scholarly journals In vivo mapping of tissue- and subcellular-specific proteomes in Caenorhabditis elegans

2016 ◽  
Author(s):  
Aaron W. Reinke ◽  
Raymond Mak ◽  
Emily R. Troemel ◽  
Eric J. Bennett
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Specific Expression ◽  
Live Animal ◽  
C Elegans ◽  
Cytoplasmic Proteins ◽  
Specific Localization ◽  
Multicellular Organisms ◽  
Subcellular Resolution

AbstractMulticellular organisms are composed of tissues that have distinct functions requiring specialized proteomes. To define the proteome of a live animal with tissue and subcellular resolution, we adapted a localized proteomics technology for use in the multicellular model organism Caenorhabditis elegans. This approach couples tissue- and location-specific expression of the enzyme ascorbate peroxidase (APX), which facilitates proximity-based protein labeling in vivo, and quantitative proteomics to identify tissue- and subcellular-restricted proteomes. We identified and localized over 3000 proteins from strains of C. elegans expressing APX in either the nucleus or cytoplasm of the intestine, epidermis, body wall muscle, or pharyngeal muscle. We also identified several hundred proteins that were specifically localized to one of the four tissues analyzed or specifically localized to the cytoplasm or the nucleus. This approach resulted in the identification of both previously characterized and unknown nuclear and cytoplasmic proteins. Further, we confirmed the tissue- and subcellular-specific localization of a subset of identified proteins using GFP-tagging and fluorescence microscopy, validating our in vivo proximity-based proteomics technique. Together, these results demonstrate a new approach that enables the tissue- and subcellular-specific identification and quantification of proteins within a live animal.

scholarly journals Normal Formation of a Subset of Intestinal Granules in Caenorhabditis elegans Requires ATP-binding Cassette Transporters HAF-4 and HAF-9, Which Are Highly Homologous to Human Lysosomal Peptide Transporter TAP-Like

2009 ◽  
Vol 20 (12) ◽  
pp. 2979-2990 ◽  
Author(s):  
Hiromi Kawai ◽  
Takahiro Tanji ◽  
Hirohisa Shiraishi ◽  
Mitsuo Yamada ◽  
Ryoko Iijima ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Model Organism ◽  
Physiological Role ◽  
Peptide Transporter ◽  
Atp Binding ◽  
Loss Of Function ◽  
Specific Expression ◽  
Atp Binding Cassette

TAP-like (TAPL; ABCB9) is a half-type ATP-binding cassette (ABC) transporter that localizes in lysosome and putatively conveys peptides from cytosol to lysosome. However, the physiological role of this transporter remains to be elucidated. Comparison of genome databases reveals that TAPL is conserved in various species from a simple model organism, Caenorhabditis elegans, to mammals. C. elegans possesses homologous TAPL genes: haf-4 and haf-9. In this study, we examined the tissue-specific expression of these two genes and analyzed the phenotypes of the loss-of-function mutants for haf-4 and haf-9 to elucidate the in vivo function of these genes. Both HAF-4 and HAF-9 tagged with green fluorescent protein (GFP) were mainly localized on the membrane of nonacidic but lysosome-associated membrane protein homologue (LMP-1)-positive intestinal granules from larval to adult stage. The mutants for haf-4 and haf-9 exhibited granular defects in late larval and young adult intestinal cells, associated with decreased brood size, prolonged defecation cycle, and slow growth. The intestinal granular phenotype was rescued by the overexpression of the GFP-tagged wild-type protein, but not by the ATP-unbound form of HAF-4. These results demonstrate that two ABC transporters, HAF-4 and HAF-9, are related to intestinal granular formation and some other physiological aspects.

scholarly journals A fast and reliable method for monitoring genomic instability in the model organism Caenorhabditis elegans

2021 ◽  
Author(s):  
Merle Marie Nicolai ◽  
Barbara Witt ◽  
Andrea Hartwig ◽  
Tanja Schwerdtle ◽  
Julia Bornhorst
Keyword(s):  
Caenorhabditis Elegans ◽  
Animal Experiments ◽  
Model Organism ◽  
Animal Testing ◽  
C Elegans ◽  
Testing Strategies ◽  
Genotoxic Agents ◽  
Genotoxicity Testing

AbstractThe identification of genotoxic agents and their potential for genotoxic alterations in an organism is crucial for risk assessment and approval procedures of the chemical and pharmaceutical industry. Classically, testing strategies for DNA or chromosomal damage focus on in vitro and in vivo (mainly rodent) investigations. In cell culture systems, the alkaline unwinding (AU) assay is one of the well-established methods for detecting the percentage of double-stranded DNA (dsDNA). By establishing a reliable lysis protocol, and further optimization of the AU assay for the model organism Caenorhabditis elegans (C. elegans), we provided a new tool for genotoxicity testing in the niche between in vitro and rodent experiments. The method is intended to complement existing testing strategies by a multicellular organism, which allows higher predictability of genotoxic potential compared to in vitro cell line or bacterial investigations, before utilizing in vivo (rodent) investigations. This also allows working within the 3R concept (reduction, refinement, and replacement of animal experiments), by reducing and possibly replacing animal testing. Validation with known genotoxic agents (bleomycin (BLM) and tert-butyl hydroperoxide (tBOOH)) proved the method to be meaningful, reproducible, and feasible for high-throughput genotoxicity testing, and especially preliminary screening.

scholarly journals Detecting changes in the Caenorhabditis elegans intestinal environment using an engineered bacterial biosensor

2019 ◽  
Author(s):  
Jack W. Rutter ◽  
Tanel Ozdemir ◽  
Leonor M. Quintaneiro ◽  
Geraint Thomas ◽  
Filipe Cabreiro ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rational Design ◽  
Model Organism ◽  
Bacterial Biosensor ◽  
Intestinal Environment ◽  
C Elegans ◽  
Ideal Candidate ◽  
Diagnostic Applications

AbstractCaenorhabditis elegans has become a key model organism within biology. In particular, the transparent gut, rapid growing time and ability to create a defined gut microbiota make it an ideal candidate organism for understanding and engineering the host microbiota. Here we present the development of an experimental model which can be used to characterise whole-cell bacterial biosensors in vivo. A dual-plasmid sensor system responding to isopropyl β-D-1-thiogalactopyranoside was developed and fully characterised in vitro. Subsequently, we show the sensor was capable of detecting and reporting on changes in the intestinal environment of C. elegans after introducing exogenous inducer into the environment. The protocols presented here may be used for aiding the rational design of engineered bacterial circuits, primarily for diagnostic applications. In addition, the model system may serve to reduce the use of current animal models and aid in the exploration of complex questions within general nematode and host-microbe biology.

scholarly journals Caenorhabditis elegans: A Useful Model for Studying Metabolic Disorders in Which Oxidative Stress Is a Contributing Factor

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Elizabeth Moreno-Arriola ◽  
Noemí Cárdenas-Rodríguez ◽  
Elvia Coballase-Urrutia ◽  
José Pedraza-Chaverri ◽  
Liliana Carmona-Aparicio ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caenorhabditis Elegans ◽  
Metabolic Disorders ◽  
Molecular Mechanisms ◽  
Second Messengers ◽  
Model Organism ◽  
Human Diseases ◽  
C Elegans ◽  
Molecular Bases

Caenorhabditis elegansis a powerful model organism that is invaluable for experimental research because it can be used to recapitulate most human diseases at either the metabolic or genomic levelin vivo. This organism contains many key components related to metabolic and oxidative stress networks that could conceivably allow us to increase and integrate information to understand the causes and mechanisms of complex diseases. Oxidative stress is an etiological factor that influences numerous human diseases, including diabetes.C. elegansdisplays remarkably similar molecular bases and cellular pathways to those of mammals. Defects in the insulin/insulin-like growth factor-1 signaling pathway or increased ROS levels induce the conserved phase II detoxification response via the SKN-1 pathway to fight against oxidative stress. However, it is noteworthy that, aside from the detrimental effects of ROS, they have been proposed as second messengers that trigger the mitohormetic response to attenuate the adverse effects of oxidative stress. Herein, we briefly describe the importance ofC. elegansas an experimental model system for studying metabolic disorders related to oxidative stress and the molecular mechanisms that underlie their pathophysiology.

Caenorhabditis elegans: A promising contrivance to study neurotoxicity and oxidative stress

2021 ◽  
Vol 16 (10) ◽  
pp. 198-206
Author(s):  
Kiran Singh ◽  
Shweta Yadav
Keyword(s):  
Oxidative Stress ◽  
Caenorhabditis Elegans ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Mechanisms Of Toxicity ◽  
C Elegans ◽  
Toxicological Studies ◽  
And Oxidative Stress

Owing to ubiquitous distribution, high abundances and ecological relevance, Caenorhabditis elegans has strong potential interest as barometer of environment and human health. Ecotoxicological methods are used to evaluate the effect of various anthropogenic contaminants on the ecosystems that circumscribe both in-vivo and in-vitro toxicities to explore the pathways and mechanisms of toxicity and to set precise toxicity thresholds. The interest in C. elegans, as a model organism in toxicological studies, has increased over the past few decades. The enticement of C. elegans comes from the ease of metabolically active digestive, sensory, endocrine, neuromuscular, reproductive systems and genetic manipulation along with the ability to fluorescently label neuronal subtypes. The study reviews the competence of Caenorhabditis elegans as a potential model organism in various toxicity assays specifically neurotoxicity and oxidative stress.

scholarly journals Identification, functional expression and enzymic analysis of two distinct CaaX proteases from Caenorhabditis elegans

2003 ◽  
Vol 370 (3) ◽  
pp. 1047-1054 ◽  
Author(s):  
Juan CADIÑANOS ◽  
Walter K. SCHMIDT ◽  
Antonio FUEYO ◽  
Ignacio VARELA ◽  
Carlos LÓPEZ-OTÍN ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Functional Expression ◽  
Cysteine Residue ◽  
C Elegans ◽  
Ras Gtpases ◽  
Acid Protein ◽  
Bona Fide

Post-translational processing of proteins such as the Ras GTPases, which contain a C-terminal CaaX motif (where C stands for cysteine, a for aliphatic and X is one of several amino acids), includes prenylation, proteolytic removal of the C-terminal tripeptide and carboxy-methylation of the isoprenyl-cysteine residue. In the present study, we report the presence of two distinct CaaX-proteolytic activities in membrane extracts from Caenorhabditis elegans, which are sensitive to EDTA and Tos-Phe-CH2Cl (tosylphenylalanylchloromethane; ‘TPCK') respectively. A protein similar to the mammalian and yeast farnesylated-proteins converting enzyme-1 (FACE-1)/Ste24p CaaX metalloprotease, encoded by a hypothetical gene (CeFACE-1/C04F12.10) found in C. elegans chromosome I, probably accounts for the EDTA-sensitive activity. An orthologue of FACE-2/Rce1p, the enzyme responsible for the proteolytic maturation of Ras oncoproteins and other prenylated substrates, probably accounts for the Tos-Phe-CH2Cl-sensitive activity, even though the gene for FACE-2/Rce1 has not been previously identified in this model organism. We have identified a previously overlooked gene in C. elegans chromosome V, which codes for a 266-amino-acid protein (CeFACE-2) with 30% sequence identity to human FACE-2/Rce1. We show that both CeFACE-1 and CeFACE-2 have the ability to promote production of the farnesylated yeast pheromone a-factor in vivo and to cleave a farnesylated peptide in vitro. These results indicate that CeFACE-1 and CeFACE-2 are bona fide CaaX proteases and support the evolutionary conservation of this proteolytic system in eukaryotes.

scholarly journals The Role of Ca2+ Signaling in Aging and Neurodegeneration: Insights from Caenorhabditis elegans Models

Cells ◽  
2020 ◽  
Vol 9 (1) ◽  
pp. 204 ◽  
Author(s):  
Javier Alvarez ◽  
Pilar Alvarez-Illera ◽  
Paloma García-Casas ◽  
Rosalba I. Fonteriz ◽  
Mayte Montero
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Ample Evidence ◽  
C Elegans ◽  
Physiological Processes ◽  
Human Proteins ◽  
Β Amyloid ◽  
Key Events

Ca2+ is a ubiquitous second messenger that plays an essential role in physiological processes such as muscle contraction, neuronal secretion, and cell proliferation or differentiation. There is ample evidence that the dysregulation of Ca2+ signaling is one of the key events in the development of neurodegenerative processes, an idea called the “calcium hypothesis” of neurodegeneration. Caenorhabditis elegans (C. elegans) is a very good model for the study of aging and neurodegeneration. In fact, many of the signaling pathways involved in longevity were first discovered in this nematode, and many models of neurodegenerative diseases have also been developed therein, either through mutations in the worm genome or by expressing human proteins involved in neurodegeneration (β-amyloid, α-synuclein, polyglutamine, or others) in defined worm tissues. The worm is completely transparent throughout its whole life, which makes it possible to carry out Ca2+ dynamics studies in vivo at any time, by expressing Ca2+ fluorescent probes in defined worm tissues, and even in specific organelles such as mitochondria. This review will summarize the evidence obtained using this model organism to understand the role of Ca2+ signaling in aging and neurodegeneration.

Relevance of bioassay of biologically active substances (BAS) with geroprotective properties in the model of the nematode Caenorhabditis elegans in experiments in vivo

2021 ◽  
Vol 14 ◽  
Author(s):  
Lyubov S. Dyshlyuk ◽  
Anastasiya I. Dmitrieva ◽  
Margarita Yu. Drozdova ◽  
Irina S. Milentyeva ◽  
Alexander Yu. Prosekov
Keyword(s):  
Caenorhabditis Elegans ◽  
Plant Extracts ◽  
Aging Process ◽  
Model Organism ◽  
Biologically Active ◽  
Model Systems ◽  
C Elegans ◽  
Living Organisms ◽  
Active Substances

: Aging is a process global in nature. The age of living organisms contributes to the appearance of chronic diseases, which not only reduce the quality of life, but also significantly damage it. Modern medicines can successfully fight multiple diseases and prolong life. At the same time, medications have a large number of side effects. New research indicates that bioactive phytochemicals have great potential for treating even the most severe diseases and can become an alternative to medicines. Despite many studies in this area, the effects of many plant ingredients on living organisms are poorly understood. Analysis of the mechanisms through which herbal preparations influence the aging process helps to select the right active substances, determine the optimal doses to obtain the maximum positive effect. It is preferable to check the effectiveness of plant extracts and biologically active components with geroprotective properties in vivo. For these purposes, live model systems such as Rattus rattus, Mus musculus, Drosophila melanogaster, and Caenorhabditis elegans are used. These models help to comprehensively study the impact of the developed new drugs on the aging process. The model organism C. elegans is gaining increasing popularity in these studies because of its many advantages. This review article discusses the advantages of the nematode C. elegans as a model organism for studying the processes associated with aging. The influence of various BAS and plant extracts on the increase in the life span of the nematode, on the increase in its stress resistance and on other markers of aging is also considered. The review showed that the nematode C. elegans has a number of advantages over other organisms and is a promising model system for studying the geroprotective properties of BAS.

scholarly journals Promoters of the dhs-21 gene encoding dicarbonyl/l-xylulose reductase in Caenorhabditis elegans

2018 ◽  
Vol 15 (2) ◽  
pp. 359-365
Author(s):  
Lê Thọ Sơn ◽  
Joohong Ahnn ◽  
Jeong Hoon Cho ◽  
Nguyễn Huy Hoàng
Keyword(s):  
Caenorhabditis Elegans ◽  
Model Organism ◽  
Biological Research ◽  
Loss Of Function ◽  
Wild Type ◽  
C Elegans ◽  
Larval Stages ◽  
Vital Functions

Dicarbonyl/L-xylulose (DCXR) was identified as a dehydrogenase. This type of enzyme was presented in various forms of lives including bacteria, fungi, plants and animals. Generally, it converts L-xylulose to xylitol in the presence of either cofactor NADH or NADPH in vitro. Previous studies reported the biochemistry properties and crystal structure but largely uncovered biological roles of DCXRs. It was impossible to dissect the functions in mice or human cells that had many DCXR homologs in their genomes. Interestingly, the wild-type Caenorhabditis elegans, a well-known model organism in biological research, has only nuclear genomic dhs-21 that encodes a unique homologous DCXR. Thus Ce.dhs-21 and the host C. elegans were relevant for investigation of the physiologically-vital functions of the DCXR. This research aimed to the expression of dhs-21 in vivo. We defined three promoters , manipulated three relative reporter-constructs that conjugated the dhs-21 gene and Green Flouresent Protein (known as GFP) one. The construct vectors were transferred into wild-type C. elegans N2 and as well as the hermaphroditic loss of function dhs-21(jh129) by microinjection. In the results, we found that the expression pattern of dhs-21 under the only p2-promoter construct was stable and similar to immunogold Electric Microscopy (EM) images. The dhs-21 gene was expressed in both sexes of at all larval stages till the deaths of worms. DHS-21 was expressed in the cytosol of the intestinal, gonad sheath and uterous seam cell (utse).

scholarly journals The Caenorhabditis elegans ABL-1 Tyrosine Kinase Is Required for Shigella flexneri Pathogenesis

2006 ◽  
Vol 72 (7) ◽  
pp. 5043-5051 ◽  
Author(s):  
Elizabeth A. Burton ◽  
Ann Marie Pendergast ◽  
Alejandro Aballay
Keyword(s):  
Caenorhabditis Elegans ◽  
Tyrosine Kinase ◽  
Diarrheal Disease ◽  
Shigella Flexneri ◽  
Model Organism ◽  
Host Susceptibility ◽  
Virulence Plasmid ◽  
Virulence Determinants ◽  
C Elegans

ABSTRACT Shigellosis is a diarrheal disease caused by the gram-negative bacterium Shigella flexneri. Following ingestion of the bacterium, S. flexneri interferes with innate immunity, establishes an infection within the human colon, and initiates an inflammatory response that results in destruction of the tissue lining the gut. Examination of host cell factors required for S. flexneri pathogenesis in vivo has proven difficult due to limited host susceptibility. Here we report the development of a pathogenesis system that involves the use of Caenorhabditis elegans as a model organism to study S. flexneri virulence determinants and host molecules required for pathogenesis. We show that S. flexneri-mediated killing of C. elegans correlates with bacterial accumulation in the intestinal tract of the animal. The S. flexneri virulence plasmid, which encodes a type III secretory system as well as various virulence determinants crucial for pathogenesis in mammalian systems, was found to be required for maximal C. elegans killing. Additionally, we demonstrate that ABL-1, the C. elegans homolog of the mammalian c-Abl nonreceptor tyrosine kinase ABL1, is required for S. flexneri pathogenesis in nematodes. These data demonstrate the feasibility of using C. elegans to study S. flexneri pathogenesis in vivo and provide insight into host factors that contribute to S. flexneri pathogenesis.

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