scholarly journals The Enigmatic Canal-Associated Neurons Regulate Caenorhabditis elegans Larval Development Through a cAMP Signaling Pathway

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1465-1478
Author(s):  
Jason Chien ◽  
Fred W. Wolf ◽  
Sarah Grosche ◽  
Nebeyu Yosef ◽  
Gian Garriga ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Adenylyl Cyclase ◽  
Larval Development ◽  
Target Cells ◽  
Specific Expression ◽  
C Elegans ◽  
Link Type ◽  
Similar Phenotype ◽  
Link Loss ◽  
Pka Pathway

Caenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development, and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29. ACY-2 is expressed predominantly in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose two models for how ACY-2 activity in the CANs regulates KIN-29 in target cells.

scholarly journals A Canal-Associated Neuron cAMP signalling pathway that regulates C. elegans larval development

2019 ◽  
Author(s):  
Jason Chien ◽  
Fred W. Wolf ◽  
Sarah Grosche ◽  
Nebeyu Yosef ◽  
Gian Garriga ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Cyclic Amp ◽  
Adenylyl Cyclase ◽  
Larval Development ◽  
Laser Microsurgery ◽  
Gain Of Function ◽  
Specific Expression ◽  
C Elegans ◽  
Similar Phenotype ◽  
Pka Pathway

ABSTRACTCaenorhabditis elegans larval development requires the function of the two Canal-Associated Neurons (CANs): killing the CANs by laser microsurgery or disrupting their development by mutating the gene ceh-10 results in early larval arrest. How these cells promote larval development, however, remains a mystery. In screens for mutations that bypass CAN function, we identified the gene kin-29, which encodes a member of the Salt-Inducible Kinase (SIK) family and a component of a conserved pathway that regulates various C. elegans phenotypes. Like kin-29 loss, gain-of-function mutations in genes that may act upstream of kin-29 or growth in cyclic-AMP analogs bypassed ceh-10 larval arrest, suggesting that a conserved adenylyl cyclase/PKA pathway inhibits KIN-29 to promote larval development and that loss of CAN function results in dysregulation of KIN-29 and larval arrest. The adenylyl cyclase ACY-2 mediates CAN-dependent larval development: acy-2 mutant larvae arrested development with a similar phenotype to ceh-10 mutants, and the arrest phenotype was suppressed by mutations in kin-29. ACY-2 is predominantly expressed in the CANs, and we provide evidence that the acy-2 functions in the CANs to promote larval development. By contrast, cell-specific expression experiments suggest that kin-29 acts in both the hypodermis and neurons, but not in the CANs. Based on our findings, we propose that cAMP produced by ACY-2 in the CANs acts in neighboring neurons and hypodermal cells where it activates PKA and inhibits KIN-29 to promote larval development. We discuss how this conserved pathway could be partitioned between two cells.

scholarly journals Insights into the Involvement of Spliceosomal Mutations in Myelodysplastic Disorders from Analysis of SACY-1/DDX41 in Caenorhabditis elegans

Genetics ◽  
2020 ◽  
Vol 214 (4) ◽  
pp. 869-893 ◽  
Author(s):  
Tatsuya Tsukamoto ◽  
Micah D. Gearhart ◽  
Seongseop Kim ◽  
Gemechu Mekonnen ◽  
Caroline A. Spike ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Myelodysplastic Syndromes ◽  
Missense Mutations ◽  
Synthetic Lethal ◽  
C Elegans ◽  
Link Type ◽  
Dead Box ◽  
Synthetic Lethal Interactions ◽  
Biochemical Analyses ◽  
Cellular Phenotypes

Mutations affecting spliceosomal proteins are frequently found in hematological malignancies, including myelodysplastic syndromes and acute myeloid leukemia (AML). DDX41/Abstrakt is a metazoan-specific spliceosomal DEAD-box RNA helicase that is recurrently mutated in inherited myelodysplastic syndromes and in relapsing cases of AML. The genetic properties and genomic impacts of disease-causing missense mutations in DDX41 and other spliceosomal proteins have been uncertain. Here, we conduct a comprehensive analysis of the Caenorhabditis elegans DDX41 ortholog, SACY-1. Biochemical analyses defined SACY-1 as a component of the C. elegans spliceosome, and genetic analyses revealed synthetic lethal interactions with spliceosomal components. We used the auxin-inducible degradation system to analyze the consequence of SACY-1 depletion on the transcriptome using RNA sequencing. SACY-1 depletion impacts the transcriptome through splicing-dependent and splicing-independent mechanisms. Altered 3′ splice site usage represents the predominant splicing defect observed upon SACY-1 depletion, consistent with a role for SACY-1 in the second step of splicing. Missplicing events appear more prevalent in the soma than the germline, suggesting that surveillance mechanisms protect the germline from aberrant splicing. The transcriptome changes observed after SACY-1 depletion suggest that disruption of the spliceosome induces a stress response, which could contribute to the cellular phenotypes conferred by sacy-1 mutant alleles. Multiple sacy-1/ddx41 missense mutations, including the R525H human oncogenic variant, confer antimorphic activity, suggesting that their incorporation into the spliceosome is detrimental. Antagonistic variants that perturb the function of the spliceosome may be relevant to the disease-causing mutations, including DDX41, affecting highly conserved components of the spliceosome in humans.

scholarly journals Dehydrated Caenorhabditis elegans Stocks Are Resistant to Multiple Freeze-Thaw Cycles

2020 ◽  
Vol 10 (12) ◽  
pp. 4505-4512
Author(s):  
Patrick D. McClanahan ◽  
Richard J. McCloskey ◽  
Melanie Ng Tung Hing ◽  
David M. Raizen ◽  
Christopher Fang-Yen
Keyword(s):  
Caenorhabditis Elegans ◽  
Freeze Thaw ◽  
C Elegans ◽  
Link Type ◽  
Defective Mutant ◽  
Genetic Stocks ◽  
Dauer Larvae

Ultracold preservation is widely used for storage of genetic stocks of Caenorhabditis elegans. Current cryopreservation protocols are vulnerable to refrigeration failures, which can result in the loss of stock viability due to damage during re-freezing. Here we present a method for preserving worms in a dehydrated and frozen form that retains viability after multiple freeze-thaw cycles. After dehydration in the presence of trehalose or glycerol, C. elegans stocks can be frozen and thawed multiple times while maintaining viability. While both dauer and non-dauer larvae survive desiccation and freezing, the dauer defective mutant daf-16 does not survive desiccation. Our technique is useful for storing stocks in a manner robust to freezer failures, and potentially for shipping strains between laboratories.

scholarly journals Rapid Degradation of Caenorhabditis elegans Proteins at Single-Cell Resolution with a Synthetic Auxin

2019 ◽  
Vol 10 (1) ◽  
pp. 267-280 ◽  
Author(s):  
Michael A. Q. Martinez ◽  
Brian A. Kinney ◽  
Taylor N. Medwig-Kinney ◽  
Guinevere Ashley ◽  
James M. Ragle ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Protein Degradation ◽  
Single Cell ◽  
Water Soluble ◽  
Growth Media ◽  
Synthetic Analog ◽  
Rapid Degradation ◽  
Target Proteins ◽  
C Elegans ◽  
Link Type

As developmental biologists in the age of genome editing, we now have access to an ever-increasing array of tools to manipulate endogenous gene expression. The auxin-inducible degradation system allows for spatial and temporal control of protein degradation via a hormone-inducible Arabidopsis F-box protein, transport inhibitor response 1 (TIR1). In the presence of auxin, TIR1 serves as a substrate-recognition component of the E3 ubiquitin ligase complex SKP1-CUL1-F-box (SCF), ubiquitinating auxin-inducible degron (AID)-tagged proteins for proteasomal degradation. Here, we optimize the Caenorhabditis elegans AID system by utilizing 1-naphthaleneacetic acid (NAA), an indole-free synthetic analog of the natural auxin indole-3-acetic acid (IAA). We take advantage of the photostability of NAA to demonstrate via quantitative high-resolution microscopy that rapid degradation of target proteins can be detected in single cells within 30 min of exposure. Additionally, we show that NAA works robustly in both standard growth media and physiological buffer. We also demonstrate that K-NAA, the water-soluble, potassium salt of NAA, can be combined with microfluidics for targeted protein degradation in C. elegans larvae. We provide insight into how the AID system functions in C. elegans by determining that TIR1 depends on C. elegansSKR-1/2, CUL-1, and RBX-1 to degrade target proteins. Finally, we present highly penetrant defects from NAA-mediated degradation of the FTZ-F1 nuclear hormone receptor, NHR-25, during C. elegans uterine-vulval development. Together, this work improves our use and understanding of the AID system for dissecting gene function at the single-cell level during C. elegans development.

scholarly journals DAF-16 and SMK-1 Contribute to Innate Immunity During Adulthood in Caenorhabditis elegans

2020 ◽  
Vol 10 (5) ◽  
pp. 1521-1539 ◽  
Author(s):  
Daniel R. McHugh ◽  
Elena Koumis ◽  
Paul Jacob ◽  
Jennifer Goldfarb ◽  
Michelle Schlaubitz-Garcia ◽  
...  
Keyword(s):  
Innate Immunity ◽  
Immune System ◽  
Caenorhabditis Elegans ◽  
Host Defense ◽  
Insulin Signaling ◽  
Bacterial Pathogens ◽  
C Elegans ◽  
Link Type ◽  
Age Dependent ◽  
Over Time

Aging is accompanied by a progressive decline in immune function termed “immunosenescence”. Deficient surveillance coupled with the impaired function of immune cells compromises host defense in older animals. The dynamic activity of regulatory modules that control immunity appears to underlie age-dependent modifications to the immune system. In the roundworm Caenorhabditis elegans levels of PMK-1 p38 MAP kinase diminish over time, reducing the expression of immune effectors that clear bacterial pathogens. Along with the PMK-1 pathway, innate immunity in C. elegans is regulated by the insulin signaling pathway. Here we asked whether DAF-16, a Forkhead box (FOXO) transcription factor whose activity is inhibited by insulin signaling, plays a role in host defense later in life. While in younger C. elegansDAF-16 is inactive unless stimulated by environmental insults, we found that even in the absence of acute stress the transcriptional activity of DAF-16 increases in an age-dependent manner. Beginning in the reproductive phase of adulthood, DAF-16 upregulates a subset of its transcriptional targets, including genes required to kill ingested microbes. Accordingly, DAF-16 has little to no role in larval immunity, but functions specifically during adulthood to confer resistance to bacterial pathogens. We found that DAF-16-mediated immunity in adults requires SMK-1, a regulatory subunit of the PP4 protein phosphatase complex. Our data suggest that as the function of one branch of the innate immune system of C. elegans (PMK-1) declines over time, DAF-16-mediated immunity ramps up to become the predominant means of protecting adults from infection, thus reconfiguring immunity later in life.

Effect of structurally related flavonoids from Zuccagnia punctata Cav. on Caenorhabditis elegans

2014 ◽  
Vol 60 (1) ◽  
Author(s):  
Romina E. D’Almeida ◽  
María R. Alberto ◽  
Phillip Morgan ◽  
Margaret Sedensky ◽  
María I. Isla
Keyword(s):  
Caenorhabditis Elegans ◽  
Larval Development ◽  
Free Living ◽  
Egg Hatching ◽  
C Elegans ◽  
Therapeutic Drugs ◽  
Aerial Parts ◽  
Free Living Nematode ◽  
Development Processes ◽  
Anthelmintic Effect

AbstractZuccagnia punctata Cav. (Fabaceae), commonly called jarilla macho or pus-pus, is being used in traditional medicine as an antiseptic, anti-inflammatory and to relieve muscle and bone pain. The aim of this work was to study the anthelmintic effects of three structurally related flavonoids present in aerial parts of Z. punctata Cav. The biological activity of the flavonoids 7-hydroxyflavanone (HF), 3,7-dihydroxyflavone (DHF) and 2´,4´-dihydroxychalcone (DHC) was examined in the free-living nematode Caenorhabditis elegans. Our results showed that among the assayed flavonoids, only DHC showed an anthelmintic effect and alteration of egg hatching and larval development processes in C. elegans. DHC was able to kill 50% of adult nematodes at a concentration of 17 μg/mL. The effect on larval development was observed after 48 h in the presence of 25 and 50 μg/mL DHC, where 33.4 and 73.4% of nematodes remained in the L3 stage or younger. New therapeutic drugs with good efficacy against drug-resistant nematodes are urgently needed. Therefore, DHC, a natural compound present in Z. punctata, is proposed as a potential anthelmintic drug.

scholarly journals An economical and highly adaptable optogenetics system for individual and population-level manipulation of Caenorhabditis elegans

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
M. Koopman ◽  
L. Janssen ◽  
E. A. A. Nollen
Keyword(s):  
Caenorhabditis Elegans ◽  
Light Stimulus ◽  
Cholinergic Neurons ◽  
Model Organisms ◽  
Parameter Study ◽  
Multiple Model ◽  
Excitable Cells ◽  
Specific Expression ◽  
C Elegans ◽  
Age Related

Abstract Background Optogenetics allows the experimental manipulation of excitable cells by a light stimulus without the need for technically challenging and invasive procedures. The high degree of spatial, temporal, and intensity control that can be achieved with a light stimulus, combined with cell type-specific expression of light-sensitive ion channels, enables highly specific and precise stimulation of excitable cells. Optogenetic tools have therefore revolutionized the study of neuronal circuits in a number of models, including Caenorhabditis elegans. Despite the existence of several optogenetic systems that allow spatial and temporal photoactivation of light-sensitive actuators in C. elegans, their high costs and low flexibility have limited wide access to optogenetics. Here, we developed an inexpensive, easy-to-build, modular, and adjustable optogenetics device for use on different microscopes and worm trackers, which we called the OptoArm. Results The OptoArm allows for single- and multiple-worm illumination and is adaptable in terms of light intensity, lighting profiles, and light color. We demonstrate OptoArm’s power in a population-based multi-parameter study on the contributions of motor circuit cells to age-related motility decline. We found that individual components of the neuromuscular system display different rates of age-dependent deterioration. The functional decline of cholinergic neurons mirrors motor decline, while GABAergic neurons and muscle cells are relatively age-resilient, suggesting that rate-limiting cells exist and determine neuronal circuit ageing. Conclusion We have assembled an economical, reliable, and highly adaptable optogenetics system which can be deployed to address diverse biological questions. We provide a detailed description of the construction as well as technical and biological validation of our set-up. Importantly, use of the OptoArm is not limited to C. elegans and may benefit studies in multiple model organisms, making optogenetics more accessible to the broader research community.

scholarly journals Loss of Pseudouridine Synthases in the RluA Family Causes Hypersensitive Nociception in Drosophila

2020 ◽  
Vol 10 (12) ◽  
pp. 4425-4438
Author(s):  
Wan Song ◽  
Susanne Ressl ◽  
W. Daniel Tracey
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Null Alleles ◽  
Rna Modification ◽  
Loss Of Function ◽  
Specific Expression ◽  
Nociceptive Neurons ◽  
Link Type ◽  
Pseudouridine Synthases ◽  
Link Loss

Nociceptive neurons of Drosophila melanogaster larvae are characterized by highly branched dendritic processes whose proper morphogenesis relies on a large number of RNA-binding proteins. Post-transcriptional regulation of RNA in these dendrites has been found to play an important role in their function. Here, we investigate the neuronal functions of two putative RNA modification genes, RluA-1 and RluA-2, which are predicted to encode pseudouridine synthases. RluA-1 is specifically expressed in larval sensory neurons while RluA-2 expression is ubiquitous. Nociceptor-specific RNAi knockdown of RluA-1 caused hypersensitive nociception phenotypes, which were recapitulated with genetic null alleles. These were rescued with genomic duplication and nociceptor-specific expression of UAS-RluA-1-cDNA. As with RluA-1, RluA-2 loss of function mutants also displayed hyperalgesia. Interestingly, nociceptor neuron dendrites showed a hyperbranched morphology in the RluA-1 mutants. The latter may be a cause or a consequence of heightened sensitivity in mutant nociception behaviors.

scholarly journals Synthesis of Gold Functionalised Nanoparticles with the Eranthis hyemalis Lectin and Preliminary Toxicological Studies on Caenorhabditis elegans

2018 ◽  
Author(s):  
Jamila Djafari ◽  
Marie T. McConnell ◽  
Hugo M. Santos ◽  
José Luis Capelo ◽  
Emilia Bertolo ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Caenorhabditis Elegans ◽  
Biological Effect ◽  
Life Stage ◽  
Target Cells ◽  
Type Ii ◽  
Biological Assays ◽  
C Elegans ◽  
Toxicological Studies ◽  
Vis Spectroscopy

The lectin found in the tubers of the Winter Aconite (Eranthis hyemalis) plant (EHL) is a Type II Ribosome Inactivating Protein (RIP); type II RIPs have shown anti-cancer properties, and have great potential as therapeutic agents. Similarly, colloidal gold nanoparticles are successfully used in biomedical applications as gold nanoparticles can be functionalised with ligands with high affinity and specificity for target cells to create therapeutic and imaging agents. Herein we present the synthesis and characterization of gold nanoparticles conjugated with EHL. The aim was to establish the viability of the conjugate and perform a set of initial assays to establish whether the biological effect of EHL is altered by the conjugation. The biological assays were performed in Caenorhabditis elegans, a free living nematode commonly used for toxicological studies; previous work from some of the authors using first life stage (L1) nematodes has shown that EHL has a strong biocidal effect on C. elegans. Gold nanoparticles functionalised with EHL (AuNPs@EHL) were successfully synthesised by bioconjugation with citrate gold nanoparticles (AuNPs@Citrate); the conjugates were analysed by UV-Vis spectroscopy, Dynamic Light Scattering (DLS), Zeta Potential analysis and Transmission Electron Microscopy (TEM). Results indicate that an optimal functionalisation was achieved with the addition of 100 µL of EHL (concentration 1090 ± 40 µg/mL) over 5 mL of AuNPs (concentration [Au0] = 0.8 mM). Biological assays on the effect of AuNPs@EHL on C. elegans were performed, using first life stage (L1) and pre-adult stage (L4) nematodes. Citrate gold nanoparticles did not have any obvious effect on the nematodes. For L1 stage nematodes, the assays show that conjugation with gold nanoparticles reduced the biological effect of EHL on C. elegans. As lectin binding activity is essential for the natural protein to bind and allow entry to cells, conformational changes due to conjugation may have affected this binding affinity. For L4 stage nematodes, both EHL alone and AuNPs@EHL showed biological activity, and reproductive delays and reduced fecundity were observed in both cases. These assays indicate that EHL can be conjugated to gold nanoparticles and retain elements of biocidal activity.

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