scholarly journals IMMUNOHISTOCHEMICAL LOCALIZATION OF CONTRACTILE PROTEINS IN LIMULUS STRIATED MUSCLE

1972 ◽  
Vol 55 (1) ◽  
pp. 221-235 ◽  
Author(s):  
Rhea J. C. Levine ◽  
Maynard M. Dewey ◽  
George W. de Villafranca
Keyword(s):  
Thin Filament ◽  
Sarcomere Length ◽  
Striated Muscle ◽  
Fluorescent Antibody ◽  
Thick Filament ◽  
Immunohistochemical Localization ◽  
Differential Staining ◽  
Indirect Fluorescent Antibody ◽  
The Core ◽  
Lateral Margins

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long (∼ 10.0 µ) and intermediate (∼ 7.0 µ) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0–∼6.0 µ. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.

scholarly journals Nature and origin of gap filaments in striated muscle

1991 ◽  
Vol 100 (4) ◽  
pp. 809-814 ◽  
Author(s):  
K. Trombitas ◽  
P.H. Baatsen ◽  
M.S. Kellermayer ◽  
G.H. Pollack
Keyword(s):  
Thin Filament ◽  
Sarcomere Length ◽  
Striated Muscle ◽  
Thick Filament ◽  
Abrupt Increase ◽  
Semitendinosus Muscle ◽  
Thin Filaments ◽  
Filament System ◽  
Anchor Points ◽  
High Degree

Immunoelectron microscopy was used to study the nature and origin of ‘gap’ filaments in frog semitendinosus muscle. Gap filaments are fine longitudinal filaments observable only in sarcomeres stretched beyond thick/thin filament overlap: they occupy the gap between the tips of thick and thin filaments. To test whether the gap filaments are part of the titin-filament system, we employed monoclonal antibodies to titin (T-11, Sigma) and observed the location of the epitope at a series of sarcomere lengths. At resting sarcomere length, the epitope was positioned in the I-band approximately 50 nm beyond the apparent ends of the thick filament. The location did not change perceptibly with increasing sarcomere length up to 3.6 microns. Above 3.6 microns, the span between the epitope and the end of the A-band abruptly increased, and above 4 microns, the antibodies could be seen to decorate the gap filaments. Between 5 and 6 microns, the epitope remained approximately in the middle of the gap. Even with this high degree of stretch, the label remained more or less aligned across the myofibril. The abrupt increase of span beyond 3.6 microns implies that the A-band domain of titin is pulled free of its anchor points along the thick filament, and moves toward the gap. Although this domain is functionally inextensible at physiological sarcomere length, the epitope movement in extremely stretched muscle shows that it is intrinsically elastic. Thus, the evidence confirms that gap filaments are clearly part of the titin-filament system. They are derived not only from the I-band domain of titin, but also from its A-band domain.

scholarly journals INTRINSIC BIREFRINGENCE OF GLYCERINATED MYOFIBRILS

1971 ◽  
Vol 51 (3) ◽  
pp. 763-771 ◽  
Author(s):  
Richard H. Colby
Keyword(s):  
Thin Filament ◽  
Striated Muscle ◽  
Thick Filament ◽  
Weight Basis ◽  
Macromolecular Structure ◽  
Form Birefringence ◽  
Sliding Filament Theory ◽  
Filament Protein ◽  
Formalin Fixed ◽  
Intrinsic Birefringence

Patterns of intrinsic birefringence were revealed in formalin-fixed, glycerinated myofibrils from rabbit striated muscle, by perfusing them with solvents of refractive index near to that of protein, about 1.570. The patterns differ substantially from those obtained in physiological salt solutions, due to the elimination of edge- and form birefringence. Analysis of myofibrils at various stages of shortening has produced results fully consistent with the sliding filament theory of contraction. On a weight basis, the intrinsic birefringence of thick-filament protein is about 2.4 times that of thin-filament protein. Nonadditivity of thick- and thin-filament birefringence in the overlap regions of A bands may indicate an alteration of macromolecular structure due to interaction between the two types of filaments.

scholarly journals Inotropic interventions do not change the resting state of myosin motors during cardiac diastole

2018 ◽  
Vol 151 (1) ◽  
pp. 53-65 ◽  
Author(s):  
Marco Caremani ◽  
Francesca Pinzauti ◽  
Joseph D. Powers ◽  
Serena Governali ◽  
Theyencheri Narayanan ◽  
...  
Keyword(s):  
Protein C ◽  
Thin Filament ◽  
Sarcomere Length ◽  
Binding Protein ◽  
Thick Filament ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Filament Activation ◽  
Myosin Binding ◽  
Myosin Motors

When striated (skeletal and cardiac) muscle is in its relaxed state, myosin motors are packed in helical tracks on the surface of the thick filament, folded toward the center of the sarcomere, and unable to bind actin or hydrolyze ATP (OFF state). This raises the question of whatthe mechanism is that integrates the Ca2+-dependent thin filament activation, making myosin heads available for interaction with actin. Here we test the interdependency of the thin and thick filament regulatory mechanisms in intact trabeculae from the rat heart. We record the x-ray diffraction signals that mark the state of the thick filament during inotropic interventions (increase in sarcomere length from 1.95 to 2.25 µm and addition of 10−7 M isoprenaline), which potentiate the twitch force developed by an electrically paced trabecula by up to twofold. During diastole, none of the signals related to the OFF state of the thick filament are significantly affected by these interventions, except the intensity of both myosin-binding protein C– and troponin-related meridional reflections, which reduce by 20% in the presence of isoprenaline. These results indicate that recruitment of myosin motors from their OFF state occurs independently and downstream from thin filament activation. This is in agreement with the recently discovered mechanism based on thick filament mechanosensing in which the number of motors available for interaction with actin rapidly adapts to the stress on the thick filament and thus to the loading conditions of the contraction. The gain of this positive feedback may be modulated by both sarcomere length and the degree of phosphorylation of myosin-binding protein C.

scholarly journals Titin switches from an extensible spring to a mechanical rectifier upon muscle activation

2021 ◽  
Author(s):  
Caterina Squarci ◽  
Pasquale Bianco ◽  
Massimo Reconditi ◽  
Marco Caremani ◽  
Theyencheri Narayanan ◽  
...  
Keyword(s):  
Thin Filament ◽  
Muscle Activation ◽  
Striated Muscle ◽  
Thick Filament ◽  
Frog Muscle ◽  
Motor Responses ◽  
Filament Activation ◽  
Single Fibres ◽  
Myosin Motors ◽  
Stimulation Of

In contracting striated muscle titin acts as a spring in parallel with the array of myosin motors in each half-sarcomere and could prevent the intrinsic instability of thousands of serially linked half-sarcomeres, if its stiffness, at physiological sarcomere lengths (SL), were ten times larger than reported. Here we define titin mechanical properties during tetanic stimulation of single fibres of frog muscle by suppressing myosin motor responses with Para-Nitro-Blebbistatin, which is able to freeze thick filament in the resting state. We discover that thin filament activation switches I-band titin spring from the large SL-dependent extensibility of the OFF-state to an ON-state in which titin acts as a SL-independent mechanical rectifier, allowing free shortening while opposing stretch with an effective stiffness 4 pN nm-1 per half-thick filament. In this way during contraction titin limits weak half-sarcomere elongation to a few % and, also, provides an efficient link for mechanosensing-based thick filament activation.

scholarly journals Aberrant myofibril assembly in tropomodulin1 null mice leads to aborted heart development and embryonic lethality

2003 ◽  
Vol 163 (5) ◽  
pp. 1033-1044 ◽  
Author(s):  
Kimberly L. Fritz-Six ◽  
Patrick R. Cox ◽  
Robert S. Fischer ◽  
Bisong Xu ◽  
Carol C. Gregorio ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Heart Development ◽  
Knockout Mouse ◽  
Thin Filament ◽  
Striated Muscle ◽  
Heart Defects ◽  
Thick Filament ◽  
Embryonic Lethality ◽  
Actin Dynamics ◽  
Filament Assembly

Tropomodulin1 (Tmod1) caps thin filament pointed ends in striated muscle, where it controls filament lengths by regulating actin dynamics. Here, we investigated myofibril assembly and heart development in a Tmod1 knockout mouse. In the absence of Tmod1, embryonic development appeared normal up to embryonic day (E) 8.5. By E9.5, heart defects were evident, including aborted development of the myocardium and inability to pump, leading to embryonic lethality by E10.5. Confocal microscopy of hearts of E8–8.5 Tmod1 null embryos revealed structures resembling nascent myofibrils with continuous F-actin staining and periodic dots of α-actinin, indicating that I-Z-I complexes assembled in the absence of Tmod1. Myomesin, a thick filament component, was also assembled normally along these structures, indicating that thick filament assembly is independent of Tmod1. However, myofibrils did not become striated, and gaps in F-actin staining (H zones) were never observed. We conclude that Tmod1 is required for regulation of actin filament lengths and myofibril maturation; this is critical for heart morphogenesis during embryonic development.

scholarly journals Decreased thin filament density and length in human atrophic soleus muscle fibers after spaceflight

2000 ◽  
Vol 88 (2) ◽  
pp. 567-572 ◽  
Author(s):  
Danny A. Riley ◽  
James L. W. Bain ◽  
Joyce L. Thompson ◽  
Robert H. Fitts ◽  
Jeffrey J. Widrick ◽  
...  
Keyword(s):  
Soleus Muscle ◽  
Thin Filament ◽  
Sarcomere Length ◽  
Muscle Fibers ◽  
Thick Filament ◽  
Thin Filaments ◽  
Contraction Velocity ◽  
Cross Bridge ◽  
Filament Spacing

Soleus muscle fibers were examined electron microscopically from pre- and postflight biopsies of four astronauts orbited for 17 days during the Life and Microgravity Sciences Spacelab Mission (June 1996). Myofilament density and spacing were normalized to a 2.4-μm sarcomere length. Thick filament density (∼1,062 filaments/μm2) and spacing (∼32.5 nm) were unchanged by spaceflight. Preflight thin filament density (2,976/μm2) decreased significantly ( P < 0.01) to 2,215/μm2 in the overlap A band region as a result of a 17% filament loss and a 9% increase in short filaments. Normal fibers had 13% short thin filaments. The 26% decrease in thin filaments is consistent with preliminary findings of a 14% increase in the myosin-to-actin ratio. Lower thin filament density was calculated to increase thick-to-thin filament spacing in vivo from 17 to 23 nm. Decreased density is postulated to promote earlier cross-bridge detachment and faster contraction velocity. Atrophic fibers may be more susceptible to sarcomere reloading damage, because force per thin filament is estimated to increase by 23%.

Effect of thin filament length on the force-sarcomere length relation of skeletal muscle

1991 ◽  
Vol 260 (5) ◽  
pp. C1060-C1070 ◽  
Author(s):  
H. L. Granzier ◽  
H. A. Akster ◽  
H. E. Ter Keurs
Keyword(s):  
Thin Filament ◽  
Sarcomere Length ◽  
Fiber Type ◽  
Thick Filament ◽  
Fiber Types ◽  
Single Region ◽  
Slow Twitch ◽  
The Difference ◽  
Slow Twitch Fibers ◽  
Fast Twitch

We studied a slow- and a fast-twitch muscle fiber type of the perch that have different thin filament lengths. The force-sarcomere length relations were measured, and it was tested whether their descending limbs were predicted by the cross-bridge theory. To determine the predicted relations, filament lengths were measured by electron microscopy. Measurements were corrected for shrinkage with the use of I-band and H-zone periodicities. Thick filament lengths of the two fiber types were found to be similar (1.63 +/- 0.06 and 1.64 +/- 0.10 microns for slow- and fast-twitch fibers, respectively), whereas the thin filament lengths were clearly different: 1.24 +/- 0.10 microns (n = 86) for the slow-twitch type and 0.94 +/- 0.04 microns (n = 94) for the fast type. The descending limbs of the two fiber types are therefore predicted to be shifted along the sarcomere length axis by approximately 0.6 microns. Sarcomere length was measured on-line by laser diffraction in a single region in the center of the fibers. The passive force-sarcomere strain relation increased much more steeply in the slow-twitch fibers. The descending limb of the active force-sarcomere length relation of fast twitch fibers was linear (r = 0.92), but was found at sarcomere lengths approximately 0.1 micron greater than predicted. The descending limb of the slow-twitch fibers was also linear (r = 0.87) but was now found at sarcomere lengths approximately 0.05 microns less than predicted. The difference in position of the descending limbs of the two fiber types amounted to 0.5 microns, approximately 0.1 micron less than predicted. The difference between measured and predicted descending limbs was statistically insignificant.

Energy utilization by Limulus telson muscle at different sarcomere and A-band lengths

1982 ◽  
Vol 242 (3) ◽  
pp. R394-R400
Author(s):  
S. Davidheiser ◽  
R. E. Davies
Keyword(s):  
Energy Expenditure ◽  
Thin Filament ◽  
Sarcomere Length ◽  
Thick Filament ◽  
Isometric Tension ◽  
Energy Utilization ◽  
External Work ◽  
Thick Filaments ◽  
Chemical Efficiency ◽  
Per Se

Phosphorylargnine utilization (delta PArg) was determined in isolated Limulus telson muscle during isometric and isovelocity contractions at long, intermediate, and short lengths when thick filaments were either long and staggered, long and aligned, or shortened. Muscles developed 30% of maximum force at a length of 0.5 Lo (Lo 7 micrometers sarcomere length) during isometric tetani of 5, 15, and 30 s; however, the rate of delta PArg was the same in both the 0.5 Lo and Lo groups. External force and delta PArg were both less during isometric tetani at lengths of 1.7 and 2.0 Lo compared with the values at Lo, as expected due to a reduction in thick-thin filament overlap. However, at lengths of 0.6 and 0.3 Lo delta PArg was the same as at Lo despite a decrease in external isometric tension. No significant delta PArg was measured when telson muscles shortened rapidly (0.8 Vmax) at long or short lengths indicating the thick filament shortening per se required little, if any, energy expenditure. The overall chemical efficiency of telson muscles for performing external work during slow isovelocity contractions was 31% in groups shortening equivalent distances (0.5 Lo) from starting lengths of 1.6 and 1.3 Lo and 15% in the group shortening from 1.1 Lo.

Orientation of actin filaments during motion in motility assay

1995 ◽  
Vol 53 ◽  
pp. 52-53
Author(s):  
J. Borejdo ◽  
S. Burlacu
Keyword(s):  
Actin Filaments ◽  
Thin Filament ◽  
Striated Muscle ◽  
Myosin Head ◽  
Classical Method ◽  
Polarization Of Fluorescence ◽  
Single Protein ◽  
Intracellular Organelles ◽  
Change Orientation ◽  
Active Entity

Polarization of fluorescence is a classical method to assess orientation or mobility of macromolecules. It has been a common practice to measure polarization of fluorescence through a microscope to characterize orientation or mobility of intracellular organelles, for example anisotropic bands in striated muscle. Recently, we have extended this technique to characterize single protein molecules. The scientific question concerned the current problem in muscle motility: whether myosin heads or actin filaments change orientation during contraction. The classical view is that the force-generating step in muscle is caused by change in orientation of myosin head (subfragment-1 or SI) relative to the axis of thin filament. The molecular impeller which causes this change resides at the interface between actin and SI, but it is not clear whether only the myosin head or both SI and actin change orientation during contraction. Most studies assume that observed orientational change in myosin head is a reflection of the fact that myosin is an active entity and actin serves merely as a passive "rail" on which myosin moves.

Rubella Antibodies in Human Serum: Detection by the Indirect Fluorescent-Antibody Technique

Science ◽  
1964 ◽  
Vol 145 (3635) ◽  
pp. 943-945 ◽  
Author(s):  
G. C. Brown ◽  
H. F. Maassab ◽  
J. A. Veronelli ◽  
T. J. Francis
Keyword(s):  
Human Serum ◽  
Fluorescent Antibody ◽  
Fluorescent Antibody Technique ◽  
Indirect Fluorescent Antibody ◽  
Antibody Technique ◽  
Serum Detection
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