Myosin V, a double-headed molecular motor, transports organelles within cells by walking processively along actin, a process that requires coordination between the heads. To understand the mechanism underlying this coordination, processive runs of single myosin V molecules were perturbed by varying nucleotide content. Contrary to current views, our results show that the two heads of a myosin V molecule communicate, not through any one mechanism but through an elaborate system of cooperative mechanisms involving multiple kinetic pathways. These mechanisms introduce redundancy and safeguards that ensure robust processivity under differing physiologic demands.A s a processive motor, a myosin V molecule carries its intracellular cargo for long distances along an actin track, taking multiple steps before detaching and deriving the energy for each step from the hydrolysis of ATP (1-3). Determining the mechanism of this processive movement is essential for explaining diseases such as Griscelli syndrome in which a mutation to human myosin Va leads to hypopigmentation and severe neurological impairment (4, 5). Each of the two heads of myosin V catalyzes the hydrolysis of ATP and after release of hydrolysis products (P i and ADP) generates motion with a rotation of its long lever arm (6-8). When both heads function together, the myosin V molecule walks along actin in a hand-over-hand fashion, taking 36-nm strides (7,(9)(10)(11)(12). One of the most critical and intriguing questions, however, remains unanswered. How do the two heads coordinate their biochemical and mechanical cycles to maintain processive movement? Solution kinetic studies to date have focused only on single-headed myosin V constructs, from which coordination between the heads can only be inferred (13-15). Therefore, we expressed double-headed, heavy meromyosin V molecules with a C-terminal yellow fluorescent protein (YFP-HMM M5 ) such that processive movement of individual myosin V molecules could be visualized by total internal reflectance fluorescence (TIRF) microscopy (16) and then described in terms of run length and velocity (17). By perturbing the biochemical cycle through addition of ATP, ADP, or P i , we assessed the impact of these ligands on myosin V processivity, identified the myosin state from which a processive run most likely terminates, and showed that the two heads of myosin V are coordinated to generate processive movement through an elaborate system of cooperative mechanisms. Methods YFP-HMMM5 Expression. YFP was cloned onto the C terminus of HMM M5 . The final construct contains the first 1,098 amino acids (G1098) of murine myosin V HMM, followed by a linker region coding for the amino acids VTGS, followed by YFP and a FLAG epitope for purification. Sf9 cells were coinfected with two recombinant viruses, one encoding for the myosin V heavy chain and one for calmodulin. The calmodulin was a mutant deficient in calcium binding (CaM⌬all) to ensure complete occupancy of all the IQ motifs (18). After 72 h of infection, cells were pelleted a...

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Unconventional myosin V (myoV) is an actin-based molecular motor that has a key function in organelle and mRNA transport, as well as in membrane trafficking. MyoV was the first member of the myosin superfamily shown to be processive, meaning that a single motor protein can 'walk' hand-over-hand along an actin filament for many steps before detaching. Full-length myoV has a low actin-activated MgATPase activity at low [Ca2+], whereas expressed constructs lacking the cargo-binding domain have a high activity regardless of [Ca2+] (refs 5-7). Hydrodynamic data and electron micrographs indicate that the active state is extended, whereas the inactive state is compact. Here we show the first three-dimensional structure of the myoV inactive state. Each myoV molecule consists of two heads that contain an amino-terminal motor domain followed by a lever arm that binds six calmodulins. The heads are followed by a coiled-coil dimerization domain (S2) and a carboxy-terminal globular cargo-binding domain. In the inactive structure, bending of myoV at the head-S2 junction places the cargo-binding domain near the motor domain's ATP-binding pocket, indicating that ATPase inhibition might occur through decreased rates of nucleotide exchange. The actin-binding interfaces are unobstructed, and the lever arm is oriented in a position typical of strong actin-binding states. This structure indicates that motor recycling after cargo delivery might occur through transport on actively treadmilling actin filaments rather than by diffusion.

Plasmodium parasites are obligate intracellular protozoa and causative agents of malaria, responsible for half a million deaths each year. The lifecycle progression of the parasite is reliant on cell motility, a process driven by myosin A, an unconventional single-headed class XIV molecular motor. Here we demonstrate that myosin A from Plasmodium falciparum (PfMyoA) is critical for red blood cell invasion. Further, using a combination of X-ray crystallography, kinetics, and in vitro motility assays, we elucidate the non-canonical interactions that drive this motor’s function. We show that PfMyoA motor properties are tuned by heavy chain phosphorylation (Ser19), with unphosphorylated PfMyoA exhibiting enhanced ensemble force generation at the expense of speed. Regulated phosphorylation may therefore optimize PfMyoA for enhanced force generation during parasite invasion or for fast motility during dissemination. The three PfMyoA crystallographic structures presented here provide a blueprint for discovery of specific inhibitors designed to prevent parasite infection.

Certain types of intracellular organelle transport to the cell periphery are thought to involve long-range movement on microtubules by kinesin with subsequent handoff to vertebrate myosin Va (myoVa) for local delivery on actin tracks. This process may involve direct interactions between these two processive motors. Here we demonstrate using single molecule in vitro techniques that myoVa is flexible enough to effectively maneuver its way through actin filament intersections and Arp2/3 branches. In addition, myoVa surprisingly undergoes a one-dimensional diffusive search along microtubules, which may allow it to scan efficiently for kinesin and/or its cargo. These features of myoVa may help ensure efficient cargo delivery from the cell center to the periphery.cytoskeleton ͉ molecular motor ͉ motility ͉ processivity C oordinated organelle transport along certain secretory pathways starts with kinesin-powered movement on microtubules and finishes with vertebrate myosin Va (myoVa)-based motility on actin (1-4). This trip requires that both motors be present when a microtubule-actin intersection is encountered, but how these two motors find one another or the cargo they share is still unknown. Another challenge is to understand how cargo is delivered to its destination by way of the complex cytoskeletal network. Vertebrate myoVa is a processive motor that can travel long distances along its actin track (5). However, the cytoskeletal intersections that myoVa must encounter along its journey may present a physical barrier to forward motion or an alternate path to its final destination.To investigate this issue from myoVa's point of view, we observed single myoVa molecules labeled with highly photostable quantum dots (Qdots) in an objective-type total internal reflectance microscope (6) as they maneuvered through a model of cytoskeletal intersections created on a microscope coverslip coated with either actin filaments (Fig. 1A) or actin filaments and microtubules (Fig. 1B). In this model system, actin-actin intersections could inhibit or alter myoVa's direction of travel while actin-microtubule intersections should act as a hurdle to myoVa's processive movement. Given myoVa's ability to take 72-nm steps as it walks processively along actin filaments in a handover-hand fashion (6-9) and its inherent flexibility (10, 11), we have observed that myoVa can easily maneuver past actin filament intersections. In contrast, when encountering a microtubule, myoVa cannot step over this physical barrier, but surprisingly can step onto the microtubule and begin a onedimensional diffusive search. Thus, myoVa has evolved to handle the challenges of the cytoskeletal network and through its interaction with the microtubule can effectively scan for its transport partner, kinesin, and/or its cargo. Results and Discussion MyoVa Dynamics at Actin Filament Intersections.To determine what happens when myoVa encounters an overlapping actin filament, we adhered Alexa Fluor 660 phalloidin-labeled actin filaments to the coverslip, followed by TRITC...

The double-headed myosin V molecular motor carries intracellular cargo processively along actin tracks in a hand-over-hand manner. To test this hypothesis at the molecular level, we observed single myosin V molecules that were differentially labeled with quantum dots having different emission spectra so that the position of each head could be identified with approximately 6-nm resolution in a total internal reflectance microscope. With this approach, the individual heads of a single myosin V molecule were observed taking 72-nm steps as they alternated positions on the actin filament during processive movement. In addition, the heads were separated by 36 nm during pauses in motion, suggesting attachment to actin along its helical repeat. The 36-nm interhead spacing, the 72-nm step size, and the observation that heads alternate between leading and trailing positions on actin are obvious predictions of the hand-over-hand model, thus confirming myosin V's mode of walking along an actin filament.

An expressed, monomeric murine myosin V construct composed of the motor domain and two calmodulinbinding IQ motifs (MD(2IQ)) was used to assess the regulatory and kinetic properties of this unconventional myosin. In EGTA, the actin-activated ATPase activity of MD(2IQ) was 7.4 ؎ 1.6 s ؊1 with a K app of ϳ1 M (37°C), and the velocity of actin movement was ϳ0.3 m/s (30°C). Calcium inhibited both of these activities, but the addition of calmodulin restored the values to ϳ70% of control, indicating that calmodulin dissociation caused inhibition. In contrast to myosin II, MD(2IQ) is highly associated with actin at physiological ionic strength in the presence of ATP, but the motor is in a weakly bound conformation based on the pyrene-actin signal. The rate of dissociation of acto-MD(2IQ) by ATP is fast (>850 s ؊1 ), and ATP hydrolysis occurs at ϳ200 s ؊1 . The affinity of acto-MD(2IQ) for ADP is somewhat higher than that of smooth S1, and ADP dissociates more slowly. Actin does not cause a large increase in the rate of ADP release, nor does the presence of ADP appreciably alter the affinity of MD(2IQ) for actin. These kinetic data suggest that monomeric myosin V is not processive.Conventional muscle myosin II polymerizes into filaments and is designed to interact with actin as part of an ensemble, and the kinetic properties of myosins isolated from these tissues reflect this role. Their so-called "duty cycle," i.e. the length of time the myosin spends in a force-or motion-producing state, is relatively low compared with the overall cycle time determined by the ATPase activity. This feature allows for speed of contraction, and much of the cycle is spent in a state that is dissociated from actin. In contrast, unconventional myosins are nonfilamentous, and most of these classes of myosin will probably operate in much smaller groups or potentially even individually. Because of these different functional roles, the kinetic properties of these motors are expected to be quite different from the well characterized myosin IIs.Murine myosin V is a member of the class of unconventional myosins that is implicated in organelle movement and membrane trafficking based on a number of cellular and genetic studies. Mutations in murine myosin V result in a range of defects, from impaired pigment granule movement, resulting in a dilute coat color, to a lack of smooth endoplasmic reticulum in the dendritic spines of Purkinje cells, which may be the cause of the neurological defect that results in early postnatal death (reviewed in Ref. 1).Myosin V is particularly interesting from several points of view. It is a dimeric molecule that has an unusually long neck, three times that of myosin II. This region of the molecule has been proposed to act as a lever arm that ultimately results in relative sliding of actin and myosin. The neck region contains six IQ motifs that have the consensus sequence (IQXXIR-GXXXR) for binding of calmodulin or myosin light chains. Thus, the potential for calcium-dependent regulation of motor activity also exists fo...

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.