High speed sliding of axonemal microtubules produced by outer arm dynein

To study dynein arm activity at high temporal resolution, axonemal sliding was measured field by field for wild type and dynein arm mutants of Tetrahymena thermophila. For wt SB255 cells, when the rate of data acquisition was 60 fps, about 5x greater than previously published observations, sliding was observed to be discontinuous with very high velocity sliding (average 196 microm/sec) for a few msec (1 or 2 fields) followed by a pause of several fields. The sliding velocities measured were an order of magnitude greater than rates previously measured by video analysis. However, when the data were analyzed at 12 fps for the same axonemes, consistent with previous observations, sliding was linear as the axonemes extended several times their original length with an average velocity of approximately 10 microm/sec. The pauses or stops occurred at approximately 200 and 300% of the initial length, suggesting that dynein arms on one axonemal doublet were initially active to the limit of extension, and then the arms on the next doublet became activated. In contrast, in a mutant where OADs are missing, sliding observed at 60 fps was continuous and slow (5 microm/sec), as opposed to the discontinuous high-velocity sliding of SB255 and of the mutant at the permissive temperature where OADs are present. High-velocity step-wise sliding was also present in axonemes from an inner arm dynein mutant (KO6). These results indicate that the high-speed discontinuous pattern of sliding is produced by the mechanochemical activity of outer arm dynein. The rate of sliding is consistent with a low duty ratio of the outer arm dynein and with the operation of each arm along a doublet once per beat.     

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.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

Inner arm dynein ATPase fraction of sea urchin sperm flagella causes active sliding of axonemal outer doublet microtubule.

In order to clarify the role of the inner arms of the axoneme in sperm flagellar movement, we prepared an ATPase fraction (12S) from the outer arm-depleted axonemes of sea urchin sperm flagella. When both arm-depleted axonemes were incubated with the 12S ATPase, they exhibited the sliding disintegration of outer doublet microtubules. Electron microscopy revealed that the ATPase rebound to the original inner arm sites of the axoneme. Therefore, it is quite likely that the 12S ATPase is one of the components of the inner arms. We referred to it as "inner arm dynein".     

Substructure of the outer dynein arm

The substructure of the outer dynein arm has been analyzed in quick-frozen deep-etch replicas of Tetrahymena and Chlamydomonas axonemes. Each arm is found to be composed of five morphologically discrete components: an elliptical head; two spherical feet; a slender stalk; and an interdynein linker. The feet make contact with the A microtubule of each doublet; the stalk contacts the B microtubule; the head lies between the feet and stalk; and the linker associates each arm with its neighbor. The spatial relationships between these five components are found to be distinctly different in rigor (ATP-depleted) versus relaxed (ATP- or vanadate plus ATP-treated) axonemes, and the stalk appears to alter its affinity for the B microtubule in the relaxed state. Images of living cilia attached to Tetrahymena cells show that the relaxed configuration is adopted in vivo. We relate our observations to morphological and experimental studies reported by others and propose several models that suggest how this newly described dynein morphology may relate to dynein function.     

Turnover of tubulin in ciliary outer doublet microtubules

Previous pulse-chase labeling studies have shown that structural proteins incorporate into fully assembled sea urchin embryonic cilia at rates approaching those of full regeneration. When all background ciliogenesis was suppressed by taxol, the turnover of most proteins, including tubulin, continued. The present study utilized chemical dissection to explore the route of tubulin incorporation in the presence of taxol and also in steady-state cilia from prism stage embryos. Surprisingly, in cilia from untreated embryos, the most heavily labeled tubulin was found in the most stable portion of the doublet microtubles, the junctional protofilaments. With taxol, this preferential incorporation was suppressed, although control-level turnover still took place in the remainder of the doublet. This paradoxical result was confirmed by pulse-chase labeling and immediately isolating steady-state cilia, then isolating two additional crops of cilia regenerated, respectively, from pools of high and then decreased label. In each case, the level of label occurring in the tubulin from the junctional protofilaments, compared with that from the remainder of the doublet, correlated with the level of pool label from which it must exchange or assemble. These data indicate that ciliary outer doublet microtubules are dynamic structures and that the junctional region is not inert. Plausible mechanisms of incorporation and turnover of tubulin in fully-assembled, fully-motile cilia can now be assessed with regared to recent discoveries, particularly intraflagellar transport, distal tip incorporation, and treadmilling.     

Three-dimensional reconstruction of axonemal outer dynein arms in situ by electron tomography

We present here for the first time a 3D reconstruction of in situ axonemal outer dynein arms. This reconstruction has been obtained by electron tomography applied to a series of tilted images collected from metal replicas of rapidly frozen, cryofractured, and metal-replicated sperm axonemes of the cecidomid dipteran Monarthropalpus flavus. This peculiar axonemal model consists of several microtubular laminae that proved to be particularly suitable for this type of analysis. These laminae are sufficiently planar to allow the visualization of many dynein molecules within the same fracture face, allowing us to recover a significant number of equivalent objects and to improve the signal-to-noise ratio of the reconstruction by applying advanced averaging protocols. The 3D model we obtained showed the following interesting structural features: First, each dynein arm has two head domains that are almost parallel and are obliquely oriented with respect to the longitudinal axis of microtubules. The two heads are therefore positioned at different distances from the surface of the A-tubule. Second, each head domain consists of a series of globular subdomains that are positioned on the same plane. Third, a stalk domain originates as a conical region from the proximal head and ends with a small globular domain that contacts the B-tubule. Fourth, the stem region comprises several globular subdomains and presents two distinct points of anchorage to the surface of the A-tubule. Finally, and most importantly, contrary to what has been observed in isolated dynein molecules adsorbed to flat surfaces, the stalk and the stem domains are not in the same plane as the head.     

The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules

Glass-adsorbed intact sea urchin outer arm dynein and its beta/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its alpha heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified alpha heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the beta/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the alpha heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine crossbridges. In contrast to the bridges formed by the intact arm, the links formed by the alpha subunit are irregularly spaced, suggesting that binding of the alpha heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the beta/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the alpha heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its alpha heavy chain. We conclude that whereas force-generation is a function of the beta/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the alpha subunit.     

Binding of axonemal dynein to microtubules comprising the cytoplasmic transport system in insect ovarioles

Dynein isolated from ciliary axonemes of Tetrahymena is shown to bind in a characteristic fashion as arms to microtubules dissected from the nutritive tubes of insect ovarioles. The microtubules in nutritive tubes are associated with the transport of cytoplasmic components along their length, and the significance of their ability to bind axonemal dynein, to the possibility that microtubule/dynein interactions are involved in microtubule-associated movements, generally, is discussed.     

Oda5p, a novel axonemal protein required for assembly of the outer dynein arm and an associated adenylate kinase

Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35-50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm.     

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.     

Cyclical interactions between two outer doublet microtubules in split flagellar axonemes

The beating of cilia and flagella is based on the localized sliding between adjacent outer doublet microtubules; however, the mechanism that produces oscillatory bending is unclear. To elucidate this mechanism, we examined the behavior of frayed axonemes of Chlamydomonas by using high-speed video recording. A pair of doublet microtubules frequently displayed association and dissociation cycles in the presence of ATP. In many instances, the dissociation of two microtubules was not accompanied by noticeable bending, suggesting that the dynein-microtubule interaction is not necessarily regulated by the microtubule curvature. On rare occasions, association and dissociation occurred simultaneously in the same interacting pair, resulting in a tip-directed movement of a stretch of gap between the pair. Based on these observations, we propose a model for cyclical bend propagation in the axoneme.     

Calcium regulates ATP-sensitive microtubule binding by Chlamydomonas outer arm dynein

The Chlamydomonas outer dynein arm contains three distinct heavy chains (alpha, beta, and gamma) that exhibit different motor properties. The LC4 protein, which binds 1-2 Ca2+ with KCa = 3 x 10-5 m, is associated with the gamma heavy chain and has been proposed to act as a sensor to regulate dynein motor function in response to alterations in intraflagellar Ca2+ levels. Here we genetically dissect the outer arm to yield subparticles containing different motor unit combinations and assess the microtubule-binding properties of these complexes both prior to and following preincubation with tubulin and ATP, which was used to inhibit ATP-insensitive (structural) microtubule binding. We observed that the alpha heavy chain exhibits a dominant Ca2+-independent ATP-sensitive MT binding activity in vitro that is inhibited by attachment of tubulin to the structural microtubule-binding domain. Furthermore, we show that ATP-sensitive microtubule binding by a dynein subparticle containing only the beta and gamma heavy chains does not occur at Ca2+ concentrations below pCa 6 but is maximally activated above pCa 5. This activity was not observed in mutant dyneins containing small deletions in the microtubule-binding region of the beta heavy chain or in dyneins that lack both the alpha heavy chain and the motor domain of the beta heavy chain. These findings strongly suggest that Ca2+ binding directly to a component of the dynein complex regulates ATP-sensitive interactions between the beta heavy chain and microtubules and lead to a model for how individual motor units are controlled within the outer dynein arm.     

Differential light chain assembly influences outer arm dynein motor function

Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.     

Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex

Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.     

Conformational change in the outer doublet microtubules from sea urchin sperm flagella

Dark-field microscopy with a high-powered light source revealed that the outer doublet microtubules (DMTs) from sea urchin (Pseudocentrotus depressus and Hemicentrotus pulcherrimus) sperm flagella assume helically coiled configurations (Miki-Noumura, T., and R. Kamiya. 1976. Exp. Cell Res. 97: 451.). We report here that the DMTs change shape when the pH or Ca-ion concentration is changed. The DMTs assumed a left-handed helical shape with a diameter of 3.7 +/- 0.5 micron and a pitch of 2.8 +/- 0.7 micron at pH 7.4 in the presence of 0.1 mM CaCl2, 1 mM MgSO4, and 10 mM Tris-HCl. When the pH was raised to 8.3, the helical diameter and pitch decreased to 2.1 +/- 0.1 micron and 1.3 +/- 0.3 micron, respectively. This transformation was a rapid and reversible process and was completed within 1 min. Between pH 7.2 and 8.3, the DMTs assumed intermediate shapes. When the Ca-ion concentration was depleted with EGTA, the helical structure became significantly larger in both pitch and diameter. For instance, the diameter was 3.8 +/- 0.4 micron at pH 8.3 in the presence of 1 mM EGTA and 2 mM MgSO4. Using a Ca-buffer system, we obtained results which suggested that this Ca-induced transformation took place at a Ca concentration of approximately 10(-7) M. These results were highly reproducible. The conformational changes in the DMT may play some role in the bending wave form of flagellar movement.     

Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.     

Molecular characterization of Ciona sperm outer arm dynein reveals multiple components related to outer arm docking complex protein 2

Using proteomic and immunochemical techniques, we have identified the light and intermediate chains (IC) of outer arm dynein from sperm axonemes of the ascidian Ciona intestinalis. Ciona outer arm dynein contains six light chains (LC) including a leucine-rich repeat protein, Tctex1- and Tctex2-related proteins, a protein similar to Drosophila roadblock and two components related to Chlamydomonas LC8. No LC with thioredoxin domains is included in Ciona outer arm dynein. Among the five ICs in Ciona, three are orthologs of those in sea urchin dynein: two are WD-repeat proteins and the third one, unique to metazoan sperm flagella, contains both thioredoxin and nucleoside diphosphate kinase modules. The remaining two Ciona ICs have extensive coiled coil structure and show sequence similarity to outer arm dynein docking complex protein 2 (DC2) that was first identified in Chlamydomonas flagella. We recently identified a third DC2-like protein with coiled coil structure, Ci-Axp66.0 that is also associated in substoichiometric amounts with Ciona outer arm dynein. In addition, Oda5p, a component of an additional complex required for assembly of outer arm dynein in Chlamydomonas flagella, also groups with this family of DC2-like proteins. Thus, the assembly of outer arm dynein onto doublet microtubules involves multiple coiled-coil proteins related to DC2.     

A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain

A novel Chlamydomonas flagellar mutant (oda-11) missing the alpha heavy chain of outer arm dynein but retaining the beta and gamma heavy chains was isolated. Restriction fragment length polymorphism analysis with an alpha heavy chain locus genomic probe indicated that the oda-11 mutation was genetically linked with the structural gene of the alpha heavy chain. In cross-section electron micrographs, the oda-11 axoneme lacked the outermost appendage of the outer arm, indicating that the alpha heavy chain should be located in this region in the wild-type outer arm. This mutant swam at 119 microns/s at 25 degrees C, i.e., at an intermediate speed between those of wild type (194 microns/s) and of oda-1 (62 microns/s), a mutant missing the entire outer dynein arm. The flagellar beat frequency (approximately 50 Hz) was also between those of wild type (approximately 60 Hz) and oda-1 (approximately 26 Hz). These results indicate that the outer dynein arm of Chlamydomonas can be assembled without the alpha heavy chain, and that the outer arm missing the alpha heavy chain retains partial function.     

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.