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.     

Sperm motility-activating complex formed by t-complex distorters

Transmission ratio distortion is a dramatic example of non-Mendelian transmission. In mice, t-haplotype males produce dysfunctional +-sperm and normal t-sperm, leading to transmission in favor of t-sperm. Genetic studies have indicated that the t-complex responder locus, Tcr, rescues t-sperm but not +-sperm from defective products of t-complex distorter loci, Tcds. Light chain 1 (LC1) and LC3 from sea urchin sperm outer arm dynein have sequence similarities to Tctex2 and Tctex1, respectively, both of which are wild-type products of Tcds. We show here that LC1 and LC3 are able to make a 1:1 complex. Since Tcr is a member of the Smok (sperm motility kinase) family and LC1 is phosphorylated at the activation of sperm motility in a cAMP-dependent manner, this complex in a dynein motor molecule might be a direct target of Smok/Tcr kinase in a signal cascade that regulates sperm motility. Thus, we designate it as Smoac (sperm motility activating complex).     

LC2, the Chlamydomonas Homologue of the t Complex-encoded Protein Tctex2, Is Essential for Outer Dynein Arm Assembly

Tctex2 is thought to be one of the distorter genes of the mouse t haplotype. This complex greatly biases the segregation of the chromosome that carries it such that in heterozygous +/t males, the t haplotype is transmitted to >95% of the offspring, a phenomenon known as transmission ratio distortion. The LC2 outer dynein arm light chain of Chlamydomonas reinhardtii is a homologue of the mouse protein Tctex2. We have identified Chlamydomonas insertional mutants with deletions in the gene encoding LC2 and demonstrate that the LC2 gene is the same as the ODA12 gene, the product of which had not been identified previously. Complete deletion of the LC2/ODA12 gene causes loss of all outer arms and a slow jerky swimming phenotype. Transformation of the deletion mutant with the cloned LC2/ODA12 gene restores the outer arms and rescues the motility phenotype. Therefore, LC2 is required for outer arm assembly. The fact that LC2 is an essential subunit of flagellar outer dynein arms allows us to propose a detailed mechanism whereby transmission ratio distortion is explained by the differential binding of mutant (t haplotype encoded) and wild-type dyneins to the axonemal microtubules of t-bearing or wild-type sperm, with resulting differences in their motility.     

Identification of the t Complex–encoded Cytoplasmic Dynein Light Chain Tctex1 in Inner Arm I1 Supports the Involvement of Flagellar Dyneins in Meiotic Drive

The cytoplasmic dynein light chain Tctex1 is a candidate for one of the distorter products involved in the non-Mendelian transmission of mouse t haplotypes. It has been unclear, however, how the t-specific mutations in this protein, which is found associated with cytoplasmic dynein in many tissues, could result in a male germ cell–specific phenotype. Here, we demonstrate that Tctex1 is not only a cytoplasmic dynein component, but is also present both in mouse sperm and Chlamydomonas flagella. Genetic and biochemical dissection of the Chlamydomonas flagellum reveal that Tctex1 is a previously undescribed component of inner dynein arm I1. Combined with the recent identification of another putative t complex distorter, Tctex2, within the outer dynein arm, these results support the hypothesis that transmission ratio distortion (meiotic drive) of mouse t haplotypes involves dysfunction of both flagellar inner and outer dynein arms but does not require the cytoplasmic isozyme.     

Solution structure of the Tctex1 dimer reveals a mechanism for dynein-cargo interactions

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.     

The Tctex1/Tctex2 class of dynein light chains. Dimerization, differential expression, and interaction with the LC8 protein family

The Tctex1/Tctex2 family of dynein light chains associates with the intermediate chains at the base of the soluble dynein particle. These components are essential for dynein assembly and participate in specific motor-cargo interactions. To further address the role of these light chains in dynein activity, the structural and biochemical properties of several members of this polypeptide class were examined. Gel filtration chromatography and native gel electrophoresis indicate that recombinant Chlamydomonas flagellar Tctex1 exists as a dimer in solution. Furthermore, yeast two-hybrid analysis suggests that this association also occurs in vivo. In contrast, both murine and Chlamydomonas Tctex2 are monomeric. To investigate protein-protein interactions involving these light chains, outer arm dynein from Chlamydomonas flagella was cross-linked using dimethylpimelimidate. Immunoblot analysis of the resulting products revealed the interaction of LC2 (Tctex2) with LC6, which is closely related to the highly conserved LC8 protein found in many enzyme systems, including dynein. Northern dot blot analysis demonstrated that Tctex1/Tctex2 family light chains are differentially expressed both in a tissue-specific and developmentally regulated manner in humans. These data provide further support for the existence of functionally distinct populations of cytoplasmic dynein with differing light chain content.     

Three members of the LC8/DYNLL family are required for outer arm dynein motor function

The highly conserved LC8/DYNLL family proteins were originally identified in axonemal dyneins and subsequently found to function in multiple enzyme systems. Genomic analysis uncovered a third member (LC10) of this protein class in Chlamydomonas. The LC10 protein is extracted from flagellar axonemes with 0.6 M NaCl and cofractionates with the outer dynein arm in sucrose density gradients. Furthermore, LC10 is specifically missing only from axonemes of those strains that fail to assemble outer dynein arms. Previously, the oda12-1 insertional allele was shown to lack the Tctex2-related dynein light chain LC2. The LC10 gene is located approximately 2 kb from that of LC2 and is also completely missing from this mutant but not from oda12-2, which lacks only the 3' end of the LC2 gene. Although oda12-1 cells assemble outer arms that lack only LC2 and LC10, this strain exhibits a flagellar beat frequency that is consistently less than that observed for strains that fail to assemble the entire outer arm and docking complex (e.g., oda1). These results support a key regulatory role for the intermediate chain/light chain complex that is an integral and highly conserved feature of all oligomeric dynein motors.     

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.     

A novel Tctex2-related light chain is required for stability of inner dynein arm I1 and motor function in the Chlamydomonas flagellum

Tctex1 and Tctex2 were originally described in mice as putative distorters/sterility factors involved in the non-Mendelian transmission of t haplotypes. Subsequently, these proteins were found to be light chains of both cytoplasmic and axonemal dyneins. We have now identified a novel Tctex2-related protein (Tctex2b) within the Chlamydomonas flagellum. Tctex2b copurifies with inner arm I1 after both sucrose gradient centrifugation and anion exchange chromatography. Unlike the Tctex2 homologue within the outer dynein arm, analysis of a Tctex2b-null strain indicates that this protein is not essential for assembly of inner arm I1. However, a lack of Tctex2b results in an unstable dynein particle that disassembles after high salt extraction from the axoneme. Cells lacking Tctex2b swim more slowly than wild type and exhibit a reduced flagellar beat frequency. Furthermore, using a microtubule sliding assay we observed that dynein motor function is reduced in vitro. These data indicate that Tctex2b is required for the stability of inner dynein arm I1 and wild-type axonemal dynein function.     

Partial purification and characterization of dynein adenosine triphosphatase from bovine sperm

Outer dynein arm polypeptides that possess Mg+2-adenosine triphosphatase (ATPase) activity have been extracted from the flagellar axonemes of demembranated bovine sperm. Electron microscopy of intact and salt-extracted sperm demonstrates a relatively selective removal of the outer dynein arms. The salt extract contains a specific ATPase activity of 55 nmoles inorganic phosphate (Pi)/min/mg protein. Sucrose density gradient centrifugation of this extract results in a 6-fold increase in specific activity of ATPase (333 nmole/Pi/min/mg protein), which sediments as a single 13S peak. Concomitant with the increase in specific activity, there is enrichment of three high molecular weight polypeptides (Mr greater than 300,000) characteristic of dynein heavy chains. ATPase activities in the initial extract and in the 13S peak are inhibited by concentrations of vanadate and erythro-9-[3-2-(hydroxynonyl)]adenine similar to those that inhibit ATPase activity in sea urchin sperm dynein. These findings indicate that outer arm dynein ATPase can be extracted and partially purified from bovine sperm.     

Is outer arm dynein intermediate chain 1 multifunctional?

The outer arm dynein of sea urchin sperm axoneme contains three intermediate chains (IC1, IC2, and IC3; M(r) 128,000, 98,000, and 74,000, respectively). IC2 and IC3 are members of the WD family; the WD motif is responsible for a protein-protein interaction. We describe here the molecular cloning of IC1. IC1 has a unique primary structure, the N-terminal part is homologous to the sequence of thioredoxin, the middle part consists of three repetitive sequences homologous to the sequence of nucleoside diphosphate kinase, and the C-terminal part contains a high proportion of negatively charged glutamic acid residues. Thus, IC1 is a novel dynein intermediate chain distinct from IC2 and IC3 and may be a multifunctional protein. The thioredoxin-related part of IC1 is more closely related to those of two redox-active Chlamydomonas light chains than thioredoxin. Antibodies were prepared against the N-terminal and middle domains of IC1 expressed as His-tagged proteins in bacteria. These antibodies cross-reacted with some dynein polypeptides (potential homologues of IC1) from distantly related species. We propose here that the three intermediate chains are the basic core units of sperm outer arm dynein because of their ubiquitous existence. The recombinant thioredoxin-related part of IC1 and outer arm dyneins from sea urchin and distantly related species were specifically bound to and eluted from a phenylarsine oxide affinity column with 2-mercaptoethanol, indicating that they contain vicinal dithiols competent to undergo reversible oxidation/reduction.     

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.     

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.     

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".     

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.     

The dynein electrophoretic bands in axonemes naturally lacking the inner or the outer arm

Two unconventional sperm models (all motile) have been studied. The first one has only the outer arm on the doublets (the gall midge, Diplolaboncus); the second one, has only a well-developed inner arm (the eel, Anguilla). Both are devoid of central tubules and radial spokes. The gall midge sperm yields a single electrophoretic band migrating similarly to the sea urchin dynein band A; a major high-molecular-weight band is obtained from eel sperm which co-migrates with the sea urchin dynein band B. The present picture is consistent with the localization of dynein in the axoneme--namely, of an A-like band in the outer arm, and of the B band in the inner arm. Moreover, the D band is present only in the eel, where gamma-links are present. ATPase activity was localized histochemically and found to be associated with both inner and outer arms, as well as with the gamma-links.     

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.     

Multivalency in the Assembly of Intrinsically Disordered Dynein Intermediate Chain

Dynein light chains are thought to increase binding efficiency of dynein intermediate chain to both dynein heavy chain and dynactin, but their exact role is not clear. Isothermal titration calorimetry and x-ray crystallography reported herein indicate that multivalency effects underlie efficient dynein assembly and regulation. For a ternary complex of a 60-amino acid segment of dynein intermediate chain (IC) bound to two homodimeric dynein light chains Tctex1 and LC8, there is a 50-fold affinity enhancement for the second light chain binding. For a designed IC construct containing two LC8 sites, observed the 1000-fold enhancement reflects a remarkably pure entropic chelate effect of a magnitude commensurate with theoretical predictions. The lower enhancement in wild-type IC is attributed to unfavorable free energy changes associated with incremental interactions of IC with Tctex1. Our results show assembled dynein IC as an elongated, flexible polybivalent duplex, and suggest that polybivalency is an important general mechanism for constructing stable yet reversible and functionally versatile complexes.     

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.