The CSC is required for complete radial spoke assembly and wild-type ciliary motility

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.     

A conserved CaM- and radial spoke associated complex mediates regulation of flagellar dynein activity

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.     

Assembly of flagellar radial spoke proteins in Chlamydomonas: identification of the axoneme binding domain of radial spoke protein 3

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 flagellar motility of Chlamydomonas pf25 mutant lacking an AKAP-binding protein is overtly sensitive to medium conditions

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 wildtype-like 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.     

The CSC connects three major axonemal complexes involved in dynein regulation

Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated 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.     

Dimeric novel HSP40 is incorporated into the radial spoke complex during the assembly process in flagella

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.     

The DPY-30 Domain and Its Flanking Sequence Mediate the Assembly and Modulation of Flagellar Radial Spoke Complexes

RIIa is known as the dimerization and docking (D/D) domain of the cyclic AMP (cAMP)-dependent protein kinase. However, numerous molecules, including radial spoke protein 2 (RSP2) in Chlamydomonas flagella, also contain an RIIa or a similar DPY-30 domain. To elucidate new roles of D/D domain-containing proteins, we investigated a panel of RSP2 mutants. An RSP2 mutant had paralyzed flagella defective in RSP2 and multiple subunits near the spokehead. New transgenic strains lacking only the DPY-30 domain in RSP2 were also paralyzed. In contrast, motility was restored in strains that lacked only RSP2's calmodulin-binding C-terminal region. These cells swam normally in dim light but could not maintain typical swimming trajectories under bright illumination. In both deletion transgenic strains, the subunits near the spokehead were restored, but their firm attachment to the spokestalk required the DPY-30 domain. We postulate that the DPY-30-helix dimer is a conserved two-prong linker, required for normal motility, organizing duplicated subunits in the radial spoke stalk and formation of a symmetrical spokehead. Further, the dispensable calmodulin-binding region appears to fine-tune the spokehead for regulation of "steering" motility in the green algae. Thus, in general, D/D domains may function to localize molecular modules for both the assembly and modulation of macromolecular complexes.     

Building a radial spoke: flagellar radial spoke protein 3 (RSP3) is a dimer

Radial spokes are critical multisubunit structures required for normal ciliary and eukaryotic flagellar motility. Experimental evidence indicates the radial spokes are mechanochemical transducers that transmit signals from the central pair apparatus to the outer doublet microtubules for local control of dynein activity. Recently, progress has been made in identifying individual components of the radial spoke, yet little is known about how the radial spoke is assembled or how it performs in signal transduction. Here we focus on radial spoke protein 3 (RSP3), a highly conserved AKAP located at the base of the radial spoke stalk and required for radial spoke assembly on the doublet microtubules. Biochemical approaches were taken to further explore the functional role of RSP3 within the radial spoke structure and for control of motility. Chemical crosslinking, native gel electrophoresis, and epitope-tagged RSP3 proteins established that RSP3 forms a dimer. Analysis of truncated RSP3 proteins indicates the dimerization domain coincides with the previously characterized axoneme binding domain in the N-terminus. We propose a model in which each radial spoke structure is built on an RSP3 dimer, and indicating that each radial spoke can potentially localize multiple PKAs or AKAP-binding proteins in position to control dynein activity and flagellar motility.     

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.     

Two anti-radial spoke monoclonal antibodies inhibit Chlamydomonas axonemal motility by different mechanisms

In the 9 + 2 axoneme, radial spokes are structural components attached to the A-tubules of the nine outer doublet microtubules. They protrude toward the central pair microtubule complex with which they have transient but regular interactions for the normal flagellar motility to occur. Flagella of Chlamydomonas mutants deficient in entire radial spokes or spoke heads are paralyzed. In this study the importance of two radial spoke proteins in the flagellar movement is exemplified by the potent inhibitory action of two monoclonal antibodies on the axonemal motility of demembranated-reactivated Chlamydomonas models. We show that one of these proteins is localized on the stalk of the radial spokes, whereas the other is a component of the head of the same structure and most likely correspond to radial spoke protein 2 and 1, respectively. Fine motility analysis by videomicrography further indicates that these two anti-radial spoke protein antibodies at low concentration affect motility of demembranated-reactivated Chlamydomonas by changing the flagellar waveform without modifying axonemal beat frequency. They also modify wave amplitude differently during motility inhibition. This brings more direct evidence for the involvement of both radial spoke stalk and head in the fine tuning of the waveform during flagellar motility.     

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly

LC8 is present in various molecular complexes. However, its role in these complexes remains unclear. We discovered that although LC8 is a subunit of the radial spoke (RS) complex in Chlamydomonas flagella, it was undetectable in the RS precursor that is converted into the mature RS at the tip of elongating axonemes. Interestingly, LC8 dimers bound in tandem to the N-terminal region of a spoke phosphoprotein, RS protein 3 (RSP3), that docks RSs to axonemes. LC8 enhanced the binding of RSP3 N-terminal fragments to purified axonemes. Likewise, the N-terminal fragments extracted from axonemes contained LC8 and putative spoke-docking proteins. Lastly, perturbations of RSP3's LC8-binding sites resulted in asynchronous flagella with hypophosphorylated RSP3 and defective associations between LC8, RSs, and axonemes. We propose that at the tip of flagella, an array of LC8 dimers binds to RSP3 in RS precursors, triggering phosphorylation, stalk base formation, and axoneme targeting. These multiple effects shed new light on fundamental questions about LC8-containing complexes and axoneme assembly.     

Flagellar motility contributes to cytokinesis in Trypanosoma brucei and is modulated by an evolutionarily conserved dynein regulatory system

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.     

A deletion variant study of the functional role of the Salmonella flagellin hypervariable domain region in motility

The eubacterial flagellum is a complex structure with an elongated extracellular filament that is composed primarily of many subunits of a flagellin protein. The highly conserved N and C termini of flagellin are important in its export and self-assembly, whereas the middle sequence region varies greatly in size and composition in different species and is known to be deletion-tolerant. In Salmonella typhimurium phase 1 flagellin, this "hypervariable" region encodes two solvent-exposed domains, D2 and D3, that form a knob-like feature on flagella fibers. The functional role of this structural feature in motility remains unclear. We investigated the structural and physiological role of the hypervariable region in flagella assembly, stability and cellular motility. A library of random internal deletion variants of S. typhimurium flagellin was constructed and screened for functional variants using a swarming agar motility assay. The relative cellular motility and propulsive force of ten representative variants were determined in semi-solid and liquid medium using colony swarming motility assays, video microscopy and optical trapping of single cells. All ten variants exhibited diminished motility, with varying extents of motility observed for internal deletions less than 75 residues and nearly complete loss of motility for deletions greater than 100 residues. The mechanical stability of the variant flagella fibers also decreased with increasing size of deletion. Comparison of the variant sequences with the wild-type sequence and structure indicated that all deletions involved loss of hydrophobic core residues, and removal of both partial and complete segments of secondary structure in the D2 and D3 domains. Homology modeling predicted disruptions of secondary structures in each variant. The hypervariable region D2 and D3 domains appear to stabilize the folded conformation of the flagellin protein and contribute to the mechanical stability and propulsive force of the flagella fibers.     

Protein tagging reveals new insights into signaling in flagella

In this issue, Oda et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201312014) use mutant analysis, protein tagging, and cryoelectron tomography to determine the detailed location of components in flagellar radial spokes—a complex of proteins that connect the peripheral microtubule doublets to the central pair. Remarkably, this approach revealed an interaction between radial spokes and the central pair based on geometry rather than a specific signaling mechanism, highlighting the importance of studying a system in three dimensions.Eukaryotic flagella and motile cilia are protrusive organelles that enable cellular motility or generate flow of extracellular fluid. This organelle, composed of more than 600 proteins (Pazour et al., 2005), retains functions such as bending and switching of waveforms by calcium ions even after isolated from the cell body, indicating that flagella and cilia can function as a closed system. Understanding this highly compartmentalized system is inherently interesting because of the striking organization and structure of this organelle and also because of its medical relevance; defects in this organelle are responsible for a group of diseases termed ciliopathies (Fliegauf et al., 2007). In this issue, Oda et al. present a 3D arrangement of a subset of flagellar/ciliary proteins and provide insight into mechanical signaling in this organelle.Most flagella and motile cilia share a common 9 + 2 axoneme structure (Fig. 1 A) with nine microtubule doublets surrounded by two singlet microtubules (called the central pair apparatus; Fig. 1 A, light green). Adjacent doublets are connected by isoforms of the microtubule-based motor protein dynein. Peripheral microtubule doublets and the central pair are linked by complexes of 23 proteins (Yang et al., 2006) called radial spokes. These structures form a beautifully ordered array along the microtubule doublets with 96-nm periodicity. Each radial spoke has a T-shaped structure with pseudo twofold symmetry (Pigino et al., 2011; Barber et al., 2012). The radial spokes and central pair are essential for the waveform behavior of cilia/flagella by regulating dynein force generation.Open in a separate windowFigure 1.3D structure of the axoneme and radial spokes. (A) Axoneme from C. reinhardtii flagella seen from the proximal end (the minus end of the microtubule). RS, radial spoke (in a blue dotted line); ODA, outer dynein arm; IDA, inner dynein arm; MTD, microtubule doublet. The central pair apparatus is shown in light green. The inset highlights one microtubule doublet, radial spoke, and central pair apparatus seen from the side (left, the proximal end). The head, neck, and stalk subdomains of the radial spoke are indicated. Modified from Bui and Ishikawa (2013). (B) 3D structure of the 96-nm periodic unit from C. reinhardtii flagella (EMDataBank accession no. EMD-2117). Proximal end is left. The color code is based on previous studies (Heuser et al., 2009; Pigino et al., 2011; Bui et al., 2012). Dark blue, radial spoke; light blue, outer dynein arm; red, inner dynein arm; green, dynein regulatory complex; yellow, intermediate and light chains of dynein f; orange, microtubule; gray, unidentified. Radial spoke proteins located in Oda et al. (2014) and dynein species (α, β, and γ are part of the outer dynein arm, and a–f form the inner dynein arm) based on Bui et al. (2012) are indicated.Undoubtedly, the bona fide study of individual proteins is essential for the understanding of the flagellar/ciliary bending mechanism. Indeed, high resolution structural biology of dynein (Burgess et al., 2003; Kon et al., 2012; Roberts et al., 2012; Schmidt et al., 2012) has provided insight into the mechanism of force generation inside flagella/cilia. However, we also need to study how individual parts are integrated into the entire system. We must clarify how the individual components interact with each other to organize the highly orchestrated bending motion. This systems biology requires three-dimensional mapping of proteins in vivo.Until now, location of component proteins in flagella/cilia has been mainly determined by comparing the structure of various deletion mutants of green algae Chlamydomonas reinhardtii using techniques of cryoelectron tomography and subtomogram averaging. With this approach, the location of all of the major dynein species was determined (Bui et al., 2008, 2012). The molecular arrangement of the radial spoke is less defined. Five proteins out of 23 are localized at the top part (called the head subdomain; Fig. 1 A, inset) of this T-shaped complex, close to the central pair apparatus. The rest of the proteins belong to the neck and stalk subdomains. Genetic depletion of one radial spoke head protein (RSP4) causes loss of the entire head, probably because it is essential for the assembly of the radial spoke head. Although a previous study using mutants (Pigino et al., 2011) identified the radial spoke proteins as a group, the exact position of the individual proteins has not been clarified. Oda et al. (2014) enabled a more detailed assignment by generating mutant strains. When RSP4 is expressed in the C. reinhardtii pf1 strain, which lacks the RSP4 gene and therefore the entire radial spoke head, the entire head domain is recovered. The authors expressed RSP4 tagged with biotin carboxyl carrier protein (BCCP). The density of the BCCP tag appears in cryoelectron tomography, indicating the position of RSP4 (Fig. 1 B). This approach enabled not only the location of the proteins to be elucidated but also provided information about protein orientations, as far as the resolution allows. The authors succeeded in identifying the location of the N and C termini of RSP4. They also applied this technology to additional radial spoke proteins and revealed how two copies of this complex of proteins are arranged in one head domain, which might support a model of preassembly proposed by Diener et al. (2011).The authors went far beyond simple structural analysis. They tagged, instead of with BCCP, with proteins of various sizes. When relatively small peptides (BSA or His tag) were used as a tag, flagellar motility appeared to be relatively normal. However, attaching a large protein (streptavidin) to the C termini of RSPs 3, 4, and 6—all of which are exposed to the interface of the central pair apparatus according to their structural analysis—caused paralysis. In contrast, expression of a large protein tag coupled to a radial spoke protein rescued the motility of paralyzed mutants that lack proteins projecting from the central pair. This remarkable finding suggests an interaction between the central pair apparatus and the radial spoke that depends on geometry, which probably modulates mechanical interaction by collision, rather than inducing a particular signal transduction mechanism. Mechanical interaction has been proposed previously as a hypothesis, based on the geometrical relationship between the radial spoke and the central pair (Warner and Satir, 1974; Lindemann, 2003), the absence of signal transduction motifs in the radial spoke head domain (Pigino and Ishikawa, 2012), and the fact that the central pair twists in the axoneme (Mitchell and Nakatsugawa, 2004), which indicates that the interface with the radial spokes is not regular along the central pair apparatus. Oda et al. (2014) provide the first experimental proof of this model. This signaling mechanism and their methodology could be applicable to other scenarios in the cell.This challenge of in vivo protein mapping of flagella/cilia will need further development and probably another breakthrough. Besides dynein and the radial spoke, only limited numbers of component proteins have been located and/or functionally analyzed even partially—dynein regulatory complex (Heuser et al., 2009), intermediate and light chains of inner arm dynein f (Heuser et al., 2012; Yamamoto et al., 2013), and an intermediate chain of the outer arm dynein (Oda and Kikkawa, 2013). More than 500 proteins of the 600 listed as flagella/cilia components are still unallocated, whereas many features in the tomogram are left unidentified. Systematic approaches to tag or label proteins in this organelle await development. Unfortunately, the design of mutants using homologous recombination in C. reinhardtii has not yet been reported. Knockdown by RNAi might be a promising approach to locate target proteins and analyze their function (Dymek et al., 2011; Rompolas et al., 2013). If knockdown and expression of mutant proteins are combined in the future, it will enable systematic mutant generation. Flagella/cilia research, which takes advantage of periodicity and symmetry/pseudosymmetry of the axoneme, will lead to in vivo structural and functional analysis of other cellular organelles, which will pave the way to three-dimensional systems biology.     

Cofactor fingerprinting with STD NMR to characterize proteins of unknown function: identification of a rare cCMP cofactor preference

Proteomics efforts have created a need for better strategies to functionally categorize newly discovered proteins. To this end, we have employed saturation transfer difference NMR with pools of closely related cofactors, to determine cofactor preferences. This approach works well for dehydrogenases and has also been applied to cyclic nucleotide-binding proteins. In the latter application, a protein (radial spoke protein-2, RSP2) that plays a central role in forming the radial spoke of Chlamydomonas reinhardtii flagella was shown to bind cCMP. cCMP-binding proteins are rare, although previous reports of their presence in sperm and flagella suggest that cCMP may have a more general role in flagellar function. 31P NMR was used to monitor the preferential hydrolysis of ATP versus GTP, suggesting that RSP2 is a kinase.     

Identification of a novel leucine-rich repeat protein as a component of flagellar radial spoke in the Ascidian Ciona intestinalis

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 ascidian Ciona 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 in Chlamydomonas 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.     

Disruption of the A-kinase anchoring domain in flagellar radial spoke protein 3 results in unregulated axonemal cAMP-dependent protein kinase activity and abnormal flagellar motility

Biochemical studies of Chlamydomonas flagellar axonemes revealed that radial spoke protein (RSP) 3 is an A-kinase anchoring protein (AKAP). To determine the physiological role of PKA anchoring in the axoneme, an RSP3 mutant, pf14, was transformed with an RSP3 gene containing a mutation in the PKA-binding domain. Analysis of several independent transformants revealed that the transformed cells exhibit an unusual phenotype: a fraction of the cells swim normally; the remainder of the cells twitch feebly or are paralyzed. The abnormal/paralyzed motility is not due to an obvious deficiency of radial spoke assembly, and the phenotype cosegregates with the mutant RSP3. We postulated that paralysis was due to failure in targeting and regulation of axonemal cAMP-dependent protein kinase (PKA). To test this, reactivation experiments of demembranated cells were performed in the absence or presence of PKA inhibitors. Importantly, motility in reactivated cell models mimicked the live cell phenotype with nearly equal fractions of motile and paralyzed cells. PKA inhibitors resulted in a twofold increase in the number of motile cells, rescuing paralysis. These results confirm that flagellar RSP3 is an AKAP and reveal that a mutation in the PKA binding domain results in unregulated axonemal PKA activity and inhibition of normal motility.     

Glutamylated and glycylated tubulin isoforms in the aberrant sperm axoneme of the gall-midge fly, Asphondylia ruebsaameni

The axonemal organization expressed in the sperm flagella of the cecidomyiid dipteran Asphondylia ruebsaameni is unconventional, being characterized by the presence of an exceedingly high number of microtubular doublets and by the absence of both the inner dynein arms and the central pair/radial spoke complex. Consequently, its motility, both in vivo and in vitro, is also peculiar. Using monoclonal antibodies directed against posttranslational modifications, we have analyzed the presence and distribution of glutamylated and glycylated tubulin isoforms in this aberrant axonemal structure, and compared them with those of a reference insect species (Apis mellifera), endowed with a conventional axoneme. Our results have shown that the unorthodox structure and motility of the Asphondylia axoneme are concomitant with: (1). a very low glutamylation extent in the alpha-tubulin subunit, (2). a high level of glutamylation in the beta-subunit, (3). an extremely low total extent of glycylation, with regard to both monoglycylated and polyglycylated sites, either in alpha- or in beta-tubulin, (4). the presence of a strong labeling of glutamylated tubulin isoforms at the proximal end of the axoneme, and (5). a uniform distribution of glutamylated as well as glycylated isoforms along the rest of the axoneme. Thus, our data indicate that tubulin molecular heterogeneity is much lower in the Asphondylia axoneme than in the conventional 9+2 axoneme with regard to both isoform content and isoform distribution along the axoneme.