Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography

Lupetti, Pietro; Lanzavecchia, Salvatore; Mercati, David; Cantele, Francesca; Dallai, Romano; Mencarelli, Caterina

doi:10.1002/cm.20084

Cited by 38 publications

(27 citation statements)

References 54 publications

(69 reference statements)

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“…These results predict that any movement of the stalk or tail will also contain a nonplanar component. A nonplanar bending of the stalk is in agreement with static in situ tomography studies, where stalks are shown to lie obliquely with respect to the dynein ring (27). Moreover, this observation suggests that previous 2D electron micrograph images in which the molecule is forced to lie flat on the substrate might not accurately capture the conformational changes that occur during force generation (27).…”

Section: ; See Si Text For Details)supporting

confidence: 83%

A structural model reveals energy transduction in dynein

Serohijos

Chen

Ding

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Intracellular active transport is driven by ATP-hydrolyzing motor proteins that move along cytoskeletal filaments. In particular, the microtubule-associated dynein motor is involved in the transport of organelles and vesicles, the maintenance of the Golgi, and mitosis. However, unlike kinesin and myosin, the mechanism by which dynein converts chemical energy into mechanical force remains largely a mystery, due primarily to the lack of a highresolution molecular structure. Using homology modeling and normal mode analysis, we propose a complete atomic structure and a mechanism for force generation by the motor protein dynein. In agreement with very recent electron microscopy (EM) reconstructions showing dynein as a ring-shaped heptamer, our model consists of six ATPases of the AAA (ATPases associated with various cellular activities) superfamily and a C-terminal domain, which is experimentally known to control motor function. Our model shows a coiled coil spanning the diameter of the motor that accounts for previously unidentified structures in EM studies and provides a potential mechanism for long-range communication between the AAA domains. Furthermore, normal mode analysis reveals that the subunits of the motor that contain the nucleotide binding sites exhibit minimal movement, whereas the rest of the motor is very mobile. Our analysis suggests the likely domain rearrangements of the motor unit that generate its power stroke. This study provides insights into the structure and function of dynein that can guide further experimental investigations into energy transduction in dynein.AAA ͉ homology modeling ͉ molecular motors ͉ electron microscopy ͉ ATP hydrolysis M otor proteins use energy derived from ATP hydrolysis to move along cytoskeletal filaments (1). In particular, the microtubule-associated molecular motor dynein is involved in the transport of organelles and vesicles, the maintenance of the Golgi, and mitosis (2, 3). Mutations in this protein have been implicated in neurodegenerative diseases (4) and polycystic kidney disease (5). Unlike the motor proteins kinesin and myosin, the mechanism by which dynein converts chemical energy into mechanical force remains largely a mystery. The primary reason for this is the lack of a high-resolution molecular structure. Cytoplasmic dynein is a large multisubunit complex (1.2 MDa) composed of two heavy chains (Ϸ0.5 MDa each) (6), making its structural characterization extremely challenging. In this study, we use homology modeling to propose a structure for the motor unit of the dynein heavy chain, which is the site for energy transduction and force generation (see Fig. 1a). Next, we perform normal mode analysis to determine the large-scale motions of the protein. Together, these results suggest a mechanism for both force generation and regulatory control in dynein.Sequence analysis of dynein's motor unit indicates that it consists of six concatenated AAA (ATPases associated with diverse cellular activities) subunits, an extended stalk that contains a microtubule bind...

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Section: ; See Si Text For Details)supporting

confidence: 83%

A structural model reveals energy transduction in dynein

Serohijos

Chen

Ding

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…This extension shares the same location as the interface between the outer and inner dynein arms in earlier work, 35 suggesting that bulb 1 is this interface. The top plate, which is assigned as the α-ring (see the next section), is connected to the rest of the outer dynein arm almost exclusively through the slender rod, whereas the middle and the bottom plates have a few direct The stalks that were visualized in freeze-fractured replicas 27,33 are not visible in our tomograms. This is probably because the coiled-coil stalks are too thin (20 Å) for the current resolution, and the variation of the stalk angles in rigor smeared the structure by averaging.…”

Section: Overall Feature Of the Outer Dynein Armmentioning

confidence: 71%

“…The quick-freeze, deep-etch technique, 4,27,29,30 the negative staining technique, 31,32 and electron tomography [33][34][35] applied to dynein arms in situ showed that head domains stack on the microtubule doublet and that there are links between the neighboring arms. In particular, the three-dimensional structure of wildtype Chlamydomonas flagella reconstructed by electron cryo-tomography and single-particle averaging 35 supported the idea that the AAA rings of the α, β and γ-heavy chains stack on the A-microtubule.…”

Section: Introductionmentioning

confidence: 99%

The Architecture of Outer Dynein Arms in Situ

Ishikawa

Sakakibara

Oiwa

2007

Journal of Molecular Biology

113

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“…Electron microscopy of dynein shows a ⌽-shaped structure, indicating that the tails of the two heads are close to each other and the ring domains partially overlap (10,25). When the heads of dynein bind to the microtubule, they also overlap (25)(26)(27). A possible model to explain the stepping movement is that the dynein heads are positioned axially along the microtubule similar to kinesin.…”

Section: Discussionmentioning

confidence: 99%

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

Toba

Watanabe

Yamaguchi-Okimoto

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

311

275

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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 nm͞s, 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 Ϸ8 nm, independent of load. It is suggested that the large dynein heads take 16-nm steps by using an overlapping hand-over-hand mechanism.microtubules ͉ motor protein ͉ optical tweezers ͉ step size ͉ nanotechnology D ynein is a molecular motor that moves along microtubules to the direction of the minus end. Cytoplasmic dynein transports cellular organelles toward the minus end of microtubules, whereas most kinesin molecules transport organelles toward the plus end (1-3). Hirakawa et al. (4) have reported that purified axonemal dynein produced maximum forces of 5 pN, and they also showed stepwise displacements of 8 nm. In contrast, another study (5) has demonstrated that single molecules of purified cytoplasmic dynein move with short steps (8 nm) at high loads (Ͼ0.8 pN) and long steps (16-32 nm) at low forces (Ͻ0.4 pN), and from these findings a molecular gear mechanism was proposed. The values for maximum force and the size of the steps differ between those two reports. Moreover, the very low velocity (Ͻ50 nm͞s) of movement of the purified cytoplasmic dynein (5) is in direct contrast to the high velocity (Ϸ1 m͞s) of dynein movement in a cell and in an in vitro motility assay (6-8). These discrepancies possibly result from inactive dynein molecules being included with the purified native dynein in the analysis.In this study a method of coating the beads with dynein was developed to keep dynein active and allow the force and step size produced by single molecules of dynein to be measured. The step size of active cytoplasmic dynein was 8 nm and was independent of both force and ATP concentration. The stall force was 7-8 pN. These values are very similar to those recorded for kinesin (9); however, the stepping manner was different from that of kinesin. ResultsActive Dynein Bound to Protein A-Coated Beads. The method used to purify cytoplasmic dynein without its accessory protein, dynactin, has been described (Fig. 1a, lane D) (10, 11). Dynein was further purified by allowing it to bind to the microtubules (Fig. 1a, lane AMP-PNP) (12). Dynein was then released from microtubules in the presence of 0.1-10 mM ATP (Fig. 1a, lane ATP).The method used to bind dynein to the coverslips for in vitro motility assay and beads for optical trap assay was modified from previously reported methods (5, 8). Beads coated with protein A were suitable for motility and force generation....

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Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography

Cited by 38 publications

References 54 publications

A structural model reveals energy transduction in dynein

A structural model reveals energy transduction in dynein

The Architecture of Outer Dynein Arms in Situ

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

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