Chlamydomonas WDR92 in association with R2TP-like complex and multiple DNAAFs to regulate ciliary dynein preassembly

Abstract The motility of cilia or eukaryotic flagella is powered by the axonemal dyneins, which are preassembled in the cytoplasm by proteins termed dynein arm assembly factors (DNAAFs) before being transported to and assembled on the ciliary axoneme. Here, we characterize the function of WDR92 in Chlamydomonas. Loss of WDR92, a cytoplasmic protein, in a mutant wdr92 generated by DNA insertional mutagenesis resulted in aflagellate cells or cells with stumpy or short flagella, disappearance of axonemal dynein arms, and diminishment of dynein arm heavy chains in the cytoplasm, suggesting that WDR92 is a DNAAF. Immunoprecipitation of WDR92 followed by mass spectrometry identified inner dynein arm heavy chains and multiple DNAAFs including RuvBL1, RPAP3, MOT48, ODA7, and DYX1C. The PIH1 domain-containing protein MOT48 formed a R2TP-like complex with RuvBL1/2 and RPAP3, while PF13, another PIH1 domain-containing protein with function in dynein preassembly, did not. Interestingly, the third PIH1 domain-containing protein TWI1 was not related to flagellar motility. WDR92 physically interacted with the R2TP-like complex and the other identified DNNAFs. Our data suggest that WDR92 functions in association with the HSP90 co-chaperone R2TP-like complex as well as linking other DNAAFs in dynein preassembly.

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

The Journal of Cell Biology ◽

10.1083/jcb.112.3.441 ◽

1991 ◽

Vol 112 (3) ◽

pp. 441-447 ◽

Cited By ~ 143

Author(s):

R Kamiya ◽

E Kurimoto ◽

E Muto

Keyword(s):

Chlamydomonas Reinhardtii ◽

Cross Section ◽

Double Mutant ◽

The Other ◽

Flagellar Motility ◽

Wild Type ◽

Single Group ◽

Heavy Chains ◽

Section Electron ◽

Dynein Arms

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.

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

The Journal of Cell Biology ◽

10.1083/jcb.126.3.737 ◽

1994 ◽

Vol 126 (3) ◽

pp. 737-745 ◽

Cited By ~ 97

Author(s):

S Takada ◽

R Kamiya

Keyword(s):

Cross Section ◽

Cross Sections ◽

Attachment Site ◽

The Other ◽

Wild Type ◽

Heavy Chains ◽

Sds Page ◽

Gamma Subunits ◽

Dynein Arms ◽

Alpha Beta

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.

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The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

10.1101/2021.05.11.443597 ◽

2021 ◽

Author(s):

Petra zur Lage ◽

Zhiyan Xi ◽

Jennifer Lennon ◽

Iain Hunter ◽

Wai Kit Chan ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Cell Types ◽

Drosophila Cell ◽

Heavy Chains ◽

Neuron Response ◽

Dynein Motor ◽

Assembly Factor ◽

Axonemal Dynein ◽

Dynein Arms ◽

Ciliary Dyskinesia

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila homologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and spermatocytes, the only two Drosophila cell types bearing motile cilia/flagella. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

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WDR92 is required for axonemal dynein heavy chain stability in cytoplasm

Molecular Biology of the Cell ◽

10.1091/mbc.e19-03-0139 ◽

2019 ◽

Vol 30 (15) ◽

pp. 1834-1845 ◽

Cited By ~ 5

Author(s):

Ramila S. Patel-King ◽

Miho Sakato-Antoku ◽

Maya Yankova ◽

Stephen M. King

Keyword(s):

Heavy Chain ◽

Beat Frequency ◽

Gel Filtration ◽

Dynein Heavy Chain ◽

Heavy Chains ◽

Assembly Factor ◽

Axonemal Dynein ◽

Dynein Arms ◽

Phylogenetic Signature ◽

Dynein Heavy Chains

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

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Phosphoregulation of an Inner Dynein Arm Complex in Chlamydomonas reinhardtii Is Altered in Phototactic Mutant Strains

The Journal of Cell Biology ◽

10.1083/jcb.136.1.177 ◽

1997 ◽

Vol 136 (1) ◽

pp. 177-191 ◽

Cited By ~ 133

Author(s):

Stephen J. King ◽

Susan K. Dutcher

Keyword(s):

Chlamydomonas Reinhardtii ◽

Kinase Inhibitor ◽

Flagellar Motility ◽

Biochemical Phenotype ◽

Phototactic Behavior ◽

Mutant Strains ◽

Axonemal Dynein ◽

Dynein Arms ◽

Intermediate Chain

To gain a further understanding of axonemal dynein regulation, mutant strains of Chlamydomonas reinhardtii that had defects in both phototactic behavior and flagellar motility were identified and characterized. ptm1, ptm2, and ptm3 mutant strains exhibited motility phenotypes that resembled those of known inner dynein arm region mutant strains, but did not have biochemical or genetic phenotypes characteristic of other inner dynein arm mutations. Three other mutant strains had defects in the f class of inner dynein arms. Dynein extracts from the pf9-4 strain were missing the entire f complex. Strains with mutations in pf9/ida1, ida2, or ida3 failed to assemble the f dynein complex and did not exhibit phototactic behavior. Fractionated dynein from mia1-1 and mia2-1 axonemes exhibited a novel f class inner dynein arm biochemical phenotype; the 138-kD f intermediate chain was present in altered phosphorylation forms. In vitro axonemal dynein activity was reduced by the mia1-1 and mia2-1 mutations. The addition of kinase inhibitor restored axonemal dynein activity concomitant with the dephosphorylation of the 138-kD f intermediate chain. Dynein extracts from uni1-1 axonemes, which specifically assemble only one of the two flagella, contained relatively high levels of the altered phosphorylation forms of the 138-kD intermediate chain. We suggest that the f dynein complex may be phosphoregulated asymmetrically between the two flagella to achieve phototactic turning.

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The light chain p28 associates with a subset of inner dynein arm heavy chains in Chlamydomonas axonemes.

Molecular Biology of the Cell ◽

10.1091/mbc.6.6.697 ◽

1995 ◽

Vol 6 (6) ◽

pp. 697-711 ◽

Cited By ~ 106

Author(s):

M LeDizet ◽

G Piperno

Keyword(s):

Heavy Chain ◽

Light Chain ◽

The Other ◽

Actin Binding ◽

Gene Encoding ◽

Dynein Heavy Chain ◽

Heavy Chains ◽

Regulatory Complex ◽

Dynein Arms ◽

Novel Protein

We show here that I2 and I3 inner dynein arm heavy chains of Chlamydomonas axonemes are resolved into two classes: one class associated with the protein p28 and the other associated with the protein caltractin/centrin. We have determined the nucleotide sequence of the gene encoding p28, a light chain that, together with actin and caltractin/centrin, is associated with inner dynein arms I2 and I3 of Chlamydomonas axonemes. p28 is a novel protein with affinity for a subset of the inner dynein arm heavy chains, but with no apparent significant homologies to tubulin- or actin-binding proteins. An antiserum specific for p28 showed that p28 is present along the entire axoneme. The same antiserum coimmunoprecipitated p28, actin, and dynein heavy chains 2' and 2. In contrast, an anti-caltractin/centrin antiserum coimmunoprecipitated caltractin/centrin, actin, and the heavy chains 2, 3, and 3'. It is likely that the dynein heavy chain 2 associated with p28, referred to as 2A, is a different polypeptide from dynein heavy chain 2 bound to caltractin/centrin, referred to as 2B. The complex formed by heavy chain 2B, actin, and caltractin/centrin is preferentially extracted by exposure to Nonidet P-40 and is missing in mutants lacking components 1 and 2 of the dynein regulatory complex.

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

The Journal of Cell Biology ◽

10.1083/jcb.118.6.1455 ◽

1992 ◽

Vol 118 (6) ◽

pp. 1455-1463 ◽

Cited By ~ 155

Author(s):

G Piperno ◽

K Mead ◽

W Shestak

Keyword(s):

Indirect Evidence ◽

Flagellar Motility ◽

Radial Spoke ◽

Recombinant Strains ◽

Heavy Chains ◽

Close Proximity ◽

Regulatory Complex ◽

Dynein Arms

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.

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Genetic Analysis of a Y-Chromosome Region That Induces Triplosterile Phenotypes and Is Essential for Spermatid Individualization in Drosophila melanogaster

Genetics ◽

10.1093/genetics/155.1.179 ◽

2000 ◽

Vol 155 (1) ◽

pp. 179-189

Author(s):

Benjamin Timakov ◽

Ping Zhang

Keyword(s):

Drosophila Melanogaster ◽

Genetic Analysis ◽

Male Sterility ◽

Y Chromosome ◽

Fluorescent Probes ◽

Male Fertility ◽

Chromosome Region ◽

The Other ◽

Axonemal Dynein ◽

Dynein Arms

Abstract The heterochromatic Y chromosome of Drosophila melanogaster contains ~40 Mb of DNA but has only six loci mutable to male sterility. Region h1-h9 on YL, which carries the kl-3 and kl-5 loci, induces male sterility when present in three copies. We show that three separate segments within the region are responsible for the triplosterility and have an additive effect on male fertility. The triplosterile males displayed pleiotropic defects, beginning at early postmeiotic stages. However, the triplosterility was unaffected by kl-3 or kl-5 alleles. These data suggest that region h1-h9 is complex and may contain novel functions in addition to those of the previously identified kl-3 and kl-5 loci. The kl-3 and kl-5 mutations as well as deficiencies within region h1-h9 result in loss of the spermatid axonemal outer dynein arms. Examination using fluorescent probes showed that males deficient for h1-h3 or h4-h9 displayed a postmeiotic lesion with disrupted individualization complexes scattered along the spermatid bundle. In contrast, the kl-3 and kl-5 mutations had no effect on spermatid individualization despite the defect in the axonemes. These results demonstrate that region h1-h9 carries genetically separable functions: one required for spermatid individualization and the other essential for assembling the axonemal dynein arms.

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The Drosophila orthologue of the primary ciliary dyskinesia-associated gene, DNAAF3, is required for axonemal dynein assembly

Biology Open ◽

10.1242/bio.058812 ◽

2021 ◽

Author(s):

Petra zur Lage ◽

Zhiyan Xi ◽

Jennifer Lennon ◽

Iain Hunter ◽

Wai Kit Chan ◽

...

Keyword(s):

Primary Ciliary Dyskinesia ◽

Cell Types ◽

Drosophila Cell ◽

Heavy Chains ◽

Neuron Response ◽

Dynein Motor ◽

Assembly Factor ◽

Axonemal Dynein ◽

Dynein Arms ◽

Ciliary Dyskinesia

Ciliary motility is powered by a suite of highly conserved axoneme-specific dynein motor complexes. In humans the impairment of these motors through mutation results in the disease, Primary Ciliary Dyskinesia (PCD). Studies in Drosophila have helped to validate several PCD genes whose products are required for cytoplasmic pre-assembly of axonemal dynein motors. Here we report the characterisation of the Drosophila orthologue of the less known assembly factor, DNAAF3. This gene, CG17669 (Dnaaf3), is expressed exclusively in developing mechanosensory chordotonal (Ch) neurons and the cells that generate spermatozoa, the only two Drosophila cell types bearing cilia/flagella containing dynein motors. Mutation of Dnaaf3 results in larvae that are deaf and adults that are uncoordinated, indicating defective Ch neuron function. The mutant Ch neuron cilia of the antenna specifically lack dynein arms, while Ca imaging in larvae reveals a complete loss of Ch neuron response to vibration stimulus, confirming that mechanotransduction relies on ciliary dynein motors. Mutant males are infertile with immotile sperm whose flagella lack dynein arms and show axoneme disruption. Analysis of proteomic changes suggest a reduction in heavy chains of all axonemal dynein forms, consistent with an impairment of dynein pre-assembly.

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Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity

The Journal of Cell Biology ◽

10.1083/jcb.201312014 ◽

2014 ◽

Vol 204 (5) ◽

pp. 807-819 ◽

Cited By ~ 71

Author(s):

Toshiyuki Oda ◽

Haruaki Yanagisawa ◽

Toshiki Yagi ◽

Masahide Kikkawa

Keyword(s):

Regulatory Mechanisms ◽

Flagellar Motility ◽

Downstream Effectors ◽

Concerted Activity ◽

Radial Spokes ◽

Axonemal Dynein ◽

Central Apparatus ◽

Dynein Arms ◽

Protein Tags ◽

Intermolecular Collision

Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella.

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