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.     

Isolated beta-heavy chain subunit of dynein translocates microtubules in vitro

Our goal was to assess the microtubule translocating ability of individual ATPase subunits of outer arm dynein. Solubilized outer arm dynein from sea urchin sperm (Stronglocentrotus purpuratus) was dissociated into subunits by low ionic strength buffer and fractionated by zonal centrifugation. Fractions were assessed by an in vitro functional assay wherein microtubules move across a glass surface to which isolated dynein fractions had been absorbed. Microtubule gliding activity was coincident with the 12-S beta-heavy chain-intermediate chain 1 ATPase fractions (beta/IC1). Neither the alpha-heavy chain nor the intermediate chains 2 and 3 fractions coincided with microtubule gliding activity. The beta/IC1 ATPase induced very rapid gliding velocities (9.7 +/- 0.88 micron/s, range 7-11.5 micron/s) in 1 mM ATP-containing motility buffers. In direct comparison, isolated intact 21-S outer arm dynein, from which the beta/IC1 fraction was derived, induced slower microtubule gliding rates (21-S dynein, 5.6 +/- 0.7 micron/s; beta/IC1, 8.7 +/- 1.2 micron/s). These results demonstrate that a single subdomain in dynein, the beta/IC1 ATPase, is sufficient for microtubule sliding activity.     

Properties of the full-length heavy chains of Tetrahymena ciliary outer arm dynein separated by urea treatment

An important challenge is to understand the functional specialization of dynein heavy chains. The ciliary outer arm dynein from Tetrahymena thermophila is a heterotrimer of three heavy chains, called alpha, beta and gamma. In order to dissect the contributions of the individual heavy chains, we used controlled urea treatment to dissociate Tetrahymena outer arm dynein into a 19S beta/gamma dimer and a 14S alpha heavy chain. The three heavy chains remained full-length and retained MgATPase activity. The beta/gamma dimer bound microtubules in an ATP-sensitive fashion. The isolated alpha heavy chain also bound microtubules, but this binding was not reversed by ATP. The 19S beta/gamma dimer and the 14S alpha heavy chain could be reconstituted into 22S dynein. The intact 22S dynein, the 19S beta/gamma dimer, and the reconstituted dynein all produced microtubule gliding motility. In contrast, the separated alpha heavy chain did not produce movement under a variety of conditions. The intact 22S dynein produced movement that was discontinuous and slower than the movement produced by the 19S dimer. We conclude that the three heavy chains of Tetrahymena outer arm dynein are functionally specialized. The alpha heavy chain may be responsible for the structural binding of dynein to the outer doublet A-tubule and/or the positioning of the beta/gamma motor domains near the surface of the microtubule track.     

The roles of noncatalytic ATP binding and ADP binding in the regulation of dynein motile activity in flagella

The regulation of dynein activity to produce microtubule sliding in flagella has not been well understood. To gain more insight into the roles of ATP and ADP in the regulation, we examined the effects of fluorescent ATP analogues and fluorescent ADP analogues on the ATPase activity and motile activity of dynein. 21S dynein purified from the outer arms of sea urchin sperm flagella hydrolyzed BODIPY(R) FL ATP (FL-ATP) at 78% of the rate for ATP hydrolysis. FL-ATP at 0.1-1 mM, however, induced neither microtubule translocation on a dynein-coated glass surface nor sliding disintegration of elastase-treated axonemes. Direct observation of single molecules of the fluorescent analogues showed that both the ATP and ADP analogues were stably bound to dynein over several minutes (dissociation rates = 0.0038-0.0082/s). When microtubule translocation on 21S dynein was induced by ATP, the initial increase of the mean velocity was accelerated by preincubation of the dynein with ADP. Similar increase was also induced by the preincubation with the ADP analogues. Even after preincubation with ADP, FL-ATP did not induce sliding disintegration of elastase-treated axonemes. After preincubation with a nonhydrolyzable ATP analogue, AMPPNP (adenosine 5'-(beta:gamma-imido)triphosphate), however, FL-ATP induced sliding disintegration in approximately 10% of the axonemes. These results indicate that both noncatalytic ATP binding and stable ADP binding, in addition to ATP hydrolysis, are involved in the regulation of the chemo-mechanical transduction in axonemal dynein.     

Inhibition of gliding movement by calcium in doublet microtubules on Tetrahymena ciliary dyneins in vitro.

We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 microns in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 microns/s. The addition of 0.1% Nonidet P-40 was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10(-9) M Ca, and at 0.05 to 1 mM ATP in the presence of 10(-3) M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2-8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility.     

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

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

Microtubule binding and translocation by inner dynein arm subtype I1

Structural, biochemical, and genetic evidence has demonstrated there are three inner dynein arm subforms, I1, I2, and I3, which differ in organization and composition (see Piperno et al.: J. Cell Biol. 110:379-389, 1990). Using dynein extracted from Chlamydomonas outer dynein armless mutant pf28, we have begun to define the structural and functional properties of isolated inner arm subforms. Inner dynein arm I1 was purified either by sucrose density gradient centrifugation or microtubule binding affinity. I1, composed of heavy chains 1 alpha and 1 beta, sedimented at 21S and selectively bound to and cross-linked purified microtubules in an ATP-sensitive manner. Deep etch electron microscopy revealed that the 21S sedimenting fraction contained two-headed structures in which large globular heads are connected by long, flexible-stem domains. In contrast, components derived from I2 and I3 sedimented as a mixture of 11S particles with single globular heads which did not bind to purified microtubules. Both the 21S and 11S sedimenting fractions supported microtubule translocation in in vitro motility assays. In 1 mM MgATP the I1-containing fraction produced very slow microtubule-gliding velocities (0.76 microns/sec) compared to the I2,I3-containing fraction (4.1 microns/sec).     

Length dependence of displacement fluctuations and velocity in microtubule sliding movement driven by sea urchin sperm outer arm beta dynein in vitro

We have studied the dependence on microtubule length of sliding velocity and positional fluctuation from recorded trajectories of microtubules sliding over sea urchin sperm outer arm beta dynein in a motility assay in vitro. The positional fluctuation was quantified by calculating the mean-square displacement deviation from the average, the calculation of which yields an effective diffusion coefficient. We have found that (1) the sliding velocity depends hyperbolically on the microtubule length, and (2) the effective diffusion coefficients do not depend on the length for sufficiently long microtubules. The length dependence of the sliding velocity indicates that the duty ratio, defined as the force producing period over the total cycle time of beta dynein interaction with microtubule, is very small. The length independence of the effective diffusion coefficient indicates that there is a correlation in the sliding movement fluctuation of microtubules.     

Two distinct isoforms of sea urchin egg dynein.

Extracts of unfertilized sea urchin eggs contain at least two isoforms of cytoplasmic dynein. One exhibits a weak affinity for microtubules and is primarily soluble. The other isoform, HMr-3, binds to microtubules in an ATP-sensitive manner, but is immunologically distinct from the soluble egg dynein (Porter et al.: Journal of Biological Chemistry 263:6759-6771, 1988). We have now further distinguished these egg dynein isoforms based on differences in NTPase activity. HMr-3 copurifies with NTPase activity, but it hydrolyzes CTP at 10 times the rate of ATP. The soluble egg dynein is similar to flagellar dynein in its nucleotide specificity; its MgCTPase activity is ca. 60% of its MgATPase activity. Non-ionic detergents and salt activate the MgATPase activities of both enzymes relative to their MgCTPase activities, but this effect is more pronounced for the soluble egg dynein than for HMr-3. Sucrose gradient-purified HMr-3 promotes an ATP-sensitive microtubule bundling, as seen with darkfield optics. We have also isolated a 20 S microtubule translocating activity by sucrose gradient fractionation of egg extracts, followed by microtubule affinity and ATP release. This 20 S fraction, which contains the HMr-3 isoform, induces a microtubule gliding activity that is distinct from kinesin. Our observations suggest that soluble dynein resembles axonemal dynein, but that HMr-2 is related to the dynein-like enzymes isolated from a variety of cell types and may represent the cytoplasmic dynein of sea urchin eggs.     

Polarized microtubule gliding and particle saltations produced by soluble factors from sea urchin eggs and embryos

In this report, we describe an in vitro system for analyzing microtubule-based movements in supernatants of sea urchin egg and embryo homogenates. Using video enhanced DIC microscopy, we have observed bidirectional saltatory particle movements on native taxol-stabilized microtubules assembled in low speed supernatants of Lytechinus egg homogenates, and gliding of these microtubules across a glass surface. A high speed supernatant of soluble proteins, depleted of organelles, microtubules, and their associated proteins supports the gliding of exogenous microtubules and translocation of polystyrene beads along these microtubules. The direction of microtubule gliding has been determined directly by observation of the gliding of flagellar axonemes in which the (+) and (-) ends could be distinguished by biased polar growth of microtubules off the ends. Microtubule gliding is toward the (-) end of the microtubule, is ATP sensitive, and inhibited only by high concentrations of vanadate. These characteristics suggest that the transport complex responsible for microtubule gliding in S2 is kinesin-like. The implications of these molecular interactions for mitosis and other motile events are discussed.     

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.     

Microtubule translocation properties of intact and proteolytically digested dyneins from Tetrahymena cilia

Tetrahymena cilia contain a three-headed 22S (outer arm) dynein and a single-headed 14S dynein. In this study, we have employed an in vitro assay of microtubule translocation along dynein-coated glass surfaces to characterize the motile properties of 14S dynein, 22S dynein, and proteolytic fragments of 22S dynein. Microtubule translocation produced by intact 22S dynein and 14S dynein differ in a number of respects including (a) the maximal velocities of movement; (b) the ability of 22S dynein but not 14S dynein to utilize ATP gamma S to induce movement; (c) the optimal pH and ionic conditions for movement; and (d) the effects of Triton X-100 on the velocity of movement. These results indicate that 22S and 14S dyneins have distinct microtubule translocating properties and suggest that these dyneins may have specialized roles in ciliary beating. We have also explored the function of the multiple ATPase heads of 22S dynein by preparing one- and two-headed proteolytic fragments of this three-headed molecule and examining their motile activity in vitro. Unlike the single-headed 14S dynein, the single-headed fragment of 22S dynein did not induce movement, even though it was capable of binding to microtubules. The two-headed fragment, on the other hand, translocated microtubules at velocities similar to those measured for intact 22S dynein (10 microns/sec). This finding indicates that the intact three-headed structure of 22S dynein is not essential for generating microtubule movement, which raises the possibility that multiple heads may serve some regulatory function or may be required for maximal force production in the beating cilium.     

Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement

The phosphorothioate analog of ATP has a sulfur atom replacing a non-bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule-dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility. It has been shown that the microtubule--22S-dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5'-[alpha-thio]triphosphate (ATP[alpha S]), Sp or Rp, by light-scattering stopped-flow methods. The dissociation by (Sp)ATP[alpha S] was rapid and the rate of the light-scattering change by (Sp)ATP[alpha S] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two-step process. On the other hand, (Rp)ATP[alpha S] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s-1). The adenosine 5'-[beta-thio]triphosphate (ATP[beta S]) isomers did not induce dissociation. The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol.mg protein-1.min-1)/apparent Km (microM) of this dynein were 400-720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[alpha S] (Sp or Rp), ATP[beta S] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at beta phosphate as the 22S dynein or myosin. The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[alpha S] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[alpha S] was one-quarter to one-half of that produced by ATP reactivation.     

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.     

Chlamydomonas outer arm dynein alters conformation in response to Ca2+

We have previously shown that Ca(2+) directly activates ATP-sensitive microtubule binding by a Chlamydomonas outer arm dynein subparticle containing the beta and gamma heavy chains (HCs). The gamma HC-associated LC4 light chain is a member of the calmodulin family and binds 1-2 Ca(2+) with K(Ca) = 3 x 10(-5) M in vitro, suggesting it may act as a Ca(2+) sensor for outer arm dynein. Here we investigate interactions between the LC4 light chain and gamma HC. Two IQ consensus motifs for binding calmodulin-like proteins are located within the stem domain of the gamma heavy chain. In vitro experiments indicate that LC4 undergoes a Ca(2+)-dependent interaction with the IQ motif domain while remaining tethered to the HC. LC4 also moves into close proximity of the intermediate chain IC1 in the presence of Ca(2+). The sedimentation profile of the gamma HC subunit changed subtly upon Ca(2+) addition, suggesting that the entire complex had become more compact, and electron microscopy of the isolated gamma subunit revealed a distinct alteration in conformation of the N-terminal stem in response to Ca(2+) addition. We propose that Ca(2+)-dependent conformational change of LC4 has a direct effect on the stem domain of the gamma HC, which eventually leads to alterations in mechanochemical interactions between microtubules and the motor domain(s) of the outer dynein arm.     

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.     

The 78,000-M(r) intermediate chain of Chlamydomonas outer arm dynein is a microtubule-binding protein

A previous study (King et al., 1991. J. Biol. Chem. 266:8401-8407) showed that the 78,000-M(r) intermediate chain (IC78) from the Chlamydomonas outer arm dynein is in direct contact with alpha-tubulin in situ, suggesting that this protein may be involved in binding the dynein to the doublet microtubules. Molecular genetic analysis of this chain recently demonstrated that it is a WD repeat protein essential for outer arm assembly (Wilkerson et al., 1995.J. Cell Biol. 129:169- 178). We have now transcribed and translated IC78 in vitro, and demonstrate that this molecule binds axonemes and microtubules, whereas a homologous protein (the 69,000-M(r) intermediate chain [IC69] of Chlamydomonas outer arm dynein) does not. Thus, IC78 is a bona fide microtubule-binding protein. Taken together with the previous results, these findings indicate that IC78 is likely to provide at least some of the adhesive force that holds the dynein to the doublet microtubule, and support the general hypothesis that the dynein intermediate chains are involved in targeting different dyneins to the specific cell organelles with which they associate. Analysis of the binding activities of various IC78 deletion constructs translated in vitro identified discrete regions of IC78 that affected the binding to microtubules; two of these regions are specifically missing in IC69. Previous studies also showed that IC78 is in direct contact with IC69; the current work indicates that the region of IC78 that mediates this interaction is coincident with two of IC78's WD repeats. This supports the hypothesis that these repeats are involved in protein-protein interactions within the dynein complex.     

Alloaffinity filtration: a general approach to the purification of dynein and dynein-like molecules

Alloaffinity filtration simply and specifically separates certain axonemal dyneins and dynein arm components from crude mixtures on the basis of their ability to bind and decorate Tetrahymena axonemal microtubules on a filter in the absence of ATP and to detach and pass into the eluate when 0.5 mM ATP is added. The procedure, which may be performed repetitively, is successful in purifying a Tetrahymena dynein that has characteristics of 30 S dynein prepared by conventional methods, while other dyneins originally present in the mixture, e.g., 14 S Tetrahymena dynein, are not found in the ATP eluate. A relatively homogeneous population of dynein oligomers is obtained. Alloaffinity-purified 30 S Tetrahymena dynein consists of heavy-, intermediate-, and light-chain polypeptides that cosediment in a sucrose gradient in fixed molar ratios and that have structural features of in situ Tetrahymena arms. Dyneins from other species will bind to Tetrahymena microtubules and can be purified by this method. Alloaffinity-purified Chlamydomonas dynein is a set of polypeptides including the four heavy chains that characterize the outer arm.     

A latent activity dynein-like cytoplasmic magnesium adenosine triphosphatase

A MgATPase has been isolated and characterized from unfertilized sea urchin eggs which is very similar, but not identical, to latent activity axonemal dynein. The cytoplasmic MgATPase activity sediments at 20 S, slightly slower than 21 S latent activity flagellar dynein. Activity is stimulated by nonionic detergent and is inhibited by sodium orthovanadate but is not as sensitive to vanadate as is 21 S flagellar dynein. The egg 20 S MgATPase is composed, at least in part, of three high molecular weight polypeptides. In addition, two intermediate-sized polypeptides appear to co-sediment with the 20 S MgATPase activity. A novel microtubule-affinity assay reveals that high molecular weight polypeptides 1 and 2 of the egg 20 S MgATPase can bind to reassembled microtubules and can be released from the microtubules with MgATP2-. Further, the apparent specific activity of the egg MgATPase is enriched 15-fold by a single microtubule binding step. The results suggest that the cytoplasmic 20 S MgATPase is a dynein-like microtubule translocator which resides in the unfertilized egg awaiting future incorporation onto microtubules in order to perform work. The egg 20 S enzyme might function in cytoplasmic microtubule-mediated movement or it might be a precursor of embryonic ciliary dynein.     

Vanadate-sensitized cleavage of dynein heavy chains by 365-nm irradiation of demembranated sperm flagella and its effect on the flagellar motility

Irradiation of demembranated flagella of sea urchin sperm at 365 nm in the presence of 0.05-1 mM MgATP and 5-10 microM vanadate (Vi) cleaves the alpha and beta heavy chains of the outer arm dynein at the same site and at about the same rate as reported previously for the solubilized dynein (Gibbons, I. R., Lee-Eiford, A., Mocz, G., Phillipson, C. A., Tang, W.-J. Y., and Gibbons, B. H. (1987) J. Biol. Chem. 262, 2780-2786). The decrease in intact alpha and beta heavy chain material is biphasic, with about 80% being lost with a half-time of 8-10 min, and the remainder more slowly. Five other axonemal polypeptides of Mr greater than 350,000 are lost similarly, concomitant with the appearance of at least 9 new peptides of Mr 150,000-250,000. The motility of irradiated sperm flagella upon subsequent dilution into reactivation medium containing 1 mM ATP and 2.5 mM catechol shows a progressive decrease in flagellar beat frequency for irradiation times that produce up to about 50% cleavage of the dynein heavy chains; more prolonged irradiation causes irreversible loss of motility. Competition between photocleaved and intact outer arm dynein for rebinding to dynein-depleted sperm flagella shows that cleavage has little effect upon the ability for rebinding, although the cleaved dynein partially inhibits subsequent motility. Substitution of MnATP for the MgATP in the irradiation medium prevents the loss of all of the axonemal polypeptides during irradiation for up to 60 min and also protects the potential for subsequent flagellar motility. It is concluded that loss of the five axonemal polypeptides upon irradiation results from a Vi-sensitized photocleavage similar to that which occurs in the alpha and beta heavy chains of outer arm dynein and that these polypeptides represent Vi-inhibitable ATPase subunits of dyneins located in the inner arms and possibly elsewhere in the flagellar axoneme.