Cryo–electron tomography of Chlamydomonas flagella reveals previously uncharacterized features of the radial spokes, including structural heterogeneity and direct interactions with dyneins and between the spoke heads. A “radial spoke 3 stand-in” occupies what would be the site of a third spoke in organisms with spoke triplets.
Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo–electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named “radial spoke 3 stand-in,” which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation.
Radial spokes are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms the 96 nm axonemal repeat unit contains only a pair of two spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pair in Chlamydomonas flagella has been well characterized, while spoke triplets have received less attention. Here, we used cryo-electron tomography and subtomogram averaging to visualize the 3D structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3 and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable radial spoke heterogeneity is conserved and that organisms with radial spoke pairs contain the basal part of RS3. This conserved radial spoke heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility.
cryo-electron tomography; axoneme; radial spoke triplet; RS3; RS3S
This study reveals the 3D structure of the CSC and its connections to three major axonemal complexes involved in dynein regulation, including the distal radial spoke and the nexin-DRC. The findings corroborate radial spoke heterogeneity and suggest a unique role for the distal spoke in calcium-mediated signal transduction and flagellar motility.
Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo–electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin–dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility.
Genetic and in vitro analyses have revealed that radial spokes play a crucial role in regulation of ciliary and flagellar motility, including control of waveform. However, the mechanisms of regulation are not understood. Here, we developed a novel procedure to isolate intact radial spokes as a step toward understanding the mechanism by which these complexes regulate dynein activity. The isolated radial spokes sediment as 20S complexes that are the size and shape of radial spokes. Extracted radial spokes rescue radial spoke structure when reconstituted with isolated axonemes derived from the radial spoke mutant pf14. Isolated radial spokes are composed of the 17 previously defined spoke proteins as well as at least five additional proteins including calmodulin and the ubiquitous dynein light chain LC8. Analyses of flagellar mutants and chemical cross-linking studies demonstrated calmodulin and LC8 form a complex located in the radial spoke stalk. We postulate that calmodulin, located in the radial spoke stalk, plays a role in calcium control of flagellar bending.
calcium; cilia; dynein; flagella; calmodulin
For virtually all cilia and eukaryotic flagella, the second messengers calcium and cyclic adenosine monophosphate are implicated in modulating dynein- driven microtubule sliding to regulate beating. Calmodulin (CaM) localizes to the axoneme and is a key calcium sensor involved in regulating motility. Using immunoprecipitation and mass spectrometry, we identify members of a CaM-containing complex that are involved in regulating dynein activity. This complex includes flagellar-associated protein 91 (FAP91), which shares considerable sequence similarity to AAT-1, a protein originally identified in testis as an A-kinase anchor protein (AKAP)– binding protein. FAP91 directly interacts with radial spoke protein 3 (an AKAP), which is located at the base of the spoke. In a microtubule sliding assay, the addition of antibodies generated against FAP91 to mutant axonemes with reduced dynein activity restores dynein activity to wild-type levels. These combined results indicate that the CaM- and spoke-associated complex mediates regulatory signals between the radial spokes and dynein arms.
Radial spokes are a conserved axonemal structural complex postulated to regulate the motility of 9 + 2 cilia and flagella via a network of phosphoenzymes and regulatory proteins. Consistently, a Chlamydomonas radial spoke protein, RSP3, has been identified by RII overlays as an A-kinase anchoring protein (AKAP) that localizes the cAMP-dependent protein kinase (PKA) holoenzyme by binding to the RIIa domain of PKA RII subunit. However, the highly conserved docking domain of PKA is also found in the N termini of several AKAP-binding proteins unrelated to PKA as well as a 24-kDa novel spoke protein, RSP11. Here, we report that RSP11 binds to RSP3 directly in vitro and colocalizes with RSP3 toward the spoke base near outer doublets and dynein motors in axonemes. Importantly, RSP11 mutant pf25 displays a spectrum of motility, from paralysis with flaccid or twitching flagella as other spoke mutants to wild-typelike swimming. The wide range of motility changes reversibly depending on the condition of liquid media without replacing defective proteins. We postulate that radial spokes use the RIIa/AKAP module to regulate ciliary and flagellar beating; absence of the spoke RIIa protein exposes a medium-sensitive regulatory mechanism that is not obvious in wild-type Chlamydomonas.
Cryo-EM tomography of wild-type and mutant cilia and flagella from Tetrahymena and Chlamydomonas reveals new information on the substructure of radial spokes.
Radial spokes (RSs) are ubiquitous components in the 9 + 2 axoneme thought to be mechanochemical transducers involved in local control of dynein-driven microtubule sliding. They are composed of >23 polypeptides, whose interactions and placement must be deciphered to understand RS function. In this paper, we show the detailed three-dimensional (3D) structure of RS in situ in Chlamydomonas reinhardtii flagella and Tetrahymena thermophila cilia that we obtained using cryoelectron tomography (cryo-ET). We clarify similarities and differences between the three spoke species, RS1, RS2, and RS3, in T. thermophila and in C. reinhardtii and show that part of RS3 is conserved in C. reinhardtii, which only has two species of complete RSs. By analyzing C. reinhardtii mutants, we identified the specific location of subsets of RS proteins (RSPs). Our 3D reconstructions show a twofold symmetry, suggesting that fully assembled RSs are produced by dimerization. Based on our cryo-ET data, we propose models of subdomain organization within the RS as well as interactions between RSPs and with other axonemal components.
Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.
cilia; intraflagellar; transport; kinesin; radial spoke
Genetic, biochemical, and structural data support a model in which axonemal radial spokes regulate dynein-driven microtubule sliding in Chlamydomonas flagella. However, the molecular mechanism by which dynein activity is regulated is unknown. We describe results from three different in vitro approaches to test the hypothesis that an axonemal protein kinase inhibits dynein in spoke-deficient axonemes from Chlamydomonas flagella. First, the velocity of dynein-driven microtubule sliding in spoke-deficient mutants (pf14, pf17) was increased to wild-type level after treatment with the kinase inhibitors HA-1004 or H-7 or by the specific peptide inhibitors of cAMP-dependent protein kinase (cAPK) PKI(6-22)amide or N alpha-acetyl-PKI(6-22)amide. In particular, the peptide inhibitors of cAPK were very potent, stimulating half-maximal velocity at 12-15 nM. In contrast, kinase inhibitors did not affect microtubule sliding in axonemes from wild- type cells. PKI treatment of axonemes from a double mutant missing both the radial spokes and the outer row of dynein arms (pf14pf28) also increased microtubule sliding to control (pf28) velocity. Second, addition of the type-II regulatory subunit of cAPK (RII) to spoke- deficient axonemes increased microtubule sliding to wild-type velocity. Addition of 10 microM cAMP to spokeless axonemes, reconstituted with RII, reversed the effect of RII. Third, our previous studies revealed that inner dynein arms from the Chlamydomonas mutants pf28 or pf14pf28 could be extracted in high salt buffer and subsequently reconstituted onto extracted axonemes restoring original microtubule sliding activity. Inner arm dyneins isolated from PKI-treated axonemes (mutant strain pf14pf28) generated fast microtubule sliding velocities when reconstituted onto both PKI-treated or control axonemes. In contrast, dynein from control axonemes generated slow microtubule sliding velocities on either PKI-treated or control axonemes. Together, the data indicate that an endogenous axonemal cAPK-type protein kinase inhibits dynein-driven microtubule sliding in spoke-deficient axonemes. The kinase is likely to reside in close association with its substrate(s), and the substrate targets are not exclusively localized to the central pair, radial spokes, dynein regulatory complex, or outer dynein arms. The results are consistent with a model in which the radial spokes regulate dynein activity through suppression of a cAMP- mediated mechanism.
Structural and functional analyses of artificial micro RNA (amiRNA) mutants reveal that the CSC plays a role not only in generating wild-type motility, but also in assembly of at least a subset of radial spokes. This study also produced the unexpected finding that, contrary to current belief, the radial spokes may not be homogeneous.
The ubiquitous calcium binding protein, calmodulin (CaM), plays a major role in regulating the motility of all eukaryotic cilia and flagella. We previously identified a CaM and Spoke associated Complex (CSC) and provided evidence that this complex mediates regulatory signals between the radial spokes and dynein arms. We have now used an artificial microRNA (amiRNA) approach to reduce expression of two CSC subunits in Chlamydomonas. For all amiRNA mutants, the entire CSC is lacking or severely reduced in flagella. Structural studies of mutant axonemes revealed that assembly of radial spoke 2 is defective. Furthermore, analysis of both flagellar beating and microtubule sliding in vitro demonstrates that the CSC plays a critical role in modulating dynein activity. Our results not only indicate that the CSC is required for spoke assembly and wild-type motility, but also provide evidence for heterogeneity among the radial spokes.
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.
Axoneme; Chaperones; Calcium; Primary ciliary dyskinesia
Previous physiological and pharmacological experiments have demonstrated that the Chlamydomonas flagellar axoneme contains a cAMP-dependent protein kinase (PKA) that regulates axonemal motility and dynein activity. However, the mechanism for anchoring PKA in the axoneme is unknown. Here we test the hypothesis that the axoneme contains an A-kinase anchoring protein (AKAP). By performing RII blot overlays on motility mutants defective for specific axonemal structures, two axonemal AKAPs have been identified: a 240-kD AKAP associated with the central pair apparatus, and a 97-kD AKAP located in the radial spoke stalk. Based on a detailed analysis, we have shown that AKAP97 is radial spoke protein 3 (RSP3). By expressing truncated forms of RSP3, we have localized the RII-binding domain to a region between amino acids 144–180. Amino acids 161–180 are homologous with the RII-binding domains of other AKAPs and are predicted to form an amphipathic helix. Amino acid substitution of the central residues of this region (L to P or VL to AA) results in the complete loss of RII binding. RSP3 is located near the inner arm dyneins, where an anchored PKA would be in direct position to modify dynein activity and regulate flagellar motility.
kinases; cell motility; flagella; dynein; AKAP
Cilia are organelles found on most eukaryotic cells, where they serve important functions in motility, sensory reception, and signaling. Recent advances in electron tomography have facilitated a number of ultrastructural studies of ciliary components that have significantly improved our knowledge of cilium architecture. These studies have produced nanometer‐resolution structures of axonemal dynein complexes, microtubule doublets and triplets, basal bodies, radial spokes, and nexin complexes. In addition to these electron tomography studies, several recently published crystal structures provide insights into the architecture and mechanism of dynein as well as the centriolar protein SAS-6, important for establishing the 9-fold symmetry of centrioles. Ciliary assembly requires intraflagellar transport (IFT), a process that moves macromolecules between the tip of the cilium and the cell body. IFT relies on a large 20-subunit protein complex that is thought to mediate the contacts between ciliary motor and cargo proteins. Structural investigations of IFT complexes are starting to emerge, including the first three‐dimensional models of IFT material in situ, revealing how IFT particles organize into larger train-like arrays, and the high-resolution structure of the IFT25/27 subcomplex. In this review, we cover recent advances in the structural and mechanistic understanding of ciliary components and IFT complexes.
► Electron tomographic structures of axonemal components. ► High‐resolution structures of dynein and SAS-6. ► Electron tomographic reconstruction of IFT particles. ► High‐resolution crystal structure of IFT complex 25/27.
2D, two‐dimensional; 3D, three‐dimensional; DIC, differential interference contrast; DRC, dynein regulatory complex; EM, electron microscopy; ET, electron tomography; IDA, inner dynein arm; IFT, intraflagellar transport; MT, microtubule; MTBD, microtubule binding domain; ODA, outer dynein arm; RS, radial spoke; RSP, radial spoke protein; cilium; intraflagellar transport; electron tomography; IFT complex; flagellum
The flagellum of Trypanosoma brucei is a multifunctional organelle with critical roles in motility and other aspects of the trypanosome life cycle. Trypanin is a flagellar protein required for directional cell motility, but its molecular function is unknown. Recently, a trypanin homologue in Chlamydomonas reinhardtii was reported to be part of a dynein regulatory complex (DRC) that transmits regulatory signals from central pair microtubules and radial spokes to axonemal dynein. DRC genes were identified as extragenic suppressors of central pair and/or radial spoke mutations. We used RNA interference to ablate expression of radial spoke (RSP3) and central pair (PF16) components individually or in combination with trypanin. Both rsp3 and pf16 single knockdown mutants are immotile, with severely defective flagellar beat. In the case of rsp3, this loss of motility is correlated with the loss of radial spokes, while in the case of pf16 the loss of motility correlates with an aberrant orientation of the central pair microtubules within the axoneme. Genetic interaction between trypanin and PF16 is demonstrated by the finding that loss of trypanin suppresses the pf16 beat defect, indicating that the DRC represents an evolutionarily conserved strategy for dynein regulation. Surprisingly, we discovered that four independent mutants with an impaired flagellar beat all fail in the final stage of cytokinesis, indicating that flagellar motility is necessary for normal cell division in T. brucei. These findings present the first evidence that flagellar beating is important for cell division and open the opportunity to exploit enzymatic activities that drive flagellar beat as drug targets for the treatment of African sleeping sickness.
To understand mechanisms of regulation of dynein activity along and around the axoneme we further characterized the "dynein regulatory complex" (drc). The lack of some axonemal proteins, which together are referred to as drc, causes the suppression of flagellar paralysis of radial spoke and central pair mutants. The drc is also an adapter involved in the ATP-insensitive binding of I2 and I3 inner dynein arms to doublet microtubules. Evidence supporting these conclusions was obtained through analyses of five drc mutants: pf2, pf3, suppf3, suppf4, and suppf5. Axonemes from drc mutants lack part of I2 and I3 inner dynein arms as well as subsets of seven drc components (apparent molecular weight from 29,000 to 192,000). In the absence of ATP-Mg, dynein-depleted axonemes from the same mutants bind I2 and I3 inner arms at both ATP-sensitive and -insensitive sites. At ATP-insensitive sites, they bind I2 and I3 inner arms to an extent that depends on the drc defect. This evidence suggested to us that the drc forms one binding site for the I2 and I3 inner arms on the A part of doublet microtubules.
The radial spoke (RS)/central pair (CP) system in cilia and flagella plays an essential role in the regulation of force generation by dynein, the motor protein that drives cilia/flagella movements. Mechanical and mechanochemical interactions between the CP and the distal part of the RS, the spokehead, should be crucial for this control; however, the details of interaction are totally unknown. As an initial step toward an understanding of the RS-CP interaction, we examined the protein-protein interactions between the five spokehead proteins (radial spoke protein (RSP)1, RSP4, RSP6, RSP9, and RSP10) and three spoke stalk proteins (RSP2, RSP5 and RSP23), all expressed as recombinant proteins. Three of them were shown to have physiological activities by electroporation-mediated protein delivery into mutants deficient in the respective proteins. Glutathione S-transferase pulldown assays in vitro detected interactions in 10 out of 64 pairs of recombinants. In addition, chemical crosslinking of axonemes using five reagents detected seven kinds of interactions between the RS subunits in situ. Finally, in the mixture of the recombinant spokehead subunits, RSP1, RSP4, RSP6, and RSP9 formed a 7-10S complex as detected by sucrose density gradient centrifugation. It may represent a partial assembly of the spokehead. From these results, we propose a model of interactions taking place between the spokehead subunits.
cilia and flagella; radial spokes; pulldown assays; chemical crosslink; protein electroporation
The radial spoke is a stable structural complex in the 9 + 2 axoneme for the control of flagellar motility. However, the spokes in Chlamydomonas mutant pf24 are heterogeneous and unstable, whereas several spoke proteins are reduced differentially. To elucidate the defective mechanism, we clone RSP16, a prominent spoke protein diminished in pf24 axonemes. Unexpectedly, RSP16 is a novel HSP40 member of the DnaJ superfamily that assists chaperones in various protein-folding-related processes. Importantly, RSP16 is uniquely excluded from the 12S spoke precursor complex that is packaged in the cell body and transported toward the flagellar tip to be converted into mature 20S axonemal spokes. Rather, RSP16, transported separately, joins the precursor complex in flagella. Furthermore, RSP16 molecules in vitro and in flagella form homodimers, a characteristic required for the cochaperone activity of HSP40. We postulate that the spoke HSP40 operates as a cochaperone to assist chaperone machinery at the flagellar tip to actively convert the smaller spoke precursor and itself into the mature stable complex; failure of the interaction between the spoke HSP40 and its target polypeptide results in heterogeneous unstable radial spokes in pf24.
Ciliary and flagellar motility is regulated by changes in intraflagellar calcium. However, the molecular mechanism by which calcium controls motility is unknown. We tested the hypothesis that calcium regulates motility by controlling dynein-driven microtubule sliding and that the central pair and radial spokes are involved in this regulation. We isolated axonemes from Chlamydomonas mutants and measured microtubule sliding velocity in buffers containing 1 mM ATP and various concentrations of calcium. In buffers with pCa > 8, microtubule sliding velocity in axonemes lacking the central apparatus (pf18 and pf15) was reduced compared with that of wild-type axonemes. In contrast, at pCa4, dynein activity in pf18 and pf15 axonemes was restored to wild-type level. The calcium-induced increase in dynein activity in pf18 axonemes was inhibited by antagonists of calmodulin and calmodulin-dependent kinase II. Axonemes lacking the C1 central tubule (pf16) or lacking radial spoke components (pf14 and pf17) do not exhibit calcium-induced increase in dynein activity in pCa4 buffer. We conclude that calcium regulation of flagellar motility involves regulation of dynein-driven microtubule sliding, that calmodulin and calmodulin-dependent kinase II may mediate the calcium signal, and that the central apparatus and radial spokes are key components of the calcium signaling pathway.
Radial spokes of the eukaryotic flagellum extend from the A tubule of each outer doublet microtubule toward the central pair microtubules. In the paralyzed flagella mutant of Chlamydomonas pf14, a mutation in the gene for one of 17 polypeptides that comprise the radial spokes results in flagella that lack all 17 spoke components. The defective gene product, radial spoke protein 3 (RSP3), is, therefore, pivotal to the assembly of the entire spoke and may attach the spoke to the axoneme. We have synthesized RSP3 in vitro and assayed its binding to axonemes from pf14 cells to determine if RSP3 can attach to spokeless axonemes. In vitro, RSP3 binds to pf14 axonemes, but not to wild-type axonemes or microtubules polymerized from purified chick brain tubulin. The sole axoneme binding domain of RSP3 is located within amino acids 1-85 of the 516 amino acid protein; deletion of these amino acids abolishes binding by RSP3. Fusion of amino acids 1-85 or 42-85 to an unrelated protein confers complete or partial binding activity, respectively, to the fusion protein. Transformation of pf14 cells with mutagenized RSP3 genes indicates that amino acids 18-87 of RSP3 are important to its function, but that the carboxy-terminal 140 amino acids can be deleted with little effect on radial spoke assembly or flagellar motility.
The ultrastructure of normal human cilia and flagella was examined and quantitatively assessed to determine the normal variations in the structure of the axoneme. Ciliated respiratory epithelial cells and spermatozoa from 10 normal, nonsmoking male volunteers who had normal semen parameters were fixed for electron microscopy. Tannic acid and MgSO4 were included during fixation to enhance, in particular, axonemal components. In 75 axonemal cross sections per sample, the number of outer doublet and central singlet microtubules, outer and inner dynein arms, and radial spokes were recorded. Statistical analysis of the results showed a marked reduction, from the expected value of nine, in the numbers of inner dynein arms (mean +/- SE, cilia, 5.31 +/- 0.13; sperm, 5.38 +/- 0.16) and radial spokes (cilia, 4.95 +/- 0.22; sperm, 5.80 +/- 0.19). The ideal axoneme with all its structural components was seen in only 0.13% of cilia and 0.80% of sperm tails. Significantly more doublet microtubules (P less than 0.05) and less central microtubules (P less than 0.01) and radial spokes (P less than 0.01) were seen in cilia than in sperm tail axonemes. Between subjects there was little variation in the mean number of a structure seen per axoneme. However, within each sample, the variation was considerably higher, particularly for the inner and outer dynein arms and radial spokes. The doublet microtubules had significantly greater standard deviations in the sperm tails compared with the cilia (P less than 0.01), and furthermore, a significantly greater number of sperm tails compared with cilia showed the incorrect number of doublet microtubules (P less than 0.02). In one semen sample, with normal semen analysis, 20% of the sperm tails showed incorrect numbers of doublet microtubules, ranging from 12 + 2 to 5 + 2 compared with only 1.3% in cilia from this subject. This study has demonstrated that the ideal axoneme is rarely seen even in normal samples, probably because of the technical difficulties in resolution and visualization, and stresses the need for thorough documentation of axonemal ultrastructure. This work provides a normal data base for comparison with patients who have chronic respiratory disease and suspected infertility.
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.
The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1–2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.
CK1 puts the brakes on dynein activity when added to purified axonemes in vitro, presumably to regulate how flagella bend.
Experimental analysis of isolated ciliary/flagellar axonemes has implicated the protein kinase casein kinase I (CK1) in regulation of dynein. To test this hypothesis, we developed a novel in vitro reconstitution approach using purified recombinant Chlamydomonas reinhardtii CK1, together with CK1-depleted axonemes from the paralyzed flagellar mutant pf17, which is defective in radial spokes and impaired in dynein-driven microtubule sliding. The CK1 inhibitors (DRB and CK1-7) and solubilization of CK1 restored microtubule sliding in pf17 axonemes, which is consistent with an inhibitory role for CK1. The phosphatase inhibitor microcystin-LR blocked rescue of microtubule sliding, indicating that the axonemal phosphatases, required for rescue, were retained in the CK1-depleted axonemes. Reconstitution of depleted axonemes with purified, recombinant CK1 restored inhibition of microtubule sliding in a DRB– and CK1-7–sensitive manner. In contrast, a purified “kinase-dead” CK1 failed to restore inhibition. These results firmly establish that an axonemal CK1 regulates dynein activity and flagellar motility.
The unicellular alga Chlamydomonas can assemble two 10 μm flagella in one hour from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are “T” shaped structures with a head of 5 proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form a 12S complex. In mutants lacking a single radial spoke protein, smaller spoke subassemblies were identified. When extracts from two such mutants were mixed in vitro the 12S complex was assembled from several smaller complexes demonstrating that portions of the stepwise assembly of radial spoke assembly can be carried out in vitro to elucidate the order of spoke assembly in the cell body.
Cilia; Flagella; Chlamydomonas; RSP3
Elegant cryoelectron tomography reveals that the nexin link between microtubule doublets in 9 + 2 axonemal structures, critical for their ability to bend, is the dynein regulatory complex.
Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending.