Laura A. Fox

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.

How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by posttranslational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification [1] that is enriched in axonemes. The enzymes responsible for this posttranslational modification, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) [2]. We show that in cilia of Tetrahymena, TTLL6 E-ligases generate glutamylation mainly on the B-tubule of outer doublet microtubules, the site of force production by ciliary dynein. Deletion of two TTLL6 paralogs caused severe deficiency in ciliary motility associated with abnormal waveform and reduced beat frequency. In isolated axonemes with a normal dynein arm composition, TTLL6 deficiency did not affect the rate of ATP-induced doublet microtubule sliding. Unexpectedly, the same TTLL6 deficiency increased the velocity of microtubule sliding in axonemes that also lack outer dynein arms, in which forces are generated by inner dynein arms. We conclude that tubulin glutamylation on the B-tubule inhibits the net force imposed on sliding doublet microtubules by inner dynein arms.

The N-DRC forms a conserved biochemical complex that maintains outer doublet alignment and limits microtubule sliding in motile axonemes

The nexin–dynein regulatory complex (N-DRC) is implicated in the control of dynein activity as a structural component of the nexin link. This study identifies several new subunits of the N-DRC and demonstrates for the first time that it forms a discrete biochemical complex that maintains outer doublet integrity and regulates microtubule sliding.

IC138 Defines a Subdomain at the Base of the I1 Dynein That Regulates Microtubule Sliding and Flagellar Motility

To understand the mechanisms that regulate the assembly and activity of flagellar dyneins, we focused on the I1 inner arm dynein (dynein f) and a null allele, bop5-2, defective in the gene encoding the IC138 phosphoprotein subunit. I1 dynein assembles in bop5-2 axonemes but lacks at least four subunits: IC138, IC97, LC7b, and flagellar-associated protein (FAP) 120--defining a new I1 subcomplex. Electron microscopy and image averaging revealed a defect at the base of the I1 dynein, in between radial spoke 1 and the outer dynein arms. Microtubule sliding velocities also are reduced. Transformation with wild-type IC138 restores assembly of the IC138 subcomplex and rescues microtubule sliding. These observations suggest that the IC138 subcomplex is required to coordinate I1 motor activity. To further test this hypothesis, we analyzed microtubule sliding in radial spoke and double mutant strains. The results reveal an essential role for the IC138 subcomplex in the regulation of I1 activity by the radial spoke/phosphorylation pathway.

The MIA complex is a conserved and novel dynein regulator essential for normal ciliary motility

The MIA complex, composed of FAP100 and FAP73, interacts with I1 dynein components and is required for normal ciliary beat frequency.

IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein--a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both alpha- and beta-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.

Distinct roles of 1α and 1β heavy chains of the inner arm dynein I1 of Chlamydomonas flagella

We took advantage of Chlmaydomonas flagellar mutant strains lacking either the 1α or 1β motor domain in I1 dynein to distinguish the functional role of each. The 1β motor domain is an effective motor required for control of microtubule sliding, whereas the 1α motor domain may restrain microtubule sliding driven by other dyneins.

bop5 mutations reveal new roles for the IC138 phosphoprotein in the regulation of flagellar motility and asymmetric waveforms

Mutations in the IC138 regulatory subunit of I1 dynein alter dynein motor activity and the flagellar waveform but do not affect phototaxis.

An axonemal PP2A B-subunit is required for PP2A localization and flagellar motility.

Analysis of Chlamydomonas axonemes revealed that the protein phosphatase, PP2A, is localized to the outer doublet microtubules and is implicated in regulation of dynein‐driven motility. We tested the hypothesis that PP2A is localized to the axoneme by a specialized, highly conserved 55‐kDa B‐type subunit identified in the Chlamydomonas flagellar proteome. The B‐subunit gene is defective in the motility mutant pf4. Consistent with our hypothesis, both the B‐ and C‐ subunits of PP2A fail to assemble in pf4 axonemes, while the dyneins and other axonemal structures are fully assembled in pf4 axonemes. Two pf4 intragenic revertants were recovered that restore PP2A to the axonemes and re‐establish nearly wild‐type motility. The revertants confirmed that the slow‐swimming Pf4 phenotype is a result of the defective PP2A B‐subunit. These results demonstrate that the axonemal B‐subunit is, in part, an anchor protein required for PP2A localization and that PP2A is required for normal ciliary motility.

Alcohol-induced ciliary dysfunction targets the outer dynein arm.

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.

Assays of axonemal dynein-driven motility

Publisher Summary This chapter examines microscopic assays of axonemal dynein function and illustrates novel observations derived from these assays. Two general types of functional motility assays—namely, (1) video microscopic analysis of ATP-induced microtubule sliding in protease-treated, isolated axonemes and (2) in vitro microtubule translocation assay are described each of which makes use of dark-field light microscopy, to directly observe active microtubule sliding in isolated axonemes. The chapter also describes microscopic setup, step-by-step procedures for each experimental approach, and data derived from these techniques. The key elements of dark-field microscopy include an intense light source, a dark-field oil immersion condenser with high numerical aperture, a high-quality 40x objective lens, and a sensitive video camera mounted on a trinocular head. An upright microscope is used for experiments, which require perfusion of samples in perfusion chambers. The inverted microscope is used for most other experiments. The sample is contained in a simple perfusion chamber constructed from a glass slide and coverslip separated by two parallel strips of double-sided tape.