Design and regulation of the AAA+ microtubule motor dynein

Dyneins are highly complex molecular motors that transport their attached cargo towards the minus end of microtubules. These enzymes are required for many essential motile activities within the cytoplasm and also power eukaryotic cilia and flagella. Each dynein contains one or more heavy chain motor units that consist of an N-terminal stem domain that is involved in cargo attachment, and six AAA+ domains (AAA1-6) plus a C-terminal globular segment that are arranged in a heptameric ring. At least one AAA+ domain (AAA1) is capable of ATP binding and hydrolysis, and the available data suggest that one or more additional domains also may bind nucleotide. The ATP-sensitive microtubule binding site is located at the tip of a 10nm coiled coil stalk that emanates from between AAA4 and AAA5. The function of this motor both in the cytoplasm and the flagellum must be tightly regulated in order to result in useful work. Consequently, dyneins also contain a series of additional components that serve to define the cargo-binding properties of the enzyme and which act as sensors to transmit regulatory inputs to the motor units. Here we describe the two basic dynein designs and detail the various regulatory systems that impinge on this motor within the eukaryotic flagellum.     

Structural insights into microtubule doublet interactions in axonemes

Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of eukaryotic cilia and flagella. Recent structural studies of the axoneme, which forms the core of cilia and flagella, have used cryo-electron tomography to reveal new details of the interactions between some of the multitude of proteins that form the axoneme and regulate its movement. Connections between the several types of dyneins, in particular, suggest ways in which their action might be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding mechanical properties related to the bending of the axoneme, and has also offered insight into the potential role of doublets in the mechanism of dynein activity regulation.     

Heterogeneity of dynein structure implies coordinated suppression of dynein motor activity in the axoneme

Axonemal dyneins provide the driving force for flagellar/ciliary bending. Nucleotide-induced conformational changes of flagellar dynein have been found both in vitro and in situ by electron microscopy, and in situ studies demonstrated the coexistence of at least two conformations in axonemes in the presence of nucleotides (the apo and the nucleotide-bound forms). The distribution of the two forms suggested cooperativity between adjacent dyneins on axonemal microtubule doublets. Although the mechanism of such cooperativity is unknown it might be related to the mechanism of bending. To explore the mechanism by which structural heterogeneity of axonemal dyneins is induced by nucleotides, we used cilia from Tetrahymena thermophila to examine the structure of dyneins in a) the intact axoneme and b) microtubule doublets separated from the axoneme, both with and without additional pure microtubules. We also employed an ATPase assay on these specimens to investigate dynein activity functionally. Dyneins on separated doublets show more activation by nucleotides than those in the intact axoneme, both structurally and in the ATPase assay, and this is especially pronounced when the doublets are coupled with added microtubules, as expected. Paralleling the reduced ATPase activity in the intact axonemes, a lower proportion of these dyneins are in the nucleotide-bound form. This indicates a coordinated suppression of dynein activity in the axoneme, which could be the key for understanding the bending mechanism.     

Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.     

Motor Regulation Results in Distal Forces that Bend Partially Disintegrated Chlamydomonas Axonemes into Circular Arcs

The bending of cilia and flagella is driven by forces generated by dynein motor proteins. These forces slide adjacent microtubule doublets within the axoneme, the motile cytoskeletal structure. To create regular, oscillatory beating patterns, the activities of the axonemal dyneins must be coordinated both spatially and temporally. It is thought that coordination is mediated by stresses or strains, which build up within the moving axoneme, and somehow regulate dynein activity. During experimentation with axonemes subjected to mild proteolysis, we observed pairs of doublets associating with each other and forming bends with almost constant curvature. By modeling the statics of a pair of filaments, we show that the activity of the motors concentrates at the distal tips of the doublets. Furthermore, we show that this distribution of motor activity accords with models in which curvature, or curvature-induced normal forces, regulates the activity of the motors. These observations, together with our theoretical analysis, provide evidence that dynein activity can be regulated by curvature or normal forces, which may, therefore, play a role in coordinating the beating of cilia and flagella.     

Computer simulation of flagellar movement VIII: coordination of dynein by local curvature control can generate helical bending waves

Computer simulations have been carried out with a model flagellum that can bend in three dimensions. A pattern of dynein activation in which regions of dynein activity propagate along each doublet, with a phase shift of approximately 1/9 wavelength between adjacent doublets, will produce a helical bending wave. This pattern can be termed "doublet metachronism." The simulations show that doublet metachronism can arise spontaneously in a model axoneme in which activation of dyneins is controlled locally by the curvature of each outer doublet microtubule. In this model, dyneins operate both as sensors of curvature and as motors. Doublet metachronism and the chirality of the resulting helical bending pattern are regulated by the angular difference between the direction of the moment and sliding produced by dyneins on a doublet and the direction of the controlling curvature for that doublet. A flagellum that is generating a helical bending wave experiences twisting moments when it moves against external viscous resistance. At high viscosities, helical bending will be significantly modified by twist unless the twist resistance is greater than previously estimated. Spontaneous doublet metachronism must be modified or overridden in order for a flagellum to generate the planar bending waves that are required for efficient propulsion of spermatozoa. Planar bending can be achieved with the three-dimensional flagellar model by appropriate specification of the direction of the controlling curvature for each doublet. However, experimental observations indicate that this "hard-wired" solution is not appropriate for real flagella.     

Autoinhibitory and other autoregulatory elements within the dynein motor domain

The dyneins are a family of microtubule motor proteins. The motor domain, which represents the C-terminal 2/3 of the dynein heavy chain, exhibits homology to the AAA family of ATPases. It consists of a ring of six related but divergent AAA+ units, with two substantial sized protruding projections, the stem, or tail, which anchors the protein to diverse subcellular sites, and the stalk, which binds microtubules. This article reviews recent efforts to probe the mechanism by which the dyneins produce force, and work from the authors' lab regarding long-range conformational regulation of dynein enzymatic activity.     

Thinking about flagellar oscillation

Bending of cilia and flagella results from sliding between the microtubular outer doublets, driven by dynein motor enzymes. This review reminds us that many questions remain to be answered before we can understand how dynein-driven sliding causes the oscillatory bending of cilia and flagella. Does oscillation require switching between two distinct, persistent modes of dynein activity? Only one mode, an active forward mode, has been characterized, but an alternative mode, either inactive or reverse, appears to be required. Does switching between modes use information from curvature, sliding direction, or both? Is there a mechanism for reciprocal inhibition? Can a localized capability for oscillatory sliding become self-organized to produce the metachronal phase differences required for bend propagation? Are interactions between adjacent dyneins important for regulation of oscillation and bend propagation? Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.     

Discrete PIH proteins function in the cytoplasmic preassembly of different subsets of axonemal dyneins

Axonemal dyneins are preassembled in the cytoplasm before being transported into cilia and flagella. Recently, PF13/KTU, a conserved protein containing a PIH (protein interacting with HSP90) domain, was identified as a protein responsible for dynein preassembly in humans and Chlamydomonas reinhardtii. This protein is involved in the preassembly of outer arm dynein and some inner arm dyneins, possibly as a cofactor of molecular chaperones. However, it is not known which factors function in the preassembly of other inner arm dyneins. Here, we analyzed a novel C. reinhardtii mutant, ida10, and found that another conserved PIH family protein, MOT48, is responsible for the formation of another subset of inner arm dyneins. A variety of organisms with motile cilia and flagella typically have three to four PIH proteins, including potential homologues of MOT48 and PF13/KTU, whereas organisms without them have no, or only one, such protein. These findings raise the possibility that multiple PIH proteins are commonly involved in the preassembly of different subsets of axonemal dyneins.     

Structural-functional relationships of the dynein,spokes, and central-pair projections predicted from an analysis of the forces acting within a flagellum

In the axoneme of eukaryotic flagella the dynein motor proteins form crossbridges between the outer doublet microtubules. These motor proteins generate force that accumulates as linear tension, or compression, on the doublets. When tension or compression is present on a curved microtubule, a force per unit length develops in the plane of bending and is transverse to the long axis of the microtubule. This transverse force (t-force) is evaluated here using available experimental evidence from sea urchin sperm and bull sperm. At or near the switch point for beat reversal, the t-force is in the range of 0.25-1.0 nN/ micro m, with 0.5 nN/ micro m the most likely value. This is the case in both beating and arrested bull sperm and in beating sea urchin sperm. The total force that can be generated (or resisted) by all the dyneins on one micron of outer doublet is also approximately 0.5 nN. The equivalence of the maximum dynein force/ micro m and t-force/ micro m at the switch point may have important consequences. Firstly, the t-force acting on the doublets near the switch point of the flagellar beat is sufficiently strong that it could terminate the action of the dyneins directly by strongly favoring the detached state and precipitating a cascade of detachment from the adjacent doublet. Secondly, after dynein release occurs, the radial spokes and central-pair apparatus are the structures that must carry the t-force. The spokes attached to the central-pair projections will bear most of the load. The central-pair projections are well-positioned for this role, and they are suitably configured to regulate the amount of axoneme distortion that occurs during switching. However, to fulfill this role without preventing flagellar bend formation, moveable attachments that behave like processive motor proteins must mediate the attachment between the spoke heads and the central-pair structure.     

Polarity and asymmetry in the arrangement of dynein and related structures in the Chlamydomonas axoneme

Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer-inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.     

One of the Nine Doublet Microtubules of Eukaryotic Flagella Exhibits Unique and Partially Conserved Structures

The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.     

The role of the dynein stalk in cytoplasmic and flagellar motility

We have recently identified a microtubule binding domain within the motor protein cytoplasmic dynein. This domain is situated at the end of a slender 10–12 nm projection which corresponds to the stalks previously observed extending from the heads of both axonemal and cytoplasmic dyneins. The stalks also correspond to the B-links observed to connect outer arm axonemal dyneins to the B-microtubules in flagella and constitute the microtubule attachment sites during dynein motility. The stalks contrast strikingly with the polymer attachment domains of the kinesins and myosins which are found on the surface of the motor head. The difference in dynein's structural design raises intriguing questions as to how the stalk functions in force production along microtubules. In this article, we attempt to integrate the myriad of biochemical and EM structural data that has been previously collected regarding dynein with recent molecular findings, in an effort to begin to understand the mechanism of dynein motility. Received: 13 March 1998 / Revised version: 17 April 1998 / Accepted: 17 April 1998     

The covalent oscillator: A paradigm accounting for the sliding/bending mechanism and wave propagation in cilia and flagella

Summary— In most models of wave propagation in eucaryotic flagella and cilia, a clear distinction is made between the dynein dependent microtubule sliding which represents the oscillatory motor and the bending mechanism which regulates wave propagation. Little is known about the physical elements regulating the latter: in the present model, the bending propagation is postulated to be supported by an open/close cyclic mechanism protease/ligase dependent, which involves transient covalent links between adjacent microtubular doublets; this open/close cycle propagates in register with the powering action of the dynein motor along the exoneme. The implications of the model are discussed in relation to previous data which involve protease/ligase in the axonemal function as well as other data which can be integrated by the proposed model.     

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.     

Domain stability in the AAA+ ATPase ClpB from Escherichia coli

ClpB is a heat-shock protein that reactivates aggregated proteins in cooperation with the DnaK chaperone system. ClpB belongs to the family of AAA+ ATPases and forms ring-shaped oligomers: heptamers in the absence of nucleotides and hexamers in the presence of nucleotides. We investigated the thermodynamic stability of ClpB in its monomeric and oligomeric forms. ClpB contains six distinct structural domains: the N-terminal domain involved in substrate binding, two AAA+ ATP-binding modules, each consisting of two domains, and a coiled-coil domain inserted between the AAA+ modules. We produced seven variants of ClpB, each containing a single Trp located in each of the ClpB domains and measured the changes in Trp fluorescence during the equilibrium urea-induced unfolding of ClpB. We found that two structural domains: the small domain of the C-terminal AAA+ module and the coiled-coil domain were destabilized in the oligomeric form of ClpB, which indicates that only those domains change their conformation and/or interactions during formation of the ClpB rings.     

Testing the geometric clutch hypothesis

The Geometric Clutch hypothesis is based on the premise that transverse forces (t-forces) acting on the outer doublets of the eukaryotic axoneme coordinate the action of the dynein motors to produce flagellar and ciliary beating. T-forces result from tension and compression on the outer doublets when a bend is present on the flagellum or cilium. The t-force acts to pry the doublets apart in an active bend, and push the doublets together when the flagellum is passively bent and thus could engage and disengage the dynein motors. Computed simulations of this working mechanism have reproduced the beating pattern of simple cilia and flagella, and of mammalian sperm. Cilia-like beating, with a clearly defined effective and recovery stroke, can be generated using one uniformly applied switching algorithm. When the mechanical properties and dimensions appropriate to a specific flagellum are incorporated into the model the same algorithm can simulate a sea urchin or bull sperm-like beat. The computed model reproduces many of the observed behaviors of real flagella and cilia. The model can duplicate the results of outer arm extraction experiments in cilia and predicted two types of arrest behavior that were verified experimentally in bull sperm. It also successfully predicted the experimentally determined nexin elasticity. Calculations based on live and reactivated sea urchin and bull sperm yielded a value of 0.5 nN/microm for the t-force at the switch-point. This is a force sufficient to overcome the shearing force generated by all the dyneins on one micron of outer doublet. A t-force of this magnitude should produce substantial distortion of the axoneme at the switch-point, especially in spoke or spoke-head deficient motile flagella. This concrete and verifiable prediction is within the grasp of recent advances in imaging technology, specifically cryoelectron microscopy and atomic force microscopy.     

Clockwise translocation of microtubules by flagellar inner-arm dyneins in vitro

Cilia and flagella are equipped with multiple species of dyneins that have diverse motor properties. To assess the properties of various axonemal dyneins of Chlamydomonas, in vitro microtubule translocation by isolated dyneins was examined with and without flow of the medium. With one inner-arm dynein species, dynein c, most microtubules became aligned parallel to the flow and translocated downstream after the onset of flow. When the flow was stopped, the gliding direction was gradually randomized. In contrast, with inner-arm dyneins d and g, microtubules tended to translocate at a shallow right angle to the flow. When the flow was stopped, each microtubule turned to the right, making a curved track. The clockwise translocation was not accompanied by lateral displacement, indicating that these dyneins generate torque that bends the microtubule. The torque generated by these dyneins in the axoneme may modulate the relative orientation between adjacent doublet microtubules and lead to more efficient functioning of total dyneins.     

Equations of Interdoublet Separation during Flagella Motion Reveal Mechanisms of Wave Propagation and Instability

The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models.     

Equations of Interdoublet Separation during Flagella Motion Reveal Mechanisms of Wave Propagation and Instability

The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models.