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Differences in the mechanical properties of mammalian smooth, skeletal, and cardiac muscle have led to the proposal that the myosin isozymes expressed by these tissues may differ in their molecular mechanics. To test this hypothesis, mixtures of fast skeletal, V1 cardiac, V3 cardiac and smooth muscle (phosphorylated and unphosphorylated) myosin were studied in an in vitro motility assay in which fluorescently-labelled actin filaments are observed moving over a myosin coated surface. Pure populations of each myosin produced actin filament velocities proportional to their actin-activated ATPase rates. Mixtures of two myosin species produced actin filament velocities between those of the faster and slower myosin alone. However, the shapes of the myosin mixture curves depended upon the types of myosins present. Analysis of myosin mixtures data suggest that: (1) the two myosins in the mixture interact mechanically and (2) the same force-velocity relationship describes a myosin's ability to operate over both positive and negative forces. These data also allow us to rank order the myosins by their average force per cross-bridge and ability to resist motion (phosphorylated smooth > skeletal = V3 cardiac > V1 cardiac). The results of our study may reflect the mechanical consequence of multiple myosin isozyme expression in a single muscle cell.

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To determine whether the apparent enhanced force-generating capabilities of smooth muscle relative to skeletal muscle are inherent to the myosin cross-bridge, the isometric steady-state force produced by myosin in the in vitro motility assay was measured. In this assay, myosin adhered to a glass surface pulls on an actin filament that is attached to an ultracompliant (50-200 nm/pN) glass microneedle. The number of myosin cross-bridge heads able to interact with a length of actin filament was estimated by measuring the density of biochemically active myosin adhered to the surface; with this estimate, the average force per cross-bridge head of smooth and skeletal muscle myosins is 0.6 pN and 0.2 pN, respectively. Surprisingly, smooth muscle myosin generates approximately three times greater average force per cross-bridge head than does skeletal muscle myosin.

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In vitro motility assays, in which fluorescently labeled actin filaments are propelled by myosin molecules adhered to a glass coverslip, require that actin filament velocity be determined. We have developed a computer-assisted filament tracking system that reduced the analysis time, minimized investigator bias, and provided greater accuracy in locating actin filaments in video images. The tracking routine successfully tracked filaments under experimental conditions where filament density, size, and extent of photobleaching varied dramatically. Videotaped images of actin filament motility were digitized and processed to enhance filament image contrast relative to background. Once processed, filament images were cross correlated between frames and a filament path was determined. The changes in filament centroid or center position between video frames were then used to calculate filament velocity. The tracking routine performance was evaluated and the sources of noise that contributed to errors in velocity were identified and quantified. Errors originated in algorithms for filament centroid determination and in the choice of sampling interval between video frames. With knowledge of these error sources, the investigator can maximize the accuracy of the velocity calculation through access to user-definable computer program parameters.

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To test the idea that the in vitro motility assay is a simplified model system for muscle contraction, the MgATP-dependent movement of actin filaments by thiophosphorylated smooth muscle myosin was characterized in the presence of the products MgADP and inorganic phosphate. The dependence of actin filament velocity on MgATP concentration was hyperbolic with a maximum velocity of 0.6 micron/s and an apparent Km = 40 microM (30 degrees C). MgADP competitively inhibited actin movement by MgATP with a Ki = 0.25 mM. Inorganic phosphate did not affect actin filament velocity in the presence of 1 mM MgATP, but competitively inhibited movement in the presence of 50 microM MgATP with a Ki = 9.5 mM. The effects of ADP and Pi on velocity agree with fiber mechanical studies, confirming that the motility assay is an excellent system to investigate the molecular mechanisms of force generation and shortening in smooth muscle. The rate at which rigor cross-bridges can be recruited to move actin filaments was observed by initiating cross-bridge cycling from rigor by flash photolysis of caged MgATP. Following the flash, which results in a rapid increase in MgATP concentration, actin filaments experienced a MgATP-dependent delay prior to achieving steady state velocity. The delay at low MgATP concentrations was interpreted as evidence that motion generating cross-bridges are slowed by a load due to a transiently high percentage of rigor cross-bridges immediately following MgATP release.

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Although it is generally believed that phosphorylation of the regulatory light chain of myosin is required before smooth muscle can develop force, it is not known if the overall degree of phosphorylation can also modulate the rate at which cross-bridges cycle. To address this question, an in vitro motility assay was used to observe the motion of single actin filaments interacting with smooth muscle myosin copolymers composed of varying ratios of phosphorylated and unphosphorylated myosin. The results suggest that unphosphorylated myosin acts as a load to slow down the rate at which actin is moved by the faster cycling phosphorylated cross-bridges. Myosin that was chemically modified to generate a noncycling analogue of the "weakly" bound conformation was similarly able to slow down phosphorylated myosin. The observed modulation of actin velocity as a function of copolymer composition can be accounted for by a model based on mechanical interactions between cross-bridges.

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Simple and general analytical expressions are derived for the acoustic radiation force on a long rigid cylinder with a small diameter, whose axis is perpendicular to the wave propagation. Results are expressed in terms of the time-averaged densities of kinetic and potential energies of the incident sound field. For the case of a standing-wave field, which was used in these experiments, the theoretical results agree well with the experimental observations.

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The shortening response of isolated single smooth muscle cells from the toad stomach "Bufo marinus", was studied using digital video microscopy. A computer program was developed to rapidly on-line digitize and store successive video images of the cell as it shortened. Single smooth muscle cells were decorated with tiny anionic exchange resin beads which served as markers for surface motion. Through an interactive software routine, the cell's outline and bead images were defined. Given this information, the program determined the beads location along the length of the cell as well as its angular position on the cell surface. The program allowed the investigator to reconstruct three dimensional images of the cell during contraction. Analysis of successive cell images revealed that during cell shortening, beads would rotate on the cell surface. These data were interpreted as evidence for corkscrew-like shortening in single smooth muscle cells.

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To understand how smooth muscle modulates its shortening velocity within the time course of a single contraction, the factors that govern crossbridge cycling in smooth muscle must be characterized. Since calcium plays an important role in regulation of the contractile apparatus, we studied the effect of lowering extracellular calcium on shortening velocity in single smooth muscle cells isolated enzymatically from the toad, Bufo marinus, stomach muscularis. Shortening velocity was estimated by three independent methods: (1) isotonic releases; (2) slack test; (3) video images of freely shortening cells. To determine the shortening velocity from isotonic releases and the slack test method, a single cell was attached at one end to an ultrasensitive force transducer and piezoelectric displacement device at its other end. At peak isometric force, in response to electrical stimulation, a series of isotonic releases to varying force levels were imposed and the resultant shortening responses used to construct force:velocity relationships in physiological saline containing low (0.18 mM) extracellular calcium. From force:velocity relationships and slack test data, an estimate of maximum shortening velocity (Vmax) was 0.19 cell lengths/s which was 3 times lower than in normal calcium (Warshaw 1987). Although Vmax was reduced in low extracellular calcium, the curvature of the force:velocity relationship estimated from the hyperbolic constant, a/Fmax, was unchanged (a/Fmax = 0.17). Additional evidence for an effect of extracellular calcium on shortening velocity was obtained from shortening responses of freely shortening cells attached at one end only. Data from cells exposed first to low (0.18 mM) and then normal (1.8 mM) calcium suggest at least a twofold decrease in free shortening velocity with lower extracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)

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The slower and more economical contraction of smooth muscle as compared to that of skeletal muscle may relate to the arrangement of its contractile apparatus. Because the arrangement of the contractile apparatus determines the manner in which a single smooth muscle cell shortens, shortening of a contracting cell was examined by tracking of marker bead movements on the cell surface by means of digital video microscopy. Smooth muscle cells were observed to freely shorten in a unique corkscrew-like fashion with a pitch of 1.4 cell lengths (that is, the length change required for one complete rotation of cell) at a rate of 27 degrees per second. Corkscrew-like shortening was interpreted in terms of a structural model in which the contractile apparatus or cytoskeleton (or both) are helically oriented within the cell. Such an arrangement of these cytoarchitectural elements may help to explain in part the contractile capabilities of smooth muscle.

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