Drosophila melanogaster Myosin-18 Represents a Highly Divergent Motor with Actin Tethering Properties

The gene encoding Drosophila myosin-18 is complex and can potentially yield six alternatively spliced mRNAs. One of the major features of this myosin is an N-terminal PDZ domain that is included in some of the predicted alternatively spliced products. To explore the biochemical properties of this protein, we engineered two minimal motor domain (MMD)-like constructs, one that contains the N-terminal PDZ (myosin-18 M-PDZ) domain and one that does not (myosin-18 M-ΔPDZ). These two constructs were expressed in the baculovirus/Sf9 system. The results suggest that Drosophila myosin-18 is highly divergent from most other myosins in the superfamily. Neither of the MMD constructs had an actin-activated MgATPase activity, nor did they even bind ATP. Both myosin-18 M-PDZ and M-ΔPDZ proteins bound to actin with Kd values of 2.61 and 1.04 μm, respectively, but only about 50–75% of the protein bound to actin even at high actin concentrations. Unbound proteins from these actin binding assays reiterated the 60% saturation maximum, suggesting an equilibrium between actin-binding and non-actin-binding conformations of Drosophila myosin-18 in vitro. Neither the binding affinity nor the substoichiometric binding was significantly affected by ATP. Optical trapping of single molecules in three-bead assays showed short lived interactions of the myosin-18 motors with actin filaments. Combined, these data suggest that this highly divergent motor may function as an actin tethering protein

Human Myosin Vc Is a Low Duty Ratio, Nonprocessive Molecular Motor

Myosin Vc is the product of one of the three genes of the class V myosin found in vertebrates. It is widely found in secretory and glandular tissues, with a possible involvement in transferrin trafficking. Transient and steady-state kinetic studies of human myosin Vc were performed using a truncated, single-headed construct. Steady-state actin-activated ATPase measurements revealed a V(max) of 1.8 +/- 0.3 s(-1) and a K(ATPase) of 43 +/- 11 microm. Unlike previously studied vertebrate myosin Vs, the rate-limiting step in the actomyosin Vc ATPase pathway is the release of inorganic phosphate (~1.5 s(-1)), rather than the ADP release step (~12.0-16.0 s(-1)). Nevertheless, the ADP affinity of actomyosin Vc (K(d) = 0.25 +/- 0.02 microm) reflects a higher ADP affinity than seen in other myosin V isoforms. Using the measured kinetic rates, the calculated duty ratio of myosin Vc was approximately 10%, indicating that myosin Vc spends the majority of the actomyosin ATPase cycle in weak actin-binding states, unlike the other vertebrate myosin V isoforms. Consistent with this, a fluorescently labeled double-headed heavy meromyosin form showed no processive movements along actin filaments in a single molecule assay, but it did move actin filaments at a velocity of approximately 24 nm/s in ensemble assays. Kinetic simulations reveal that the high ADP affinity of actomyosin Vc may lead to elevations of the duty ratio of myosin Vc to as high as 64% under possible physiological ADP concentrations. This, in turn, may possibly imply a regulatory mechanism that may be sensitive to moderate changes in ADP concentration

Myosin-5 varies its steps along the irregular F-actin track

Molecular motors employ chemical energy to generate unidirectional mechanical output against a track. By contrast to the majority of macroscopic machines, they need to navigate a chaotic cellular environment, potential disorder in the track and Brownian motion. Nevertheless, decades of nanometer-precise optical studies suggest that myosin-5a, one of the prototypical molecular motors, takes uniform steps spanning 13 subunits (36 nm) along its F-actin track. Here, we use high-resolution interferometric scattering (iSCAT) microscopy to reveal that myosin takes strides spanning 22 to 34 actin subunits, despite walking straight along the helical actin filament. We show that cumulative angular disorder in F-actin accounts for the observed proportion of each stride length, akin to crossing a river on variably-spaced stepping stones. Electron microscopy revealed the structure of the stepping molecule. Our results indicate that both motor and track are soft materials that can adapt to function in complex cellular conditions

Probing the mechano-chemical transduction mechanism of skeletal muscle myosin II using a feedback enhanced optical trap

Skeletal muscle uses more energy when it is shortening rapidly and less energy when it is maintaining a static load, a phenomenon discovered in 1923 by W. O. Fenn and A. V. Hill. This physiological feedback, termed the “Fenn effect”, was one of the key observations leading to the actomyosin cross-bridge theory of muscle contraction by A. F. Huxley in 1957. The efficient control of energy liberation in isometric vs. shortening skeletal muscle implies that one or more steps in the actomyosin ATPase cycle are controlled by mechanical load borne by the molecular motor, myosin. The objective of this study is to obtain a precise correlation between the mechanical and biochemical aspects of actomyosin interactions at the single molecule level to understand the molecular mechanism of this load adaptive molecular motor. In this work, a novel feedback enhanced infrared laser-based optical gradient trap, the “isometric clamp”, was constructed. The isometric clamp was used to study the mechanics of individual skeletal muscle myosin in order to detect the reaction steps that depend on the dynamic properties of the external load. The method also enabled reliable and quantitative measurement of the mechanical properties such as the isometric force, power stroke displacement and stiffness, produced by single actomyosin interactions. The results obtained from this work indicate that single myosin molecules transduce energy as efficiently as whole muscle and that a component of the Fenn effect is reversal of the force-generating actomyosin transition under high load without net utilization of ATP. Our results, together with the earlier studies of muscle contraction suggest that the actomyosin enzymatic cycle has multiple strain dependent steps, and that two factors contribute toward the Fenn effect: at low shortening velocities, the economical reversal of the force-generating actomyosin transition saves ATP and at high shortening velocities strain-dependent acceleration of ADP release increases ATP turnover. Both of these effects probably occur in muscle contraction and thus contribute to its efficient control of energy utilization

Probing the mechano-chemical transduction mechanism of skeletal muscle myosin II using a feedback enhanced optical trap

Skeletal muscle uses more energy when it is shortening rapidly and less energy when it is maintaining a static load, a phenomenon discovered in 1923 by W. O. Fenn and A. V. Hill. This physiological feedback, termed the “Fenn effect”, was one of the key observations leading to the actomyosin cross-bridge theory of muscle contraction by A. F. Huxley in 1957. The efficient control of energy liberation in isometric vs. shortening skeletal muscle implies that one or more steps in the actomyosin ATPase cycle are controlled by mechanical load borne by the molecular motor, myosin. The objective of this study is to obtain a precise correlation between the mechanical and biochemical aspects of actomyosin interactions at the single molecule level to understand the molecular mechanism of this load adaptive molecular motor. In this work, a novel feedback enhanced infrared laser-based optical gradient trap, the “isometric clamp”, was constructed. The isometric clamp was used to study the mechanics of individual skeletal muscle myosin in order to detect the reaction steps that depend on the dynamic properties of the external load. The method also enabled reliable and quantitative measurement of the mechanical properties such as the isometric force, power stroke displacement and stiffness, produced by single actomyosin interactions. The results obtained from this work indicate that single myosin molecules transduce energy as efficiently as whole muscle and that a component of the Fenn effect is reversal of the force-generating actomyosin transition under high load without net utilization of ATP. Our results, together with the earlier studies of muscle contraction suggest that the actomyosin enzymatic cycle has multiple strain dependent steps, and that two factors contribute toward the Fenn effect: at low shortening velocities, the economical reversal of the force-generating actomyosin transition saves ATP and at high shortening velocities strain-dependent acceleration of ADP release increases ATP turnover. Both of these effects probably occur in muscle contraction and thus contribute to its efficient control of energy utilization

EHENとウラシルの同時投与によるラット腎腫瘍の性差および系統差

各群30匹, 8群合計240匹のラットを用いた.第1～第4群が薬剤投与群, 第5～第8群が薬剤非投与の対照群とした.薬剤投与群には飲料水中に0.05%EHENを, 粉末飼料中に3%ウラシルを混入して, これを3週間同時に投与した.全実験期間は33週間とした.腎腫瘍の発生率は, 第1, 2, 3, 4群でそれぞれ100%, 95%, 71%, 60%であり, WistarラットでF344ラットよりも有意に高率に腎腫瘍の発生がみられた.性差による発生率では, 雄に高率に発生する傾向があったが, 有意差はなかった.対照群には腎腫瘍は発生しなかった.Wistar雄ラットに, EHENとウラシルを同時に投与すれば, 短期間に高頻度に腎腫瘍を誘発させうるWe earlier demonstrated that simultaneous administration of EHEN and uracil for 3 weeks resulted in enhancement of renal carcinogenesis in F344 female rats. Therefore, to establish a model of renal carcinogenesis in rats that can induce advanced renal carcinoma at a high incidence, differences in the susceptibility to N-ethyl-N-hydroxyethylnitrosamine (EHEN) and uracil of the kidneys in male and female rats of two strains were examined. Group 1 (male Wistar rats), group 2 (female Wistar rats), group 3 (male F344 rats), and group 4 (female F344 rats) received a 3-week simultaneous administration of 0.05% EHEN in the drinking water and 3% uracil in the diet after one week's acclimation. In all the above four groups, the rats were thereafter given a basal diet and water without chemical addition for a 29-week period. Group 5 (male Wistar rats), group 6 (female Wistar rats), group 7 (male F344 rats) and group 8 (female F344 rats) received no chemicals for the entire 33 weeks. At the end of the experiment, renal adenocarcinomas were found in 85, 68, 14 and 0% of the rats in groups 1, 2, 3 and 4, respectively. The incidence of adenomas and adenocarcinomas in Wistar rats were significantly greater than in F344 rats (p < 0.0001). These findings indicate strain and possibly sex differences in kidney carcinogenesis in rats treated with EHEN and uracil, and simultaneous administration of the two agents to male Wistar rats might have an advantage for models to induce advanced renal carcinoma at a high incidence

Divergent diagnosis from arthroscopic findings and identification of CPII and C2C for detection of cartilage degradation in horses

The objective of this study was to investigate the changes in synovial fluid concentration of collagen type II cleavage site (C2C) and procollagen II C-propeptide (CPII), markers of joint cartilage degeneration and synthesis, respectively, in horses with intraarticular fracture or osteochondrosis dissecans (OCD), and to examine the relationship between arthroscopic findings and these biomarker levels. Synovial fluid was collected from 36 joints in 18 horses (6 fractures and 12 OCDs). Samples from contralateral normal joints, when available, served as controls (n = 12). Concentrations of C2C and CPII were measured using enzyme-linked immunosorbant assays. Moreover, the severity of the cartilage degradation was graded arthroscopically in 16 horses, and the correlation between the C2C and CPII levels and the arthroscopic scores were investigated. Compared to the control, the concentration of C2C was increased in OCD joints but not in fracture joints, whereas the concentration of CPII was increased in fracture joints but not in OCD joints. Within each disease group there was no correlation between biomarker levels and arthroscopic findings. Therefore, although C2C and CPII have diagnostic potential further knowledge is required to provide accurate analysis

The usefulness of shear wave elastography in evaluating erectile dysfunction severity before and after prostaglandin E1 test

Prostaglandin E1 intracavernous injection test is an established method for diagnosing erectile dysfunction. However, the evaluation is non-objective and often influenced by the evaluator’s subjectivity. Herein, we measured and objectively evaluated shear wave elastography results of the corpus cavernosum before and after injection in 16 patients who underwent prostaglandin E1 testing. The response score of prostaglandin E1 tests were “1” in 2 cases, “2” in 2 cases, and “3” in 12 cases. The average transmission velocity before the injection and at the time of maximum erection after the injection were 2.21 m/s and 1.57 m/s, respectively. Transmission velocity decreased during erection in 14 of 16 cases (87.5%). The overall rate of change in transmission velocity due to injection was –26.7% and was significantly different between the poor (responses 1 and 2: –16.1%) and good erection (response 3: –30.2%) groups. To the best of our knowledge, this is the first attempt to evaluate erectile phenomenon using percutaneous ultrasonic elastography in Japan. Rate of change in shear wave transmission velocity due to prostaglandin E1 injection in the corpus cavernosum penis was associated with the degree of erection. Therefore, the rate of change in shear wave transmission velocity in the corpus cavernosum penis could be used as an objective index of erectile phenomenon. Percutaneous ultrasonic elastography is a non-invasive and useful test method for diagnosing erectile dysfunction, determining the therapeutic effect, and predicting prognosis

Behavioral and physiological correlates of kinetically tracking a chaotic target.

Humans can innately track a moving target by anticipating its future position from a brief history of observations. While ballistic trajectories can be readily extrapolated, many natural and artificial systems are governed by more general nonlinear dynamics and, therefore, can produce highly irregular motion. Yet, relatively little is known regarding the behavioral and physiological underpinnings of prediction and tracking in the presence of chaos. Here, we investigated in lab settings whether participants could manually follow the orbit of a paradigmatic chaotic system, the Rössler equations, on the (x,y) plane under different settings of a control parameter, which determined the prominence of transients in the target position. Tracking accuracy was negatively related to the level of unpredictability and folding. Nevertheless, while participants initially reacted to the transients, they gradually learned to anticipate it. This was accompanied by a decrease in muscular co-contraction, alongside enhanced activity in the theta and beta EEG bands for the highest levels of chaoticity. Furthermore, greater phase synchronization of breathing was observed. Taken together, these findings point to the possible ability of the nervous system to implicitly learn topological regularities even in the context of highly irregular motion, reflecting in multiple observables at the physiological level