Myosin VI is an actin-based motor that moves backwards.

Myosins and kinesins are molecular motors that hydrolyse ATP to track along actin filaments and microtubules, respectively. Although the kinesin family includes motors that move towards either the plus or minus ends of microtubules, all characterized myosin motors move towards the barbed (+) end of actin filaments. Crystal structures of myosin II (refs 3-6) have shown that small movements within the myosin motor core are transmitted through the 'converter domain' to a 'lever arm' consisting of a light-chain-binding helix and associated light chains. The lever arm further amplifies the motions of the converter domain into large directed movements. Here we report that myosin VI, an unconventional myosin, moves towards the pointed (-) end of actin. We visualized the myosin VI construct bound to actin using cryo-electron microscopy and image analysis, and found that an ADP-mediated conformational change in the domain distal to the motor, a structure likely to be the effective lever arm, is in the opposite direction to that observed for other myosins. Thus, it appears that myosin VI achieves reverse-direction movement by rotating its lever arm in the opposite direction to conventional myosin lever arm movement.     

Molecular engineering of a backwards-moving myosin motor

All members of the diverse myosin superfamily have a highly conserved globular motor domain that contains the actin- and nucleotide-binding sites and produces force and movement. The light-chain-binding domain connects the motor domain to a variety of functionally specialized tail domains and amplifies small structural changes in the motor domain through rotation of a lever arm. Myosins move on polarized actin filaments either forwards to the barbed (+) or backwards to the pointed (-) end. Here, we describe the engineering of an artificial backwards-moving myosin from three pre-existing molecular building blocks. These blocks are: a forward-moving class I myosin motor domain, a directional inverter formed by a four-helix bundle segment of human guanylate-binding protein-1 and an artificial lever arm formed by two alpha-actinin repeats. Our results prove that reverse-direction movement of myosins can be achieved simply by rotating the direction of the lever arm 180 degrees.     

The structure of the myosin VI motor reveals the mechanism of directionality reversal

Here we solve a 2.4-A structure of a truncated version of the reverse-direction myosin motor, myosin VI, that contains the motor domain and binding sites for two calmodulin molecules. The structure reveals only minor differences in the motor domain from that in plus-end directed myosins, with the exception of two unique inserts. The first is near the nucleotide-binding pocket and alters the rates of nucleotide association and dissociation. The second unique insert forms an integral part of the myosin VI converter domain along with a calmodulin bound to a novel target motif within the insert. This serves to redirect the effective 'lever arm' of myosin VI, which includes a second calmodulin bound to an 'IQ motif', towards the pointed (minus) end of the actin filament. This repositioning largely accounts for the reverse directionality of this class of myosin motors. We propose a model incorporating a kinesin-like uncoupling/docking mechanism to provide a full explanation of the movements of myosin VI.     

A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd

Kinesins are microtubule-based motor proteins that power intracellular transport. Most kinesin motors, exemplified by Kinesin-1, move towards the microtubule plus end, and the structural changes that govern this directional preference have been described. By contrast, the nature and timing of the structural changes underlying the minus-end-directed motility of Kinesin-14 motors (such as Drosophila Ncd) are less well understood. Using cryo-electron microscopy, here we demonstrate that a coiled-coil mechanical element of microtubule-bound Ncd rotates approximately 70 degrees towards the minus end upon ATP binding. Extending or shortening this coiled coil increases or decreases velocity, respectively, without affecting ATPase activity. An unusual Ncd mutant that lacks directional preference shows unstable nucleotide-dependent conformations of its coiled coil, underscoring the role of this mechanical element in motility. These results show that the force-producing conformational change in Ncd occurs on ATP binding, as in other kinesins, but involves the swing of a lever-arm mechanical element similar to that described for myosins.     

Bidirectional movement of actin filaments along tracks of myosin heads

It is well established that muscle contraction results from the relative sliding of actin and myosin filaments. Both filaments have definite polarities and well-ordered structures. Thick filaments, however, are not vital for supporting movement in vitro. Previously we have demonstrated that actin filaments can move continuously on myosin fragments (subfragment-1 or heavy meromyosin (HMM] that are bound to a nitrocellulose surface. Here we report that actin filaments can move in opposite directions on tracks of myosin heads formed when actin filaments decorated with HMM are placed on a nitrocellulose surface. The actin filaments always move forward, frequently changing the direction of the movement, but never move backward reversing the polarity of the movement. The direction of movement is therefore determined by the polarity of the actin filament. These results indicate that myosin heads have considerable flexibility.     

The motor domain determines the large step of myosin-V.

Class-V myosin proceeds along actin filaments with large ( approximately 36 nm) steps. Myosin-V has two heads, each of which consists of a motor domain and a long (23 nm) neck domain. In accordance with the widely accepted lever-arm model, it was suggested that myosin-V steps to successive (36 nm) target zones along the actin helical repeat by tilting its long neck (lever-arm). To test this hypothesis, we measured the mechanical properties of single molecules of myosin-V truncation mutants with neck domains only one-sixth of the native length. Our results show that the processivity and step distance along actin are both similar to those of full-length myosin-V. Thus, the long neck domain is not essential for either the large steps or processivity of myosin-V. These results challenge the lever-arm model. We propose that the motor domain and/or the actomyosin interface enable myosin-V to produce large processive steps during translocation along actin.     

A mutant of the motor protein kinesin that moves in both directions on microtubules

Molecular motors move directionally to either the plus or the minus end of microtubules or actin filaments. Kinesin moves towards microtubule plus ends, whereas the kinesin-related Ncd motor moves to the minus ends. The 'neck'--the region between the stalk and motor domain--is required for Ncd to move to microtubule minus ends, but the mechanism underlying directional motor movement is not understood. Here we show that a single amino-acid change in the Ncd neck causes the motor to reverse directions and move with wild-type velocities towards the plus or minus end; thus, the neck is functional but directionality is defective. Mutation of a motor-core residue that touches the neck residue in crystal structures also results in movement in both directions, indicating that directed movement to the minus end requires interactions of the neck and motor core. Low-density laser-trap assays show that a conformational change or working stroke of the Ncd motor is directional and biased towards the minus end, whereas that of the neck mutant occurs in either direction. We conclude that the directional bias of the working stroke is dependent on neck/motor core interactions. Absence of these interactions removes directional constraints and permits movement in either direction.     

The cargo-binding domain regulates structure and activity of myosin 5

Myosin 5 is a two-headed motor protein that moves cargoes along actin filaments. Its tail ends in paired globular tail domains (GTDs) thought to bind cargo. At nanomolar calcium levels, actin-activated ATPase is low and the molecule is folded. Micromolar calcium concentrations activate ATPase and the molecule unfolds. Here we describe the structure of folded myosin and the GTD's role in regulating activity. Electron microscopy shows that the two heads lie either side of the tail, contacting the GTDs at a lobe of the motor domain (approximately Pro 117-Pro 137) that contains conserved acidic side chains, suggesting ionic interactions between motor domain and GTD. Myosin 5 heavy meromyosin, a constitutively active fragment lacking the GTDs, is inhibited and folded by a dimeric GST-GTD fusion protein. Motility assays reveal that at nanomolar calcium levels heavy meromyosin moves robustly on actin filaments whereas few myosins bind or move. These results combine to show that with no cargo, the GTDs bind in an intramolecular manner to the motor domains, producing an inhibited and compact structure that binds weakly to actin and allows the molecule to recycle towards new cargoes.     

Direct observation of motion of single F-actin filaments in the presence of myosin

Actin is found in almost all kinds of non-muscle cells where it is thought to have an important role in cell motility. A proper understanding of that role will only be possible when reliable in vitro systems are available for investigating the interaction of cellular actin and myosin. A start has been made on several systems, most recently by Sheetz and Spudich who demonstrated unidirectional movement of HMM-coated beads along F-actin cables on arrays of chloroplasts exposed by dissection of a Nitella cell. As an alternative approach, we report here the direct observation by fluorescence microscopy of the movements of single F-actin filaments interacting with soluble myosin fragments energized by Mg2+-ATP.     

Myosin head movements are synchronous with the elementary force-generating process in muscle.

Motor proteins such as myosin, dynein and kinesin use the free energy of ATP hydrolysis to produce force or motion, but despite recent progress their molecular mechanism is unknown. The best characterized system is the myosin motor which moves actin filaments in muscle. When an active muscle fibre is rapidly shortened the force first decreases, then partially recovers over the next few milliseconds. This elementary force-generating process is thought to be due to a structural 'working stroke' in the myosin head domain, although structural studies have not provided definitive support for this. X-ray diffraction has shown that shortening steps produce a large decrease in the intensity of the 14.5 nm reflection arising from the axial repeat of the myosin heads along the filaments. This was interpreted as a structural change at the end of the working stroke, but the techniques then available did not allow temporal resolution of the elementary force-generating process itself. Using improved measurement techniques, we show here that myosin heads move by about 10 nm with the same time course as the elementary force-generating process.     

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.     

Myosin-V is a processive actin-based motor.

Class-V myosins, one of 15 known classes of actin-based molecular motors, have been implicated in several forms of organelle transport, perhaps working with microtubule-based motors such as kinesin. Such movements may require a motor with mechanochemical properties distinct from those of myosin-II, which operates in large ensembles to drive high-speed motility as in muscle contraction. Based on its function and biochemistry, it has been suggested that myosin-V may be a processive motor like kinesin. Processivity means that the motor undergoes multiple catalytic cycles and coupled mechanical advances for each diffusional encounter with its track. This allows single motors to support movement of an organelle along its track. Here we provide direct evidence that myosin-V is indeed a processive actin-based motor that can move in large steps approximating the 36-nm pseudo-repeat of the actin filament.     

Formation of reverse rigor chevrons by myosin heads

The uniform angle and conformation of myosin subfragment 1 (S1) bound to actin filaments (F-actin) attest to the precise alignment and stereospecificity of the binding of these two contractile proteins. Because actin filaments are polar, myosin heads must swing or rotate about the head-tail junction in order to bind. Electron microscopy of isolated thick filaments and of myosin molecules suggests that the molecules are flexible, but myosin fragments and crossbridges have been reported not to interact with inappropriately oriented actin filaments. Here we describe myofibrillar defects engendered by a site-directed mutation within the flight-muscle-specific actin gene of the fruitfly Drosophila. The mutation apparently retards sarcomere assembly: peripheral thick and thin filaments are misregistered and not incorporated into the Z-line. Therefore, a myosin filament encounters thin filaments with the 'wrong' polarity. We show that myosin heads tethered in a single thick filament can bind with opposite rigor crossbridge angles to flanking thin filaments, which are apparently of opposite polarities. Preservation of identical actomyosin interfaces requires that sets of heads originating from opposite sides of the thick filament swivel 180 degrees relative to each other, implying that myosin crossbridges are as flexible as isolated molecules.     

Direct observation of the mechanochemical coupling in myosin Va during processive movement

Myosin Va transports intracellular cargoes along actin filaments in cells. This processive, two-headed motor takes multiple 36-nm steps in which the two heads swing forward alternately towards the barbed end of actin driven by ATP hydrolysis. The ability of myosin Va to move processively is a function of its long lever arm, the high duty ratio of its kinetic cycle and the gating of the kinetics between the two heads such that ADP release from the lead head is greatly retarded. Mechanical studies at the multiple- and the single-molecule level suggest that there is tight coupling (that is, one ATP is hydrolysed per power stroke), but this has not been directly demonstrated. We therefore investigated the coordination between the ATPase mechanism of the two heads of myosin Va and directly visualized the binding and dissociation of single fluorescently labelled nucleotide molecules, while simultaneously observing the stepping motion of the fluorescently labelled myosin Va as it moved along an actin filament. Here we show that preferential ADP dissociation from the trail head of mouse myosin Va is followed by ATP binding and a synchronous 36-nm step. Even at low ATP concentrations, the myosin Va molecule retained at least one nucleotide (ADP in the lead head position) when moving. Thus, we directly demonstrate tight coupling between myosin Va movement and the binding and dissociation of nucleotide by simultaneously imaging with near nanometre precision.     

Video imaging of walking myosin V by high-speed atomic force microscopy

The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative 'visual evidence' for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action.     

Movement of myosin-coated beads on oriented filaments reconstituted from purified actin

Although the biochemical properties of the actin/myosin interaction have been studied extensively using actin activation of myosin ATPase as an assay, until recently no well-defined assay has been available to measure the mechanical properties of ATP-dependent movement of myosin along actin filaments. The first direct measurements of the rate of myosin movement in vitro used a naturally occurring, biochemically ill-defined array of actin filaments from the alga Nitella. We report here the construction of an oriented array of filaments reconstituted from purified muscle actin and the use of this array in a biochemically defined quantitative assay for the directed movement of myosin-coated polystyrene beads. We demonstrate for the first time that actin alone, linked to a substratum by a protein anchor, is sufficient to support movement of myosin at rates consistent with the speeds of muscle contraction and other forms of cell motility.     

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.     

Propulsion of organelles isolated from Acanthamoeba along actin filaments by myosin-I

Eukaryotic cells are dependent on their ability to translocate membraneous elements about the cytoplasm. In many cells long translocations of organelles are associated with microtubules. In other cases, such as the rapid cytoplasmic streaming in some algae, organelles appear to be propelled along actin filaments. It has been assumed, but not proven, that myosin produces these movements. We have tested vesicles from another eukaryotic cell for their ability to move on the exposed actin bundles of Nitella as an indiction that actin-based organelle movements may be a general property of cells. We found that organelles from Acanthamoeba castellanii can move along Nitella actin filaments. Here, we report two different experiments indicating that the single-headed non-polymerizable myosin isozyme myosin-I is responsible for this organelle motility. First, monoclonal antibodies to myosin-I inhibit movement, but antibodies that inhibit double-headed myosin-II do not. Second, approximately 20% of the myosin-I in homogenates co-migrates with motile vesicles during Percoll density-gradient ultracentrifugation. This is the first indication of a role for myosin-I within the cell and supports the suggestion of Albanesi et al. that myosin-I moves vesicles in this way.     

Retrograde transport by the microtubule-associated protein MAP 1C

Microtubules are involved in several forms of intracellular motility, including mitosis and organelle movement. Fast axonal transport is a highly ordered form of organelle motility that operates in both the anterograde (outwards from the cell body) and retrograde (from the periphery towards the cell body) direction. Similar microtubule-associated movement is observed in non-neuronal cells, and might be involved in secretion, endocytosis and the positioning of organelles within the cell. Kinesin is a mechanochemical protein that produces force along microtubules in an anterograde direction. We recently found that the brain microtubule-associated protein MAP 1C (ref. 7) is a microtubule-activated ATPase and, like kinesin, can translocate microtubules in an in vitro assay for microtubule-associated motility. MAP 1C seemed to be related to the ciliary and flagellar ATPase, dynein, which is thought to produce force in a direction opposite to that observed for kinesin. Here we report that MAP 1C, in fact, acts in a direction opposite to kinesin, and has the properties of a retrograde translocator.     

Regulation of non-muscle myosin assembly by calmodulin-dependent light chain kinase

The presence of actin and myosin in non-muscle cells suggests that they may be involved in a wide range of cellular contractile activities. The generally accepted view is that interaction between actin and myosin in these cells and in vertebrate smooth muscle, is regulated by the level of phosphorylation of the 20,000-molecular weight (MW) light chain. In the absence of calcium, this light chain is not phosphorylated and the myosin cannot interact with actin. Calcium activates a specific calmodulin-dependent kinase which phosphorylates the light chain, initiating actin-myosin interaction. Although most studies on the role of phosphorylation have concentration on the regulation of actin-activated myosin Mg-ATPase activity, phosphorylation of the light chain also seems to control the assembly of smooth muscle myosin into filaments. Using purified smooth muscle light chain kinase, we have confirmed this observation. We report here studies of myosins isolated from the two non-muscle sources, thymus cells and platelets. We observed that these myosins are assembled into filaments at physiological ionic strength and Mg-ATP concentrations, only when the 20,000-MW light chain is phosphorylated.