Of rings and levers: the dynein motor comes of age

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Of rings and levers: the dynein motor comes of age Michael P. Koonce1,2 and Montserrat Samso´3 1

Division of Molecular Medicine, Wadsworth Center, Albany, NY 12201-0509, USA Department of Biomedical Sciences, University at Albany, NY 12201-0509, USA 3 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA 2

After nearly four decades of investigation, the dynein motor is finally on the verge of revealing its inner secrets. This multisubunit ATPase participates in several important microtubule-based motilities in eukaryotic cells. Numerous recent articles have advanced the understanding of the dynein motor substructure and its mechanism of force production, revealing both similarities to other motors and some surprises. We are now in a position to summarize a basic blueprint for dynein. At its core, the motor is a ring-shaped object with two protruding levers: one engages cargo and might provide much of the force for movement, and the other interacts with the microtubule track. The activities of both levers are linked through nucleotide-dependent conformational changes in the ring. Three families of cytoskeleton-based molecular motors together drive much of the motility of and within eukaryotic cells. Myosin, kinesin and dynein have prominent roles in muscle contraction, organelle transport, and endomembrane and cytoskeletal organization – processes that are essential for cell shape, division and motility [1–3]. Understanding how these motors work – that is, how they convert the energy of nucleotide hydrolysis into force production – has been a long-standing, fundamental goal in cell biology. The three motors have several functional parallels with one another [4,5], and they might seem to perform in similar ways. Members of each family are characterized by a distinctive motor domain (Figure 1) that functions through cycles of nucleotide binding, hydrolysis and release. Each motor is connected to its cargo-binding region through a thin, filamentous linkage. Typically, this link also connects an additional motor, and these dimeric ATPases coordinate their activities in a type of hand-overhand fashion to move along a filament or microtubule [6,7]. There are exceptions to this general plan that include singleheaded motors, and homo- or heterodimeric, trimeric and tetrameric assemblies. Superficially, the three types of motor follow the same actions: the motor core hydrolyzes ATP and coordinates this catalytic cycle to generate conformational changes that both produce work (movement or other actions such as tubule disassembly) and modulate the affinity for actin Corresponding author: Michael P. Koonce ([email protected]). Available online 12 October 2004

filaments (myosin) or microtubules (dynein, kinesin). Tight synchronization between movement and substrate affinity is essential for efficient movement. Surprisingly, however, these two activities are not necessarily ‘hardwired’ together, because there are examples of mutations that can uncouple this coordination, and nucleotides that can be cleaved without producing force [8–10]. Step sizes for the three motor families are in the low nanometer range, and the motors generate forces on the low piconewton scale. Functionality within the same family of motors is specified through differences in how they are targeted and docked to specific cargoes, and through the actions of additional regulatory proteins. Despite the superficial similarities noted above, there are several differences in the details of nucleotide catalysis, filament and microtubule affinity, and power stroke that show that there are many ways in which to run the same basic engine [11]. Myosins and kinesins have received considerable structural attention, and their mechanisms of action have

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Figure 1. Representation of motor domains from the conventional kinesin, dynein and myosin families. The complete lengths of the cargo-binding tails are not shown, but the motors are drawn to the same scale. The geometry of dynein largely results from the asymmetrical arrangement of seven domains around a hollow core with two functional extensions: a stalk to connect microtubules (MT stalk) and a tail for cargo binding. In the dynein structure, the red sphere suggests the location for the nucleotide catalytic site in AAA domain 1 (AAA-1), and the green sphere at the end of the MT stalk represents the contact site for ATP-sensitive interactions with microtubules.

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been described in detail. From analyses of atomicresolution crystal structures [12,13] and high-resolution electron microscopy (EM) imaging of motor-decorated filaments or microtubules [14–19], it seems clear that both motors generate force through a rotation that pivots the motor relative to a connecting rod (e.g. a swinginglever arm) and moves cargo forward (or sometimes ‘backward’ [20,21]). The sophistication of these studies is impressive, revealing that family members have different ranges of lever-arm activity and that the overall step sizes are a function of how the second motor in a complex is positioned. Substrate affinity is modulated by structural rearrangements within a surface region near the catalytic site that binds the filament or microtubule; for myosins, this rearrangement includes the opening and closing of a cleft. Even more impressive is that the level of structural detail enables custom alterations to affect motor direction or to generate sufficient room in the nucleotide-binding pocket to accommodate nucleotide analogs that act as isoform-specific inhibitors [22–26]. Dynein, by contrast, has long been the odd motor out. Although it was first identified in 1965 as the forceproducing element in axonemes [27] and subsequently as a universal motor that functions in the cytoplasmic compartment [28,29] (Box 1), structural studies directed at understanding how this motor works have been slow to advance. The relatively large size of the dynein molecule

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and the complexities associated with its expression have represented formidable barriers to progress. Molecular genetic investigations have shown that the minimal size necessary for complete motor activity (w380 kDa) is nearly ten times larger than that required for kinesin [30,31], raising the obvious question: why does the dynein motor need to be so big? An initial three-dimensional (3D) reconstruction from EM images suggested that the motor has a ring-like organization formed by roughly seven protein densities that surround a central cavity [32]. Sequence comparisons have indicated that the motor is more likely to have arisen from a different evolutionary ancestor (the AAA ATPase family [33]) than to have shared a common origin with myosin and kinesin [34–36]. In fact, at least six repeated AAA-like domains (denoted AAA-1 to AAA-6) are found in the sequence of the dynein motor. These differences in size, shape and sequence give dynein plenty of potential to move in a manner that is completely distinct from the movement of kinesin or myosin. In the past two years, several studies have begun to unravel the architectural and mechanistic complexities of dynein. EM combined with image processing (Box 2) has provided striking views of its motor activities with much improved resolution. The two-dimensional (2D) analysis of a single-headed inner-arm dynein from Chlamydomonas axonemes has shown, with impressive clarity, different

Box 1. The dynein family of motors Eukaryotic organisms contain upwards of 14 different genes that encode dynein heavy chains (DHCs) [66,67]. Most of the gene products are relegated to axonemal functions and their genes are absent in simple organisms that lack cilia and flagella. Whereas cytoplasmic dynein isoforms (typically 1–2 per organism) are homodimers of the same DHC, axonemal dyneins come in single-headed, dimeric and trimeric variants containing different DHCs (Figure I). The motor domains of all dyneins are generally well conserved in sequence (with 40–80% similarity) and are largely indistinguishable by electron microscopy at the single-particle level. As far as it is known, the motors are thought to function in the same conserved fashion; there are differences in the details, however, including varied rates of motion and motors that impart a torque on the polymer as it moves, and these differences might yet yield structural surprises. The N-terminal one-third of the DHC is responsible for motor assembly,

Cytoplasmic dynein

targeting and binding to cargo (Figure I); not surprisingly, this domain is the most variable region of the DHC. The tail region of cytoplasmic dynein binds several intermediate and lighter mass polypeptides, which in turn link to several different targets. In this way, the same motor might function in many different types of organelle transport. By contrast, axonemal dyneins contain a second, ATP-insensitive, microtubule linkage at their tail to fix their position in the core structure of cilia and flagella. This principal subset of dyneins must work together as a group to effect a single outcome, coordinating their efforts along the length and on both the inner and outer positions of the axonemal subfiber-A microtubule to propagate a bending motion of the axoneme [68]. To complicate matters further, axonemal dyneins also respond to different physiological stimuli to regulate both beat frequency and waveform.

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Figure I. Cartoon representation of the dynein family of motors. The various dynein heavy chains (DHCs) found in axonemal dyneins are represented by distinct colors. The N-terminal one-third of the DHC is shown in black. www.sciencedirect.com

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Box 2. Single-particle image processing of electron micrographs Electron micrographs of macromolecules attached to a carbon film (as in the case of negatively stained samples) or macromolecules suspended in a thin layer of ice (as in cryo-EM), are digitized and individual images of the macromolecule are identified on the computer and windowed out to generate single-particle views, which can be then subjected to two consecutive levels of processing. † Two-dimensional (2D) averaging. To increase the signal-to-noise ratio and to produce a clearer picture of the macromolecule, many such single particles (in this case, dynein) are rotationally aligned to each other and averaged, thereby producing a 2D average. The images contributing to such averages must correspond to the macromolecule lying on the EM grid in the same orientation and conformation. Classification schemes are used to distinguish groups of particles representing different orientations (if present), and thus often generate several 2D averages, each corresponding to a different view or conformation of the particle. † Three-dimensional (3D) reconstruction. To gain information in three dimensions, views of the macromolecule lying on the EM grid in

conformations of both the microtubule-connecting stalk (MT stalk) and the cargo-connecting tail [37]. The 3D reconstruction of the cytoplasmic motor from Dictyostelium has confirmed that the motor core has a seven-domain ring architecture and suggests how the successive AAA domains are arranged [38]. Molecular genetic manipulations are beginning to address the significance of the multiple ATP-binding motifs present in the motor [39,40]. Moreover, recent biophysical analyses of dynein movement suggest that the step size of the motor varies as a function of its load [41]. Taken together, these findings represent a crucial threshold in our understanding of how dynein works. The difficulties inherent in working with a polypeptide the size of dynein are being overcome, and the recent EM data demonstrate that this motor is amenable to structural characterization. Notably, these studies show just how architecturally distinct dynein is from kinesin and myosin and they highlight the similarities and differences that underlie the mechanical properties of motility generation. In this review, we aim to summarize the recent structural information and attempt to develop a working plan of how the dynein motor might work. We focus first on the structural components and then on the mechanical activities of the motor.

different orientations are required. The 3D reconstruction algorithms find the angles that define the different orientations of the macromolecule on the EM grid and combine this information to produce a density map of the protein in three dimensions. A 2D average represents the projection of the protein in one direction, reflecting the orientation of the 3D object on the EM grid. The 2D averages discussed in this review correspond to the projection of dynein oriented either as in Figure 2b (main text) or flipped 1808 respect to that orientation. These are the more frequent orientations that dynein adopts on the EM grid. A 3D reconstruction affords information on the internal structure and surface of the molecule, thereby providing deeper insight into its overall configuration. By using these methods under optimal conditions, macromolecule resolutions of 10–20 A˚ are now common. The tremendous power of the EM and 3D reconstruction methods lies in their ability to generate medium-resolution images of macromolecules or complexes that are not amenable to X-ray or NMR analysis. (For further information on both the methodology and the applications, see Refs [69,70].)

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The dynein ring A single dynein motor domain is huge, with a mass of almost 400 kDa. Most of this mass is contained in seven protein densities that encircle a cavity to produce a ringlike architecture [38] (Figure 2). At least six of the seven densities probably correspond to the previously described AAA domains. The structural nature of the seventh density is unknown, but it could be accounted for by coding sequence on either the amino- (N) or carboxy (C)-terminal sides of the repeated AAA domains [42,43]. Additional mass on the motor forms non-identical cap-like features on the sides of the ring plane, which partially enclose the cavity and perhaps provide stability to the ring. The first attempt at providing atomic-level details of the dynein motor substructure was made by Mocz and Gibbons [42], who developed homology models for each of www.sciencedirect.com

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250 aa Figure 2. Structure of the cytoplasmic dynein motor domain from Dictyostelium determined by electron microscopy (EM) and image processing. (a–d) Images show the same orientation of the structure, but represent a 2D average (a), a 3D reconstruction (b), a 3D reconstruction with a higher threshold to show the ring-like order of the seven densities (c), and proposed fitting of the individual AAA domain models from Ref. [42] (d). In (a), the microtubule-connecting stalk (MT stalk) is weakly visible, protruding off the top of the motor. The numbers in (d) represent the ordering of the six AAA domains (where 1 is AAA-1, 2 is AAA-2, and so on), as well as position of the seventh density (see Ref. [38] for more details). Note that AAA-6 is the shortest domain in sequence and corresponds to the smallest of the densities in (c). (e) Linear diagram of the motor sequence (amino acids 1384–4725 of the dynein heavy chain), indicating the positions of the six AAA domains and the MT stalk (MTB). Vertical red bars indicate the positions of the nucleotide-binding P-loop motifs. Panels (a–e) modified, with permission, from Ref. [38].

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the AAA domains of sea urchin axonemal dynein, which they then docked together to produce a hexameric core for the motor. Superficially, there are striking similarities between the hexameric model and the EM 3D reconstructions, including the existence of an inner cavity, peripheral extensions where the AAA domains fit together and tangential ridges across the ring perimeter [38]. However, most dyneins probably have an additional density on their ring that disrupts the hexameric symmetry. By fitting the AAA models into 3D envelopes of the Dictyostelium motor, a working idea has been proposed for how the AAA domains might be arranged around a heptameric ring [38] (Figure 2d). Although such models are far from conclusive, they generate some interesting points. For example, the sequence of the MT stalk lies between the fourth and the fifth AAA domain. In the 3D map, the two domains that flank the position where the MT stalk merges with the motor must therefore represent domains AAA-4 and AAA-5. Assuming that the AAA domains are arranged in order (Figure 2e), the catalytic domain (AAA-1) must be located on the side of the ring that is directly opposite the MT stalk. This position remains the same whether the numbering of the AAA densities is clockwise or counterclockwise, or whether the seventh density is located at the N or the C terminus of the six AAA motifs (Figure 2). This ring architecture is distinctly different from the single densities that comprise the myosin and kinesin motors, and offers an explanation for why the motor cannot be trimmed into a smaller functional unit. Proper closure of the ring at the N and C termini of the seven densities is evidently necessary to establish a geometry that allows the propagation of conformational changes for movement. Dynein levers In contrast to myosin and kinesin, dynein contains two distinct extended structures that project from the motor (Figure 1). A tail of about 20–30 nm forms the linkage with the cargo-binding region, and a unique stalk of about 10–15 nm supports a small globular tip that forms the ATP-sensitive contact with microtubules [44]. The sequence boundaries of the MT stalk are well marked by conserved proline residues in the heavy chain, which form a distinctive helical-globular-helical motif of about 380 amino acids between domains AAA-4 and AAA-5 [31,35]. The two helices probably form an antiparallel coiled-coil stalk, which positions the small globular tip away from the main mass of the motor [45]. By contrast, the architecture of the cargo-binding tail is not so well resolved. The cargo-binding sequence does not have a strong probability of forming an extended conventional a-helix as it does in myosin or kinesin, and in the EM images of dynein it seems thicker than would be expected for a typical coiled coil. There are, however, interspersed regions of predicted helical structure that could be organized into a higher-order assembly; such a structure could account for both the thickness and the elongated appearance of the cargo-binding tail [46]. Both the extended MT stalk and the extended cargo-binding tail have crucial roles in dynein activity and thus must be functionally integrated into the ring. www.sciencedirect.com

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The dynein engine For most dynein heavy chains, the first four of the six AAA-like domains are relatively well conserved and contain intact phosphate-loop (P-loop) motifs that are essential for binding the g trinucleotide phosphate [47]. In sea urchin axonemal dynein at least, each of the first four AAA domains binds nucleotide [48,49]. Domain AAA-1 binds ATP tightly and constitutes the active catalytic core of the motor [50]. Nucleotide binding in domains AAA-2, AAA-3 and AAA-4 has been generally considered to regulate the activity of dynein rather than to drive the production of force. This consideration is based, in large part, on the lack of significant, measurable catalytic activity at these three sites. Recent work has directly tested this assertion. By mutating the P-loop in AAA-3, Silvanovich et al. [39] produced a Drosophila motor that is capable of nucleotide hydrolysis and microtubule binding, but is apparently unable to undergo release from a microtubule. A similar behavior has been recently demonstrated for Saccharomyces cerevisiae dynein [40], suggesting that the P-loop in AAA-3 in this motor also has an essential role in coordinating nucleotide hydrolysis with microtubule dissociation. However, a mutation in yeast AAA-2 or AAA-4, or surprisingly a triple mutation in AAA-2, AAA-3 and AAA-4, does not produce obvious defects in dynein function in vivo, indicating that only one site (AAA-1) is absolutely required to drive the dynein engine. Finally, there is intriguing evidence from in vitro analyses of Chlamydomonas inner-arm dynein that nucleotide binding (in this case, ADP) at the non-catalytic sites can regulate the speed of microtubule gliding [51]. This result supports previous findings indicating that ADP can affect the ATPase activity and translocation of dynein (e.g. see Ref. [52]). Thus, an important follow up to the yeast mutations might be to measure the microtubule gliding rates and catalytic activities of the yeast mutant dynein or similarly mutated motors in other organisms. Although the dynein engine contains a single catalytic site for force production, it differs from myosin or kinesin by containing at least three additional nucleotide-binding sites that could regulate other aspects of movement. Dynein motor mechanics Dynein has two potential levers, located on opposite sides of a ring, which raises the significant question: which components of the motor undergo movement during nucleotide hydrolysis? This issue has been recently addressed for a single-headed axonemal dynein from Chlamydomonas. Burgess et al. [37] have demonstrated that the MT stalk and the tail undergo impressive movements, with both structural features showing considerable flexibility. From a comparison of two dynein preparations that probably represent the two maximally different states of its catalytic cycle [either lacking nucleotide (post-stroke) or containing ADP–vanadate to mimic an ATP-bound state (pre-stroke)], Burgess et al. concluded that a distinct pivoting occurs around the junction between the motor and the tail (Figure 3). In this way, dynein might act similarly to myosin and kinesin in using its cargo-binding linkage as a lever arm to produce

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Figure 3. Two-dimensional averages of inner-arm axonemal dynein in two distinct catalytic states. (a) Both the tail and the microtubule-connecting stalk (MT stalk) of this single-headed dynein can be clearly seen extending from the core of the motor. The main difference between the two averages, ADP–vanadate bound (pre-stroke; left) and apo (no nucleotide, post-stroke; right), is an apparent pivoting of the motor core with respect to the tail, as shown in (b). Numbers 1–4 indicate an ordering of the nucleotide-binding AAA domains 1–4. It is important to note that their orientation relative to each other might change as a function of nucleotide binding or release. These changes help to drive the movement of the ring relative to the cargo-binding stem attached at AAA-1, in addition to modifying microtubule affinity at the tip of the MT stalk that extends from the junction between AAA domain 4 and domain 5 (not labeled). Abbreviation: Pi, inorganic phosphate. Reproduced, with permission, from Ref. [37].

force. If this conclusion holds true for other dyneins, then it suggests that the three types of cytoskeletal motor share a universal mechanism for force production. If dynein generates force through a tail-based leverarm action, then the mechanism by which the tail engages the catalytic subunit becomes a fundamental issue. First, nothing about dynein has been simple and, to complicate matters further, the tail (at least for the inner-arm isoform) does not seem to emerge directly out of the motor. Rather, it seems to arc across one face of the ring and forms a distinctive, almost hairpin, loop as it extends away from the motor [37] (Figure 3). In some images, the segment lying across the ring face seems to be detached, suggesting that this portion of the tail, or linker region, is tethered in place. Such a tether might provide an interesting twist in models of how force might be directed. This loop is not obvious in other types of dynein (e.g. see Ref. [53]) and could represent a unique feature associated www.sciencedirect.com

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with specific axonemal function or might simply illustrate the difficulties in imaging the fine features of this motor. Second, if the seventh density of the ring were located at the N-terminal side of the AAA domains, then any nucleotide-driven conformational change would have to be transmitted through this density to effect tail rotation. This would seem to be a rather inefficient mechanism to couple force production with the small structural rearrangements associated with nucleotide binding and release. It would be more reasonable if the seventh density were positioned at the C-terminal end of the sequence, thereby providing a more ‘direct-drive’ linkage at the N terminus (see also Ref. [37]). Notably, Mocz and Gibbons [42] have pointed out that in fungal dyneins (from yeasts and filamentous fungi) the heavy chain sequences are shortened, terminating just after the sixth AAA domain. These proteins would thus make interesting structural candidates for comparing the number of ring densities with non-fungal dyneins in order to determine the structural contributions of the C terminus to the ring architecture. On the other side of the ring, Burgess et al. [37] have shown that the MT stalk seems to be flexible. Notably, the range of MT-stalk flexion varies as a function of the nucleotide-bound state of the motor, with an approximate 118 range in conformation in the nucleotide-free condition and a 208 range when ADP–vanadate is bound. By contrast, 2D images of cytoplasmic dynein can be classified into three subsets that reflect distinct orientations of the MT stalk and an overall range of 408 in conformation [40]. This apparent difference between cytoplasmic and axonemal dyneins could be due to several reasons but, at a minimum, the work of both research groups shows that the MT stalk is capable of movement relative to the bulk of the motor domain. Distinguishing between MT-stalk flexibility and active positioning is an important issue that needs to be resolved because it will indicate whether this feature also enhances force production or step size, or simply functions as a mechanism for microtubule binding. Linking motor activity to microtubule affinity In addition to forward movement, the motor must coordinate nucleotide catalysis with substrate-binding activity. In this aspect, dynein has a far greater hurdle to overcome than do the other two motors: changes in the catalytic site must be propagated over a much greater distance, around the ring, through several globular densities to alter the relative positions between domains AAA-4 and AAA-5. Such structural changes will probably affect the proximal architecture of the coiled coil that forms the MT stalk. But how these changes are propagated another 10–15 nm along the length of the coiled coil to modify microtubule affinity at the distal tip (e.g. by local unwinding or changes in helical pitch) is unknown. Finally, what happens at the tip to confer tight or weak microtubule-binding states is also unknown. Site-directed mutagenesis of the tip region has shown that the microtubule interaction is sensitive to perturbations in local structure [54], suggesting that even slight changes to the coiled coil could be sufficient to modify microtubule affinity. Thus, although dynein, in common with myosin or kinesin, might generate force through a swinging lever-arm action, its substrate-binding

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Figure 4. Gallery of negatively stained, native cytoplasmic dynein molecules. The motor domains (pseudocolored blue) are connected to the cargo-binding tail domain (yellow) by apparently flexible stems (green) that also constitute the force-producing lever arm. Note that the microtubule-connecting stalk (MT stalk) is not visible above background staining, highlighting a potential difference in diameter between the MT stalk and the tail levers. Scale bar, 20 nm.

mechanism, which acts through an extended coiled-coil, is significantly different. Ironically (or perhaps by design), dynein’s footprint on the microtubule seems to occur in the same region as kinesin’s footprint [55]. Cryo-EM imaging of the distal tip of the MT stalk reveals that it binds to both a- and b-tubulin at the protofilament junction of the microtubule, in the same location as kinesin. This overlap is supported by biochemical competition and crosslinking experiments, and suggests that even microtubules might be subject to occasional traffic jams. Beyond the motor: taking a step The lever-arm rotation described for the inner-arm dynein could accommodate a potential nucleotide-dependent displacement of up to 15 nm [37] and could easily account for the 8-nm steps of the same motor, which have been measured under load in an optical trap [56]. At least one other axonemal dynein [57] and a cytoplasmic dynein [41] have also been measured under load to make 8-nm steps along a microtubule – a distance that is similar to the measured steps of kinesin (8 nm) and close to the steps of some myosins (5 nm) [4]. Notably, under no-load conditions, the same cytoplasmic dynein seems to move predominantly by a combination of 24- and 32-nm steps, implying that the motor mechanics are sensitive to pulling force [41]. For the unusual myosin V, even larger-sized steps (36–37 nm) have been reported [58] that require an extended lever arm of 24 nm and an angular change of up to 1008. However, no analogous load-dependent variations in step size have been reported for kinesin or myosin. Mallik et al. [41] have suggested that coordination between the pulling force and nucleotide occupancy in domains AAA-2, AAA-3 and AAA-4 alters the geometry of the power stroke of dynein. It is difficult to understand how nucleotide binding in a motor of dynein’s dimensions (14!12!9 nm3) alone can effect a 24-nm variation in step size; thus, the alteration must indeed have an effect on the angular range or length of any lever-arm action. Perhaps unappreciated for dynein are the length and flexibility of the tail region that forms at least part of the lever arm. Whereas kinesin and myosin both have definite neck regions that limit and coordinate the movement of their heads, this is not the case for dynein. A typical EM preparation of native, two-headed dynein shows that the linked heads can lie in various orientations, indicating that there is tremendous flexibility in the shape of the native molecule [53,59,60] (Figure 4). Some hints of this www.sciencedirect.com

difference between kinesin and dynein might have been seen in earlier in vitro work that compared the tracks that these two motors make along a microtubule (either straight along a protofilament or wobbling between two or three protofilaments, respectively [61,62]). The dynein wobble might reflect the structural flexibility of the tail connections or MT stalk, and it would be interesting to determine whether it is reduced under load. Thus, for dynein there are several issues that might affect its step size: the geometry of the motor when bound to its cargo, the load that the motor is pulling against, and even the binding of accessory proteins. For example, Lis-1, a protein that is known to regulate some of the activities of dynein, has been shown to bind the heavy chain in a region that could indeed influence a crucial neck-like feature to limit or to stabilize the lever-arm motion of the tail [63]. Concluding remarks The recent convergence of genetic, EM, single-molecule, biophysical and cell imaging studies have finally given rise to a testable blueprint for dynein, one in which nucleotide hydrolysis gives rise to conformational changes that move the tail in a lever-arm fashion – akin to the movement in myosin and kinesin – to produce the motive force. Coupled with this force, structural changes must also propagate around the ring to modify the affinity of dynein for microtubules. The physical distance between the catalytic site and microtubule action is significantly greater and its linkage is mechanically more complex in dynein than in myosin or kinesin. The ring mechanics are probably influenced by nucleotide occupancy in domains AAA-2, AAA-3 and AAA-4, which might affect the rate or the extent of the conformational changes. Thus, the potential exists in dynein to regulate microtubule interaction at a site that is separate from the catalytic cycle at AAA-1 – a feature that distinguishes dynein from myosin or kinesin. Although the tail seems flexible on an EM grid, it could interact with several regulatory or even cargo-binding proteins in situ to achieve a stiffness that determines or modulates its step size. If the step size of dynein varies as a function of load in vitro, then the distance covered per cycle of nucleotide hydrolysis might also vary in situ. Unlike what has been reported for kinesin or myosin, perhaps a single dynein motor can run at variable speeds, depending on its load and level of activation. With regard to the future, an immediate wish list for dynein includes results from studies applying cryo-EM, domain-tagging and single-molecule methods to modified

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motors. In the absence of an atomic structure, the extension of dynein imaging to ice-embedded samples should lead to a significant enhancement in the resolution of 3D reconstructions; the crucial advantage of cryo-EM lies in its preservation of the native structure of the molecule in an unstained or unperturbed condition. Work by Burgess et al. [64] and others [55] (M. Samso´ and M.P. Koonce, unpublished) has shown that dynein does have sufficient density and structural asymmetry to facilitate image processing of unstained molecules. The placement and localization of sequence-specific tags on the motor will be important for confirming the ordering of the ring densities. The use of motor tags will also provide more-selective ‘handles’ for single-molecule work. Both structural and biophysical approaches will lead to a better understanding of how the ring is organized and will enable the derivation of details of how the motor moves during nucleotide hydrolysis. Examination of the step sizes of motors with mutated AAA domains should help to distinguish between the mechanical contributions of a tail-based lever arm and the linkage between AAA-1 and the MTstalk. Finally, a global understanding of whether the two heads in a dimer coordinate their activities [65] and whether pulling a load forces the motor to straighten out along a protofilament will be crucial for resolving the apparent paradox of how a flexible tail connection could serve as a force-producing lever arm. All in all, it is an interesting time for the dynein motor.

Acknowledgements We are grateful to Alexey Khodjakov and Adriana Verschoor for critically reading the manuscript. Work in our laboratory is supported by a grant from the National Institutes of Health (GM51532) to M.P.K.

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