Identification of dynein heavy chain genes expressed in human and mouse testis: chromosomal localization of an axonemal dynein gene

Dynein heavy chains are involved in microtubule-dependent transport processes. While cytoplasmic dyneins are involved in chromosome or vesicle movement, axonemal dyneins are essential for motility of cilia and flagella. Here we report the isolation of dynein heavy chain (DHC)-like sequences in man and mouse. Using polymerase chain reaction and reverse-transcribed human and mouse testis RNA cDNA fragments encoding the conserved ATP binding region of dynein heavy chains were amplified. We identified 11 different mouse and eight human dynein-like sequences in testis which show high similarity to known dyneins of different species such as rat, sea urchin or green algae. Sequence similarities suggest that two of the mouse clones and one human clone encode putative cytoplasmic dynein heavy chains, whereas the other sequences show higher similarity to axonemal dyneins. Two of nine axonemal dynein isoforms identified in the mouse testis are more closely related to known outer arm dyneins, while seven clones seem to belong to the inner arm dynein group. Of the isolated human isoforms three clones were classified as outer arm and four clones as inner arm dynein heavy chains. Each of the DHC cDNAs corresponds to an individual gene as determined by Southern blot experiments. The alignment of the deduced protein sequences between human (HDHC) and mouse (MDHC) dynein fragments reveals higher similarity between single human and mouse sequences than between two sequences of the same species. Human and mouse cDNA fragments were used to isolate genomic clones. Two of these clones, gHDHC7 and gMDHC7, are homologous genes encoding axonemal inner arm dyneins. While the human clone is assigned to 3p21, the mouse gene maps to chromosome 14.     

Twenty-five dyneins in Tetrahymena: A re-examination of the multidynein hypothesis

Dyneins are responsible for essential movements in eukaryotic cells. The motor activity of each dynein complex resides in its complement of heavy chains. In the present study, we examined 136 heavy chain sequences from the completed genomes of 11 diverse model organisms, including examples from Viridiplantae, Excavata, Chromalveolata, and Metazoa. In many cases, we discovered dynein heavy chains previously not identified. For example, Tetrahymena expresses a total of 25 DYH genes rather than the previously identified 14. The Tetrahymena DYH genes are nonaxonemal DYH1 and DYH2; axonemal outer arm alpha, beta, and gamma; axonemal two-headed inner arm 1alpha and 1beta; and 18 single-headed inner arm heavy chains. The heavy chains divide into nine classes; six of these are highly conserved in sequence and number of isoforms in a given organism. The other three are single-headed inner arm dyneins, whose numbers vary significantly in different organisms. These findings lead to two conclusions. One, the last common ancestor of all eukaryotes expressed nine different dynein heavy chains. Two, subsequent to the divergences leading to different organisms, additional dynein heavy chains emerged. These newer dyneins are not well conserved across species and the variation may reflect different motility requirements in different organisms. Together, these results suggest that each of the nine classes of dyneins is functionally distinct, but members within some of the classes are not specialized. An understanding of the relationships among the various dynein heavy chains is important when deducing functions across species.     

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.     

Dynein of sperm flagella of oyster beloning to protostomia also has a two-headed structure

Summary— An axonemal dynein was purified from the sperm of Crassostrea gigas, an oyster belonging to Protostomia. The molecular masses of component polypeptides were almost equivalent to those of other dyneins. Biochemical and biophysical properties were also quite similar. For example, UV-cleavage, inhibition of ATPase by vanadate and induction of microtubule gliding were observed with the axonemal dynein. The oyster dynein had a two-headed structure as had the outer arm dynein of Deuterostomia such as sea urchin, rainbow trout and bull spermatozoa. On the other hand, dyneins of Protozoa are three-headed particles. From the evolutional point of view, it is likely that the number of heads of dynein molecule decreased when Metazoa evolved from Protozoa.     

The dynein heavy chain family

Dynein is the large molecular motor that translocates to the (-) ends of microtubules. Dynein was first isolated from Tetrahymena cilia four decades ago. The analysis of the primary structure of the dynein heavy chain and the discovery that many organisms express multiple dynein heavy chains have led to two insights. One, dynein, whose motor domain comprises six AAA modules and two potential mechanical levers, generates movement by a mechanism that is fundamentally different than that which underlies the motion of myosin and kinesin. And two, organisms with cilia or flagella express approximately 14 different dynein heavy chain genes, each gene encodes a distinct dynein protein isoform, and each isoform appears to be functionally specialized. Sequence comparisons demonstrate that functionally equivalent isoforms of dynein heavy chains are well conserved across species. Alignments of portions of the motor domain result in seven clusters: (i) cytoplasmic dynein Dyhl; (ii) cytoplasmic dynein Dyh2; (iii) axonemal outer arm dynein alpha; (iv) outer arm dyneins beta and gamma; (v) inner arm dynein 1alpha; (vi) inner arm dynein 1beta; and (vii) a group of apparently single-headed inner arm dyneins. Some of the dynein groups contained more than one representative from a single organism, suggesting that these may be tissue-specific variants.     

The proximal portion of Chlamydomonas flagella contains a distinct set of inner dynein arms

A specific type of inner dynein arm is located primarily or exclusively in the proximal portion of Chlamydomonas flagella. This dynein is absent from flagella less than 6 microns long, is assembled during the second half of flagellar regeneration time and is resistant to extraction under conditions causing complete solubilization of two inner arm heavy chains and partial solubilization of three other heavy chains. This and other evidence described in this report suggest that the inner arm row is composed of five distinct types of dynein arms. Therefore, the units of three inner arms that repeat every 96 nm along the axoneme are composed of different dyneins in the proximal and distal portions of flagella.     

Conformational changes of dynein: mapping and sequence analysis of ATP/Vanadate-dependent trypsin-sensitive sites on the outer arm dynein b heavy chain from sea urchin sperm flagella

Conformational changes of dynein during ATP hydrolysis are demonstrated by the difference in the tryptic fragments of the dynein heavy chain between in the absence and presence of ATP and vanadate. Here tryptic sites in the presence of ATP and vanadate (Tav sites) have been mapped on the betaheavy chain of outer arm dynein from sea urchin sperm flagella. Tav sites are located not only near the central catalytic domain which includes four P-loops, but also near the carboxyl-terminal coiled-coil region. The Tav2 site is located in the most carboxyl-terminal region, which is nearly 850 amino acid residues apart from the the fourth P-loop (P4 site). The region from the most amino-terminal Tav site (Tav1 site) to the Tav2 site covers approximately 2,100 amino acid residues, which is almost half the whole betaheavy chain. Comparison of the sequences around the tryptic sites of the sea urchin b chain and those of the dynein heavy chains from other organisms reveals that the sequence around the Tav1 site is highly conserved in both cytoplasmic and axonemal dyneins but that around Tav2 sites is only conserved in axonemal dyneins, suggesting functional differences in the Tav2 region between the two subfamilies of dynein.     

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.     

Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties

Extraction of isolated axonemes from trout (Salmo gairdneri) sperm with 0.6 M NaCl removed 97% of the outer arms, approximately 12% of the protein, and approximately 50% of the MgATPase activity. Fractionation of this high salt extract by sucrose density gradient centrifugation yielded a single peak of ATPase activity with an apparent sedimentation coefficient of 19 S. Electrophoretic analysis showed that this 19 S particle was composed of two heavy chains (termed alpha and beta; Mr 430,000 and 415,000, respectively), five intermediate molecular weight chains (IC1-IC5; Mr 85,000, 73,000, 65,000, 63,000, and 57,000), and six light chains (LC1-LC6; Mr 22,000-6,000). A similar complex was obtained following further purification by DEAE-Sephacel column chromatography. Quantitative densitometry of Coomassie Blue-stained gels indicated that the heavy and intermediate chains were present in equimolar amounts. Electron microscopic examination of the 19 S particles revealed that it consisted of two globular heads joined together by a Y-shaped stem. The 19 S particle had a specific MgATPase activity of 1.1 +/- 0.3 mumol of phosphate released/min/mg and exhibited an apparent Km for MgATP2- of 40 +/- 16 microM. MnATP2- and CaATP2- were hydrolyzed at rates 100 and 80% that of MgATP2-, respectively. The Mg-ATPase activity was inhibited by vanadate, but not by ouabain or oligomycin, and exhibited a high activity between pH 7.0 and 10.0 with a maximum at pH 9.0-9.5. ATP was the preferred nucleotide, although GTP and CTP (but not ITP) did interact with the dynein to a minor extent. Based on its origin, sedimentation coefficient, polypeptide composition, and enzymatic properties, we conclude that this two-headed 19 S particle represents the entire trout sperm axonemal outer arm dynein. This dynein is probably exemplary of the outer arm dyneins of other vertebrates.     

Flagellar quiescence in Chlamydomonas: Characterization and defective quiescence in cells carrying sup-pf-1 and sup-pf-2 outer dynein arm mutations

Chlamydomonas reinhardtii can use their flagella for two distinct types of movement: swimming through liquid or gliding on a solid substrate. Cells switching from swimming to gliding motility undergo a reversible flagellar quiescence. This phenomenon appears to involve the outer dynein arms, since mutants having altered outer arm beta and gamma dyneins (sup-pf-1 and sup-pf-2) show a diminished ability to quiesce. Sup-pf-1 and sup-pf-2 were originally isolated as gain-of-function mutations that suppress the flagellar paralysis resulting from radial spoke or central pair defects. Defective quiescence is also a gain-of-function phenomenon, as cells completely lacking outer arm heavy chains show a normal quiescence phenotype. These data suggest that regulation of outer arm dynein activity is essential for flagellar quiescence and furthermore that regulation of quiescence involves a signal transduction pathway that shares elements with the radial spoke/central pair system.     

HMW-2, the Sertoli cell cytoplasmic dynein from rat testis, is a dimer composed of nearly identical subunits

The ultrastructure and biochemical characteristics of HMW-2, the Sertoli cell cytoplasmic dynein isolated from rat testes, were analyzed. Electron microscopic studies revealed a two-headed two-stem structure with dimensions very similar to other dyneins. We found that, like other cytoplasmic dyneins, both heads have an approximately spherical shape with a central cavity. Heavy chain analysis suggested the presence of only one type of heavy chain, a finding that was supported by the simple Michaelis-Menten kinetics displayed by the HMW-2-associated ATPase activity. In addition, dissociation of the HMW-2 complex resulted in a single type of dynein subunit sedimenting at 11.8 S. This fraction contained all the polypeptides present in the undissociated HMW-2. Ultrastructurally the HMW-2 subunits were composed of one globular domain with a tail. The simplest interpretation is that HMW-2 is a dimer of nearly identical subunits, each containing one heavy chain, one 90-kDa intermediate chain, and two light chains.     

Chlamydomonas flagellar outer row dynein assembly protein ODA7 interacts with both outer row and I1 inner row dyneins

We previously found that a mutation at the ODA7 locus in Chlamydomonas prevents axonemal outer row dynein assembly by blocking association of heavy chains and intermediate chains in the cytoplasm. We have now cloned the ODA7 locus by walking in the Chlamydomonas genome from nearby molecular markers, confirmed the identity of the gene by rescuing the mutant phenotype with genomic clones, and identified the ODA7 gene product as a 58-kDa leucine-rich repeat protein unrelated to outer row dynein LC1. Oda7p is missing from oda7 mutant flagella but is present in flagella of other outer row or inner row dynein assembly mutants. However, Oda7 levels are greatly reduced in flagella that lack both outer row dynein and inner row I1 dynein. Biochemical fractionation and rebinding studies support a model in which Oda7 participates in a previously uncharacterized structural link between inner and outer row dyneins.     

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.     

Computer simulation of flagellar movement: VII. Conventional but functionally different cross-bridge models for inner and outer arm dyneins can explain the effects of outer arm dynein removal

Outer arm dynein removal from flagella by genetic or chemical methods causes decreased frequency and power, but little change in bending pattern. These results suggest that outer arm dynein operates within bends to increase the speed of bend propagation, but does not produce forces that alter the bending pattern established by inner arm dyneins. A flagellar model incorporating different cross-bridge models for inner and outer arm dyneins has been examined. The inner arm dynein model has a hyperbolic force-velocity curve, with a maximum average force at 0 sliding velocity of about 14 pN for each 96 nm group of inner arm dyneins. The outer arm dynein model has a very different force-velocity curve, with a maximum force at about 10-15% of V(max). The outer arm dynein model is adjusted so that the unloaded sliding velocity for a realistic mixture of inner and outer arm dyneins is twice the unloaded sliding velocity for the inner arm dynein model alone. With these cross-bridge models, a flagellar model can be obtained that reduces its sliding velocity and frequency by approximately 50% when outer arm dyneins are removed, with little change in bending pattern. The addition of outer arm dyneins, therefore, gives an approximately 4-fold increase in power output against viscous resistances, and outer arm dyneins may generate 90% or more of the power output. Cell Motil.     

Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA+ molecular motor

Cytoplasmic dynein is an AAA(+)-type molecular motor whose major components are two identical heavy chains containing six AAA(+) modules in tandem. It moves along a single microtubule in multiple steps which are accompanied with multiple ATP hydrolysis. This processive sliding is crucial for cargo transports in vivo. To examine how cytoplasmic dynein exhibits this processivity, we performed in vitro motility assays of two-headed full-length or truncated single-headed heavy chains. The results indicated that four to five molecules of the single-headed heavy chain were required for continuous microtubule sliding, while approximately one molecule of the two-headed full-length heavy chain was enough for the continuous sliding. The ratio of the stroking time to the total ATPase cycle time, which is a quantitative indicator of the processivity, was approximately 0.2 for the single-headed heavy chain, while it was approximately 0.6 for the full-length molecule. When two single-headed heavy chains were artificially linked by a coiled-coil of myosin, the processivity was restored. These results suggest that the two heads of a single cytoplasmic dynein communicate with each other to take processive steps along a microtubule.     

Structure and mass analysis of 12S and 19S dynein obtained from bull sperm flagella

The Brookhaven scanning transmission electron microscope (STEM) was used to elucidate the structures and masses of 12S and 19S dynein extracted from bull sperm flagella. The 12S particle was a single globular particle with an average mass of 311 +/- 10 kdaltons. The 19S dynein particles consisted of two globular heads joined to a common base. The average mass of the 19S particle was 1.6 +/- 0.04 X 10(6) daltons. Thus, with the exception of the larger mass, the bull sperm 19S dynein molecule resembles the two-headed 21S dynein obtained from sea urchin sperm flagella and the 18S dynein obtained from Chlamydomonas with the possibility of a third head giving rise to the 12S particle. The structure, mass and polypeptide composition of bull sperm flagella dynein is compared with outer arm dyneins previously obtained from Chlamydomonas, Tetrahymena, and sea urchin sperm flagella.     

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.     

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.     

Association between actin and light chains in Chlamydomonas flagellar inner-arm dyneins

Inner dynein arms in cilia and flagella contain actin as a subunit; however, the function of this actin is totally unknown. Here we performed chemical crosslinking experiments to examine the interaction of actin with other subunits. Six of the seven Chlamydomonas inner-arm dynein species separated by anion-exchange chromatography contain actin and either one of the two previously identified light chains, p28 and centrin, in a mutually exclusive manner. Western blotting of chemically crosslinked dyneins indicated that actin is directly associated with p28 and centrin but not with the dynein heavy chains (HCs). In contrast, p28 and centrin both appeared to interact directly with the N-terminal half of the HCs. Thus it is likely that actin is associated with the heavy chains through p28/centrin. These light chains may well function in the assembly or targeting of the inner arm to the correct axonemal location.     

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.