Regulation of flagellar dynein by the axonemal central apparatus

Numerous studies indicate that the central apparatus, radial spokes, and dynein regulatory complex form a signaling pathway that regulates dynein activity in eukaryotic flagella. This regulation involves the action of several kinases and phosphatases anchored to the axoneme. To further investigate the role of the central apparatus in this signaling pathway, we have taken advantage of a microtubule-sliding assay to assess dynein activity in central apparatus defective mutants of Chlamydomonas. Axonemes isolated from both pf18 and pf15 (lacking the entire central apparatus) and from pf16 (lacking the C1 central microtubule) have reduced microtubule-sliding velocity compared with wild-type axonemes. Based on functional analyses of axonemes isolated from radial spokeless mutants, we hypothesized that inhibitors of casein kinase 1 (CK1) and cAMP dependent protein kinase (PKA) would rescue dynein activity and increase microtubule-sliding velocity in central pairless mutants. Treatment of axonemes isolated from both pf18 and pf16 with DRB, a CK1 inhibitor, but not with PKI, a PKA inhibitor, restored dynein activity to wild-type levels. The DRB-induced increase in dynein-driven microtubule sliding was inhibited if axonemes were first incubated with the phosphatase inhibitor, microcystin. Inhibiting CK1 in pf15 axonemes, which lack the central pair as well as PP2A [Yang et al., 2000: J. Cell Sci. 113:91-102], did not increase microtubule-sliding velocity. These data are consistent with a model in which the central apparatus, and specifically the C1 microtubule, regulate dynein through interactions with the radial spokes that ultimately alter the activity of CK1 and PP2A. These data are also consistent with localization of axonemal CK1 and PP2A near the dynein arms.     

Regulation of flagellar dynein by calcium and a role for an axonemal calmodulin and calmodulin-dependent kinase

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.     

Casein kinase I is anchored on axonemal doublet microtubules and regulates flagellar dynein phosphorylation and activity

Flagellar dynein activity is regulated by phosphorylation. One critical phosphoprotein substrate in Chlamydomonas is the 138-kDa intermediate chain (IC138) of the inner arm dyneins (Habermacher, G., and Sale, W. S. (1997) J. Cell Biol. 136, 167-176). In this study, several approaches were used to determine that casein kinase I (CKI) is physically anchored in the flagellar axoneme and regulates IC138 phosphorylation and dynein activity. First, using a videomicroscopic motility assay, selective CKI inhibitors rescued dynein-driven microtubule sliding in axonemes isolated from paralyzed flagellar mutants lacking radial spokes. Rescue of dynein activity failed in axonemes isolated from these mutant cells lacking IC138. Second, CKI was unequivocally identified in salt extracts from isolated axonemes, whereas casein kinase II was excluded from the flagellar compartment. Third, Western blots indicate that within flagella, CKI is anchored exclusively to the axoneme. Analysis of multiple Chlamydomonas motility mutants suggests that the axonemal CKI is located on the outer doublet microtubules. Finally, CKI inhibitors that rescued dynein activity blocked phosphorylation of IC138. We propose that CKI is anchored on the outer doublet microtubules in position to regulate flagellar dynein.     

Regulation of flagellar assembly by glycogen synthase kinase 3 in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii controls flagellar assembly such that flagella are of an equal and predetermined length. Previous studies demonstrated that lithium, an inhibitor of glycogen synthase kinase 3 (GSK3), induced flagellar elongation, suggesting that a lithium-sensitive signal transduction pathway regulated flagellar length (S. Nakamura, H. Takino, and M. K. Kojima, Cell Struct. Funct. 12:369-374, 1987). Here, we demonstrate that lithium treatment depletes the pool of flagellar proteins from the cell body and that the heterotrimeric kinesin Fla10p accumulates in flagella. We identify GSK3 in Chlamydomonas and demonstrate that its kinase activity is inhibited by lithium in vitro. The tyrosine-phosphorylated, active form of GSK3 was enriched in flagella and GSK3 associated with the axoneme in a phosphorylation-dependent manner. The level of active GSK3 correlated with flagellar length; early during flagellar regeneration, active GSK3 increased over basal levels. This increase in active GSK3 was rapidly lost within 30 min of regeneration as the level of active GSK3 decreased relative to the predeflagellation level. Taken together, these results suggest a possible role for GSK3 in regulating the assembly and length of flagella.     

ODA16p, a Chlamydomonas flagellar protein needed for dynein assembly

Dynein motors of cilia and flagella function in the context of the axoneme, a very large network of microtubules and associated proteins. To understand how dyneins assemble and attach to this network, we characterized two Chlamydomonas outer arm dynein assembly (oda) mutants at a new locus, ODA16. Both oda16 mutants display a reduced beat frequency and altered swimming behavior, similar to previously characterized oda mutants, but only a partial loss of axonemal dyneins as shown by both electron microscopy and immunoblots. Motility studies suggest that the remaining outer arm dyneins on oda16 axonemes are functional. The ODA16 locus encodes a 49-kDa WD-repeat domain protein. Homologues were found in mammalian and fly databases, but not in yeast or nematode databases, implying that this protein is only needed in organisms with motile cilia or flagella. The Chlamydomonas ODA16 protein shares 62% identity with its human homologue. Western blot analysis localizes more than 90% of ODA16p to the flagellar matrix. Because wild-type axonemes retain little ODA16p but can be reactivated to a normal beat in vitro, we hypothesize that ODA16p is not an essential dynein subunit, but a protein necessary for dynein transport into the flagellar compartment or assembly onto the axoneme.     

Evidence for axonemal distortion during the flagellar beat of Chlamydomonas

In order to understand the working mechanism that governs the flagellar beat it is essential to know if the axoneme undergoes distortion during the course of the beat cycle. The rapid fixation method employed by Mitchell was able to preserve the waveform of Chlamydomonas flagella much as it appears during normal flagellar beating [Mitchell, Cell Motil Cytoskeleton 2003;56:120-129]. This conservation of the waveform suggests that the stress responsible for the production of bending is also trapped by the fixation procedure. Longitudinal sections of these well-preserved flagella were used to document variations in the relative axonemal diameter. Sections aligned to the plane of bending, showing both the central pair microtubules and outer doublets, were examined for this purpose. Micrographs were selected that continuously showed both the outer doublets and the central pair from a straight region to a curved region of the flagellum. Axoneme diameters measured from these select micrographs showed an increase in relative diameter that averaged 39 nm greater at the crest of the bent region. This constituted a 24% increase in the axoneme diameter in the bends. The transverse stress acting across the axoneme during bending was calculated from the Geometric Clutch computer model for a simulated Chlamydomonas-like flagellar beat. If we assume that this is representative of the transverse stress acting in a real flagellum, then the Young's modulus of the intact axoneme is approximately 0.02 MPa. The possibility that the distortion of the axoneme during the beat could play a significant role in regulating dynein function is discussed.     

Regulation of flagellar length in Chlamydomonas

Chlamydomonas reinhardtii has two apically localized flagella that are maintained at an equal and appropriate length. Assembly and maintenance of flagella requires a microtubule-based transport system known as intraflagellar transport (IFT). During IFT, proteins destined for incorporation into or removal from a flagellum are carried along doublet microtubules via IFT particles. Regulation of IFT activity therefore is pivotal in determining the length of a flagellum. Reviewed is our current understanding of the role of IFT and signal transduction pathways in the regulation of flagellar length.     

An axonemal dynein particularly important for flagellar movement at high viscosity. Implications from a new Chlamydomonas mutant deficient in the dynein heavy chain gene DHC9

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

A motile Chlamydomonas flagellar mutant that lacks outer dynein arms

A new Chlamydomonas flagellar mutant, pf-28, which swims more slowly than wild-type cells, was selected. Thin-section electron microscopy revealed the complete absence of outer-row dynein arms in this mutant, whereas inner-row arms and other axonemal structures appeared normal. SDS PAGE analysis also indicated that polypeptides previously identified as outer-arm dynein components are completely absent in pf-28. The two ATPases retained by this mutant sediment at 17.7S and 12.7S on sucrose gradients that contain 0.6 M KCl. Overall swimming patterns of pf-28 differ little from wild-type except that forward swimming speed is reduced to 35% of the wild-type value, and cells show little or no backward movement during photophobic avoidance. Mutant cells will respond to phototactic stimuli, and their flagella will beat in either the forward or reverse mode. This is the first report of a mutant that lacks dynein arms that can swim.     

Regulation of flagellar motility by temperature-dependent phosphorylation of a 43 kDa axonemal protein in fowl spermatozoa.

Phosphorylation of fowl sperm proteins was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) after incubating the demembranated spermatozoa with [gamma-32P]ATP at 30 degrees C or 40 degrees C. A marked difference of phosphorylation between 30 degrees C and 40 degrees C was observed in a 43 kDa protein. This protein was slightly phosphorylated at 40 degrees C, but strongly phosphorylated at 30 degrees C in a cAMP-independent manner. The motility of demembranated spermatozoa was negligible at 40 degrees C, but vigorous movement was observed at 30 degrees C. These results showed that phosphorylation of a 43 kDa protein is likely to be a regulatory step in the maintenance of fowl sperm motility.     

Slow ADP-dependent acceleration of microtubule translocation produced by an axonemal dynein

Dynein has four nucleotide binding sites, of which the functional significance is unknown except for the single catalytic site. To obtain clues to the function of non-catalytic nucleotide binding, we examined the effect of ADP on the in vitro motility of Chlamydomonas inner-arm dynein species 'a'. Upon continuous perfusion with ATP and ADP, microtubules glided on a dynein-coated glass surface with a velocity that gradually increased over a few minutes. The velocity increased faster at higher ADP concentrations. These results suggest that this dynein is activated by nucleotide binding to regulatory site(s) through an extremely slow process.     

Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein

Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.     

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.     

Oda5p, a novel axonemal protein required for assembly of the outer dynein arm and an associated adenylate kinase

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

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.     

Molecular cloning and expression of flagellar radial spoke and dynein genes of Chlamydomonas

Several flagellar dynein ATPase and radial spokehead genes have been isolated from a Chlamydomonas genomic expression library in lambda gt11. The library was probed with polyclonal and monoclonal antibodies raised against purified flagellar polypeptides, and recombinant phage giving positive signals were cloned. In vitro translation of mRNAs hybrid-selected by the cloned sequences from whole cell RNA provided confirmation of identity for three of the four clones. Evidence supporting the identification of the fourth, which encodes a dynein heavy chain, was provided by antibody selection; the fusion protein produced by this clone selected heavy chain-specific antibodies from a complex polyclonal antiserum recognizing many dynein determinants. One of the radial spoke sequences isolated here is of particular interest because it encodes the wild-type allele of a locus which was defined previously by temperature-sensitive paralyzed flagella mutation pf-26ts (Huang, B., G. Piperno, Z. Ramanis, and D. J. L. Luck, 1981, J. Cell Biol., 88:80-88). The cloned sequence was used to hybrid-select mRNA from mutant pf-26ts cells, and when translated in vitro, the selected mRNA produced a mutant spokehead polypeptide with an altered electrophoretic mobility. This confirms that the pf-26ts mutation alters the primary structure of a radial spokehead polypeptide. To quantify spokehead and dynein mRNAs during flagellar regeneration, all of the cloned sequences were used as hybridization probes in RNA dot experiments. Levels increased rapidly and coordinately after deflagellation, peaked 3-10-fold above nondeflagellated controls, and then returned to control values within 2 h. This accumulation pattern was similar to that of flagellar alpha-tubulin mRNA.     

A novel MAP kinase regulates flagellar length in Chlamydomonas

Little is known about the molecular basis of organelle size control in eukaryotes. Cells of the biflagellate alga Chlamydomonas reinhardtii actively maintain their flagella at a precise length. Chlamydomonas mutants that lose control of flagellar length have been isolated and used to demonstrate that a dynamic process keeps flagella at an appropriate length. To date, none of the proteins required for flagellar length control have been identified in any eukaryotic organism. Here, we show that a novel MAP kinase is crucial to enforcing wild-type flagellar length in C. reinhardtii. Null mutants of LF4 [2], a gene encoding a protein with extensive amino acid sequence identity to a mammalian MAP kinase of unknown function, MOK [3], are unable to regulate the length of their flagella. The LF4 protein (LF4p) is localized to the flagella, and in vitro enzyme assays confirm that the protein is a MAP kinase. The long-flagella phenotype of lf4 cells is rescued by transformation with the cloned LF4 gene. The demonstration that a novel MAP kinase helps enforce flagellar length control indicates that a previously unidentified signal transduction pathway controls organelle size in C. reinhardtii.     

Regulation of flagellar glycoprotein movements by protein phosphorylation

Cross-linking of surface exposed domains on certain Chlamydomonas flagellar membrane glycoproteins induces their movement within the plane of the flagellar membrane. A number of observations suggest that active movements of flagellar membrane glycoproteins are associated with the processes of whole cell gliding motility and the early events of fertilization in Chlamydomonas. Protein redistribution is totally inhibited if the free calcium concentration in the medium is 10(-7) M or below or in the presence of a number of calcium channel blockers (Bloodgood, R. A., N. L. Salomonsky, J. Cell Sci. 96, 27-33 (1990]. The present report demonstrates that glycoprotein redistribution in vivo is inhibited reversibly by three different protein kinase inhibitors: H-7, H-8 and staurosporine. Taken together, these observations suggest that the flagellum uses a signaling pathway that involves calcium influx induced by glycoprotein cross-linking, calcium activation of a protein kinase and specific protein phosphorylation to initiate flagellar surface dynamics.