Kinetic and spectroscopic evidence for three actomyosin:ADP states in smooth muscle.
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Smooth muscle myosin II undergoes an additional movement of the regulatory domain with ADP release that is not seen with fast skeletal muscle myosin II. In this study, we have examined the interactions of smooth muscle myosin subfragment 1 with ADP to see if this additional movement corresponds to an identifiable state change. These studies indicate that for this myosin:ADP, both the catalytic site and the actin-binding site can each assume one of two conformations. Relatively loose coupling between these two binding sites leads to three discrete actin-associated ADP states. Following an initial, weakly bound state, binding of myosin:ADP to actin shifts the equilibrium toward a mixture of two states that each bind actin strongly but differ in the conformation of their catalytic sites. By contrast, fast myosins, including Dictyostelium myosin II, have reciprocal coupling between the actin- and ADP-binding sites, so that either actin or nucleotide, but not both, can be tightly bound. This uncoupling, which generates a second strongly bound actomyosin ADP state in smooth muscle, would prolong the fraction of the ATPase cycle time that this actomyosin spends in a force-generating conformation and may be central to explaining the physiologic differences between this and other myosins. (+info)
Comparative single-molecule and ensemble myosin enzymology: sulfoindocyanine ATP and ADP derivatives.
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Single-molecule and macroscopic reactions of fluorescent nucleotides with myosin have been compared. The single-molecule studies serve as paradigms for enzyme-catalyzed reactions and ligand-receptor interactions analyzed as individual stochastic processes. Fluorescent nucleotides, called Cy3-EDA-ATP and Cy5-EDA-ATP, were derived by coupling the dyes Cy3.29.OH and Cy5.29.OH (compounds XI and XIV, respectively, in, Bioconjug. Chem. 4:105-111)) with 2'(3')-O-[N-(2-aminoethyl)carbamoyl]ATP (EDA-ATP). The ATP(ADP) analogs were separated into their respective 2'- and 3'-O-isomers, the interconversion rate of which was 30[OH(-)] s(-1) (0.016 h(-1) at pH 7.1) at 22 degrees C. Macroscopic studies showed that 2'(3')-O-substituted nucleotides had properties similar to those of ATP and ADP in their interactions with myosin, actomyosin, and muscle fibers, although the ATP analogs did not relax muscle as well as ATP did. Significant differences in the fluorescence intensity of Cy3-nucleotide 2'- and 3'-O-isomers in free solution and when they interacted with myosin were evident. Single-molecule studies using total internal reflection fluorescence microscopy showed that reciprocal mean lifetimes of the nucleotide analogs interacting with myosin filaments were one- to severalfold greater than predicted from macroscopic data. Kinetic and equilibrium data of nucleotide-(acto)myosin interactions derived from single-molecule microscopy now have a biochemical and physiological framework. This is important for single-molecule mechanical studies of motor proteins. (+info)
Influence of ionic strength on the actomyosin reaction steps in contracting skeletal muscle fibers.
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Muscle contraction occurs as the result of actin-myosin interaction, which is mediated by the intermolecular forces exerted at the actin-myosin interface. To obtain information about the nature of these intermolecular forces, we tested the sensitivity of various contractile parameters of skinned skeletal muscle fibers to ionic strength (IS) at 3-5 degrees C; IS variation is a useful technique for distinguishing between ionic and nonionic (primarily hydrophobic) types of intermolecular forces. The most striking effect of elevated IS was the strong suppression of isometric tension. However, none of the measured parameters suggested a corresponding decrease in the number of force-generating myosin heads on actin. The rate of actin-myosin association seemed to be only modestly IS-sensitive. The following force-generating isomerization was apparently IS-insensitive. The dissociation of the force-generating actomyosin complex was decelerated by elevated IS, contrary to the expectation from the suppressed isometric tension. These results led us to conclude that an IS-sensitive step, responsible for the large suppression of tension, occurs after force-generating isomerization but before dissociation. The present study suggests that the actomyosin interaction is generally nonionic in nature, but there are at least two ionic processes, one at the beginning and the other close to the end of the actomyosin interaction. (+info)
Single-motor mechanics and models of the myosin motor.
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Recent progress in single-molecule detection techniques is remarkable. These techniques have allowed the accurate determination of myosin-head-induced displacements and how mechanical cycles are coupled to ATP hydrolysis, by measuring individual mechanical events and chemical events of actomyosin directly at the single-molecule level. Here we review our recent work in which we have made detailed measurements of myosin step size and mechanochemical coupling, and propose a model of the myosin motor. (+info)
Fission yeast Rng3p: an UCS-domain protein that mediates myosin II assembly during cytokinesis.
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Cell division in many eukaryotes, including the fission yeast Schizosaccharomyces pombe, utilizes a contractile actomyosin ring. In S. pombe, the actomyosin ring is assembled at the medial cortex upon entry into mitosis and constricts at the end of anaphase to guide the centripetal deposition of the septum. Despite identification of several structural components essential for actomyosin ring assembly, the interdependencies between these gene-products in the process of ring assembly are unknown. This study investigates the role of Rng3p, a member of the UCS-domain containing protein family (Unc-45p, Cro1p, She4p), in actomyosin ring assembly. Null mutants in rng3 resemble deletion mutants in the type II myosin heavy chain (myo2) and rng3(ts) mutants show strong negative interactions with the myo2-E1 mutant, suggesting that Rng3p is involved in modulating aspects of type II myosin function. Interestingly, a green fluorescent protein (GFP) tagged Rng3p fusion is detected at the division site in the myo2-E1 mutant, but not in other myo2-alleles, wild-type cells or in 18 other cytokinesis mutants. Assembly and maintenance of Rng3p at the division site in the myo2-E1 mutant requires F-actin. Rng3p is also required for the proper assembly of Myo2p and F-actin into a functional actomyosin ring but is not necessary for their accumulation at the division site. We conclude that Rng3p is a novel component of the F-actin cytoskeleton essential for a late step in actomyosin ring assembly and that it might monitor some aspect of type II myosin assembly during actomyosin ring construction. (+info)
Microtubules remodel actomyosin networks in Xenopus egg extracts via two mechanisms of F-actin transport.
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Interactions between microtubules and filamentous actin (F-actin) are crucial for many cellular processes, including cell locomotion and cytokinesis, but are poorly understood. To define the basic principles governing microtubule/F-actin interactions, we used dual-wavelength digital fluorescence and fluorescent speckle microscopy to analyze microtubules and F-actin labeled with spectrally distinct fluorophores in interphase Xenopus egg extracts. In the absence of microtubules, networks of F-actin bundles zippered together or exhibited serpentine gliding along the coverslip. When microtubules were nucleated from Xenopus sperm centrosomes, they were released and translocated away from the aster center. In the presence of microtubules, F-actin exhibited two distinct, microtubule-dependent motilities: rapid ( approximately 250-300 nm/s) jerking and slow ( approximately 50 nm/s), straight gliding. Microtubules remodeled the F-actin network, as F-actin jerking caused centrifugal clearing of F-actin from around aster centers. F-actin jerking occurred when F-actin bound to motile microtubules powered by cytoplasmic dynein. F-actin straight gliding occurred when F-actin bundles translocated along the microtubule lattice. These interactions required Xenopus cytosolic factors. Localization of myosin-II to F-actin suggested it may power F-actin zippering, while localization of myosin-V on microtubules suggested it could mediate interactions between microtubules and F-actin. We examine current models for cytokinesis and cell motility in light of these findings. (+info)
Characterization of single actomyosin rigor bonds: load dependence of lifetime and mechanical properties.
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Load dependence of the lifetime of the rigor bonds formed between a single myosin molecule (either heavy meromyosin, HMM, or myosin subfragment-1, S1) and actin filament was examined in the absence of nucleotide by pulling the barbed end of the actin filament with optical tweezers. For S1, the relationship between the lifetime (tau) and the externally imposed load (F) at absolute temperature T could be expressed as tau(F) = tau(0).exp(-F.d/k(B)T) with tau(0) of 67 s and an apparent interaction distance d of 2.4 nm (k(B) is the Boltzmann constant). The relationship for HMM was expressed by the sum of two exponentials, with two sets of tau(0) and d being, respectively, 62 s and 2.7 nm, and 950 s and 1.4 nm. The fast component of HMM coincides with tau(F) for S1, suggesting that the fast component corresponds to single-headed binding and the slow component to double-headed binding. These large interaction distances, which may be a common characteristic of motor proteins, are attributed to the geometry for applying an external load. The pulling experiment has also allowed direct estimation of the number of myosin molecules interacting with an actin filament. Actin filaments tethered to a single HMM molecule underwent extensive rotational Brownian motion, indicating a low torsional stiffness for HMM. From these results, we discuss the characteristics of interaction between actin and myosin, with the focus on the manner of binding of myosin. (+info)
An oncogenic epidermal growth factor receptor signals via a p21-activated kinase-caldesmon-myosin phosphotyrosine complex.
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Many ligand-independent receptor tyrosine kinases are tumorigenic. The biochemical signals that mediate ligand-independent transformation of cells by these transmembrane receptors are poorly defined. In this report, we demonstrate that a constitutively activated mutant epidermal growth factor receptor (v-ErbB) induces the formation of a transformation-specific signaling module that complexes with myosin II. The components of this signaling complex include the signal adapter proteins Shc, Grb2, and Nck, and tyrosine-phosphorylated forms of p21-activated kinase (Pak), caldesmon, and myosin light chain kinase. Transformation-specific, tyrosine phosphorylation of Pak enhances the catalytic activity of this serine/threonine kinase. Furthermore, the tyrosine phosphorylation of Pak is Rho-, but not Ras-, Rac-, or Cdc42-dependent. These results demonstrate that a ligand-independent epidermal growth factor receptor mutant can transduce oncogenic signals that are distinct from ligand-dependent, mitogenic signals. In addition, these data provide evidence for the coupling of oncogenic receptor tyrosine kinases with the actomyosin molecular motor. This myosin-associated signaling module may mediate some of the biochemical changes of myosin found in v-ErbB- transformed fibroblasts, thereby contributing to the regulation of the mechanical forces governing cellular adhesion, cytoskeletal tension, and, hence, anchorage-independent cell growth. (+info)