Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene

We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant.     

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

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

Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms

To clarify the functional differentiation between the outer and inner dynein arms in eukaryotic flagella, their mechanochemical properties were assessed by measuring the sliding velocities of outer-doublet microtubules in disintegrating axonemes of Chlamydomonas, using wild-type and mutant strains that lack either of the arms. A special procedure was developed to induce sliding disintegration in Chlamydomonas axonemes which is difficult to achieve by ordinary methods. The flagella were first fragmented by sonication, demembranated by Nonidet P-40, and then perfused under a microscope with Mg-ATP and nagarse, a bacterial protease with broad substrate specificity. The sliding velocity varied with the Mg-ATP concentration in a Michaelis-Menten manner in the axonemes from the wild type and a motile mutant lacking the outer dynein arm (oda38). The maximal sliding velocity and apparent Michaelis constant for Mg-ATP were measured to be 13.2 +/- 1.0 micron/s and 158 +/- 36 microM for the wild type and 2.0 +/- 0.1 micron/s and 64 +/- 18 microM for oda38. These maximal sliding velocities were significantly smaller than those estimated in beating axonemes; the reason is not clear. The velocities in the presence or absence of 10(-5) M Ca2+ did not differ noticeably. The axonemes of nonmotile mutants lacking either outer arms (pf13A, pf22) or inner arms (pf23) were examined for their ability to undergo sliding disintegration in the presence of 0.1 mM Mg-ATP. Whereas pf13A axonemes underwent normal sliding disintegration, the other two species displayed it only very poorly. The poor ability of pf23 axonemes to undergo sliding disintegration raises the possibility that the outer dynein arm cannot function well in the absence of the inner arm.     

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.     

The LC7 light chains of Chlamydomonas flagellar dyneins interact with components required for both motor assembly and regulation

Members of the LC7/Roadblock family of light chains (LCs) have been found in both cytoplasmic and axonemal dyneins. LC7a was originally identified within Chlamydomonas outer arm dynein and associates with this motor's cargo-binding region. We describe here a novel member of this protein family, termed LC7b that is also present in the Chlamydomonas flagellum. Levels of LC7b are reduced approximately 20% in axonemes isolated from strains lacking inner arm I1 and are approximately 80% lower in the absence of the outer arms. When both dyneins are missing, LC7b levels are diminished to <10%. In oda9 axonemal extracts that completely lack outer arms, LC7b copurifies with inner arm I1, whereas in ida1 extracts that are devoid of I1 inner arms it associates with outer arm dynein. We also have observed that some LC7a is present in both isolated axonemes and purified 18S dynein from oda1, suggesting that it is also a component of both the outer arm and inner arm I1. Intriguingly, in axonemal extracts from the LC7a null mutant, oda15, which assembles approximately 30% of its outer arms, LC7b fails to copurify with either dynein, suggesting that it interacts with LC7a. Furthermore, both the outer arm gamma heavy chain and DC2 from the outer arm docking complex completely dissociate after salt extraction from oda15 axonemes. EDC cross-linking of purified dynein revealed that LC7b interacts with LC3, an outer dynein arm thioredoxin; DC2, an outer arm docking complex component; and also with the phosphoprotein IC138 from inner arm I1. These data suggest that LC7a stabilizes both the outer arms and inner arm I1 and that both LC7a and LC7b are involved in multiple intradynein interactions within both dyneins.     

Partially functional outer-arm dynein in a novel Chlamydomonas mutant expressing a truncated gamma heavy chain

The outer dynein arm of Chlamydomonas flagella contains three heavy chains (alpha, beta, and gamma), each of which exhibits motor activity. How they assemble and cooperate is of considerable interest. Here we report the isolation of a novel mutant, oda2-t, whose gamma heavy chain is truncated at about 30% of the sequence. While the previously isolated gamma chain mutant oda2 lacks the entire outer arm, oda2-t retains outer arms that contain alpha and beta heavy chains, suggesting that the N-terminal sequence (corresponding to the tail region) is necessary and sufficient for stable outer-arm assembly. Thin-section electron microscopy and image analysis localize the gamma heavy chain to a basal region of the outer-arm image in the axonemal cross section. The motility of oda2-t is lower than that of the wild type and oda11 (lacking the alpha heavy chain) but higher than that of oda2 and oda4-s7 (lacking the motor domain of the beta heavy chain). Thus, the outer-arm dynein lacking the gamma heavy-chain motor domain is partially functional. The availability of mutants lacking individual heavy chains should greatly facilitate studies on the structure and function of the outer-arm dynein.     

Functional reconstitution of Chlamydomonas outer dynein arms from alpha- beta and gamma subunits: requirement of a third factor

The outer dynein arm of Chlamydomonas flagella, when isolated under Mg(2+)-free conditions, tends to dissociate into an 11 to 12S particle (12S dynein) containing the gamma heavy chain and a 21S particle (called 18S dynein) containing the alpha and beta heavy chains. We show here that functional outer arms can be reconstituted by the addition of 12S and 18S dyneins to the axonemes of the outer armless mutants oda1- oda6. A third factor that sediments at integral 7S is required for efficient reconstitution of the outer arms on the axonemes of oda1 and oda3. However, this factor is not necessary for reconstitution on the axonemes of oda2, oda4, oda5, and oda6. SDS-PAGE analysis indicates that the axonemes of the former two mutants lack a integral of 70-kD polypeptide that is present in those of the other mutants as well as in the 7S fraction from the wild-type extract. Furthermore, electron micrographs of axonemal cross sections revealed that the latter four mutants, but not oda1 or oda3, have small pointed structures on the outer doublets, at a position in cross section where outer arms normally occur. We suggest that the 7S factor constitutes the pointed structure on the outer doublets and facilitates attachment of the outer arm. The discovery of this structure raises a new question as to how the attachment site for the outer arm dynein is determined within the axoneme.     

Differential light chain assembly influences outer arm dynein motor function

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

Transport and arrangement of the outer-dynein-arm docking complex in the flagella of Chlamydomonas mutants that lack outer dynein arms

The outer dynein arms of Chlamydomonas flagella are attached to a precise site on the outer doublet microtubules and repeat at a regular interval of 24 nm. This binding is mediated by the outer dynein arm docking complex (ODA-DC), which is composed of three protein subunits. In this study, antibodies against the 83- and 62-kD subunits (DC83 and DC62) of the ODA-DC were used to analyze its state of association with outer arm components within the cytoplasm, and its localization in the axonemes of oda mutants. Immunoprecipitation indicates that DC83 and DC62 are preassembled within the cytoplasm, but that they are not associated with outer arm dynein. Both proteins are lost or greatly diminished in oda1 and oda3, mutants in the structural genes of DC62 and DC83, respectively, demonstrating that their association is necessary for their stable presence in the cytoplasm. Immunoelectron microscopy indicates that DC83 repeats at 24-nm intervals along the length of the doublet microtubules of oda6, which lacks outer arms; thus, outer arm periodicity may be determined by the ODA-DC. Flagellar regeneration and temporary dikaryon experiments indicate that the ODA-DC can be rapidly transported into the flagellum and assembled on the doublet microtubules independently of the outer arms and independently of flagellar growth. Unexpectedly, the intensity of ODA-DC labeling decreased toward the distal ends of axonemes of oda6 but not wild-type cells, suggesting that the outer arms reciprocally contribute to the assembly/stability of the ODA-DC.     

The inner dynein arms I2 interact with a "dynein regulatory complex" in Chlamydomonas flagella.

We provide indirect evidence that six axonemal proteins here referred to as "dynein regulatory complex" (drc) are located in close proximity with the inner dynein arms I2 and I3. Subsets of drc subunits are missing from five second-site suppressors, pf2, pf3, suppf3, suppf4, and suppf5, that restore flagellar motility but not radial spoke structure of radial spoke mutants. The absence of drc components is correlated with a deficiency of all four heavy chains of inner arms I2 and I3 from axonemes of suppressors pf2, pf3, suppf3, and suppf5. Similarly, inner arm subunits actin, p28, and caltractin/centrin, or subsets of them, are deficient in pf2, pf3, and suppf5. Recombinant strains carrying one of the mutations pf2, pf3, or suppf5 and the inner arm mutation ida4 are more defective for I2 inner arm heavy chains than the parent strains. This evidence indicates that at least one subunit of the drc affects the assembly of and interacts with the inner arms I2.     

Inner dynein arms but not outer dynein arms require the activity of kinesin homologue protein KHP1(FLA10) to reach the distal part of flagella in Chlamydomonas

Inner dynein arms, but not outer dynein arms, require the activity of KHP1(FLA10) to reach the distal part of axonemes before binding to outer doublet microtubules. We have analyzed the rescue of inner or outer dynein arms in quadriflagellate dikaryons by immunofluorescence microscopy of p28(IDA4), an inner dynein arm light chain, or IC69(ODA6), an outer dynein arm intermediate chain. In dikaryons two strains with different genetic backgrounds share the cytoplasm. As a consequence, wild-type axonemal precursors are transported to and assembled in mutant axonemes to complement the defects. The rescue of inner dynein arms containing p28 in ida4-wild-type dikaryons progressively occurred from the distal part of the axonemes and with time was extended towards the proximal part. In contrast, the rescue of outer dynein arms in oda2-wild-type dikaryons progressively occurred along the entire length of the axoneme. Rescue of inner dynein arms containing p28 in ida4fla10-fla10 dikaryons was similar to the rescue observed in ida4-wild-type dikaryons at 21 degrees C, whereas it was inhibited at 32 degrees C, a nonpermissive temperature for KHP1(FLA10). In contrast, rescue of outer dynein arms in oda2fla10-fla10 dikaryons was similar to the rescue observed in oda2-wild-type dikaryons at both 21 degrees and 32 degrees C and was not inhibited at 32 degrees C. Positioning of substructures in the internal part of the axonemal shaft requires the activity of kinesin homologue protein 1.     

The outer dynein arm-docking complex: composition and characterization of a subunit (oda1) necessary for outer arm assembly

To learn more about how dyneins are targeted to specific sites in the flagellum, we have investigated a factor necessary for binding of outer arm dynein to the axonemal microtubules of Chlamydomonas. This factor, termed the outer dynein arm-docking complex (ODA-DC), previously was shown to be missing from axonemes of the outer dynein armless mutants oda1 and oda3. We have now partially purified the ODA-DC, determined that it contains equimolar amounts of M(r) approximately 105,000 and approximately 70,000 proteins plus a third protein of M(r) approximately 25,000, and found that it is associated with the isolated outer arm in a 1:1 molar ratio. We have cloned a full-length cDNA encoding the M(r) approximately 70,000 protein; the sequence predicts a 62.5-kDa protein with potential homologs in higher ciliated organisms, including humans. Sequencing of corresponding cDNA from strain oda1 revealed it has a mutation resulting in a stop codon just downstream of the initiator ATG; thus, it is unable to make the full-length M(r) approximately 70,000 protein. These results demonstrate that the ODA1 gene encodes the M(r) approximately 70,000 protein, and that the protein is essential for assembly of the ODA-DC and the outer dynein arm onto the doublet microtubule.     

Arrangement of inner dynein arms in wild-type and mutant flagella of Chlamydomonas

We have used computer averaging of electron micrographs from longitudinal and cross-sections of wild-type and mutant axonemes to determine the arrangement of the inner dynein arms in Chlamydomonas reinhardtii. Based on biochemical and morphological data, the inner arms have previously been described as consisting of three distinct subspecies, I1, I2, and I3. Our longitudinal averages revealed 10 distinguishable lobes of density per 96-nm repeating unit in the inner row of dynein arms. These lobes occurred predominantly but not exclusively in two parallel rows. We have analyzed mutant strains that are missing I1 and I2 subspecies. Cross-sectional averages of pf9 axonemes, which are missing the I1 subspecies, showed a loss of density in both the inner and outer portions of the inner arm. Averages from longitudinal images showed that three distinct lobes were missing from a single region; two of the lobes were near the outer arms but one was more inward. Serial 24-nm cross-sections of pf9 axonemes showed a complete gap at the proximal end of the repeating unit, confirming that the I1 subunit spans both inner and outer portions of the inner arm region. Examination of pf23 axonemes, which are missing both I1 and I2 subspecies, showed an additional loss almost exclusively in the inner portion of the inner arm. In longitudinal view, this additional loss occurred in three separate locations and consisted of three inwardly placed lobes, one adjacent to each of the two radial spokes and the third at the distal end of the repeating unit. These same lobes were absent ida4 axonemes, which lack only the I2 subspecies. The I2 subspecies thus does not consist of a single dynein arm subunit in the middle of the repeating unit. The radial spoke suppressor mutation, pf2, is missing four polypeptides of previously unknown location. Averages of these axonemes were missing a portion of the structures remaining in pf23 axonemes. This result suggests that polypeptides of the radial spoke control system are close to the inner dynein arms.     

A Chlamydomonas outer arm dynein mutant with a truncated beta heavy chain

A new allele of the Chlamydomonas oda4 flagellar mutant (oda4-s7) possessing abnormal outer dynein arms was isolated. Unlike the previously described oda4 axoneme lacking all three (alpha, beta, and gamma) outer-arm dynein heavy chains, the oda4-s7 axoneme contains the alpha and gamma heavy chains and a novel peptide with a molecular mass of approximately 160 kD. The peptide reacts with a mAb (18 beta B) that recognizes an epitope on the NH2-terminal part of the beta heavy chain. These observations indicate that this mutant has a truncated beta heavy chain, and that the NH2-terminal part of the beta heavy chain is important for the stable assembly of the outer arms. In averaged electron microscopic images of outer arms from cross sections of axonemes, the mutant outer arm lacks its mid-portion, producing a forked appearance. Together with our previous finding that the mutant oda11 lacks the alpha heavy chain and the outermost portion of the arm (Sakakibara, H., D. R. Mitchell, and R. Kamiya. 1991. J. Cell Biol. 113:615-622), this result defines the approximate locations of the three outer arm heavy chains in the axonemal cross section. The swimming velocity of oda4-s7 is 65 +/- 8 microns/s, close to that of oda4 which lacks the entire outer arm (62 +/- 8 microns/s) but significantly lower than the velocities of wild type (194 +/- 23 microns/s) and oda11 (119 +/- 17 microns/s). Thus, the lack of the beta heavy chain impairs outer-arm function more seriously than does the lack of the alpha heavy chain, suggesting that the alpha and beta chains play different roles in outer arm function.     

Bending patterns of Chlamydomonas flagella: IV. Mutants with defects in inner and outer dynein arms indicate differences in dynein arm function

Mutants with outer dynein arm defects or deficiencies all show a major reduction in beat frequency to about half the normal value; some of these mutants show an additional decrease in sliding velocity associated with reduced shear amplitude and an additional reduction in beat frequency, as well as other more minor modifications of the normal forward mode bending pattern. New mutants (ida98, pf30), which appear to be deficient in a subset of inner dynein arms show a reduction in sliding velocity that is primarily associated with a reduction in shear amplitude, with only a small reduction in beat frequency. These differences in motility phenotype between inner and outer dynein arm mutants suggest that inner and outer dynein arms may have distinct functions. The relatively large decrease in sliding velocity associated with partial loss of inner arms is consistent with earlier observations on pf23, a nonmotile mutant lacking inner arms, suggesting that inner arms may have an essential function in motility. The ability to generate reverse mode bending patterns is retained in some inner or outer dynein arm mutants, but appears to be decreased in those mutants which show reduced shear amplitude for the forward mode bending pattern.     

Microtubule sliding in flagellar axonemes of Chlamydomonas mutants missing inner- or outer-arm dynein: velocity measurements on new types of mutants by an improved method.

To help understand the functional properties of inner and outer dynein arms in axonemal motility, sliding velocities of outer doublets were measured in disintegrating axonemes of Chlamydomonas mutants lacking either of the arms. Measurements under improved solution conditions yielded significantly higher sliding velocities than those observed in a previous study [Okagaki and Kamiya, 1986, J. Cell Biol. 103:1895-1902]. As in the previous study, it was found that the velocities in axonemes of wild type (wt) and a mutant (oda1) missing the outer arm differ greatly: 18.5 +/- 4.1 microns/sec for wt and 4.4 +/- 2.3 microns/sec for oda1 at 0.5 mM Mg-ATP. In contrast, axonemes of two types of mutants (ida2 and ida4) that lacked different sets of two inner-arm heavy chains displayed velocities almost identical with the wild-type velocity. Moreover, axonemes of a non-motile double mutant ida2 X ida4 underwent sliding disintegration at a similar high velocity, although less frequently than in axonemes of single mutants. These observations support the hypothesis that the inner and outer dynein arms in disintegrating axonemes drive microtubules at different speeds and it is the faster outer arm that determines the overall speed when both arms are present. The inner arm may be important for the initiation of sliding. The axoneme thus appears to be equipped with two (or more) types of motors with different intrinsic speeds.     

The bop2-1 mutation reveals radial asymmetry in the inner dynein arm region of Chlamydomonas reinhardtii

Strains of Chlamydomonas reinhardtii with a mutant allele at the BOP2 locus swim slowly and have an abnormal flagellar waveform similar to previously identified strains with defects in the inner arm region. Double mutant strains with the bop2-1 allele and any of 17 different mutations that affect the dynein arm region swim more slowly than either parent, which suggests that the bop2-1 mutation does not affect solely the outer dynein arms, the I1 or ida4 inner dynein arms, or the dynein regulatory complex. Flagellar axonemes isolated from bop2-1 cells are missing a phosphorylated polypeptide of 152 kD. Electron microscopic analysis shows that bop2-1 axonemes are missing density in the inner dynein arm region. Surprisingly, two populations of images were observed in longitudinal sections of axonemes from the bop2-1 strain. In the 10 longitudinal axonemes examined, a portion of the dynein regulatory complex and a newly identified structure, the projection, are affected. In five of these 10 longitudinal axonemes examined, two lobes of the ida4 inner arm are also missing. By examining the cross-sectional images of wild-type and bop2-1 axonemes at each outer doublet position around the axoneme, we have determined that the bop2-1 mutation affects the assembly of inner arm region components in a doublet specific manner. Doublets 5, 6, and 8 have the most severe deficiency, doublet 9 has an intermediate phenotype, and doublets 2, 3, 4, and 7 have the least severe phenotype. The bop2-1 mutation provides the first evidence of radial asymmetry in the inner dynein arm region.     

Regulation of Chlamydomonas flagellar dynein by an axonemal protein kinase

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.     

Functional binding of inner-arm dyneins with demembranated flagella of Chlamydomonas mutants

Experiments were carried out to see if isolated inner arm dyneins could functionally combine with axonemes lacking them. High-salt extract from the axoneme of Chlamydomonas oda1 mutant lacking outer-arm dynein was added to the demembranated cell models of ida1oda1 lacking inner arm dynein f (dynein I1) and outer arm dynein. After incubation, the originally paralyzed ida1oda1 axonemes recovered the ability to beat in the presence of ATP. A similar good motility recovery after incubation with crude oda1 extract was observed in ida9oda2 lacking outer arm and inner arm dynein c, and partial recovery in ida4oda1 lacking outer arm and inner arm species a, c, and d. These observations indicate that dynein f and dynein c can functionally bind with mutant axonemes lacking them. A method for combining isolated inner arm dyneins with axonemes in a functionally active manner should provide a powerful experimental tool with which to study the mechanism of beating.