Three-headed outer arm dynein from Chlamydomonas that can functionally combine with outer-arm-missing axonemes.

A procedure was developed for isolating Chlamydomonas outer-arm dynein that can functionally combine with the axoneme of an outer-arm-missing mutant, oda1. Previous studies showed that the outer-arm dynein of this organism, containing three heavy chains (alpha, beta, gamma), dissociates upon extraction with a high-salt-concentration buffer solution into an 18-S particle containing the alpha and beta heavy chains and a 12-S particle containing the gamma heavy chain. It was found, however, that the three heavy chains did not dissociate if the high-salt extract was centrifuged in the presence of Mg2+; the three chains constituted a single species (23-S dynein) sedimenting at about 23 S and displayed a three-headed bouquet configuration in electron micrographs. Furthermore, the 23-S dynein had the activity to bind to the axonemes of oda1 and increase the reactivated motility of detergent-extracted cell models; its addition increased the beat frequency from 28 Hz to 53 Hz, a frequency comparable to that of wild-type axoneme. The 18-S and 12-S dyneins, on the other hand, were unable to increase the motility of oda1 axonemes even when added together. The new protocol thus enables purification of outer-arm dynein that retains its functional activity. It will provide a useful experimental system with which to study the mechanism of outer-arm function.     

Structural conformation of the ciliary ATPase dynein.

The ATPase dynein forms part of a mechanoohemically active complex responsible for the sliding filament mechanism of ciliary and flagellar motion. Extraction of demembranated cilia from the lamellibranch mollusc Unio or the protozoan Tetrahymena by 0.5 m-KCl solubilizes the outer rows of dynein and, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, releases the A form (Mr 360,000) of dynein into solution. Negative contrast electron microscopy of the solubilized dynein fraction reveals an homogeneous array of 93 Å particles that we identify as the ATPase dynein in its monomeric form. Because of the method of dynein extraction, the conformation of the molecule, and the size and shape of the outer arms in situ, we suggest that monomeric dynein is only one part of a larger, non-covalently joined molecular complex that forms the entire arm. When KCl-extracted axonemes are viewed by negative contrast but prior to fractionation of the dynein, individual arms can be seen that comprise three to four of the 93 Å subunits, thus suggesting that each arm is a multisubunit polymer of dynein or dynein-like molecules.     

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.     

Two-headed outer- and inner-arm dyneins of Leishmania sp bear conserved IQ-like motifs

Dyneins are high molecular weight microtubule based motor proteins responsible for beating of the flagellum. The flagellum is important for the viability of trypanosomes like Leishmania. However, very little is known about dynein and its role in flagellar motility in such trypanosomatid species. Here, we have identified genes in five species of Leishmania that code for outer-arm dynein (OAD) heavy chains α and β, and inner-arm dynein (IAD) heavy chains 1α and 1β using BLAST and MSA. Our sequence analysis indicates that unlike the three-headed outer-arm dyneins of Chlamydomonas and Tetrahymena, the outer-arm dyneins of the genus Leishmania are two-headed, lacking the γ chain like that of metazoans. N-terminal sequence analysis revealed a conserved IQ-like calmodulin binding motif in the outer-arm α and inner-arm 1α dynein heavy chain in the five species of Leishmania similar to Chlamydomonas reinhardtii outer-arm γ. It was predicted that both motifs were incapable of binding calmodulin. Phosphorylation site prediction revealed conserved serine and threonine residues in outer-arm dynein α and inner-arm 1α as putative phosphorylation sites exclusive to Leishmania but not in Trypanosoma brucei suggesting that regulation of dynein activity might be via phosphorylation of these IQ-like motifs in Leishmania sp.     

The substructure of isolated and in situ outer dynein arms of sea urchin sperm flagella

Outer-arm dynein from the sperm of the sea urchin S. purpuratus was adsorbed to mica flakes and visualized by the quick-freeze, deep-etch technique. Replicas reveal particles comprised of two globular heads joined by two irregularly shaped stems which make contact along their length. One head is pear-shaped (18.5 X 12.5 nm) and the other is spherical (14.5-nm diam). The stems are decorated by a complex of bead-like subunits. The same two-headed protein is found in the 21S dynein-1 fraction of sucrose gradients. The beta-heavy chain/intermediate chain 1 (beta/IC-1) dynein subfraction, produced by low-salt dialysis and zonal centrifugation of the high-salt-extracted dynein-1, contains only single-headed molecules with single stems. These heads are predominantly pear-shaped (18.5 X 12.5 nm). Since 21S dynein-1 contains two heavy chains (alpha and beta), and the beta/IC-1 subfraction is comprised of only the beta-heavy chain (Tang et al., 1982, J. Biol. Chem. 257: 508-515), we conclude that each head is formed by a heavy chain, that the pear-shaped head contains the beta-heavy chain, and that the spherical head contains the alpha-heavy chain. The in situ outer dynein arms of demembranated sperm were also studied by the quick-freeze, deep-etch method. When frozen in reactivation buffer devoid of ATP, each arm consists of a large globular head that attaches to the A-microtubule by distally skewed subunits and attaches to the B-microtubule by a slender stalk. In ATP, this head shifts its orientation such that it can be seen to be constructed from two globular domains. We offer possible correlates between the in situ and the in vitro images, and we compare the structure of sea-urchin dynein with dynein previously described from Chlamydomonas and Tetrahymena.     

ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.     

High-pressure liquid chromatography fractionation of Chlamydomonas dynein extracts and characterization of inner-arm dynein subunits

A rapid procedure for fractionating salt-stable dynein subunits from high-salt extracts of Chlamydomonas axonemes has been developed using a high-pressure liquid chromatography system with an anion exchange column and gradient salt elution. Five distinct fractions are shown to be highly enriched for five distinct subunits or subunit complexes by SDS/polyacrylamide gel electrophoresis. ATPase activity and electron microscopy. Peaks 1 and 4 contain, respectively, the single-headed gamma-subunit and the two-headed alpha/beta-heteropolymer that form the outer arm in situ and are dissociated by salt exposure; both peaks are absent from the outer arm-less mutant pf-28. Peaks 2, 3 and 5 contain, respectively, two distinct single-headed species and a double-headed species that derive from inner arms; all three peaks are missing from the inner arm-less mutant pf-23. Sucrose-gradient sedimentation analysis confirms these assignments and provides additional information on the intermediate-chain and light-chain composition of the inner-arm species. Electron microscopy of the purified inner-arm species visualized by the quick-freeze deep-etch technique complements a previous analysis of outer-arm species. Each protein is shown to have a unique morphology, and both the inner- and outer-arm proteins clearly belong to a common family whose structural divergence presumably reflects functional specialization.     

Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein

Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.     

Some properties of bound and soluble dynein from sea urchin sperm flagella

Axonemes were isolated from sperm of Colobocentrotus by a procedure involving two extractions with 1% Triton X-100 and washing The isolated axonemes contained 7 x 10¹⁵ g protein per µm of their length. Treatment of the axonemes with 0 5 M KCl for 30 min extracted 50–70% of the flagellar ATPase protein, dynein, and removed preferentially the outer arms from the doublet tubules. Almost all of the dynein (85–95%) could be extracted from the axonemes by dialysis at low ionic strength. In both cases the extracted dynein sedimented through sucrose gradients at 12–14S, and no 30S form was observed The enzymic properties of dynein changed when it was extracted from the axonemes into solution. Solubilization had a particularly marked effect on the KCl- and pH-dependence of the ATPase activity. The pH-dependence of soluble dynein was fairly simple with a single peak extending from about pH 6 to pH 10. The pH-dependence of bound dynein was more complex. In 0.1 M KCl, the bound activity appeared to peak at about pH 9, and dropped off rapidly with decreasing pH, reaching almost zero at pH 7; an additional peak at pH 10 0 resulted from the breakdown of the axonemal structure and solubilization of dynein that occurred at about this pH. A similar curve was obtained in the absence of KCl, except for the presence of a further large peak at pH 8 Measurement of the kinetic parameters of soluble dynein showed that both K_m and V_max increased with increasing concentrations of KCl up to 0.5 M When bound dynein was assayed under conditions that would induce motility in reactivated sperm (0 15 M KCl with Mg⁺⁺ activation), it did not obey Michaelis-Menten kinetics, although it did when assayed under other conditions. The complex enzyme-kinetic behavior of bound dynein, and the differences between its enzymic properties and those of soluble dynein, may result from its interactions with tubulin and other axonemal proteins     

A biochemical study of flagellar dynein from starfish spermatozoa: protein components of the arm structure.

Half of the adenosine triphosphatase (dynein) activity of starfish sperm tail axonemes was extracted with 0.6 m KCl-10 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (KCl-EDTA), while with 1 mm Tris · HCl (pH 7.8)-0.1 mm EDTA-0.5 mm dithiothreitol (Tris-EDTA) around 90% of the activity was extracted. The main adenosine triphosphatase (ATPase) in the KCl-EDTA extract had a sedimentation coefficient of 20S and that in the Tris-EDTA extract had a sedimentation coefficient of 12S. The effects of divalent cations, pH, and an SH-blocking reagent and the K_m for ATP were different for the activities of the two forms of dynein ATPase. These two forms of dynein can interconvert to some extent when the ionic strength of the medium is changed. In a medium suitable for recombination of dynein to outer doublet microtubules (recombination buffer, 20 mm Tris-HCl (pH 7.6)-2 mm MgCl₂-0.5 mm dithiothreitol), the 20S ATPase converted to a 24S ATPase. Recombination of the ATPase activity from the KCl-EDTA extract was almost complete while that from the Tris-EDTA extract was around 50%. Outer arms disappeared preferentially by the treatment with KCl-EDTA, and the extracted arms could be reconstituted in the recombination buffer. In the case of the Tris-EDTA extraction, both the outer and inner arms disappeared and the reconstitution of the arms could not be confirmed. From the above results it can be considered that the 20 or 24S dynein represented the arm structure. The 20 or 24S ATPase fraction contained two large polypeptide chains as main components having electrophoretic mobilities in the presence of sodium dodecylsulfate similar to those of Tetrahymena ciliary dyneins and of sea urchin sperm flagellar dyneins. The relationship between these chains and dynein subunits is discussed.     

Interactions of Tetrahymena dynein with microtubule protein. Tubulin-induced stimulation of dynein ATPase activity

The ATPase (EC 3.6.1.3) activity of 30 S dynein from Tetrahymena cilia was remarkably stimulated by porcine brain tubulin at pH 10. The activity increased with increasing concentration of tubulin until the molar ratio of tubulin dimer to 30 S dynein reached approx. 10. The optimum of the ATPase activity of 30 S dynein in the presence of tubulin was 1?2 mM for MgCl₂ and 2 mM for CaCl₂. Increasing ionic strength gradually inhibited the stimulation effects of tubulin. Activation energies of 30 S dynein in the presence and absence of tubulin were almost the same. At the temperatures beyond 25 °C stimulation effects of tubulin disappeared. ATP was a specific substrate even in the presence of tubulin. In kinetic investigations parallel reciprocal plots were observed in a constant ratio of divalent cations to ATP of 2, indicating that tubulin was less tightly bound to 30 S dynein in the presence of ATP than in the absence. The similar results were obtained at pH 8.2. 14 S dynein and the 12 S fragment which have poor ability to recombine with outer fibers were also activated with brain tubulin.     

Functional binding of inner-arm dyneins with demembranated flagella of Chlamydomonas mutants

Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating.     

The salt-extractable fraction of dynein from sea urchin sperm flagella: An analysis by gel electrophoresis and by adenosine triphosphatase activity

Previous work has shown that the dynein from axonemes of sea urchin sperm consists of two distinct fractions which differ substantially in their extractability by salt. Upon gel electrophoresis of whole demembranated axonemes solubilized with sodium dodecyl sulfate, the dynein fraction shows two closely spaced bands with apparent molecular weights of 520,000 and 460,000; the proteins in these bands are termed the A and B components of the dynein. Similar electrophoresis of the soluble fraction obtained by extracting the axonemes with 0.5 M NaCl shows a single prominent band containing approximately half of the A component of the dynein (A₁ component). The residue of extracted axonemes contain the other half of the A component of the dynein (A₂ component) and all the B component. Densitometry of the bands indicates that the A₁, A₂ and B components of the dynein are present in approximately equal molar quantity. Electron microscopic studies show that the A₁ component of the dynein constitutes the outer arms on the doublet tubules. Assay of ATPase activity in 0.05 M KCl and l mM ATP indicates about 65% of the total ATPase activity becomes soluble when the A₁ component of the dynein is extracted with salt.     

Substructure of sea urchin egg cytoplasmic dynein

The substructure of the cytoplasmic dynein molecule was studied using the quick-freeze, deep-etch technique. Cytoplasmic dynein purified as a 12 S form from the eggs of the sea urchin Hemicentrotus pulcherrimus was composed of a single high molecular weight polypeptide. Rotary shadowing images of cytoplasmic dynein either sprayed on to a mica surface or quick-frozen on mica flakes demonstrated a single-headed molecule, in contrast to the two-headed molecule of sea urchin sperm flagellar 21 S dynein. More detailed substructure was visualized by rotary shadowing after quick-freeze deep-etching. Cytoplasmic dynein consisted of a head and a stem. The head was pear-shaped (16 nm X 11 nm) and a little smaller than the pear-shaped head of 21 S dynein (18 nm X 14 nm). The form of the stem was irregular, and its apparent length varied from 0 to 32 nm. Binding of cytoplasmic dynein to brain microtubule in the solution was observed by negative staining, and that in the precipitate was examined by the quick-freeze, deep-etch method as well. Both methods revealed the presence of two kinds of microtubules, one a fully decorated microtubule and the other a non-decorated microtubule. Cytoplasmic dynein bound to microtubule also appeared as a globular particle. Neither the periodic binding nor the crossbridges that were observed with 21 S dynein were formed by cytoplasmic dynein, although cytoplasmic dynein appeared to bind to microtubules co-operatively.     

Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.     

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.     

ATPase activity of Tetrahymena cilia before and after extraction of dynein

Cilia from Tetrahymena pyriformis were extracted twice with Tris-EDTA. The first extraction increased the total ATPase activity by about one-third. No increase in activity occurred as a result of the second extraction, but 40% of the original ATPase activity remained in the pellet. The activity remaining in the pellet differed in its substrate specificity, its thermostability, and its sensitivity to monovalent cation chlorides from the solubilized dynein. Several of the properties of the ATPase activity of whole cilia differed from those computed for a mixture of 40% pellet ATPase + 60% solubilized dynein ATPase. From these differences it was deduced that dynein in situ is more thermostable than is solubilized dynein and, in contrast to solubilized dynein, is slightly inhibited by KCl, NaCl, LiCl, and NH₄Cl. The increase in total activity upon solubilization of the dynein and the changes in thermostability and in sensitivity to monovalent cations indicates that dynein ATPase in situ is modified by interaction with other components of the axonemal bend generating system.The pellet remaining after extraction of dynein by two dialyses against Tris-EDTA was treated with 0.4% Triton X-100 to solubilize ciliary membranes. Less than half of the ATPase activity was solubilized by this treatment. The possibility that the activity remaining in the Tris-EDTA- and Triton X-100-extracted residue represents an additional ATPase of cilia is discussed.     

Effects of trypsin-digested outer-arm dynein fragments on the velocity of microtubule sliding in elastase-digested flagellar axonemes

Flagellar movement is caused by the coordinated activity of outer and inner dynein arms, which induces sliding between doublet microtubules. In trypsin-treated flagellar axonemes, microtubule sliding induced by ATP is faster in the presence than in the absence of the outer arms. To elucidate the mechanism by which the outer arms regulate microtubule sliding, we studied the effect of trypsin-digested outer-arm fragments on the velocity of microtubule sliding in elastase-treated axonemes of sea urchin sperm flagella. We found that microtubule sliding was significantly slower in elastase-treated axonemes than in trypsin-treated axonemes, and that this difference disappeared after the complete removal of the outer arms. After about 95% of the outer arms were removed, however, the velocity of sliding induced by elastase and ATP increased significantly by adding outer arms that had been treated with trypsin in the presence of ATP. The increase in sliding velocity did not occur in the elastase-treated axonemes from which the outer arms had been completely removed. Among the outer arm fragments obtained by trypsin treatment, a polypeptide of about 350 kDa was found to be possibly involved in the regulation of sliding velocity. These results suggest that the velocity of sliding in the axonemes with only inner arms is similar to that in the axonemes with both inner and outer arms, and that the 350 kDa fragment, probably of the alpha heavy chains, increases the sliding activity of the intact outer and inner arms on the doublet microtubules.     

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase

Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild- type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke- deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP- mediated mechanism.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.