Simulation of Cyclic Dynein-Driven Sliding, Splitting, and Reassociation in an Outer Doublet Pair

A regular cycle of dynein-driven sliding, doublet separation, doublet reassociation, and resumption of sliding was previously observed by Aoyama and Kamiya in outer doublet pairs obtained after partial dissociation of Chlamydomonas flagella. In the work presented here, computer programming based on previous simulations of oscillatory bending of microtubules was extended to simulate the cycle of events observed with doublet pairs. These simulations confirm the straightforward explanation of this oscillation by inactivation of dynein when doublets separate and resumption of dynein activity after reassociation. Reassociation is augmented by a dynein-dependent “adhesive force” between the doublets. The simulations used a simple mathematical model to generate velocity-dependent shear force, and an independent elastic model for adhesive force. Realistic results were obtained with a maximum adhesive force that was 36% of the maximum shear force. Separation between a pair of doublets is the result of a buckling instability that also initiates a period of uniform sliding that enlarges the separation. A similar instability may trigger sliding initiation events in flagellar bending cycles.     

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.     

Effects of imposed bending on microtubule sliding in sperm flagella

The movement of eukaryotic flagella is characterized by its oscillatory nature. In sea urchin sperm, for example, planar bends are formed in alternating directions at the base of the flagellum and travel toward the tip as continuous waves. The bending is caused by the orchestrated activity of dynein arms to induce patterned sliding between doublet microtubules of the flagellar axoneme. Although the mechanism regulating the dynein activity is unknown, previous studies have suggested that the flagellar bending itself is important in the feedback mechanism responsible for the oscillatory bending. If so, experimentally bending the microtubules would be expected to affect the sliding activity of dynein. Here we report on experiments with bundles of doublets obtained by inducing sliding in elastase-treated axonemes. Our results show that bending not only "switches" the dynein activity on and off but also affects the microtubule sliding velocity, thus supporting the idea that bending is involved in the self-regulatory mechanism underlying flagellar oscillation.     

Self-Sustained Oscillatory Sliding Movement of Doublet Microtubules and Flagellar Bend Formation

It is well established that the basis for flagellar and ciliary movements is ATP-dependent sliding between adjacent doublet microtubules. However, the mechanism for converting microtubule sliding into flagellar and ciliary movements has long remained unresolved. The author has developed new sperm models that use bull spermatozoa divested of their plasma membrane and midpiece mitochondrial sheath by Triton X-100 and dithiothreitol. These models enable the observation of both the oscillatory sliding movement of activated doublet microtubules and flagellar bend formation in the presence of ATP. A long fiber of doublet microtubules extruded by synchronous sliding of the sperm flagella and a short fiber of doublet microtubules extruded by metachronal sliding exhibited spontaneous oscillatory movements and constructed a one beat cycle of flagellar bending by alternately actuating. The small sliding displacement generated by metachronal sliding formed helical bends, whereas the large displacement by synchronous sliding formed planar bends. Therefore, the resultant waveform is a half-funnel shape, which is similar to ciliary movements.     

Computer simulation of bend propagation by axoplasmic microtubules

The generation of bending waves by microtubules in squid nerve axoplasm has been modelled using appropriately modified versions of computer programs developed previously for simulation of flagellar bending waves. The results confirm that a constant longitudinal force directed along the axis of the microtubule is sufficient to cause the generation of regular oscillations and propagated bending waves when the forward gliding movement of the microtubule is obstructed. No control mechanism is required to modulate the active force-generating system. In order to obtain bending waves similar to those observed experimentally, it was necessary to use a model for the force-generating system in which the active force decreases with increasing sliding velocity. If the elastic bending resistance of axoplasmic microtubules is similar to that of microtubules in sperm terminal filaments, the longitudinal force per unit length generated by the axoplasmic microtubules must be of the same order of magnitude as the force generated by dynein arms along the doublet microtubules of eukaryotic flagella.     

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.     

Effects of various treatments on microtubules and axial units of lung-fluke spermatozoa

Summary Sperm of the frog lung-fluke, Haematoloechus medioplexus, were treated in various ways and their microtubules and axial units were subsequently studied in sectioned and negatively-stained material. Microtubules and axial units were generally unaffected by exposure to colchicine, cold, and KCl, although with KCl certain lateral projections from doublet tubule A appeared more prominent in negatively-stained preparations. Both mercaptoethanol and urea have a dissociative effect on doublet tubules and microtubules, with doublet tubules being the more sensitive. Pepsin-HCl initially digests the dense region associated with the A tubule of a doublet pair and the core of the axial unit. Microtubules and B tubules of doublet units are later digested; in microtubules, there appears to be a proteinaceous material in the lucent central region which is digested before disappearance of the wall of the microtubule. Further evidence is presented indicating that the characteristically helical wall of the microtubules is made up of spherical subunits about 50 Å in diameter, with about 8 subunits in one turn of the helix. Under certain conditions, the helical structure may be altered to form a wall comprised of longitudinal filaments. It is emphasized that not all microtubules are structurally and chemically equivalent, and it follows that all microtubules do not share a common function.This research was supported by U.S. Public Health Service Grant AI-06448 and an institutional grant from the American Cancer Society.     

A Model Describing Bending in Flagella

In Part I of this paper, we present a modelto account for the force generationproducing bending, and the formation of awaveform in sperm flagella. The model isbased on the observation that dimers, andhence microtubules, possess dipole moments.The electric field these dipoles produce isthe source for storing mechanical work indynein arms. The mechanical work is thenreleased and act on the doublets to producea distally directed force with the resultthat bending occurs. The model described isconsistent with experimental observationsreported in the literature. The flexuralrigidity of a dynein arm is alsocalculated. In Part II of this paper, theconsequences of the bending mechanism arediscussed. It is shown that the sum offorces from dynein arms acting distallyalong doublet microtubules in a flagellumis essentially zero when all dyneins areattached thus resulting in the rigor state.The waveform in a flagellum occurs if oneof the sets of bending moments is zero,that is, a row of dyneins are detached oversome distance along the flagellum. Thedirection of the bend in the waveform isdetermined by which set of dynein arms aredetached with respect to the verticalmedian plane of the flagellum. Thepropagation of a bending wave is the resultof a moving region in which alternate sidesfrom the vertical median plane haveinactive dynein arms. The processes bywhich this moving region occurs and therelationship of the above results to thepropulsion of the flagellum are notconsidered.     

Structure and organization of radial spokes in cilia of Tetrahymena pyriformis.

The structure and organization of radial spokes, the principal components between each of the peripheral doublet microtubules and the central sheath which surrounds the central pair of microtubules have been described in Tetrahymena pyriformis cilia. The radial spokes are grouped in triplets and are attached to the A-microtubule of each peripheral doublet at intervals of 200/280/360 A, the 200 A spacing being most distal to the base of the cilium. The radial spoke triplets are organized in the axoneme in a double helix with a pitch of 4,680 A. A method for determining the helical disposition by correcting for doublet sliding is presented.     

Polarity of dynein-microtubule interactions in vitro: cross-bridging between parallel and antiparallel microtubules

Ciliary doublet microtubules produced by sliding disintegration in 20 muM MgATP2-reassociate in the presence of exogenous 30S dynein and 6 mM MgSO4. The doublets form overlapping arrays, held together by dynein cross-bridges. Dynein arms on both A and B subfibers serve as unambiguous markers of microtubule polarity within the arrays. Doublets reassociate via dynein cross-bridges in both parallel and antiparallel modes, although parallel interactions are favored 2:1. When 20 muM ATP is added to the arrays, the doublets undergo both vanadate-sensitive and insensitive forms of secondary disintegration to reproduce the original population of doublets. The results demonstrate that both parallel and antiparallel doublet cross-bridging is sensitive to dissociation by ATP even though normal ciliary motion depends strictly on dynein interactions between parallel microtubules.     

Axoneme-specific beta-tubulin specialization: a conserved C-terminal motif specifies the central pair.

Axonemes are ancient organelles that mediate motility of cilia and flagella in animals, plants, and protists. The long evolutionary conservation of axoneme architecture, a cylinder of nine doublet microtubules surrounding a central pair of singlet microtubules, suggests all motile axonemes may share common assembly mechanisms. Consistent with this, alpha- and beta-tubulins utilized in motile axonemes fall among the most conserved tubulin sequences [1, 2], and the beta-tubulins contain a sequence motif at the same position in the carboxyl terminus [3]. Axoneme doublet microtubules are initiated from the corresponding triplet microtubules of the basal body [4], but the large macromolecular "central apparatus" that includes the central pair microtubules and associated structures [5] is a specialization unique to motile axonemes. In Drosophila spermatogenesis, basal bodies and axonemes utilize the same alpha-tubulin but different beta-tubulins [6--13]. beta 1 is utilized for the centriole/basal body, and beta 2 is utilized for the motile sperm tail axoneme. beta 2 contains the motile axoneme-specific sequence motif, but beta 1 does not [3]. Here, we show that the "axoneme motif" specifies the central pair. beta 1 can provide partial function for axoneme assembly but cannot make the central microtubules [14]. Introducing the axoneme motif into the beta 1 carboxyl terminus, a two amino acid change, conferred upon beta 1 the ability to assemble 9 + 2 axonemes. This finding explains the conservation of the axoneme-specific sequence motif through 1.5 billion years of evolution.     

Space-Dependent Formation of Central Pair Microtubules and Their Interactions with Radial Spokes

Cilia and flagella contain nine outer doublet microtubules and a pair of central microtubules. The central pair of microtubules (CP) is important for cilia/flagella beating, as clearly shown by primary ciliary dyskinesia resulting from the loss of the CP. The CP is thought to regulate axonemal dyneins through interaction with radial spokes (RSs). However, the nature of the CP-RS interaction is poorly understood. Here we examine the appearance of CPs in the axonemes of a Chlamydomonas mutant, bld12, which produces axonemes with 8 to 11 outer-doublets. Most of its 8-doublet axonemes lack CPs. However, in the double mutant of bld12 and pf14, a mutant lacking the RS, most 8-doublet axonemes contain the CP. Thus formation of the CP apparently depends on the internal space limited by the outer doublets and RSs. In 10- or 11-doublet axonemes, only 3–5 RSs are attached to the CP and the doublet arrangement is distorted most likely because the RSs attached to the CP pull the outer doublets toward the axonemal center. The CP orientation in the axonemes varies in double mutants formed between bld12 and mutants lacking particular CP projections. The mutant bld12 thus provides the first direct and visual information about the CP-RS interaction, as well as about the mechanism of CP formation.     

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.     

Flagellar elongation and shortening in Chlamydomonas. III. structures attached to the tips of flagellar microtubules and their relationship to the directionality of flagellar microtubule assembly

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microbutule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the A-tubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of sublnits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.     

A Role for Katanin-mediated Axonemal Severing during Chlamydomonas Deflagellation

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca²⁺, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of the Chlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate that Chlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca²⁺-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.     

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.     

Direct observation of microtubule pushing by cortical dynein in living cells

Microtubules are under the influence of forces mediated by cytoplasmic dynein motors associated with the cell cortex. If such microtubules are free to move, they are rapidly transported inside cells. Here we directly observe fluorescent protein–labeled cortical dynein speckles and motile microtubules. We find that several dynein complex subunits, including the heavy chain, the intermediate chain, and the associated dynactin subunit Dctn1 (also known as p150glued) form spatially resolved, dynamic speckles at the cell cortex, which are preferentially associated with microtubules. Measurements of bleaching and dissociation kinetics at the cell cortex reveal that these speckles often contain multiple labeled dynein heavy-chain molecules and turn over rapidly within seconds. The dynamic behavior of microtubules, such as directional movement, bending, or rotation, is influenced by association with dynein speckles, suggesting a direct physical and functional interaction. Our results support a model in which rapid turnover of cell cortex–associated dynein complexes facilitates their search to efficiently capture and push microtubules directionally with leading plus ends.     

Axoneme beta-tubulin sequence determines attachment of outer dynein arms

Axonemes of motile eukaryotic cilia and flagella have a conserved structure of nine doublet microtubules surrounding a central pair of microtubules. Outer and inner dynein arms on the doublets mediate axoneme motility [1]. Outer dynein arms (ODAs) attach to the doublets at specific interfaces [2-5]. However, the molecular contacts of ODA-associated proteins with tubulins of the doublet microtubules are not known. We report here that attachment of ODAs requires glycine 56 in the beta-tubulin internal variable region (IVR). We show that in Drosophila spermatogenesis, a single amino acid change at this position results in sperm axonemes markedly deficient in ODAs. Moreover, we found that axonemal beta-tubulins throughout the phylogeny have invariant glycine 56 and a strongly conserved IVR, whereas nonaxonemal beta-tubulins vary widely in IVR sequences. Our data reveal a deeply conserved physical requirement for assembly of the macromolecular architecture of the motile axoneme. Amino acid 56 projects into the microtubule lumen [6]. Imaging studies of axonemes indicate that several proteins may interact with the doublet-microtubule lumen [3, 4, 7, 8]. This region of beta-tubulin may determine the conformation necessary for correct attachment of ODAs, or there may be sequence-specific interaction between beta-tubulin and a protein involved in ODA attachment or stabilization.     

A physical model of microtubule sliding in ciliary axonemes.

Ciliary movement is caused by coordinated sliding interactions between the peripheral doublet microtubules of the axoneme. In demembranated organelles treated with trypsin and ATP, this sliding can be visualized during progressive disintegration. In this paper, microtubule sliding behavior resulting from various patterns of dynein arm activity and elastic link breakage is determined using a simplified model of the axoneme. The model consists of a cylindrical array of microtubules joined, initially, by elastic links, with the possibility of dynein arm interaction between microtubules. If no elastic links are broken, sliding can produce stable distortion of the model, which finds application to straight sections of a motile cilium. If some elastic links break, the model predicts a variety of sliding patterns, some of which match, qualitatively, the observed disintegration behavior of real axonemes. Splitting of the axoneme is most likely to occur between two doublets N and N + 1 when either the arms on doublet N + 1 are active and arms on doublet N are inactive or arms on doublet N - 1 are active while arms on doublet N are inactive. The analysis suggests further experimental studies which, in conjunction with the model, will lead to a more detailed understanding of the sliding mechanism, and will allow the mechanical properties of some axonemal components to be evaluated.