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.     

Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner- or outer-arm dynein: velocity measurements on new types of mutants by an improved method.

To help understand the functional properties of inner and outer dynein arms in axonemal motility, sliding velocities of outer doublets were measured in disintegrating axonemes of Chlamydomonas mutants lacking either of the arms. Measurements under improved solution conditions yielded significantly higher sliding velocities than those observed in a previous study [Okagaki and Kamiya, 1986, J. Cell Biol. 103:1895-1902]. As in the previous study, it was found that the velocities in axonemes of wild type (wt) and a mutant (oda1) missing the outer arm differ greatly: 18.5 +/- 4.1 microns/sec for wt and 4.4 +/- 2.3 microns/sec for oda1 at 0.5 mM Mg-ATP. In contrast, axonemes of two types of mutants (ida2 and ida4) that lacked different sets of two inner-arm heavy chains displayed velocities almost identical with the wild-type velocity. Moreover, axonemes of a non-motile double mutant ida2 X ida4 underwent sliding disintegration at a similar high velocity, although less frequently than in axonemes of single mutants. These observations support the hypothesis that the inner and outer dynein arms in disintegrating axonemes drive microtubules at different speeds and it is the faster outer arm that determines the overall speed when both arms are present. The inner arm may be important for the initiation of sliding. The axoneme thus appears to be equipped with two (or more) types of motors with different intrinsic speeds.     

The bop2-1 mutation reveals radial asymmetry in the inner dynein arm region of Chlamydomonas reinhardtii

Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Flagellar axonemes isolated from bop2-1 cells are missing a phosphorylated polypeptide of 152 kD. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes examined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region.     

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.     

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.     

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.     

Inner dynein arms but not outer dynein arms require the activity of kinesin homologue protein KHP1(FLA10) to reach the distal part of flagella in Chlamydomonas

Inner dynein arms, but not outer dynein arms, require the activity of KHP1(FLA10) to reach the distal part of axonemes before binding to outer doublet microtubules. We have analyzed the rescue of inner or outer dynein arms in quadriflagellate dikaryons by immunofluorescence microscopy of p28(IDA4), an inner dynein arm light chain, or IC69(ODA6), an outer dynein arm intermediate chain. In dikaryons two strains with different genetic backgrounds share the cytoplasm. As a consequence, wild-type axonemal precursors are transported to and assembled in mutant axonemes to complement the defects. The rescue of inner dynein arms containing p28 in ida4-wild-type dikaryons progressively occurred from the distal part of the axonemes and with time was extended towards the proximal part. In contrast, the rescue of outer dynein arms in oda2-wild-type dikaryons progressively occurred along the entire length of the axoneme. Rescue of inner dynein arms containing p28 in ida4fla10-fla10 dikaryons was similar to the rescue observed in ida4-wild-type dikaryons at 21 degrees C, whereas it was inhibited at 32 degrees C, a nonpermissive temperature for KHP1(FLA10). In contrast, rescue of outer dynein arms in oda2fla10-fla10 dikaryons was similar to the rescue observed in oda2-wild-type dikaryons at both 21 degrees and 32 degrees C and was not inhibited at 32 degrees C. Positioning of substructures in the internal part of the axonemal shaft requires the activity of kinesin homologue protein 1.     

Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.     

A tektin homologue is decreased in chlamydomonas mutants lacking an axonemal inner-arm dynein

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found approximately 80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a-g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment.     

A novel subunit of axonemal dynein conserved among lower and higher eukaryotes

To elucidate the subunit composition of axonemal inner-arm dynein, we examined a 38 kDa protein (p38) co-purified with a Chlamydomonas inner arm subspecies, dynein d. We found it is a novel protein conserved among a variety of organisms with motile cilia and flagella. Immunoprecipitation using specific antibody verified its association with a heavy chain, actin and a previously identified light chain (p28). Unexpectedly, mutant axonemes lacking dynein d and other dyneins retained reduced amounts of p38. This finding suggests that p38 is involved in the docking of dynein d to specific loci.     

Solubilization of dynein from Tetrahymena ssp. axonemes using phosphate analogues

One major protein was selectively solubilized when phosphate analogues, such as inorganic vanadate (Vi), beryllium fluoride (BeFx) or aluminum fluoride (AlFx), were added to ciliary axonemes of Tetrahymena ssp. (T. pyriformis or T. thermophila) in the presence of ATP. This protein contains three high molecular weight polypeptides, characteristic of an outer arm dynein. Electron microscopic observation of the axonemes after solubilization using ATP and Vi revealed axonemes partially lacking outer arm dyneins. These results suggest that the solubilized protein is an outer arm dynein and also that a dynein-ADP-phosphate complex decreases its affinity with the adjacent microtubules within axonemes. Limited digestion with chymotrypsin revealed that each solubilized dynein has a similar conformation, but it is markedly different from that of dynein in the absence of ATP or a phosphate analogue. The solubilized dynein obtained by the addition of Vi and ATP to axonemes was digested by UV irradiation to yield at least five new polypeptides (240, 230, 225, 180 and 160 kDa) but the dyneins solubilized by BeFx (or AlFx) in the presence of ATP did not produce any photocleavage products under the same conditions.     

Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms

To clarify the functional differentiation between the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were assessed by measuring the sliding velocities of outer-doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild-type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which is difficult to achieve by ordinary methods. The flagella were first fragmented by sonication, demembranated by Nonidet P-40, and then perfused under a microscope with Mg-ATP and nagarse, a bacterial protease with broad substrate specificity. The sliding velocity varied with the Mg-ATP concentration in a Michaelis-Menten manner in the axonemes from the wild type and a motile mutant lacking the outer dynein arm (oda38). The maximal sliding velocity and apparent Michaelis constant for Mg-ATP were measured to be 13.2 +/- 1.0 micron/s and 158 +/- 36 microM for the wild type and 2.0 +/- 0.1 micron/s and 64 +/- 18 microM for oda38. These maximal sliding velocities were significantly smaller than those estimated in beating axonemes; the reason is not clear. The velocities in the presence or absence of 10(-5) M Ca2+ did not differ noticeably. The axonemes of nonmotile mutants lacking either outer arms (pf13A, pf22) or inner arms (pf23) were examined for their ability to undergo sliding disintegration in the presence of 0.1 mM Mg-ATP. Whereas pf13A axonemes underwent normal sliding disintegration, the other two species displayed it only very poorly. The poor ability of pf23 axonemes to undergo sliding disintegration raises the possibility that the outer dynein arm cannot function well in the absence of the inner arm.     

High speed sliding of axonemal microtubules produced by outer arm dynein

To study dynein arm activity at high temporal resolution, axonemal sliding was measured field by field for wild type and dynein arm mutants of Tetrahymena thermophila. For wt SB255 cells, when the rate of data acquisition was 60 fps, about 5x greater than previously published observations, sliding was observed to be discontinuous with very high velocity sliding (average 196 microm/sec) for a few msec (1 or 2 fields) followed by a pause of several fields. The sliding velocities measured were an order of magnitude greater than rates previously measured by video analysis. However, when the data were analyzed at 12 fps for the same axonemes, consistent with previous observations, sliding was linear as the axonemes extended several times their original length with an average velocity of approximately 10 microm/sec. The pauses or stops occurred at approximately 200 and 300% of the initial length, suggesting that dynein arms on one axonemal doublet were initially active to the limit of extension, and then the arms on the next doublet became activated. In contrast, in a mutant where OADs are missing, sliding observed at 60 fps was continuous and slow (5 microm/sec), as opposed to the discontinuous high-velocity sliding of SB255 and of the mutant at the permissive temperature where OADs are present. High-velocity step-wise sliding was also present in axonemes from an inner arm dynein mutant (KO6). These results indicate that the high-speed discontinuous pattern of sliding is produced by the mechanochemical activity of outer arm dynein. The rate of sliding is consistent with a low duty ratio of the outer arm dynein and with the operation of each arm along a doublet once per beat.     

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.     

Strikingly low ATPase activities in flagellar axonemes of a Chlamydomonas mutant missing outer dynein arms

The ATPase activities in Chlamydomonas axonemes were compared between wild type and a mutant (oda) that lacks entire outer dynein arms, at various ionic strengths and pH values, and in the presence of different concentrations of high-molecular-mass dextran. Over a 0-0.2 M KCl concentration range, the ATPase activity of oda axonemes was found to be 5-12 times lower than that of the wild-type axonemes. The low activity in oda is surprising since outer arm-depleted axonemes of sea urchin sperm have been reported to retain about 50% of the normal activity. In both wild type and oda, the ATPase activity of dynein was higher when contained within the axoneme than when released from it with 0.6 M KCl. The ATPase activation within the wild-type axoneme was inhibited by high ionic strengths or by the presence of dextran. The activation in oda axonemes, on the other hand, was not inhibited by these factors. These significantly different ATPase properties suggest that the inner and outer dynein arms perform somewhat different functions in this organism.     

Mammalian cells express three distinct dynein heavy chains that are localized to different cytoplasmic organelles

We describe two dynein heavy chain (DHC)-like polypeptides (DHCs 2 and 3) that are distinct from the heavy chain of conventional cytoplasmic dynein (DHC1) but are expressed in a variety of mammalian cells that lack axonemes. DHC2 is a distant member of the "cytoplasmic" branch of the dynein phylogenetic tree, while DHC3 shares more sequence similarity with dynein-like polypeptides that have been thought to be axonemal. Each cytoplasmic dynein is associated with distinct cellular organelles. DHC2 is localized predominantly to the Golgi apparatus. Moreover, the Golgi disperses upon microinjection of antibodies to DHC2, suggesting that this motor is involved in establishing proper Golgi organization. DCH3 is associated with as yet unidentified structures that may represent transport intermediates between two or more cytoplasmic compartments. Apparently, specific cytoplasmic dyneins, like individual members of the kinesin superfamily, play unique roles in the traffic of cytomembranes.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila

Ciliary beat frequency is primarily regulated by outer arm dyneins (22 S dynein). Chilcote and Johnson (Chilcote, T. J., and Johnson, K. A. (1990) J. Biol. Chem. 256, 17257-17266) previously studied isolated Tetrahymena 22 S dynein, identifying a protein p34, which showed cAMP-dependent phosphorylation. Here, we characterize the molecular biochemistry of p34 further, demonstrating that it is the functional ortholog of the 22 S dynein regulatory light chain, p29, in Paramecium. p34, thiophosphorylated in isolated axonemes in the presence of cAMP, co-purified with 22 S dynein and not with inner arm dynein (14 S dynein). Isolated 22 S dynein containing phosphorylated p34 showed approximately 70% increase in in vitro microtubule translocation velocity compared with its unphosphorylated counterpart. Extracted p34 rebound to isolated 22 S dynein from either Tetrahymena or Paramecium but not to 14 S dynein from either ciliate. Binding of radiolabeled p34 to 22 S dynein was competitive with p29. Phosphorylated p34 was not present in axonemes isolated from a mutant lacking outer arms. Two-dimensional gel electrophoresis followed by phosphorimaging revealed at least five phosphorylated p34-related spots, consistent with multiple phosphorylation sites in p34 or perhaps multiple isoforms of p34. These new features suggest that a class of outer arm dynein light chains including p34 regulates microtubule sliding velocity and consequently ciliary beat frequency through phosphorylation.     

ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.