Dynein Heads Research Articles

ATP‐induced conformational change of axonemal outer dynein arms revealed by cryo‐electron tomography

Axonemal outer dynein arm (ODA) motors generate force for ciliary beating. We analyzed three states of the ODA during the power stroke cycle using in situ cryo‐electron tomography, subtomogram averaging, and classification. These states of force generation depict the prepower stroke, postpower stroke, and intermediate state conformations. Comparison of these conformations to published in vitro atomic structures of cytoplasmic dynein, ODA, and the Shulin–ODA complex revealed differences in the orientation and position of the dynein head. Our analysis shows that in the absence of ATP, all dynein linkers interact with the AAA3/AAA4 domains, indicating that interactions with the adjacent microtubule doublet B‐tubule direct dynein orientation. For the prepower stroke conformation, there were changes in the tail that is anchored on the A‐tubule. We built models starting with available high‐resolution structures to generate a best‐fitting model structure for the in situ pre‐ and postpower stroke ODA conformations, thereby showing that ODA in a complex with Shulin adopts a similar conformation as the active prepower stroke ODA in the axoneme.

Atomistic analysis of ATP hydrolysis effect on a dynein walking mechanism

Dynein is a microtubule motor protein that is highly involved in cell motility. Although its atomic structure was recently reported, it has been believed that dynein, like other motor proteins, generates a large swing motion powered by ATP hydrolysis. In this paper, we have analyzed ATP hydrolysis effect on the walking mechanism of dynein at the atomic level using a Ca coarse-grained elastic network model. Normal mode analysis (NMA) revealed that the detachment of the linker from the head of dynein, which is triggered by ATP hydrolysis, facilitates the main swing motion of dynein in two ways: allowing the bending motion as an extra degree-of-freedom (topologically) and lowering the energy barrier between the closed and open forms (energetically). This large conformational transition pathway is successfully generated by Elastic network interpolation (ENI). Comparison between the ENI pathway and the NMA results clearly elucidates the role of ATP hydrolysis in motor proteins as well as the swing motion of dynein in atomic detail.

Cytoplasmic dynein transports cargos via load-sharing between the heads.

Cytoplasmic dynein is a motor protein that walks along microtubules (MTs) and performs mechanical work to power a variety of cellular processes. It remains unclear how a dynein dimer is able to transport cargos against load without coordinating the stepping cycles of its two heads. Here, by using a DNA-tethered optical trapping geometry, we find that the force-generating step of a head occurs in the MT-bound state, while the ‘primed’ unbound state is highly diffusional and only weakly biased to step towards the MT minus end. The stall forces of the individual heads are additive, with both heads contributing equally to the maximal force production of the dimer. Based on these results, we propose that the heads of dynein utilize a ‘load-sharing’ mechanism, unlike kinesin and myosin. This mechanism may allow dynein to work against hindering forces larger than the maximal force produced by a single head.

Structure of dimeric axonemal dynein in cilia suggests an alternative mechanism of force generation.

The mechanism by which the two different heads of the ciliary outer dynein arm produce force to translocate the microtubule during beating is still unknown. In this report we use cryo-electron tomography and image processing to analyze the conformational changes and the relative abundance of each conformation of the two dynein heads from mouse respiratory cilia. In the absence of nucleotides the majority of dynein dimers are in the apo form and both heads are tightly packed, whereas they are dissociated and move independently in the presence of nucleotides. The head of the external outer arm dynein heavy chain has a diagonal shift toward both the neighboring B-tubule and the proximal end of the axoneme, while the head of the internal heavy chain shifts only longitudinally toward the proximal end. In the presence of nucleotides a significant number of the dynein dimers have two heads overlapped in the proximal shifting form or overlapped in the apo form. During ciliary bending axonemal dynein translocates microtubules by moving with short steps and two heads stay at the same position longer than cytoplasmic dynein. This demonstrates that the step of the outer arm dynein dimer is not dominated by the hand-over-hand motion, but also indicates the difference between axonemal dynein and cytoplasmic dynein.

Load-Sharing Mechanism of Cytoplasmic Dynein

Cytoplasmic dynein is a homodimeric AAA+ motor protein responsible for minus end-directed transport along microtubules. The two heads of dynein are not strictly coordinated during processive motility, unlike those of dimeric kinesins and myosins. Therefore, tail-tethered optical trapping experiments yield insufficient information about the force production of individual heads. Here, we connected a trapped bead to a dynein dimer through a rigid DNA tether and directly measured the stepping motion of a single head under load. We observed that even though the step size of a head decreases under load, the head only stalls at half the stall force of the dimer and must rely on its partner head to walk against near-stall loads. The bulk of the force is produced when a head is bound to the microtubule, and stepping direction in the unbound state can be reversed by as little as 0.5 pN of backward load. Force-velocity curves indicate that the forward stepping rate of a dynein head is strongly inhibited by external force, while the backward rate is slow and nearly force-independent. Our results provide evidence for tension gating in dynein motility and lead us to propose a load-sharing mechanism between the two heads of a dynein dimer.

Microtubule Doublet Curvature and its Role in Cilia Actuation

Motile cilia are hair-like organelles that beat to produce fluid flow. It is not fully understood how these incredibly complex molecular machines function at the protein level to produce complicated beat patterns. The mechanical properties of microtubule doublets, the main structural element of the cilium, have not been well studied. Here we show that doublet microtubules separated from human airway cilia curl up with an average radius of curvature of 1.39 +- 0.01 µm, determined from transmission electron and atomic force imaging. This result is surprising given that cytoplasmic microtubules have a persistence length on the order of millimeters, but is consistent with curvature seen in the literature. Doublet microtubules were isolated by digesting the axoneme with trypsin and inducing sliding by the addition of ATP. After sliding, transmission electron images showed that dynein motors were still attached, which is consistent with prior studies. We prepared samples with a fraction of the outer dynein arms removed using a 0.6M potassium chloride salt extraction. Removal of dynein was confirmed by SDS-PAGE. Samples with removed dynein had an increased radius of curvature, 2.3 +- 0.1 µm. The straightening of the doublet microtubules with the removal of dynein motors implies that the asymmetric distribution of dynein motors causes a stress that bends the doublet microtubule. Our results suggest an alternative source for curvature in the beating cilium. The distribution of dynein heads bound to a microtubule doublet is known to change dynamically during the cilium beat. Changing distribution of dynein heads bound to the microtubules could induce changes in the curvature of the cilium without the need for the motors to apply a shear force.

The Mechanism of Cytoplasmic Dynein Motility

Cytoplasmic dynein is a homodimeric AAA+ motor that transports a multitude of cargos towards the microtubule minus end. The mechanism of dynein processivity and interhead coordination remain unclear due to its large size (2.6 MDa) and the complexity of its structure. In contrast to kinesins and myosins, we directly observed that dynein heads move independently along the microtubule. Stepping behavior of the heads varies as a function of interhead separation and establishing the basis of high variability in dynein step size. By engineering the mechanical and catalytic properties of the dynein motor domain, we show that a rigid linkage between monomers and dimerization between N-terminal tail domains are not essential for processive movement. Instead, dynein processivity minimally requires the linker domain of one active monomer to be attached to an inert MT tether retaining only the MT-binding domain. The release of a dynein monomer from the MT can be mediated either by nucleotide binding or external load. However, nucleotide dependent release is inhibited when force was applied to the linker domain. Force dependent release is significantly asymmetric, with faster release towards the minus-end. On the basis of these measurements, we developed a model that describes the basis of dynein processivity, directionality and force generation.

DYNEIN'S NETWORK OF CHEMOMECHANICAL MOTOR CYCLES

An eight-state network model of dynein's chemomechanical motor cycles is developed, in which the states of an effective single dynein head are represented by the number of ATP binding at the primary site and the number of ATP binding at other three secondary sites. The binding and unbinding of ATP, as well as the hydrolysis of ATP and the reverse process, are characterized by transition rates between certain states. Our results show that the stall force of dynein increases fast with ATP up to 1 mM ATP, beyond which it increases slowly to a saturated value, and that load and ATP concentration can adjust the step size of dynein, i.e., dynein can shift gears according to conditions. These results are in agreement with experiments [R. Mallik, B. C. Carter, S. A. Lex, S. J. King and S. P. Gross, Nature427, 649 (2004)].

Detailed Analysis of the Dynein Stepping Mechanism using Multicolor Tracking

Cytoplasmic dynein is a motor protein that moves processively towards the microtubule minus end. To characterize dynein's step size at both limiting (5 µM) and saturating (1 mM) ATP concentrations, we have improved the temporal resolution of FIONA to 2 milliseconds and tracked the movement of single yeast cytoplasmic dynein motors, each labeled with one quantum dot. In contrast to kinesin, dynein's step size is highly variable, and backwards and sideways steps are frequently observed. As the tail domain takes ∼8 nm steps, the heads advance by taking both short (∼8 nm) and long (∼16 nm) steps. These data indicate that the heads do not move in a strictly alternating manner, as is the case with kinesin and myosin. To further characterize how the dynein heads move relative to each other, we have tracked the movement of both heads simultaneously. To precisely measure the head-head separation vector, we have developed novel small quantum dots (10 nm diameter) that specifically attach to each dynein head. The fluorescent signals of the differently-colored quantum dots attached to dynein are then registered with 2 nm precision. Using this technique, we find the heads to be widely separated, mostly in the off-axis, indicating that the two heads walk along separate filaments. Our two-color stepping data also show that the dynein heads advance mostly by taking alternating steps, as is the case with kinesin. However, the heads may also move independently of each other, and one head may take multiple steps before its partner moves forward. Both the leading head and the trailing head may initiate a step. Taken together, these unprecedented behaviors indicate that cytoplasmic dynein achieves processive movement via a mechanism unique among the molecular motors.

Single Protofilament is Enough to Support Unidirectional Walking of Cytoplasmic Dynein

Cytoplasmic dynein moves on microtubules toward its minus end with 8nm stepping. Dynein motor domain has a ring structure which consists of six AAA modules, and the diameter of the ring is 14 nm. This size is so large as compared with the 8 nm step size, and then question arises whether dynein can walk on a single protofirament of microtubule or dynein uses multiple protofilaments. To address this point, we examined dynein motility using zinc-induced tubulin sheet (Zn-sheet) which has an arrangement of adjacent protofilaments with anti-parallel and opposite orientations. From the structural analysis, the dynein binding sites are revealed to be exposed only at either edge of a Zn-sheet. Previously, we have shown that the Zn-sheet move on the glass surface coated with the dynein or kinesin molecules. Unlike microtubules, Zn-sheets followed the winding path and the tracks are often circular. In this study, we aimed to observe the processive movement of single dynein molecules on the edge of a Zn-sheet by TIRF microscopy. As a result of deliberate preparations, we have successfully observed that single dynein molecules walk on Zn-sheets. The velocity of the movement on Zn-sheets was almost the same as that on microtubules. These results demonstrate that the single protofilament is enough to support dynein motility, and suggest that dynein uses only one protofilament of microtubules as well as one protofilament at the edge of Zn-sheets. Considering the large size of the dynein head, it is hard for the two heads of the dynein molecules to move on a single protofilament by the hand-over-hand mechanism advocated for the two heads of kinesin, and it is necessary to investigate the coordination of the two head of dynein.

Time-dependent measure of a nanoscale force-pulse driven by the axonemal dynein motors in individual live sperm cells

Nanoscale mechanical forces generated by motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces is important to developing motile biomimetic nanodevices powered by biological motors for nanomedicine. Axonemal dynein motors positioned inside the sperm flagellum drive microtubule sliding and give rise to rhythmic beating. This force-generating action pushes the sperm cell through viscous media. Here we report new nanoscale information on how the propulsive force is generated by the sperm flagellum and how this force varies over time. Using a modified atomic force microscope, single-cell recordings reveal discrete ∼50-ms pulses oscillating with amplitude 9.8 ± 2.6 nN independent of pulse frequency (3.5–19.5 Hz). The average work carried out by each cell is 4.6 × 10 -16 J per pulse, equivalent to the hydrolysis of ∼5500 molecules of adenosine triphosphate. The mechanochemical coupling at each active dynein head is ∼2.2 pN per adenosine triphosphate molecule and ∼3.9 pN per dynein arm. From the Clinical Editor In this paper, nanoscale mechanical forces generated by axonemal dynein motors derived from sperm flagellum are examined and reported. These motor proteins are crucial to normal cellular and organismal functioning. The ability to measure and exploit such forces is important to developing motile biomimetic nanodevices powered by biological motors for nanomedicine.

3D Arrangement, Conformation and ATP-Induced Structural Change of Inner and Outer Dynein Arms Revealed by Electron Cryo-Tomography

Axonemal dyneins form inner and outer arms, cause sliding among nine microtubule doublets to generate bending motion of eukaryotic flagella/cilia. However, it is not clear how the linear motion of dynein is integrated into well-coordinated bending. To understand the mechanism of flagellar/ciliary bending motion, we reconstructed 3D structure of inner and outer dynein arms at various nucleotide states using the technique of electron cryo-tomography and single particle averaging. Our averaged tomogram visualized three heavy chains of outer arms dyneins from Chlamydomonas stack vertically, while eight inner arm dyneins make a horizontal array (Ishikawa et al. (2007) JMB; Bui et al. (2008) JCB) (figure). We also found that the arrangement of inner dyneins and other linkers is not symmetrical among nine microtubule doublets (Bui et al. (2009) JCB). By further image analysis we revealed the shift of the ATPase head of dynein toward the tip of flagella during Pi release. The orientation of the coild-coil stalk is constant. This shift can winch adjacent microtubule. Interestingly apo and nucleotide-bound forms of dynein coexist and they make clusters in flagella, which could explain torsion for bending.

Steps, Force And Motile Mechanism Of Cytoplasmic Dynein

Native cytoplasimic dynein moves with 8-nm steps by overlapping hand-over-hand mechanism (Toba et al. PNAS 2006). The 8-nm steps were confirmed in vesicle transport using FIONA method of quantum labeled vesicles. The contribution of individual dynein head to each step of the dynein is unclear because the step size is always masked by 8-nm of microtubule pitch. To avoid the masking, a recombinant single-headed dynein was prepared and bound separately onto a bead. The movement of dynein heads on the bead was characterized by laser trap and measurement with nanometer accuracy.

Multiscale approaches for studying energy transduction in dynein.

Cytoplasmic dynein is an important motor that drives all minus-end directed movement along microtubules. Dynein is a complex motor whose processive motion is driven by ATP-hydrolysis. Dynein's run length has been measured to be several millimetres with typical velocities in the order of a few nanometres per second. Therefore, the average time between steps is a fraction of a second. When this time scale is compared with typical time scales for protein side chain and backbone movements (approximately 10(-9) s and approximately 10(-5) s, respectively), it becomes clear that a multi-timescale modelling approach is required to understand energy transduction in this protein. Here, we review recent efforts to use computational and mathematical modelling to understand various aspects of dynein's chemomechanical cycle. First, we describe a structural model of dynein's motor unit showing a heptameric organization of the motor subunits. Second, we describe our molecular dynamics simulations of the motor unit that are used to investigate the dynamics of the various motor domains. Third, we present a kinetic model of the coordination between the two dynein heads. Lastly, we investigate the various potential geometries of the dimer during its hydrolytic and stepping cycle.

Kinetic model for dynein oscillatory activity

A kinetic model for dynein, a molecular motor, is considered. This model explains the oscillatory behaviour, observed by Chikako Shingyoji et al. [Ch. Shingyoji, H. Higuchi, M. Yoshimura, E. Katayama, T. Yanagida, Dynein arms are oscillatory force generators, Nature 393 (1998) 711–714.] and by Susumu Aoyama and Ritsu Kamiya [S. Aoyama, R. Kamiya, Cyclical interactions between two outer doublet microtubules in split flagellar axonemes, Biophys. J. 89 (2005) 3261–3268.] in surprisingly simple axonemal fragments. The model shows that sustained oscillations can be generated due to the obligate cooperative interaction of the two dynein heads in the axonemal fragments. No other feedback control interactions are involved in the model to explain oscillations, similar to those observed experimentally, for realistic dynein rate constants. The modified model shows how the ATP hydrolytic exhaustion influences the amplitude and frequency of dynein oscillatory activity.

Three-dimensional structure of cytoplasmic dynein bound to microtubules

Cytoplasmic dynein is a large, microtubule-dependent molecular motor (1.2 MDa). Although the structure of dynein by itself has been characterized, its conformation in complex with microtubules is still unknown. Here, we used cryoelectron microscopy (cryo-EM) to visualize the interaction between dynein and microtubules. Most dynein molecules in the nucleotide-free state are bound to the microtubule in a defined conformation and orientation. A 3D image reconstruction revealed that dynein's head domain, formed by a ring-like arrangement of AAA+ domains, is located approximately 280 A away from the center of the microtubule. The order of the AAA+ domains in the ring was determined by using recombinant markers. Furthermore, a 3D helical image reconstruction of microtubules with a dynein's microtubule binding domain [dynein stalk (DS)] revealed that the stalk extends perpendicular to the microtubule. By combining the 3D maps of the dynein-microtubule and DS-microtubule complexes, we present a model for how dynein in the nucleotide-free state binds to microtubules and discuss models for dynein's power stroke.

2P177 単頭ダイニン2分子による連続的運動中の個々の分子の寄与(分子モーター,口頭発表,第45回日本生物物理学会年会)

2P177 単頭ダイニン2分子による連続的運動中の個々の分子の寄与(分子モーター,口頭発表,第45回日本生物物理学会年会)

Overlapping hand-over-hand mechanism of single molecular motility of cytoplasmic dynein

Structural differences between dynein and kinesin suggest a unique molecular mechanism of dynein motility. Measuring the mechanical properties of a single molecule of dynein is crucial for revealing the mechanisms underlying its movement. We measured the step size and force produced by single molecules of active cytoplasmic dynein by using an optical trap and fluorescence imaging with a high temporal resolution. The velocity of dynein movement, 800 nm/s, is consistent with that reported in cells. The maximum force of 7-8 pN was independent of the ATP concentration and similar to that of kinesin. Dynein exhibited forward and occasional backwards steps of approximately 8 nm, independent of load. It is suggested that the large dynein heads take 16-nm steps by using an overlapping hand-over-hand mechanism.

2P206 The contribution of individual dynein head to the processive movement generated by two single headed dynein(37. Molecular motor (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

2P206 The contribution of individual dynein head to the processive movement generated by two single headed dynein(37. Molecular motor (II),Poster Session,Abstract,Meeting Program of EABS & BSJ 2006)

Optically-controllable, micron-sized motor based on live cells

We demonstrate rotation of live Chlamydomonas reinhardtii cells in an optical trap; the speed and direction of rotation are amenable to control by varying the optical trapping force. Cells rotate with a frequency of 60-100 rpm; functional flagella are shown to play a decisive role in rotation. The rotating cells generate torque (typically ~7500-12000 pN nm) that is much larger than that generated chemically by a dynein head in vitro (40 pN nm). The total force associated with a rotating live cell (~10 pN) suggests that activity of only a small fraction (~5%) of dynein molecules per beat cycle is sufficient to generate flagellar motion.