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 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.

scholarly journals Type IV Collagen Is Detectable in Most, but Not All, Basement Membranes of Caenorhabditis elegans and Assembles on Tissues That Do Not Express It

1997 ◽  
Vol 137 (5) ◽  
pp. 1171-1183 ◽  
Author(s):  
Patricia L. Graham ◽  
Jeffrey J. Johnson ◽  
Shaoru Wang ◽  
Marion H. Sibley ◽  
Malini C. Gupta ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Type Iv Collagen ◽  
Fluorescent Protein ◽  
Muscle Cells ◽  
Basement Membranes ◽  
Body Wall Muscle ◽  
Collagen Molecule ◽  
C Elegans ◽  
Type Iv

Type IV collagen in Caenorhabditis elegans is produced by two essential genes, emb-9 and let-2, which encode α1- and α2-like chains, respectively. The distribution of EMB-9 and LET-2 chains has been characterized using chain-specific antisera. The chains colocalize, suggesting that they may function in a single heterotrimeric collagen molecule. Type IV collagen is detected in all basement membranes except those on the pseudocoelomic face of body wall muscle and on the regions of the hypodermis between body wall muscle quadrants, indicating that there are major structural differences between some basement membranes in C. elegans. Using lacZ/green fluorescent protein (GFP) reporter constructs, both type IV collagen genes were shown to be expressed in the same cells, primarily body wall muscles, and some somatic cells of the gonad. Although the pharynx and intestine are covered with basement membranes that contain type IV collagen, these tissues do not express either type IV collagen gene. Using an epitope-tagged emb-9 construct, we show that type IV collagen made in body wall muscle cells can assemble into the pharyngeal, intestinal, and gonadal basement membranes. Additionally, we show that expression of functional type IV collagen only in body wall muscle cells is sufficient for C. elegans to complete development and be partially fertile. Since type IV collagen secreted from muscle cells only assembles into some of the basement membranes that it has access to, there must be a mechanism regulating its assembly. We propose that interaction with a cell surface–associated molecule(s) is required to facilitate type IV collagen assembly.

scholarly journals A screen for genetic loci required for body-wall muscle development during embryogenesis in Caenorhabditis elegans.

Genetics ◽  
1994 ◽  
Vol 137 (2) ◽  
pp. 483-498
Author(s):  
J Ahnn ◽  
A Fire
Keyword(s):  
Caenorhabditis Elegans ◽  
Myosin Heavy Chain ◽  
Muscle Cell ◽  
Heavy Chain ◽  
Body Wall ◽  
Muscle Development ◽  
Contractile Function ◽  
The Body ◽  
Body Wall Muscle ◽  
Genetic Loci

Abstract We have used available chromosomal deficiencies to screen for genetic loci whose zygotic expression is required for formation of body-wall muscle cells during embryogenesis in Caenorhabditis elegans. To test for muscle cell differentiation we have assayed for both contractile function and the expression of muscle-specific structural proteins. Monoclonal antibodies directed against two myosin heavy chain isoforms, the products of the unc-54 and myo-3 genes, were used to detect body-wall muscle differentiation. We have screened 77 deficiencies, covering approximately 72% of the genome. Deficiency homozygotes in most cases stain with antibodies to the body-wall muscle myosins and in many cases muscle contractile function is observed. We have identified two regions showing distinct defects in myosin heavy chain gene expression. Embryos homozygous for deficiencies removing the left tip of chromosome V fail to accumulate the myo-3 and unc-54 products, but express antigens characteristic of hypodermal, pharyngeal and neural development. Embryos lacking a large region on chromosome III accumulate the unc-54 product but not the myo-3 product. We conclude that there exist only a small number of loci whose zygotic expression is uniquely required for adoption of a muscle cell fate.

scholarly journals Mutations Affecting Nerve Attachment of Caenorhabditis elegans

Genetics ◽  
2001 ◽  
Vol 157 (4) ◽  
pp. 1611-1622 ◽  
Author(s):  
Go Shioi ◽  
Michinari Shoji ◽  
Masashi Nakamura ◽  
Takeshi Ishihara ◽  
Isao Katsura ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nerve Cord ◽  
Ventral Nerve Cord ◽  
Body Wall ◽  
The Body ◽  
Genetic Loci ◽  
Muscle Attachment ◽  
C Elegans ◽  
Ventral Cord ◽  
Excretory Canal

Abstract Using a pan-neuronal GFP marker, a morphological screen was performed to detect Caenorhabditis elegans larval lethal mutants with severely disorganized major nerve cords. We recovered and characterized 21 mutants that displayed displacement or detachment of the ventral nerve cord from the body wall (Ven: ventral cord abnormal). Six mutations defined three novel genetic loci: ven-1, ven-2, and ven-3. Fifteen mutations proved to be alleles of previously identified muscle attachment/positioning genes, mup-4, mua-1, mua-5, and mua-6. All the mutants also displayed muscle attachment/positioning defects characteristic of mua/mup mutants. The pan-neuronal GFP marker also revealed that mutants of other mua/mup loci, such as mup-1, mup-2, and mua-2, exhibited the Ven defect. The hypodermis, the excretory canal, and the gonad were morphologically abnormal in some of the mutants. The pleiotropic nature of the defects indicates that ven and mua/mup genes are required generally for the maintenance of attachment of tissues to the body wall in C. elegans.

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.

Duels without obvious sense: counteracting inductions involved in body wall muscle development in the Caenorhabditis elegans embryo

Development ◽  
1995 ◽  
Vol 121 (7) ◽  
pp. 2219-2232 ◽  
Author(s):  
R. Schnabel
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Muscle Development ◽  
Early Embryo ◽  
Cell Interactions ◽  
Body Wall Muscle ◽  
Cellular Interactions ◽  
Classical Criterion ◽  
Founder Cells ◽  
Cell Cell

During the first four cleavage rounds of the Caenorhabditis elegans embryo, five somatic founder cells AB, MS, E, C and D are born, which later form the tissues of the embryo. The classical criterion for a cell-autonomous specification of a tissue is the capability of primordial cells to produce this tissue in isolation from the remainder of the embryo. By this criterion, the somatic founder cells MS, C and D develop cell-autonomously. Laser ablation experiments, however, reveal that within the embryonic context these blastomeres form a network of duelling cellular interactions. During normal development, the blastomere D inhibits muscle specification in the MS and the C lineage inhibits muscle specification in the D lineage. These inhibitory interactions are counteracted by two activating inductions. As described before the inhibition of body wall muscle in MS is counteracted by an activating signal from the ABa lineage. Body wall muscle in the D lineage is induced by MS descendants, which suppress an inhibitory activity of the C lineage. The interaction between the D and the MS lineage occurs through the C lineage. An interesting feature of these cell-cell interactions is that they do not serve to discriminate between equivalent cells but are permissive or nonpermissive inductions. No evidence was found that the C-derived body wall muscle also depends on an induction, which suggests that possibly three different pathways coexist in the early embryo to specify body wall muscle, two of which are, in different ways, influenced by cell-cell interactions and a third that is autonomous. This work supplies evidence that cells may acquire transient states during embryogenesis that influence the specification of other cells in the embryo. These states, however, may not be reflected in the developmental potentials of the cells themselves. They can only be scored indirectly by their action on the specification of other cells in the embryo. Blastomeres that behave cell-autonomously in isolation are nevertheless subjected to cell-cell interactions in the embryonic context. Why this should be is an intriguing question. The classical notion has been that blastomeres are specified autonomously in nematodes. In recent years, it was established that at least five inductions are required to determine the AB descendants of C. elegans, whereas the P1 descendants have been typically viewed to develop more autonomously. It appears now that inductions also play a major role during the determination of P1-derived blastomeres.

scholarly journals Ca^-binding to site IV and TnI-binding of body-wall muscle troponin C are essential for development in Caenorhabditis elegans

2004 ◽  
Vol 44 (supplement) ◽  
pp. S65
Author(s):  
T. Takaya ◽  
H. Terami ◽  
M. Sohda ◽  
T. Iio ◽  
H. Kagawa
Keyword(s):  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Troponin C ◽  
Body Wall Muscle

Broad oxygen tolerance in the nematode Caenorhabditis elegans

2000 ◽  
Vol 203 (16) ◽  
pp. 2467-2478 ◽  
Author(s):  
W.A. Van Voorhies ◽  
S. Ward
Keyword(s):  
Caenorhabditis Elegans ◽  
Metabolic Rate ◽  
Developmental Stages ◽  
Small Body ◽  
Metabolic Cost ◽  
Soil Nematode ◽  
Nematode Caenorhabditis Elegans ◽  
Oxygen Levels ◽  
C Elegans ◽  
Short Period

This study examined the effects of oxygen tensions ranging from 0 to 90 kPa on the metabolic rate (rate of carbon dioxide production), movement and survivorship of the free-living soil nematode Caenorhabditis elegans. C. elegans requires oxygen to develop and survive. However, it can maintain a normal metabolic rate at oxygen levels of 3.6 kPa and has near-normal metabolic rates at oxygen levels as low as 2 kPa. The ability to withstand low ambient oxygen levels appears to be a consequence of the small body size of C. elegans, which allows diffusion to supply oxygen readily to the cells without requiring any specialized respiratory or metabolic adaptations. Thus, the small size of this organism pre-adapts C. elegans to living in soil environments that commonly become hypoxic. Movement in C. elegans appears to have a relatively minor metabolic cost. Several developmental stages of C. elegans were able to withstand up to 24 h of anoxia without major mortality. Longer periods of anoxia significantly increased mortality, particularly for eggs. Remarkably, long-term exposure to 100 % oxygen had no effect on the metabolic rate of C. elegans, and populations were able to survive for a least 50 generations in 100 % (90 kPa) oxygen. Such hyperoxic conditions are fatal to most organisms within a short period.

Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene

Development ◽  
1991 ◽  
Vol 111 (3) ◽  
pp. 667-681 ◽  
Author(s):  
P.Y. Goh ◽  
T. Bogaert
Keyword(s):  
Extracellular Matrix ◽  
Body Wall ◽  
Early Stage ◽  
Muscle Growth ◽  
The Body ◽  
Body Wall Muscle ◽  
C Elegans ◽  
Extracellular Matrix Molecule ◽  
Extracellular Matrix Components ◽  
Body Wall Muscles

As part of a general study of genes specifying a pattern of muscle attachments, we identified and genetically characterised mutants in the mup-1 gene. The body wall muscles of early stage mup-1 embryos have a wild-type myofilament pattern but may extend ectopic processes. Later in embryogenesis, some body wall muscles detach from the hypodermis. Genetic analysis suggests that mup-1 has both a maternal and a zygotic component and is not required for postembryonic muscle growth and attachment. mup-1 mutants are suppressed by mutations in several genes that encode extracellular matrix components. We propose that mup-1 may encode a cell surface/extracellular matrix molecule required both for the positioning of body wall muscle attachments in early embryogenesis and the subsequent maintenance of these attachments to the hypodermis until after cuticle synthesis.

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