exchanger NHX-2. We here provide evidence for a central mechanism by which the PEPT-1/NHX-2 system strongly influences the in vivo fat content of C. elegans. Loss of PEPT-1 decreases intestinal proton influx leading to a higher uptake of free fatty acids with fat accumulation whereas loss of NHX-2 causes intracellular acidification by the PEPT-1 mediated proton/dipeptide symport with an almost abolished uptake of fatty acids and a lean phenotype.
Acid-sensitive K+ channels of the tandem P-domain K+-channel family (TASK-1 and TASK-3) have been implicated in peripheral and central respiratory chemosensitivity; however, because of the lack of decisive pharmacological agents, the final proof of the role of the TASK channel in the chemosensory control of breathing has been missing. In the mouse, TASK-1 and TASK-3 channels are dispensable for central respiratory chemosensitivity (Mulkey et al., 2007). Here, we have used knock-out animals to determine whether TASK-1 and TASK-3 channels play a role in the carotid body function and chemosensory control of breathing exerted by the carotid body chemoreceptors. Ventilatory responses to hypoxia (10% O2 in inspired air) and moderate normoxic hypercapnia (3-6% CO2 in inspired air) were significantly reduced in TASK-1 knock-out mice. In contrast, TASK-3-deficient mice showed responses to both stimuli that were similar to those developed by their wild-type counterparts. TASK-1 channel deficiency resulted in a marked reduction of the hypoxia (by 49%)- and CO2 (by 68%)-evoked increases in the carotid sinus nerve chemoafferent discharge recorded in the in vitro superfused carotid body/carotid sinus nerve preparations. Deficiency in both TASK-1 and TASK-3 channels increased baseline chemoafferent activity but did not cause a further reduction of the carotid body chemosensory responses. These observations provide direct evidence that TASK-1 channels contribute significantly to the increases in the carotid body chemoafferent discharge in response to a decrease in arterial P(O2) or an increase in P(CO2)/[H+]. TASK-1 channels therefore play a key role in the control of ventilation by peripheral chemoreceptors.
The genes encoding the mitogen-activated protein kinases DRL-1 and FLR-4 are required for growth and lipid homeostasis in C. elegans . Interestingly, the flr-3 ( ut9 ) mutant, which was previously isolated in a forward genetic screen for mutations that confer fluoride resistance, phenocopies the drl-1 and flr-4 loss-of-function mutants; however, the genetic identity of flr-3 is unknown. Through whole genome sequencing, we found that the flr-3 ( ut9 ) mutation is an insertion in the drl-1 locus and disrupts drl-1 gene function, resulting in dramatic growth defects and impaired vitellogenin production.
Ion channels and transporters are ubiquitously expressed on cell membrane, which involve in a plethora of physiological process such as contraction, neurotransmission, secretion and so on. Ion channels and transporters is of great importance to maintaining membrane potential homeostasis, which is essential to absorption of nutrients in gastrointestinal tract. Most of nutrients are electrogenic and require ion channels and transporters to absorb. This review summarizes the latest research on the role of ion channels and transporters in regulating nutrient uptake such as K+ channels, Ca2+ channels and ion exchangers. Revealing the mechanism of ion channels and transporters associated with nutrient uptake will be helpful to provide new methods to diagnosis and find potential targets for diseases like diabetes, inflammatory bowel diseases, etc. Even though some of study still remain ambiguous and in early stage, we believe that ion channels and transporters will be novel therapeutic targets in the future.
Defecation in the nematode Caenorhabditis elegans is achieved by a cyclical stereotyped motor program. The first step in each cycle is contraction of a set of posterior body muscles (pBoc), followed by contraction of a set of anterior body muscles (aBoc), and finally contraction of specialized anal muscles that open the anus and expel intestinal contents (Exp). By testing existing behavioral mutants and screening for new mutants that become constipated due to defects in defecation, I have identified 18 genes that are involved in defecation. Mutations in 16 of these genes affect specific parts of the motor program: mutations in two genes specifically affect the pBoc step; mutations in four genes affect the aBoc step; mutations in four genes affect the Exp step; and mutations in six genes affect both aBoc and Exp. Mutations in two other genes affect the defecation cycle period but have a normal motor program. Sensory inputs that regulate the cycle timing in the wild type are also described. On the basis of the phenotypes of the defecation mutants and of double mutants, I suggest a formal genetic pathway for the control of the defecation motor program.
ERM proteins are conserved regulators of cortical membrane specialization, that function as membrane-actin linkers and molecular hubs. Activity of ERM proteins requires a conformational switch from an inactive cytoplasmic form into an active membrane- and actin-bound form, which is thought to be mediated by sequential PIP2-binding and phosphorylation of a conserved C-terminal threonine residue. Here, we use the single C. elegans ERM ortholog, ERM-1, to study the contribution of these regulatory events to ERM activity and tissue formation in vivo Using CRISPR/Cas9-generated erm-1 mutant alleles we demonstrate that a PIP2-binding site is critically required for ERM-1 function. In contrast, dynamic regulation of C-terminal T544 phosphorylation is not essential but modulates ERM-1 apical localization and dynamics in a tissue-specific manner, to control cortical actin organization and support lumen formation in epithelial tubes. Our work highlights the dynamic nature of ERM protein regulation during tissue morphogenesis and the importance of C-terminal phosphorylation in fine-tuning ERM activity in a tissue-specific context.
The molecular basis of how animals integrate metabolic, developmental, and environmental information before committing resources to reproduction is an unresolved issue in developmental biology. In C. elegans, adult animals reallocate fat stores from intestinal cells to the germline via low-density lipoprotein (LDL)-like particles to promote embryogenesis. Here, I demonstrate that two conserved homeodomain transcription factors, CEH-60/PBX and UNC-62/MEIS, coordinate a transcriptional network that supports reproduction while suppressing longevity and stress-response pathways. The CEH-60:UNC-62 heterodimer serves an unanticipated dual function in intestinal nuclei by directly activating the expression of lipoprotein genes while directly repressing stress-responsive genes. Consequently, ceh-60 mutants display fat storage defects, a dramatic lifespan extension, and hyper-activation of innate immunity genes. Finally, CEH-60 associates with PQM-1 at the DAF-16-associated element within the promoters of stress-responsive genes to control gene expression. Thus, CEH-60 governs an elaborate transcriptional network that balances stress responses and longevity against reproduction during developmental transitions. Reproductive success often occurs at the expense of longevity and stress resistance. Dowen identifies two key molecular regulators of this trade-off, CEH-60/PBX and UNC-62/MEIS, in C. elegans. These transcription factors function together to balance the allocation of lipid resources between somatic and germline functions to ensure organismal energy homeostasis.
The ability to distinguish harmful and beneficial microbes is critical for the survival of an organism. Here, we show that bloating of the intestinal lumen of Caenorhabditis elegans caused by microbial colonization elicits a microbial aversion behavior. Bloating of the intestinal lumen also activates a broad innate immune response, even in the absence of bacterial pathogens or live bacteria. Neuroendocrine pathway genes are upregulated by intestinal bloating and are required for microbial aversion behavior. We propose that microbial colonization and bloating of the intestine may be perceived as a danger signal that activates an immune fight-and-flight response. These results reveal how inputs from the intestine can aid in the recognition of a broad range of microbes and modulate host behavior via neuroendocrine signaling.
Adjusting the efficiency of movement in response to environmental cues is an essential integrative characteristic of adaptive locomotion behavior across species. However, the modulatory molecules and the pathways involved are largely unknown. Recently, we demonstrated that in Caenorhabditis elegans, a loss of function of the two-pore-domain potassium (K2P) channel TWK-7 causes a fast, coordinated, persistent forward crawling behavior in which five central aspects of stimulated locomotion -- velocity, direction, wave parameters, duration and straightness -- are affected. Here, we isolated the reduction-of-function allele cau1 of the C. elegans gene kin-2 in a forward genetic screen and showed that it phenocopies the locomotion activity and locomotion behavior of twk-7(null) animals. Kin-2 encodes the negative regulatory subunit of protein kinase A (KIN-1/PKA). Consistently, we found that other gain-of-function mutants of the G?S-KIN-1/PKA pathway resemble kin-2(cau1) and twk-7(null) in locomotion phenotype. Using the powerful genetics of the C. elegans system in combination with cell-type-specific approaches and detailed locomotion analyses, we identified TWK-7 as a putative downstream target of the G?S-KIN-1/PKA pathway at the level of the GABAergic D-type motor neurons. Due to this epistatic interaction, we suggest that KIN-1/PKA and TWK-7 may share a common pathway that is probably involved in the modulation of both locomotor activity and locomotion behavior during forward crawling.
Inflammatory bowel disease (IBD) is a chronic inflammatory disorder with a complex pathogenesis. Diarrhea is a highly prevalent and often debilitating symptom of IBD patients that results, at least in part, from an intestinal hydro-electrolytic imbalance. Evidence suggests that reduced electrolyte absorption is more relevant than increased secretion to this disequilibrium. This systematic review analyses and integrates the current evidence on the roles of epithelial Na(+),K(+)-ATPase (NKA), Na(+)/H(+) exchangers (NHE), epithelial Na(+) channels (ENaC) and K(+) channels (KC) in IBD-associated diarrhea. NKA is the key driving force of the transepithelial ionic transport and its activity is decreased in IBD. In addition, the downregulation of apical NHE and ENaC and the upregulation of apical large-conductance KC all contribute to the IBD-associated diarrhea by lowering sodium absorption and/or increasing potassium secretion.
The ~100 MB genome of C. elegans codes for ~20,000 protein-coding genes many of which are required for the function of the nervous system, composed of 302 neurons in the adult hermaphrodite and of 383 neurons in the adult male. In addition to housekeeping genes, a differentiated neuron is thought to express many hundreds if not thousands of genes that define its functionally properties. These genes code for ion channels, G-protein-coupled receptors, neurotransmitter-synthesizing enzymes, transporters and receptors, neuropeptides and their receptors, cell adhesion molecules, motor proteins, signaling molecules and many others. Collectively such genes have been referred to as "terminal differentiation genes" or "effector genes". The differential expression of distinct combinations of terminal differentiation genes define different neuron types. This paper provides a compendium of more than 2,800 putative terminal differentiation genes. One pervasive theme revealed by the analysis of many gene families is the nematode-specific expansions of many neuron function-related gene families, including, for example, many types of ion channel families, sensory receptors and neurotransmitter receptors. The gene lists provided here can serve multiple purposes. They can serve as quick reference guides for individual gene families or they can be used to mine large datasets (e.g., expression datasets) for genes with likely functions in the nervous system. They also serve as a starting point for future projects. For example, a comprehensive understanding of the regulation of the often complex expression patterns of these genes in the nervous system will eventually explain how nervous systems are built.
Background:
Rhythmic behaviors are driven by endogenous biological clocks in pacemakers, which must reliably transmit timing information to target tissues that execute rhythmic outputs. During the defecation motor program in C. elegans, calcium oscillations in the pacemaker (intestine), which occur about every 50 s, trigger rhythmic enteric muscle contractions through downstream GABAergic neurons that innervate enteric muscles. However, the identity of the timing signal released by the pacemaker and the mechanism underlying the delivery of timing information to the GABAergic neurons are unknown.
Results:
Here, we show that a neuropeptide-like protein (NLP-40) released by the pacemaker triggers a single rapid calcium transient in the GABAergic neurons during each defecation cycle. We find that mutants lacking nlp-40 have normal pacemaker function, but lack enteric muscle contractions. NLP-40 undergoes calcium-dependent release that is mediated by the calcium sensor, SNT-2/synaptotagmin. We identify AEX-2, the G-protein-coupled receptor on the GABAergic neurons, as the receptor for NLP-40. Functional calcium imaging reveals that NLP-40 and AEX-2/GPCR are both necessary for rhythmic activation of these neurons. Furthermore, acute application of synthetic NLP-40-derived peptide depolarizes the GABAergic neurons in vivo.
Conclusions:
Our results show that NLP-40 carries the timing information from the pacemaker via calcium-dependent release and delivers it to the GABAergic neurons by instructing their activation. Thus, we propose that rhythmic release of neuropeptides can deliver temporal information from pacemakers to downstream neurons to execute rhythmic behaviors.
The mammalian target of rapamycin (MTOR) protein kinase complex is a key component of a pathway that regulates cell growth and proliferation in response to energy levels, hypoxia, nutrients and insulin. Inhibition of MTORC1 strongly induces autophagy by regulating the activity of the ULK protein kinase complex that is required for the formation of autophagosomes. However, the participation of MTORC1 in the expression of autophagy genes has not been characterized. Here we show that MTORC1 regulates nuclear localization and activity of the transcription factor EB (TFEB), a member of the bHLH leucine-zipper family of transcription factors that drives expression of autophagy and lysosomal genes. Under normal nutrient conditions, TFEB is phosphorylated in Ser211 in an MTORC1-dependent manner. This phosphorylation promotes association of TFEB with members of the YWHA (14-3-3) family of proteins and retention of the transcription factor in the cytosol. Pharmacological or genetic inhibition of MTORC1 causes dissociation of the TFEB/YWHA complex and rapid transport of TFEB to the nucleus where it increases transcription of multiple genes implicated in autophagy and lysosomal function. Active TFEB also associates with late endosomal/lysosomal membranes through interaction with the LAMTOR/RRAG/MTORC1 complex. Our results unveil a novel role for MTORC1 in the maintenance of cellular homeostasis by regulating autophagy at the transcriptional level.
Upon nutrient deprivation, cells are thought to suppress biosynthesis but activate catabolic pathways to provide alternative energy sources and nutrients. However, here we provide evidence that in adult male C. elegans, both biosynthesis and degradation activities, including ribosome biogenesis and turnover, are enhanced during early starvation and appear to depend on the availability of intestinal lipid stores. Upon depletion of the intestinal lipids, further food deprivation results in a significant reduction in metabolic activity in the starved male worms. Our data show that adult C. elegans exhibits a two-phase metabolic response to starvation stress: an initial phase with enhanced metabolic activity that rapidly exhausts the lipid stores, followed by a phase with low metabolic activity, which outlasts the life of fed control worms. DAF-2 insulin/IGF-1 receptor signaling to the RAS pathway is required for the starvation-induced ribosome biogenesis and rapid lipid depletion in the initial phase of starvation.
Defecation behavior in Caenorhabditis elegans is driven by an endogenous ultradian clock in the intestine. Its periods are positively regulated by FLR-1, an ion channel of the epithelial sodium channel/degenerin superfamily, and FLR-4, a protein kinase with a hydrophobic domain at the carboxyl terminus. FLR-1 has many putative phosphorylation sites in the C-terminal intracellular region. This structure implies that the periods may be regulated by the phosphorylation of FLR-1 by FLR-4, but it remains to be clarified. Here, we show that a truncated FLR-1 lacking the C-terminal intracellular region resulted in longer periods, suggesting that this region is involved in the negative regulation of defecation cycle periods. Contrary to our expectation, FLR-4 was still necessary for the function of the truncated FLR-1. Furthermore, FLR-4 containing a kinase-dead mutation or lacking the whole kinase domain was sufficient for normal defecation cycle periods. FLR-4 was necessary for the stable expression of FLR-1::GFP, and its hydrophobic domain was sufficient also for this function. FLR-1 and FLR-4 are often colocalized in the plasma membrane. These data showed an unexpected role of FLR-4: its hydrophobic domain stabilizes the FLR-1 ion channel, a key regulator of defecation cycle periods in the intestine.
The intestine plays an essential role in organism-wide regulatory networks in both vertebrates and invertebrates. In Caenorhabditis elegans, class 1 flr genes (flr-1, flr-3 and flr-4) act in the intestine and control growth rates and defecation cycle periods, while class 2 flr genes (flr-2, flr-5, flr-6 and flr-7) are characterized by mutations that suppress the slow growth of class 1 flr mutants. This study revealed that flr-2 gene controls antibacterial defense and intestinal color, confirming that flr-2 regulates intestinal functions. flr-2 encoded the only glycoprotein hormone alpha subunit in C. elegans and was expressed in certain neurons. Furthermore, FLR-2 bound to another secretory protein GHI-1, which belongs to a family of lipid- and lipopolysaccharide-binding proteins. A ghi-1 deletion mutation partially suppressed the short defecation cycle periods of class 1 flr mutants, and this effect was enhanced by flr-2 mutations. Thus, FLR-2 acts as a signaling molecule for the neural control of intestinal functions, which is achieved in a functional network involving class 1 and class 2 flr genes as well as ghi-1. These results are informative to studies of glycoprotein hormone signaling in higher animals.