The Chlamydomonas reinhardtii oda8 mutation blocks assembly of flagellar outer dynein arms (ODAs), and interacts genetically with ODA5 and ODA10, which encode axonemal proteins thought to aid dynein binding onto axonemal docking sites. We positionally cloned ODA8 and identified the gene product as the algal homolog of vertebrate LRRC56. Its flagellar localization depends on ODA5 and ODA10, consistent with genetic interaction studies, but phylogenomics suggests that LRRC56 homologs play a role in intraflagellar transport (IFT)-dependent assembly of outer row dynein arms, not axonemal docking. ODA8 distribution between cytoplasm and flagella is similar to that of IFT proteins and about half of flagellar ODA8 is in the soluble matrix fraction. Dynein extracted in vitro from wild type axonemes will rebind efficiently to oda8 mutant axonemes, without re-binding of ODA8, further supporting a role in dynein assembly or transport, not axonemal binding. Assays comparing preassembled ODA complexes from the cytoplasm of wild type and mutant strains show that dynein in oda8 mutant cytoplasm has not properly preassembled and cannot bind normally onto oda axonemes. We conclude that ODA8 plays an important role in formation and transport of mature dynein complexes during flagellar assembly.
assembly; cilia; dynein; flagella; intraflagellar transport; motility.
© 2014 The Authors. Cytoskeleton Published by Wiley Periodicals, Inc.
Molecular identification of the
oda8 locus. (A) To positionally clone ODA8, based on its map location on the right arm of chromosome 1, the segregation of markers GBP1 and CNA73 was followed in six meiotic products with crossovers in the ARG7-ODA8 interval. Numbers indicate the number of crossover events observed in each sub-interval. (B) Location of BAC and lambda phage clones that rescued the slow swimming phenotype of an oda8 mutant. Arrow indicates the orientation of the ODA8 gene. (C) The location of cloned subfragments of the ODA8-B phage insert that rescued the oda8 phenotype are shown below a diagram of the ODA8 gene structure, which also shows the frame-shift mutation identified at codons 35–36 of the oda8-1 allele.
Phylogenomic distribution of ODA8/LRRC56 gene orthologs. The presence of orthologs of genes important for ciliary assembly (IFT-A and IFT-B proteins), ODA assembly (ODA-DC2, ODA16 and ODA8) and axonemal dynein subunits (ODA, IDA-I1) is diagrammed in selected organisms spanning diverse eukaryotic clades. Specific proteins used in these comparisons as representative of each multi-subunit complex are listed in a key across the top, with each sector of a circle representing a single protein. Orthologs were identified as sequences generating reciprocal BLAST best hits, starting with the
Chlamydomonas protein sequence; stronger similarity is represented by dark shading, weaker but significant similarity by light shading, and failure to identify any ortholog is shown as unshaded segments. The presence or absence of cilia and the motility state of cilia, if present, is diagrammed along the right margin. Actual E-values for each comparison are available in Supporting Information Table I.
ODA8 distribution is similar to that of transport factors. (A) HA-tagged ODA8 expressed from an integrated transgene rescues the beat frequency (bf) of
oda8 cells to wild type levels (∼60 Hz), and the expressed protein migrates at ∼104 kDa on blots of transformant oda8::ODA8HA (WT*) whole cell protein samples. (B) Expression of the HA-tagged transgene rescues the assembly of flagellar ODA subunit IC2 to wild type levels. Anti-IC140 recognizes inner arm I1 dynein, which is not affected by the oda8 mutation and serves as a loading control. C. Samples of whole cells (WC), deflagellated CB and flagella (FL) from equal numbers of cells (1:1) and with flagella at 50-fold excess (50:1) were probed with the indicated antibodies. ODA8HA abundance ratios are similar to those of IFT-B subunit IFT46 and IFT-associated transport factor ODA16, not those of ODA subunits such as IC2. Anti-NAB1, which recognizes a 26 kDa cytoplasmic protein, was used as a loading control in A and C.
Interacting mutants alter ODA8 distribution. (A) Blots of flagella from
WT* and from doublet mutant oda5, oda8 and oda10, oda8 strains that express ODA8HA ( oda5* and oda10*) were probed with antibodies to HA and IC2. Both oda5 and oda10 block ODA assembly, and both greatly reduce the flagellar abundance of ODA8HA. B. Blots of flagella from WT* and from a double mutant oda6,oda8 strain expressing ODA8HA ( oda6*) show that flagellar abundance of ODA8HA is not affected by the failure of ODA assembly in oda6. C. Blots of cell extracts show that cytoplasmic abundance of ODA8HA is unaffected by the oda5 and oda10 mutations. Acetylated tubulin (AcTub) is used as a loading control for flagellar samples, and NAB1 for cell extract samples. All three lanes in both blots in C. were from the same blot images. Vertical spaces indicate removal of intervening lanes.
Flagellar ODA8 is in both matrix and axonemal fractions. (A) Coomassie stained gel (upper panel) and blot probed for HA (lower panel) of
WT* FL, supernatant fractions (S) and pellets (P) after three sequential extractions of flagella with solutions containing 0.8% octylglucopyranoside detergent (OG), by treating flagella to a freeze/thaw (F/T), or to freeze/thaw followed by 0.8% OG (+ OG). About half of the flagellar ODA8HA is solubilized by detergent or freeze/thaw treatment. (B) Coomassie stained gel and blots of whole FL, 0.1% NP-40 detergent-extracted axonemes (AX) and the supernatant and pellet fractions of axonemes extracted with the indicated salt concentrations. About half of axonemal ODA8HA resists extraction with 0.6 M NaCl, unlike ODA subunit IC2. The sizes in kDa of protein standards are shown along the gel margins.
ODA8 is not needed for ODA binding to axonemes
in vitro. (A) Axonemes from the indicated strains were mixed with a dialyzed salt extract from wild type axonemes (HSE). After 1 h, axonemes were pelleted and the presence of bound dynein was tested by blotting for IC2. (B) Coomassie stained gel and corresponding blots of oda8 axonemes (Axo), a salt extract of WT* axonemes (HSE), and the supernatant (S) and pellet (P) fractions prepared 1 h after mixing oda8 axonemes and HSE at a 1:2 ratio. IC2 pellets with the oda8 axonemes, whereas ODA8HA remains soluble. (C) The stability of ODAs bound to oda8 axonemes (as in A) was tested by resuspending the pellet fraction for the indicated times before re-pelleting. IC2 remains tightly bound after a 60 min wash. Acetylated tubulin (Ac tub) blots show equal loading of axonemal protein (A and C).
ODA complexes are defective in
oda8 cytoplasm. (A) Blots of flagella from WT*, oda8, and double mutant strain oda8, oda16 that expresses the ODA8HA transgene ( oda16*) probed with the indicated antibodies. Neither mutation prevents flagellar assembly of the other protein. IC140 is used as a loading control. (B) Stained gel and corresponding blot of cell extracts from wild type and oda8 cells, loaded for equal amounts of protein. Blots show the relative abundance of ODA subunits in each extract. (C) Binding experiment in which extract samples shown in B, adjusted to contain equal ODA subunit concentrations, were mixed with axonemes from the indicated oda mutants. After 1 h, axonemes were pelleted, washed, and prepared as gel samples. An immunoblot shows the relative amount of IC2 bound. (D) Gel and corresponding blots of flagella from oda6 (IC2-null) , oda6 transformed with a MYC-tagged IC2 gene ( IC2MYC), and untransformed wild type cells ( WT), probed for MYC and IC2, show that MYC-tagged IC2 supports normal levels of ODA assembly. (E) Blots of anti-MYC IP samples, from cell extracts of IC2MYC and oda8, IC2MYC, probed with the indicated antibodies. Both IC1 and IC2-MYC are present at equal abundance, whereas the relative amount of each heavy chain is reduced in the oda8 sample to about 55% of its wild type abundance.
Diagram summarizing hypothesized steps in ODA assembly and transport, and a proposed role for ODA8. Initial synthesis of ODA subunits and dimerization of intermediate chains is followed by folding and co-assembly of heavy chains with intermediate chains, dependent on the action of ODA7 [Duquesnoy et al., 2009] and PF13 [Omran et al., 2008]. Several other cytoplasmic dynein assembly factors may function at this step. Exposure of a heavy chain epitope in a PF22-dependent step [Mitchison et al., 2012] is modeled as chaperone removal. ODA8 transforms an assembled, but unstable intermediate into a stable cytoplasmic storage form, which is then able to interact with IFT particles, aided by IFT-associated assembly factor ODA16. An ODA5/10 complex is hypothesized to interact with ODAs in the cytoplasm and aid ODA8 function. Once ODAs are transported to flagella, they are released from IFT particles, dissociate from ODA5/10 and ODA8, and bind through interaction of the IC dimer with ODA-DC complexes that have assembled independently on A-tubules of outer doublets.
All figures (8)
Chlamydomonas ODA10 is a conserved axonemal protein that plays a unique role in outer dynein arm assembly.
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ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery.
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