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. 2013 Dec;24(23):3689-96.
doi: 10.1091/mbc.E13-06-0310. Epub 2013 Oct 2.

Chlamydomonas ODA10 Is a Conserved Axonemal Protein That Plays a Unique Role in Outer Dynein Arm Assembly

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Free PMC article

Chlamydomonas ODA10 Is a Conserved Axonemal Protein That Plays a Unique Role in Outer Dynein Arm Assembly

Anudariya B Dean et al. Mol Biol Cell. .
Free PMC article

Abstract

Assembly of outer dynein arms (ODAs) requires multiple steps and involves multiple proteins in addition to dynein subunits. The Chlamydomonas ODA10, ODA5, and ODA8 loci genetically interact and are hypothesized to function as an axonemal accessory complex, but only ODA5p was previously characterized. We positionally cloned ODA10 and identified the gene by rescuing an oda10 mutant with a hemagglutinin-tagged cDNA. ODA10 sequence predicts a conserved coiled-coil protein homologous to mouse ccdc151. ODA10p is present in cytoplasm and flagella, remains axonemal after detergent treatment, and is extracted with 0.6 M NaCl. Both outer arm dynein and ODA10p rebound to the axonemes when desalted extracts are mixed with oda10-mutant axonemes. Sucrose gradient separation of these extracts shows that ODA10p sediments near the top of the gradient, not with 23S outer dynein arm proteins. Unexpectedly, dynein and ODA10p fractions are able to bind individually to oda10 axonemes. ODA10p is present on oda8-mutant flagella at wild-type levels. However, ODA10p does not assemble into oda5 flagella and is absent from oda5 cytoplasm, suggesting a necessity of ODA5p for stability of ODA10p in vivo. The results suggest that ODA10p does not function as a part of a traditionally defined docking complex.

Figures

FIGURE 1:
FIGURE 1:
ODA10p is a conserved coiled-coil protein. (A) A Coomassie-stained gel (top) and Western blot (bottom), probed with an antibody against HA epitope, of equally loaded (by cell number) whole-cell samples of oda10-2,ODA10HA and oda10-2 cells show expression of ODA10HAp in a strain with rescued beat frequency. (B) A Coomassie-stained gel (top) and western blot (bottom), probed with antibodies against HA epitope and IC2, of flagellar samples of mutant (oda10-2), wild type (137c), and rescued transformants (oda10-2,ODA10HA 1-9) show restored assembly of outer dynein arms to wild-type levels in transformant cells expressing the highest levels of ODA10HAp. (C) The distribution of predicted coiled-coil domains of ODA1p, ODA3p, ODA5p, and ODA10p. (D) All homologues of ODA10p and of three Chlamydomonas ODA10p paralogues (ODA5p, ODA1p [ = DC2], ODA3p [ = DC1]) in an alga (Micromonas), a basal chordate (Ciona), a fish (Danio), and a mammal (Mus) were aligned, and an unrooted tree was generated to display relative sequence similarities. Green background highlights homologues of ODA1p; yellow background highlights homologues of ODA10p. Protein sequences used in the alignments that are not given in the figure were Q8CDV6 (ccdc63), AAI60348 (ccdc114), AAH57069 (ccdc151), NP_001027646 (Ciona p66), NP_001072015 (Ciona IC4), NP_001071888 (Ciona IC5), AAC49732 (ODA3), AAK72125 (ODA1), AAS10183 (ODA5), and AGW18228 (ODA10).
FIGURE 2:
FIGURE 2:
ODA10p is a salt-extractable axonemal protein that does not cofractionate with dynein. (A) Western blots of cytoplasmic extract and flagellar samples from equal numbers of cells, probed with antibodies against HA (ODA10p) and NAB1 (a cytoplasmic protein used as a control for cell body contamination in the flagellar fraction), show ODA10p distribution in cytoplasm and flagella. (B) Western blots of flagellar, detergent-resistant axonemal, and detergent-soluble membrane and matrix fractions, probed with antibodies against HA and IC2, show that ODA10p remains microtubule associated, similar to IC2. (C) A Coomassie-stained gel (top) and Western blot of soluble and insoluble fractions of wild-type axonemes after exposure to increasing salt concentrations, probed with an antibody against HA, show extraction of ODA10p >0.4 M NaCl. (D) A Coomassie-stained gel (top) and Western blot (bottom) of sucrose gradient fractions of a 0.6 M NaCl extract of ODA10HA axonemes. Outer row dynein (IC2) does not cosediment with ODA10p (ODA10HA).
FIGURE 3:
FIGURE 3:
ODA10p is unstable in oda5 mutant. (A) A Coomassie-stained gel (top) and Western blot (bottom) of flagellar samples from the indicated strains, probed with a polyclonal antibody against ODA10p, show that ODA10p is missing from oda5 as well as oda10 flagella. (B) Flagellar and axonemal samples of oda8,ODA10HA oda5,ODA10HA and oda10,ODA10HA were probed with antibody against the HA epitope. The tubulin region of a Coomassie-stained gel (bottom) serves as a loading control. (C) A Western blot (top) of deflagellated cell body samples of oda10,ODA10HA oda8,ODA10HA and oda5,ODA10HA probed for ODA10HAp. The amount of ODA10p in cytoplasm of oda5 was greatly reduced but was near wild type in oda8. A portion of the Coomassie-stained gel serves as a loading control.
FIGURE 4:
FIGURE 4:
ODA10p and outer arm dynein stably assemble onto oda10 axonemes independently in vitro. Western blots probed with anti-IC2 and anti-HA detect proteins bound in vitro to oda10 axonemes. (A) oda10 axonemes were incubated with buffer alone (–) or a dialyzed HSE of wild-type axonemes from ODA10HA cells (+) for 1 h, and pellet fractions were examined. Both IC2 and ODA10HAp are present in the pellet. (B) oda10 axonemes were incubated on ice for 1 h with separate sucrose gradient fractions of outer row dyneins (∼23 S) or ODA10HAp (∼6 S) or buffer alone. Both pellet (P) and supernatant (S) were analyzed. (C, D) The binding experiment in B was repeated in presence of 1 mM ATP, and pellets were probed for ODA10HA or IC2. ODA10HA and outer dynein arms pellet with axonemes, regardless of presence of ATP. (E, F) oda10 axonemes previously incubated with either 6S (E) or 23S (F) fractions were pelleted, and half of each sample was resuspended in fresh buffer, incubated on ice for 2 h, and pelleted again. Equal amounts of ODA10HAp (E) and IC2 (F) are associated with oda10 axonemes before (0) and after (2) incubation.

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