scholarly journals Purified thick filaments from the nematode Caenorhabditis elegans: evidence for multiple proteins associated with core structures.

1988 ◽  
Vol 106 (6) ◽  
pp. 1985-1995 ◽  
Author(s):  
H F Epstein ◽  
G C Berliner ◽  
D L Casey ◽  
I Ortiz
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Gradient Centrifugation ◽  
Blue Staining ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Thick Filaments ◽  
Cell Biol ◽  
Associated Proteins ◽  
Body Wall Muscles

The thick filaments of the nematode, Caenorhabditis elegans, arising predominantly from the body-wall muscles, contain two myosin isoforms and paramyosin as their major proteins. The two myosins are located in distinct regions of the surfaces, while paramyosin is located within the backbones of the filaments. Tubular structures constitute the cores of the polar regions, and electron-dense material is present in the cores of the central regions (Epstein, H.F., D.M. Miller, I. Ortiz, and G.C. Berliner. 1985. J. Cell Biol. 100:904-915). Biochemical, genetic, and immunological experiments indicate that the two myosins and paramyosin are not necessary core components (Epstein, H.F., I. Ortiz, and L.A. Traeger Mackinnon. 1986. J. Cell Biol. 103:985-993). The existence of the core structures suggests, therefore, that additional proteins may be associated with thick filaments in C. elegans. To biochemically detect minor associated proteins, a new procedure for the isolation of thick filaments of high purity and structural preservation has been developed. The final step, glycerol gradient centrifugation, yielded fractions that are contaminated by, at most, 1-2% with actin, tropomyosin, or ribosome-associated proteins on the basis of Coomassie Blue staining and electron microscopy. Silver staining and radioautography of gel electrophoretograms of unlabeled and 35S-labeled proteins, respectively, revealed at least 10 additional bands that cosedimented with thick filaments in glycerol gradients. Core structures prepared from wild-type thick filaments contained at least six of these thick filament-associated protein bands. The six proteins also cosedimented with thick filaments purified by gradient centrifugation from CB190 mutants lacking myosin heavy chain B and from CB1214 mutants lacking paramyosin. For these reasons, we propose that the six associated proteins are potential candidates for putative components of core structures in the thick filaments of body-wall muscles of C. elegans.

Differential assembly of alpha- and gamma-filagenins into thick filaments in Caenorhabditis elegans

2000 ◽  
Vol 113 (22) ◽  
pp. 4001-4012 ◽  
Author(s):  
F. Liu ◽  
I. Ortiz ◽  
A. Hutagalung ◽  
C.C. Bauer ◽  
R.G. Cook ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Immunoelectron Microscopy ◽  
Body Wall ◽  
Developmental Stages ◽  
Body Wall Muscle ◽  
Developmentally Regulated ◽  
C Elegans ◽  
Novel Proteins ◽  
Thick Filaments ◽  
Associated Proteins

Muscle thick filaments are highly organized supramolecular assemblies of myosin and associated proteins with lengths, diameters and flexural rigidities characteristic of their source. The cores of body wall muscle thick filaments of the nematode Caenorhabditis elegans are tubular structures of paramyosin sub-filaments coupled by filagenins and have been proposed to serve as templates for the assembly of native thick filaments. We have characterized alpha- and gamma-filagenins, two novel proteins of the cores with calculated molecular masses of 30,043 and 19,601 and isoelectric points of 10.52 and 11.49, respectively. Western blot and immunoelectron microscopy using affinity-purified antibodies confirmed that the two proteins are core components. Immunoelectron microscopy of the cores revealed that they assemble with different periodicities. Immunofluorescence microscopy showed that alpha-filagenin is localized in the medial regions of the A-bands of body wall muscle cells whereas gamma-filagenin is localized in the flanking regions, and that alpha-filagenin is expressed in 1.5-twofold embryos while gamma-filagenin becomes detectable only in late vermiform embryos. The expression of both proteins continues throughout later stages of development. C. elegans body wall muscle thick filaments of these developmental stages have distinct lengths. Our results suggest that the differential assembly of alpha- and gamma-filagenins into thick filaments of distinct lengths may be developmentally regulated.

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

1986 ◽  
Vol 103 (1) ◽  
pp. 23-31 ◽  
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.

scholarly journals Muscle cell attachment in Caenorhabditis elegans.

1991 ◽  
Vol 114 (3) ◽  
pp. 465-479 ◽  
Author(s):  
R Francis ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Basement Membrane ◽  
Muscle Cell ◽  
Body Wall ◽  
Cell Attachment ◽  
Muscle Cells ◽  
The Body ◽  
Nematode Caenorhabditis Elegans ◽  
Hypodermal Cell ◽  
Body Wall Muscles

In the nematode Caenorhabditis elegans, the body wall muscles exert their force on the cuticle to generate locomotion. Interposed between the muscle cells and the cuticle are a basement membrane and a thin hypodermal cell. The latter contains bundles of filaments attached to dense plaques in the hypodermal cell membranes, which together we have called a fibrous organelle. In an effort to define the chain of molecules that anchor the muscle cells to the cuticle we have isolated five mAbs using preparations enriched in these components. Two antibodies define a 200-kD muscle antigen likely to be part of the basement membrane at the muscle/hypodermal interface. Three other antibodies probably identify elements of the fibrous organelles in the adjacent hypodermis. The mAb IFA, which reacts with mammalian intermediate filaments, also recognizes these structures. We suggest that the components recognized by these antibodies are likely to be involved in the transmission of tension from the muscle cell to the cuticle.

Quantitative fluorescence imaging of mitochondria in body wall muscles of Caenorhabditis elegans under hyperglycemic conditions using a microfluidic chip

2020 ◽  
Vol 12 (6) ◽  
pp. 150-160 ◽  
Author(s):  
Samuel Sofela ◽  
Sarah Sahloul ◽  
Sukanta Bhattacharjee ◽  
Ambar Bose ◽  
Ushna Usman ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Fluorescence Imaging ◽  
Microfluidic Device ◽  
Microfluidic Chip ◽  
Body Wall ◽  
The Body ◽  
C Elegans ◽  
Technological Advantage ◽  
Body Wall Muscles ◽  
Quantitative Fluorescence

Abstract Type 2 diabetes is the most common metabolic disease, and insulin resistance plays a role in the pathogenesis of the disease. Because completely functional mitochondria are necessary to obtain glucose-stimulated insulin from pancreatic beta cells, dysfunction of mitochondrial oxidative pathway could be involved in the development of diabetes. As a simple animal model, Caenorhabditis elegans renders itself to investigate such metabolic mechanisms because it possesses insulin/insulin-like growth factor-1 signaling pathway similar to that in humans. Currently, the widely spread agarose pad-based immobilization technique for fluorescence imaging of the mitochondria in C. elegans is laborious, batchwise, and does not allow for facile handling of the worm. To overcome these technical challenges, we have developed a single-channel microfluidic device that can trap a C. elegans and allow to image the mitochondria in body wall muscles accurately and in higher throughput than the traditional approach. In specific, our microfluidic device took advantage of the proprioception of the worm to rotate its body in a microfluidic channel with an aspect ratio above one to gain more space for its undulation motion that was favorable for quantitative fluorescence imaging of mitochondria in the body wall muscles. Exploiting this unique feature of the microfluidic chip-based immobilization and fluorescence imaging, we observed a significant decrease in the mitochondrial fluorescence intensity under hyperglycemic conditions, whereas the agarose pad-based approach did not show any significant change under the same conditions. A machine learning model trained with these fluorescence images from the microfluidic device could classify healthy and hyperglycemic worms at high accuracy. Given this significant technological advantage, its easiness of use and low cost, our microfluidic imaging chip could become a useful immobilization tool for quantitative fluorescence imaging of the body wall muscles in C. elegans.

scholarly journals The Caenorhabditis elegans vab-10 spectraplakin isoforms protect the epidermis against internal and external forces

2003 ◽  
Vol 161 (4) ◽  
pp. 757-768 ◽  
Author(s):  
Julia M. Bosher ◽  
Bum-Soo Hahn ◽  
Renaud Legouis ◽  
Satis Sookhareea ◽  
Robby M. Weimer ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Epidermal Thickness ◽  
C Elegans ◽  
Attachment Structures ◽  
Body Wall Muscles ◽  
Embryonic Morphogenesis ◽  
External Forces ◽  
New Mutations

Morphogenesis of the Caenorhabditis elegans embryo is driven by actin microfilaments in the epidermis and by sarcomeres in body wall muscles. Both tissues are mechanically coupled, most likely through specialized attachment structures called fibrous organelles (FOs) that connect muscles to the cuticle across the epidermis. Here, we report the identification of new mutations in a gene known as vab-10, which lead to severe morphogenesis defects, and show that vab-10 corresponds to the C. elegans spectraplakin locus. Our analysis of vab-10 reveals novel insights into the role of this plakin subfamily. vab-10 generates isoforms related either to plectin (termed VAB-10A) or to microtubule actin cross-linking factor plakins (termed VAB-10B). Using specific antibodies and mutations, we show that VAB-10A and VAB-10B have distinct distributions and functions in the epidermis. Loss of VAB-10A impairs the integrity of FOs, leading to epidermal detachment from the cuticle and muscles, hence demonstrating that FOs are functionally and molecularly related to hemidesmosomes. We suggest that this isoform protects against forces external to the epidermis. In contrast, lack of VAB-10B leads to increased epidermal thickness during embryonic morphogenesis when epidermal cells change shape. We suggest that this isoform protects cells against tension that builds up within the epidermis.

scholarly journals Caenorhabditis elegans Unc-45 Is a Component of Muscle Thick Filaments and Colocalizes with Myosin Heavy Chain B, but Not Myosin Heavy Chain a

2000 ◽  
Vol 148 (2) ◽  
pp. 375-384 ◽  
Author(s):  
Wanyuan Ao ◽  
Dave Pilgrim
Keyword(s):  
Caenorhabditis Elegans ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Body Wall ◽  
Thick Filament ◽  
The Body ◽  
Temperature Sensitive ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Body Wall Muscles

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B–dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.

PTL-1, a microtubule-associated protein with tau-like repeats from the nematode Caenorhabditis elegans

1996 ◽  
Vol 109 (11) ◽  
pp. 2661-2672 ◽  
Author(s):  
M. Goedert ◽  
C.P. Baur ◽  
J. Ahringer ◽  
R. Jakes ◽  
M. Hasegawa ◽  
...  
Keyword(s):  
Amino Acid ◽  
Caenorhabditis Elegans ◽  
Tandem Repeats ◽  
Microtubule Assembly ◽  
Nematode Caenorhabditis Elegans ◽  
Microtubule Associated Proteins ◽  
C Elegans ◽  
Functional Characterisation ◽  
Associated Proteins ◽  
Mammalian Origin

Tau, MAP2 and MAP4 are structural microtubule-associated proteins (MAPs) that promote the assembly and stability of microtubules. They share three or four imperfect tandem repeats of an amino acid motif, which is involved in the binding to microtubules. All sequences to data containing this motif are of mammalian origin. We report here the cloning and functional characterisation of a new member of this family of proteins from the nematode Caenorhabditis elegans. This protein exists as two isoforms of 413 and 453 amino acids with four or five tandem repeats that are 50% identical to the tau/MAP2/MAP4 repeats. Both isoforms bind to microtubules and promote microtubule assembly, with the five-repeat isoform being more effective at promoting assembly than the four-repeat isoform. When expressed in COS cells, the five-repeat isoform co-localises with microtubules and induces the formation of microtubule bundles, whereas its expression in Sf9 cells leads to the extension of long unipolar processes. In view of its length, amino acid sequence and functional characteristics, we have named this invertebrate structural MAP ‘Protein with Tau-Like Repeats’ (PTL-1). In C. elegans PTL-1 is expressed in two places known to require microtubule function. It is first seen in the embryonic epidermis, when circumferentially oriented microtubules help to distribute forces generated during elongation. Later, it is found in mechanosensory neurons which contain unusual 15 protofilament microtubules required for the response to touch. These findings indicate that MAPs of the tau/MAP2/MAP4 family are found throughout much of the animal kingdom, where they may play a role in specialised processes requiring microtubules.

Nematicidal effect of plumbagin on Caenorhabditis elegans: a model for testing a nematicidal drug

2016 ◽  
Vol 71 (5-6) ◽  
pp. 121-131 ◽  
Author(s):  
Phantip Chaweeborisuit ◽  
Chinnawut Suriyonplengsaeng ◽  
Worawit Suphamungmee ◽  
Prasert Sobhon ◽  
Krai Meemon
Keyword(s):  
Caenorhabditis Elegans ◽  
Traditional Medicine ◽  
Plant Species ◽  
Body Length ◽  
Body Wall ◽  
Parasitic Nematodes ◽  
Intestinal Cells ◽  
C Elegans ◽  
Body Wall Muscles ◽  
Anthelmintic Effect

Abstract Plumbagin, (5-hydroxy-2-methyl-1,4-naphthoquinone), a natural substance found in the roots of plant species in the genus Plumbago, has been used as a traditional medicine against many diseases. In this study, Caenorhabditis elegans was used as a model for testing the anthelmintic effect of plumbagin. The compound exhibited a nematicidal effect against all stages of C. elegans: L4 was least susceptible, while L1 was most susceptible to plumbagin with an LC50 of 220 and 156 μM, respectively. Plumbagin inhibited C. elegans development from L1 to adult stages with an IC50 of 235 μM, and body length was also reduced at concentrations of 25 and 50 μg/ml. Brood sizes decreased from 203±6 to 43±6 and 18±3 eggs per hatch in plumbagin-treated worms at 10, 25, 50 μg/ml, respectively. Furthermore, plumbagin was lethal to strains resistant to the nematicides levamisole, albendazole, and ivermectin, indicating that it possesses a strong and unique nematicidal action. Plumbagin decreased the number of mitochondria in hypodermal and intestinal cells and body wall muscles and damaged the ultrastructure of these tissues. Taken together, plumbagin may be a new drug against parasitic nematodes.

scholarly journals Sperm Competition in the Absence of Fertilization in Caenorhabditis elegans

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 201-208 ◽  
Author(s):  
Andrew Singson ◽  
Katherine L Hill ◽  
Steven W L’Hernault
Keyword(s):  
Caenorhabditis Elegans ◽  
Sperm Competition ◽  
Sperm Function ◽  
Sperm Precedence ◽  
Wild Type ◽  
Nematode Caenorhabditis Elegans ◽  
C Elegans ◽  
Normal Sperm ◽  
Self Fertilization ◽  
Competition Assays

Abstract Hermaphrodite self-fertilization is the primary mode of reproduction in the nematode Caenorhabditis elegans. However, when a hermaphrodite is crossed with a male, nearly all of the oocytes are fertilized by male-derived sperm. This sperm precedence during reproduction is due to the competitive superiority of male-derived sperm and results in a functional suppression of hermaphrodite self-fertility. In this study, mutant males that inseminate fertilization-defective sperm were used to reveal that sperm competition within a hermaphrodite does not require successful fertilization. However, sperm competition does require normal sperm motility. Additionally, sperm competition is not an absolute process because oocytes not fertilized by male-derived sperm can sometimes be fertilized by hermaphrodite-derived sperm. These results indicate that outcrossed progeny result from a wild-type cross because male-derived sperm are competitively superior and hermaphrodite-derived sperm become unavailable to oocytes. The sperm competition assays described in this study will be useful in further classifying the large number of currently identified mutations that alter sperm function and development in C. elegans.

scholarly journals Genetic, Behavioral and Environmental Determinants of Male Longevity in Caenorhabditis elegans

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1597-1610 ◽  
Author(s):  
David Gems ◽  
Donald L Riddle
Keyword(s):  
Caenorhabditis Elegans ◽  
Life Span ◽  
Wild Type ◽  
Neuronal Function ◽  
Nematode Caenorhabditis Elegans ◽  
Wild Isolate ◽  
Young Males ◽  
C Elegans ◽  
Single Sex ◽  
Solitary Males

Abstract Males of the nematode Caenorhabditis elegans are shorter lived than hermaphrodites when maintained in single-sex groups. We observed that groups of young males form clumps and that solitary males live longer, indicating that male-male interactions reduce life span. By contrast, grouped or isolated hermaphrodites exhibited the same longevity. In one wild isolate of C. elegans, AB2, there was evidence of copulation between males. Nine uncoordinated (unc) mutations were used to block clumping behavior. These mutations had little effect on hermaphrodite life span in most cases, yet many increased male longevity even beyond that of solitary wild-type males. In one case, the neuronal function mutant unc-64(e246), hermaphrodite life span was also increased by up to 60%. The longevity of unc-4(e120), unc-13(e51), and unc-32(e189) males exceeded that of hermaphrodites by 70–120%. This difference appears to reflect a difference in sex-specific life span potential revealed in the absence of male behavior that is detrimental to survival. The greater longevity of males appears not to be affected by daf-2, but is influenced by daf-16. In the absence of male-male interactions, median (but not maximum) male life span was variable. This variability was reduced when dead bacteria were used as food. Maintenance on dead bacteria extended both male and hermaphrodite longevity.

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