The 78,000 M(r) intermediate chain of Chlamydomonas outer arm dynein isa WD-repeat protein required for arm assembly

We have isolated and sequenced a full-length cDNA clone encoding the 78,000 Mr intermediate chain (IC78) of the Chlamydomonas outer arm dynein. This protein previously was shown to be located at the base of the solubilized dynein particle and to interact with alpha tubulin in situ, suggesting that it may be involved in binding the outer arm to the doublet microtubule. The sequence predicts a polypeptide of 683 amino acids having a mass of 76.5 kD. Sequence comparison indicates that IC78 is homologous to the 69,000 M(r) intermediate chain (IC69) of Chlamydomonas outer arm dynein and to the 74,000 M(r) intermediate chain (IC74) of cytoplasmic dynein. The similarity between the chains is greatest in their COOH-terminal halves; the NH(2)-terminal halves are highly divergent. The COOH-terminal half of IC78 contains six short imperfect repeats, termed WD repeats, that are thought to be involved in protein-protein interactions. Although not previously reported, these repeated elements also are present in IC69 and IC74. Using the IC78 cDNA as a probe, we screened a group of slow-swimming insertional mutants and identified one which has a large insertion in the IC78 gene and seven in which the IC78 gene is completely deleted. Electron microscopy of three of these IC78 mutants revealed that each is missing the outer arm, indicating that IC78 is essential for arm assembly or attachment to the outer doublet. Restriction fragment length polymorphism mapping places the IC78 gene on the left arm of chromosome XII/XIII, at or near the mutation oda9, which also causes loss of the outer arm. Mutants with defects in the IC78 gene do not complement the oda9 mutation in stable diploids, strongly suggesting that ODA9 is the structural gene for IC78.     

The Mr 78,000 intermediate chain of Chlamydomonas outer arm dynein interacts with alpha-tubulin in situ

We have used the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to examine protein-protein associations within purified outer arm dynein and axonemes from Chlamydomonas flagella. When axonemes were treated with 0.5-1 mM EDC in either the presence or absence of ATP/vanadate, a polypeptide band of Mr 127,000 recognized by monoclonal antibody 1878A (specific for the Mr 78,000 intermediate chain (IC78) of outer arm dynein) was generated. This conjugate was not obtained when purified dynein was treated with EDC. Further immunological analysis demonstrated that this complex also contained alpha- (but not beta-) tubulin. These results indicate that IC78 interacts with alpha-tubulin in situ in an ATP-insensitive manner. Identification of this interface between dynein and tubulin suggests that IC78, which probably is located at the base of the dynein particle (King, S. M., and Witman, G. B. (1990) J. Biol. Chem. 265, 19807-19811), contributes to the structural attachment of the dynein arms to the A-tubules of the outer doublet microtubules. Analysis of the cross-linked products from the purified dynein revealed several additional interactions involving the intermediate chains; these adducts provide further evidence for an intermediate chain/light chain complex within dynein and confirm that IC78 and IC69 associate directly.     

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.     

The Mr 140,000 Intermediate Chain of Chlamydomonas Flagellar Inner Arm Dynein Is a WD-Repeat Protein Implicated in Dynein Arm Anchoring

Previous structural and biochemical studies have revealed that the inner arm dynein I1 is targeted and anchored to a unique site located proximal to the first radial spoke in each 96-nm axoneme repeat on flagellar doublet microtubules. To determine whether intermediate chains mediate the positioning and docking of dynein complexes, we cloned and characterized the 140-kDa intermediate chain (IC140) of the I1 complex. Sequence and secondary structural analysis, with particular emphasis on β-sheet organization, predicted that IC140 contains seven WD repeats. Reexamination of other members of the dynein intermediate chain family of WD proteins indicated that these polypeptides also bear seven WD/β-sheet repeats arranged in the same pattern along each intermediate chain protein. A polyclonal antibody was raised against a 53-kDa fusion protein derived from the C-terminal third of IC140. The antibody is highly specific for IC140 and does not bind to other dynein intermediate chains or proteins in Chlamydomonas flagella. Immunofluorescent microscopy of Chlamydomonas cells confirmed that IC140 is distributed along the length of both flagellar axonemes. In vitro reconstitution experiments demonstrated that the 53-kDa C-terminal fusion protein binds specifically to axonemes lacking the I1 complex. Chemical cross-linking indicated that IC140 is closely associated with a second intermediate chain in the I1 complex. These data suggest that IC140 contains domains responsible for the assembly and docking of the I1 complex to the doublet microtubule cargo.     

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.     

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.     

DC3, the 21-kDa subunit of the outer dynein arm-docking complex (ODA-DC), is a novel EF-hand protein important for assembly of both the outer arm and the ODA-DC

The outer dynein arm-docking complex (ODA-DC) is a microtubule-associated structure that targets the outer dynein arm to its binding site on the flagellar axoneme (Takada et al. 2002. Mol. Biol. Cell 13, 1015-1029). The ODA-DC of Chlamydomonas contains three proteins, referred to as DC1, DC2, and DC3. We here report the isolation and sequencing of genomic and full-length cDNA clones encoding DC3. The sequence predicts a 21,341 Da protein with four EF-hands that is a member of the CTER (calmodulin, troponin C, essential and regulatory myosin light chains) group and is most closely related to a predicted protein from Plasmodium. The DC3 gene, termed ODA14, is intronless. Chlamydomonas mutants that lack DC3 exhibit slow, jerky swimming because of loss of some but not all outer dynein arms. Some outer doublet microtubules without arms had a "partial" docking complex, indicating that DC1 and DC2 can assemble in the absence of DC3. In contrast, DC3 cannot assemble in the absence of DC1 or DC2. Transformation of a DC3-deletion strain with the wild-type DC3 gene rescued both the motility phenotype and the structural defect, whereas a mutated DC3 gene was incompetent to rescue. The results indicate that DC3 is important for both outer arm and ODA-DC assembly.     

Localization of an intermediate chain of outer arm dynein by immunoelectron microscopy

We have used immunoelectron microscopy to determine the location of an intermediate chain in the isolated outer arm dynein from Chlamydomonas flagella. When the purified alpha beta dimer of the outer arm was incubated with antibodies recognizing two distinct epitopes on its 69-kDa intermediate chain and then negatively stained and examined by electron microscopy, both antibodies appeared to have bound to the base of the Y-shaped stem that connects the two heads of the particle. These results indicate that this intermediate chain is located at the base of the stem. Inasmuch as this polypeptide is tightly associated with the 78-kDa intermediate chain and several light chains in an intermediate chain-light chain complex, it is likely that this entire assemblage is located at the base of the particle. Thus, these polypeptides are in a potentially important position with regard to the ATP-insensitive (structural end) binding of dynein to microtubules and to dynein-dynein interactions within the axoneme.     

Cloning of dynein intermediate chain cDNA of the newt Cynops pyrrhogaster.

To isolate genes whose expression is up-regulated after initiation of meiosis, we employed an mRNA differential display method using RNA extracted from newt testis fragments in the spermatogonial and spermatocyte stages. We report here isolation of a spermatocyte stage-specific cDNA clone encoding a newt homologue of dynein intermediate chain (IC). The newt dynein IC cDNA was found to encode a polypeptide consisting of 694 amino acid residues with 66.8% and 45.8% amino acid sequence similarity to sea urchin dynein IC3 and Chlamydomonas IC69, respectively. The predicted protein contains five WD repeats and a novel repeated motif in the C-terminal region. Northern blot analysis revealed that newt dynein IC mRNA was expressed in the spermatocyte and round spermatid stages, suggesting that dynein IC plays a role in formation of flagella as well as in meiotic events.     

The alpha subunit of sea urchin sperm outer arm dynein mediates structural and rigor binding to microtubules

Glass-adsorbed intact sea urchin outer arm dynein and its beta/IC1 subunit supports movement of microtubules, yet does not form a rigor complex upon depletion of ATP (16). We show here that rigor is a feature of the isolated intact outer arm, and that this property subfractionates with its alpha heavy chain. Intact dynein mediates the formation of ATP-sensitive microtubule bundles, as does the purified alpha heavy chain, indicating that both particles are capable of binding to microtubules in an ATP-sensitive manner. In contrast, the beta/IC1 subunit does not bundle microtubules. Bundles formed with intact dynein are composed of ribbon-like sheets of parallel microtubules that are separated by 54 nm (center-to-center) and display the same longitudinal repeat (24 nm) and cross-sectional geometry of dynein arms as do outer doublets in situ. Bundles formed by the alpha heavy chain are composed of microtubules with a center-to-center spacing of 43 nm and display infrequent, fine crossbridges. In contrast to the bridges formed by the intact arm, the links formed by the alpha subunit are irregularly spaced, suggesting that binding of the alpha heavy chain to the microtubules is not cooperative. Cosedimentation studies showed that: (a) some of the intact dynein binds in an ATP-dependent manner and some binds in an ATP-independent manner; (b) the beta/IC1 subunit does not cosediment with microtubules under any conditions; and (c) the alpha heavy chain cosediments with microtubules in the absence or presence of MgATP2-. These results suggest that the structural binding observed in the intact arm also is a property of its alpha heavy chain. We conclude that whereas force-generation is a function of the beta/IC1 subunit, both structural and ATP-sensitive (rigor) binding of the arm to the microtubule are mediated by the alpha subunit.     

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.     

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.     

The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly

To learn more about how dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of M(r) approximately 105,000 and approximately 70,000 proteins plus a third protein of M(r) approximately 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the M(r) approximately 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length M(r) approximately 70,000 protein. These results demonstrate that the ODA1 gene encodes the M(r) approximately 70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule.     

The motile beta/IC1 subunit of sea urchin sperm outer arm dynein does not form a rigor bond

We used in vitro translocation and cosedimentation assays to study the microtubule binding properties of sea urchin sperm outer arm dynein and its beta/IC1 subunit. Microtubules glided on glass-absorbed sea urchin dynein for a period of time directly proportional to the initial MgATP2- concentration and then detached when 70-95% of the MgATP2- was hydrolyzed. Detachment resulted from MgATP2- depletion, because (a) perfusion with fresh buffer containing MgATP2- reconstituted binding and gliding, (b) microtubules glided many minutes with an ATP-regenerating system at ATP concentrations which alone supported gliding for only 1-2 min, and (c) microtubules detached upon total hydrolysis of ATP by an ATP-removal system. The products of ATP hydrolysis antagonized binding and gliding; as little as a threefold excess of ADP/Pi over ATP resulted in complete loss of microtubule binding and translocation by the beta/IC1 subunit. In contrast to the situation with sea urchin dynein, microtubules ceased gliding but remained bound to glass-absorbed Tetrahymena outer arm dynein when MgATP2- was exhausted. Cosedimentation assays showed that Tetrahymena outer arm dynein sedimented with microtubules in an ATP-sensitive manner, as previously reported (Porter, M.E., and K. A. Johnson. J. Biol. Chem. 258: 6575-6581). However, the beta/IC1 subunit of sea urchin dynein did not cosediment with microtubules in the absence of ATP. Thus, this subunit, while capable of generating motility, lacks both structural and rigor-type microtubule binding.     

Ap58: a novel in situ outer dynein arm-binding protein

Outer arm dynein is a molecular motor that is positioned at 24 nm intervals on outer doublet microtubules in cilia and flagella. In the present paper, we report identification of a 58 kDa novel protein with a tetratricopeptide repeat (TPR), referred to as ap58 (for 58 kDa axonemal protein) in sea urchin sperm axonemes. Ap58 is extracted along with the outer arm dynein by a high salt solution from axonemes. Sucrose density gradient centrifugation or gel filtration of the extract separates the outer arm dynein core from ap58. Most ap58 sediments to the lower density fraction or elutes in fractions of smaller molecules. However, immunogold localization reveals that ap58 is distributed at approximately 25 nm intervals on doublet microtubules, suggesting that in situ it is associated with the outer dynein arm. Thus, ap58 with the TPR motif is a new member of outer dynein arm-binding proteins distinct from the outer dynein arm-docking complex.     

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.     

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.     

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.     

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.