Molecular Motor Myosin Research Articles

Searching, Stepping, and Stomping: What Polymer Theory can teach us about the Molecular Motor Myosin V

Though myosin V is among the most extensively studied of motor proteins, improvements in experimental techniques continue to provide new insights into the details of its dynamics. High-speed atomic force microscopy has visualized not only the expected hand-over-hand stepping of the two-headed motor along actin filaments, but additional, less well understood behaviors like “foot stomping”, where one head detaches and rebinds to the same site. A comprehensive picture of myosin motility needs to account for all the kinetic pathways, including backstepping and foot stomping, how they vary under load, and their relationship to the structural and chemical parameters of the motor. Starting from a simple polymer model, we develop an analytical theory to understand the minimal physical properties that govern motor dynamics. In particular, we solve the first-passage problem of the head reaching the target binding site, investigating the competing effects of load pulling back at the motor, strain in the leading head that biases the diffusion in the direction of the target, and the possibility of preferential binding to the forward site due to the recovery stroke. The theory reproduces a variety of experimental data, including the power stroke and slow diffusive search regimes in the mean trajectory of the detached head, and the force dependence of the forward-to-backward step ratio, run length, and velocity. The analytical approach yields a formula for the stall force, identifying the relative contributions of the chemical cycle rates versus mechanical features like the bending rigidities of the lever arms. Moreover we can fully explore the parameter phase space, to determine the robustness of the dynamical behavior to perturbations, and the natural constraints that dictate the structure of the motor.

A Metal Switch for Controlling the Activity of Molecular Motor Proteins

Molecular motors, such as kinesins and myosins, as well as molecular switches such as G-proteins, use the energy from nucleotide hydrolysis to carry out cellular tasks. In addition to the P-loop, these proteins use similar structural motifs, called switch 1 and switch 2, to sense and respond to the presence or absence of the gamma-phosphate of the nucleotides and coordinate nucleotide hydrolysis. We have developed a strategy to probe metal interactions within kinesin and myosin molecular motors, by taking advantage of the differential affinities of Mg(II) and Mn(II) for serine (-OH) and cysteine (−SH). We present the crystal structure of a kinesin motor domain bound to MnADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn(II) to Mg(II). This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg(II). In kinesin-1, kinesin-5, kinesin-10 and kinesin-14, this defect is rescued by Mn(II), providing a way to control both the enzymatic activity and force-generating ability of these nanomachines. We also present ATPase data for an analogous substitution in the switch 1 of non-muscle myosin-2 from Dictyostelium discoideum. This mutant myosin shows aberrant actin interaction whereby actomyosin dissociation becomes slow and rate-limiting in the presence of Mg(II), yet is rescued to wild-type activity by Mn(II). These data are consistent with switch 1 being unable to efficiently close upon MgATP binding, thus affecting the opening of the upper 50K domain. There are several relevant and important applications to this metal switch technology that will allow further biophysical characterization of molecular motors and molecular switch proteins.

The Cortical Acto-Myosin Network: From Diffusion Barrier to Functional Gateway in the Transport of Neurosecretory Vesicles to the Plasma Membrane

Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma, and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca2+ to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones. Here we discuss the functions of the cortical actin network, myosins, and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.

Myosin-Driven Intercellular Transportation of Wheat Germ Agglutinin Mediated by Membrane Nanotubes between Human Lung Cancer Cells

Membrane nanotubes can facilitate direct intercellular communication between cells and provide a unique channel for intercellular transfer of cellular contents. However, the transport mechanisms of membrane nanotubes remain poorly understood between cancer cells. Also largely unknown is the transport pattern mediated by membrane nanotubes. In this work, wheat germ agglutinin (WGA), a widely used drug carrier and potential antineoplastic drug, was labeled with quantum dots (QDs-WGA) as a model for exploring the intercellular transportation via membrane nanotubes. We found that membrane nanotubes allowed effective transfer of QDs-WGA. Long-term single-particle tracking indicated that the movements of QDs-WGA exhibited a slow and directed motion pattern in nanotubes. Significantly, the transport of QDs-WGA was driven by myosin molecular motors in an active and unidirectional manner. These results contribute to a better understanding of cell-to-cell communication for cancer research.

One path to understanding energy transduction in biological systems

Who is not fascinated by the myriad biological movements that define life? From cell migration, cell division and a network of translocation activities within cells to highly specialized muscle contraction, molecular motors operate by burning ATP as fuel. Three types of molecular motors—myosin, kinesin and dynein—and nearly 100 different subtypes transduce that chemical energy into mechanical movements to carry out a wide variety of cellular tasks. Understanding the molecular basis of energy transduction by these motors has taken decades.

Dynamic Exchange of Myosin VI on Endocytic Structures

The actin-based molecular motor myosin VI functions in the endocytic uptake pathway, both during the early stages of clathrin-mediated uptake and in later transport to/from early endosomes. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover rate of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures with a characteristic half-life common to both the large insert isoform of myosin VI on clathrin-coated structures and the no-insert isoform on early endosomes. This half-life is shared by the myosin VI-binding partner Dab2 and is identical for full-length myosin VI and the cargo-binding tail region. The 4-fold slower half-life of an artificially dimerized construct of myosin VI on clathrin-coated structures suggests that wild type myosin VI does not function as a stable dimer, but either as a monomer or in a monomer/dimer equilibrium. Taken together, these FRAP results offer insight into both the basic turnover dynamics and the monomer/dimer nature of myosin VI.

Origin of Motion in the Human Ureter: Mechanics, Energetics and Kinetics of the Myosin Molecular Motors

Ureteral peristalsis is the result of coordinated mechanical motor performance of longitudinal and circular smooth muscle layer of the ureter wall. The main aim of this study was to characterize in smooth muscle of proximal segments of human ureter, the mechanical properties at level of muscle tissue and at level of myosin molecular motors. Ureteral samples were collected from 15 patients, who underwent nephrectomy for renal cancer. Smooth muscle strips longitudinally and circularly oriented from proximal segments of human ureter were used for the in vitro experiments. Mechanical indices including the maximum unloaded shortening velocity (Vmax), and the maximum isometric tension (P0) normalized per cross-sectional area, were determined in vitro determined in electrically evoked contractions of longitudinal and circular smooth muscle strips. Myosin cross-bridge (CB) number per mm2 (Ψ) the elementary force per single CB (Ψ) and kinetic parameters were calculated in muscle strips, using Huxley's equations adapted to nonsarcomeric muscles. Longitudinal smooth muscle strips exhibited a significantly (p<0.05) faster Vmax (63%) and a higher P0 (40%), if compared to circular strips. Moreover, longitudinal muscle strips showed a significantly higher unitary force (Ψ) per CB. However, no significant differences were observed in CB number, the attachment (f1) and the detachment (g2) rate constants between longitudinal and circular muscle strips. The main result obtained in the present work documents that the mechanical, energetic and unitary forces per CB of longitudinal layer of proximal ureter are better compared to the circular one; these preliminary findings suggested, unlike intestinal smooth muscle, a major role of longitudinal smooth muscle layer in the ureter peristalsis.

Myrip Couples the Capture of Secretory Granules by the Actin-Rich Cell Cortex and Their Attachment to the Plasma Membrane

Exocytosis of secretory granules (SGs) requires their delivery to the actin-rich cell cortex followed by their attachment to the plasma membrane (PM). How these reactions are executed and coordinated is still unclear. Myrip, which is also known as Slac-2c, binds to the SG-associated GTPase Rab27 and is thought to promote the delivery of SGs to the PM by recruiting the molecular motor myosin Va. Myrip also interacts with actin and the exocyst complex, suggesting that it may exert multiple roles in the secretory process. By combining total internal reflection fluorescence microscopy, single-particle tracking, a photoconversion-based assay, and mathematical modeling, we show that, in human enterochromaffin cells, Myrip (1) inhibits a class of SG motion characterized by fast and directed movement, suggesting that it facilitates the dissociation of SGs from microtubules; (2) enhances their motion toward the PM and the probability of SG attachment to the PM; and (3) increases the characteristic time of immobilization at the PM, indicating that it is a component of the molecular machinery that tether SGs to the PM. Remarkably, while the first two effects of Myrip depend on its ability to recruit myosin Va on SGs, the third is myosin Va independent but relies on the C-terminal domain of Myrip. We conclude that Myrip couples the retention of SGs in the cell cortex, their transport to the PM, and their attachment to the PM, and thus promotes secretion. These three steps of the secretory process are thus intimately coordinated.

Myosin-V as a Mechanical Sensor: An Elastic Network Study

According to recent experiments, the molecular-motor myosin behaves like a strain sensor, exhibiting different functional responses when loads in opposite directions are applied to its tail. Within an elastic-network model, we explore the sensitivity of the protein to the forces acting on the tail and find, in agreement with experiments, that such forces invoke conformational changes that should affect filament binding and ADP release. Furthermore, conformational responses of myosin to the application of forces to individual residues in its principal functional regions are systematically investigated and a detailed sensitivity map of myosin-V is thus obtained. The results suggest that the strain-sensor behavior is involved in the intrinsic operation of this molecular motor.

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.

2PS015 ミオシン首振り運動の分子動力学シミュレーション(日本生物物理学会第50回年会(2012年度))

2PS015 ミオシン首振り運動の分子動力学シミュレーション(日本生物物理学会第50回年会(2012年度))

RNA Granules and Stress Granules in Virus Systems

Viruses initiate a number of cellular stress responses and modulate gene regulation and compartmentalization of RNA upon infection to be successful parasites. Virus infections may induce or impair stress granule (SG) formation to maximize replication efficiency. SGs and processing bodies (PBs) are the RNA granules, which contain translationally inactive pool of transcripts as the mRNA silencing foci. PBs and SGs, the highly conserved macromolecular aggregates, can release mRNAs to allow their translations. Unlike constitutively existing PBs that can respond to stimuli and affect mRNA translation and decay, SGs are specifically induced upon cellular stress and can triggers a global translational silencing by several pathways, including phosphorylation of the key translation initiation factor eIF2alpha, tRNA cleavage, and sequestration of cellular components and so on. The dynamics of PBs and SGs are regulated by several signaling pathways, including histone deacetylase 6, and depend on microfilaments and microtubules, and the cognate molecular motors myosin, dynein, and kinesin. SGs share features with aggresomes and related aggregates of unfolded proteins and may play a role in the pathology. The recent advances in understanding the relationship between viruses and mRNA stress granules are summarized.

Examination of the Kinetics of Myosin VI during Endocytosis

The molecular motor myosin VI has been implicated in endocytosis, a trafficking pathway mediating intracellular uptake of items such as membrane receptors and nutrients. Previous studies demonstrate the localization of the large insert isoform of myosin VI to clathrin-coated vesicles and the no insert isoform of myosin VI to early endosomes. The kinetics of myosin VI functionality on these endocytic structures has not yet been examined, however. This study uses fluorescence recovery after photobleaching (FRAP) to examine the turnover kinetics of the no insert and large insert isoforms of myosin VI during endocytosis. The results demonstrate that myosin VI turns over dynamically on endocytic structures and that different isoforms on different intracellular compartments have distinct turnover rates. The FRAP assay system is then implemented with a novel live cell expression of an artificial dimer of myosin VI to demonstrate the dimeric functionality of myosin VI on endocytic structures in vivo. Further studies of the turnover rates of the myosin VI binding partner Dab2 on clathrin-coated structures demonstrate that Dab2 turns over more rapidly than full length myosin VI, a novel indication of the relative turnover rates of a motor protein versus its binding partner on a given cellular compartment. The turnover kinetics of specific myosin VI motor domain mutants on endocytic structures are also examined, e.g. the D179Y mutant implicated in progressive hearing loss, a study which offers insight into the functional source of myosin VI-related deafness. In addition to providing insight into the endocytic functionality of myosin VI, these FRAP studies offer general insight into the dynamics of myosin motor proteins on intracellular structures, the functional differences between isoforms of given myosin proteins, and the comparative turnover rates of myosin proteins and their binding partners.

Interactions between Leishmania braziliensis and Macrophages Are Dependent on the Cytoskeleton and Myosin Va.

Leishmaniasis is a neglected tropical disease with no effective vaccines. Actin, microtubules and the actin-based molecular motor myosin Va were investigated for their involvement in Leishmania braziliensis macrophage interactions. Results showed a decrease in the association index when macrophages were without F-actin or microtubules regardless of the activation state of the macrophage. In the absence of F-actin, the production of NO in non-activated cells increased, while in activated cells, the production of NO was reduced independent of parasites. The opposite effect of an increased NO production was observed in the absence of microtubules. In activated cells, the loss of cytoskeletal components inhibited the release of IL-10 during parasite interactions. The production of IL-10 also decreased in the absence of actin or microtubules in non-activated macrophages. Only the disruption of actin altered the production of TNF-α in activated macrophages. The expression of myosin Va tail resulted in an acute decrease in the association index between transfected macrophages and L. braziliensis promastigotes. These data reveal the importance of F-actin, microtubules, and myosin-Va suggesting that modulation of the cytoskeleton may be a mechanism used by L. braziliensis to overcome the natural responses of macrophages to establish infections.

Comparative statistical mechanics of myosin molecular motors in rat heart, diaphragm and tracheal smooth muscle

Purpose Statistical mechanics establishes a link between microscopic properties of matter and its bulk properties. A. Huxley's equations (1957) [1] provide the necessary phenomenological formalism to use statistical mechanics. Methods We compared statistical mechanics in rat diaphragm in tetanus (tet; n = 10) and twitch (tw; n = 12) modes, in heart in twitch mode ( n = 20), and in tracheal smooth muscle in tetanus mode (TSM; n = 10). This powerful tool makes it possible to determine: (i) statistical entropy (S) which is related to the dispersal of energy and represents a measure of the degree of disorder in muscular system; (ii) thermodynamic force A/T (chemical affinity A and temperature T); (iii) thermodynamic flow (υ); (iv) entropy production rate (A/T × υ), which quantifies irreversible chemical processes generated by myosin crossbridge (CB) molecular motors. Results All muscles studied operated near equilibrium, i.e., A << 2500 J/mol and in a stationary linear regime, i.e., A/T varied linearly with υ. The heart operated farther from equilibrium than both diaphragm (tet and tw) and TSM, as attested by its high entropy production rate. S was of the same order of magnitude in heart and TSM but lower in diaphragm (tet and tw). Conclusion CB kinetics derived from A. Huxley's equations conferred a characteristic profile in terms of statistical mechanics on each muscle type. All studied muscles differed in terms of statistical entropy, chemical affinity, and entropy production rate. Stimulation mode (tet and tw) modulated CB kinetics and statistical mechanics. All muscle types operated near equilibrium and in a stationary linear regime.

Mechanics and energetics of myosin molecular motors from nonpregnant human myometrium

Mechanical properties of spontaneously contracting isolated nonpregnant human myometrium (NPHM) were investigated throughout the whole continuum of load from zero load up to isometry. This made it possible to assess the three-dimensional tension-velocity-length (T-V-L) relationship characterizing the level of contractility and to determine crossbridge (CB) kinetics of myosin molecular motors. Seventy-seven muscle strips were obtained from hysterectomy in 42 nonpregnant patients. Contraction and relaxation parameters were measured during spontaneous mechanical activity. The isotonic tension-peak velocity (T-V) relationship was hyperbolic in 30 cases and nonhyperbolic in 47 cases. When the T-V relationship was hyperbolic, the Huxley formalism could be used to calculate CB kinetics and CB unitary force. At the whole muscle level and for a given isotonic load level, part of the V-L phase plane showed a common pathway, so that a given instantaneous length corresponded to only one possible instantaneous velocity, independent of time and initial length. At the molecular level, rate constants for CB attachment and detachment were dramatically low, ∼100 times lower than those of striated muscles, and ∼5 to 10 times lower than those of other smooth muscles. The CB unitary force was ∼1.4 ± 0.1 pN. NPHM shared similar basic contractile properties with striated muscles, reflected in the three-dimensional T-V-L relationship characterizing the contractile level. Low CB attachment and detachment rate constants made it possible to generate normal CB unitary force and normal muscle tension in NPHM, even though it contracted extremely slowly compared with other muscles.

Active fluctuation in the cortical cytoskeleton observed by high-speed live-cell scanning probe microscopy

We investigated the dynamics of the cortical cytoskeleton in living cells by analyzing the motion of the endogenous components of the cytoskeleton using scanning probe microscopy (SPM). We performed molecular characterization of the microgranules visualized by SPM in living cells and analyzed the motion of these microgranules via particle tracking. Simultaneous SPM and epifluorescence microscopy observations showed that the microgranules recruited not only actin but also cortactin, which can bind to actin filaments. This indicates condensation of actin filaments at microgranules, leading us to identify them as “cytoskeletal microdomains”. High-speed SPM observation and particle-tracking analysis showed that these cytoskeletal microdomains exhibit random walk-like diffusive fluctuations over a timescale of seconds. Inhibition of the molecular motor myosin II, which drives actin filaments, led to subdiffusive fluctuations of the microdomains. These results can be explained by longitudinal sliding of actin filaments stochastically driven by myosin II and the bending motion of the actin filaments in the absence of sliding. Analysis of the cytoskeletal microdomains thus revealed the intrinsic dynamics of the cortical cytoskeleton.

How Molecular Motors Are Arranged on a Cargo Is Important for Vesicular Transport

The spatial organization of the cell depends upon intracellular trafficking of cargos hauled along microtubules and actin filaments by the molecular motor proteins kinesin, dynein, and myosin. Although much is known about how single motors function, there is significant evidence that cargos in vivo are carried by multiple motors. While some aspects of multiple motor function have received attention, how the cargo itself —and motor organization on the cargo—affects transport has not been considered. To address this, we have developed a three-dimensional Monte Carlo simulation of motors transporting a spherical cargo, subject to thermal fluctuations that produce both rotational and translational diffusion. We found that these fluctuations could exert a load on the motor(s), significantly decreasing the mean travel distance and velocity of large cargos, especially at large viscosities. In addition, the presence of the cargo could dramatically help the motor to bind productively to the microtubule: the relatively slow translational and rotational diffusion of moderately sized cargos gave the motors ample opportunity to bind to a microtubule before the motor/cargo ensemble diffuses out of range of that microtubule. For rapidly diffusing cargos, the probability of their binding to a microtubule was high if there were nearby microtubules that they could easily reach by translational diffusion. Our simulations found that one reason why motors may be approximately 100 nm long is to improve their ‘on’ rates when attached to comparably sized cargos. Finally, our results suggested that to efficiently regulate the number of active motors, motors should be clustered together rather than spread randomly over the surface of the cargo. While our simulation uses the specific parameters for kinesin, these effects result from generic properties of the motors, cargos, and filaments, so they should apply to other motors as well.

Regulation of Cardiac Contractility

ABSTRACT Contractility describes the relative ability of the heart to eject a stroke volume (SV) at a given prevailing afterload (arterial pressure) and preload (end-diastolic volume; EDV). Various measures of contractility are related to the fraction as the SV/EDV or the ejection fraction, and the dynamics of ejection as determined from maximum pressure rise in the ventricles or arteries or from aortic flow velocities determined by echocardiography. At the cellular level, the ultimate determinant of contractility is the relative tension generation and shortening capability of the molecular motors (myosin cross-bridges) of the sarcomeres as determined by the rates and extent of Ca activation, the turnover kinetics of the cross-bridges, and the relative Ca responsiveness of the sarcomeres. Engagement of the regulatory signaling cascades controlling contractility occurs with occupancy and signal transduction by receptors for neurohumors of the autonomic nervous system as well as growth and stress signaling ...

Chemomechanical Coupling and Motor Cycles of Myosin V

The molecular motor myosin V has been studied extensively both in bulk and single molecule experiments. Based on the chemical states of the motor, we construct a systematic network theory that includes experimental observations about the stepping behavior of myosin V. We utilize constraints arising from nonequilibrium thermodynamics to determine motor parameters and demonstrate that the motor behavior is governed by three chemomechanical motor cycles. The competition between these cycles can be understood via the influence of external load forces onto the chemical transition rates for the binding of adenosine triphosphate and adenosine diphosphate. In addition, we also investigate the functional dependence of the mechanical stepping rates on these forces. For substall forces, the dominant pathway of the motor network is profoundly different from the one for superstall forces, which leads to a stepping behavior that is in agreement with the experimental observations. Our theory provides a unified description of the experimental data as obtained for myosin V in single motor experiments.