Melanosomes Transported by Myosin-V in Xenopus Melanophores Perform Slow 35 nm Steps

We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes’ motion.     

Extracting the Stepping Dynamics of Molecular Motors in Living Cells from Trajectories of Single Particles

Molecular motors are responsible of transporting a wide variety of cargos in the cytoplasm. Current efforts are oriented to characterize the biophysical properties of motors in cells with the aim of elucidating the mechanisms of these nanomachines in the complex cellular environment. In this study, we present an algorithm designed to extract motor step sizes and dwell times between steps from trajectories of motors or cargoes driven by motors in cells. The algorithm is based on finding patterns in the trajectory compatible with the behavior expected for a motor step, i.e., a region of confined motion followed by a jump in the position to another region of confined motion with similar characteristics to the previous one. We show that this algorithm allows the analysis of 2D trajectories even if they present complex motion patterns such as active transport interspersed with diffusion and does not require the assumption of a given step size or dwell period. The confidence on the step detection can be easily obtained and allows the evaluation of the confidence of the dwell and step size distributions. To illustrate the possible applications of this algorithm, we analyzed trajectories of myosin-V driven organelles in living cells.     

The diffusive search mechanism of processive myosin class-V motor involves directional steps along actin subunits

It is widely accepted that the vesicle-transporter myosin-V moves processively along F-actin with large steps of approximately 36 nm using a hand-over-hand mechanism. A key question is how does the rear head of two-headed myosin-V search for the forward actin target in the forward direction. Scanning probe nanometry was used to resolve this underlying search process, which was made possible by attaching the head to a relatively large probe. One-headed myosin-V undergoes directional diffusion with approximately 5.5 nm substeps to develop an average displacement of approximately 20 nm, which was independent of the neck length (2IQ and 6IQ motifs). Two-headed myosin-V showed several approximately 5.5 nm substeps within each processive approximately 36 nm step. These results suggest that the myosin-V head searches in the forward direction for the actin target using directional diffusion on the actin subunits according to a potential slope created along the actin helix.     

Force-dependent stepping kinetics of myosin-V

Myosin-V is a processive two-headed actin-based motor protein involved in many intracellular transport processes. A key question for understanding myosin-V function and the communication between its two heads is its behavior under load. Since in vivo myosin-V colocalizes with other much stronger motors like kinesins, its behavior under superstall forces is especially relevant. We used optical tweezers with a long-range force feedback to study myosin-V motion under controlled external forward and backward loads over its full run length. We find the mean step size remains constant at approximately 36 nm over a wide range of forces from 5 pN forward to 1.5 pN backward load. We also find two force-dependent transitions in the chemomechanical cycle. The slower ADP-release is rate limiting at low loads and depends only weakly on force. The faster rate depends more strongly on force. The stronger force dependence suggests this rate represents the diffusive search of the leading head for its binding site. In contrast to kinesin motors, myosin-V's run length is essentially independent of force between 5 pN of forward to 1.5 pN of backward load. At superstall forces of 5 pN, we observe continuous backward stepping of myosin-V, indicating that a force-driven reversal of the power stroke is possible.     

Mechanical Characterization of One-Headed Myosin-V Using Optical Tweezers

Class V myosin (myosin-V) is a cargo transporter that moves along an actin filament with large (∼36-nm) successive steps. It consists of two heads that each includes a motor domain and a long (23 nm) neck domain. One of the more popular models describing these steps, the hand-over-hand model, assumes the two-headed structure is imperative. However, we previously succeeded in observing successive large steps by one-headed myosin-V upon optimizing the angle of the acto-myosin interaction. In addition, it was reported that wild type myosin-VI and myosin-IX, both one-headed myosins, can also generate successive large steps. Here, we describe the mechanical properties (stepsize and stepping kinetics) of successive large steps by one-headed and two-headed myosin-Vs. This study shows that the stepsize and stepping kinetics of one-headed myosin-V are very similar to those of the two-headed one. However, there was a difference with regards to stability against load and the number of multisteps. One-headed myosin-V also showed unidirectional movement that like two-headed myosin-V required 3.5 k_BT from ATP hydrolysis. This value is also similar to that of smooth muscle myosin-II, a non-processive motor, suggesting the myosin family uses a common mechanism for stepping regardless of the steps being processive or non-processive. In this present paper, we conclude that one-headed myosin-V can produce successive large steps without following the hand-over-hand mechanism.     

Dynamics of myosin-V processivity

Myosin-V is an actin-associated processive molecular motor. Single molecule experiments revealed that myosin-V walks in a stepwise fashion with occasional backward steps. By combining the mechanical structure of the motor with the ATP hydrolysis kinetics, we construct a dynamical model that accounts for the stepwise processivity. The molecular properties of the protein chains connecting the myosin heads are important. A simple elastic model demonstrates that the stress transmitted from the leading head to the trailing head leads to net forward motion. The step-sizes are non-uniform. We also predict there are several substeps. The translational speed and step-size distributions are computed for several different conditions. The computed force-versus-velocity curve shows that under an external load, myosin-V slows down. However, the sizes of the steps remain the same.     

A kinetic model describing the processivity of myosin-V

The precise details of how myosin-V coordinates the biochemical reactions and mechanical motions of its two head elements to engineer effective processive molecular motion along actin filaments remain unresolved. We compare a quantitative kinetic model of the myosin-V walk, consisting of five basic states augmented by two further states to allow for futile hydrolysis and detachments, with experimental results for run lengths, velocities, and dwell times and their dependence on bulk nucleotide concentrations and external loads in both directions. The model reveals how myosin-V can use the internal strain in the molecule to synchronize the motion of the head elements. Estimates for the rate constants in the reaction cycle and the internal strain energy are obtained by a computational comparison scheme involving an extensive exploration of the large parameter space. This scheme exploits the fact that we have obtained analytic results for our reaction network, e.g., for the velocity but also the run length, diffusion constant, and fraction of backward steps. The agreement with experiment is often reasonable but some open problems are highlighted, in particular the inability of such a general model to reproduce the reported dependence of run length on ADP concentration. The novel way that our approach explores parameter space means that any confirmed discrepancies should give new insights into the reaction network model.     

Myosin-V makes two brownian 90 degrees rotations per 36-nm step

Myosin-V processively walks on actin filaments in a hand-over-hand fashion. The identical structures of the heads predict a symmetric hand-over-hand mechanism where regular, unidirectional rotation occurs during a 36-nm step. We investigated this by observing how fixed myosin-V rotates actin filaments. Actin filaments randomly rotated 90 degrees both clockwise and counter-clockwise during each step. Furthermore, ATP-dependent rotations were regularly followed by ATP-independent ones. Kinetic analysis indicated that the two 90 degrees rotations relate to the coordinated unbinding and rebinding of the heads with actin. We propose a 'brownian rotation hand-over-hand' model, in which myosin-V randomly rotates by thermally twisting its elastic neck domains during the 36-nm step. The brownian rotation may be advantageous for cargo transport through a crowded actin meshwork and for carrying cargoes reliably via multiple myosin-V molecules in the cell.     

Random Walk of Processive, Quantum Dot-Labeled Myosin Va Molecules within the Actin Cortex of COS-7 Cells

Myosin Va (myoVa) is an actin-based intracellular cargo transporter. In vitro experiments have established that a single myoVa moves processively along actin tracks, but less is known about how this motor operates within cells. Here we track the movement of a quantum dot (Qdot)-labeled myoVa HMM in COS-7 cells using total internal reflectance fluorescence microscopy. This labeling approach is unique in that it allows myoVa, instead of its cargo, to be tracked. Single-particle analysis showed short periods (≤0.5 s) of ATP-sensitive linear motion. The mean velocity of these trajectories was 604 nm/s and independent of the number of myoVa molecules attached to the Qdot. With high time (16.6 ms) and spatial (15 nm) resolution imaging, Qdot-labeled myoVa moved with sequential 75 nm steps per head, at a rate of 16 s⁻¹, similarly to myoVa in vitro. Monte Carlo modeling suggests that the random nature of the trajectories represents processive myoVa motors undergoing a random walk through the dense and randomly oriented cortical actin network.     

A simple kinetic model describes the processivity of myosin-v

Myosin-V is a motor protein responsible for organelle and vesicle transport in cells. Recent single-molecule experiments have shown that it is an efficient processive motor that walks along actin filaments taking steps of mean size close to 36 nm. A theoretical study of myosin-V motility is presented following an approach used successfully to analyze the dynamics of conventional kinesin but also taking some account of step-size variations. Much of the present experimental data for myosin-V can be well described by a two-state chemical kinetic model with three load-dependent rates. In addition, the analysis predicts the variation of the mean velocity and of the randomness-a quantitative measure of the stochastic deviations from uniform, constant-speed motion-with ATP concentration under both resisting and assisting loads, and indicates a substep of size d(0) approximately 13-14 nm (from the ATP-binding state) that appears to accord with independent observations.     

Short-range axonal/dendritic transport by myosin-V: A model for vesicle delivery to the synapse

Myosin-V is a versatile motor involved in short-range axonal/dendritic transport of vesicles in the actin-rich cortex and synaptic regions of nerve cells. It binds to several different kinds of neuronal vesicles by its globular tail domain but the mechanism by which it is recruited to these vesicles is not known. In this study, we used an in vitro motility assay derived from axoplasm of the squid giant axon to study the effects of the globular tail domain on the transport of neuronal vesicles. We found that the globular tail fragment of myosin-V inhibited actin-based vesicle transport by displacing native myosin-V and binding to vesicles. The globular tail domain pulled down kinesin, a known binding partner of myosin-V, in affinity isolation experiments. These data confirmed earlier evidence that kinesin and myosin-V interact to form a hetero-motor complex. The formation of a kinesin/myosin-V hetero-motor complex on vesicles is thought to facilitate the coordination of long-range movement on microtubules and short-range movement on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated. These results are the first demonstration that the recombinant tail of myosin-V inhibits vesicle transport in an in vitro motility assay. Future experiments are designed to determine the functional significance of the interaction between myosin-V and kinesin and to identify other proteins that bind to the globular tail domain of myosin-V.     

Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications

BACKGROUND: Cytoplasmic dynein is the molecular motor responsible for most retrograde microtubule-based vesicular transport. In vitro single-molecule experiments suggest that dynein function is not as robust as that of kinesin-1 or myosin-V because dynein moves only a limited distance (approximately 800 nm) before detaching and can exert a modest (approximately 1 pN) force. However, dynein-driven cargos in vivo move robustly over many microns and exert forces of multiple pN. To determine how to go from limited single-molecule function to robust in vivo transport, we began to build complexity in a controlled manner by using in vitro experiments. RESULTS: We show that a single cytoplasmic dynein motor frequently transitions into an off-pathway unproductive state that impairs net transport. Addition of a second (and/or third) dynein motor, so that cargos are moved by two (or three) motors rather than one, is sufficient to recover several properties of in vivo motion; such properties include long cargo travels, robust motion, and increased forces. Part of this improvement appears to arise from selective suppression of the unproductive state of dynein rather than from a fundamental change in dynein's mechanochemical cycle. CONCLUSIONS: Multiple dyneins working together suppress shortcomings of a single motor and generate robust motion under in vitro conditions. There appears to be no need for additional cofactors (e.g., dynactin) for this improvement. Because cargos are often driven by multiple dyneins in vivo, our results show that changing the number of dynein motors could allow modulation of dynein function from the mediocre single-dynein limit to robust in vivo-like dynein-driven motion.     

The force-velocity relationship for the actin-based motility of Listeria monocytogenes

The intracellular movement of the bacterial pathogen Listeria monocytogenes has helped identify key molecular constituents of actin-based motility (recent reviews ). However, biophysical as well as biochemical data are required to understand how these molecules generate the forces that extrude eukaryotic membranes. For molecular motors and for muscle, force-velocity curves have provided key biophysical data to distinguish between mechanistic theories. Here we manipulate and measure the viscoelastic properties of tissue extracts to provide the first force-velocity curve for Listeria monocytogenes. We find that the force-velocity relationship is highly curved, almost biphasic, suggesting a high cooperativity between biochemical catalysis and force generation. Using high-resolution motion tracking in low-noise extracts, we find long trajectories composed exclusively of molecular-sized steps. Robust statistics from these trajectories show a correlation between the duration of steps and macroscopic Listeria speed, but not between average step size and speed. Collectively, our data indicate how the molecular properties of the Listeria polymerization engine regulate speed, and that regulation occurs during molecular-scale pauses.     

Improved hidden Markov models for molecular motors, part 2: extensions and application to experimental data

Unbiased interpretation of noisy single molecular motor recordings remains a challenging task. To address this issue, we have developed robust algorithms based on hidden Markov models (HMMs) of motor proteins. The basic algorithm, called variable-stepsize HMM (VS-HMM), was introduced in the previous article. It improves on currently available Markov-model based techniques by allowing for arbitrary distributions of step sizes, and shows excellent convergence properties for the characterization of staircase motor timecourses in the presence of large measurement noise. In this article, we extend the VS-HMM framework for better performance with experimental data. The extended algorithm, variable-stepsize integrating-detector HMM (VSI-HMM) better models the data-acquisition process, and accounts for random baseline drifts. Further, as an extension, maximum a posteriori estimation is provided. When used as a blind step detector, the VSI-HMM outperforms conventional step detectors. The fidelity of the VSI-HMM is tested with simulations and is applied to in vitro myosin V data where a small 10 nm population of steps is identified. It is also applied to an in vivo recording of melanosome motion, where strong evidence is found for repeated, bidirectional steps smaller than 8 nm in size, implying that multiple motors simultaneously carry the cargo.     

Anomalous Dynamics of Melanosomes Driven by Myosin-V in Xenopus laevis Melanophores

The organization of the cytoplasm is regulated by molecular motors, which transport organelles and other cargoes along cytoskeleton tracks. In this work, we use single particle tracking to study the in vivo regulation of the transport driven by myosin-V along actin filaments in Xenopus laevis melanophores. Melanophores have pigment organelles or melanosomes, which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. We followed the motion of melanosomes in cells treated to depolymerize microtubules during aggregation and dispersion, focusing the analysis on the dynamics of these organelles in a time window not explored before to our knowledge. These data could not be explained by previous models that only consider active transport. We proposed a transport-diffusion model in which melanosomes may detach from actin tracks and reattach to nearby filaments to resume the active motion after a given time of diffusion. This model predicts that organelles spend ∼70% and 10% of the total time in active transport during dispersion and aggregation, respectively. Our results suggest that the transport along actin filaments and the switching from actin to microtubule networks are regulated by changes in the diffusion time between periods of active motion driven by myosin-V.     

Robust single-particle tracking in live-cell time-lapse sequences

Single-particle tracking (SPT) is often the rate-limiting step in live-cell imaging studies of subcellular dynamics. Here we present a tracking algorithm that addresses the principal challenges of SPT, namely high particle density, particle motion heterogeneity, temporary particle disappearance, and particle merging and splitting. The algorithm first links particles between consecutive frames and then links the resulting track segments into complete trajectories. Both steps are formulated as global combinatorial optimization problems whose solution identifies the overall most likely set of particle trajectories throughout a movie. Using this approach, we show that the GTPase dynamin differentially affects the kinetics of long- and short-lived endocytic structures and that the motion of CD36 receptors along cytoskeleton-mediated linear tracks increases their aggregation probability. Both applications indicate the requirement for robust and complete tracking of dense particle fields to dissect the mechanisms of receptor organization at the level of the plasma membrane.     

A hand-over-hand diffusing model for myosin-VI molecular motors

Single molecules of dimeric myosin-VI have been demonstrated to be able to move processively towards the pointed end of actin filament with a mean step size of approximately 36 nm. Here we present a hand-over-hand diffusing mechanism for this unidirectional movement. Based on this mechanism, its dynamical behaviors such as the step-size distribution, dwell-time distributions and mean dwell time at various ATP and ADP concentrations and under various loads are studied in detail. The calculated results show good agreement with previous experimental results. The processive movement of mutant myosin-V with its neck domains truncated to only one IQ motif can also be explained by using this hand-over-hand diffusing model.     

A cellular automaton model for the migration of glioma cells

We present a study of in vitro cell migration in two dimensions as a first step towards understanding the mechanisms governing the motility of glioma cells. Our study is based on a cellular automaton model which aims at reproducing the kinetics of a lump of glioma cells deposited on a substrate of collagen. The dynamical effects of cell attraction and motion inertia are introduced through adequate automaton rules. We compare the density profiles given by the model to those obtained experimentally. The result of the best fit indicates a substantial cell-cell attraction due to cell-cell communication through gap junctions (or chemotaxis) and negligible inertia effects during migration. Tracking of individual migrating cells indicates highly convoluted cell trajectories.     

Step Detection in Single-Molecule Real Time Trajectories Embedded in Correlated Noise

Single-molecule real time trajectories are embedded in high noise. To extract kinetic or dynamic information of the molecules from these trajectories often requires idealization of the data in steps and dwells. One major premise behind the existing single-molecule data analysis algorithms is the Gaussian ‘white’ noise, which displays no correlation in time and whose amplitude is independent on data sampling frequency. This so-called ‘white’ noise is widely assumed but its validity has not been critically evaluated. We show that correlated noise exists in single-molecule real time trajectories collected from optical tweezers. The assumption of white noise during analysis of these data can lead to serious over- or underestimation of the number of steps depending on the algorithms employed. We present a statistical method that quantitatively evaluates the structure of the underlying noise, takes the noise structure into account, and identifies steps and dwells in a single-molecule trajectory. Unlike existing data analysis algorithms, this method uses Generalized Least Squares (GLS) to detect steps and dwells. Under the GLS framework, the optimal number of steps is chosen using model selection criteria such as Bayesian Information Criterion (BIC). Comparison with existing step detection algorithms showed that this GLS method can detect step locations with highest accuracy in the presence of correlated noise. Because this method is automated, and directly works with high bandwidth data without pre-filtering or assumption of Gaussian noise, it may be broadly useful for analysis of single-molecule real time trajectories.     

Brain myosin-V, a calmodulin-carrying myosin, binds to calmodulin-dependent protein kinase II and activates its kinase activity.

Myosin-V, an unconventional myosin, has two notable structural features: (i) a regulatory neck domain having six IQ motifs that bind calmodulin and light chains, and (ii) a structurally distinct tail domain likely responsible for its specific intracellular interactions. Myosin-V copurifies with synaptic vesicles via its tail domain, which also is a substrate for calmodulin-dependent protein kinase II. We demonstrate here that myosin-V coimmunoprecipitates with CaM-kinase II from a Triton X-100-solubilized fraction of isolated nerve terminals. The purified proteins also coimmunoprecipitate from dilute solutions and bind in overlay experiments on Western blots. The binding region on myosin-V was mapped to its proximal and medial tail domains. Autophosphorylated CaM-kinase II binds to the tail domain of myosin-V with an apparent Kd of 7.7 nM. Surprisingly, myosin-V activates CaM-kinase II activity in a Ca2+-dependent manner, without the need for additional CaM. The apparent activation constants for the autophosphorylation of CaM-kinase II were 10 and 26 nM, respectively, for myosin-V versus CaM. The maximum incorporation of 32P into CaM-kinase II activated by myosin-V was twice that for CaM, suggesting that myosin-V binding to CaM-kinase II entails alterations in kinetic and/or phosphorylation site parameters. These data suggest that myosin-V, a calmodulin-carrying myosin, binds to and delivers CaM to CaM-kinase II, a calmodulin-dependent enzyme.