scholarly journals Molecular and ultrastructural defects in a Drosophila myosin heavy chain mutant: differential effects on muscle function produced by similar thick filament abnormalities.

1988 ◽  
Vol 107 (6) ◽  
pp. 2601-2612 ◽  
Author(s):  
P T O'Donnell ◽  
S I Bernstein
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Muscle Function ◽  
Flight Muscle ◽  
Thick Filament ◽  
Differential Sensitivity ◽  
Indirect Flight Muscle ◽  
Molecular Defect ◽  
Thick Filaments ◽  
Nuclease Mapping

We have determined the molecular defect of the Drosophila melanogaster myosin heavy chain (MHC) mutation Mhc and the mutation's effect on indirect flight muscle, jump muscle, and larval intersegmental muscle. We show that the Mhc1 mutation is essentially a null allele which results in the dominant-flightless and recessive-lethal phenotypes associated with this mutant (Mogami, K., P. T. O'Donnell, S. I. Bernstein, T. R. F. Wright, C. P. Emerson, Jr. 1986. Proc. Natl. Acad. Sci. USA. 83:1393-1397). The mutation is a 101-bp deletion in the MHC gene which removes most of exon 5 and the intron that precedes it. S1 nuclease mapping indicates that mutant transcripts follow two alternative processing pathways. Both pathways result in the production of mature transcripts with altered reading frames, apparently yielding unstable, truncated MHC proteins. Interestingly, the preferred splicing pathway uses the more distal of two available splice donor sites. We present the first ultrastrutural characterization of a completely MHC-null muscle and show that it lacks any discernable thick filaments. Sarcomeres in these muscles are completely disorganized suggesting that thick filaments play a critical role in sarcomere assembly. To understand why the Mhc1 mutation severely disrupts indirect flight muscle and jump muscle function in heterozygotes, but does not seriously affect the function of other muscle types, we examined the muscle ultrastructure of Mhc1/+ heterozygotes. We find that these organisms have a nearly 50% reduction in the number of thick filaments in indirect flight muscle, jump muscle, and larval intersegmental muscle. In addition, aberrantly shaped thick filaments are common in the jump muscle and larval intersegmental muscle. We suggest that the differential sensitivity of muscle function to the Mhc1 mutation is a consequence of the unique myofilament arrays in each of these muscles. The highly variable myofilament array of larval intersegmental muscle makes its function relatively insensitive to changes in thick filament number and morphology. Conversely, the rigid double hexagonal lattice of the indirect flight muscle, and the organized lattice of the jump muscle cannot be perturbed without interfering with the specialized and evolutionarily more complex functions they perform.

scholarly journals Ifm(2)2 is a myosin heavy chain allele that disrupts myofibrillar assembly only in the indirect flight muscle of Drosophila melanogaster.

1988 ◽  
Vol 107 (6) ◽  
pp. 2613-2621 ◽  
Author(s):  
M Chun ◽  
S Falkenthal
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Microscopic Analysis ◽  
Thick Filament ◽  
Indirect Flight Muscle ◽  
Chain Gene ◽  
Wild Type ◽  
Electron Microscopic ◽  
Sarcomere Assembly

Using a combination of molecular and genetic techniques we demonstrate that Ifm(2)2 is an allele of the single-copy sarcomeric myosin heavy chain gene. Flies homozygous for this allele accumulate wild-type levels of mRNA and protein in tubular muscle of adults, but fail to accumulate detectable amounts of myosin heavy chain mRNA or protein in the indirect flight muscle. We propose that the mutation interferes with either transcription of the gene or splicing of the primary transcript in the indirect flight muscle and not in other muscle tissues. Biochemical and electron microscopic analysis of flies homozygous for this mutation has revealed that thick filament assembly is abolished in the indirect flight muscle resulting in the instability of wild-type thick filament proteins. In contrast, thin filament and Z disc assembly are marginally affected. We discuss a working hypothesis for sarcomere assembly and define and experimental approach to test the predictions of this proposed pathway for sarcomere assembly.

scholarly journals The Drosophila indirect flight muscle myosin heavy chain isoform is insufficient to transform the jump muscle into a highly stretch-activated muscle type

2017 ◽  
Vol 312 (2) ◽  
pp. C111-C118 ◽  
Author(s):  
Cuiping Zhao ◽  
Douglas M. Swank
Keyword(s):  
Power Generation ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Flight Muscle ◽  
Fold Increase ◽  
Isometric Tension ◽  
Indirect Flight Muscle ◽  
Muscle Type ◽  
Myosin Heavy Chain Isoform ◽  
Muscle Types

Stretch activation (SA) is a delayed increase in force that enables high power and efficiency from a cyclically contracting muscle. SA exists in various degrees in almost all muscle types. In Drosophila, the indirect flight muscle (IFM) displays exceptionally high SA force production ( FSA), whereas the jump muscle produces only minimal FSA. We previously found that expressing an embryonic (EMB) myosin heavy chain (MHC) isoform in the jump muscle transforms it into a moderately SA muscle type and enables positive cyclical power generation. To investigate whether variation in MHC isoforms is sufficient to produce even higher FSA, we substituted the IFM MHC isoform (IFI) into the jump muscle. Surprisingly, we found that IFI only caused a 1.7-fold increase in FSA, less than half the increase previously observed with EMB, and only at a high Pi concentration, 16 mM. This IFI-induced FSA is much less than what occurs in IFM, relative to isometric tension, and did not enable positive cyclical power generation by the jump muscle. Both isometric tension and FSA of control fibers decreased with increasing Pi concentration. However, for IFI-expressing fibers, only isometric tension decreased. The rate of FSA generation was ~1.5-fold faster for IFI fibers than control fibers, and both rates were Pi dependent. We conclude that MHC isoforms can alter FSA and hence cyclical power generation but that isoforms can only endow a muscle type with moderate FSA. Highly SA muscle types, such as IFM, likely use a different or additional mechanism.

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 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 Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells

1997 ◽  
Vol 8 (12) ◽  
pp. 2605-2615 ◽  
Author(s):  
James H. Sabry ◽  
Sheri L. Moores ◽  
Shannon Ryan ◽  
Ji-Hong Zang ◽  
James A. Spudich
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Fluorescent Protein ◽  
Molecular Motor ◽  
Thick Filament ◽  
Live Cells ◽  
Tail Region ◽  
Thick Filaments ◽  
Dividing Cells ◽  
Green Fluorescent

Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.

scholarly journals The effect of heavy chain phosphorylation and solution conditions on the assembly of Acanthamoeba myosin-II.

1989 ◽  
Vol 109 (4) ◽  
pp. 1529-1535 ◽  
Author(s):  
J H Sinard ◽  
T D Pollard
Keyword(s):  
Light Scattering ◽  
Ionic Strength ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Critical Concentration ◽  
Myosin Ii ◽  
Acidic Ph ◽  
Thick Filaments ◽  
Low Ionic Strength

At low ionic strength, Acanthamoeba myosin-II polymerizes into bipolar minifilaments, consisting of eight molecules, that scatter about three times as much light as monomers. With this light scattering assay, we show that the critical concentration for assembly in 50-mM KCl is less than 5 nM. Phosphorylation of the myosin heavy chain over the range of 0.7 to 3.7 P per molecule has no effect on its KCl dependent assembly properties: the structure of the filaments, the extent of assembly, and the critical concentration for assembly are the same. Sucrose at a concentration above a few percent inhibits polymerization. Millimolar concentrations of MgCl2 induce the lateral aggregation of fully formed minifilaments into thick filaments. Compared with dephosphorylated minifilaments, minifilaments of phosphorylated myosin have a lower tendency to aggregate laterally and require higher concentrations of MgCl2 for maximal light scattering. Acidic pH also induces lateral aggregation, whereas basic pH leads to depolymerization of the myosin-II minifilaments. Under polymerizing conditions, millimolar concentrations of ATP only slightly decrease the light scattering of either phosphorylated or dephosphorylated myosin-II. Barring further modulation of assembly by unknown proteins, both phosphorylated and dephosphorylated myosin-II are expected to be in the form of minifilaments under the ionic conditions existing within Acanthamoeba.

scholarly journals Specific Myosin Heavy Chain Mutations Suppress Troponin I Defects in Drosophila Muscles

1999 ◽  
Vol 144 (5) ◽  
pp. 989-1000 ◽  
Author(s):  
William A. Kronert ◽  
Angel Acebes ◽  
Alberto Ferrús ◽  
Sanford I. Bernstein
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Thin Filament ◽  
Troponin I ◽  
Point Mutations ◽  
Thick Filament ◽  
Actin Binding ◽  
Filament Protein ◽  
Phenotypic Rescue

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.

scholarly journals Structure of Thick Filaments from Drosophila Indirect Flight Muscle by Cryo-EM

2019 ◽  
Vol 116 (3) ◽  
pp. 404a
Author(s):  
Nadia Daneshparvar ◽  
Dianne Taylor ◽  
Hamidreza Rahmani ◽  
Kenneth A. Taylor
Keyword(s):  
Flight Muscle ◽  
Indirect Flight Muscle ◽  
Thick Filaments

scholarly journals Point Mutations in Human β Cardiac Myosin Heavy Chain Have Differential Effects on Sarcomeric Structure and Assembly: An ATP Binding Site Change Disrupts Both Thick and Thin Filaments, Whereas Hypertrophic Cardiomyopathy Mutations Display Normal Assembly

1997 ◽  
Vol 137 (1) ◽  
pp. 131-140 ◽  
Author(s):  
K. David Becker ◽  
Kim R. Gottshall ◽  
Reed Hickey ◽  
Jean-Claude Perriard ◽  
Kenneth R. Chien
Keyword(s):  
Hypertrophic Cardiomyopathy ◽  
Subcellular Localization ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Point Mutations ◽  
Neonatal Rat ◽  
Atp Binding ◽  
Cardiac Myosin ◽  
Wild Type ◽  
Thick Filaments

Hypertrophic cardiomyopathy is a human heart disease characterized by increased ventricular mass, focal areas of fibrosis, myocyte, and myofibrillar disorganization. This genetically dominant disease can be caused by mutations in any one of several contractile proteins, including β cardiac myosin heavy chain (βMHC). To determine whether point mutations in human βMHC have direct effects on interfering with filament assembly and sarcomeric structure, full-length wild-type and mutant human βMHC cDNAs were cloned and expressed in primary cultures of neonatal rat ventricular cardiomyocytes (NRC) under conditions that promote myofibrillogenesis. A lysine to arginine change at amino acid 184 in the consensus ATP binding sequence of human βMHC resulted in abnormal subcellular localization and disrupted both thick and thin filament structure in transfected NRC. Diffuse βMHC K184R protein appeared to colocalize with actin throughout the myocyte, suggesting a tight interaction of these two proteins. Human βMHC with S472V mutation assembled normally into thick filaments and did not affect sarcomeric structure. Two mutant myosins previously described as causing human hypertrophic cardiomyopathy, R249Q and R403Q, were competent to assemble into thick filaments producing myofibrils with well defined I bands, A bands, and H zones. Coexpression and detection of wild-type βMHC and either R249Q or R403Q proteins in the same myocyte showed these proteins are equally able to assemble into the sarcomere and provided no discernible differences in subcellular localization. Thus, human βMHC R249Q and R403Q mutant proteins were readily incorporated into NRC sarcomeres and did not disrupt myofilament formation. This study indicates that the phenotype of myofibrillar disarray seen in HCM patients which harbor either of these two mutations may not be directly due to the failure of the mutant myosin heavy chain protein to assemble and form normal sarcomeres, but may rather be a secondary effect possibly resulting from the chronic stress of decreased βMHC function.

scholarly journals Co-expression of multiple myosin heavy chain genes, in addition to a tissue-specific one, in extraocular musculature.

1985 ◽  
Vol 101 (2) ◽  
pp. 618-629 ◽  
Author(s):  
D F Wieczorek ◽  
M Periasamy ◽  
G S Butler-Browne ◽  
R G Whalen ◽  
B Nadal-Ginard
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Mrna Level ◽  
S1 Nuclease ◽  
Tissue Specific ◽  
Adult Rat ◽  
Mhc Genes ◽  
The Individual ◽  
Genomic Probes ◽  
Nuclease Mapping

We have investigated the developmental transitions of myosin heavy chain (MHC) gene expression in the rat extraocular musculature (EOM) at the mRNA level using S1-nuclease mapping techniques and at the protein level by polypeptide mapping and immunochemistry. We have isolated a genomic clone, designated lambda 10B3, corresponding to an MHC gene which is expressed in the EOM fibers (recti and oblique muscles) of the adult rat but not in hind limb muscles. Using cDNA and genomic probes for MHC genes expressed in skeletal (embryonic, neonatal, fast oxidative, fast glycolytic, and slow/cardiac beta-MHC), cardiac (alpha-MHC), and EOM (lambda 10B3) muscles, we demonstrate the concomitant expression at the mRNA level of at least six different MHC genes in adult EOM. Protein and immunochemical analyses confirm the presence of at least four different MHC types in EOM. Immunocytochemistry demonstrates that different myosin isozymes tend to segregate into individual myofibers, although some fibers seem to contain more than one MHC type. The results also show that the EOM fibers exhibit multiple patterns of MHC gene regulation. One set of fibers undergoes a sequence of isoform transitions similar to the one described for limb skeletal muscles, whereas other EOM myofiber populations arrest the MHC transition at the embryonic, neonatal/adult, or adult EOM-specific stage. Thus, the MHC gene family is not under the control of a strict developmental clock, but the individual genes can modify their expression by tissue-specific and/or environmental factors.

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