Orcokinin neuropeptides regulate sleep in Caenorhabditis elegans.

Orcokinin neuropeptides are conserved among ecdysozoans, but their functions are incompletely understood. Here, we report a role for orcokinin neuropeptides in the regulation of sleep in the nematode Caenorhabditis elegans. The C. elegans orcokinin peptides, which are encoded by the nlp-14 and nlp-15 genes, are necessary and sufficient for quiescent behaviors during developmentally timed sleep (DTS) as well as during stress-induced sleep (SIS). The five orcokinin neuropeptides encoded by nlp-14 have distinct but overlapping functions in the regulation of movement and defecation quiescence during SIS. We suggest that orcokinins may regulate behavioral components of sleep-like states in nematodes and other ecdysozoans.

Structure-guided design and functional characterization of an artificial red light-regulated guanylate/adenylate cyclase for optogenetic applications.

Genetically targeting biological systems to control cellular processes with light is the concept of optogenetics. Despite impressive developments in this field, underlying molecular mechanisms of signal transduction of the employed photoreceptor modules are frequently not sufficiently understood to rationally design new optogenetic tools. Here, we investigate the requirements for functional coupling of red light-sensing phytochromes with non-natural enzymatic effectors by creating a series of constructs featuring the Deinococcus radiodurans bacteriophytochrome linked to a Synechocystis guanylate/adenylate cyclase. Incorporating characteristic structural elements important for cyclase regulation in our designs, we identified several red light-regulated fusions with promising properties. We provide details of one light-activated construct with low dark-state activity and high dynamic range that outperforms previous optogenetic tools in vitro and expands our in vivo toolkit, as demonstrated by manipulation of Caenorhabditis elegans locomotor activity. The full-length crystal structure of this phytochrome-linked cyclase revealed molecular details of photoreceptor-effector coupling, highlighting the importance of the regulatory cyclase element. Analysis of conformational dynamics by hydrogen-deuterium exchange in different functional states enriched our understanding of phytochrome signaling and signal integration by effectors. We found that light-induced conformational changes in the phytochrome destabilize the coiled-coil sensor-effector linker, which releases the cyclase regulatory element from an inhibited conformation, increasing cyclase activity of this artificial system. Future designs of optogenetic functionalities may benefit from our work, indicating that rational considerations for the effector improve the rate of success of initial designs to obtain optogenetic tools with superior properties.

Distinct Mechanisms Underlie Quiescence during Two Caenorhabditis elegans Sleep-Like States.

Electrophysiological recordings have enabled identification of physiologically distinct yet behaviorally similar states of mammalian sleep. In contrast, sleep in nonmammals has generally been identified behaviorally and therefore regarded as a physiologically uniform state characterized by quiescence of feeding and locomotion, reduced responsiveness, and rapid reversibility. The nematode Caenorhabditis elegans displays sleep-like quiescent behavior under two conditions: developmentally timed quiescence (DTQ) occurs during larval transitions, and stress-induced quiescence (SIQ) occurs in response to exposure to cellular stressors. Behaviorally, DTQ and SIQ appear identical. Here, we use optogenetic manipulations of neuronal and muscular activity, pharmacology, and genetic perturbations to uncover circuit and molecular mechanisms of DTQ and SIQ. We find that locomotion quiescence induced by DTQ- and SIQ-associated neuropeptides occurs via their action on the nervous system, although their neuronal target(s) and/or molecular mechanisms likely differ. Feeding quiescence during DTQ results from a loss of pharyngeal muscle excitability, whereas feeding quiescence during SIQ results from a loss of excitability in the nervous system. Together these results indicate that, as in mammals, quiescence is subserved by different mechanisms during distinct sleep-like states in C. elegans.

FMRFamide signaling promotes stress-induced sleep in Drosophila.

Enhanced sleep in response to cellular stress is a conserved adaptive behavior across multiple species, but the mechanism of this process is poorly understood. Drosophila melanogaster increases sleep following exposure to septic or aseptic injury, and Caenorhabditis elegans displays sleep-like quiescence following exposure to high temperatures that stress cells. We show here that, similar to C. elegans, Drosophila responds to heat stress with an increase in sleep. In contrast to Drosophila infection-induced sleep, heat-induced sleep is not sensitive to the time-of-day of the heat pulse. Moreover, the sleep response to heat stress does not require Relish, the NFκB transcription factor that is necessary for infection-induced sleep, indicating that sleep is induced by multiple mechanisms from different stress modalities. We identify a sleep-regulating role for a signaling pathway involving FMRFamide neuropeptides and their receptor FR. Animals mutant for either FMRFamide or for the FMRFamide receptor (FR) have a reduced recovery sleep in response to heat stress. FR mutants, in addition, show reduced sleep responses following infection with Serratia marcescens, and succumb to infection at a faster rate than wild-type controls. Together, these findings support the hypothesis that FMRFamide and its receptor promote an adaptive increase in sleep following stress. Because an FMRFamide-like neuropeptide plays a similar role in C. elegans, we propose that FRMFamide neuropeptide signaling is an ancient regulator of recovery sleep which occurs in response to cellular stress.

A bow-tie genetic architecture for morphogenesis suggested by a genome-wide RNAi screen in Caenorhabditis elegans.

During animal development, cellular morphogenesis plays a fundamental role in determining the shape and function of tissues and organs. Identifying the components that regulate and drive morphogenesis is thus a major goal of developmental biology. The four-celled tip of the Caenorhabditis elegans male tail is a simple but powerful model for studying the mechanism of morphogenesis and its spatiotemporal regulation. Here, through a genome-wide post-embryonic RNAi-feeding screen, we identified 212 components that regulate or participate in male tail tip morphogenesis. We constructed a working hypothesis for a gene regulatory network of tail tip morphogenesis. We found regulatory roles for the posterior Hox genes nob-1 and php-3, the TGF-ß pathway, nuclear hormone receptors (e.g. nhr-25), the heterochronic gene blmp-1, and the GATA transcription factors egl-18 and elt-6. The majority of the pathways converge at dmd-3 and mab-3. In addition, nhr-25 and dmd-3/mab-3 regulate each others' expression, thus placing these three genes at the center of a complex regulatory network. We also show that dmd-3 and mab-3 negatively regulate other signaling pathways and affect downstream cellular processes such as vesicular trafficking (e.g. arl-1, rme-8) and rearrangement of the cytoskeleton (e.g. cdc-42, nmy-1, and nmy-2). Based on these data, we suggest that male tail tip morphogenesis is governed by a gene regulatory network with a bow-tie architecture.

New alleles of nlp-2 , nlp-22 , and nlp-23 demonstrate that they are dispensable for stress-induced sleep in C. elegans.

Sleep is ancient and genetically conserved across phylogeny. Neuropeptide signaling plays a fundamental role in the regulation of sleep for mammals, fish, and invertebrates like Caenorhabditis elegans . Developmentally timed-sleep and stress-induced sleep of C. elegans are controlled by distinct and overlapping neuropeptide pathways. The RPamide neuropeptides nlp-2 , nlp-22 , and nlp-23 , play antagonistic roles during the regulation of developmentally-timed sleep, however, their role in stress-induced sleep has not been explored. These genes are linked on the X chromosome, which has made genetic analyses challenging. Here we used CRISPR to generate new alleles of nlp-22 and nlp-23 , nlp-22 ; nlp-23 double mutants, and nlp-2 ; nlp-22 ; nlp-23 triple mutants. Confirming previous studies, we find that nlp-22 is required for developmentally-timed sleep, and show that nlp-23 is also required. However, all three genes are dispensable for stress-induced sleep.

The neuropeptide receptor npr-38 regulates avoidance and stress-induced sleep in Caenorhabditis elegans.

Although essential and conserved, sleep is not without its challenges that must be overcome; most notably, it renders animals vulnerable to threats in the environment. Infection and injury increase sleep demand, which dampens sensory responsiveness to stimuli, including those responsible for the initial insult. Stress-induced sleep in Caenorhabditis elegans occurs in response to cellular damage following noxious exposures the animals attempted to avoid. Here, we describe a G-protein-coupled receptor (GPCR) encoded by npr-38, which is required for stress-related responses including avoidance, sleep, and arousal. Overexpression of npr-38 shortens the avoidance phase and causes animals to initiate movement quiescence and arouse early. npr-38 functions in the ADL sensory neurons, which express neuropeptides encoded by nlp-50, also required for movement quiescence. npr-38 regulates arousal by acting on the DVA and RIS interneurons. Our work demonstrates that this single GPCR regulates multiple aspects of the stress response by functioning in sensory and sleep interneurons.

An AMPK biosensor for Caenorhabditis elegans.

Adenosine monophosphate-activated kinase (AMPK) functions in a broad spectrum of cellular stress response pathways. Investigation of AMPK activity has been limited to whole-organism analyses in Caenorhabditis elegans which does not allow for observations of cellular heterogeneity, temporal dynamics, or correlation with physiological states in real time. We codon adapted the genetically-coded AMPK biosensor, called AMPKAR-EV, for use in C. elegans . We report heterogeneity of activation in different tissues (intestine, neurons, muscle) and test the biosensor in the context of two missense mutations affecting residues T243 and S244 on the AMPK α subunit, AAK-2, which are predicted regulatory sites.

Analysis of detergent-free lipid rafts isolated from CD4+ T cell line: interaction with antigen presenting cells promotes coalescing of lipid rafts.

BACKGROUND: Lipid rafts present on the plasma membrane play an important role in spatiotemporal regulation of cell signaling. Physical and chemical characterization of lipid raft size and assessment of their composition before, and after cell stimulation will aid in developing a clear understanding of their regulatory role in cell signaling. We have used visual and biochemical methods and approaches for examining individual and lipid raft sub-populations isolated from a mouse CD4+ T cell line in the absence of detergents. RESULTS: Detergent-free rafts were analyzed before and after their interaction with antigen presenting cells. We provide evidence that the average diameter of lipid rafts isolated from un-stimulated T cells, in the absence of detergents, is less than 100 nm. Lipid rafts on CD4+ T cell membranes coalesce to form larger structures, after interacting with antigen presenting cells even in the absence of a foreign antigen. CONCLUSIONS: Findings presented here indicate that lipid raft coalescence occurs during cellular interactions prior to sensing a foreign antigen.

Comparative analysis of CAX2-like cation transporters indicates functional and regulatory diversity.

Internal compartmentalization of metals is an important metal tolerance mechanism in many organisms. In plants and fungi, sequestration into the vacuole is a major detoxification mechanism for metals. Cation transport into the vacuole can be mediated by CAX (cation exchanger) transporters. The Arabidopsis thaliana AtCAX2 transporter was shown previously to transport Ca(2+), Cd(2+) and Mn(2+). To assess the conservation of the functional and regulatory characteristics of CAX2-like transporters in higher plants, we have characterized AtCAX2 orthologues from Arabidopsis (AtCAX5), tomato (LeCAX2) and barley (HvCAX2). Substrate specificity and regulatory activity were assessed using a yeast heterologous-expression assay. Each CAX could transport Ca(2+) and Mn(2+) into the yeast vacuole, but they each had different cation transport kinetics. Most notably, there was variation in the regulation of the transporters. As found with AtCAX2 previously, only expression of an N-terminally truncated form of AtCAX5 in yeast was able to mediate Ca(2+) and Mn(2+) transport, indicating that activity may be controlled by an autoregulatory region at the N-terminus. In contrast, either full-length or truncated LeCAX2 could efficiently transport Ca(2+), although Mn(2+) transport was controlled by the N-terminus. HvCAX2 did not appear to possess an N-terminal regulatory domain. Expression of AtCAX2 was not significantly modulated by metal stress; however, AtCAX5 and HvCAX2 were transcriptionally up-regulated by high Mn(2+) treatment, and by Ca(2+) and Na(+) stress respectively. It is therefore apparent that, despite the high sequence identity between plant CAX2 orthologues, there is significant diversity in their functional characteristics, particularly with regard to regulatory mechanisms.

Teething during sleep: Ultrastructural analysis of pharyngeal muscle and cuticular grinder during the molt in Caenorhabditis elegans.

Complex extracellular structures exist throughout phylogeny, but the dynamics of their formation and dissolution are often opaque. One example is the pharyngeal grinder of the nematode Caenorhabditis elegans, an extracellular structure that ruptures bacteria during feeding. During each larval transition stage, called lethargus, the grinder is replaced with one of a larger size. Here, we characterize at the ultrastructural level the deconstruction of the larval grinder and the construction of the adult grinder during the fourth larval stage (L4)-to-adult transition. Early in L4 lethargus, pharyngeal muscle cells trans-differentiate from contractile to secretory cells, as evidenced by the appearance of clear and dense core vesicles and disruptions in sarcomere organization. This is followed, within minutes, by the dissolution of the L4 grinder and the formation and maturation of the adult grinder. Components of the nascent adult grinder are deposited basally, and are separated from the dissolving larval grinder by a visible apical layer. The complete grinder is a lamellated extracellular matrix comprised of five layers. Following grinder formation, pharyngeal muscle cells regain ultrastructural contractile properties, and muscle contractions resume. Our findings add to our understanding of how complex extracellular structures assemble and dissemble.

RPamide neuropeptides NLP-22 and NLP-2 act through GnRH-like receptors to promote sleep and wakefulness in C. elegans.

Sleep and wakefulness are fundamental behavioral states of which the underlying molecular principles are becoming slowly elucidated. Transitions between these states require the coordination of multiple neurochemical and modulatory systems. In Caenorhabditis elegans sleep occurs during a larval transition stage called lethargus and is induced by somnogenic neuropeptides. Here, we identify two opposing neuropeptide/receptor signaling pathways: NLP-22 promotes behavioral quiescence, whereas NLP-2 promotes movement during lethargus, by signaling through gonadotropin-releasing hormone (GnRH) related receptors. Both NLP-2 and NLP-22 belong to the RPamide neuropeptide family and share sequence similarities with neuropeptides of the bilaterian GnRH, adipokinetic hormone (AKH) and corazonin family. RPamide neuropeptides dose-dependently activate the GnRH/AKH-like receptors GNRR-3 and GNRR-6 in a cellular receptor activation assay. In addition, nlp-22-induced locomotion quiescence requires the receptor gnrr-6. By contrast, wakefulness induced by nlp-2 overexpression is diminished by deletion of either gnrr-3 or gnrr-6. nlp-2 is expressed in a pair of olfactory AWA neurons and cycles with larval periodicity, as reported for nlp-22, which is expressed in RIA. Our data suggest that the somnogenic NLP-22 neuropeptide signals through GNRR-6, and that both GNRR-3 and GNRR-6 are required for the wake-promoting action of NLP-2 neuropeptides.

Interneurons Regulate Locomotion Quiescence via Cyclic Adenosine Monophosphate Signaling During Stress-Induced Sleep in Caenorhabditis elegans.

Sleep is evolutionarily conserved, thus studying simple invertebrates such as Caenorhabditis elegans can provide mechanistic insight into sleep with single cell resolution. A conserved pathway regulating sleep across phylogeny involves cyclic adenosine monophosphate (cAMP), a ubiquitous second messenger that functions in neurons by activating protein kinase A. C. elegans sleep in response to cellular stress caused by environmental insults [stress-induced sleep (SIS)], a model for studying sleep during sickness. SIS is controlled by simple neural circuitry, thus allowing for cellular dissection of cAMP signaling during sleep. We employed a red-light activated adenylyl cyclase, IlaC22, to identify cells involved in SIS regulation. We found that pan-neuronal activation of IlaC22 disrupts SIS through mechanisms independent of the cAMP response element binding protein. Activating IlaC22 in the single DVA interneuron, the paired RIF interneurons, and in the CEPsh glia identified these cells as wake-promoting. Using a cAMP biosensor, epac1-camps, we found that cAMP is decreased in the RIF and DVA interneurons by neuropeptidergic signaling from the ALA neuron. Ectopic overexpression of sleep-promoting neuropeptides coded by flp-13 and flp-24, released from the ALA, reduced cAMP in the DVA and RIFs, respectively. Overexpression of the wake-promoting neuropeptides coded by pdf-1 increased cAMP levels in the RIFs. Using a combination of optogenetic manipulation and in vivo imaging of cAMP we have identified wake-promoting neurons downstream of the neuropeptidergic output of the ALA. Our data suggest that sleep- and wake-promoting neuropeptides signal to reduce and heighten cAMP levels during sleep, respectively.

The RFamide receptor DMSR-1 regulates stress-induced sleep in C. elegans.

In response to environments that cause cellular stress, animals engage in sleep behavior that facilitates recovery from the stress. In Caenorhabditis elegans, stress-induced sleep(SIS) is regulated by cytokine activation of the ALA neuron, which releases FLP-13 neuropeptides characterized by an amidated arginine-phenylalanine (RFamide) C-terminus motif. By performing an unbiased genetic screen for mutants that impair the somnogenic effects of FLP-13 neuropeptides, we identified the gene dmsr-1, which encodes a G-protein coupled receptor similar to an insect RFamide receptor. DMSR-1 is activated by FLP-13 peptides in cell culture, is required for SIS in vivo, is expressed non-synaptically in several wake-promoting neurons, and likely couples to a Gi/o heterotrimeric G-protein. Our data expand our understanding of how a single neuroendocrine cell coordinates an organism-wide behavioral response, and suggest that similar signaling principles may function in other organisms to regulate sleep during sickness.

FRPR-4 Is a G-Protein Coupled Neuropeptide Receptor That Regulates Behavioral Quiescence and Posture in Caenorhabditis elegans.

Neuropeptides signal through G-protein coupled receptors (GPCRs) to regulate a broad array of animal behaviors and physiological processes. The Caenorhabditis elegans genome encodes approximately 100 predicted neuropeptide receptor GPCRs, but in vivo roles for only a few have been identified. We describe here a role for the GPCR FRPR-4 in the regulation of behavioral quiescence and locomotive posture. FRPR-4 is activated in cell culture by several neuropeptides with an amidated isoleucine-arginine-phenylalanine (IRF) motif or an amidated valine-arginine-phenylalanine (VRF) motif at their carboxy termini, including those encoded by the gene flp-13. Loss of frpr-4 function results in a minor feeding quiescence defect after heat-induced cellular stress. Overexpression of frpr-4 induces quiescence of locomotion and feeding as well as an exaggerated body bend posture. The exaggerated body bend posture requires the gene flp-13. While frpr-4 is expressed broadly, selective overexpression of frpr-4 in the proprioceptive DVA neurons results in exaggerated body bends that require flp-13 in the ALA neuron. Our results suggest that FLP-13 and other neuropeptides signal through FRPR-4 and other receptors to regulate locomotion posture and behavioral quiescence.

FMRFamide-like FLP-13 neuropeptides promote quiescence following heat stress in Caenorhabditis elegans.

Among the most important decisions an animal makes is whether to engage in active movement and feeding behavior or to become quiescent. The molecular signaling mechanisms underlying this decision remain largely unknown. The nematode Caenorhabditis elegans displays sleep-like quiescence following exposures that result in cellular stress. The neurosecretory ALA neuron is required for this stress-induced recovery quiescence, but the mechanisms by which ALA induces quiescence have been unknown. We report here that quiescence induced by heat stress requires ALA depolarization and release of FMRFamide-like neuropeptides encoded by the flp-13 gene. Optogenetic activation of ALA reduces feeding and locomotion in a FLP-13-dependent manner. Overexpression of flp-13 is sufficient to induce quiescent behavior during normally active periods. We have here identified a major biological role for FMRFamide-like neuropeptides in nematodes, and we suggest that they may function in a similar capacity in other organisms.

A sleep state during C. elegans development.

Caenorhabditis elegans is the simplest animal shown to sleep. It sleeps during lethargus, a larval transition stage. Behavior during lethargus has the sleep properties of a specific quiescent posture and elevated arousal threshold that are reversible to strong stimulation and of increased sleep drive following sleep deprivation. Genetic similarities between sleep regulation during C. elegans lethargus and sleep regulation in other animals point to a sleep state that was an evolutionarily ancestor to sleep both in C. elegans and other animals. Recent publications have shed light on key questions in sleep biology: First, How is sleep regulated? Second, How is sensory information gated during sleep? Third, How is sleep homeostasis mediated? Fourth, What is the core function of sleep?

Overlap extension PCR: an efficient method for transgene construction.

Combining genes or regulatory elements to make hybrid genes is a widely used methodology throughout the biological sciences. Here, we describe an optimized approach for hybrid gene construction called overlap extension PCR. In this method, the polymerase chain reaction (PCR) is employed for efficient and reliable construction of hybrid genes. A PCR-based approach does not rely on available restriction sites or other specific sequences, an advantage over more conventional cloning or recombineering methods. With the use of high-fidelity DNA polymerase, this method can be used for making even very large constructs (>20 kb) with minimal unwanted mutations. Finally, overlap extension-PCR can be used as a means for site-directed mutagenesis, introducing desired mutations to the final hybrid gene.

A novel approach to examining compositional heterogeneity of detergent-resistant lipid rafts.

Lipid rafts play an important role in cell signalling, cell adhesion and other cellular functions. Compositional heterogeneity of lipid rafts provides one mechanism of how lipid rafts provide the spatial and temporal regulation of cell signalling and cell adhesion. The constitutive presence of some signalling receptors/molecules and accumulation of others in the lipid raft allows them to interact with each other and thereby facilitate relay of signals from the plasma membrane to the cell interior. Devising a method that can analyze these lipid microdomains for the presence of signalling receptors/molecules on an individual raft basis is required to address the issue of lipid raft heterogeneity. SDS-PAGE analysis, currently used for analyses of detergent-resistant lipid rafts, does not address this question. We have designed a cell-free assay that captures detergent-resistant lipid rafts with an antibody against a raft-resident molecule and detects the presence of another lipid raft molecule. Our results suggest that detergent-resistant lipid rafts, also known as detergent-resistant membranes, are heterogeneous populations on an immortalized mouse T-cell plasma membrane with respect to antigen receptor/signalling complex and other signalling/adhesion proteins. This cell-free assay provides a simple and quick way to examine the simultaneous presence of two proteins in the lipid rafts and has the potential to estimate trafficking of molecules in and out of the lipid microdomains during cell signalling on a single detergent-resistant lipid raft basis.