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.     

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.     

Structural conformation of ciliary dynein arms and the generation of sliding forces in Tetrahymena cilia

The sliding tubule model of ciliary motion requires that active sliding of microtubules occur by cyclic cross-bridging of the dynein arms. When isolated, demembranated Tetrahymena cilia are allowed to spontaneously disintegrate in the presence of ATP, the structural conformation of the dynein arms can be clearly resolved by negative contrast electron microscopy. The arms consist of three structural subunits that occur in two basic conformations with respect to the adjacent B subfiber. The inactive conformation occurs in the absence of ATP and is characterized by a uniform, 32 degrees base-directed polarity of the arms. Inactive arms are not attached to the B subfiber of adjacent doublets. The bridged conformation occurs strictly in the presence of ATP and is characterized by arms having the same polarity as inactive arms, but the terminal subunit of the arms has become attached to the B subfiber. In most instances the bridged conformation is accompanied by substantial tip-directed sliding displacement of the bridged doublets. Because the base-directed polarity of the bridged arms is opposite to the direction required for force generation in these cilia and because the bridges occur in the presence of ATP, it is suggested that the bridged conformation may represent the initial attachment phase of the dynein cross-bridge cycle. The force-generating phase of the cycle would then require a tip-directed deflection of the arm subunit attached to the B subfiber.     

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.     

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.     

Are the local adjustments of the relative spatial frequencies of the dynein arms and the beta-tubulin monomers involved in the regulation of the "9+2" axoneme?

The “9+2” axoneme is a highly specific cylindrical machine whose periodic bending is due to the cumulative shear of its 9 outer doublets of microtubules. Because of the discrete architecture of the tubulin monomers and the active appendices that the outer doublets carry (dynein arms, nexin links and radial spokes), this movement corresponds to the relative shear of these topological verniers, whose characteristics depend on the geometry of the wave train. When an axonemal segment bends, this induces the compressed and dilated conformations of the tubulin monomers and, consequently, the modification of the spatial frequencies of the appendages that the outer doublets carry. From a dynamic point of view, the adjustments of the spatial frequencies of the elements of the two facing verniers that must interact create different longitudinal periodic patterns of distribution of the joint probability of the molecular interaction as a function of the location of the doublet pairs around the axonemal cylinder and their spatial orientation within the axonemal cylinder. During the shear, these patterns move along the outer doublet intervals at a speed that ranges from one to more than a thousand times that of sliding, in two opposite directions along the two opposite halves of the axoneme separated by the bending plane, respecting the polarity of the dynein arms within the axoneme. Consequently, these waves might be involved in the regulation of the alternating activity of the dynein arms along the flagellum, because they induce the necessary intermolecular dialog along the axoneme since they could be an element of the local dynamic stability/instability equilibrium of the axoneme. This complements the geometric clutch model [Lindemann, C., 1994. A “geometric clutch” hypothesis to explain oscillations of the axoneme of cilia and flagella. J. Theor. Biol. 168, 175-189].     

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.     

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 STRUCTURAL BASIS OF CILIARY BEND FORMATION : Radial Spoke Positional Changes Accompanying Microtubule Sliding

The sliding microtubule model of ciliary motility predicts that cumulative local displacement (Δl) of doublet microtubules relative to one another occurs only in bent regions of the axoneme. We have now tested this prediction by using the radial spokes which join the A subfiber of each doublet to the central sheath as markers of microtubule alignment to measure sliding displacements directly. Gill cilia from the mussel Elliptio complanatus have radial spokes lying in groups of three which repeat at 860 Å along the A subfiber. The spokes are aligned with the two rows of projections along each of the central microtubules that form the central sheath. The projections repeat at 143 Å and form a vernier with the radial spokes in the precise ratio of 6 projection repeats to 1 spoke group repeat. In straight regions of the axoneme, either proximal or distal to a bend, the relative position of spoke groups between any two doublets remains constant for the length of that region. However, in bent regions, the position of spoke groups changes systematically so that Δl (doublet 1 vs. 5) can be seen to accumulate at a maximum of 122 Å per successive 860-Å spoke repeat. Local contraction of microtubules is absent. In straight regions of the axoneme, the radial spokes lie in either of two basic configurations: (a) the parallel configuration where spokes 1–3 of each group are normal (90°) to subfiber A, and (b) the tilted spoke 3 configuration where spoke 3 forms an angle (θ) of 9–20°. Since considerable sliding of doublets relative to the central sheath (~650 Å) has usually occurred in these regions, the spokes must be considered, functionally, as detached from the sheath projections. In bent regions of the axoneme, two additional spoke configurations occur where all three spokes of each group are tilted to a maximum of ± 33° from normal. Since the spoke angles do not lie on radii through the center of bend curvature, and Δl accumulates in the bend, the spokes must be considered as attached to the sheath when bending occurs. The observed radial spoke configurations strongly imply that there is a precise cycle of spoke detachment-reattachment to the central sheath which we conclude forms the main part of the mechanism converting active interdoublet sliding into local bending.     

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.     

Flagellate spermatozoa of Protura (Insecta,Apterygota) are motile

A new model of sperm axoneme with 16 + 0 doublets is described. The spermatozoon of Acerentulus confinis (Apterygota : Protura) has a short conical acrosome, a long helicoidal nucleus, well-developed centriolar adjunct material, and a long flagellum. Using fixation with a glutaraldehyde-tannic acid mixture, without osmium post-fixation, doublet protofilaments, inner dynein arms, radial spokes, nexin bridges, and Y-links of the sperm axoneme of A. confinis and Acerentomon italicum were clearly observed. Optical observation shows that the proturan flagellate spermatozoa are motile cells. The process involving the transformation of the spermatozoa from a coiled to an elongated swimming form was studied by scanning electron microscope. The findings confirmed that flagellar motility is due to the presence of a single dynein arm on doublets in spite of the unusual axonemal pattern.     

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.     

Direction of force generated by the inner row of dynein arms on flagellar microtubules

Our goal was to determine the direction of force generation of the inner dynein arms in flagellar axonemes. We developed an efficient means of extracting the outer row of dynein arms in demembranated sperm tail axonemes, leaving the inner row of dynein arms structurally and functionally intact. Sperm tail axonemes depleted of outer arms beat at half the beat frequency of sperm tails with intact arms over a wide range of ATP concentrations. The isolated, outer arm-depleted axonemes were induced to undergo microtubule sliding in the presence of ATP and trypsin. Electron microscopic analysis of the relative direction of microtubule sliding (see Sale, W. S. and P. Satir, 1977, Proc. Natl. Acad. Sci. USA, 74:2045-2049) revealed that the doublet microtubule with the row of inner dynein arms, doublet N, always moved by sliding toward the proximal end of the axoneme relative to doublet N + 1. Therefore, the inner arms generate force such that doublet N pushes doublet N + 1 tipward. This is the same direction of microtubule sliding induced by ATP and trypsin in axonemes having both inner and outer dynein arms. The implications of this result for the mechanism of ciliary bending and utility in functional definition of cytoplasmic dyneins are discussed.     

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.     

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 functional flagella with a 6 + 0 pattern]

The male gamete of the Gregarine Lecudina tuzetae has been studied with transmission electron microscopy and microcinematography. It is characterized by a flagellar axoneme of 6 + 0 pattern, a reduction of the chondriome, and the abundance of storage polysaccharide or lipid bodies. The movements of the flagella are of the undulating type and they are performed in the three dimensions of space. They are very slow, with a cycle time of about 2s. The structure of the axoneme components are similar to those of flagella with a 9 + 2 pattern. Each doublet has overall dimensions of 350 x 220 A; the space between the adjacent doublets is about 160 A. The A subfiber bears arms like dynein arms. The diameter of the axoneme is about 1,000 A. The basal body consists of a cylinder of dense material 2,500 A long and 1,300-1,400 A in diameter; a microtubule 200 A in diameter is present in the axis. This study shows that a 6 + 0 pattern can generate a flagellar movement. The mechanism of the flagellar movement of the male gamete of L. tuzetae does not require the presence of central microtubules and it would include molecular interactions of the dynein-tubulin type between the adjacent peripheric doublets. The slowness of the movements is discussed in terms of the axoneme's structure and its energy supply. Finally, the phylogenetic significance of this flagella is examined on the basis of the morphopoietic potentialities of the centriolar structures.     

Sperm axoneme of two rows of doublets reversely oriented in the gall-midge Lestremia (Diptera, Cecidomyiidae)

The spermatozoon of Lestremia lacks an acrosome and has a giant centriole that gives origin to a giant axoneme with about 150 doublets. The axonemal doublets, disposed in two opposite rows oriented antiparallel, have A doublets with two dynein arms and a B tubule filled with dense proteinaceous material. Mitochondria fuse in two derivatives and show cristae and a longitudinal crystallized axis. The probable origin of the giant axoneme is hypothesized and the more prolonged motility of Lestremia sperm in comparison with that of other gall midges is related to the presence of a more precise axonemal organization. The spermatological results agree with the systematic position of Lestremiinae at the base of the evolutionary trend of the family Cecidomyiidae.     

High-frequency vibration in flagellar axonemes with amplitudes reflecting the size of tubulin

Flagellar axonemes of sea urchin sperm display high-frequency (approximately 300 Hz) vibration with nanometer-scale amplitudes in the presence of ATP (Kamimura, S., and R. Kamiya. 1989. Nature (Lond.). 340:476-478). The vibration appears to represent normal mechanochemical interaction between dynein and microtubules because the dependence of the frequency on MgATP concentration is similar to that of the axonemal motility, and because it is inhibited by micromolar concentrations of vanadate. In this study a two-dimensional photo-sensor was used to characterize this phenomenon in detail. Several new features were revealed. First, the vibration was found to be due to a back-and-forth movement of the doublet microtubules along the axonemal length. Two beads attached to different parts of the same axoneme vibrated in unison, i.e., synchronized exactly in phase. This suggested that the outer doublet can be regarded as a stiff rod in vibrating axonemes. Second, evidence was obtained that the amplitude of the vibration reflected the number of active dynein arms. Third, under certain conditions, the vibration amplitude took stepwise values of 8 x N + 4 nm (N = 0, 1, 2, 3, or 4), indicating that the amplitude of microtubule sliding was limited by the size of tubulin dimer (8 nm) or monomer (4 nm). To explain this phenomenon, a model is presented based on an assumption that the force production by dynein is turned off when dynein is subjected to tensile force; i.e., dynein is assumed to be equipped with a feedback mechanism necessary for oscillation.     

Bending of the “9+2” axoneme analyzed by the finite element method

Many data demonstrate that the regulation of the bending polarity of the “9+2” axoneme is supported by the curvature itself, making the internal constraints central in this process, adjusting either the physical characteristics of the machinery or the activity of the enzymes involved in different pathways. Among them, the very integrated Geometric Clutch model founds this regulation on the convenient adjustments of the probability of interaction between the dynein arms and the β-tubulin monomers of the outer doublet pairs on which they walk. Taking into consideration (i) the deviated bending of the outer doublets pairs (Cibert, C., Heck, J.-V., 2004. Cell Motil. Cytoskeleton 59, 153-168), (ii) the internal tensions of the radial spokes and the tangential links (nexin links, dynein arms), (iii) a theoretical 5 μm long proximal segment of the axoneme and (iv) the short proximal segment of the axoneme, we have reevaluated the adjustments of these intervals using a finite element approach. The movements we have calculated within the axonemal cylinder are consistent with the basic hypothesis that found the Geometric Clutch model, except that the axonemal side where the dynein arms are active increases the intervals between the two neighbor outer doublet pairs. This result allows us to propose a mechanism of bending reversion of the axoneme, involving the concerted ignition of the molecular engines along the two opposite sides of the axoneme delineated by the bending plane.     

Functional reconstitution of Chlamydomonas outer dynein arms from alpha- beta and gamma subunits: requirement of a third factor

The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1- oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.