Factors affecting movement of F-actin filaments propelled by skeletal muscle heavy meromyosin

1992 ◽  
Vol 262 (3) ◽  
pp. C714-C723 ◽  
Author(s):  
E. Homsher ◽  
F. Wang ◽  
J. R. Sellers
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Actin Filament ◽  
Molecular Motors ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Shortening Velocity ◽  
Weakly Bound ◽  
Factors Affecting ◽  
Cross Bridges

The measurement of fluorescent-labeled actin filament movement driven by mechanoenzymes (e.g., myosin) is an important methodology for the study of molecular motors. It is assumed that the filament velocity (Vf) is analogous to the unloaded shortening velocity (Vu) seen in muscle fibers. Methods are described to reproducibly quantitate the movement of these filaments and to select uniformly moving filaments and specify their Vf. Use of these techniques allowed comparison of Vf to literature values for Vu with regard to [ATP], [ADP], [Pi], pH, ionic strength (10-150 mM), and temperature (15-30 degrees C). Vf and Vu are quantitatively similar with respect to the effects of substrate and product concentrations and temperatures greater than 20 degrees C. However, Vf is more sensitive to decreases in pH and temperatures less than 20 degrees C than Vu. At ionic strengths of 50-150 mM, Vf and Vu exhibit similar ionic strength dependencies (decreasing with ionic strength). At ionic strengths less than 50 mM, Vf is markedly reduced. Results of experiments using adenosine 5'-O-(3-thiotriphosphate) suggest that increasing the number of weakly bound cross bridges does not seriously affect Vf. Thus, although Vf is a good analogue for Vu under certain conditions (elevated ionic strength and temperatures greater than 20 degrees C), under others it is not. The results of motility assays must be cautiously interpreted.

Basis of passive tension and stiffness in isolated rabbit myofibrils

1997 ◽  
Vol 273 (1) ◽  
pp. C266-C276 ◽  
Author(s):  
M. L. Bartoo ◽  
W. A. Linke ◽  
G. H. Pollack
Keyword(s):  
Ionic Strength ◽  
Dynamic Stiffness ◽  
Passive Tension ◽  
Passive Force ◽  
Force Response ◽  
Weakly Bound ◽  
Strain Limit ◽  
Sinusoidal Oscillations ◽  
Cross Bridges ◽  
Low Ionic Strength

By examining the mechanical properties of isolated skeletal and cardiac myofibrils in calcium-free, ATP-containing solution, we attempted to separate the stiffness contribution of titin filaments from that of weakly bound cross bridges. Efforts to enhance weak cross-bridge binding by lowering ionic strength were met by clear contractile responses. Even at low temperature, myofibrils bathed in low-ionic-strength relaxing solution generated increased force and exhibited sarcomere shortening, apparently caused by active contraction. At normal ionic strength, myofibril stiffness, estimated from the force response to rapid sinusoidal oscillations, increased steadily with sarcomere extension up to a strain limit. No obvious stiffness contribution from weak cross bridges was detectable. Instead, the stiffness response, which was frequency dependent at all sarcomere lengths, was apparently generated by the viscoelastic titin filaments. During imposed stretch-hold ramps, both peak force/stiffness and the amount of subsequent stress relaxation increased with higher stretch rates, larger stretch amplitudes, and longer sarcomere lengths. We conclude that, for a truly relaxed myofibril, both passive force and dynamic stiffness principally reflect the intrinsic viscoelastic properties of the titin filaments.

Nonmuscle tropomyosin-4 requires coexpression with other low molecular weight isoforms for binding to thin filaments in cardiomyocytes

1999 ◽  
Vol 112 (3) ◽  
pp. 371-380 ◽  
Author(s):  
D.M. Helfman ◽  
C. Berthier ◽  
J. Grossman ◽  
M. Leu ◽  
E. Ehler ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Actin Filament ◽  
Actin Filaments ◽  
Fluorescent Protein ◽  
Synergistic Effects ◽  
Neonatal Rat ◽  
Low Molecular Weight ◽  
Rat Cardiomyocytes ◽  
Filament Dynamics

Vertebrate tropomyosins (TMs) are expressed from four genes, and at least 18 distinct isoforms are generated via a complex pattern of alternative RNA splicing and alternative promoters. The functional significance of this isoform diversity is largely unknown and it remains to be determined whether specific isoforms are required for assembly and integration into distinct actin-containing structures. The ability of nonmuscle (TM-1, -2, -3, -4, -5(NM1), -5a or -5b) and striated muscle (skeletal muscle (α)-TM) isoforms to incorporate into actin filaments of neonatal rat cardiomyocytes (NRCs) was studied using expression plasmids containing TM-fusions with GFP (green fluorescent protein) as well as with VSV- or HA-epitope tags. All isoforms, except of fibroblast TM-4, were able to incorporate into the I-band of NRCs. When TM-4 was co-transfected with other low molecular weight (LMW) isoforms of TM (TM-5, TM-5a and TM-5b), it was able to incorporate into sarcomeres of NRCs. This result was not obtained when TM-4 was co-transfected with high molecular weight (HMW) TMs (TM-1, TM-2 or skeletal muscle (α)-TM). These data demonstrate that the ability of TM-4 to bind to actin filaments can be specifically influenced by its interaction with other LMW TM isoforms. In addition, cells that incorporated the muscle or nonmuscle GFP-TMs into their sarcomeres continued to beat and exhibited sarcomeric contraction. These studies provide the first in vivo demonstration of synergistic effects between TM isoforms for binding to actin filaments. These results have important implications in understanding actin filament dynamics in nonmuscle cell systems, especially during development and in transformed cells, where alterations in the ratio of different LMW isoforms might lead to changes in their interactions with actin filaments. Furthermore, these studies demonstrate that GFP-TM can be used to study thin-filament dynamics in muscle cells and actin filament dynamics in nonmuscle cells.

scholarly journals FILAMENT ULTRASTRUCTURE AND ORGANIZATION IN VERTEBRATE SMOOTH MUSCLE

1967 ◽  
Vol 35 (2) ◽  
pp. 303-321 ◽  
Author(s):  
Bernard J. Panner ◽  
Carl R. Honig
Keyword(s):  
Electron Microscopy ◽  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Actin Filaments ◽  
Heavy Meromyosin ◽  
Dense Bodies ◽  
Contraction Mechanism ◽  
Dark Staining ◽  
Technical Factors ◽  
Low Ionic Strength

Using a variety of preparative techniques for electron microscopy, we have obtained evidence for the disposition of actin and myosin in vertebrate smooth muscle. All longitudinal myofilaments seen in sections appear to be actin. Previous reports of two types of longitudinal filaments in sections are accounted for by technical factors, and by differentiated areas of opacity along individual filaments. Dense bodies with actin emerging from both ends have been identified in homogenates, and resemble Z discs from skeletal muscle (Huxley, 1963). In sections, short, dark-staining lateral filaments 15–25 A in diameter link adjacent actin filaments within dense bodies and in membrane dense pataches. They appear homologous with Z-disc filaments. Similar lateral filaments connect actin to plasma membrane. Dense bodies and dense patches, therefore, are attachment points and denote units analogous to sarcomeres. In glycerinated, methacrylate-embedded sections, lateral processes different in length and staining characteristics from lateral filaments in dense bodies exist at intervals along actin filaments. These processes are about 30 A wide and resemble heavy meromyosin from skeletal muscle. They also resemble heads of whole molecules of myosin in negatively stained material from gizzard homogenates. Intact single myosin molecules and dimers have been found, both free and attached to actin, even in media of very low ionic strength. Myosin can, therefore, exist in relatively disaggregated form. Models of the contraction mechanism of smooth muscle are proposed. The unique features are: (1) Myosin exists as small functional units. (2) Movement occurs by interdigitation and sliding of actin filaments.

scholarly journals High ionic strength and low pH detain activated skinned rabbit skeletal muscle crossbridges in a low force state.

1993 ◽  
Vol 101 (4) ◽  
pp. 487-511 ◽  
Author(s):  
C Y Seow ◽  
L E Ford
Keyword(s):  
Skeletal Muscle ◽  
Ionic Strength ◽  
Isometric Force ◽  
Maximum Velocity ◽  
Low Ph ◽  
High Ionic Strength ◽  
Rabbit Skeletal Muscle ◽  
Shortening Velocity ◽  
Force Ratio ◽  
Force Difference

The effects of varying pH and ionic strength on the force-velocity relations and tension transients of skinned rabbit skeletal muscle were studied at 1-2 degrees C. Both decreasing pH from 7.35 to 6.35 and raising ionic strength from 125 to 360 mM reduced isometric force by about half and decreased sarcomere stiffness by about one-fourth, so that the stiffness/force ratio was increased by half. Lowering pH also decreased maximum shortening velocity by approximately 29%, while increasing ionic strength had little effect on velocity. These effects on velocity were correlated with asymmetrical effects on stiffness. The increase in the stiffness/force ratio with both interventions was manifest as a greater relative force change associated with a sarcomere length step. This force difference persisted for a variable time after the step. At the high ionic strength the force difference was long-lasting after stretches but relaxed quickly after releases, suggesting that the structures responsible would not impose much resistance to steady-state shortening. The opposite was found in the low pH experiments. The force difference relaxed quickly after stretches but persisted for a long time after releases. Furthermore, this force difference reached a constant value of approximately 8% of isometric force with intermediate sizes of release, and was not increased with larger releases. This value was almost identical to the value of an internal load that would be sufficient to account for the reduction in maximum velocity seen at the low pH. The results are interpreted as showing that both low pH and high ionic strength inhibit the movement of crossbridges into the force-generating parts of their cycle after they have attached to the actin filaments, with very few other effects on the cycle. The two interventions are different, however, in that detained bridges can be detached readily by shortening when the detention is caused by high ionic strength but not when it is caused by low pH.

scholarly journals Ionic Strength and the Contraction Kinetics of Skinned Muscle Fibers

1974 ◽  
Vol 63 (4) ◽  
pp. 509-530 ◽  
Author(s):  
Marc D. Thames ◽  
Louis E. Teichholz ◽  
Richard J. Podolsky
Keyword(s):  
Ionic Strength ◽  
Muscle Fibers ◽  
Bathing Solution ◽  
Shortening Velocity ◽  
Skinned Muscle ◽  
Cross Bridges ◽  
Low Ionic Strength ◽  
Contraction Cycle ◽  
Kinetics Of ◽  
Contraction Kinetics

The influence of KCl concentration on the contraction kinetics of skinned frog muscle fibers at 5–7°C was studied at various calcium levels. The magnitude of the calcium-activated force decreased continuously as the KCl concentration of the bathing solution was increased from 0 to 280 mM. The shortening velocity at a given relative load was unaffected by the level of calcium activation at 140 mM KCl, as has been previously reported by Podolsky and Teichholz (1970. J. Physiol. [Lond.]. 211: 19), and was independent of ionic strength when the KCl concentration was increased from 140 to 280 mM. In contrast, the shortening velocity decreased as the KCl concentration was reduced below 140 mM; the decrease in velocity was enhanced when the fibers were only partially activated. In the low KCl range, the resting tension of the fibers increased after the first contraction cycle. The results suggest that in fibers activated at low ionic strength some of the cross bridges that are formed are abnormal in the sense that they retard shortening and persist in relaxing solution.

scholarly journals Myosin Cross-bridge Behaviour in Contracting Muscle – The T1 Curve of Huxley & Simmons (1971) Revisited

2019 ◽  
Author(s):  
Carlo Knupp ◽  
John M. Squire
Keyword(s):  
Actin Filaments ◽  
Striated Muscle ◽  
X Ray Diffraction ◽  
Step Size ◽  
Weakly Bound ◽  
X Ray ◽  
Cross Bridge ◽  
Key Factor ◽  
Length Changes ◽  
Cross Bridges

The stiffness of the myosin cross-bridges is a key factor in analysing possible scenarios to explain myosin head changes during force generation in active muscles.  The seminal study of Huxley and Simmons (1971: Nature 233: 533) suggested that most of the observed half-sarcomere instantaneous compliance (=1/stiffness) resides in the myosin heads.    They showed with a so-called T1 plot that, after a very fast release, the half-sarcomere tension reduced to zero after a step size of about 60Å (later with improved experiments reduced to 40Å).   However, later X-ray diffraction studies showed that myosin and actin filaments themselves stretch slightly under tension, which means that most (at least two-thirds) of the half sarcomere compliance comes from the filaments and not from cross-bridges.    Here we have used a different approach, namely to model the compliances in a virtual half sarcomere structure in silico.   We confirm that the T1 curve comes almost entirely from length changes in the myosin and actin filaments, because the calculated cross-bridge stiffness (probably greater than 0.4 pN/Å) is higher than previous studies have suggested.    In the light of this, we present a plausible modified scenario to describe aspects of the myosin cross-bridge cycle in active muscle.   In particular, we suggest that, apart from the filament compliances, most of the cross-bridge contribution to the instantaneous T1 response comes from weakly-bound myosin heads, not myosin heads in strongly attached states.   The strongly attached heads would still contribute to the T1 curve, but only in a very minor way, with a stiffness that we postulate could be around 0.1 pN/Å, a value which would generate a working stroke close to 100 Å from the hydrolysis of one ATP molecule.  The new program can serve as a tool to calculate sarcomere elastic properties for any vertebrate striated muscle once various parameters have been determined (e.g. tension, T1 intercept, temperature, X-ray diffraction spacing results).

Dimethyl sulphoxide enhances the effects of Pi in myofibrils and inhibits the activity of rabbit skeletal muscle contractile proteins

2001 ◽  
Vol 358 (3) ◽  
pp. 627-636 ◽  
Author(s):  
Ana C. MARIANO ◽  
Giani M. C. ALEXANDRE ◽  
Leonardo C. SILVA ◽  
Alexandre ROMEIRO ◽  
Luiz Claudio CAMERON ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Ethylene Glycol ◽  
Structural Changes ◽  
Catalytic Cycle ◽  
Skinned Fibres ◽  
Weakly Bound ◽  
In Vitro Motility Assay ◽  
Skeletal Muscle Myosin ◽  
Cross Bridges

In the catalytic cycle of skeletal muscle, myosin alternates between strongly and weakly bound cross-bridges, with the latter contributing little to sustained tension. Here we describe the action of DMSO, an organic solvent that appears to increase the population of weakly bound cross-bridges that accumulate after the binding of ATP, but before Pi release. DMSO (5–30%, v/v) reversibly inhibits tension and ATP hydrolysis in vertebrate skeletal muscle myofibrils, and decreases the speed of unregulated F-actin in an in vitro motility assay with heavy meromyosin. In solution, controls for enzyme activity and intrinsic tryptophan fluorescence of myosin subfragment 1 (S1) in the presence of different cations indicate that structural changes attributable to DMSO are small and reversible, and do not involve unfolding. Since DMSO depresses S1 and acto–S1MgATPase activities in the same proportions, without altering acto–S1 affinity, the principal DMSO target apparently lies within the catalytic cycle rather than with actin–myosin binding. Inhibition by DMSO in myofibrils is the same in the presence or the absence of Ca2+ and regulatory proteins, in contrast with the effects of ethylene glycol, and the Ca2+ sensitivity of isometric tension is slightly decreased by DMSO. The apparent affinity for Pi is enhanced markedly by DMSO (and to a lesser extent by ethylene glycol) in skinned fibres, suggesting that DMSO stabilizes cross-bridges that have ADP·Pi or ATP bound to them.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

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