Myosin VI regulates actin structure specialization through conserved cargo-binding domain sites

Actin structures are often stable, remaining unchanged in organization for the lifetime of a differentiated cell. Little is known about stable actin structure formation, organization, or maintenance. During Drosophila spermatid individualization, long-lived actin cones mediate cellular remodeling. Myosin VI is necessary for building the dense meshwork at the cones' fronts. We test several ideas for myosin VI's mechanism of action using domain deletions or site-specific mutations of myosin VI. The head (motor) and globular tail (cargo-binding) domains were both needed for localization at the cone front and dense meshwork formation. Several conserved partner-binding sites in the globular tail previously identified in vertebrate myosin VI were critical for function in cones. Localization and promotion of proper actin organization were separable properties of myosin VI. A vertebrate myosin VI was able to localize and function, indicating that functional properties are conserved. Our data eliminate several models for myosin VI's mechanism of action and suggest its role is controlling organization and action of actin assembly regulators through interactions at conserved sites. The Drosophila orthologues of interaction partners previously identified for vertebrate myosin VI are likely not required, indicating novel partners mediate this effect. These data demonstrate that generating an organized and functional actin structure in this cell requires multiple activities coordinated by myosin VI.     

Myosin V is a left-handed spiral motor on the right-handed actin helix

Myosin V is a two-headed, actin-based molecular motor implicated in organelle transport. Previously, a single myosin V molecule has been shown to move processively along an actin filament in discrete approximately 36 nm steps. However, 36 nm is the helical repeat length of actin, and the geometry of the previous experiments may have forced the heads to bind to, or halt at, sites on one side of actin that are separated by 36 nm. To observe unconstrained motion, we suspended an actin filament in solution and attached a single myosin V molecule carrying a bead duplex. The duplex moved as a left-handed spiral around the filament, disregarding the right-handed actin helix. Our results indicate a stepwise walking mechanism in which myosin V positions and orients the unbound head such that the head will land at the 11th or 13th actin subunit on the opposing strand of the actin double helix.     

Sequence and phylogenetic analysis of squid myosin-V: a vesicle motor in nerve cells

We have shown that vesicles in the axoplasm of the squid giant axon move on actin filaments and that movement is inhibited by myosin V-specific antibodies [Tabb et al., 1998]. In the study reported in this article, experiments were performed to clone and sequence the cDNA for squid brain myosin V. Five proteolytic fragments of purified squid brain myosin V were analyzed by direct protein sequencing [Tabb et al., 1998]. Based on this sequence information, degenerate primers were constructed and used to isolate cDNA clones by PCR. Five clones, representing overlapping segments of the gene, were sequenced. The sequence data and the previous biochemical characterization of the molecule support the classification of this vesicle-associated myosin as a member of the class V myosins. Motif analysis of the head, neck, and tail domains revealed that squid MyoV has consensus sequences for all the motifs found in vertebrate members of the myosin V family of motor proteins. A phylogenetic tree was constructed from a sequence alignment by the neighbor-joining method, using Megalign (DNAStar, Madison, WI); the resulting phylogenetic tree showed that squid MyoV is more closely related to vertebrate MyoV (mouse dilute, chicken dilute, rat myr6, and human myo5a) than Drosophila and yeast (myo2, and myo4) myosins V. These new data on the phylogenetic relationships of squid myosin V to vertebrate myosin V strengthens the argument that myosin V functions as a vesicle motor in vertebrate neurons.     

Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.     

Human myosin Vc is a low duty ratio nonprocessive motor

There are three distinct members of the myosin V family in vertebrates, and each isoform is involved in different membrane trafficking pathways. Both myosin Va and Vb have demonstrated that they are high duty ratio motors that are consistent with the processive nature of these motors. Here we report that the ATPase cycle mechanism of the single-headed construct of myosin Vc is quite different from those of other vertebrate myosin V isoforms. K(ATPase) of the actin-activated ATPase was 62 microm, which is much higher than that of myosin Va ( approximately 1 mum). The rate of ADP release from actomyosin Vc was 12.7 s(-1), which was 2 times greater than the entire ATPase cycle rate, 6.5 s(-1). P(i) burst size was 0.31, indicating that the equilibrium of the ATP hydrolysis step is shifted to the prehydrolysis form. Our kinetic model, based on all kinetic data we determined in this study, suggests that myosin Vc spends the majority of the ATPase cycle time in the weak actin binding state in contrast to myosin Va and Vb. Consistently, the two-headed myosin Vc construct did not show processive movement in total internal reflection fluorescence microscope analysis, demonstrating that myosin Vc is a nonprocessive motor. Our findings suggest that myosin Vc fulfills its function as a cargo transporter by different mechanisms from other myosin V isoforms.     

The UCS domain protein She4p binds to myosin motor domains and is essential for class I and class V myosin function

BACKGROUND: Myosins are motor proteins involved in processes like cell motility, vesicle transport, or cytokinesis. In a variety of organisms, a novel group of proteins forming the UCS (UNC-45/CRO1/SHE4) domain-containing family are essential for proper myosin function. The Saccharomyces cerevisae UCS domain protein She4p is involved in two myosin-requiring events, endocytosis and mRNA localization. RESULTS: In contrast to UCS domain proteins from other organisms that interact with class II myosins, we demonstrate that She4p associates with yeast class I and class V myosins. She4p binds to motor domains of class V myosin Myo4p and class I myosin Myo5p, and this binding depends on She4p's UCS domain. In vivo, She4p is essential for the function and localization of Myo3p, Myo4p, and Myo5p (but not of Myo2p) and for colocalization of class I myosins with cortical actin patches. In vitro, She4p stimulates binding of Myo5p to filamentous actin. Wild-type She4p, but not a mutant lacking the UCS domain, accumulates in a cap-like structure at the bud tip. This localization requires Myo2p and actin, suggesting a Myo2-dependent mechanism by which She4p is targeted to the bud cap. Localization of She4p is essential for proper positioning and myosin-actin association of cortical Myo5p. CONCLUSIONS: Our results suggest that She4p is a novel myosin motor domain binding protein and operates as a localized regulator of myosin function of class I and likely class V myosins.     

Magnesium regulates ADP dissociation from myosin V

Processivity in myosin V is mediated through the mechanical strain that results when both heads bind strongly to an actin filament, and this strain regulates the timing of ADP release. However, what is not known is which steps that lead to ADP release are affected by this mechanical strain. Answering this question will require determining which of the several potential pathways myosin V takes in the process of ADP release and how actin influences the kinetics of these pathways. We have addressed this issue by examining how magnesium regulates the kinetics of ADP release from myosin V and actomyosin V. Our data support a model in which actin accelerates the release of ADP from myosin V by reducing the magnesium affinity of a myosin V-MgADP intermediate. This is likely a consequence of the structural changes that actin induces in myosin to release phosphate. This effect on magnesium affinity provides a plausible explanation for how mechanical strain can alter this actin-induced acceleration. For actomyosin V, magnesium release follows phosphate release and precedes ADP release. Increasing magnesium concentration to within the physiological range would thus slow both the ATPase activity and the velocity of movement of this motor.     

The structural basis of myosin V processive movement as revealed by electron cryomicroscopy

The processive motor myosin V has a relatively high affinity for actin in the presence of ATP and, thus, offers the unique opportunity to visualize some of the weaker, hitherto inaccessible, actin bound states of the ATPase cycle. Here, electron cryomicroscopy together with computer-based docking of crystal structures into three-dimensional (3D) reconstructions provide the atomic models of myosin V in both weak and strong actin bound states. One structure shows that ATP binding opens the long cleft dividing the actin binding region of the motor domain, thus destroying the strong binding actomyosin interface while rearranging loop 2 as a tether. Nucleotide analogs showed a second new state in which the lever arm points upward, in a prepower-stroke configuration (lever arm up) bound to actin before phosphate release. Our findings reveal how the structural elements of myosin V work together to allow myosin V to step along actin for multiple ATPase cycles without dissociating.     

ADP inhibition of myosin V ATPase activity

The kinetic mechanism of myosin V is of great interest because recent evidence indicates that the two-headed myosin V molecule functions as a processive motor, i.e., myosin V is capable of moving along an actin filament for many catalytic cycles of the motor without dissociating. Three recent publications assessing the kinetics of single-headed myosin V provide different conclusions regarding the mechanism, particularly the rate-limiting step of the cycle. One study (, Proc. Natl. Acad. Sci. USA. 96:13726-13731) identifies ADP release as the rate-limiting step and provides a kinetic explanation for myosin V processivity. The others (, J. Biol. Chem. 274:27448-27456;, J. Biol. Chem. 275:4329-4335) do not identify the rate-limiting step but conclude that it is not ADP release. We show experimental and simulated data demonstrating that the inconsistencies in the reports may be due to difficulties in the measurement of the steady-state ATPase rate. Under standard assay conditions, ADP competes with ATP, resulting in product inhibition of the ATPase rate. This presents technical problems in analyzing and interpreting the kinetics of myosin V and likely of other members of the myosin family with high ADP affinities.     

Walking with myosin V

The cytoplasm of cells is teaming with vesicles and other cargo that are moving along tracks of microtubules or actin filaments, powered by myosins, kinesins and dyneins. Myosin V has been implicated in several types of intracellular transport. The mechanism by which myosin V moves processively along actin filaments has been the subject of many biophysical and biochemical studies and a consensus is starting to emerge about how this minute molecular motor operates.     

Myosin IXb is a single-headed minus-end-directed processive motor

Myosin is an actin-based molecular motor that constitutes a diverse superfamily. In contrast to conventional myosin, which binds to actin for only a short time during cross-bridge cycling, recent studies have demonstrated that class V myosin moves along actin filaments for a long distance without dissociating. This would make it suitable for supporting cargo movement in cells. Because myosin V has a two-headed structure with an expanded neck domain, it has been postulated to 'walk' along the 36-nm helical repeat of the actin filament, with one head attached to the actin and leading the other head to the neighbouring helical pitch. Here, we report that myosin IXb, a single-headed myosin, moves processively on actin filaments. Furthermore, we found that myosin IXb is a minus-end-directed motor. In addition to class VI myosin, this is the first myosin superfamily member identified that moves in the reverse direction. The processive movement of the single-headed myosin IXb cannot be explained by a 'hand-over-hand' mechanism. This suggests that an alternative mechanism must be operating for the processive movement of single-headed myosin IXb.     

Engineering the processive run length of Myosin V

The processive motor myosin V has a high affinity for actin in the weak binding states when compared with non-processive myosins. Here we test whether this feature is essential for myosin V to walk processively along an actin filament. The net charge of loop 2, a surface loop implicated in the initial weak binding between myosin and actin, was increased or decreased to correspondingly change the affinity of myosin V for actin in the weak binding state, without changing the velocity of movement. Processive run lengths of single molecules were determined by total internal reflection fluorescence microscopy. Reducing the net positive charge of loop 2 significantly decreased both the affinity of myosin V for actin and the processive run length. Conversely, the addition of positive charge to loop 2 increased actin affinity and processive run length. We hypothesize that a high affinity for actin allows the detached head of a stepping myosin V to find its next actin binding site more quickly, thus decreasing the probability of run termination.     

Actin and light chain isoform dependence of myosin V kinetics

Recent studies on myosin V report a number of kinetic differences that may be attributed to the different heavy chain (chicken vs mouse) and light chain (essential light chains vs calmodulin) isoforms used. Understanding the extent to which individual light chain isoforms contribute to the kinetic behavior of myosin V is of critical importance, since it is unclear which light chains are bound to myosin V in cells. In addition, all studies to date have used alpha-skeletal muscle actin, whereas myosin V is in nonmuscle cells expressing beta- and gamma-actin. Therefore, we characterized the actin and light chain dependence of single-headed myosin V kinetics. The maximum actin-activated steady-state ATPase rate (V(max)) of a myosin V construct consisting of the motor domain and first light chain binding domain is the same when either of two essential light chain isoforms or calmodulin is bound. However, with bound calmodulin, the K(ATPase) is significantly higher and there is a reduction in the rate and equilibrium constants for ATP hydrolysis, indicating that the essential light chain favors formation of the M. ADP.P(i) state. No kinetic parameters of myosin V are strongly influenced by the actin isoform. ADP release from the actin-myosin complex is the rate-limiting step in the ATPase cycle with all actin and light chain isoforms. We postulate that although there are significant light-chain-dependent alterations in the kinetics that could affect myosin V processivity in in vitro assays, these differences likely are minimized under physiological conditions.     

How myosin VI coordinates its heads during processive movement

A processive molecular motor must coordinate the enzymatic state of its two catalytic domains in order to prevent premature detachment from its track. For myosin V, internal strain produced when both heads of are attached to an actin track prevents completion of the lever arm swing of the lead head and blocks ADP release. However, this mechanism cannot work for myosin VI, since its lever arm positions are reversed. Here, we demonstrate that myosin VI gating is achieved instead by blocking ATP binding to the lead head once it has released its ADP. The structural basis for this unique gating mechanism involves an insert near the nucleotide binding pocket that is found only in class VI myosin. Reverse strain greatly favors binding of ADP to the lead head, which makes it possible for myosin VI to function as a processive transporter as well as an actin-based anchor. While this mechanism is unlike that of any other myosin superfamily member, it bears remarkable similarities to that of another processive motor from a different superfamily--kinesin I.     

Myosin V motor proteins: marching stepwise towards a mechanism

Mammalian myosin V motors transport cargo processively along actin filaments. Recent biophysical and structural studies have led to a detailed understanding of the mechanism of myosin V, making it perhaps the best understood cytoskeletal motor. In addition to describing the mechanism, this review will illustrate how "dynamic" single molecule measurements can synergize with "static" protein structural studies to produce amazingly clear information on the workings of a nanometer-scale machine.     

A model of myosin V processivity

Cytoplasmic transport is mediated by a group of molecular motors that typically work in isolation, under conditions where they must move their cargos long distances without dissociating from their tracks. This processive behavior requires specific adaptations of motor enzymology to meet these unique physiologic demands. One of these involves the ability of the two heads of a processive motor to communicate their structural states to each other. In this study, we examine a processive motor from the myosin superfamily myosin V. We have measured the kinetics of nucleotide release, of phosphate release, and of the weak-to-strong transition, as this motor interacts with actin, and we have used these studies to develop a model of how myosin V functions as a transport motor. Surprisingly, both heads release phosphate rapidly upon the initial encounter with an actin filament, suggesting that there is little or no intramolecular strain associated with this step. However, ADP release can be affected by both forward and rearward strain, and under steady-state conditions it is essentially prevented in the lead head until the rear head detaches. Many of these features are remarkably like those underlying the processive movement of kinesin on microtubules, supporting our hypothesis that different molecular motors satisfy the requirement for processive movement in similar ways, regardless of their particular family of origin.     

Step-size is determined by neck length in myosin V

The highly processive motor, myosin V, has an extremely long neck containing six calmodulin-binding IQ motifs that allows it to take multiple 36 nm steps corresponding to the pseudo-repeat of actin. To further investigate how myosin V moves processively on actin filaments, we altered the length of the neck by adding or deleting IQ motifs in myosin constructs lacking the globular tail domain. These myosin V IQ mutants were fluorescently labeled by exchange of a single Cy3-labeled calmodulin into the neck region of one head. We measured the step-size of these individual IQ mutants with nanometer precision and subsecond resolution using FIONA. The step-size was proportional to neck length for constructs containing 2, 4, 6, and 8 IQ motifs, providing strong support for the swinging lever-arm model of myosin motility. In addition, the kinetics of stepping provided additional support for the hand-over-hand model whereby the two heads alternately assume the leading position. Interestingly, the 8IQ myosin V mutant gave a broad distribution of step-sizes with multiple peaks, suggesting that this mutant has many choices of binding sites on an actin filament. These data demonstrate that the step-size of myosin V is affected by the length of its neck and is not solely determined by the pseudo-repeat of the actin filament.     

Functional role of loop 2 in myosin V

Myosin V is molecular motor that is capable of moving processively along actin filaments. The kinetics of monomeric myosin V containing a single IQ domain (MV 1IQ) differ from nonprocessive myosin II in that actin affinity is higher, phosphate release is extremely rapid, and ADP release is rate-limiting. We generated two mutants of myosin V by altering loop 2, a surface loop in the actin-binding region thought to alter actin affinity and phosphate release in myosin II, to determine the role that this loop plays in the kinetic tuning of myosin V. The loop 2 mutants altered the apparent affinity for actin (K(ATPase)) without altering the maximum ATPase rate (V(MAX)). Transient kinetic analysis determined that the rate of binding to actin, as well as the affinity for actin, was dependent on the net positive charge of loop 2, while other steps in the ATPase cycle were unchanged. The maximum rate of phosphate release was unchanged, but the affinity for actin in the M.ADP.Pi-state was dramatically altered by the mutations in loop 2. Thus, loop 2 is important for allowing myosin V to bind to actin with a relatively high affinity in the weak binding states but does not play a direct role in the product release steps. The ability to maintain a high affinity for actin in the weak binding states may prevent diffusion away from the actin filament and increase the degree of processive motion of myosin V.     

Kinetic analysis of Drosophila muscle myosin isoforms suggests a novel mode of mechanochemical coupling

The molecular mechanism of myosin function was addressed by measuring transient kinetic parameters of naturally occurring and chimeric Drosophila muscle myosin isoforms. We assessed the native embryonic isoform, the native indirect flight muscle isoform, and two chimeric isoforms containing converter domains exchanged between the indirect flight muscle and embryonic isoforms. Myosin was purified from the indirect flight muscles of transgenic flies, and S1 was produced by alpha-chymotryptic digestion. Previous studies in vertebrate and scallop myosins have shown a correlation between actin filament velocity in motility assays and cross-bridge detachment rate, specifically the rate of ADP release. In contrast, our study showed no correlation between the detachment rate and actin filament velocity in Drosophila myosin isoforms and further that the converter domain does not significantly influence the biochemical kinetics governing the detachment of myosin from actin. We suggest that evolutionary pressure on a single muscle myosin gene may maintain a fast detachment rate in all isoforms. As a result, the attachment rate and completion of the power stroke or the equilibrium between actin.myosin.ADP states may define actin filament velocity for these myosin isoforms.     

Myosin VI stabilizes an actin network during Drosophila spermatid individualization

Here, we demonstrate a new function of myosin VI using observations of Drosophila spermatid individualization in vivo. We find that myosin VI stabilizes a branched actin network in actin structures (cones) that mediate the separation of the syncytial spermatids. In a myosin VI mutant, the cones do not accumulate F-actin during cone movement, whereas overexpression of myosin VI leads to bigger cones with more F-actin. Myosin subfragment 1-fragment decoration demonstrated that the actin cone is made up of two regions: a dense meshwork at the front and parallel bundles at the rear. The majority of the actin filaments were oriented with their pointed ends facing in the direction of cone movement. Our data also demonstrate that myosin VI binds to the cone front using its motor domain. Fluorescence recovery after photobleach experiments using green fluorescent protein-myosin VI revealed that myosin VI remains bound to F-actin for minutes, suggesting its role is tethering, rather than transporting cargo. We hypothesize that myosin VI protects the actin cone structure either by cross-linking actin filaments or anchoring regulatory molecules at the cone front. These observations uncover a novel mechanism mediated by myosin VI for stabilizing long-lived actin structures in cells.