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. 2015 Mar 15;308(6):L569-76.
doi: 10.1152/ajplung.00257.2014. Epub 2015 Jan 16.

Alcohol-induced Ciliary Dysfunction Targets the Outer Dynein Arm

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Free PMC article

Alcohol-induced Ciliary Dysfunction Targets the Outer Dynein Arm

Fan Yang et al. Am J Physiol Lung Cell Mol Physiol. .
Free PMC article

Abstract

Alcohol abuse results in an increased incidence of pulmonary infection, in part attributable to impaired mucociliary clearance. Analysis of motility in mammalian airway cilia has revealed that alcohol impacts the ciliary dynein motors by a mechanism involving altered axonemal protein phosphorylation. Given the highly conserved nature of cilia, it is likely that the mechanisms for alcohol-induced ciliary dysfunction (AICD) are conserved. Thus we utilized the experimental advantages offered by the model organism, Chlamydomonas, to determine the precise effects of alcohol on ciliary dynein activity and identify axonemal phosphoproteins that are altered by alcohol exposure. Analysis of live cells or reactivated cell models showed that alcohol significantly inhibits ciliary motility in Chlamydomonas via a mechanism that is part of the axonemal structure. Taking advantage of informative mutant cells, we found that alcohol impacts the activity of the outer dynein arm. Consistent with this finding, alcohol exposure results in a significant reduction in ciliary beat frequency, a parameter of ciliary movement that requires normal outer dynein arm function. Using mutants that lack specific heavy-chain motor domains, we have determined that alcohol impacts the β- and γ-heavy chains of the outer dynein arm. Furthermore, using a phospho-threonine-specific antibody, we determined that the phosphorylation state of DCC1 of the outer dynein arm-docking complex is altered in the presence of alcohol, and its phosphorylation correlates with AICD. These results demonstrate that alcohol targets specific outer dynein arm components and suggest that DCC1 is part of an alcohol-sensitive mechanism that controls outer dynein arm activity.

Keywords: alcohol; cilia; dynein; ethanol.

Figures

Fig. 1.
Fig. 1.
Alcohol treatment reduces ciliary beat frequency (CBF) and alters outer dynein arm (ODA) function via the β- and γ-heavy chain (HC). A: wild-type (WT) cells were treated with increasing levels of alcohol. Swimming speeds were measured and show a dose-dependent decrease in response to increasing alcohol concentration. B: motility analyses of WT cells and mutants strains defective in the inner dynein arm (IDA) and ODA motors (left) show that alcohol significantly reduces swimming velocities in WT cells and in mutant strains defective in the IDA motors [dyneins f (ida3) and dyneins a, b, and c (ida4)]. In contrast, the alcohol-induced decrease in swimming speed is not observed in oda3 (lacking the ODA). Motility analyses of oda HC mutants (right) show that alcohol reduces forward swimming speeds in oda11 (lacking the α-HC). In contrast, alcohol shows no ciliary-slowing effect in oda4-s7 (lacks β-HC motor domain), sup-pf1 [lacking 7 amino acids in the coiled-coil 1 (CC1) region of the dynein motor stalk domain], and oda2-t (lacks γ-HC motor domain). Thus WT, ida mutants, and oda11 are susceptible (S) to alcohol-induced ciliary dysfunction (AICD), whereas oda3 and the β- and γ-HC mutants are alcohol resistant (R). C: AICD is recapitulated in WT cell models, where the ciliary membrane is removed. Thus AICD is mediated by mechanisms intrinsic to the ciliary axoneme. Importantly, oda2 cell models are resistant to AICD and confirm that AICD targets the ODA motors. D: in the presence of 10 mM alcohol, CBF is significantly reduced in WT cells over the course of 15 min (*P ≤ 0.0001). CBF is not reduced by alcohol in a mutant that lacks the ODA (oda3). Measurements were performed using Sisson-Ammons Video Analysis (42).
Fig. 2.
Fig. 2.
Alcohol affects the phosphorylation of axonemal proteins. A: isolated axonemes were treated with phosphatase or incubated with 1 mM ATP in the absence or presence of 100 mM alcohol. Samples were analyzed by Western blot using an antibody to detect phosphorylated threonine. A prominent 100-kDa axonemal phospho-protein is detected in untreated axonemes (Axo), which becomes dephosphorylated by phosphatase treatment (calf intestinal phosphatase, CIP). In the presence of ATP, this phospho-protein becomes heavily phosphorylated (ATP; note the increased smearing of the band marked by the arrow). In the presence of ATP and alcohol, this increased phosphorylation is not observed (ATP + Alc). Alcohol exposure results in altered phosphorylation of several additional proteins as well. B: to better separate phosphorylated proteins, axoneme samples were analyzed by the Phos-Tag SDS-PAGE method. Untreated axonemes show 2 distinct bands (1 and 2). In axonemes treated with ATP, bands 1 and 2 become more heavily phosphorylated, and 2 new bands are detected (3 and 4). In the presence of alcohol, all 4 bands are detected with decreased phosphorylation. Bottom: Coomassie Brilliant Blue (CBB)-stained gel demonstrating equivalent protein loading. C: densitometry of the bands detected by Western blot show the increase in phosphorylation with ATP and the corresponding decrease in phosphorylation in the presence of alcohol. Densitometry of the protein-stained gel (* in B) demonstrates equivalent loading of all samples. D: p100 is reduced or has less threonine phosphorylation in mutants that lack the central pair (CP) (pf18) and the ODA (oda2), whereas p100 phosphorylation is unaffected by loss of the radial spoke (RS) (pf14), nexin-dynein regulatory complex (N-DRC) (pf3), or IDAs (ida1, ida4). Tubulin is shown as a loading control. E: Western blots of isolated axonemes from WT and oda HC mutants. Phosphorylated p100 (pThr) is detected in WT and oda11 (lacking the α-HC) but is absent in oda3 (missing DCC1 and ODA), the β-HC mutants (oda4-s7 and sup-pf1), and the γ-HC mutant (oda2-t). DCC1 antibodies (DCC1) detect the presence of DCC1 in WT and the HC mutants but not oda3. CBB shows protein-stained tubulin bands for loading controls.
Fig. 3.
Fig. 3.
The p100 phosphoprotein is DCC1. A: isolated WT axonemes containing p100 (axo) were extracted with high-salt buffers, and the fractions were analyzed by Western blot using the anti-phosphothreonine antibody. p100 is solubilized from the axoneme with high salt and is detected in the high-salt extract (HSE). B: p100 is immunoprecipitated from HSEs with the anti-phosphothreonine antibody but not with control IgG. C: protein-stained gel showing a band at the expected size for p100 in WT immune complexes, which is absent in immune complexes from an ODA mutant, oda3. The p100 region in both WT and oda3 were excised and analyzed by tandem MS/MS. D: blots in B were stripped and reprobed with the DCC1 antibody, which shows a band for DCC1 in the immune pellet (p100 immunoprecipitation, IP) which comigrates with the p100 band. E: p100 and DCC1 comigrate on 2D gels. WT axonemes were separated on 2D gels followed by Western blotting with the anti-phosphothreonine and DCC1 antibodies. The spot patterns detected by both antibodies comigrate at the same molecular weight and pI range (∼6.1–7.2).
Fig. 4.
Fig. 4.
Model for regulation of ODA activity. The CP contains appendage structures (dark gray) that are asymmetrically oriented within the CP apparatus. The rotation of the CP (black round arrow) mechanically activates signaling pathways built into the CP and RS structures. The signal initiated in the CP is transmitted through the RS (arrow) to signaling molecules at the base of the RS and on the outer doublet microtubules. The predicted locations of signal transduction proteins [PKA, CK1, protein phosphatase 1 (PP1), PP2A, and calmodulin complexes] are shown. The ODA-docking complex (ODA-DC) at the base of the ODA contains phosphorylated DCC1. Upon activation of motility, DCC1 is further phosphorylated and may regulate the activity of the β-and γ-HCs. Alcohol may interfere in this pathway.

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