scholarly journals SOME ASPECTS OF THE STRUCTURAL ORGANIZATION OF THE MYOFIBRIL AS REVEALED BY ANTIBODY-STAINING METHODS

1966 ◽  
Vol 28 (3) ◽  
pp. 505-525 ◽  
Author(s):  
Frank A. Pepe
Keyword(s):  
Structural Changes ◽  
Fluorescent Antibody ◽  
Thick Filament ◽  
Myosin Molecule ◽  
Weakly Bound ◽  
Antibody Staining ◽  
Thick Filaments ◽  
Antigenic Sites ◽  
Myosin Filaments ◽  
Staining Methods

From observations of fluorescent antibody staining and antibody staining in electron microscopy, evidence is presented for the following: (a) Direct contact of the actin and myosin filaments occurs at all stages of contraction. This results in inhibition of antibody staining of the H-meromyosin portion of the myosin molecule in the region of overlap of the thin and thick filaments. (b) Small structural changes occur in the thick filaments during contraction. This leads to exposure of antigenic sites of the L-meromyosin portion of the myosin molecule. The accessibility of these antigenic sites is dependent upon the sarcomere length. (c) The M line is composed of a protein which is weakly bound to the center of the thick filament and is not actin, myosin, or tropomyosin. (d) Tropomyosin as well as actin is present in the I band. (e) If actin or tropomyosin is present in the Z line, it is masked and unavailable for staining with antibody.

Myosin thick filament lability induced by mechanical strain in airway smooth muscle

2001 ◽  
Vol 90 (5) ◽  
pp. 1811-1816 ◽  
Author(s):  
Kuo-Hsing Kuo ◽  
Lu Wang ◽  
Peter D. Paré ◽  
Lincoln E. Ford ◽  
Chun Y. Seow
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Cross Sections ◽  
Structural Changes ◽  
Isometric Force ◽  
Mechanical Strain ◽  
Thick Filament ◽  
Functional Changes ◽  
Thick Filaments ◽  
Length Changes

Airway smooth muscle adapts to different lengths with functional changes that suggest plastic alterations in the filament lattice. To look for structural changes that might be associated with this plasticity, we studied the relationship between isometric force generation and myosin thick filament density in cell cross sections, measured by electron microscope, after length oscillations applied to the relaxed porcine trachealis muscle. Muscles were stimulated regularly for 12 s every 5 min. Between two stimulations, the muscles were submitted to repeated passive ±30% length changes. This caused tetanic force and thick-filament density to fall by 21 and 27%, respectively. However, in subsequent tetani, both force and filament density recovered to preoscillation levels. These findings indicate that thick filaments in airway smooth muscle are labile, depolymerization of the myosin filaments can be induced by mechanical strain, and repolymerization of the thick filaments underlies force recovery after the oscillation. This thick-filament lability would greatly facilitate plastic changes of lattice length and explain why airway smooth muscle is able to function over a large length range.

scholarly journals LOCALIZATION OF MYOSIN FILAMENTS IN SMOOTH MUSCLE

1968 ◽  
Vol 37 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Robert E. Kelly ◽  
Robert V. Rice
Keyword(s):  
Smooth Muscle ◽  
Cross Section ◽  
Striated Muscle ◽  
Thick Filament ◽  
Longitudinal Axis ◽  
Thin Filaments ◽  
Chicken Gizzard ◽  
Thick Filaments ◽  
Myosin Filaments ◽  
Muscle Myosin

Thick myosin filaments, in addition to actin filaments, were found in sections of glycerinated chicken gizzard smooth muscle when fixed at a pH below 6.6. The thick filaments were often grouped into bundles and run in the longitudinal axis of the smooth muscle cell. Each thick filament was surrounded by a number of thin filaments, giving the filament arrangement a rosette appearance in cross-section. The exact ratio of thick filaments to thin filaments could not be determined since most arrays were not so regular as those commonly found in striated muscle. Some rosettes had seven or eight thin filaments surrounding a single thick filament. Homogenates of smooth muscle of chicken gizzard also showed both thick and thin filaments when the isolation was carried out at a pH below 6.6, but only thin filaments were found at pH 7.4. No Z or M lines were observed in chicken gizzard muscle containing both thick and thin filaments. The lack of these organizing structures may allow smooth muscle myosin to disaggregate readily at pH 7.4.

scholarly journals THE M BAND

1971 ◽  
Vol 48 (2) ◽  
pp. 340-347 ◽  
Author(s):  
Ellen Kundrat ◽  
Frank A. Pepe
Keyword(s):  
Strong Evidence ◽  
Structural Integrity ◽  
Staining Pattern ◽  
Fluorescent Antibody ◽  
Extraction Procedure ◽  
Thick Filament ◽  
Tris Buffer ◽  
Antibody Staining ◽  
Band Material ◽  
Fluorescent Antibody Staining

The M band can be extracted from fibrils suspended in 5 mM Tris buffer, pH 8.0, for 15 min. The M band is completely removed only from fibrils of sarcomere lengths greater than 2.1 µ. Extraction does not alter the fluorescent antimyosin staining pattern of the A band, thus providing strong evidence that no alteration of the structural integrity of the thick filament has occurred. Fluorescent antibody staining of the M band of unextracted fibrils can be prevented specifically by absorbing the fluorescent antibody with extracted M band material prior to staining. This verifies the specificity of the extraction procedure.

scholarly journals Monoclonal antibodies against seven sites on the head and tail of Dictyostelium myosin.

1985 ◽  
Vol 100 (4) ◽  
pp. 1016-1023 ◽  
Author(s):  
G Peltz ◽  
J A Spudich ◽  
P Parham
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Heavy Chain ◽  
Light Chain ◽  
Binding Sites ◽  
Myosin Molecule ◽  
Dictyostelium Myosin ◽  
Antigenic Sites ◽  
Myosin Filaments ◽  
A Site

Ten monoclonal antibodies (My1-10) against Dictyostelium discoideum myosin were prepared and characterized. Nine bound to the 210-kD heavy chain and one (My8) bound to the 18-kD light chain. They defined six topographically distinct antigenic sites of the heavy chain. Five binding sites (the My1, My5, My10 site, and the My2, My3, My4, and My9 sites) are located on the rod portion of the myosin molecule. The position of the sixth site (the My6 and My7 site) is less certain, but it appears to be near the junction of the globular heads and the rod. Three of the antibodies (My2, My3, and My6) bound to myosin filaments in solution and could be sedimented in stoichiometric amounts with the filamentous myosin. In contrast, My4, which recognized a site on the rod, inhibited the polymerization of monomeric myosin into filaments. A single antibody (My6) affected the actin-activated ATPase of myosin. The nature of the effect depended on the valency of the antibody and the myosin. Bivalent IgG and F(ab')2 fragments of My6 inhibited the actin-activated ATPase of filamentous myosin by 50% whereas univalent Fab' fragments increased the activity by 50%. The actin-activated ATPase activity of the soluble chymotryptic fragment of myosin was increased 80-90% by both F(ab')2 and Fab' of My6.

The self-assembly of synthetic filaments of myosin isolated from Chaos carolinensis and Amoeba proteus

1977 ◽  
Vol 25 (1) ◽  
pp. 387-402
Author(s):  
J.S. Condeelis
Keyword(s):  
Ionic Strength ◽  
Self Assembly ◽  
Divalent Cations ◽  
Central Zone ◽  
Thick Filament ◽  
Amoeba Proteus ◽  
High Ionic Strength ◽  
Thick Filaments ◽  
Myosin Filaments ◽  
Low Ionic Strength

Synthetic myosin thick filaments were formed from preparations of electrophoretically homogeneous myosin isolated from Chaos carolinensis and Amoeba proteus when dialysed to physiological ionic strength and pH. Myosin dialysed directly against low ionic strength buffers formed native-like thick filaments in the presence and absence of exogenous divalent cations. The average dimensions of the synthetic filaments grown under these conditions were 455 nm long and 16 nm wide with a distinct bare central zone 174 nm long. Myosin predialysed against EGTA-EDTA solutions at high ionic strength and then dialysed to low ionic strength formed native-like filaments only in the presence of 1mM Mg2+. 1 mM Ca2+ could not be substituted for Mg2+ under these conditions to achieve native-like filaments. Filaments grown from predialysed myosin in the absence of Mg2+ resembled EGTA-dissociated myosin filaments observed in EGTA-treated cytoplasm and were highly branched, poorly formed filaments lacking a distinct bare central zone. The average dimensions of the filaments grown from predialysed myosin in the absence of Mg2+ were 328 nm long, 13 nm wide with a bare central zone 111 nm long. Under the conditions tested, myosin isolated from these amoebae did not demonstrate a divalent cation requirement for thick filament formation. The results obtained with myosin isolated from the 2 organisms were identical.

scholarly journals Distinct contributions of the thin and thick filaments to length-dependent activation in heart muscle

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Xuemeng Zhang ◽  
Thomas Kampourakis ◽  
Ziqian Yan ◽  
Ivanka Sevrieva ◽  
Malcolm Irving ◽  
...  
Keyword(s):  
Heart Muscle ◽  
Structural Changes ◽  
Muscle Cells ◽  
Thick Filament ◽  
Calcium Sensitivity ◽  
Cellular Level ◽  
Thin Filaments ◽  
Thick Filaments ◽  
Filament Structure ◽  
Heart Muscle Cells

The Frank-Starling relation is a fundamental auto-regulatory property of the heart that ensures the volume of blood ejected in each heartbeat is matched to the extent of venous filling. At the cellular level, heart muscle cells generate higher force when stretched, but despite intense efforts the underlying molecular mechanism remains unknown. We applied a fluorescence-based method, which reports structural changes separately in the thick and thin filaments of rat cardiac muscle, to elucidate that mechanism. The distinct structural changes of troponin C in the thin filaments and myosin regulatory light chain in the thick filaments allowed us to identify two aspects of the Frank-Starling relation. Our results show that the enhanced force observed when heart muscle cells are maximally activated by calcium is due to a change in thick filament structure, but the increase in calcium sensitivity at lower calcium levels is due to a change in thin filament structure.

scholarly journals Dynamics of myosin replacement in skeletal muscle cells

2015 ◽  
Vol 309 (10) ◽  
pp. C669-C679 ◽  
Author(s):  
Koichi Ojima ◽  
Emi Ichimura ◽  
Yuya Yasukawa ◽  
Jun-ichi Wakamatsu ◽  
Takanori Nishimura
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Dynamic Equilibrium ◽  
Muscle Cells ◽  
Thick Filament ◽  
Skeletal Muscle Cells ◽  
Myosin Molecule ◽  
Thick Filaments ◽  
Cultured Myotubes ◽  
Exchange Unit

Highly organized thick filaments in skeletal muscle cells are formed from ∼300 myosin molecules. Each thick-filament-associated myosin molecule is thought to be constantly exchanged. However, the mechanism of myosin replacement remains unclear, as does the source of myosin for substitution. Here, we investigated the dynamics of myosin exchange in the myofibrils of cultured myotubes by fluorescent recovery after photobleaching and found that myofibrillar myosin is actively replaced with an exchange half-life of ∼3 h. Myosin replacement was not disrupted by the absence of the microtubule system or by actomyosin interactions, suggesting that known cytoskeletal systems are dispensable for myosin substitution. Intriguingly, myosin replacement was independent of myosin binding protein C, which links myosin molecules together to form thick filaments. This implies that an individual myosin molecule rather than a thick filament functions as an exchange unit. Furthermore, the myosin substitution rate was decreased by the inhibition of protein synthesis, suggesting that newly synthesized myosin, as well as preexisting cytosolic myosin, contributes to myosin replacement in myofibrils. Notably, incorporation and release of myosin occurred simultaneously in myofibrils, but rapid myosin release from myofibrils was observed without protein synthesis. Collectively, our results indicate that myosin shuttles between myofibrils and the nonmyofibrillar cytosol to maintain a dynamic equilibrium in skeletal muscle cells.

Hemocytic rickettsia-like organisms inIxodes scapularis: transovarial and transstadial transmission

1995 ◽  
Vol 73 (7) ◽  
pp. 1380-1383
Author(s):  
Louis A. Magnarelli ◽  
Thomas N. Mather ◽  
Min-Tsung Yeh
Keyword(s):  
Rhode Island ◽  
Ixodes Scapularis ◽  
Spotted Fever ◽  
Fluorescent Antibody ◽  
Rocky Mountain ◽  
Rocky Mountain Spotted Fever ◽  
Antibody Staining ◽  
Host Seeking ◽  
Staining Methods ◽  
Fluorescent Antibody Staining

Unengorged, host-seeking female Ixodes scapularis were collected from the northeastern United States (Rhode Island and Massachusetts) in April 1992. Hemolymph preparations from F1males and females were tested to determine if hemocytic rickettsia-like organisms, having shared antigens with ehrlichiae, are passed transovarially and transstadially. Fluorescein-labeled dog anti-Ehrlichia canis antiserum was used to detect infected hemocytes by means of direct fluorescent antibody staining methods. In duplicate tests of F1ticks, hemocytes from 25 males and 25 females were tested by indirect fluorescent antibody staining methods with antisera from two persons who had Rocky Mountain spotted fever. These hemolymph preparations were nonreactive, but those from cohorts of ticks reared from five infected females and tested with anti-E. canis antiserum contained rickettsia-like organisms in the cytoplasm of hemocytes. Thirty-four (54.8%) of 62 males and 71 (63.4%) of 112 females were positive. Prevalence of positive males among the five cohorts ranged from 18.8 to 80%, while results for females varied between 21.2 and 91.7%. The unidentified rickettsia-like hemocytic organisms, which have shared antigens with Ehrlichia species, are transmitted transovarially and transstadially in I. scapularis.

scholarly journals Structural changes that occur in scallop myosin filaments upon activation.

1985 ◽  
Vol 101 (3) ◽  
pp. 830-837 ◽  
Author(s):  
P Vibert ◽  
R Craig
Keyword(s):  
Calcium Binding ◽  
Structural Changes ◽  
Striated Muscle ◽  
Thick Filament ◽  
Light Chains ◽  
Free Calcium ◽  
Thin Filaments ◽  
Regulatory Light Chains ◽  
Myosin Filaments ◽  
Free Calcium Concentration

Myosin filaments isolated from scallop striated muscle have been activated by calcium-containing solutions, and their structure has been examined by electron microscopy after negative staining. The orderly helical arrangement of myosin projections characteristic of the relaxed state is largely lost upon activation. The oblique striping that arises from alignment of elongated projections along the long-pitched helical tracks is greatly weakened, although a 145 A axial periodicity is sometimes partially retained. The edges of the filaments become rough, and the myosin heads move outwards as their helical arrangement becomes disordered. Crossbridges at various angles appear to link thick and thin filaments after activation. The transition from order to disorder is reversible and occurs over a narrow range of free calcium concentration near pCa 5.7. Removal of nucleotide, as well as dissociation of regulatory light chains, also disrupts the ordered helical arrangement of projections. We suggest that the relaxed arrangement of the projections is probably maintained by intermolecular interactions between myosin molecules, which depend on the regulatory light chains. Calcium binding changes the interactions between light chains and the rest of the head, activating the myosin molecule. Intermolecular contacts between molecules may thus be altered and may propagate activation cooperatively throughout the thick filament.

scholarly journals Myosin molecule packing within the vertebrate skeletal muscle thick filaments. A complete bipolar model.

2002 ◽  
Vol 49 (4) ◽  
pp. 829-840 ◽  
Author(s):  
Ludmila Skubiszak ◽  
Leszek Kowalczyk
Keyword(s):  
Skeletal Muscle ◽  
Computer Modelling ◽  
Thick Filament ◽  
Myosin Molecule ◽  
Bipolar Structure ◽  
Cross Bridge ◽  
Filament Axis ◽  
Thick Filaments ◽  
C Terminus ◽  
Vertebrate Skeletal Muscle

Computer modelling related to the real dimensions of both the whole filament and the myosin molecule subfragments has revealed two alternative modes for myosin molecule packing which lead to the head disposition similar to that observed by EM on the surface of the cross-bridge zone of the relaxed vertebrate skeletal muscle thick filaments. One of the modes has been known for three decades and is usually incorporated into the so-called three-stranded model. The new mode differs from the former one in two aspects: (1) myosin heads are grouped into asymmetrical cross-bridge crowns instead of symmetrical ones; (2) not the whole myosin tail, but only a 43-nm C-terminus of each of them is straightened and near-parallel to the filament axis, the rest of the tail is twisted. Concurrent exploration of these alternative modes has revealed their influence on the filament features. The parameter values for the filament models as well as for the building units depicting the myosin molecule subfragments are verified by experimental data found in the literature. On the basis of the new mode for myosin molecule packing a complete bipolar structure of the thick filament is created.

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