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.     

Dynein heavy chain isoforms and axonemal motility

The translocation of dynein along microtubules is the basis for a wide variety of essential cellular movements. Dynein was first discovered in the ciliary axoneme, where it causes the directed sliding between outer doublet microtubules that underlies ciliary bending. The initiation and propagation of ciliary bends are produced by a precisely located array of different dyneins containing eight or more different dynein heavy chain isoforms. The detailed clarification of the structural and functional diversity of axonemal dynein heavy chains will not only provide the key to understanding how cilia function, but also give insights applicable to the study of non-axonemal microtubule motors.     

CCDC151 Mutations Cause Primary Ciliary Dyskinesia by Disruption of the Outer Dynein Arm Docking Complex Formation

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151^ts272a and mouse Ccdc151^Snbl mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.     

Mice deficient in the axonemal protein Tektin-t exhibit male infertility and immotile-cilium syndrome due to impaired inner arm dynein function

The haploid germ cell-specific Tektin-t protein is a member of the Tektin family of proteins that form filaments in flagellar, ciliary, and axonemal microtubules. To investigate the physiological role of Tektin-t, we generated mice with a mutation in the tektin-t gene. The homozygous mutant males were infertile, while the females were fully fertile. Sperm morphology and function were abnormal, with frequent bending of the sperm flagella and marked defects in motility. In vitro fertilization assays showed that the defective spermatozoa were able to fertilize eggs. Electron microscopic examination showed that the dynein inner arm structure was disrupted in the sperm flagella of tektin-t-deficient mice. Furthermore, homozygous mutant mice had functionally defective tracheal cilia, as evidenced by altered dynein arm morphology. These results indicate that Tektin-t participates in dynein inner arm formation or attachment and that the loss of Tektin-t results in impaired motility of both flagella and cilia. Therefore, the tektin-t gene is one of the causal genes for immotile-cilium syndrome/primary ciliary dyskinesia.     

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.     

Kinesin motors and primary cilia

Cilia and flagella play important roles in human health by contributing to cellular motility as well as sensing and responding to environmental cues. Defects in ciliary assembly and/or function can lead to a range of human diseases, collectively known as the ciliopathies, including polycystic kidney, liver and pancreatic diseases, sterility, obesity, situs inversus, hydrocephalus and retinal degeneration. A basic understanding of how cilia form and function is essential for deciphering ciliopathies and generating therapeutic treatments. The cilium is a unique compartment that contains a distinct complement of protein and lipid. However, the molecular mechanisms by which soluble and membrane protein components are targeted to and trafficked into the cilium are not well understood. Cilia are generated and maintained by IFT (intraflagellar transport) in which IFT cargoes are transported along axonemal microtubules by kinesin and dynein motors. A variety of genetic, biochemical and cell biological approaches has established the heterotrimeric kinesin-2 motor as the 'core' IFT motor, whereas other members of the kinesin-2, kinesin-3 and kinesin-4 families function as 'accessory' motors for the transport of specific cargoes in diverse cell types. Motors of the kinesin-9 and kinesin-13 families play a non-IFT role in regulating ciliary beating or axonemal length, respectively. Entry of kinesin motors and their cargoes into the ciliary compartment requires components of the nuclear import machinery, specifically importin-β2 (transportin-1) and Ran-GTP (Ran bound to GTP), suggesting that similar mechanisms may regulate entry into the nuclear and ciliary compartments.     

A monoclonal antibody against the dynein IC1 peptide of sea urchin spermatozoa inhibits the motility of sea urchin, dinoflagellate, and human flagellar axonemes.

To investigate the role of axonemal components in the mechanics and regulation of flagellar movement, we have generated a series of monoclonal antibodies (mAb) against sea urchin (Lytechinus pictus) sperm axonemal proteins, selected for their ability to inhibit the motility of demembranated sperm models. One of these antibodies, mAb D1, recognizes an antigen of 142 kDa on blots of sea urchin axonemal proteins and of purified outer arm dynein, suggesting that it acts by binding to the heaviest intermediate chain (IC1) of the dynein arm. mAb D1 blocks the motility of demembranated sea urchin spermatozoa by modifying the beating amplitude and shear angle without affecting the ATPase activity of purified dynein or of demembranated immotile spermatozoa. Furthermore, mAb D1 had only a marginal effect on the velocity of sliding microtubules in trypsin-treated axonemes. This antibody was also capable of inhibiting the motility of flagella of Oxyrrhis marina, a primitive dinoflagellate, and those of demembranated human spermatozoa. Localization of the antigen recognized by mAb D1 by immunofluorescence reveals its presence on the axonemes of flagella from sea urchin spermatozoa and O. marina but not on the cortical microtubule network of the dinoflagellate. These results are consistent with a dynamic role for the dynein intermediate chain IC1 in the bending and/or wave propagation of flagellar axonemes.     

The Motility of Axonemal Dynein Is Regulated by the Tubulin Code

Microtubule diversity, arising from the utilization of different tubulin genes and from posttranslational modifications, regulates many cellular processes including cell division, neuronal differentiation and growth, and centriole assembly. In the case of cilia and flagella, multiple cell biological studies show that microtubule diversity is important for axonemal assembly and motility. However, it is not known whether microtubule diversity directly influences the activity of the axonemal dyneins, the motors that drive the beating of the axoneme, nor whether the effects on motility are indirect, perhaps through regulatory pathways upstream of the motors, such as the central pair, radial spokes, or dynein regulatory complex. To test whether microtubule diversity can directly regulate the activity of axonemal dyneins, we asked whether in vitro acetylation or deacetylation of lysine 40 (K40), a major posttranslational modification of α-tubulin, or whether proteolytic cleavage of the C-terminal tail (CTT) of α- and β-tubulin, the location of detyrosination, polyglutamylation, and polyglycylation modifications as well as most of the genetic diversity, can influence the activity of outer arm axonemal dynein in motility assays using purified proteins. By quantifying the motility with displacement-weighted velocity analysis and mathematically modeling the results, we found that K40 acetylation increases and CTTs decrease axonemal dynein motility. These results show that axonemal dynein directly deciphers the tubulin code, which has important implications for eukaryotic ciliary beat regulation.     

Rotation and translocation of microtubules in vitro induced by dyneins from Tetrahymena cilia

Dynein, the force-generating enzyme that powers the movement of cilia and flagella, has been characterized biochemically, but no simple system has been available for examining its motile properties. Here we describe a quantitative in vitro motility assay in which dynein adsorbed onto a glass surface induces linear translocation of purified bovine microtubules. Using this assay, we show that both 22S and 14S dyneins from Tetrahymena cilia induce movement but have distinct motile properties. A unique property of 14S dynein, which has not been described for other motility proteins, is its ability to generate torque that causes microtubules to rotate during forward translocation. In the axoneme, 14S dynein-induced torque may induce rotation of central-pair microtubules and may play an important role in generating three-dimensional ciliary beating patterns.     

An Outer Arm Dynein Conformational Switch Is Required for Metachronal Synchrony of Motile Cilia in Planaria

Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.     

Remodeling and activation mechanisms of outer arm dyneins revealed by cryo‐EM

Cilia are thin microtubule‐based protrusions of eukaryotic cells. The swimming of ciliated protists and sperm cells is propelled by the beating of cilia. Cilia propagate the flow of mucus in the trachea and protect the human body from viral infections. The main force generators of ciliary beating are the outer dynein arms (ODAs) which attach to the doublet microtubules. The bending of cilia is driven by the ODAs'' conformational changes caused by ATP hydrolysis. Here, we report the native ODA complex structure attaching to the doublet microtubule by cryo‐electron microscopy. The structure reveals how the ODA complex is attached to the doublet microtubule via the docking complex in its native state. Combined with coarse‐grained molecular dynamic simulations, we present a model of how the attachment of the ODA to the doublet microtubule induces remodeling and activation of the ODA complex.     

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.     

CFAP53 regulates mammalian cilia-type motility patterns through differential localization and recruitment of axonemal dynein components

Motile cilia can beat with distinct patterns, but how motility variations are regulated remain obscure. Here, we have studied the role of the coiled-coil protein CFAP53 in the motility of different cilia-types in the mouse. While node (9+0) cilia of Cfap53 mutants were immotile, tracheal and ependymal (9+2) cilia retained motility, albeit with an altered beat pattern. In node cilia, CFAP53 mainly localized at the base (centriolar satellites), whereas it was also present along the entire axoneme in tracheal cilia. CFAP53 associated tightly with microtubules and interacted with axonemal dyneins and TTC25, a dynein docking complex component. TTC25 and outer dynein arms (ODAs) were lost from node cilia, but were largely maintained in tracheal cilia of Cfap53^-/- mice. Thus, CFAP53 at the base of node cilia facilitates axonemal transport of TTC25 and dyneins, while axonemal CFAP53 in 9+2 cilia stabilizes dynein binding to microtubules. Our study establishes how differential localization and function of CFAP53 contributes to the unique motion patterns of two important mammalian cilia-types.     

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.     

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.     

A model for the oscillatory motion of single dynein molecules

Axonemal dynein is the molecular motor responsible for the rhythmic beating of eukaryotic cilia and flagella. An individual axonemal dynein molecule is capable of both unidirectional and oscillatory motion along a microtubule (Nature 393 (1998) 711). We propose a model which links the physical motion of a two-headed dynein molecule to its ATP hydrolysis cycle, and which exhibits both processive and oscillatory behaviors. A mathematical analysis of the model is used to make experimentally testable predictions.     

Functional architecture of the outer arm dynein conformational switch

Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia.     

The motor activity of mammalian axonemal dynein studied in situ on doublet microtubules

Flagellar dynein generates forces that produce relative shearing between doublet microtubules in the axoneme; this drives propagated bending of flagella and cilia. To better understand dynein's role in coordinated flagellar and ciliary motion, we have developed an in situ assay in which polymerized single microtubules glide along doublet microtubules extruded from disintegrated bovine sperm flagella at a pH of 7.8. The exposed, active dynein remain attached to their respective doublet microtubules, allowing gliding of individual microtubules to be observed in an environment that allows direct control of chemical conditions. In the presence of ATP, translocation of microtubules by dynein exhibits Michaelis-Menten type kinetics, with V(max) = 4.7 +/- 0.2 microm/s and K(m) = 124 +/- 11 microM. The character of microtubule translocation is variable, including smooth gliding, stuttered motility, oscillations, buckling, complete dissociation from the doublet microtubule, and occasionally movements reversed from the physiologic direction. The gliding velocity is independent of the number of dynein motors present along the doublet microtubule, and shows no indication of increased activity due to ADP regulation. These results reveal fundamental properties underlying cooperative dynein activity in flagella, differences between mammalian and non-mammalian flagellar dynein, and establish the use of natural tracks of dynein arranged in situ on the doublet microtubules of bovine sperm as a system to explore the mechanics of the dynein-microtubule interactions in mammalian flagella.