The methylome of motile cilia.

Cilia are highly complex motile, sensory, and secretory organelles that contain perhaps 1000 or more distinct protein components, many of which are subject to various posttranslational modifications such as phosphorylation, N-terminal acetylation, and proteolytic processing. Another common modification is the addition of one or more methyl groups to the side chains of arginine and lysine residues. These tunable additions delocalize the side-chain charge, decrease hydrogen bond capacity, and increase both bulk and hydrophobicity. Methylation is usually mediated by S-adenosylmethionine (SAM)-dependent methyltransferases and reversed by demethylases. Previous studies have identified several ciliary proteins that are subject to methylation including axonemal dynein heavy chains that are modified by a cytosolic methyltransferase. Here, we have performed an extensive proteomic analysis of multiple independently derived cilia samples to assess the potential for SAM metabolism and the extent of methylation in these organelles. We find that cilia contain all the enzymes needed for generation of the SAM methyl donor and recycling of the S-adenosylhomocysteine and tetrahydrofolate byproducts. In addition, we find that at least 155 distinct ciliary proteins are methylated, in some cases at multiple sites. These data provide a comprehensive resource for studying the consequences of methyl marks on ciliary biology.

Methylation of ciliary dynein motors involves the essential cytosolic assembly factor DNAAF3/PF22.

Axonemal dynein motors drive ciliary motility and can consist of up to twenty distinct components with a combined mass of ~2 MDa. In mammals, failure of dyneins to assemble within the axonemal superstructure leads to primary ciliary dyskinesia. Syndromic phenotypes include infertility, rhinitis, severe bronchial conditions, and situs inversus. Nineteen specific cytosolic factors (Dynein Axonemal Assembly Factors; DNAAFs) are necessary for axonemal dynein assembly, although the detailed mechanisms involved remain very unclear. Here, we identify the essential assembly factor DNAAF3 as a structural ortholog of S-adenosylmethionine-dependent methyltransferases. We demonstrate that dynein heavy chains, especially those forming the ciliary outer arms, are methylated on key residues within various nucleotide-binding sites and on microtubule-binding domain helices directly involved in the transition to low binding affinity. These variable modifications, which are generally missing in a Chlamydomonas null mutant for the DNAAF3 ortholog PF22 (DAB1), likely impact on motor mechanochemistry fine-tuning the activities of individual dynein complexes.

Heme-binding protein CYB5D1 is a radial spoke component required for coordinated ciliary beating.

Coordinated beating is crucial for the function of multiple cilia. However, the molecular mechanism is poorly understood. Here, we characterize a conserved ciliary protein CYB5D1 with a heme-binding domain and a cordon-bleu ubiquitin-like domain. Mutation or knockdown of Cyb5d1 in zebrafish impaired coordinated ciliary beating in the otic vesicle and olfactory epithelium. Similarly, the two flagella of an insertional mutant of the CYB5D1 ortholog in Chlamydomonas (Crcyb5d1) showed an uncoordinated pattern due to a defect in the cis-flagellum. Biochemical analyses revealed that CrCYB5D1 is a radial spoke stalk protein that binds heme only under oxidizing conditions. Lack of CrCYB5D1 resulted in a reductive shift in flagellar redox state and slowing down of the phototactic response. Treatment of Crcyb5d1 with oxidants restored coordinated flagellar beating. Taken together, these data suggest that CrCYB5D1 may integrate environmental and intraciliary signals and regulate the redox state of cilia, which is crucial for the coordinated beating of multiple cilia.

The outer dynein arm assembly factor CCDC103 forms molecular scaffolds through multiple self-interaction sites.

CCDC103 is a small protein with unusual biophysical properties that is required for outer dynein arm assembly on ciliary axonemes. Mutations in both human and zebrafish CCDC103 proteins lead to primary ciliary dyskinesia. Previous studies revealed that this protein can oligomerize and appears to be arrayed along the entire length of the ciliary axoneme. CCDC103 also binds purified microtubules directly and indeed stabilizes them. Here we use biochemical approaches to identify two regions of CCDC103 that mediate self-interaction. In both cases, these associations are stable to heating in the presence of detergent and are not disrupted by strong reducing agents. One interaction region consists of a 27-residue inherently disorder segment that can mediate heat/detergent-resistant dimerization when attached to unrelated monomeric proteins. The second interface includes the C-terminal RPAP3_C alpha helical domain. Our data suggest that CCDC103 can form an unconventional polymer and we propose models for how the monomers might be organized. We also use molecular modeling of the RPAP3_C domain to determine the structural consequences of the pathogenic H154P mutation found in human PCD patients.

WDR92 is required for axonemal dynein heavy chain stability in cytoplasm.

WDR92 associates with a prefoldin-like cochaperone complex and known dynein assembly factors. WDR92 has been very highly conserved and has a phylogenetic signature consistent with it playing a role in motile ciliary assembly or activity. Knockdown of WDR92 expression in planaria resulted in ciliary loss, reduced beat frequency and dyskinetic motion of the remaining ventral cilia. We have now identified a Chlamydomonas wdr92 mutant that encodes a protein missing the last four WD repeats. The wdr92-1 mutant builds only ∼0.7-µm cilia lacking both inner and outer dynein arms, but with intact doublet microtubules and central pair. When cytoplasmic extracts prepared by freeze/thaw from a control strain were fractionated by gel filtration, outer arm dynein components were present in several distinct high molecular weight complexes. In contrast, wdr92-1 extracts almost completely lacked all three outer arm heavy chains, while the IFT dynein heavy chain was present in normal amounts. A wdr92-1 tpg1-2 double mutant builds ∼7-µm immotile flaccid cilia that completely lack dynein arms. These data indicate that WDR92 is a key assembly factor specifically required for the stability of axonemal dynein heavy chains in cytoplasm and suggest that cytoplasmic/IFT dynein heavy chains use a distinct folding pathway.

The oligomeric outer dynein arm assembly factor CCDC103 is tightly integrated within the ciliary axoneme and exhibits periodic binding to microtubules.

CCDC103 is an ∼29-kDa protein consisting of a central RPAP3_C domain flanked by N- and C-terminal coiled coils. Defects in CCDC103 lead to primary ciliary dyskinesia caused by the loss of outer dynein arms. This protein is present along the entire length of the ciliary axoneme and does not require other dynein or docking complex components for its integration. Unlike other known dynein assembly factors within the axoneme, CCDC103 is not solubilized by 0.6 M NaCl and requires more chaotropic conditions, such as 0.5 M KI. Alternatively, it can be extracted using 0.3% sarkosyl. CCDC103 forms stable dimers and other oligomers in solution through interactions involving the central domain. The smallest particle observed by dynamic light scattering has a hydrodynamic diameter of ∼25 nm. Furthermore, CCDC103 binds microtubules directly, forming ∼9-nm diameter particles that exhibit a 12-nm spacing on the microtubule lattice, suggesting that there may be two CCDC103 units per outer arm dynein repeat. Although the outer dynein arm docking complex is necessary to form arrays of dyneins along microtubules, it is not sufficient to set up a single array in a precise location on each axonemal doublet. We propose that CCDC103 helps generate a high-affinity site on the doublets for outer arm assembly, either through direct interactions or indirectly, perhaps by modifying the underlying microtubule lattice.

Zebrafish Ciliopathy Screen Plus Human Mutational Analysis Identifies C21orf59 and CCDC65 Defects as Causing Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.

WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia.

Retrograde intraflagellar transport (IFT) is required for assembly of cilia. We identify a Chlamydomonas flagellar protein (flagellar-associated protein 163 [FAP163]) as being closely related to the D1bIC(FAP133) intermediate chain (IC) of the dynein that powers this movement. Biochemical analysis revealed that FAP163 is present in the flagellar matrix and is actively trafficked by IFT. Furthermore, FAP163 copurified with D1bIC(FAP133) and the LC8 dynein light chain, indicating that it is an integral component of the retrograde IFT dynein. To assess the functional role of FAP163, we generated an RNA interference knockdown of the orthologous protein (WD60) in planaria. The Smed-wd60(RNAi) animals had a severe ciliary assembly defect that dramatically compromised whole-organism motility. Most cilia were present as short stubs that had accumulated large quantities of IFT particle-like material between the doublet microtubules and the membrane. The few remaining approximately full-length cilia had a chaotic beat with a frequency reduced from 24 to ∼10 Hz. Thus WD60/FAP163 is a dynein IC that is absolutely required for retrograde IFT and ciliary assembly.

Association of Lis1 with outer arm dynein is modulated in response to alterations in flagellar motility.

The cytoplasmic dynein regulatory factor Lis1, which induces a persistent tight binding to microtubules and allows for transport of cargoes under high-load conditions, is also present in motile cilia/flagella. We observed that Lis1 levels in flagella of Chlamydomonas strains that exhibit defective motility due to mutation of various axonemal substructures were greatly enhanced compared with wild type; this increase was absolutely dependent on the presence within the flagellum of the outer arm dynein α heavy chain/light chain 5 thioredoxin unit. To assess whether cells might interpret defective motility as a "high-load environment," we reduced the flagellar beat frequency of wild-type cells through enhanced viscous load and by reductive stress; both treatments resulted in increased levels of flagellar Lis1, which altered the intrinsic beat frequency of the trans flagellum. Differential extraction of Lis1 from wild-type and mutant axonemes suggests that the affinity of outer arm dynein for Lis1 is directly modulated. In cytoplasm, Lis1 localized to two punctate structures, one of which was located near the base of the flagella. These data reveal that the cell actively monitors motility and dynamically modulates flagellar levels of the dynein regulatory factor Lis1 in response to imposed alterations in beat parameters.

An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria.

Motile cilia mediate the flow of mucus and other fluids across the surface of specialized epithelia in metazoans. Efficient clearance of peri-ciliary fluids depends on the precise coordination of ciliary beating to produce metachronal waves. The role of individual dynein motors and the mechanical feedback mechanisms required for this process are not well understood. Here we used the ciliated epithelium of the planarian Schmidtea mediterranea to dissect the role of outer arm dynein motors in the metachronal synchrony of motile cilia. We demonstrate that animals that completely lack outer dynein arms display a significant decline in beat frequency and an inability of cilia to coordinate their oscillations and form metachronal waves. Furthermore, lack of a key mechanosensitive regulatory component (LC1) yields a similar phenotype even though outer arms still assemble in the axoneme. The lack of metachrony was not due simply to a decrease in ciliary beat frequency, as reducing this parameter by altering medium viscosity did not affect ciliary coordination. In addition, we did not observe a significant temporal variability in the beat cycle of impaired cilia. We propose that this conformational switch provides a mechanical feedback system within outer arm dynein that is necessary to entrain metachronal synchrony.

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

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

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

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

Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor.

Intraflagellar transport (IFT) is the bi-directional movement of particles along the length of axonemal outer doublet microtubules and is needed for the assembly and maintenance of eukaryotic cilia and flagella. Retrograde IFT requires cytoplasmic dynein 1b, a motor complex whose organization, structural composition and regulation is poorly understood. We have characterized the product of the Chlamydomonas FAP133 gene that encodes a new WD-repeat protein similar to dynein intermediate chains and homologous to the uncharacterized vertebrate protein WD34. FAP133 is located at the peri-basal body region as well as in punctate structures along the flagella. This protein is associated with the IFT machinery because it is specifically depleted from the flagella of cells with defects in anterograde IFT. Fractionation of flagellar matrix proteins indicates that FAP133 associates with both the LC8 dynein light chain and the IFT dynein heavy chain and light intermediate chain (DHC1b-D1bLIC) motor complex. In the absence of DHC1b or D1bLIC, FAP133 fails to localize at the peri-basal body region but, rather, is concentrated in a region of the cytoplasm near the cell center. Furthermore, we found that FAP133, LC8, DHC1b, D1bLIC, the FLA10 kinesin-2 necessary for anterograde IFT and other IFT scaffold components associate to form a large macromolecular assembly.

Differential light chain assembly influences outer arm dynein motor function.

Tctex1 and Tctex2 were originally described as potential distorters/sterility factors in the non-Mendelian transmission of t-haplotypes in mice. These proteins have since been identified as subunits of cytoplasmic and/or axonemal dyneins. Within the Chlamydomonas flagellum, Tctex1 is a subunit of inner arm I1. We have now identified a second Tctex1-related protein (here termed LC9) in Chlamydomonas. LC9 copurifies with outer arm dynein in sucrose density gradients and is missing only in those strains completely lacking this motor. Zero-length cross-linking of purified outer arm dynein indicates that LC9 interacts directly with both the IC1 and IC2 intermediate chains. Immunoblot analysis revealed that LC2, LC6, and LC9 are missing in an IC2 mutant strain (oda6-r88) that can assemble outer arms but exhibits significantly reduced flagellar beat frequency. This defect is unlikely to be due to lack of LC6, because an LC6 null mutant (oda13) exhibits only a minor swimming abnormality. Using an LC2 null mutant (oda12-1), we find that although some outer arm dynein components assemble in the absence of LC2, they are nonfunctional. In contrast, dyneins from oda6-r88, which also lack LC2, retain some activity. Furthermore, we observed a synthetic assembly defect in an oda6-r88 oda12-1 double mutant. These data suggest that LC2, LC6, and LC9 have different roles in outer arm assembly and are required for wild-type motor function in the Chlamydomonas flagellum.

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.

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

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

The Roadblock light chains are ubiquitous components of cytoplasmic dynein that form homo- and heterodimers.

The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella.

A bipartite Ca2+-regulated nucleoside-diphosphate kinase system within the Chlamydomonas flagellum. The regulatory subunit p72.

Regulation of flagellar activity in Chlamydomonas involves both Ca(2+) and cAMP-mediated signaling pathways. However, Chlamydomonas and sea urchin sperm flagella also exhibit nucleoside-diphosphate kinase (NDK) activity, suggesting a requirement for GTP within this highly conserved organelle. In sea urchin sperm, the NDK catalytic subunit is an integral component of the outer dynein arm. Here we describe a modular protein (p72) from the Chlamydomonas flagellum that consists of three domains closely related to the presumptive regulatory segment of rat NDK-7 followed by two EF-hands that are predicted to bind Ca(2+). There are close homologues of p72 in both mammalian and insect genomes. The p72 protein is tightly associated with the flagellar axoneme and is located along the entire length except at the transition zone. Cross-linking experiments suggest that p72 interacts with two or three additional axonemal polypeptides. The sensitivity of p72 to tryptic digestion differed considerably in the presence and the absence of Ca(2+), suggesting that it indeed binds this ligand. These studies indicate that the flagellar NDK system is bipartite with the regulatory and catalytic components residing on different polypeptides. We propose that Ca(2+) regulation of flagellar motility in Chlamydomonas may be achieved in part through a downstream GTP-mediated signaling pathway.

Redox-based control of the gamma heavy chain ATPase from Chlamydomonas outer arm dynein.

The outer dynein arm from Chlamydomonas flagella contains two redox-active thioredoxin-related light chains associated with the alpha and beta heavy chains; these proteins belong to a distinct subgroup within the thioredoxin family. This observation suggested that some aspect of dynein activity might be modulated through redox poise. To test this, we have examined the effect of sulfhydryl oxidation on the ATPase activity of isolated dynein and axonemes from wildtype and mutant strains lacking various heavy chain combinations. The outer, but not inner, dynein arm ATPase was stimulated significantly following treatment with low concentrations of dithionitrobenzoic acid; this effect was readily reversible by dithiol, and to a lesser extent, monothiol reductants. Mutational and biochemical dissection of the outer arm revealed that ATPase activation in response to DTNB was an exclusive property of the gamma heavy chain, and that enzymatic enhancement was modulated by the presence of other dynein components. Furthermore, we demonstrate that the LC5 thioredoxin-like light chain binds to the N-terminal stem domain of the alpha heavy chain and that the beta heavy chain-associated LC3 protein also interacts with the gamma heavy chain. These data suggest the possibility of a dynein-associated redox cascade and further support the idea that the gamma heavy chain plays a key regulatory role within the outer arm.