In Vivo and in Vitro Evidence that the Four Essential Intermediate Filament (IF) Proteins A1, A2, A3 and B1 of the Nematode Caenorhabditis elegans Form an Obligate Heteropolymeric IF System

Exposure of the nematode Caenorhabditis elegans to elevated temperatures induces the preferential synthesis of eight major polypeptides of approximate molecular weights 81 000, 70 000, 41 000, 38 000, 29 000, 19 000, 18 000, and 16 000. In pulse-labelled worms these peptides first appear at 29 °C and continue to be synthesized up to lethal temperatures. They are heat inducible at every stage of development. While temperature elevation induces the synthesis of the heat-shock polypeptides, the in vivo synthesis of most other proteins present before heat shock is suppressed. In contrast, in vitro translation of mRNA from heat-shocked worms shows no alteration from the pattern of normal 20 °C mRNAs except for the appearance of the heat-shock mRNAs. An in vitro study of RNA from control and heat-shocked dauer larvae shows that this developmental variant possesses little translatable mRNA but, upon heat shock, synthesizes a set of messages corresponding to the heat-shock polypeptides. The low background of this system will be especially useful in the analysis and purification of heat-shock mRNA for molecular cloning experiments. Extensive similarities between the Drosophila and C. elegans heat-shock responses are shown, including homology between the 70-kdalton heat-shock genes of the two organisms.

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Talin requires beta-integrin, but not vinculin, for its assembly into focal adhesion-like structures in the nematode Caenorhabditis elegans.

Molecular Biology of the Cell ◽

10.1091/mbc.7.8.1181 ◽

1996 ◽

Vol 7 (8) ◽

pp. 1181-1193 ◽

Cited By ~ 49

Author(s):

G L Moulder ◽

M M Huang ◽

R H Waterston ◽

R J Barstead

Keyword(s):

Caenorhabditis Elegans ◽

Focal Adhesion ◽

Focal Adhesions ◽

The Body ◽

Nematode Caenorhabditis Elegans ◽

C Elegans ◽

Beta Integrin ◽

Cell Shapes

In cultured cells, the 230-kDa protein talin is found at discrete plasma membrane foci known as focal adhesions, sites that anchor the intracellular actin cytoskeleton to the extracellular matrix. The regulated assembly of focal adhesions influences the direction of cell migrations or the reorientation of cell shapes. Biochemical studies of talin have shown that it binds to the proteins integrin, vinculin, and actin in vitro. To understand the function of talin in vivo and to correlate its in vitro and in vivo biochemical properties, various genetic approaches have been adopted. With the intention of using genetics in the study of talin, we identified a homologue to mouse talin in a genetic model system, the nematode Caenorhabditis elegans. C. elegans talin is 39% identical and 59% similar to mouse talin. In wild-type adult C. elegans, talin colocalizes with integrin, vinculin, and alpha-actinin in the focal adhesion-like structures found in the body-wall muscle. By examining the organization of talin in two different C. elegans mutant strains that do not make either beta-integrin or vinculin, we were able to determine that talin does not require vinculin for its initial organization at the membrane, but that it depends critically on the presence of integrin for its initial assembly at membrane foci.

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Faculty Opinions recommendation of In vitro and in vivo reconstitution of the cadherin-catenin-actin complex from Caenorhabditis elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.4846956.5753065 ◽

2010 ◽

Author(s):

Alpha Yap

Keyword(s):

Caenorhabditis Elegans

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Caenorhabditis elegans as an in vivo Model to Assess Amphetamine Tolerance

Brain Behavior and Evolution ◽

10.1159/000514858 ◽

2021 ◽

pp. 1-9

Author(s):

Dayana Torres Valladares ◽

Sirisha Kudumala ◽

Murad Hossain ◽

Lucia Carvelli

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Drugs Of Abuse ◽

Genetic Model ◽

In Vivo Model ◽

C Elegans ◽

Hyperactivity Disorder ◽

In Vitro Data

Amphetamine is a potent psychostimulant also used to treat attention deficit/hyperactivity disorder and narcolepsy. In vivo and in vitro data have demonstrated that amphetamine increases the amount of extra synaptic dopamine by both inhibiting reuptake and promoting efflux of dopamine through the dopamine transporter. Previous studies have shown that chronic use of amphetamine causes tolerance to the drug. Thus, since the molecular mechanisms underlying tolerance to amphetamine are still unknown, an animal model to identify the neurochemical mechanisms associated with drug tolerance is greatly needed. Here we took advantage of a unique behavior caused by amphetamine in <i>Caenorhabditis elegans</i> to investigate whether this simple, but powerful, genetic model develops tolerance following repeated exposure to amphetamine. We found that at least 3 treatments with 0.5 mM amphetamine were necessary to see a reduction in the amphetamine-induced behavior and, thus, to promote tolerance. Moreover, we found that, after intervals of 60/90 minutes between treatments, animals were more likely to exhibit tolerance than animals that underwent 10-minute intervals between treatments. Taken together, our results show that <i>C. elegans</i> is a suitable system to study tolerance to drugs of abuse such as amphetamines.

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Microtubules and microtubule-associated proteins from the nematode Caenorhabditis elegans: periodic cross-links connect microtubules in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.103.1.23 ◽

1986 ◽

Vol 103 (1) ◽

pp. 23-31 ◽

Cited By ~ 35

Author(s):

E J Aamodt ◽

J G Culotti

Keyword(s):

Caenorhabditis Elegans ◽

Apparent Molecular Weight ◽

Cross Link ◽

Nematode Caenorhabditis Elegans ◽

Microtubule Associated Proteins ◽

C Elegans ◽

Associated Proteins ◽

Cross Links ◽

The Cross

The nematode Caenorhabditis elegans should be an excellent model system in which to study the role of microtubules in mitosis, embryogenesis, morphogenesis, and nerve function. It may be studied by the use of biochemical, genetic, molecular biological, and cell biological approaches. We have purified microtubules and microtubule-associated proteins (MAPs) from C. elegans by the use of the anti-tumor drug taxol (Vallee, R. B., 1982, J. Cell Biol., 92:435-44). Approximately 0.2 mg of microtubules and 0.03 mg of MAPs were isolated from each gram of C. elegans. The C. elegans microtubules were smaller in diameter than bovine microtubules assembled in vitro in the same buffer. They contained primarily 9-11 protofilaments, while the bovine microtubules contained 13 protofilaments. The principal MAP had an apparent molecular weight of 32,000 and the minor MAPs were 30,000, 45,000, 47,000, 50,000, 57,000, and 100,000-110,000 mol wt as determined by SDS-gel electrophoresis. The microtubules were observed, by electron microscopy of negatively stained preparations, to be connected by stretches of highly periodic cross-links. The cross-links connected the adjacent protofilaments of aligned microtubules, and occurred at a frequency of one cross-link every 7.7 +/- 0.9 nm, or one cross-link per tubulin dimer along the protofilament. The cross-links were removed when the MAPs were extracted from the microtubules with 0.4 M NaCl. The cross-links then re-formed when the microtubules and the MAPs were recombined in a low salt buffer. These results strongly suggest that the cross-links are composed of MAPs.

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Interaction of plakophilins with desmoplakin and intermediate filament proteins: an in vitro analysis

Journal of Cell Science ◽

10.1242/jcs.113.13.2471 ◽

2000 ◽

Vol 113 (13) ◽

pp. 2471-2483 ◽

Cited By ~ 3

Author(s):

I. Hofmann ◽

C. Mertens ◽

M. Brettel ◽

V. Nimmrich ◽

M. Schnolzer ◽

...

Keyword(s):

Epithelial Cells ◽

Intermediate Filament ◽

Solid Phase ◽

Specific Interaction ◽

Binding Assays ◽

Intermediate Filament Proteins ◽

Amino Terminal ◽

Vitro Analysis

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm-repeat protein family. They are both constitutively expressed in most vertebrate cells, in two splice forms named a and b, and display a remarkable dual location: they occur in the nuclei of cells and, in epithelial cells, at the plasma membrane within the desmosomal plaques. We have shown by solid phase-binding assays that both PKP1a and PKP2a bind to intermediate filament (IF) proteins, in particular to cytokeratins (CKs) from epidermal as well as simple epithelial cells and, to some extent, to vimentin. In line with this we show that recombinant PKP1a binds strongly to IFs assembled in vitro from CKs 8/18, 5/14, vimentin or desmin and integrates them into thick (up to 120 nm in diameter) IF bundles extending for several microm. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation experiments. In particular, the binding of PKP1a to IF proteins is saturable at an approximately equimolar ratio. In extracts from HaCaT cells, distinct soluble complexes containing PKP1a and desmoplakin I (DPI) have been identified by co-immunoprecipitation and sucrose density fractionation. The significance of these interactions of PKP1a with IF proteins on the one hand and desmoplakin on the other is discussed in relation to the fact that PKP1a is not bound - and does not bind - to extended IFs in vivo. We postulate that (1) effective cellular regulatory mechanisms exist that prevent plakophilins from unscheduled IF-binding, and (2) specific desmoplakin interactions with either PKP1, PKP2 or PKP3, or combinations thereof, are involved in the selective recruitment of plakophilins to the desmosomal plaques.

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Vimentin in a cold-water fish, the rainbow trout: highly conserved primary structure but unique assembly properties

Journal of Cell Science ◽

10.1242/jcs.109.3.569 ◽

1996 ◽

Vol 109 (3) ◽

pp. 569-578 ◽

Cited By ~ 2

Author(s):

H. Herrmann ◽

M.D. Munick ◽

M. Brettel ◽

B. Fouquet ◽

J. Markl

Keyword(s):

Rainbow Trout ◽

Intermediate Filament ◽

Cold Water ◽

White Blood Cells ◽

Cellular Functions ◽

Filamentous Form ◽

Rainbow Trout Oncorhynchus Mykiss ◽

Bona Fide

We have isolated from a rainbow trout (Oncorhynchus mykiss) spleen cDNA library a clone coding for vimentin. The deduced amino acid sequence reveals a high degree of identity with vimentin from carp (81%), frog (71%), chick and human (73% each). Large stretches in the central alpha-helical rod are identical within all four classes of vertebrates, but in 17 residues spread over the entire rod, the two fish differ distinctly from the tetrapod species. In addition, in the more diverged non-helical head domain, a nonapeptide motif previously shown to be important for regular filament formation is conserved. Recombinant trout vimentin assembles into bona fide filaments in vitro, with a temperature optimum between 18 and 24 degrees C. Above 27 degrees C, however, filament assembly is abruptly abolished and short filaments with thickened ends as well as structures without typical intermediate filament appearance are formed. This distinguishes its assembly properties significantly from amphibian, avian and mammalian vimentin. Also in vivo, after cDNA transfection into vimentin-free mammalian epithelial cells, trout vimentin does not form typical intermediate filament arrays at 37 degrees C. At 28 degrees C, and even more pronounced at 22 degrees C, the vimentin-positive material in the transfected cells is reorganized in the perinuclear region with a partial fibrillar appearance, but typical intermediate filament arrays are not formed. Together with immunoblotting and immunolocalization data from trout tissues, where vimentin is predominantly found in glial and white blood cells, we conclude that vimentin is indeed important in its filamentous form in fish and other vertebrates, possibly fulfilling cellular functions not directly evident in gene targeting experiments carried out in mice.

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Identification of gmhA, a Yersinia pestis Gene Required for Flea Blockage, by Using a Caenorhabditis elegans Biofilm System

Infection and Immunity ◽

10.1128/iai.73.11.7236-7242.2005 ◽

2005 ◽

Vol 73 (11) ◽

pp. 7236-7242 ◽

Cited By ~ 55

Author(s):

Creg Darby ◽

Sandya L. Ananth ◽

Li Tan ◽

B. Joseph Hinnebusch

Keyword(s):

Caenorhabditis Elegans ◽

Yersinia Pestis ◽

Biofilm Formation ◽

Yersinia Pseudotuberculosis ◽

C Elegans ◽

Transposon Insertion ◽

Insertion Mutants ◽

Random Screening

ABSTRACT Yersinia pestis, the cause of bubonic plague, blocks feeding by its vector, the flea. Recent evidence indicates that blockage is mediated by an in vivo biofilm. Y. pestis and the closely related Yersinia pseudotuberculosis also make biofilms on the cuticle of the nematode Caenorhabditis elegans, which block this laboratory animal's feeding. Random screening of Y. pseudotuberculosis transposon insertion mutants with a C. elegans biofilm assay identified gmhA as a gene required for normal biofilms. gmhA encodes phosphoheptose isomerase, an enzyme required for synthesis of heptose, a conserved component of lipopolysaccharide and lipooligosaccharide. A Y. pestis gmhA mutant was constructed and was severely defective for C. elegans biofilm formation and for flea blockage but only moderately defective in an in vitro biofilm assay. These results validate use of the C. elegans biofilm system to identify genes and pathways involved in Y. pestis flea blockage.

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Rosemary Flowers as Edible Plant Foods: Phenolic Composition and Antioxidant Properties in Caenorhabditis elegans

Antioxidants ◽

10.3390/antiox9090811 ◽

2020 ◽

Vol 9 (9) ◽

pp. 811

Author(s):

Cristina Moliner ◽

Víctor López ◽

Lillian Barros ◽

Maria Inês Dias ◽

Isabel C. F. R. Ferreira ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Antioxidant Properties ◽

Ethanolic Extract ◽

Food Ingredient ◽

Edible Plant ◽

Phenolic Profile ◽

C Elegans ◽

Rosmarinus Officinalis L

Rosmarinus officinalis L., commonly known as rosemary, has been largely studied for its wide use as food ingredient and medicinal plant; less attention has been given to its edible flowers, being necessary to evaluate their potential as functional foods or nutraceuticals. To achieve that, the phenolic profile of the ethanolic extract of R. officinalis flowers was determined using LC-DAD-ESI/MSn and then its antioxidant and anti-ageing potential was studied through in vitro and in vivo assays using Caenorhabditis elegans. The phenolic content was 14.3 ± 0.1 mg/g extract, trans rosmarinic acid being the predominant compound in the extract, which also exhibited a strong antioxidant capacity in vitro and increased the survival rate of C. elegans exposed to lethal oxidative stress. Moreover, R. officinalis flowers extended C. elegans lifespan up to 18%. Therefore, these findings support the potential use of R. officinalis flowers as ingredients to develop products with pharmaceutical and/or nutraceutical potential.

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