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HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella. Disruption of the gene by the MRC1 transposon in Chlamydomonas resulted in jerky flagella. Traditional electron microscopy, cryo-electron tomography, and sub-tomogram analysis revealed RSs of various altered morphologies that, unexpectedly, differed between the two RS species. This indicates that HSP40 locks the RS into a functionally rigid conformation, facilitating its interactions with the adjacent central pair apparatus for transducing locally varied mechanical feedback, which permits rhythmic beating. Missing HSP40, like missing RSs, could be restored in a tip-to-base direction when HSP40 mutants fused with a HSP40 donor cell. However, without concomitant de novo RS assembly, the repair was exceedingly slow, suggesting HSP40/RS-coupled intraflagellar trafficking and assembly. Biochemical analysis and modeling uncovered spoke HSP40’s cochaperone traits. On the basis of our data, we propose that HSP40 accompanies its client RS precursor when traveling to the flagellar tip. Upon arrival, both refold in concert to assemble into the mature configuration. HSP40’s roles in chaperoning and structural maintenance shed new light on its versatility and flagellar biology.

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The single-cell green algaChlamydomonas reinhardtiipossesses two α-tubulin genes (tua1andtua2) and two β-tubulin genes (tub1andtub2), with the two genes in each pair encoding identical amino acid sequences. Here, we used anaphVIIIgene cassette insertional library to establish eight disruptants with defectivetua2, tub1, ortub2expression. None of the disruptants exhibited apparent defects in cell growth, flagellar length, or flagellar regeneration after amputation. Because few tubulin mutants ofC. reinhardtiihave been reported to date, we then used our disruptants, together with atua1disruptant obtained from theChlamydomonasLibrary Project (CLiP), to isolate novel tubulin-mutants resistant to the anti-tubulin agents propyzamide and oryzalin. As a result of several trials, we obtained 8 strains bearing 7 different α-tubulin mutations and 24 strains bearing 12 different β-tubulin mutations. Some of these mutations are known to confer drug resistance in human cancer cells. Thus, single-tubulin-gene disruptants are an efficient means of isolating novelC. reinhardtiitubulin mutants.IMPORTANCEChlamydomonas reinhardtiiis a useful organism for the study of tubulin function; however, only five kinds of tubulin mutations have been reported to date. This scarcity is partly due toC. reinhardtiipossessing two tubulin genes each for α- and β-tubulin. Here, we obtained several strains in which one of the α- or β-tubulin genes was disrupted, and then used those disruptants to isolate 32 strains bearing 19 mostly novel tubulin mutations that conferred differing degrees of resistance to two anti-tubulin compounds. The majority of the tubulin mutations were located outside of the drug-binding sites in the three-dimensional tubulin structure, suggesting that structural changes underlie the drug resistance conferred by these mutations. Thus, single-tubulin-gene disruptants are an efficient means of generating tubulin mutants for the study of the structure–function relationship of tubulin and for the development of novel therapies based on anti-tubulin agents.

We determined how the ciliary motor I1 dynein is transported. A specialized adapter, IDA3, facilitates I1 dynein attachment to the ciliary transporter called intraflagellar transport (IFT). Loading of IDA3 and I1 dynein on IFT is regulated by ciliary length., Axonemal dyneins, including inner dynein arm I1, assemble in the cytoplasm prior to transport into cilia by intraflagellar transport (IFT). How I1 dynein interacts with IFT is not understood. We take advantage of the Chlamydomonas reinhardtii ida3 mutant, which assembles the inner arm I1 dynein complex in the cytoplasm but fails to transport I1 into the cilium, resulting in I1 dynein-deficient axonemes with abnormal motility. The IDA3 gene encodes an ∼115-kDa coiled-coil protein that primarily enters the cilium during ciliary growth but is not an axonemal protein. During growth, IDA3, along with I1 dynein, is transported by anterograde IFT to the tip of the cilium. At the tip, IDA3 uncouples from IFT and diffuses within the cilium. IFT transport of IDA3 decreases as cilia lengthen and subsides once full length is achieved. IDA3 is the first example of an essential and selective IFT adapter that is regulated by ciliary length.

Publisher Summary This chapter elucidates the role of genetic studies in understanding the axonemal dynein function in various organisms. Forward and reverse genetic studies of axonemal dynein indicate that the functional characteristics of various types of dyneins are well conserved among organisms. Genetic studies of axonemal dynein have been most extensively carried out using Chlamydomonas, a unicellular organism that yields a variety of flagella-deficient mutants. Most dynein components identified in Chlamydomonas have homologs in vertebrates and other ciliated organisms. Therefore, the forward genetic approach using Chlamydomonas has greatly contributed to the studies of axonemal dyneins in general. The study begins with discussing the genetic research carried out on Chlamydomonas axonemal dyneins. Chlamydomonas dynein-deficient mutants are isolated from cells displaying slow swimming or non-motile phenotypes, and the isolation mechanism is explained. Chlamydomonas axonemal dyneins are comprised of three distinct types of dyneins: an outer-arm dynein containing three HCs, a two-headed inner-arm dynein (containing two HCs), and multiple species of single-headed dyneins. Other than Chlamydomonas, mutants deficient in axonemal dyneins have been obtained in several unicellular and multicellular organisms, such as, tetrahymena, trypanosoma, zebrafish, medaka, frog, mouse, and, finally, human. In most cases, mutants were generated by reverse genetics techniques, using information from genome databases. The summaries of each of the research studies carried out on these organisms are presented.

Dynein light chain 1 (LC1) of the outer arm dynein (OAD) complex associates with the microtubule-binding domain (MTBD) of γ heavy chain inside the complex. LC1 is considered to regulate the OAD activity and ciliary/flagellar motion by modulating γ MTBD's affinity to the B-tubule of the doublet microtubule in the axoneme., The outer arm dynein (OAD) complex is the main propulsive force generator for ciliary/flagellar beating. In Chlamydomonas and Tetrahymena, the OAD complex comprises three heavy chains (α, β, and γ HCs) and >10 smaller subunits. Dynein light chain-1 (LC1) is an essential component of OAD. It is known to associate with the Chlamydomonas γ head domain, but its precise localization within the γ head and regulatory mechanism of the OAD complex remain unclear. Here Ni-NTA-nanogold labeling electron microscopy localized LC1 to the stalk tip of the γ head. Single-particle analysis detected an additional structure, most likely corresponding to LC1, near the microtubule-binding domain (MTBD), located at the stalk tip. Pull-down assays confirmed that LC1 bound specifically to the γ MTBD region. Together with observations that LC1 decreased the affinity of the γ MTBD for microtubules, we present a new model in which LC1 regulates OAD activity by modulating γ MTBD's affinity for the doublet microtubule.

Existence of cellular structures with specific size raises a fundamental question in biology: How do cells measure length? One conceptual answer to this question is by a molecular ruler, but examples of such rulers in eukaryotes are lacking. In this work, we identified a molecular ruler in eukaryotic cilia and flagella. Using cryo-electron tomography, we found that FAP59 and FAP172 form a 96–nanometer (nm)–long complex in Chlamydomonas flagella and that the absence of the complex disrupted 96-nm repeats of axonemes. Furthermore, lengthening of the FAP59/172 complex by domain duplication resulted in extension of the repeats up to 128 nm, as well as duplication of specific axonemal components. Thus, the FAP59/172 complex is the molecular ruler that determines the 96-nm repeat length and arrangements of components in cilia and flagella.

This review outlines the current knowledge of the functional diversity of axonemal dyneins, as revealed by studies with the model organism Chlamydomonas. Axonemal dyneins, which comprise outer and inner dynein arms, power cilia and flagella beating by producing sliding movements between adjacent outer-doublet microtubules. Outer- and inner-arm dyneins have traditionally been considered similar in structure and function. However, recent evidence suggests that they differ rather strikingly in subunit composition, axonemal arrangement, and molecular motor properties. We posit that these arms make up two largely independent motile systems; whereas outer-arm dynein can generate axonemal beating by itself under certain conditions, inner-arm dynein can generate beating only in cooperation with the central pair/radial spokes. This conclusion is supported by genome analyses of various organisms. Outer-arm dynein appears to be particularly important for nodal cilia of mammalian embryos that function for determination of left-right body asymmetry.

Outer arm dynein (OAD) is bound to specific loci on outer-doublet-microtubules by interactions at two sites: via intermediate chain 1 (IC1) and the outer dynein arm docking complex (ODA-DC). Studies using Chlamydomonas mutants have suggested that the individual sites have rather weak affinities for microtubules, and therefore strong OAD attachment to microtubules is achieved by their cooperation. To test this idea, we examined interactions between IC1, IC2 (another intermediate chain) and ODA-DC using recombinant proteins. Recombinant IC1 and IC2 were found to form a 1:1 complex, and this complex associated with ODA-DC in vitro. Binding of IC1 to mutant axonemes revealed that there are specific binding sites for IC1. From these data, we propose a novel model of OAD-outer doublet association.

The MIA complex, composed of FAP100 and FAP73, interacts with I1 dynein components and is required for normal ciliary beat frequency., Axonemal dyneins must be precisely regulated and coordinated to produce ordered ciliary/flagellar motility, but how this is achieved is not understood. We analyzed two Chlamydomonas reinhardtii mutants, mia1 and mia2, which display slow swimming and low flagellar beat frequency. We found that the MIA1 and MIA2 genes encode conserved coiled-coil proteins, FAP100 and FAP73, respectively, which form the modifier of inner arms (MIA) complex in flagella. Cryo–electron tomography of mia mutant axonemes revealed that the MIA complex was located immediately distal to the intermediate/light chain complex of I1 dynein and structurally appeared to connect with the nexin–dynein regulatory complex. In axonemes from mutants that lack both the outer dynein arms and the MIA complex, I1 dynein failed to assemble, suggesting physical interactions between these three axonemal complexes and a role for the MIA complex in the stable assembly of I1 dynein. The MIA complex appears to regulate I1 dynein and possibly outer arm dyneins, which are both essential for normal motility.

The tpg1 mutant of Chlamydomonas lacks the tubulin polyglutamylase TTLL9 and is deficient in flagellar tubulin polyglutamylation. It exhibits slow swimming, whereas the double mutant with oda2 (a slow-swimming mutant that lacks outer-arm dynein) is completely nonmotile. Thus, tubulin polyglutamylation must be important for the functioning of inner-arm dynein(s). In this study, we show that the tpg1 mutation only slightly affects the motility of mutants that lack dynein "e," one of the seven species of major inner-arm dyneins, whereas it greatly reduces the motility of mutants lacking other inner-arm dynein species. This suggests that dynein e is the main target of motility regulation by tubulin polyglutamylation. Furthermore, the motility of various mutants in the background of the tpg1 mutation raises the possibility that tubulin polyglutamylation also affects the dynein regulatory complex, a dynein e-associated key regulator of flagellar motility, which possibly constitutes the interdoublet (nexin) link. Tubulin polyglutamylation thus may play a central role in the regulation of ciliary and flagellar motility. © 2012 Wiley Periodicals, Inc.

Cryoelectron tomography and subtomogram averaging reveal a high degree of structural asymmetry and polarization in dynein localization in the Chlamydomonas flagella., Understanding the molecular architecture of the flagellum is crucial to elucidate the bending mechanism produced by this complex organelle. The current known structure of the flagellum has not yet been fully correlated with the complex composition and localization of flagellar components. Using cryoelectron tomography and subtomogram averaging while distinguishing each one of the nine outer doublet microtubules, we systematically collected and reconstructed the three-dimensional structures in different regions of the Chlamydomonas flagellum. We visualized the radial and longitudinal differences in the flagellum. One doublet showed a distinct structure, whereas the other eight were similar but not identical to each other. In the proximal region, some dyneins were missing or replaced by minor dyneins, and outer–inner arm dynein links were variable among different microtubule doublets. These findings shed light on the intricate organization of Chlamydomonas flagella, provide clues to the mechanism that produces asymmetric flagellar beating, and pose a new challenge for the functional study of the flagella.

An inducible dynein heavy chain 1b mutant reveals that robust retrograde intraflagellar transport is required for flagellar assembly and function but not the maintenance of flagellar length., The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.

The formation and function of eukaryotic cilia/flagella require the action of a large array of dynein microtubule motor complexes. Due to genetic, biochemical, and microscopic tractability, Chlamydomonas reinhardtii has become the premier model system in which to dissect the role of dyneins in flagellar assembly, motility, and signaling. Currently, fifty-four proteins have been described as components of various Chlamydomonas flagellar dyneins or as factors required for their assembly in the cytoplasm and/or transport into the flagellum; orthologues of nearly all these components are present in other ciliated organisms including humans. For historical reasons, the nomenclature of these diverse dynein components and their corresponding genes, mutant alleles and orthologues has become extraordinarily confusing. Here, we unify Chlamydomonas dynein gene nomenclature and establish a systematic classification scheme based on structural properties of the encoded proteins. Furthermore, we provide detailed tabulations of the various mutant alleles and protein aliases that have been used and explicitly define the correspondence with orthologous components in other model organisms and humans.

Mutations in the IC138 regulatory subunit of I1 dynein alter dynein motor activity and the flagellar waveform but do not affect phototaxis., I1 dynein, or dynein f, is a highly conserved inner arm isoform that plays a key role in the regulation of flagellar motility. To understand how the IC138 IC/LC subcomplex modulates I1 activity, we characterized the molecular lesions and motility phenotypes of several bop5 alleles. bop5-3, bop5-4, and bop5-5 are null alleles, whereas bop5-6 is an intron mutation that reduces IC138 expression. I1 dynein assembles into the axoneme, but the IC138 IC/LC subcomplex is missing. bop5 strains, like other I1 mutants, swim forward with reduced swimming velocities and display an impaired reversal response during photoshock. Unlike mutants lacking the entire I1 dynein, however, bop5 strains exhibit normal phototaxis. bop5 defects are rescued by transformation with the wild-type IC138 gene. Analysis of flagellar waveforms reveals that loss of the IC138 subcomplex reduces shear amplitude, sliding velocities, and the speed of bend propagation in vivo, consistent with the reduction in microtubule sliding velocities observed in vitro. The results indicate that the IC138 IC/LC subcomplex is necessary to generate an efficient waveform for optimal motility, but it is not essential for phototaxis. These findings have significant implications for the mechanisms by which IC/LC complexes regulate dynein motor activity independent of effects on cargo binding or complex stability.

Chlamydomonas flagella display surface motility such that small polystyrene beads (microspheres) attached to the flagellar membrane move bidirectionally along the flagellum. This surface motility enables cells to glide on a solid substrate to which they are attached by the flagellar surface. Previous studies suggested that microsphere movement and gliding motility result from the movement of transmembrane glycoprotein(s) within the plane of the plasma membrane, driven by intraflagellar transport (IFT), which utilizes cytoplasmic dynein and kinesin-2. However, it is not well understood how a cell can continuously glide in one direction further than a single flagellar length. Here we show that, during microsphere translocation on the flagella of a non-motile mutant, pf18, some flagellar glycoproteins, including FMG-1B and FAP113, detach from the membrane and attach to the microspheres. We propose that such relocation of surface glycoproteins underlies the ability to glide over a long distance. Surface motility is likely common to cilia/flagella of various organisms, as a similar microsphere movement is observed in the apical ciliary tuft in sea urchin embryos.

Outer arm dynein (OAD) in cilia and flagella contains two to three nonidentical heavy chains (HCs) that possess motor activity. In Chlamydomonas, flagellar OAD contains three HCs, alpha-, beta-, and gamma-HCs, each appearing to have a distinct role. To determine the precise molecular mechanism of their function, cross-sectional electron micrographs of wild-type and single HC-disruption mutants were compared and statistically analyzed. While the alpha-HC mutant displayed an OAD of lower density, which was attributed to a lack of alpha-HC, the OAD of beta- and gamma-HC mutants not only lacked the corresponding HC, but was also significantly affected in its structure, particularly with respect to the localization of alpha-HC. The lack of beta-HC induced mislocalization of alpha-HC, while a disruption of the gamma-HC gene resulted in the synchronized movement of alpha-HC and beta-HC in the manners for stacking. Interestingly, using cryo-electron microscopy, purified OADs were typically observed consisting of two stacked heads and an independent single head, which presumably corresponded to gamma-HC. This conformation is different from previous reports in which the three HCs displayed a stacked form in flagella observed by cryo-electron tomography and a bouquet structure on mica in deep-etch replica images. These results suggest that gamma-HC supports the tight stacking arrangement of inter or intra alpha-/beta-HC to facilitate the proper functioning of OAD.

How ciliary and flagellar motility is regulated is a challenging problem. The flagellar movement in Chlamydomonas reinhardtii is in part regulated by phosphorylation of a 138 kD intermediate chain (IC138) of inner arm dynein f (also called I1). In the present study, we found that the axoneme of mutants lacking dynein f lacks a novel protein having ankyrin repeat motifs, registered as FAP120 in the flagellar proteome database. FAP120 is also missing or decreased in the axonemes of bop5, a mutant that has a mutation in the structural gene of IC138 but assembles the dynein f complex. Intriguingly, the amounts of FAP120 in the axonemes of different alleles of bop5 and several dynein f-lacking mutants roughly parallel their contents of IC138. These results suggest a weak but stoichiometric interaction between FAP120 and IC138. We propose that FAP120 functions in the regulatoryprocess as part of a protein complex involving IC138. Cell Motil. Cytoskeleton 2008. (c) 2008 Wiley-Liss, Inc.

The movements of cilia and flagella are driven by multiple species of dynein heavy chains (DHCs), which constitute inner- and outer-dynein arms. In Chlamydomonas, 11 DHC proteins have been identified in the axoneme, but 14 genes encoding axonemal DHCs are present in the genome. Here, we assigned each previously unassigned DHC gene to a particular DHC protein and found that DHC3, DHC4 and DHC11 encode novel, relatively low abundance DHCs. Immunofluorescence microcopy revealed that DHC11 is localized exclusively to the proximal approximately 2 microm region of the approximately 12 microm long flagellum. Analyses of growing flagella suggested that DHC3 and DHC4 are also localized to the proximal region. By contrast, the DHC of a previously identified inner-arm dynein, dynein b, displayed an inverse distribution pattern. Thus, the proximal portion of the flagellar axoneme apparently differs in dynein composition from the remaining portion; this difference might be relevant to the special function performed by the flagellar base.

Outer arm dynein (OAD) of cilia and flagella contains two or three distinct heavy chains, each having a motor function. To elucidate their functional difference, we compared the in vitro motile properties of Chlamydomonas wild-type OAD containing the alpha, beta, and gamma heavy chains and three kinds of mutant OADs, each lacking one of the three heavy chains. For systematic comparison, a method was developed to introduce a biotin tag into a subunit, LC2, which served as the specific anchoring site on an avidin-coated glass surface. Wild-type OAD displayed microtubule gliding in the presence of ATP and ADP, with a maximal velocity of 5.0 mum/s, which is approximately 1/4 of the microtubule sliding velocity in the axoneme. The duty ratio was estimated to be as low as 0.08. The absence of the beta heavy chain lowered both the gliding velocity and ATPase activity, whereas the absence of the gamma heavy chain increased both activities. Strikingly, the absence of the alpha heavy chain lowered the gliding velocity but increased the ATPase activity. Thus, the three heavy chains are likely to play distinct roles and regulate each other to achieve coordinated force production.

Flagellar beating in Chlamydomonas was found to be activated by mechanical stimulation. Immediately after a wild-type cell suspension was vortexed, the average swimming velocity of cells increased from 130 mum/second to 150 mum/second, due to an elevation of flagellar beat frequency from approximately 60 Hz to approximately 70 Hz without detectable change in the flagellar waveforms. This response required outer arm dynein. Treatment with EGTA, Ca(2+)-channel blockers, or mechanosensitive-channel blockers inhibited it. In demembranated and reactivated cell models, a modest increase in Ca(2+) concentration elevated the axonemal beat frequency. These data indicate that the mechanical agitation increases beat frequency because it causes Ca(2+) influx into flagella, which then activates outer arm dynein.

Cilia and flagella are highly conserved organelles that have diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia and flagella often result in primary ciliary dyskinesia. However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a new gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in primary ciliary dyskinesia patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.

The outer dynein arm of Chlamydomonas flagella contains three heavy chains (α, β, and γ), 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 γ heavy chain is truncated at about 30% of the sequence. While the previously isolated γ chain mutant oda2 lacks the entire outer arm, oda2 - t retains outer arms that contain α and β 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 γ 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 α heavy chain) but higher than that of oda2 and oda4 - s7 (lacking the motor domain of the β heavy chain). Thus, the outer-arm dynein lacking the γ 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.

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580 :6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved.

Centrioles/basal bodies have a characteristic cylindrical structure consisting of nine triplet microtubules arranged in a rotational symmetry. How this elaborate structure is formed is a major unanswered question in cell biology [1, 2]. We previously identified a 170 kDa coiled-coil protein essential for the centriole formation in Chlamydomonas. This protein, Bld10p, is the first protein shown to localize to the cartwheel, a 9-fold symmetrical structure possibly functioning as the scaffold for the centriole-microtubule assembly [3]. Here, we report results by using a series of truncated Bld10p constructs introduced into a bld10 null mutant. Remarkably, a transformant (DeltaC2) in which 35% of Bld10p at the C terminus was deleted assembled centrioles with eight symmetrically arranged triplets, in addition to others with the normal nine triplets. The cartwheels in these eight-membered centrioles had spokes approximately 24% shorter than those in the wild-type, suggesting that the eight-triplet centrioles were formed because the cartwheel's smaller diameter. From the morphology of the cartwheel spoke in the DeltaC2 centriole and immunoelectron-microscope localization, we conclude that Bld10p is a major spoke-tip component that extends the cartwheel diameter and attaches triplet microtubules. These results provide the first experimental evidence for the crucial function of the cartwheel in centriolar assembly.

Motile cilia and flagella rapidly propagate bending waves and produce water flow over the cell surface. Their function is important for the physiology and development of various organisms including humans. The movement is based on the sliding between outer doublet microtubules driven by axonemal dyneins, and is regulated by various axonemal components and environmental factors. For studies aiming to elucidate the mechanism of cilia/flagella movement and regulation, Chlamydomonas is an invaluable model organism that offers a variety of mutants. This chapter introduces standard methods for studying Chlamydomonas flagellar motility including analysis of swimming paths, measurements of swimming speed and beat frequency, motility reactivation in demembranated cells (cell models), and observation of microtubule sliding in disintegrating axonemes. Most methods may be easily applied to other organisms with slight modifications of the medium conditions.

Tubulin polyglutamylation is a posttranslational modification known to affect ciliary/flagellar motility and assembly. Investigation of Chlamydomonas mutants deficient in axonemal polyglutamylation shows that polyglutamylation functions by increasing tubulin turnover at the flagellar tip and reducing axonemal stability., Ciliary length control is an incompletely understood process essential for normal ciliary function. The flagella of Chlamydomonas mutants lacking multiple axonemal dyneins are shorter than normal; previously it was shown that this shortness can be suppressed by the mutation suppressor of shortness 1 (ssh1) via an unknown mechanism. To elucidate this mechanism, we carried out genetic analysis of ssh1 and found that it is a new allele of TPG2 (hereafter tpg2-3), which encodes FAP234 functioning in tubulin polyglutamylation in the axoneme. Similar to the polyglutamylation-deficient mutants tpg1 and tpg2-1, tpg2-3 axonemal tubulin has a greatly reduced level of long polyglutamate side chains. We found that tpg1 and tpg2-1 mutations also promote flagellar elongation in short-flagella mutants, consistent with a polyglutamylation-dependent mechanism of suppression. Double mutants of tpg1 or tpg2-1 and fla10-1, a temperature-sensitive mutant of intraflagellar transport, underwent slower flagellar shortening than fla10-1 at restrictive temperatures, indicating that the rate of tubulin disassembly is decreased in the polyglutamylation-deficient flagella. Moreover, α-tubulin incorporation into the flagellar tips in temporary dikaryons was retarded in polyglutamylation-deficient flagella. These results show that polyglutamylation deficiency stabilizes axonemal microtubules, decelerating axonemal disassembly at the flagellar tip and shifting the axonemal assembly/disassembly balance toward assembly.

Motile cilia and flagella rapidly propagate bending waves and produce water flow over the cell surface. Their function is important for the physiology and development of various organisms including humans. The movement is based on the sliding between outer doublet microtubules driven by axonemal dyneins, and is regulated by various axonemal components and environmental factors. For studies aiming to elucidate the mechanism of cilia/flagella movement and regulation, Chlamydomonas is an invaluable model organism that offers a variety of mutants. This chapter introduces standard methods for studying Chlamydomonas flagellar motility including analysis of swimming paths, measurements of swimming speed and beat frequency, motility reactivation in demembranated cells (cell models), and observation of microtubule sliding in disintegrating axonemes. Most methods may be easily applied to other organisms with slight modifications of the medium conditions.

The radial spoke is a ubiquitous component of `9+2' cilia and flagella, and plays an essential role in the control of dynein arm activity by relaying signals from the central pair of microtubules to the arms. The Chlamydomonas reinhardtii radial spoke contains at least 23 proteins, only 8 of which have been characterized at the molecular level. Here, we use mass spectrometry to identify 10 additional radial spoke proteins. Many of the newly identified proteins in the spoke stalk are predicted to contain domains associated with signal transduction, including Ca2+-, AKAP- and nucleotide-binding domains. This suggests that the spoke stalk is both a scaffold for signaling molecules and itself a transducer of signals. Moreover, in addition to the recently described HSP40 family member, a second spoke stalk protein is predicted to be a molecular chaperone, implying that there is a sophisticated mechanism for the assembly of this large complex. Among the 18 spoke proteins identified to date, at least 12 have apparent homologs in humans, indicating that the radial spoke has been conserved throughout evolution. The human genes encoding these proteins are candidates for causing primary ciliary dyskinesia, a severe inherited disease involving missing or defective axonemal structures, including the radial spokes.

Ciliary and flagellar axonemes contain multiple inner arm dyneins of which the functional difference is largely unknown. In this study, a Chlamydomonas mutant, ida9, lacking inner arm dynein c was isolated and shown to carry a mutation in the DHC9 dynein heavy chain gene. The cDNA sequence of DHC9 was determined, and its information was used to show that >80% of it is lost in the mutant. Electron microscopy and image analysis showed that the ida9 axoneme lacked electron density near the base of the S2 radial spoke, indicating that dynein c localizes to this site. The mutant ida9 swam only slightly slower than the wild type in normal media. However, swimming velocity was greatly reduced when medium viscosity was modestly increased. Thus, dynein c in wild type axonemes must produce a significant force when flagella are beating in viscous media. Because motility analyses in vitro have shown that dynein c is the fastest among all the inner arm dyneins, we can regard this dynein as a fast yet powerful motor.

In the mechanism underlying the phototactic behavior of Chlamydomonas, Ca2+ has been thought to control the dominance between the two flagella so as to steer the cell to correct directions. A newly isolated mutant, lsp1, that displays weak phototaxis was found to be defective in this Ca2+-dependent shift in flagellar dominance; in demembranated and reactivated cell models, the trans flagellum (the flagellum farthest from the eyespot) beat more strongly than the other (the cis flagellum) in about half of the cells regardless of the Ca2+ concentration between

Of the uncloned ODA genes required for outer dynein arm assembly in Chlamydomonas, ODA5 and ODA10 are of particular interest because they do not encode known subunits of the outer arm or the outer dynein arm-docking complex (ODA-DC), and because genetic studies suggest their products interact. Beginning with a tagged oda5 allele, we isolated genomic and cDNA clones of the wild-type gene. ODA5 predicts a novel, 66-kDa coiled-coil protein. Immunoblotting indicates Oda5p is an axonemal component that assembles onto the axoneme independently of the outer arm and ODA-DC and is uniquely missing in oda5 and oda10 axonemes. Oda5p is released from the axoneme by extraction with 0.6 M KCl, but the soluble Oda5p does not cosediment with the outer dynein arm/ODA-DC in sucrose gradients. Quantitative mass spectrometry by using isotope coded affinity tagging revealed that a previously unidentified adenylate kinase is reduced 35–50% in oda5 flagella. Direct enzymatic assays demonstrated a comparable reduction in adenylate kinase activity in oda5 flagella, and also in oda10 flagella, but not in flagella of other oda mutants. We propose that Oda5p is part of a novel axonemal complex that is required for outer arm assembly and anchors adenylate kinase in proximity to the arm.

How centrioles and basal bodies assemble is a long-standing puzzle in cell biology. To address this problem, we analyzed a novel basal body-defective Chlamydomonas reinhardtii mutant isolated from a collection of flagella-less mutants. This mutant, bld10, displayed disorganized mitotic spindles and cytoplasmic microtubules, resulting in abnormal cell division and slow growth. Electron microscopic observation suggested that bld10 cells totally lack basal bodies. The product of the BLD10 gene (Bld10p) was found to be a novel coiled-coil protein of 170 kD. Immunoelectron microscopy localizes Bld10p to the cartwheel, a structure with ninefold rotational symmetry positioned near the proximal end of the basal bodies. Because the cartwheel forms the base from which the triplet microtubules elongate, we suggest that Bld10p plays an essential role in an early stage of basal body assembly. A viable mutant having such a severe basal body defect emphasizes the usefulness of Chlamydomonas in studying the mechanism of basal body/centriole assembly by using a variety of mutants.

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 ∼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.

Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.

The outer dynein arm-docking complex (ODA-DC) targets the outer dynein arm to its correct binding site on the flagellar axoneme. The Chlamydomonas ODA-DC contains three proteins; loss of any one prevents normal assembly of the outer arm, leading to a slow, jerky swimming phenotype. We showed previously that the smallest ODA-DC subunit, DC3, has four EF-hands (Casey, D. M., Inaba, K., Pazour, G. J., Takada, S., Wakabayashi, K., Wilkerson, C. G., Kamiya, R., and Witman, G. B. (2003) Mol. Biol. Cell 14, 3650-3663). Two of the EF-hands fit the consensus pattern for calcium binding, and one of these contains two cysteine residues within its binding loop. To determine whether the predicted EF-hands are functional, we purified bacterially expressed wild-type DC3 and analyzed its calcium-binding potential in the presence and absence of dithiothreitol and Mg2+. The protein bound one calcium ion with an affinity (Kd) of approximately 1 x 10-5 m. Calcium binding was observed only in the presence of dithiothreitol and thus is redox-sensitive. DC3 also bound Mg2+ at physiological concentrations but with a much lower affinity. Changing the essential glutamate to glutamine in both EF-hands eliminated the calcium binding activity of the bacterially expressed protein. To investigate the role of the EF-hands in vivo, we transformed the modified DC3 gene into a Chlamydomonas insertional mutant lacking DC3. The transformed strain swam normally, assembled a normal number of outer arms, and had a normal photoshock response, indicating that the Glu to Gln mutations did not affect ODA-DC assembly, outer arm assembly, or Ca2+-mediated outer arm activity. Thus, DC3 is a true calcium-binding protein, but the function of this activity remains unknown.

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.

Chlamydomonas has two actin genes, one coding for a conventional actin and the other coding for a highly divergent actin. The divergent actin NAP (for “novel actin-like protein”) is expressed only negligibly in wild-type cells but abundantly in a null mutant of conventional actin, the ida5 mutant. The presence of the dormant NAP gene suggests that NAP may also have its own function in wild-type cells under some conditions. However, no specific functions have been suggested. In this study, we examined the expression of actin and NAP in wild-type and ida5 cells under conditions where actin function has been shown to be important. We found that deflagellation induces the expression of NAP as well as that of actin in wild-type cells. The expressed NAP becomes localized to the regrown flagella, apparently without being associated with dynein. Mating of gametes also increased the expression of actin in wild-type cells and that of NAP in ida5 cells, resulting in accumulation of these proteins in flagella (in both wild-type and ida5 cells) and the fertilization tubule (only in wild-type cells). However, it did not induce significant NAP expression in wild-type cells. These and other observations suggest that the expression of actin and NAP mRNAs is controlled by two discrete mechanisms and that NAP plays a role in flagellar formation in wild-type cells.

Ciliary and flagellar axonemes are basically composed of nine outer doublet microtubules and several functional components, e.g. dynein arms, radial spokes, and interdoublet links. Each A-tubule of the doublet contains a specialized "ribbon" of three protofilaments composed of tubulin and other proteins postulated to specify the three-dimensional arrangement of the various axonemal components. The interdoublet links hold the doublet microtubules together and limit their sliding during the flagellar beat. In this study on Chlamydomonas reinhardtii, we cloned a cDNA encoding a 71,985-Da polypeptide with three DM10 repeats, two C-terminal EF-hand motifs, and homologs extending to humans. This polypeptide, designated as Rib72, is a novel component of the ribbon compartment of flagellar microtubules. It remained associated with 9-fold arrays of doublet tubules following extraction under high and low ionic conditions, and anti-Rib72 antibodies revealed an approximately 96-nm periodicity along axonemes, consistent with Rib72 associating with interdoublet links. Following proteolysis- and ATP-dependent disintegration of axonemes, the rate of cleavage of Rib72 correlated closely with the rate of sliding disintegration. These observations identify a ribbon-associated protein that may function in the structural assembly of the axoneme and in the mechanism and regulation of ciliary and flagellar motility.

Axonemes are highly organized microtubule-based structures conserved in many eukaryotes. In an attempt to study axonemes by a proteomics approach, we selectively cloned cDNAs of axonemal proteins by immunoscreening the testis cDNA library from the ascidianCiona intestinalis by using an antiserum against whole axonemes. We report here a 37-kDa protein of which cDNA occurred most frequently among total positive clones. This protein, named LRR37, belongs to the class of SDS22+ leucine-rich repeat (LRR) family. LRR37 is different from the LRR outer arm dynein light chain reported inChlamydomonas and sea urchin flagella, and thus represents a novel axonemal LRR protein. Immunoelectron microscopy by using a polyclonal antibody against LRR37 showed that it is localized on the tip of the radial spoke, most likely on the spoke head. The LRR37 protein in fact seems to form a complex together with radial spoke protein 3 in a KI extract of the axonemes. These results suggest that LRR37 is a component of the radial spoke head and is involved in the interaction with other radial spoke components or proteins in the central pair projection.