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.     

An outer arm dynein light chain acts in a conformational switch for flagellar motility

A system distinct from the central pair–radial spoke complex was proposed to control outer arm dynein function in response to alterations in the mechanical state of the flagellum. In this study, we examine the role of a Chlamydomonas reinhardtii outer arm dynein light chain that associates with the motor domain of the γ heavy chain (HC). We demonstrate that expression of mutant forms of LC1 yield dominant-negative effects on swimming velocity, as the flagella continually beat out of phase and stall near or at the power/recovery stroke switchpoint. Furthermore, we observed that LC1 interacts directly with tubulin in a nucleotide-independent manner and tethers this motor unit to the A-tubule of the outer doublet microtubules within the axoneme. Therefore, this dynein HC is attached to the same microtubule by two sites: via both the N-terminal region and the motor domain. We propose that this γ HC–LC1–microtubule ternary complex functions as a conformational switch to control outer arm activity.     

Solution structure of a dynein motor domain associated light chain

Dyneins are molecular motors that translocate towards the minus ends of microtubules. In Chlamydomonas flagellar outer arm dynein, light chain 1 (LC1) associates with the nucleotide binding region within the gamma heavy chain motor domain and consists of a central leucine-rich repeat section that folds as a cylindrical right handed spiral formed from six beta-beta-alpha motifs. This central cylinder is flanked by terminal helical subdomains. The C-terminal helical domain juts out from the cylinder and is adjacent to a hydrophobic surface within the repeat region that is proposed to interact with the dynein heavy chain. The position of the C-terminal domain on LC1 and the unexpected structural similarity between LC1 and U2A' from the human spliceosome suggest that this domain interacts with the dynein motor domain.     

Partially functional outer-arm dynein in a novel Chlamydomonas mutant expressing a truncated gamma heavy chain

The outer dynein arm of Chlamydomonas flagella contains three heavy chains (alpha, beta, and gamma), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose gamma heavy chain is truncated at about 30% of the sequence. While the previously isolated gamma chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain alpha and beta heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the gamma heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the alpha heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the beta heavy chain). Thus, the outer-arm dynein lacking the gamma heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.     

The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation

Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.     

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.     

Relaxation-based structure refinement and backbone molecular dynamics of the dynein motor domain-associated light chain

The light chain 1 (LC1) polypeptide is a member of the leucine-rich repeat protein family and binds at or near the ATP hydrolytic site within the motor domain of the gamma heavy chain from Chlamydomonas outer arm dynein. It consists of an N-terminal helix, a central barrel formed from six leucine-rich repeats that fold as beta beta alpha units, and a C-terminal helical domain that protrudes from the main axis defined by the leucine-rich repeats. Interaction with the gamma heavy chain is likely mediated through a hydrophobic patch on the larger beta sheet face, and the C-terminal region is predicted to insert into the dynein ATP hydrolytic site. Here we have used 1H-15N heteronuclear relaxation measurements obtained at 500 and 600 MHz to refine and validate the LC1 solution structure. In this refined structure, the C-terminal helix is significantly reoriented by more than 20 degrees as compared to the control and provides a more precise understanding of the potential regulatory role of this domain. We also employed the refined structure to perform a dynamic analysis of LC1 using the 600 MHz data set. These results, which were cross validated using the 500 MHz data set, strongly support identification of the predicted LC1 binding surfaces and provide additional insight into the interaction mechanisms of leucine-rich repeat proteins.     

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.     

Functional architecture of the outer arm dynein conformational switch

Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia.     

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.     

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.     

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.     

Interaction mapping of a dynein heavy chain. Identification of dimerization and intermediate-chain binding domains.

Cytoplasmic dynein is a multisubunit microtubule-based motor protein that is involved in several eukaryotic cell motilities. Two dynein heavy chains each form a motor domain that connects to a common cargo-binding tail. Although this tail domain is composed of multiple polypeptides, subunit organization within this region is poorly understood. Here we present an in vitro dissection of the tail-forming region of the dynein heavy chain from Dictyostelium. Our work identifies a sequence important for dimerization and for binding the dynein intermediate chain. The core of this motif localizes within an approximately 150-amino acid region that is strongly conserved among other cytoplasmic dyneins. This level of conservation does not extend to the axonemal dynein heavy chains, suggesting functional differences between the two. Dimerization appears to occur through a different mechanism than the heavy chain-intermediate chain interaction. We corroborate the in vitro interactions with in vivo expression of heavy chain fragments in Dictyostelium. Fragments lacking the interaction domain express well, without an obvious phenotype. On the other hand, the region crucial for both interactions appears to be lethal when overexpressed.     

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.     

Mutations in the SUP-PF-1 locus of Chlamydomonas reinhardtii identify a regulatory domain in the beta-dynein heavy chain

We have characterized a group of regulatory mutations that alter the activity of the outer dynein arms. Three mutations were obtained as suppressors of the paralyzed central pair mutant pf6 (Luck, D.J.L., and G. Piperno. 1989. Cell Movement. pp. 49-60), whereas two others were obtained as suppressors of the central pair mutant pfl6. Recombination analysis and complementation tests indicate that all five mutations are alleles at the SUP-PF-1/ODA4 locus and that each allele can restore motility to radial spoke and central pair defective strains. Restriction fragment length polymorphism analysis with a genomic probe for the beta-dynein heavy chain (DHC) gene confirms that this locus is tightly linked to the beta-DHC gene. Although all five mutant sup-pf-1 alleles alter the activity of the outer dynein arm as assayed by measurements of flagellar motility, only two alleles have a discernable polypeptide defect by SDS-PAGE. We have used photolytic and proteolytic cleavage procedures to localize the polypeptide defect to an approximately 100-kD domain downstream from the last putative nucleotide binding site. This region is encoded by approximately 5 kb of genomic DNA (Mitchell, D.R., and K. Brown. 1994. J. Cell Sci. 107:653-644). PCR amplification of wild-type and mutant DNA across this region identified one PCR product that was consistently smaller in the sup-pf-1 DNA. Direct DNA sequencing of the PCR products revealed that two of the sup-pf-1 mutations are distinct, in-frame deletions. These deletions occur within a region that is predicted to encode a small alpha-helical coiled-coil domain of the beta-DHC. This domain may play a role in protein-protein interactions within the outer dynein arm. Since both the size and location of this domain have been conserved in all axonemal and cytoplasmic DHCs sequenced to date, it presumably performs a common function in all dynein isoforms.     

A Chlamydomonas outer arm dynein mutant missing the alpha heavy chain

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

Chromosomal Mapping of Two Members of the Human Dynein Gene Family to Chromosome Regions 7p15 and 11q13 near the Deafness Loci DFNA 5 and DFNA 11

We mapped expressed tagged sequences (ESTs) corresponding to two human dynein heavy chain genes: β heavy chain of the outer dynein arm and heavy chain isotype 1B (DYH1B), by using somatic cell hybrids and radiation hybrid panels. The EST for the β heavy chain of the outer dynein arm mapped to chromosome region 7p15, and the EST for DYH1B mapped to 11q13.5. Two loci for nonsyndromic forms of deafness, DFNA5 and DFNA11, have previously been mapped to these two chromosomal regions. Including the gene for the axonemal light chain, hp28, we have mapped three different dynein genes near loci for different forms of nonsyndromic deafness. The hypothesis that mutations in some dynein genes are associated with nonsyndromic deafness should now be tested.     

A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.     

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.     

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.