High Duty Ratio Research Articles

Isoforms Confer Characteristic Force Generation and Mechanosensation by Myosin II Filaments

Myosin II isoforms with varying mechanochemistry and filament size interact with filamentous actin (F-actin) arrays to generate contractile forces in muscle and nonmuscle cells. How myosin II force production is shaped by isoform-specific motor properties and environmental stiffness remains poorly understood. Here, we used computer simulations to analyze force production by an ensemble of myosin motors against an elastically tethered actin filament. We found that force output depends on two timescales: the duration of F-actin attachment, which varies sharply with the ensemble size, motor duty ratio, and external load; and the time to build force, which scales with the ensemble stall force, gliding speed, and environmental stiffness. Although force-dependent kinetics were not required to sense changes in stiffness, the myosin catch bond produced positive feedback between the attachment time and force to trigger switch-like transitions from transient attachments, generating small forces, to high-force-generating runs. Using parameters representative of skeletal muscle myosin, nonmuscle myosin IIB, and nonmuscle myosin IIA revealed three distinct regimes of behavior, respectively: 1) large assemblies of fast, low-duty ratio motors rapidly build stable forces over a large range of environmental stiffness; 2) ensembles of slow, high-duty ratio motors serve as high-affinity cross-links with force buildup times that exceed physiological timescales; and 3) small assemblies of low-duty ratio motors operating at intermediate speeds are poised to respond sharply to changes in mechanical context—at low force or stiffness, they serve as low-affinity cross-links, but they can transition to force production via the positive-feedback mechanism described above. Together, these results reveal how myosin isoform properties may be tuned to produce force and respond to mechanical cues in their environment.

A kinetic model for estimating net photosynthetic rates of cos lettuce leaves under pulsed light.

Time-averaged net photosynthetic rate (P n) under pulsed light (PL) is known to be affected by the PL frequency and duty ratio, even though the time-averaged photosynthetic photon flux density (PPFD) is unchanged. This phenomenon can be explained by considering that photosynthetic intermediates (PIs) are pooled during light periods and then consumed by partial photosynthetic reactions during dark periods. In this study, we developed a kinetic model to estimate P n of cos lettuce (Lactuca sativa L. var. longifolia) leaves under PL based on the dynamics of the amount of pooled PIs. The model inputs are average PPFD, duty ratio, and frequency; the output is P n. The rates of both PI accumulation and consumption at a given moment are assumed to be dependent on the amount of pooled PIs at that point. Required model parameters and three explanatory variables (average PPFD, frequency, and duty ratio) were determined for the simulation using P n values under PL based on several combinations of the three variables. The model simulation for various PL levels with a wide range of time-averaged PPFDs, frequencies, and duty ratios further demonstrated that P n under PL with high frequencies and duty ratios was comparable to, but did not exceed, P n under continuous light, and also showed that P n under PL decreased as either frequency or duty ratio was decreased. The developed model can be used to estimate P n under various light environments where PPFD changes cyclically.

Kinetic mechanism of Nicotiana tabacum myosin-11 defines a new type of a processive motor.

The 175-kDa myosin-11 from Nicotiana tabacum (Nt(175kDa)myosin-11) is exceptional in its mechanical activity as it is the fastest known processive actin-based motor, moving 10 times faster than the structurally related class 5 myosins. Although this ability might be essential for long-range organelle transport within larger plant cells, the kinetic features underlying the fast processive movement of Nt(175kDa)myosin-11 still remain unexplored. To address this, we generated a single-headed motor domain construct and carried out a detailed kinetic analysis. The data demonstrate that Nt(175kDa)myosin-11 is a high duty ratio motor, which remains associated with actin most of its enzymatic cycle. However, different from other processive myosins that establish a high duty ratio on the basis of a rate-limiting ADP-release step, Nt(175kDa)myosin-11 achieves a high duty ratio by a prolonged duration of the ATP-induced isomerization of the actin-bound states and ADP release kinetics, both of which in terms of the corresponding time constants approach the total ATPase cycle time. Molecular modeling predicts that variations in the charge distribution of the actin binding interface might contribute to the thermodynamic fine-tuning of the kinetics of this myosin. Our study unravels a new type of a high duty ratio motor and provides important insights into the molecular mechanism of processive movement of higher plant myosins.

Design of high duty ratio nonlinear photonic crystal fiber with near-zero flattened dispersion after 1.205 μm

The paper presents a high nonlinear photonic crystal fiber (HN-PCF) with highly duty ratio for time-stretch analog-to-digital conversion (TSADC). The simulation results show that a nonlinear of 42.26W−1km−1 and the flattened dispersion of less than 1.0ps/(nmkm) are obtained in more than 495nm waveband (1205–1700nm). Owing to its high nonlinear coefficient and flattened dispersion, the high nonlinear PCF is expected to be suitable for supercontinuum (SC) generation. The numerical simulation results demonstrate that the proposed high duty ratio HN-PCF can generate wideband SC.

Chaperone-enhanced purification of unconventional myosin 15, a molecular motor specialized for stereocilia protein trafficking.

Unconventional myosin 15 is a molecular motor expressed in inner ear hair cells that transports protein cargos within developing mechanosensory stereocilia. Mutations of myosin 15 cause profound hearing loss in humans and mice; however, the properties of this motor and its regulation within the stereocilia organelle are unknown. To address these questions, we expressed a subfragment 1-like (S1) truncation of mouse myosin 15, comprising the predicted motor domain plus three light-chain binding sites. Following unsuccessful attempts to express functional myosin 15-S1 using the Spodoptera frugiperda (Sf9)-baculovirus system, we discovered that coexpression of the muscle-myosin-specific chaperone UNC45B, in addition to the chaperone heat-shock protein 90 (HSP90) significantly increased the yield of functional protein. Surprisingly, myosin 15-S1 did not bind calmodulin with high affinity. Instead, the IQ domains bound essential and regulatory light chains that are normally associated with class II myosins. We show that myosin 15-S1 is a barbed-end-directed motor that moves actin filaments in a gliding assay (∼ 430 nm · s(-1) at 30 °C), using a power stroke of 7.9 nm. The maximum ATPase rate (k(cat) ∼ 6 s(-1)) was similar to the actin-detachment rate (k(det) = 6.2 s(-1)) determined in single molecule optical trapping experiments, indicating that myosin 15-S1 was rate limited by transit through strongly actin-bound states, similar to other processive myosin motors. Our data further indicate that in addition to folding muscle myosin, UNC45B facilitates maturation of an unconventional myosin. We speculate that chaperone coexpression may be a simple method to optimize the purification of other myosin motors from Sf9 insect cells.

Magnesium Modulates Actin Binding and ADP Release in Myosin Motors

We examined the magnesium dependence of five class II myosins, including fast skeletal muscle myosin, smooth muscle myosin, β-cardiac myosin (CMIIB), Dictyostelium myosin II (DdMII), and nonmuscle myosin IIA, as well as myosin V. We found that the myosins examined are inhibited in a Mg(2+)-dependent manner (0.3-9.0 mm free Mg(2+)) in both ATPase and motility assays, under conditions in which the ionic strength was held constant. We found that the ADP release rate constant is reduced by Mg(2+) in myosin V, smooth muscle myosin, nonmuscle myosin IIA, CMIIB, and DdMII, although the ADP affinity is fairly insensitive to Mg(2+) in fast skeletal muscle myosin, CMIIB, and DdMII. Single tryptophan probes in the switch I (Trp-239) and switch II (Trp-501) region of DdMII demonstrate these conserved regions of the active site are sensitive to Mg(2+) coordination. Cardiac muscle fiber mechanic studies demonstrate cross-bridge attachment time is increased at higher Mg(2+) concentrations, demonstrating that the ADP release rate constant is slowed by Mg(2+) in the context of an activated muscle fiber. Direct measurements of phosphate release in myosin V demonstrate that Mg(2+) reduces actin affinity in the M·ADP·Pi state, although it does not change the rate of phosphate release. Therefore, the Mg(2+) inhibition of the actin-activated ATPase activity observed in class II myosins is likely the result of Mg(2+)-dependent alterations in actin binding. Overall, our results suggest that Mg(2+) reduces the ADP release rate constant and rate of attachment to actin in both high and low duty ratio myosins.

High step-up isolated efficient single switch DC-DC converter for renewable energy source

In this paper, an isolated high step-up single switch DC-DC converter for renewable energy source is proposed. In the proposed converter high step-up voltage is obtained by single power switching technique that operates low duty cycle with isolated transformer inductors and switched capacitors and power diodes. The disadvantage of conventional converters is that it has high duty ratio and high voltage stress on power devices with less efficiency. The proposed converter eliminates the switching losses and recycles the leakage energy which includes reverse recovery energy of the power diode by using passive clamp circuit. To achieve high output voltage gain, the isolated transformer primary terminal and secondary terminal are connected in series during switching operation. PSIM software has been used for simulation. Simulation circuit is analyzed at 40Vdc/400Vdc, 200W and this operation is validated by implementing in the hardware model at 12Vdc/120Vdc, 60W.

Mouse Myosin-19 Is a Plus-end-directed, High-duty Ratio Molecular Motor

Class XIX myosin (Myo19) is a vertebrate-specific unconventional myosin, responsible for the transport of mitochondria. To characterize biochemical properties of Myo19, we prepared recombinant mouse Myo19-truncated constructs containing the motor domain and the IQ motifs using the baculovirus/Sf9 expression system. We identified regulatory light chain (RLC) of smooth muscle/non-muscle myosin-2 as the light chain of Myo19. The actin-activated ATPase activity and the actin-gliding velocity of Myo19-truncated constructs were about one-third and one-sixth as those of myosin-5a, respectively. The apparent affinity of Myo19 to actin was about the same as that of myosin-5a. The RLCs bound to Myo19 could be phosphorylated by myosin light chain kinase, but this phosphorylation had little effect on the actin-activated ATPase activity and the actin-gliding activity of Myo19-truncated constructs. Using dual fluorescence-labeled actin filaments, we determined that Myo19 is a plus-end-directed molecular motor. We found that, similar to that of the high-duty ratio myosin, such as myosin-5a, ADP release rate was comparable with the maximal actin-activated ATPase activity of Myo19, indicating that ADP release is a rate-limiting step for the ATPase cycle of acto-Myo19. ADP strongly inhibited the actin-activated ATPase activity and actin-gliding activity of Myo19-truncated constructs. Based on the above results, we concluded that Myo19 is a high-duty ratio molecular motor moving to the plus-end of the actin filament.

A Switched-Capacitor-Based Active-Network Converter With High Voltage Gain

The voltage gain of traditional boost converter is limited due to the high current ripple, high voltage stress across active switch and diode, and low efficiency associated with large duty ratio operation. High voltage gain is required in applications, such as the renewable energy power systems with low input voltage. A high step-up voltage gain active-network converter with switched-capacitor technique is proposed in this paper. The proposed converter can achieve high voltage gain without extremely high duty ratio. In addition, the voltage stress of the active switches and output diodes is low. Therefore, low voltage components can be adopted to reduce the conduction loss and cost. The operating principle and steady-state analysis are discussed in detail. A prototype with 20-40-V input voltage, 200-V output voltage, and 200-W output power has been established in the laboratory. Experimental results are given to verify the analysis and advantages of the proposed converter.

Molecular Characterization and Subcellular Localization of Arabidopsis Class VIII Myosin, ATM1

Land plants possess myosin classes VIII and XI. Although some information is available on the molecular properties of class XI myosins, class VIII myosins are not characterized. Here, we report the first analysis of the enzymatic properties of class VIII myosin. The motor domain of Arabidopsis class VIII myosin, ATM1 (ATM1-MD), and the motor domain plus one IQ motif (ATM1-1IQ) were expressed in a baculovirus system and characterized. ATM1-MD and ATM1-1IQ had low actin-activated Mg(2+)-ATPase activity (Vmax = 4 s(-1)), although their affinities for actin were high (Kactin = 4 μM). The actin-sliding velocities of ATM1-MD and ATM1-1IQ were 0.02 and 0.089 μm/s, respectively, from which the value for full-length ATM1 is calculated to be ∼0.2 μm/s. The results of actin co-sedimentation assay showed that the duty ratio of ATM1 was ∼90%. ADP dissociation from the actin·ATM1 complex (acto-ATM1) was extremely slow, which accounts for the low actin-sliding velocity, low actin-activated ATPase activity, and high duty ratio. The rate of ADP dissociation from acto-ATM1 was markedly biphasic with fast and slow phase rates (5.1 and 0.41 s(-1), respectively). Physiological concentrations of free Mg(2+) modulated actin-sliding velocity and actin-activated ATPase activity by changing the rate of ADP dissociation from acto-ATM1. GFP-fused full-length ATM1 expressed in Arabidopsis was localized to plasmodesmata, plastids, newly formed cell walls, and actin filaments at the cell cortex. Our results suggest that ATM1 functions as a tension sensor/generator at the cell cortex and other structures in Arabidopsis.

Enhanced light extraction in n-GaN-based light-emitting diodes with three-dimensional semi-spherical structure

Three-dimensional (3D) periodic micro/nanostructures can have a remarkable enhancement effect on light-emitting diodes (LEDs). However, simple, high-throughput and large-area fabrication of 3D periodic micro/nanostructures with a high duty ratio is difficult. In this Letter, high-duty-ratio 3D semi-spherical structures were fabricated on the surface of n-GaN-based vertical-structure LEDs by under-exposure ultraviolet lithography and dry etching. The resulting LEDs provide about 200% more light output power than those with a flat surface. This method of fabricating high-duty-ratio 3D semi-spherical structures could be used in other optical devices and shows potential for industrial production and commercialization.

Myosin-10 Produces its Power-Stroke in Two Phases and Moves Processively along a Single Actin Filament under Low-Load

Myosin-10 is an actin-activated ATPase that participates in essential intracellular processes such as filopodia formation/extension, phagocytosis, cell migration and mitotic spindle maintenance. The myosin-10 duty-cycle ratio - i.e. the fraction of time the motor remains tightly bound to actin during its total ATPase cycle - from previous biochemical studies are inconclusive, thus whether this myosin displays intermediate or high duty ratio is still under debate. To study this motor protein's mechano-chemical properties we have used a recombinant, truncated form of myosin-10 consisting of the first 940 amino acids, followed by a GCN4 leucine zipper motif to force dimerization. Negative-stain electron microscopy reveals that the majority of molecules (∼87%) are dimeric with a head-to-head contour distance of ∼50 nm. In vitro motility assays show that myosin-10 moves actin filaments smoothly with a velocity of 150 - 400 nm s−1. Steady-state and transient kinetic analysis of the ATPase cycle shows that the ADP release rate (∼13 s−1) is similar to the maximum ATPase activity (∼12 - 14 s−1) and, therefore, contributes to rate-limitation of the enzymatic cycle. Single molecule optical tweezers experiments show that under intermediate load (∼0.5 pN) myosin-10 interacts intermittently with actin and produces a working stroke of ∼17 nm, composed of an initial 15 nm and subsequent 2 nm movement. At low optical trap loads, we observed staircase-like processive movements of myosin-10 interacting with the actin filament, consisting of up to six, ∼35 nm, steps per binding interaction. Here we describe the kinetics and mechanics of myosin-10, interrogating bulk and single molecule biophysical/biochemical properties to further our understanding of its ATP-driven, motor mechanism and how this relates to its cellular functions.

Influence of duty ratio of pulsed bias on structure and properties of silicon-doped diamond-like carbon films by plasma deposition

We have investigated the influence of the duty ratio of pulsed substrate bias on the structure and properties of Si-doped diamond-like carbon (Si-DLC) films deposited by radio frequency plasma-enhanced chemical vapor deposition using CH4, Ar, and monomethylsilane (CH3SiH3) as the Si source. The Si/(Si+C) ratios in the Si-DLC films deposited using pulsed bias were higher than that of the dc-biased Si-DLC film, and the Si fraction increased with decreasing pulse duty ratio. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy analyses revealed that Si-C, Si-Hn, and C-Hn bonds in the Si-DLC films increased with decreasing duty ratio. The internal stress decreased as the duty ratio decreased, which is probably due to the increase in Si-C, Si-Hn, and C-Hn bonds in the films. The Si-DLC films deposited using pulsed bias had higher adhesion strength than the dc-biased Si-DLC film because of the further reduction of internal stress. At higher duty ratios, although the Si fractions of the pulse-biased Si-DLC films were higher than that of the dc-biased Si-DLC film, the wear rates of the former were less than that of the latter.

Global Fit Analysis of Myosin-5b Motility Reveals Thermodynamics of Mg2+-Sensitive Acto-Myosin-ADP States

Kinetic and thermodynamic studies of the mechanochemical cycle of myosin motors are essential for understanding the mechanism of energy conversion. Here, we report our investigation of temperature and free Mg2+-ion dependencies of sliding velocities of a high duty ratio class-5 myosin motor, myosin-5b from D. discoideum using in vitro motility assays. Previous studies have shown that the sliding velocity of class-5 myosins obeys modulation by free Mg2+-ions. Free Mg2+-ions affect ADP release kinetics and the dwell time of actin-attached states. The latter determines the maximal velocity of actin translocation in the sliding filament assay. We measured the temperature dependence of sliding velocity in the range from 5 to 55°C at two limiting free Mg2+-ion concentrations. Arrhenius plots demonstrated non-linear behavior. Based on this observation we propose a kinetic model, which explains both sensitivity towards free Mg2+-ions and non-linearity of the temperature dependence of sliding velocity. According to this model, velocity is represented as a simple analytical function of temperature and free Mg2+-ion concentrations. This function has been applied to global non-linear fit analysis of three data sets including temperature and magnesium (at 20°C) dependence of sliding velocity. As a result we obtain thermodynamic parameters (ΔHMg and ΔSMg) of a fast equilibrium between magnesium free (AM·D) and magnesium bound acto-myosin-ADP (AM· Mg2+D) states and the corresponding enthalpic barriers associated with ADP release (ΔH1 ‡ and ΔH2 ‡). The herein presented integrative approach of data analysis based on global fitting can be applied to the remaining steps of the acto-myosin ATPase cycle facilitating the determination of energetic parameters and thermodynamics of acto-myosin interactions.

Analysis and implementation of an integrated electronic ballast for high‐intensity‐discharge lamps featuring high‐power factor

This paper proposes a high-power-factor electronic ballast for metal-halide (MH) lamps. In the proposed circuit, two buck–boost converters and a buck converter are integrated with a full-bridge inverter by sharing the four active switches of the full-bridge inverter. Two active switches are switched at high frequencies, while the other two are switched at lower frequencies. The buck converter is used to step down the DC-link voltage of the full-bridge inverter and filter out the high-frequency components to drive lamps with a low-frequency square-wave (LFSW) current. The buck–boost converters are operated at discontinuous-conduction mode to perform the function of power-factor correction (PFC) to ensure almost unity power factor at the input line. By tactful arrangement of diodes, the DC-link capacitors are discharged in parallel; this helps to operate the high-frequency active switches at a high duty ratio, achieving a small peak value of the PFC-converter current and correspondingly smaller conduction losses. Detailed operation modes and the design equations are provided. A prototype electronic ballast for a 70 W MH lamp is built and tested. Experimental measurements have proven that the proposed electronic ballast has the advantages of high-power factor and being free of acoustic resonance. By integrating the active switches of the converters and the inverter, the proposed electronic ballast is advantageous in terms of cost-effectiveness and high energy-conversion efficiency.

Biochemical and bioinformatic analysis of the myosin‐XIX motor domain

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin‐XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well‐conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19‐specific mechanochemistry. Steady‐state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell‐expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin‐activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin‐co‐sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP‐MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild‐type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high‐duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment. © 2013 Wiley Periodicals, Inc

Mechanisms of Mechanochemical Coupling in Low and High Duty Ratio Myosins

All myosins undergo the same ATP dependent biochemical cycle but with variations in the lifetime of the individual states of the cycle and the fraction of the total cycle time spent in each state. E.g., processive class-5 myosins are high duty ratio motors that stay most of the ATPase cycle time (> 0.5) attached to actin, whereas fast myosins such as muscle myosin-2 and some class-1 myosin members have a low duty ratio (< 0.1). The members of the respective groups are assumed to use different ways to couple conformational changes at the nucleotide binding regions to changes that occur at the actin binding sites. Conserved active-site elements termed switch-1 and switch-2 play a major role in the coupling mechanism; however, whether and to which extent small variations in the sequence of switch-1 and switch-2 affect the duty ratio of a given myosin, thus determining its ability for processive movement, fast contractility or tension bearing remains unresolved. Based on structural considerations and confirmed by mutational analyses, we identified key residues in the nucleotide binding pocket that are responsible for making ADP dissociation kinetics dependent on the concentration of free magnesium ions. The exchange of a single amino acid in switch-2 affected the motor properties of all myosins tested, but also transformed low duty ratio motors into high duty ratio and vice versa. In addition, x-ray structural analyses and molecular modeling allowed us to relate the observed changes to altered coupling between the active sites and actin binding regions. These results, together with our cell biological studies demonstrating for the first time that magnesium ions have a regulatory role on motor protein function in vivo reveal that, switch-2 can act as magnesium sensor critically determining the mechanochemical properties of myosins.

Effect of pro-proximity pulse voltage on the third-generation low light level image intensifiers halo effect

In order to explore the proper working voltage for the third-generation low light level image intensifier, the influence of pro-proximity pulse voltage on image intensifier halo effect is investigated. The pulse voltage is applied to photocathode of image intensifier. Respectively change the high and low level voltage and duty ratio, image intensifier halo images are collected by high-resolution charge-coupled device (CCD). The gray distributions for pixel points on halo image central line are given and comparatively analyzed. The results show that as high level voltage and duty ratio increase, the number of pixel points whose gray value is 255 increases and the border between signal and background becomes clear. When high level voltage is above 200 V and duty ratio is above 60%, the pro-proximity voltage has not great influence on image intensifier halo effect. When low level voltage is above 2 V, photoelectrons escaping from photocathode cannot reach microchannel plate under low level voltage stage. The present investigation is beneficial to the exploration of the optimal working voltage for image intensifier and energy range of photoelectrons escaping from photocathode, and provides an experimental support for the improvement of the third-generation low light level image intensifier performance.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ~14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (~20-25%) motor. The ADP release step (~0.35 s(-1)) of NMIIB is only ~3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s(-1)). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of single- and double-headed myosin fragments and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished using total internal reflection fluorescence (TIRF) with fluorescence imaging with 1-nm accuracy (FIONA) and dual-beam optical trapping. At a physiological ATP concentration (1 mm), the rate of detachment of the single-headed and double-headed molecules was similar (~0.4 s(-1)). Using optical tweezers we found that the power stroke sizes of single- and double-headed heavy meromyosin (HMM) were each ~6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast, robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady-state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.

Kinetic Characterization of Nonmuscle Myosin IIB at the Single Molecule Level

Nonmuscle myosin IIB (NMIIB) is a cytoplasmic myosin, which plays an important role in cell motility by maintaining cortical tension. It forms bipolar thick filaments with ∼14 myosin molecule dimers on each side of the bare zone. Our previous studies showed that the NMIIB is a moderately high duty ratio (∼20-25%) motor. The ADP release step (∼0.35 s-1), of NMIIB is only ∼3 times faster than the rate-limiting phosphate release (0.13 ± 0.01 s-1), and as a result acto-NMIIB has the highest ADP-affinity reported so far for the myosin superfamily (<0.15 μM). The aim of this study was to relate the known in vitro kinetic parameters to the results of single molecule experiments and to compare the kinetic and mechanical properties of a single- and double-headed myosin fragments, and nonmuscle IIB thick filaments. Examination of the kinetics of NMIIB interaction with actin at the single molecule level was accomplished by the use of TIRF using FIONA and dual-beam optical trapping. At a physiological ATP concentration (1 mM), the rate of detachment of the single-headed and double-headed molecules was similar (∼0.4 s-1). using optical tweezers we found that the power-stroke sizes of single- and double-headed HMM were ∼6 nm. No signs of processive stepping at the single molecule level were observed in the case of NMIIB-HMM in optical tweezers or TIRF/in vitro motility experiments. In contrast robust motility of individual fluorescently labeled thick filaments of full-length NMIIB was observed on actin filaments. Our results are in good agreement with the previous steady state and transient kinetic studies and show that the individual nonprocessive nonmuscle myosin IIB molecules form a highly processive unit when polymerized into filaments.