Dynein arm attachment probed with a non-hydrolyzable ATP analog. Structural evidence for patterns of activity

The dynein arms that power ciliary motility are normally permanently attached by one end exclusively to subfiber A of each axonemal doublet (N) while the other (head) end transiently attaches to the subfiber B of the adjacent doublet (N + 1) to produce sliding of the doublets. In Tetrahymena axonemes, sliding of contiguous groups of doublets is induced by ATP suggesting that, in the absence of exogenous protease, there may be sets of potentially active and potentially inactive or refractory arms in a single axoneme. In the presence of a non-hydrolyzable analog of ATP, beta,gamma-methylene adenosine 5'-triphosphate (AMP-PCP), about half the doublets in an axonemal preparation retain all arms bound to subfiber A, but half the doublets show long regions where some arms are pulled away from subfiber A of doublet N and attached to subfiber B of doublet N + 1 by their head ends. In AMP-PCP-induced splaying, positional information regarding arm state is retained. Analysis reveals that throughout regions where B subfiber attachment is found, small groups of about four subfiber B attached arms alternate with groups of about four arms that remain attached to subfiber A. This unique pattern of attachment suggests that arms function co-operatively in groups of four. Further, the repetition of the pattern is reminiscent of metachronal activity seen at higher levels of biological organization. This suggests that in these regions we have instantaneously preserved groups of arms capable of attaching to and detaching from doublet N + 1 in rapid succession. This appearance could be used to delineate the potentially active sets of arm, primed for mechanochemical activity, within an axoneme.     

Cation-induced attachment of ciliary dynein cross-bridges

Isolated, demembranated Unio gill cilia that have been activated and fixed for thin-section electron microscopy in the presence of 2 mM MgSO4 have 87% of their outer dynein arms attached to an adjacent B subfiber. The distribution of attached arms is uniform with respect to doublet position in the cilium. When both 0.1 mM ATP and Mg++ are added to the activation and fixation solutions, the frequency of bridged arms is reduced to 48%. At the same time, the distribution of the attached arms appears to have been systematically modified with respect to doublet position and the active bend plane. Those doublet pairs positioned in the bend plane where interdoublet sliding is minimal retain a greater number of bridged arms than those doublet pairs positioned outside the bend plane where sliding is maximal. These observations imply a functional coupling of the Mg++-induced bridging of the dynein arms and the subsequent binding and hydrolysis of ATP that results in a force-generating cross-bridge cycle.     

Dynein as a microtubule translocator in ciliary motility: current studies of arm structure and activity pattern

The dynein arms of ciliary doublet microtubules cause adjacent axonemal doublets to slide apart with fixed polarity. This suggests that there is a unique mechanochemistry to the dynein arm with unidirectional force generation in all active arms and also that not all arms are active at once during a ciliary beat. Negative stain and thin-section images of arms in axonemes treated with beta, gamma methylene adenosine triphosphate (AMP-PCP) show a consistent subunit construction where the globular head of the arm interacts with subfiber B of doublet N+1. This interpretation differs from that provided by freeze etch and STEM interpretations of in situ arm construction and has implications for the mechanochemical cycle of the arm. A computer model of the arms in relation to other axonemal structures has been constructed to test these interpretations. Attachment of the head of the arm subfiber B is directly demonstrable in splayed axonemes in AMP-PCP. About half of the doublets in an axoneme show such attachments, while half do not. This might imply that about half the doublets in an axoneme are active at any given instant and can be identified as such. This information may be useful in probing questions of how active arms differ biochemically from inactive arms and of how microtubule translocators in general become active.     

Interactions of dynein arms with b subfibers of Tetrahymena cilia: quantitation of the effects of magnesium and adenosine triphosphate

Tetrahymena 30S dynein was extracted with 0.5 M KCl and tested for retention of several functional properties associated wtih its in situ force-generating capacity. The dynein fraction will rebind to extracted outer doublets in the presence of Mg2+ to restore dynein arms. The arms attach at one end to the A subfiber and form bridges at the other end to the B subfiber of an adjacent doublet. Recombined arms retain an ATPase activity that remains coupled to potential generation of interdoublet sliding forces. To examine important aspects of the dynein- tubulin interaction that we presume are directly related to the dynein force-generating cross-bridge cycle, a simple and quantitative spectrophotometric assay was devised for monitoring the associations between isolated 30S dynein and the B subfiber. Utilizing this assay, the binding of dynein to B subfibers was found to be dependent upon divalent cations, saturating at 3 mM Mg2+. Micromolar concentrations of MgATP2- cause the release of dynein from the B subfiber; however, not all of the dynein bound under these conditions is released by ATP. ATP- insensitive dynein binding results from dynein interactions with non-B- tubule sites on outer-doublet and central-pair microtubules and from ATP-insensitive binding to sites on the B subfiber. Vanadate over a wide concentration range (10(-6)-10(-3) M) has no effect on the Mg2+- induced binding of dynein or its release by MgATP2-, and was used to inhibit secondary doublet disintegration in the suspensions. In the presence of 10 microM vanadate, dynein is maximally dissociated by MgATP2- concentrations greater than or equal to 1 microM with half- maximal release at 0.2 microM. These binding properties of isolated dynein arms closely resemble the cross-bridging behavior of in situ dynein arms reported previously, suggesting that quantitative studies such as those presented here may yield reliable information concerning the mechanism of force generation in dynein-microtubule motile systems. The results also suggest that vanadate may interact with an enzyme- product complex that has a low affinity for tubulin.     

Binding of dynein 21 S ATPase to microtubules. Effects of ionic conditions and substrate analogs

Binding of 21 S dynein ATPase isolated from Tetrahymena cilia to B subfibers of microtubule doublets was used as a model system to study dynein-tubulin interactions and their relationship to the microtubule-based sliding filament mechanism. Binding of 21 S dynein to both A and B microtubule subfibers is supported by monovalent as well as divalent ions. Monovalent cation chlorides support dynein binding to B subfibers with the specificity Li greater than Na congruent to K congruent to Rb congruent to Cs congruent to choline. The corresponding sodium or potassium halides follow the order F greater than Cl greater than Br greater than I. However, an optimal binding concentration of 40 mM KCl supports only 55% of the protein binding which takes place in 3 mM MgSO4 and does not stabilize dynein cross-bridges when whole axonemes are fixed for electron microscopy. Divalent metal ion chlorides (MgCl2, CaCl2, SrCl2, and BaCl2) have nearly equivalent effects at a concentration of 6 mM; all support about 140% of the binding observed in 6 mM MgSO4. The binding data suggest negative cooperativity or the presence of more than one class of dynein binding sites on the microtubule lattice. Low concentrations of MgATP2- induce dissociation of dynein bound to B subfibers in either 6 mM MgSO4 or 40 mM KCl. ADP, Pi, PPi, and AMP-PCH2P are unable to induce dynein dissociation, while AMP-PNHP and ATP4- both cause dynein release from B subfiber sites. The half-maximal sensitivities of the tubulin-dynein complex to MgATP2-, ATP4-, and adenylyl-imidodiphosphate (AMP.PNP) are 1.3 X 10(-8) M, 3.6 X 10(-5) M, and 4.7 X 10(-4) M respectively. Incubation of doublets or 21 S dynein in N-ethylmaleimide (NEM), which can inhibit active sliding, has no effect on either association of dynein with the B subfiber or on dissociation of the resulting dynein-B subfiber complex by MgATP2-.     

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.     

Dynein arm substructure and the orientation of arm-microtubule attachments

In the presence of AMP-PCP (beta, gamma-methyleneadenosine 5'-triphosphate), a non-hydrolyzable analog of ATP, negative stain images of increased morphological detail indicate that the dynein arm, attached to ciliary doublet microtubules, is composed of subunits including a cape, an elongated body and a head. The arrangement of these subunits makes it possible to distinguish A from B subfiber binding sites on a single arm and to demonstrate that the head of an extended arm on subfiber A of one ciliary doublet is capable of binding to subfiber B of an adjacent doublet in a specific orientation, which supports a key step in a current model of the mechanochemical cycle by which the arm produces microtubule sliding in the ciliary axoneme.     

Splitting the ciliary axoneme: implications for a "switch-point" model of dynein arm activity in ciliary motion

In the presence of specific inhibitors of beat. 20 microM VO4(3-) or pCa 4, mussel gill lateral (L) cilia can be arrested in two positions--"hands down" or "hands up"--at opposite ends of the stroke cycle. Cilia move to these positions by doublet microtubule sliding. Axonemes of arrested cilia, still tethered to the cell, are intact after demembranation and protease treatment. When reactivated by 4 mM ATP with inhibitors present, about 40% split apart. Splits are not random but occur preferentially between different specific doublets in the two opposite arrest positions. Several different related patterns of splitting are observed; for every pattern in "hands down" axonemes, there is a corresponding complementary split pattern in "hands up" axonemes. In some split patterns two doublets remain firmly attached to the central pair; these also differ depending on axonemal position. Although some of the patterns seen may be artifactual or difficult to explain, the complementary splitting patterns are predictable with simple assumptions by a "switch point" hypothesis of ciliary activity where, during each recovery stroke, doublets 6-8 have active dynein arms, while during each effective stroke, arms on doublets 1-4 become active, and arms 6-8 are turned off. Because of a difference between the patterns seen and the predictions, the status of the arms on doublet 9 is unresolved. The patterns also suggest that a spoke-central sheath attachment cycle may correlate with switching of arm activity during the generation of an asymmetric beat.     

EFFECTS OF TRYPSIN DIGESTION ON FLAGELLAR STRUCTURES AND THEIR RELATIONSHIP TO MOTILITY

Flagellar axonemes isolated from sea urchin sperm were digested with trypsin for various time periods. The course of digestion was monitored turbidimetrically and was found to take two different courses depending on the presence or absence of ATP in the digestion mixture. It was found that ATP induced active disintegration of the axonemes after slight digestion. Samples of the digested axonemes were examined with the electron microscope to determine the effects of trypsin digestion on the substructures of the axonemes. The rate at which trypsin sensitized the axonemes to ATP paralleled the rate at which it damaged the radial spokes and the nexin links, while the dynein arms were removed much more slowly. The results suggest that inactive dynein arms form cross bridges between the adjacent doublet tubules in digested axonemes, and that when activated by the addition of ATP, they induce an active shearing force between adjacent doublets. The radial spokes and the nexin links are not directly involved in the production of mechanical force, but they may participate in regulating the sliding between tubules to produce a propagated bending wave.     

Tails of Tetrahymena

SYNOPSIS. The source of force generation of beating cilia and flagella is an interaction between the doublet microtubules mediated by the dynein-1 arms which cause the doublets to slide relative to one another. Previously, we demonstrated direct sliding of Tetrahymena ciliary axonemes by dark field light microscopy. In this paper, the results of such an experiment have been captured on a polylysine-coated grid surface for whole-mount electron microscopy. Images in which sliding between doublets has taken place can be identified. We conclude that doublets slide relative to one another with a constant polarity. To produce the observed displacement, the direction of the dynein-1 arm force generation must be from base to tip, so that the doublet (n), to which the arms are attached, pushes the next doublet (n+ 1) toward the tip. In addition to the functional polarity, the dynein-1 arms are found to have a structural polarity: they tilt toward the base when viewed along the edges of the A-subfiber. A scheme is presented which reconciles the finding of a single polarity of active sliding with the geometry of microtubule tip displacement of bent cilia.     

Binding of 30s dynein with the B-tubule of the outer doublet of axonemes from Tetrahymena pyriformis and adenosine triphosphate-induced dissociation of the complex.

The binding properties of dynein arms to the A- and B-tubules of outer doublets of cilia from Tetrahymena pyriformis were examined, with the following results: 1. When 30s dynein purified from Tetrahymena cilia was added to doublets deficient in dynein arms, it bound to both A- and B-tubules almost equally and formed arms along the edges. The overall length of arms bound to the A-tubule was 22 +/- 3 nm, and that of arms bound to the B-tubule was 24 +/- 3 nm. Each arm bound to the A- and B-tubules was pointed toward the base at angles of 55 degrees +/- 7 degrees and 48 degrees +/- 7 degrees, respectively. In the presence of sufficient amounts of dynein, the arms along the A- and B-tubules were located at intervals of 22.8 +/- 1.5 nm and 22.5 +/- 1.7 nm, respectively. 2. On adding ATP, only the arms bound to the B-tubule were dissociated from the doublet decorated with arms on both sides. The dissociated arms rebound themselves to the B-tubule after hydrolysis of the ATP. When several doublets decorated with arms along both A- and B-tubules were arrayed side by side, the interdoublet spacing increased from 14 +/- 2 nm to 17 +/- 2 nm on addition of ATP. 3. The turbidity of a suspension of trypsin [EC 3.4.21.4]-treated axonemes decreased rapidly on addition of ATP, then recovered partially. Observations by dark-field microscopy and electron microscopy showed that the doublets which had slid out from the axonemes on ATP addition formed large aggregates after hydrolysis of the ATP. The dynein arms were also solubilized from the axonemes upon addition of ATP, and rebound themselves to the B-tubule after hydrolysis of the added ATP. 4. The double-reciprocal plot for the ATPase [EC 3.6.1.3] activity of the trypsin-treated axonemes against ATP concentration was composed of two straight lines, from which the Km values were estimated to be 1.0 and 12.7 micrometer. The dependence of the decrease in turbidity of the axonemal suspension on ATP concentration indicated that the binding of ATP to sites with an apparent dissociation constant of 1 micrometer induced dissociation of the arms from the B-tubule.     

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.     

Ciliary inter-microtubule bridges.

Electron micrographs of both negatively contrasted and thin-sectioned lamellibranch gill cilia reveal several new features of ciliary fine structure, particularly in regard to those structures forming intermittent or permanent crossbridges between microtubules. Negative-contrasting reveals the presence of a 14-5-nm repeating bridge between the central microtubules. Frontal views of negatively contrasted dynein arm rows along subfibre A show that the arms (23-nm repeat) in the outer row are displaced in a left-handed manner by 3-4nm with respect to those in the inner row. This displacement is probably a direct reflexion of the helical tubulin subunit lattice of the subfibre. Interdoublet (nexin) links are seen connecting adjacent A and B subfibres at intervals of 86 nm along the doublet. Negative-contrasting shows thin, highly elastic connexions holding the doublets together. When seen in longitudinal thin sections, the interdoublet links are often tilted to considerable angles, indicating they may have an elastic response to interdoublet sliding.     

Effects of the central pair apparatus on microtubule sliding velocity in sea urchin sperm flagella.

To produce oscillatory bending movement in cilia and flagella, the activity of dynein arms must be regulated. The central-pair microtubules, located at the centre of the axoneme, are often thought to be involved in the regulation, but this has not been demonstrated definitively. In order to determine whether the central-pair apparatus are directly involved in the regulation of the dynein arm activity, we analyzed the movement of singlet microtubules that were brought into contact with dynein arms on bundles of doublets obtained by sliding disintegration of elastase-treated flagellar axonemes. An advantage of this new assay system was that we could distinguish the bundles that contained the central pair apparatus from those that did not, the former being clearly thicker than the latter. We found that microtubule sliding occurred along both the thinner and the thicker bundles, but its velocity differed between the two kinds of bundles in an ATP concentration dependent manner. At high ATP concentrations, such as 0.1 and 1 mM, the sliding velocity on the thinner bundles was significantly higher than that on the thicker bundles, while at lower ATP concentrations the sliding velocity did not change between the thinner and the thicker bundles. We observed similar bundle width-related differences in sliding velocity after removal of the outer arms. These results provide first evidence suggesting that the central pair and its associated structures may directly regulate the activity of the inner (and probably also the outer) arm dynein.     

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.     

Kinetic properties of dynein ATPase from Tetrahymena pyriformis. The initial phosphate burst of dynein ATPase and its interaction with ATP analogs.

1. Dynein was extracted with 0.5 M KCl from Tetrahymena axonemes. SDS-gel electrophoresis of the extract indicated that about 50% of the extracted protein had a molecular weight of about 3.5 X 10(5), and that 90% of the proteins with this weight had been extracted. 2. The ATPase [EC 3.6.1.3] reaction of the KCl-extracted dynein fraction was enhanced by 60-80% by addition of the outer doublet fraction. It showed an initial burst of Pi liberation of about 1 mol per mol of proteins with a molecular weight of 3.5 X 10(5). 3. We examined the interaction of the dynein-tubulin system from Tetrahymena cilia with ten ATP analogs [2'-dATP, 3'-dATP, epsilonATP, FTP, 8-NH(CH3)-ATP, 8,3'-S-cyclo-ATP, 8-Br-ATP, 8-OCH3-ATP, 8-SCH3-ATP, and AMPPNP]. Among them, 2'-dATP and 3'-dATP were good substrates for dynein ATPase, as they induced the dissociation of dynein arms from the B-tubule of outer doublets, the sliding movement between outer doublets, and the bending movement of axonemes. The other analogs did not induce the dissociation or the sliding movement. 4. Among the ATP analogs tested, only 2'-dATP and 3'-dATP induced the reorientation of cilia on the Triton model of Tetrahymena; the reorientation rates were smaller than that induced by ATP.     

ATP-dependent structural changes of the outer dynein arm in Tetrahymena cilia: a freeze-etch replica study

With the rapid-freeze, deep-etch replica technique, the structural conformations of outer dynein arms in demembranated cilia from Tetrahymena were analyzed under two different conditions, i.e., in the absence of ATP and in the presence of ATP and vanadate. In the absence of ATP, the lateral view of axonemes was characterized by the egg- shaped outer dynein arms, which showed a slightly baseward tilt with a mean inclination of 11.1 degrees +/- 3.4 degrees SD from the perpendicular to the doublet microtubules. On the other hand, in the presence of 1 mM ATP and 100 microM vanadate, the outer arms were extended and slender and showed an increased baseward tilt with a mean inclination of 31.6 degrees +/- 4.9 degrees SD. In ATP-activated axonemes, these two types of arms coexisted, each type occurring in groups along one row of outer arms. These findings strongly suggest that the interdoublet sliding is caused by dynamic structural changes of dynein arms that follow the hydrolysis of ATP.     

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.     

Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.     

The role of axonemal components in ciliary motility

1. The axoneme is the detergent-insoluble cytoskeleton of the cilium. 2. All axonemes generate movement by the same fundamental mechanism: microtubule sliding utilizing ATP hydrolysis during a mechanochemical cycling of dynein arms on the axonemal doublets. 3. Structure, fundamental biochemistry and physiology of the axoneme are conserved evolutionarily, but the phenotypes of beating movements and the responses to specific cytoplasmic signals differ greatly from organism to organism. 4. A model of asynchronous dynein arm activity--the switch point hypothesis--has been proposed to account for cyclic beating in the face of unidirectional sliding. The model suggests that the diversity of beat phenotype may be explicable by changes in the timing of switching between active and inactive states of doublet arm activity. Evidence of axonemal splitting in arrested axonemes provides new support for the hypothesis.