scholarly journals The alteration of myosin isoform compartmentation in specific mutants of Caenorhabditis elegans.

1986 ◽  
Vol 103 (3) ◽  
pp. 985-993 ◽  
Author(s):  
H F Epstein ◽  
I Ortiz ◽  
L A Mackinnon
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
Body Wall ◽  
Thick Filament ◽  
Polar Regions ◽  
Myosin Isoforms ◽  
Intrinsic Properties ◽  
Specific Antibodies ◽  
Thick Filaments ◽  
Filament Protein

Myosin isoforms A and B are located at the surface of the central and polar regions, respectively, of thick filaments in body muscle cells of Caenorhabditis elegans, whereas paramyosin and a distinct core structure comprise the backbones of these filaments. Thick filaments and related structures were isolated from nematode mutants that have altered thick filament protein compositions. These mutant filaments and their complexes with specific antibodies were studied by electron microscopy to determine the distribution of the two myosins. The compartmentation of the two myosin isoforms in body wall muscle thick filaments depends not only upon the intrinsic properties of the myosins but their interactions with other components such as paramyosin and their relative quantities determined by synthesis.

scholarly journals Thick filament substructures in Caenorhabditis elegans: evidence for two populations of paramyosin.

1993 ◽  
Vol 123 (2) ◽  
pp. 303-311 ◽  
Author(s):  
P R Deitiker ◽  
H F Epstein
Keyword(s):  
Electron Microscopy ◽  
Caenorhabditis Elegans ◽  
Structural Analysis ◽  
Heavy Chain ◽  
Thick Filament ◽  
Myosin Heavy Chain Isoforms ◽  
Nematode Caenorhabditis Elegans ◽  
Thick Filaments ◽  
Nacl Concentration ◽  
Two Populations

The thick filaments of the nematode Caenorhabditis elegans contain two myosin heavy chain isoforms A and B and paramyosin, the products of the myo-3, unc-54, and unc-15 genes, respectively. Dissociation of paramyosin from native thick filaments at pH 6.36 shows a biphasic function with respect to NaCl concentration. Electron microscopy of the remaining structures shows 15-nm core structures that label with monoclonal anti-paramyosin antibody at 72.5-nm intervals. Purified core structures also show 72.5 nm repeats by negative staining. Structural analysis of native thick filaments and dissociated structures suggests that the more dissociable paramyosin is removed radially as well as processively from the filament ends. Minor proteins with masses of 20, 28, and 30 kD cosediment stoichiometrically with paramyosin in purified core structures.

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.

scholarly journals Myosin and paramyosin are organized about a newly identified core structure.

1985 ◽  
Vol 100 (3) ◽  
pp. 904-915 ◽  
Author(s):  
H F Epstein ◽  
D M Miller ◽  
I Ortiz ◽  
G C Berliner
Keyword(s):  
Thick Filament ◽  
Core Structure ◽  
Polar Regions ◽  
Myosin Isoforms ◽  
The Core ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Filament Structure ◽  
Intermediate Domain

Myosin isoforms A and B are differentially localized to the central and polar regions, respectively, of thick filaments in body wall muscle cells of Caenorhabditis elegans (Miller, D. M. III, I. Ortiz, G. C. Berliner, and H. F. Epstein, 1983, Cell, 34:477-490). Biochemical and electron microscope studies of KCl-dissociated filaments show that the myosin isoforms occupy a surface domain, paramyosin constitutes an intermediate domain, and a newly identified core structure exists. The diameters of the thick filaments vary significantly from 33.4 nm centrally to 14.0 nm near the ends. The latter value is comparable to the 15.2 nm diameter of the core structures. The internal density of the filament core appears solid medially and hollow at the poles. The differentiation of thick filament structure into supramolecular domains possessing specific substructures of characteristic stabilities suggests a sequential mode for thick filament assembly. In this model, the two myosin isoforms have distinct roles in assembly. The behavior of the myosins, including nucleation of assembly and determination of filament length, depend upon paramyosin and the core structure as well as their intrinsic molecular properties.

scholarly journals Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

1996 ◽  
Vol 135 (2) ◽  
pp. 371-382 ◽  
Author(s):  
P E Hoppe ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Thick Filament ◽  
Surface Hydrophobicity ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Characteristic Variation ◽  
Muscle Myosin

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

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 Axial arrangement of the myosin rod in vertebrate thick filaments: immunoelectron microscopy with a monoclonal antibody to light meromyosin.

1985 ◽  
Vol 101 (3) ◽  
pp. 1115-1123 ◽  
Author(s):  
T Shimizu ◽  
J E Dennis ◽  
T Masaki ◽  
D A Fischman
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Solid Phase ◽  
Strong Support ◽  
Thick Filament ◽  
Repeat Distance ◽  
Thick Filaments ◽  
Axial Translation

A monoclonal antibody, MF20, which has been shown previously to bind the myosin heavy chain of vertebrate striated muscle, has been proven to bind the light meromyosin (LMM) fragment by solid phase radioimmune assay with alpha-chymotryptic digests of purified myosin. Epitope mapping by electron microscopy of rotary-shadowed, myosin-antibody complexes has localized the antibody binding site to LMM at a point approximately 92 nm from the C-terminus of the myosin heavy chain. Since this epitope in native thick filaments is accessible to monoclonal antibodies, we used this antibody as a high affinity ligand to analyze the packing of LMM along the backbone of the thick filament. By immunofluorescence microscopy, MF20 was shown to bind along the entire A-band of chicken pectoralis myofibrils, although the epitope accessibility was greater near the ends than at the center of the A-bands. Thin-section, transmission electron microscopy of myofibrils decorated with MF20 revealed 50 regularly spaced, cross-striations in each half A-band, with a repeat distance of approximately 13 nm. These were numbered consecutively, 1-50, from the A-band to the last stripe, approximately 68 nm from the filament tips. These same striations could be visualized by negative staining of native thick filaments labeled with MF20. All 50 striations were of a consecutive, uninterrupted repeat which approximated the 14-15-nm axial translation of cross-bridges. Each half M-region contained five MF20 striations (approximately 13 nm apart) with a distance between stripes 1 and 1', on each half of the bare zone, of approximately 18 nm. This is compatible with a packing model with full, antiparallel overlap of the myosin rods in the bare zone region. Differences in the spacings measured with negatively stained myofilaments and thin-sectioned myofibrils have been shown to arise from specimen shrinkage in the fixed and embedded preparations. These observations provide strong support for Huxley's original proposal for myosin packing in thick filaments of vertebrate muscle (Huxley, H. E., 1963, J. Mol. Biol., 7:281-308) and, for the first time, directly demonstrate that the 14-15-nm axial translation of LMM in the thick filament backbone corresponds to the cross-bridge repeat detected with x-ray diffraction of living muscle.

scholarly journals Role of stress fiber-like structures in assembling nascent myofibrils in myosheets recovering from exposure to ethyl methanesulfonate.

1986 ◽  
Vol 102 (4) ◽  
pp. 1464-1479 ◽  
Author(s):  
P B Antin ◽  
S Tokunaka ◽  
V T Nachmias ◽  
H Holtzer
Keyword(s):  
Electron Microscopy ◽  
Sarcoplasmic Reticulum ◽  
Intermediate Filaments ◽  
Alkylating Agent ◽  
Temporal Correlation ◽  
Stress Fiber ◽  
Ethyl Methanesulfonate ◽  
Myosin Isoforms ◽  
Thick Filaments

When day 1 cultures of chick myogenic cells were exposed to the mutagenic alkylating agent ethyl methanesulfonate (EMS) for 3 d, 80% of the replicating cells were killed, but postmitotic myoblasts survived. The myoblasts fused to form unusual multinucleated "myosheets": extraordinarily wide, flattened structures that were devoid of myofibrils but displayed extensive, submembranous stress fiber-like structures (SFLS). Immunoblots of the myosheets indicated that the carcinogen blocked the synthesis and accumulation of the myofibrillar myosin isoforms but not that of the cytoplasmic myosin isoform. When removed from EMS, widely spaced nascent myofibrils gradually emerged in the myosheets after 3 d. Striking co-localization of fluorescent reagents that stained SFLS and those that specifically stained myofibrils was observed for the next 2 d. By both immunofluorescence and electron microscopy, individual nascent myofibrils appeared to be part of, or juxtaposed to, preexisting individual SFLS. By day 6, all SFLS had disappeared, and the definitive myofibrils were displaced from their submembranous site into the interior of the myosheet. Immunoblots from recovering myosheets demonstrated a temporal correlation between the appearance of the myofibrillar myosin isoforms and the assembly of thick filaments. The assembly of definitive myofibrils did not appear to involve desmin intermediate filaments, but a striking aggregation of sarcoplasmic reticulum elements was seen at the level of each I-Z-band. Our findings suggest that SFLS in the EMS myosheets function as early, transitory assembly sites for nascent myofibrils.

scholarly journals Caenorhabditis elegansUNC-96 Is a New Component of M-Lines That Interacts with UNC-98 and Paramyosin and Is Required in Adult Muscle for Assembly and/or Maintenance of Thick Filaments

2006 ◽  
Vol 17 (9) ◽  
pp. 3832-3847 ◽  
Author(s):  
Kristina B. Mercer ◽  
Rachel K. Miller ◽  
Tina L. Tinley ◽  
Seema Sheth ◽  
Hiroshi Qadota ◽  
...  
Keyword(s):  
Body Wall ◽  
Polarized Light ◽  
Thick Filament ◽  
Immunofluorescent Staining ◽  
Molecular Architecture ◽  
Adult Muscle ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Reduced Temperature ◽  
Insight Into

To gain further insight into the molecular architecture, assembly, and maintenance of the sarcomere, we have carried out a molecular analysis of the UNC-96 protein in the muscle of Caenorhabditis elegans. By polarized light microscopy of body wall muscle, unc-96 mutants display reduced myofibrillar organization and characteristic birefringent “needles.” By immunofluorescent staining of known myofibril components, unc-96 mutants show major defects in the organization of M-lines and in the localization of a major thick filament component, paramyosin. In unc-96 mutants, the birefringent needles, which contain both UNC-98 and paramyosin, can be suppressed by starvation or by exposure to reduced temperature. UNC-96 is a novel ∼47-kDa polypeptide that has no recognizable domains. Antibodies generated to UNC-96 localize the protein to the M-line, a region of the sarcomere in which thick filaments are cross-linked. By genetic and biochemical criteria, UNC-96 interacts with UNC-98, a previously described component of M-lines, and paramyosin. Additionally, UNC-96 copurifies with native thick filaments. A model is presented in which UNC-96 is required in adult muscle to promote thick filament assembly and/or maintenance.

Distribution of c-protein isoforms in sarcomeres of developing chick skeletal muscle

1988 ◽  
Vol 46 ◽  
pp. 226-227
Author(s):  
D. A. Fischman ◽  
J. E. Dennis ◽  
T. Obinata ◽  
H. Takano-Ohmuro
Keyword(s):  
Skeletal Muscles ◽  
Thick Filament ◽  
Protein Isoforms ◽  
Actin Binding ◽  
Striated Muscles ◽  
C Protein ◽  
Sarcomeric Protein ◽  
Thick Filaments ◽  
Cross Bridges ◽  
Bridge Bearing

C-protein is a 150 kDa protein found within the A bands of all vertebrate cross-striated muscles. By immunoelectron microscopy, it has been demonstrated that C-protein is distributed along a series of 7-9 transverse stripes in the medial, cross-bridge bearing zone of each A band. This zone is now termed the C-zone of the sarcomere. Interest in this protein has been sparked by its striking distribution in the sarcomere: the transverse repeat between C-protein stripes is 43 nm, almost exactly 3 times the 14.3 nm axial repeat of myosin cross-bridges along the thick filaments. The precise packing of C-protein in the thick filament is still unknown. It is the only sarcomeric protein which binds to both myosin and actin, and the actin-binding is Ca-sensitive. In cardiac and slow, but not fast, skeletal muscles C-protein is phosphorylated. Amino acid composition suggests a protein of little or no αhelical content. Variant forms (isoforms) of C-protein have been identified in cardiac, slow and embryonic muscles.

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