scholarly journals WDR92 is required for axonemal dynein heavy chain stability in cytoplasm

2019 ◽  
Vol 30 (15) ◽  
pp. 1834-1845 ◽  
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.

scholarly journals Identification of seven rat axonemal dynein heavy chain genes: expression during ciliated cell differentiation.

1996 ◽  
Vol 7 (1) ◽  
pp. 71-79 ◽  
Author(s):  
K L Andrews ◽  
P Nettesheim ◽  
D J Asai ◽  
L E Ostrowski
Keyword(s):  
Cell Differentiation ◽  
Heavy Chain ◽  
Ciliated Cell ◽  
Northern Analysis ◽  
Ciliated Cells ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Cells In Culture ◽  
Axonemal Dynein ◽  
Dynein Heavy Chains

Axonemal dyneins are molecular motors that drive the beating of cilia and flagella. We report here the identification and partial cloning of seven unique axonemal dynein heavy chains from rat tracheal epithelial (RTE) cells. Combinations of axonemal-specific and degenerate primers to conserved regions around the catalytic site of dynein heavy chains were used to obtain cDNA fragments of rat dynein heavy chains. Southern analysis indicates that these are single copy genes, with one possible exception, and Northern analysis of RNA from RTE cells shows a transcript of approximately 15 kb for each gene. Expression of these genes was restricted to tissues containing axonemes (trachea, testis, and brain). A time course analysis during ciliated cell differentiation of RTE cells in culture demonstrated that the expression of axonemal dynein heavy chains correlated with the development of ciliated cells, while cytoplasmic dynein heavy chain expression remained constant. In addition, factors that regulate the development of ciliated cells in culture regulated the expression of axonemal dynein heavy chains in a parallel fashion. These are the first mammalian dynein heavy chain genes shown to be expressed specifically in axonemal tissues. Identification of the mechanisms that regulate the cell-specific expression of these axonemal dynein heavy chains will further our understanding of the process of ciliated cell differentiation.

scholarly journals Molecular analysis of the gamma heavy chain of Chlamydomonas flagellar outer-arm dynein

1994 ◽  
Vol 107 (3) ◽  
pp. 497-506 ◽  
Author(s):  
C.G. Wilkerson ◽  
S.M. King ◽  
G.B. Witman
Keyword(s):  
Heavy Chain ◽  
Coiled Coil ◽  
Cytoplasmic Dynein ◽  
Complete Sequence ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Microtubule Binding Domain ◽  
Helical Content ◽  
Axonemal Dynein ◽  
Dynein Heavy Chains

We report here the complete sequence of the gamma dynein heavy chain of the outer arm of the Chlamydomonas flagellum, and partial sequences for six other dynein heavy chains. The gamma dynein heavy chain sequence contains four P-loop motifs, one of which is the likely hydrolytic site based on its position relative to a previously mapped epitope. Comparison with available cytoplasmic and flagellar dynein heavy chain sequences reveals regions that are highly conserved in all dynein heavy chains sequenced to date, regions that are conserved only among axonemal dynein heavy chains, and regions that are unique to individual dynein heavy chains. The presumed hydrolytic site is absolutely conserved among dyneins, two other P loops are highly conserved among cytoplasmic dynein heavy chains but not in axonemal dynein heavy chains, and the fourth P loop is invariant in axonemal dynein heavy chains but not in cytoplasmic dynein. One region that is very highly conserved in all dynein heavy chains is similar to a portion of the ATP-sensitive microtubule-binding domain of kinesin. Two other regions present in all dynein heavy chains are predicted to have high alpha-helical content and have a high probability of forming coiled-coil structures. Overall, the central one-third of the gamma dynein heavy chain is most conserved whereas the N-terminal one-third is least conserved; the fact that the latter region is divergent between the cytoplasmic dynein heavy chain and two different axonemal dynein heavy chains suggests that it is involved in chain-specific functions.

scholarly journals Asymmetry of inner dynein arms and inter-doublet links in Chlamydomonas flagella

2009 ◽  
Vol 186 (3) ◽  
pp. 437-446 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Heavy Chain ◽  
Single Particle ◽  
Heavy Chains ◽  
Molecular Arrangement ◽  
Dynein Arms ◽  
Electron Cryotomography ◽  
Microtubule Doublet ◽  
Asymmetrical Bending ◽  
Dynein Heavy Chains

Although the widely shared “9 + 2” structure of axonemes is thought to be highly symmetrical, axonemes show asymmetrical bending during planar and conical motion. In this study, using electron cryotomography and single particle averaging, we demonstrate an asymmetrical molecular arrangement of proteins binding to the nine microtubule doublets in Chlamydomonas reinhardtii flagella. The eight inner arm dynein heavy chains regulate and determine flagellar waveform. Among these, one heavy chain (dynein c) is missing on one microtubule doublet (this doublet also lacks the outer dynein arm), and another dynein heavy chain (dynein b or g) is missing on the adjacent doublet. Some dynein heavy chains either show an abnormal conformation or were replaced by other proteins, possibly minor dyneins. In addition to nexin, there are two additional linkages between specific pairs of doublets. Interestingly, all these exceptional arrangements take place on doublets on opposite sides of the axoneme, suggesting that the transverse functional asymmetry of the axoneme causes an in-plane bending motion.

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

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.

scholarly journals The sup-pf-2 mutations of Chlamydomonas alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

1996 ◽  
Vol 135 (6) ◽  
pp. 1853-1865 ◽  
Author(s):  
G Rupp ◽  
E O'Toole ◽  
L C Gardner ◽  
B F Mitchell ◽  
M E Porter
Keyword(s):  
Linkage Analysis ◽  
Heavy Chain ◽  
Biochemical Analysis ◽  
Beat Frequency ◽  
Wild Type ◽  
Dynein Heavy Chain ◽  
Flagellar Beat ◽  
Regulatory Mutations ◽  
Components Analysis ◽  
Dynein Arms

The sup-pf-2 mutation is a member of a group of dynein regulatory mutations that are capable of restoring motility to paralyzed central pair or radial spoke defective strains. Previous work has shown that the flagellar beat frequency is reduced in sup-pf-2, but little else was known about the sup-pf-2 phenotype (Huang, B., Z. Ramanis, and D.J.L. Luck. 1982. Cell. 28:115-125; Brokaw, C.J., and D.J.L. Luck. 1985. Cell Motil. 5:195-208). We have reexamined sup-pf-2 using improved biochemical and structural techniques and by the analysis of additional sup-pf-2 alleles. We have found that the sup-pf-2 mutations are associated with defects in the outer dynein arms. Biochemical analysis of sup-pf-2-1 axonemes indicates that both axonemal ATPase activity and outer arm polypeptides are reduced by 40-50% when compared with wild type. By thin-section EM, these defects correlate with an approximately 45% loss of outer dynein arm structures. Interestingly, this loss is biased toward a subset of outer doublets, resulting in a radial asymmetry that may reflect some aspect of outer arm assembly. The defects in outer arm assembly do not appear to result from defects in either the outer doublet microtubules or the outer arm docking structures, but rather appear to result from defects in outer dynein arm components. Analysis of new sup-pf-2 mutations indicates that the severity of the outer arm assembly defects varies with different alleles. Complementation tests and linkage analysis reveal that the sup-pf-2 mutations are alleles of the PF28/ODA2 locus, which is thought to encode the gamma-dynein heavy chain subunit of the outer arm. The sup-pf-2 mutations therefore appear to alter the activity of the outer dynein arms by modification of the gamma-dynein heavy chain.

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

1995 ◽  
Vol 6 (6) ◽  
pp. 697-711 ◽  
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.

scholarly journals Evidence for Four Cytoplasmic Dynein Heavy Chain Isoforms in Rat Testis

1998 ◽  
Vol 9 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Peggy S. Criswell ◽  
David J. Asai
Keyword(s):  
Heavy Chain ◽  
Broad Band ◽  
Cytoplasmic Dynein ◽  
Peptide Sequence ◽  
Gene Encoding ◽  
Rat Testis ◽  
Dynein Heavy Chain ◽  
Heavy Chains ◽  
Low Ionic Strength ◽  
Dynein Heavy Chains

Recent studies have revealed the expression of multiple putative cytoplasmic dynein heavy chain (DHC) genes in several organisms, with each gene encoding a separate protein isoform. This finding is consistent with the hypothesis that different isoforms do different things, as is the case for the axonemal dyneins. Furthermore, the large number of tasks ascribed to cytoplasmic dynein suggests that there may be additional isoforms not yet identified. Two of the mammalian cytoplasmic dynein heavy chains are DHC1a and DHC1b. DHC1a is conventional cytoplasmic dynein and is found in all organisms examined. DHC1b is expressed in organisms that have multiple dyneins, and has been implicated in the intracellular trafficking of molecules in unciliated and ciliated cells. In the present study, we examined the DHC1b protein from rat testis. Testis cytoplasmic dynein contains a large amount of dynein heavy chain reactive with an antibody raised against a peptide sequence of rat DHC1b. The testis anti-DHC1b immunoreactive protein is slightly smaller than testis DHC1a, as assessed by SDS-PAGE. In Northern blots, the DHC1b mRNA is smaller than the DHC1a mRNA. In sucrose gradients made in low ionic strength, DHC1a sedimented at approximately 20S, and the anti-1b immunoreactive heavy chains sedimented in a broad band centered at approximately 14S. The V1-photolysis reaction of individual sucrose gradient fractions revealed three distinct patterns of photolysis, suggesting that there are at least three separate 1b-like heavy chain isoforms in testis. Using a high-stringency Western blotting protocol, the anti-1b antibody and the anti-DHC2 antibody recognized the same heavy chain and specifically bound to one of the three 1b-like heavy chains. We conclude that rat testis contains three 1b-like dynein heavy chains, and one of these is the product of the DHC1b/DHC2 gene previously identified.

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

Biology Open ◽  
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.

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

2018 ◽  
Vol 11 (9) ◽  
pp. 770-780 ◽  
Author(s):  
Guang Liu ◽  
Limei Wang ◽  
Junmin Pan
Keyword(s):  
Mass Spectrometry ◽  
Insertional Mutagenesis ◽  
The Other ◽  
Cytoplasmic Protein ◽  
Flagellar Motility ◽  
Heavy Chains ◽  
The Third ◽  
Axonemal Dynein ◽  
Dynein Arms ◽  
Dna Insertional Mutagenesis

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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