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 Rapid degradation of C. elegans proteins at single-cell resolution with a synthetic auxin

2019 ◽  
Author(s):  
Michael A. Q. Martinez ◽  
Brian A. Kinney ◽  
Taylor N. Medwig-Kinney ◽  
Guinevere Ashley ◽  
James M. Ragle ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Single Cell ◽  
Single Cells ◽  
Nuclear Hormone Receptor ◽  
Water Soluble ◽  
Growth Media ◽  
Synthetic Analog ◽  
Rapid Degradation ◽  
Target Proteins ◽  
C Elegans

ABSTRACTAs 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, functioning through the activity of 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 method, 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 minutes 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 interacts with C. elegans SKR-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 provides a conceptual improvement to the AID system for dissecting gene function at the single-cell level during C. elegans development.

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 Combining single-cell RNA-sequencing with a molecular atlas unveils new markers for Caenorhabditis elegans neuron classes

2020 ◽  
Author(s):  
Ramiro Lorenzo ◽  
Michiho Onizuka ◽  
Matthieu Defrance ◽  
Patrick Laurent
Keyword(s):  
Caenorhabditis Elegans ◽  
Single Cell ◽  
Rna Sequencing ◽  
Cell Fate ◽  
Single Neuron ◽  
Genetic Manipulations ◽  
C Elegans ◽  
Single Cell Rna Sequencing ◽  
Normal Differentiation

Abstract Single-cell RNA-sequencing (scRNA-seq) of the Caenorhabditis elegans nervous system offers the unique opportunity to obtain a partial expression profile for each neuron within a known connectome. Building on recent scRNA-seq data and on a molecular atlas describing the expression pattern of ∼800 genes at the single cell resolution, we designed an iterative clustering analysis aiming to match each cell-cluster to the ∼100 anatomically defined neuron classes of C. elegans. This heuristic approach successfully assigned 97 of the 118 neuron classes to a cluster. Sixty two clusters were assigned to a single neuron class and 15 clusters grouped neuron classes sharing close molecular signatures. Pseudotime analysis revealed a maturation process occurring in some neurons (e.g. PDA) during the L2 stage. Based on the molecular profiles of all identified neurons, we predicted cell fate regulators and experimentally validated unc-86 for the normal differentiation of RMG neurons. Furthermore, we observed that different classes of genes functionally diversify sensory neurons, interneurons and motorneurons. Finally, we designed 15 new neuron class-specific promoters validated in vivo. Amongst them, 10 represent the only specific promoter reported to this day, expanding the list of neurons amenable to genetic manipulations.

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.

scholarly journals A lineage-resolved molecular atlas of C. elegans embryogenesis at single-cell resolution

Science ◽  
2019 ◽  
Vol 365 (6459) ◽  
pp. eaax1971 ◽  
Author(s):  
Jonathan S. Packer ◽  
Qin Zhu ◽  
Chau Huynh ◽  
Priya Sivaramakrishnan ◽  
Elicia Preston ◽  
...  
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Single Cell ◽  
Molecular Basis ◽  
Cell Types ◽  
Embryonic Cells ◽  
Cell Type ◽  
C Elegans ◽  
Exact Position ◽  
Sister Cells

Caenorhabditis elegans is an animal with few cells but a wide diversity of cell types. In this study, we characterize the molecular basis for their specification by profiling the transcriptomes of 86,024 single embryonic cells. We identify 502 terminal and preterminal cell types, mapping most single-cell transcriptomes to their exact position in C. elegans’ invariant lineage. Using these annotations, we find that (i) the correlation between a cell’s lineage and its transcriptome increases from middle to late gastrulation, then falls substantially as cells in the nervous system and pharynx adopt their terminal fates; (ii) multilineage priming contributes to the differentiation of sister cells at dozens of lineage branches; and (iii) most distinct lineages that produce the same anatomical cell type converge to a homogenous transcriptomic state.

scholarly journals Signaling by AWC Olfactory Neurons Is Necessary for Caenorhabditis elegans’ Response to Prenol, an Odor Associated with Nematode-Infected Insects

Genetics ◽  
2020 ◽  
Vol 216 (1) ◽  
pp. 145-157
Author(s):  
Tiffany Baiocchi ◽  
Kyle Anesko ◽  
Nathan Mercado ◽  
Heenam Park ◽  
Kassandra Kin ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Behavioral Ecology ◽  
Natural Variation ◽  
Isoamyl Alcohol ◽  
Life Cycles ◽  
C Elegans ◽  
Link Type ◽  
Genome Wide ◽  
Ecological Differences

Chemosensation plays a role in the behaviors and life cycles of numerous organisms, including nematodes. Many guilds of nematodes exist, ranging from the free-living Caenorhabditis elegans to various parasitic species such as entomopathogenic nematodes (EPNs), which are parasites of insects. Despite ecological differences, previous research has shown that both EPNs and C. elegans respond to prenol (3-methyl-2-buten-1-ol), an odor associated with EPN infections. However, it is unclear how C. elegans responds to prenol. By utilizing natural variation and genetic neuron ablation to investigate the response of C. elegans to prenol, we found that the AWC neurons are involved in the detection of prenol and that several genes (including dcap-1, dcap-2, and clec-39) influence response to this odorant. Furthermore, we identified that the response to prenol is mediated by the canonically proposed pathway required for other AWC-sensed attractants. However, upon testing genetically diverse isolates, we found that the response of some strains to prenol differed from their response to isoamyl alcohol, suggesting that the pathways mediating response to these two odorants may be genetically distinct. Further, evaluations leveraging natural variation and genome wide association revealed specific genes that influence nematode behavior and provide a foundation for future studies to better understand the role of prenol in nematode behavioral ecology.

The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation

1993 ◽  
Vol 13 (3) ◽  
pp. 1371-1377
Author(s):  
M Zhen ◽  
R Heinlein ◽  
D Jones ◽  
S Jentsch ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Elevated Temperatures ◽  
Yeast Cells ◽  
Amino Acid Sequence Similarity ◽  
Western Blots ◽  
Target Proteins ◽  
C Elegans ◽  
Cell Functions ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

scholarly journals The ubc-2 gene of Caenorhabditis elegans encodes a ubiquitin-conjugating enzyme involved in selective protein degradation.

1993 ◽  
Vol 13 (3) ◽  
pp. 1371-1377 ◽  
Author(s):  
M Zhen ◽  
R Heinlein ◽  
D Jones ◽  
S Jentsch ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Elevated Temperatures ◽  
Yeast Cells ◽  
Amino Acid Sequence Similarity ◽  
Western Blots ◽  
Target Proteins ◽  
C Elegans ◽  
Cell Functions ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzyme

The ubiquitin-protein conjugation system is involved in a variety of eukaryotic cell functions, including the degradation of abnormal and short-lived proteins, chromatin structure, cell cycle progression, and DNA repair. The ubiquitination of target proteins is catalyzed by a ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzymes (E2s) and in some cases also requires auxiliary substrate recognition proteins (E3s). Multiple E2s have been found, and these likely possess specificity for different classes of target proteins. Here we report the cloning and characterization of a gene (ubc-2) encoding a ubiquitin-conjugating enzyme which is involved in the selective degradation of abnormal and short-lived proteins in the nematode Caenorhabditis elegans. The nematode ubc-2 gene encodes a 16.7-kDa protein with striking amino acid sequence similarity to Saccharomyces cerevisiae UBC4 and UBC5 and Drosophila UbcD1. When driven by the UBC4 promoter, ubc-2 can functionally substitute for UBC4 in yeast cells; it rescues the slow-growth phenotype of ubc4 ubc5 mutants at normal temperature and restores their ability to grow at elevated temperatures. Western blots (immunoblots) of ubc4 ubc5 yeast cells transformed with ubc-2 reveal a protein of the expected size, which cross-reacts with anti-Drosophila UbcD1 antibody. C. elegans ubc-2 is constitutively expressed at all life cycle stages and, unlike yeast UBC4 and UBC5, is not induced by heat shock. Both trans and cis splicing are involved in the maturation of the ubc-2 transcript. These data suggest that yeast UBC4 and UBC5, Drosophila UbcD1, and C. elegans ubc-2 define a highly conserved gene family which plays fundamental roles in all eukaryotic cells.

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