Biased Brownian motion mechanism for processivity and directionality of single-headed myosin-VI

Conventional form to function as a vesicle transporter is not a 'single molecule' but a coordinated 'two molecules'. The coordinated two molecules make it complicated to reveal its mechanism. To overcome the difficulty, we adopted a single-headed myosin-VI as a model protein. Myosin-VI is an intracellular vesicle and organelle transporter that moves along actin filaments in a direction opposite to most other known myosin classes. The myosin-VI was expected to form a dimer to move processively along actin filaments with a hand-over-hand mechanism like other myosin organelle transporters. However, wild-type myosin-VI was demonstrated to be monomer and single-headed, casting doubt on its processivity. Using single molecule techniques, we show that green fluorescent protein (GFP)-fused single-headed myosin-VI does not move processively. However, when coupled to a 200 nm polystyrene bead (comparable to an intracellular vesicle in size) at a ratio of one head per bead, single-headed myosin-VI moves processively with large (40 nm) steps. Furthermore, we found that a single-headed myosin-VI-bead complex moved more processively in a high-viscous solution (40-fold higher than water) similar to cellular environment. Because diffusion of the bead is 60-fold slower than myosin-VI heads alone in water, we propose a model in which the bead acts as a diffusional anchor for the myosin-VI, enhancing the head's rebinding following detachment and supporting processive movement of the bead-monomer complex. This investigation will help us understand how molecular motors utilize Brownian motion in cells.     

The Lever Arm Effects a Mechanical Asymmetry of the Myosin-V-Actin Bond

Myosin-V is a two-headed molecular motor taking multiple ATP-dependent steps toward the plus end (forward) of actin filaments. At high mechanical loads, the motor processively steps toward the minus end (backward) even in the absence of ATP, whereas analogous forward steps cannot be induced. The detailed mechanism underlying this mechanical asymmetry is not known. We investigate the effect of force on individual single headed myosin-V constructs bound to actin in the absence of ATP. If pulled forward, the myosin-V head dissociates at forces twice as high than if pulled backward. Moreover, backward but not forward distances to the unbinding barrier are dependent on the lever arm length. This asymmetry of unbinding force distributions in a single headed myosin forms the basis of the two-headed asymmetry. Under load, the lever arm functions as a true lever in a mechanical sense.     

Mental Representation of Arm Motion Dynamics in Children and Adolescents

Motor imagery, i.e., a mental state during which an individual internally represents an action without any overt motor output, is a potential tool to investigate action representation during development. Here, we took advantage of the inertial anisotropy phenomenon to investigate whether children can generate accurate motor predictions for movements with varying dynamics. Children (9 and 11 years), adolescents (14 years) and young adults (21 years) carried-out actual and mental arm movements in two different directions in the horizontal plane: rightwards (low inertia) and leftwards (high inertia). We recorded and compared actual and mental movement times. We found that actual movement times were greater for leftward than rightward arm movements in all groups. For mental movements, differences between leftward versus rightward movements were observed in the adults and adolescents, but not among the children. Furthermore, significant differences between actual and mental times were found at 9 and 11 years of age in the leftward direction. The ratio R/L (rightward direction/leftward direction), which indicates temporal differences between low inertia and high inertia movements, was inferior to 1 at all ages, except for the mental movements at 9 years of age, indicating than actual and mental movements were shorter for the rightward than leftward direction. Interestingly, while the ratio R/L of actual movements was constant across ages, it gradually decreased with age for mental movements. The ratio A/M (actual movement/mental movement), which indicates temporal differences between actual and mental movements, was near to 1 in the adults'' groups, denoting accurate mental timing. In children and adolescents, an underestimation of mental movement times appeared for the leftward movements only. However, this overestimation gradually decreased with age. Our results showed a refinement in the motor imagery ability during development. Action representation reached maturation at adolescence, during which mental actions were tightly related to their actual production.     

Membrane-induced Lever Arm Expansion Allows Myosin VI to Walk with Large and Variable Step Sizes

Myosin VI, the only known minus-ended actin filament-dependent motor, plays diverse cellular roles both as a processive motor and as a mechanical anchor. Although myosin VI has a short lever arm containing only one “IQ-motif” and a unique insertion for CaM binding, the motor walks with large and variable step sizes of ∼30–36 nm. Here, we show that the previously predicted coiled-coil domain immediately following the IQ-motifs (referred to as the lever arm extension (LAE)) adopts a stable monomeric, three-helix bundle fold in solution. Importantly, the LAE can undergo reversible, lipid membrane-dependent conformational changes. Upon exposure to lipid membranes, the LAE adopts a partially extended rod shape, and the removal of lipids from the LAE converts it back into the compact helix bundle structure. Molecular dynamics simulations indicate that lipid membrane binding may initiate unfolding and thereby trigger the LAE expansion. This reversible, lipid membrane-dependent expansion of the LAE provides a mechanistic base for myosin VI to walk with large and variable step sizes.     

Effect of Nonreciprocal Forces on Brownian Motion of Plasma Crystal

Plasma Physics Reports - The effect is studied theoretically of nonreciprocity of interparticle forces on the Brownian motion of a single-layer plasma crystal with the hexagonal lattice. The...     

Effects of Aging on Arm Swing during Gait: The Role of Gait Speed and Dual Tasking

Healthy walking is characterized by pronounced arm swing and axial rotation. Aging effects on gait speed, stride length and stride time variability have been previously reported, however, less is known about aging effects on arm swing and axial rotation and their relationship to age-associated gait changes during usual walking and during more challenging conditions like dual tasking. Sixty healthy adults between the ages of 30–77 were included in this study designed to address this gap. Lightweight body fixed sensors were placed on each wrist and lower back. Participants walked under 3 walking conditions each of 1 minute: 1) comfortable speed, 2) walking while serially subtracting 3’s (Dual Task), 3) walking at fast speed. Aging effects on arm swing amplitude, range, symmetry, jerk and axial rotation amplitude and jerk were compared between decades of age (30–40; 41–50; 51–60; 61–77 years). As expected, older adults walked slower (p = 0.03) and with increased stride variability (p = 0.02). Arm swing amplitude decreased with age under all conditions (p = 0.04). In the oldest group, arm swing decreased during dual task and increased during the fast walking condition (p<0.0001). Similarly, arm swing asymmetry increased during the dual task in the older groups (p<0.004), but not in the younger groups (p = 0.67). Significant differences between groups and within conditions were observed in arm swing jerk (p<0.02), axial rotation amplitude (p<0.02) and axial jerk (p<0.001). Gait speed, arm swing amplitude of the dominant arm, arm swing asymmetry and axial rotation jerk were all independent predictors of age in a multivariate model. These findings suggest that the effects of gait speed and dual tasking on arm swing and axial rotation during walking are altered among healthy older adults. Follow-up work is needed to examine if these effects contribute to reduced stability in aging.     

Stability of Phase Relationships While Coordinating Arm Reaches with Whole Body Motion

The human movement repertoire is characterized by the smooth coordination of several body parts, including arm movements and whole body motion. The neural control of this coordination is quite complex because the various body parts have their own kinematic and dynamic properties. Behavioral inferences about the neural solution to the coordination problem could be obtained by examining the emerging phase relationship and its stability. Here, we studied the phase relationships that characterize the coordination of arm-reaching movements with passively-induced whole-body motion. Participants were laterally translated using a vestibular chair that oscillated at a fixed frequency of 0.83 Hz. They were instructed to reach between two targets that were aligned either parallel or orthogonal to the whole body motion. During the first cycles of body motion, a metronome entrained either an in-phase or an anti-phase relationship between hand and body motion, which was released at later cycles to test phase stability. Results suggest that inertial forces play an important role when coordinating reaches with cyclic whole-body motion. For parallel reaches, we found a stable in-phase and an unstable anti-phase relationship. When the latter was imposed, it readily transitioned or drifted back toward an in-phase relationship at cycles without metronomic entrainment. For orthogonal reaches, we did not find a clear difference in stability between in-phase and anti-phase relationships. Computer simulations further show that cost models that minimize energy expenditure (i.e. net torques) or endpoint variance of the reach cannot fully explain the observed coordination patterns. We discuss how predictive control and impedance control processes could be considered important mechanisms underlying the rhythmic coordination of arm reaches and body motion.     

Actomyosin: law and order in motility

The crystal structures of smooth muscle and scallop striated muscle myosin have both been completed in the past 18 months. Structural studies of unconventional myosins, in particular the stunning discovery that myosin VI moves backwards on actin, are starting to have deep impact on the field and have induced new ways of thinking about actin-based motility. Sophisticated genetic, biochemical and biophysical studies were used to test and refine hypotheses of the molecular mechanism of motility that were developed in the past. Although all these studies confirmed some aspects of these hypotheses, they also raised many new unresolved questions. Much of the evidence points to the importance of the actin-myosin binding process and an associated disorder-to-order transition.     

A Model Coupling Vibrational and Rotational Motion for the DNA Molecule

We investigate a mechanical model for the DNA molecule using an extension of the Peyrard and Bishop model. In the present model, there are two chains of oscillators linked by a Morse potential, which represent the hydrogen bonds. The rotational and vibrational motions of each base pair are considered and the coupling for these motions are introduced by a nonlinear combination of them in the Morse potential. In this context, thermodynamics and structural properties are discussed.     

Nanoblinker: Brownian Motion Powered Bio-Nanomachine for FRET Detection of Phagocytic Phase of Apoptosis

We describe a new type of bio-nanomachine which runs on thermal noise. The machine is solely powered by the random motion of water molecules in its environment and does not ever require re-fuelling. The construct, which is made of DNA and vaccinia virus topoisomerase protein, can detect DNA damage by employing fluorescence. It uses Brownian motion as a cyclic motor to continually separate and bring together two types of fluorescent hairpins participating in FRET. This bio-molecular oscillator is a fast and specific sensor of 5′OH double-strand DNA breaks present in phagocytic phase of apoptosis. The detection takes 30 s in solution and 3 min in cell suspensions. The phagocytic phase is critical for the effective execution of apoptosis as it ensures complete degradation of the dying cells’ DNA, preventing release of pathological, viral and tumor DNA and self-immunization. The construct can be used as a smart FRET probe in studies of cell death and phagocytosis.     

Auditory Hair Cell Centrioles Undergo Confined Brownian Motion Throughout the Developmental Migration of the Kinocilium

Planar polarization of the forming hair bundle, the mechanosensory antenna of auditory hair cells, depends on the poorly characterized center-to-edge displacement of a primary cilium, the kinocilium, at their apical surface. Taking advantage of the gradient of hair cell differentiation along the cochlea, we reconstituted a map of the kinocilia displacements in the mouse embryonic cochlea. We then developed a cochlear organotypic culture and video-microscopy approach to monitor the movements of the kinocilium basal body (mother centriole) and its daughter centriole, which we analyzed using particle tracking and modeling. We found that both hair cell centrioles undergo confined Brownian movements around their equilibrium positions, under the apparent constraint of a radial restoring force of ∼0.1 pN. This magnitude depended little on centriole position, suggesting nonlinear interactions with constraining, presumably cytoskeletal elements. The only dynamic change observed during the period of kinocilium migration was a doubling of the centrioles’ confinement area taking place early in the process. It emerges from these static and dynamic observations that kinocilia migrate gradually in parallel with the organization of hair cells into rows during cochlear neuroepithelium extension. Analysis of the confined motion of hair cell centrioles under normal and pathological conditions should help determine which structures contribute to the restoring force exerting on them.     

The Contractile and Control Sites of Natural Actomyosin

The various contractile and control sites of natural actomyosin gel were studied by comparing the kinetics of ATP hydrolysis with those of gel contraction, measured as an increase in turbidity. Contraction of actomyosin gel seems to require the cooperative reaction of ATP (with Mg) at two different sites. One of these sites catalyzes the hydrolysis of ATP and most probably contributes the driving force for contraction; the binding of ATP to the other site appears to break certain links that retard movement of the gel components. At limiting concentrations of ATP, the rate of contraction seems to depend on the rate of breaking these links as well as on the rate of ATP hydrolysis. But when both sites are saturated, the rate of contraction appears to be limited only by the rate of ATP hydrolysis. In addition to these two contractile sites, there are also two different control sites. At one, the relaxing site, the binding of ATP with Mg inhibits ATP hydrolysis and gel contraction. At the other, the binding of calcium activates contraction by overcoming the inhibitory action of Mg and ATP at the relaxing site. This control system—inhibition by substrate and disinhibition by calcium—can be selectively inactivated by heat and reactivated by dithiothreitol, a disulfide-reducing agent. These observations on the isolated contractile system are discussed in relation to the contraction and relaxation of muscle.     

平滑肌肌球蛋白磷酸化与肌动球蛋白功能调节

在有Ｃａ２＋和钙调蛋白存在时,肌球蛋白轻链激酶催化肌球蛋白磷酸化,促使肌动蛋白激活的肌球蛋白（肌动球蛋白）Ｍｇ２＋－ＡＴＰ酶活性显著增加．然而,肌球蛋白磷酸化水平与Ｍｇ２＋－ＡＴＰ酶之间的关系是非线性的,原肌球蛋白可以进一步增加Ｍｇ２＋－ＡＴＰ酶的活性,但仍不改变它们之间的非线性关系．肌球蛋白轻链激酶的合成肽抑制剂抑制了肌球蛋白磷酸化和Ｍｇ２＋－ＡＴＰ酶活性,并导致平滑肌去膜肌纤维的等长收缩张力与速度的降低．结果提示肌球蛋白轻链激酶参与脊椎动物平滑肌收缩的调节过程,肌球蛋白轻链磷酸化作用会引起平滑肌收缩     

One-Dimensional Brownian Motion of Charged Nanoparticles along Microtubules: A Model System for Weak Binding Interactions

Various proteins are known to exhibit one-dimensional Brownian motion along charged rodlike polymers, such as microtubules (MTs), actin, and DNA. The electrostatic interaction between the proteins and the rodlike polymers appears to be crucial for one-dimensional Brownian motion, although the underlying mechanism has not been fully clarified. We examined the interactions of positively-charged nanoparticles composed of polyacrylamide gels with MTs. These hydrophilic nanoparticles bound to MTs and displayed one-dimensional Brownian motion in a charge-dependent manner, which indicates that nonspecific electrostatic interaction is sufficient for one-dimensional Brownian motion. The diffusion coefficient decreased exponentially with an increasing particle charge (with the exponent being 0.10 k_BT per charge), whereas the duration of the interaction increased exponentially (exponent of 0.22 k_BT per charge). These results can be explained semiquantitatively if one assumes that a particle repeats a cycle of binding to and movement along an MT until it finally dissociates from the MT. During the movement, a particle is still electrostatically constrained in the potential valley surrounding the MT. This entire process can be described by a three-state model analogous to the Michaelis-Menten scheme, in which the two parameters of the equilibrium constant between binding and movement, and the rate of dissociation from the MT, are derived as a function of the particle charge density. This study highlights the possibility that the weak binding interactions between proteins and rodlike polymers, e.g., MTs, are mediated by a similar, nonspecific charge-dependent mechanism.     

Probing Muscle Myosin Motor Action: X-Ray (M3 and M6) Interference Measurements Report Motor Domain Not Lever Arm Movement

The key question in understanding how force and movement are produced in muscle concerns the nature of the cyclic interaction of myosin molecules with actin filaments. The lever arm of the globular head of each myosin molecule is thought in some way to swing axially on the actin-attached motor domain, thus propelling the actin filament past the myosin filament. Recent X-ray diffraction studies of vertebrate muscle, especially those involving the analysis of interference effects between myosin head arrays in the two halves of the thick filaments, have been claimed to prove that the lever arm moves at the same time as the sliding of actin and myosin filaments in response to muscle length or force steps. It was suggested that the sliding of myosin and actin filaments, the level of force produced and the lever arm angle are all directly coupled and that other models of lever arm movement will not fit the X-ray data. Here, we show that, in addition to interference across the A-band, which must be occurring, the observed meridional M3 and M6 X-ray intensity changes can all be explained very well by the changing diffraction effects during filament sliding caused by heads stereospecifically attached to actin moving axially relative to a population of detached or non-stereospecifically attached heads that remain fixed in position relative to the myosin filament backbone. Crucially, and contrary to previous interpretations, the X-ray interference results provide little direct information about the position of the myosin head lever arm; they are, in fact, reporting relative motor domain movements. The implications of the new interpretation are briefly assessed.     

Actomyosin ATPase activity of middle ear muscles in the cat

Summary When incubated for histochemical demonstration of actomyosin ATPase both the tensor tympani and the stapedius were found to contain two types of muscle fibres, one with high actomyosin ATPase activity, indicating a high speed of contraction, and one with low actomyosin ATPase activity, indicating a low speed of contraction. In the tensor tympani 59% and 41% of the muscle fibres had a high and low ATPase activity, respectively. The corresponding numbers in the stapedius muscle were 82% and 18%. These findings are discussed with reference to previous physiological and morphological studies.