ATP-induced Detachment Research Articles

Kinetic Model for Myosin Gating and Backward Stepping Mechanisms

Members of the myosin superfamily form “processive” dimers, meaning they take many consecutive mechanical steps per diffusional encounter with an actin filament. Processivity requires synchronization between the two heads, or gating, to ensure that the trailing head steps first and binds forward, which is necessary for unidirectional motility. Synchronization, which is thought to occur through “biochemical gating” due to inter-head tension created by the strain in the two-headed attachment, modifies the rates for nucleotide binding, hydrolysis and/or release to favor detachment of the trailing head. Alternatively, the myosin power stroke may bias reattachment of the free head to bind to a forward actin subunit - a process we refer to as “mechanical gating”. Evidence exists to support both the mechanisms, but the dominant one is both highly debated and could be myosin isoform specific. Myosin motors also take occasional backward steps that may or may not be initiated by ATP binding. To utilize existing experimental data and infer the mechanisms governing gating, we present a general kinetic model for the processivity of myosin dimers. By including multiple pathways for forward and backward stepping, and both spontaneous and ATP-induced detachment from actin, the model predicts the consequences of different types of gating on processivity. Further, by fitting recently published data for myosin VI, we are able to predict which gating mechanisms are compatible with the data. Thus, the kinetic model serves as a generalized tool for researchers to use experimentally derived metrics of motility (processivity, backwards stepping, velocity, run length, and their nucleotide dependencies) to infer the molecular gating mechanisms of each myosin motor.

Nonlinear Actomyosin Elasticity in Muscle?

Cyclic interactions between myosin II motor domains and actin filaments that are powered by turnover of ATP underlie muscle contraction and have key roles in motility of nonmuscle cells. The elastic characteristics of actin-myosin cross-bridges are central in the force-generating process, and disturbances in these properties may lead to disease. Although the prevailing paradigm is that the cross-bridge elasticity is linear (Hookean), recent single-molecule studies suggest otherwise. Despite convincing evidence for substantial nonlinearity of the cross-bridge elasticity in the single-molecule work, this finding has had limited influence on muscle physiology and physiology of other ordered cellular actin-myosin ensembles. Here, we use a biophysical modeling approach to close the gap between single molecules and physiology. The model is used for analysis of available experimental results in the light of possible nonlinearity of the cross-bridge elasticity. We consider results obtained both under rigor conditions (in the absence of ATP) and during active muscle contraction. Our results suggest that a wide range of experimental findings from mechanical experiments on muscle cells are consistent with nonlinear actin-myosin elasticity similar to that previously found in single molecules. Indeed, the introduction of nonlinear cross-bridge elasticity into the model improves the reproduction of key experimental results and eliminates the need for force dependence of the ATP-induced detachment rate, consistent with observations in other single-molecule studies. The findings have significant implications for the understanding of key features of actin-myosin-based production of force and motion in living cells, particularly in muscle, and for the interpretation of experimental results that rely on stiffness measurements on cells or myofibrils.

Towards a Unified Theory of Muscle Contraction. I: Foundations

Molecular models of contractility in striated muscle require an integrated description of the action of myosin motors, firstly in the filament lattice of the half-sarcomere. Existing models do not adequately reflect the biochemistry of the myosin motor and its sarcomeric environment. The biochemical actin-myosin-ATP cycle is reviewed, and we propose a model cycle with two 4- to 5-nm working strokes, where phosphate is released slowly after the first stroke. A smaller third stroke is associated with ATP-induced detachment from actin. A comprehensive model is defined by applying such a cycle to all myosin-S1 heads in the half-sarcomere, subject to generic constraints as follows: (a) all strain-dependent kinetics required for actin-myosin interactions are derived from reaction-energy landscapes and applied to dimeric myosin, (b) actin-myosin interactions in the half-sarcomere are controlled by matching rules derived from the structure of the filaments, so that each dimer may be associated with a target zone of three actin sites, and (c) the myosin and actin filaments are treated as elastically extensible. Numerical predictions for such a model are presented in the following paper.

Isolation and kinetic characterisation of myosin and myosin S1 from the Drosophila indirect flight muscles.

The potential to explore myosin function through the alternative exons and mutations of the single muscle myosin heavy chain gene, Mhc of Drosophila requires detailed kinetic analysis of the myosins. We have obtained microgram quantities of enzymatically active Drosophila myosin and subfragment 1 (S1) from dissected indirect flight muscles. Using recent developments in stopped-flow and flash-photolysis methods combined with fluorescent/light scattering technologies we have determined some of the key kinetic parameters of actin-myosin and myosin-nucleotide interactions. The rate of ATP-induced dissociation of actin from Drosophila myosin (0.23 microM(-1) s(-1)) and subfragment 1 (S1, 0.82 microM(-1) s(-1)) are both fast and similar to values measured for mammalian skeletal muscle myosins and S1 fragments respectively. The ATP-induced cross bridge dissociation of Drosophila acto.S1 is expected to be fast since, for a rapidly contracting muscle like the Drosophila flight muscle, the post power stroke cross bridge must detach rapidly from actin or become a drag on the contracting filament. ATP-induced detachment is preceded by ADP release and this is proposed as the rate-limiting step that defines muscle shortening velocity. We show that the affinity of ADP for acto.S1 at 400 microM is 2-3 fold weaker than fast vertebrate myosins. This leads to an estimate of the ADP release rate constant of 4000 s(-1). We show that this predicts a maximum shortening velocity very similar to that obtained from in vivo estimates of indirect flight muscle shortening. The data is therefore compatible with ADP dissociation limiting the in vivo shortening velocity.

Evidence for cooperative interactions between the two motor domains of cytoplasmic dynein

Cytoplasmic dynein is a force-transducing ATPase that powers the movement of cellular cargoes along microtubules. Two identical heavy chain polypeptides (> 500 kDa) of the cytoplasmic dynein complex contain motor domains that possess the ATPase and microtubule-binding activities required for force production [1]. It is of great interest to determine whether both heavy chains (DHCs) in the dynein complex are required for progression of the mechanochemical cycle and motility, as observed for other dimeric motors. We have used transgenic constructs to investigate cooperative interactions between the two motor domains of the Drosophila cytoplasmic dynein complex. We show that 138 kDa and 180 kDa amino-terminal fragments of DHC can assemble with full-length DHC to form heterodimeric complexes containing only a single motor domain. The single-headed dynein complexes can bind and hydrolyze ATP, yet do not show the ATP-induced detachment from microtubules that is characteristic of wild-type homodimeric dynein. These results suggest that cooperative interactions between the monomeric units of the dimer are required for efficient ATP-induced detachment of dynein and unidirectional movement along the microtubule.

The influence of ionic strength upon relaxation from rigor induced by flash photolysis of caged-ATP in skinned murine skeletal muscle fibres

The influence of ionic strength upon relaxation kinetics from rigor in skinned murine extensor digitorum longus (EDL) skeletal muscle fibres was examined using photolysis of caged-ATP at low Ca2+. The ionic strength was adjusted with either KMeSO3 or ethylene glycolbis-(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, dipotassium salt (K2EGTA) in the range of tau /2 = 65-215mM, or I.E. 49-194mM, where I.E. denotes ionic equivalent. Following rigor development at a tau /2 of 165-215mM (I.E. 144-194mM), the liberation of approximately 0.5mM ATP resulted in an initial 6-to 10-ms detachment phase with a decline in force of approximately 10-20% followed by a 10-to 30-ms reattachment with up to a 60% increase compared to the corresponding rigor level and a final detachment leading to complete relaxation. Interestingly, when similar ATP concentrations were liberated at lower ionic strengths between a tau /2 of 65mM and 110mM (I.E. 60-100mM), the initial detachment phase was shortened and force decreased by only approximately 5-10%, while the following reattachment phase was lengthened and led to an increased steady-state force of approximately 20-80% without final relaxation. ATP-induced detachment and subsequent reattachment were mainly determined by the currently present ionic strength and were relatively independent of the preceding rigor state which had been developed at higher or lower ionic strengths. The effects of phosphate and apyrase on the force transient suggest that reattachment of ADP- binding crossbridges may contribute to the increase in tension at high and even more at low ionic strengths. The study shows that the kinetics of initial fast relaxation and subsequent redevelopment of force following flash photolysis of similar ATP concentrations are markedly modified by the ionic strength in the narrow range of between 65mM and 215mM.

Thin filament regulation of force activation is not essential in single vascular smooth muscle cells.

To investigate thin filament regulation of force activation in smooth muscle, we recorded force and stiffness of alpha-toxin-permeabilized single smooth muscle cells. At pCa 9, the rigor state was characterized by high in-phase stiffness, low force, and low quadrature stiffness, suggesting that the attachment of rigor cross bridges does not depend on either Ca2+ or myosin light chain (MLC) phosphorylation, and cross bridges can enter a rigor state without producing force. At pCa 4, 20 microM ATP increased force, in-phase stiffness, and quadrature stiffness, while 20 microM CTP did not increase any of these parameters, suggesting that although MLC phosphorylation is not required for the formation of rigor cross bridges, MLC phosphorylation is required for detached cross bridges to attach to actin and undergo a force-producing isomerization. These results also suggest that for smooth muscle, force activation is regulated by myosin light-chain kinase. From rigor, 20 microM ATP (pCa 9) increased force and quadrature without changing in-phase stiffness. This force increase could be explained if in rigor solution both actomyosin (AM) and AM.ADP cross bridges exist (2, 32), and ATP-induced detachment of AM cross bridges is accompanied by AM.ADP cross bridges undergoing a force-producing isomerization in combination with cooperative cross-bridge reattachment (36). Thus results of our experiments suggest that thin filament-based regulation of force activation is not essential in smooth muscle, and a population of cross bridges must begin in an attached state for force to be produced in the absence of MLC phosphorylation.

Relaxation from rigor of skinned trabeculae of the guinea pig induced by laser photolysis of caged ATP

The kinetics of ATP-induced rigor cross-bridge detachment were studied by initiating relaxation in chemically skinned trabeculae of the guinea pig heart using photolytic release of ATP in the absence of calcium ions (pCa > 8). The time course of the fall in tension exhibited either an initial plateau phase of variable duration with little change in tension or a rise in tension, followed by a decrease to relaxed levels. The in-phase component of tissue stiffness initially decreased. The rate then slowed near the end of the tension plateau, indicating transient cross-bridge rebinding, before falling to relaxed levels. Estimates of the apparent second-order rate constant for ATP-induced detachment of rigor cross-bridges based on the half-time for relaxation or on the half-time to the convergence of tension records to a common time course were similar at 3 x 10(3) M-1 s-1. Because the characteristics of the mechanical transients observed during relaxation from rigor were markedly similar to those reported from studies of rabbit psoas fibers in the presence of MgADP (Dantzig, J. A., M. G. Hibberd, D. R. Trentham, and Y. E. Goldman. 1991. Cross-bridge kinetics in the presence of MgADP investigated by photolysis of caged ATP in rabbit psoas muscle fibres. J. Physiol. 432:639-680), direct measurements of MgADP using [3H]ATP in cardiac tissue in rigor were made. Results indicated that during rigor, nearly 18% of the cross-bridges in skinned trabeculae had [3H]MgADP bound. Incubation of the tissue during rigor with apyrase, an enzyme with both ADPase and ATPase activity, reduced the level of [3H]MgADP to that measured following a 2-min chase in a solution containing 5 mM unlabeled MgATP. Apyrase incubation also significantly reduced the tension and stiffness transients, so that both time courses became monotonic and could be fit with a simple model for cross-bridge detachment. The apparent second-order rate constant for ATP-induced rigor cross-bridge detachment measured in the apyrase treated tissue at 4 x 10(4) M-1 s-1 was faster than that measured in untreated tissue. Nevertheless, this rate was still over an order of magnitude slower than the analogous rate measured in previous studies of isolated cardiac actomyosin-S1. These results are consistent with the hypothesis that the presence of MgADP bound cross-bridges suppresses the inhibition normally imposed by the thin filament regulatory system in the absence of calcium ions and allows cross-bridge rebinding and force production during relaxation from rigor.

Mechanical transients initiated by photolysis of caged ATP within fibers of insect fibrillar flight muscle.

Kinetics of the cross-bridge cycle in insect fibrillar flight muscle have been measured using laser pulse photolysis of caged ATP and caged inorganic phosphate (Pi) to produce rapid step increases in the concentration of ATP and Pi within single glycerol-extracted fibers. Rapid photochemical liberation of 100 microM-1 mM ATP from caged ATP within a fiber caused relaxation in the absence of Ca2+ and initiated an active contraction in the presence of approximately 30 microM Ca2+. The apparent second order rate constant for detachment of rigor cross-bridges by ATP was between 5 x 10(4) and 2 x 10(5) M-1s-1. This rate is not appreciably sensitive to the Ca2+ or Pi concentrations or to rigor tension level. The value is within an order of magnitude of the analogous reaction rate constant measured with isolated actin and insect myosin subfragment-1 (1986. J. Muscle Res. Cell Motil. 7:179-192). In both the absence and presence of Ca2+ insect fibers showed evidence of transient cross-bridge reattachment after ATP-induced detachment from rigor, as found in corresponding experiments on rabbit psoas fibers. However, in contrast to results with rabbit fibers, tension traces of insect fibers starting at different rigor tensions did not converge to a common time course until late in the transients. This result suggests that the proportion of myosin cross-bridges that can reattach into force-generating states depends on stress or strain in the filament lattice. A steady 10-mM concentration of Pi markedly decreased the transient reattachment phase after caged ATP photolysis. Pi also decreased the amplitude of stretch activation after step stretches applied in the presence of Ca2+ and ATP. Photolysis of caged Pi during stretch activation abruptly terminated the development of tension. These results are consistent with a linkage between Pi release and the steps leading to force production in the cross-bridge cycle.

Cytochalasin B selectively releases ovalbumin mRNA precursors but not the mature ovalbumin mRNA from hen oviduct nuclear matrix.

Hen oviduct nuclear matrix-bound mature ovalbumin mRNA is released from the matrix in the presence of ATP, while the ovalbumin mRNA precursors remain bound to this structure. Detachment of the mature mRNA from the matrix by ATP as well as ATP-dependent efflux of mRNA from isolated nuclei were found to be inhibited by cytochalasin B. On the other hand, in the absence of ATP, cytochalasin B exclusively caused the release (and nucleocytoplasmic efflux) of the ovalbumin messenger precursors, but not of the mature mRNA. After cytochalasin B treatment, actin could be detected in the matrix supernatant. Phalloidin which stabilizes actin filaments did not cause RNA liberation in the absence of ATP, but inhibited the ATP-induced detachment of mature mRNA. RNA release was also achieved with a monoclonal antibody against actin but not with monoclonal antibodies against tubulin and intermediate filaments. These results suggest that actin-containing filaments are involved in the restriction of immature messengers to the cell nucleus.