Reiko Ohkura

AAA+ Ring and linker swing mechanism in the dynein motor.

Summary Dynein ATPases power diverse microtubule-based motilities. Each dynein motor domain comprises a ring-like head containing six AAA+ modules and N- and C-terminal regions, together with a stalk that binds microtubules. How these subdomains are arranged and generate force remains poorly understood. Here, using electron microscopy and image processing of tagged and truncated Dictyostelium cytoplasmic dynein constructs, we show that the heart of the motor is a hexameric ring of AAA+ modules, with the stalk emerging opposite the primary ATPase site (AAA1). The C-terminal region is not an integral part of the ring but spans between AAA6 and near the stalk base. The N-terminal region includes a lever-like linker whose N terminus swings by ∼17 nm during the ATPase cycle between AAA2 and the stalk base. Together with evidence of stalk tilting, which may communicate changes in microtubule binding affinity, these findings suggest a model for dyneins structure and mechanism.

Swing of the lever arm of a myosin motor at the isomerization and phosphate-release steps

In muscle, the myosin head (‘crossbridge’) performs the ‘working stroke’, in which ATP is hydrolysed to generate the sliding of actin and myosin filaments. The myosin head consists of a globular motor domain and a long lever-arm domain. The ‘lever-arm hypothesis’ predicts that during the working stroke, the lever-arm domain tilts against the motor domain, which is bound to actin in a fixed orientation. To detect this working stroke in operation, we constructed fusion proteins by connecting Aequorea victoria green fluorescent protein and blue fluorescent protein to the amino and carboxyl termini of the motor domain of myosin II of Dictyostelium discoideum, a soil amoeba, and measured the fluorescence resonance energy transfer between the two fluorescent proteins. We show here that the carboxy-terminal fluorophore swings at the isomerization step of the ATP hydrolysis cycle, and then swings back at the subsequent step in which inorganic phosphate is released, thereby mimicking the swing of the lever arm. The swing at the phosphate-release step may correspond to the working stroke, and the swing at the isomerization step to the recovery stroke.

ATP hydrolysis cycle–dependent tail motions in cytoplasmic dynein

The motor protein dynein is predicted to move the tail domain, a slender rod-like structure, relative to the catalytic head domain to carry out its power stroke. Here, we investigated ATP hydrolysis cycle–dependent conformational dynamics of dynein using fluorescence resonance energy transfer analysis of the dynein motor domain labeled with two fluorescent proteins. We show that dynein adopts at least two conformational states (states I and II), and the tail undergoes ATP-induced motions relative to the head domain during transitions between the two states. Our measurements also suggest that in the course of the ATP hydrolysis cycle of dynein, the tail motion from state I to state II takes place in the ATP-bound state, whereas the motion from state II to state I occurs in the ADP-bound state. The latter tail motion may correspond to the predicted power stroke of dynein.

Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding

Coupling between ATPase and track binding sites is essential for molecular motors to move along cytoskeletal tracks. In dynein, these sites are separated by a long coiled coil stalk that must mediate communication between them, but the underlying mechanism remains unclear. Here we show that changes in registration between the two helices of the coiled coil can perform this function. We locked the coiled coil at three specific registrations using oxidation to disulfides of paired cysteine residues introduced into the two helices. These trapped ATPase activity either in a microtubule-independent high or low state, and microtubule binding activity either in an ATP-insensitive strong or weak state, depending on the registry of the coiled coil. Our results provide direct evidence that dynein uses sliding between the two helices of the stalk to couple ATPase and microtubule binding activities during its mechanochemical cycle.

The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein

The dynein motor domain is composed of a tail, head, and stalk and is thought to generate a force to microtubules by swinging the tail against the head during its ATPase cycle. For this “power stroke,” dynein has to coordinate the tail swing with microtubule association/dissociation at the tip of the stalk. Although a detailed picture of the former process is emerging, the latter process remains to be elucidated. By using the single-headed recombinant motor domain of Dictyostelium cytoplasmic dynein, we address the questions of how the interaction of the motor domain with a microtubule is modulated by ATPase steps, how the two mechanical cycles (the microtubule association/dissociation and tail swing) are coordinated, and which ATPase site among the multiple sites in the motor domain regulates the coordination. Based on steady-state and pre-steady-state measurements, we demonstrate that the two mechanical cycles proceed synchronously at most of the intermediate states in the ATPase cycle: the motor domain in the poststroke state binds strongly to the microtubule with a Kd of ≈0.2 μM, whereas most of the motor domains in the prestroke state bind weakly to the microtubule with a Kd of >10 μM. However, our results suggest that the timings of the microtubule affinity change and tail swing are staggered at the recovery stroke step in which the tail swings from the poststroke to the prestroke position. The ATPase site in the AAA1 module of the motor domain was found to be responsible for the coordination of these two mechanical processes.

Hybrid nanotransport system by biomolecular linear motors

We have demonstrated a novel micro/nanotransport system using biomolecular motors driven by adenosine triphosphate (ATP). For the driving mechanism, microtubule-kinesin system, which is one of the linear biomolecular motor systems was investigated. ATP dissolved in an aqueous condition is hydrolyzed to adenosine diphosphate (ADP) to energize the bionanoactuators in this mechanism. This means the system does not require an external electrical or mechanical energy source. Therefore, a purely chemical system which is similar to the in vivo transport will be realized. This paper reports some fundamental studies to integrate biomaterials and MEMS. The microtubules, or rail molecules, were patterned on a glass substrate with poly(dimethyl siloxane) (PDMS) using a regular soft lithography technique. Microbeads (320 nm in diameter) and a micromachined structure (2/spl times/3 /spl mu/m, 2 /spl mu/m in thickness) coated with kinesin molecules were transported along the microtubules at an average speed of 476/spl plusmn/56 and 308 nm/s, respectively. While ATP injection activated the transport system we have also managed to provide repetitive on/off control using hexokinase as an inhibitor. For the minimum response time in the repetitive control, the optimized concentration for ATP was 10/sup 2/ /spl mu/M and 10/sup 3/ U/L for hexokinase.

Two modes of microtubule sliding driven by cytoplasmic dynein.

Dynein is a huge multisubunit microtubule (MT)-based motor, whose motor domain resides in the heavy chain. The heavy chain comprises a ring of six AAA (ATPases associated with diverse cellular activities) modules with two slender protruding domains, the tail and stalk. It has been proposed that during the ATP hydrolysis cycle, this tail domain swings against the AAA ring as a lever arm to generate the power stroke. However, there is currently no direct evidence to support the model that the tail swing is tightly linked to dynein motility. To address the question of whether the power stroke of the tail drives MT sliding, we devised an in vitro motility assay using genetically biotinylated cytoplasmic dyneins anchored on a glass surface in the desired orientation with a biotin–streptavidin linkage. Assays on the dyneins with the site-directed biotin tag at eight different locations provided evidence that robust MT sliding is driven by the power stroke of the tail. Furthermore, the assays revealed slow MT sliding independent of dynein orientation on the glass surface, which is mechanically distinct from the sliding driven by the power stroke of the tail.

ATP-Driven Remodeling of the Linker Domain in the Dynein Motor

Summary Dynein ATPases are the largest known cytoskeletal motors and perform critical functions in cells: carrying cargo along microtubules in the cytoplasm and powering flagellar beating. Dyneins are members of the AAA+ superfamily of ring-shaped enzymes, but how they harness this architecture to produce movement is poorly understood. Here, we have used cryo-EM to determine 3D maps of native flagellar dynein-c and a cytoplasmic dynein motor domain in different nucleotide states. The structures show key sites of conformational change within the AAA+ ring and a large rearrangement of the “linker” domain, involving a hinge near its middle. Analysis of a mutant in which the linker “undocks” from the ring indicates that linker remodeling requires energy that is supplied by interactions with the AAA+ modules. Fitting the dynein-c structures into flagellar tomograms suggests how this mechanism could drive sliding between microtubules, and also has implications for cytoplasmic cargo transport.

Molecular mechanism of force generation by dynein, a molecular motor belonging to the AAA+ family

Dynein is an AAA+ (ATPase associated with various cellular activities)-type motor complex that utilizes ATP hydrolysis to actively drive microtubule sliding. The dynein heavy chain (molecular mass >500 kDa) contains six tandemly linked AAA+ modules and exhibits full motor activities. Detailed molecular dissection of this motor with unique architecture was hampered by the lack of an expression system for the recombinant heavy chain, as a result of its large size. However, the recent success of recombinant protein expression with full motor activities has provided a method for advances in structure-function studies in order to elucidate the molecular mechanism of force generation.

BREK/LMTK2 is a myosin VI‐binding protein involved in endosomal membrane trafficking

Myosin VI is involved in a wide range of endocytic and exocytic membrane trafficking pathways; clathrin‐mediated endocytosis, intracellular transport of clathrin‐coated and ‐uncoated vesicles, AP‐1B‐dependent basolateral sorting in polarized epithelial cells and secretion from the Golgi complex to the cell surface. In this study, using a yeast two‐hybrid screen, we identified brain‐enriched kinase/lemur tyrosine kinase 2 (BREK/LMTK2), a transmembrane serine/threonine kinase with previously unknown cellular functions, as a myosin VI‐interacting protein. Several binding experiments confirmed the interaction of myosin VI with BREK in vivo and in vitro. Immunocytochemical analyses revealed that BREK localizes to cytoplasmic membrane vesicles and to perinuclear recycling endosomes. Notably, cells in which BREK was depleted by siRNA were still able to internalize transferrin molecules and to transport them to early endosomes, but were unable to transport them to perinuclear recycling endosomes. Our results show that BREK is critical for the transition of endocytosed membrane vesicles from early endosomes to recycling endosomes and also suggest an involvement of myosin VI in this pathway.