Tubulin Polyglutamylation Regulates Axonemal Motility by Modulating Activities of Inner-Arm Dyneins

SummaryTubulin polyglutamylation is a modification that adds multiple glutamates to the γ-carboxyl group of a glutamate residue in the C-terminal tails of α- and β-tubulin [1, 2]. This modification has been implicated in the regulation of axonal transport and ciliary motility. However, its molecular function in cilia remains unknown. Here, using a novel Chlamydomonas reinhardtii mutant (tpg1) that lacks a homolog of human TTLL9, a glutamic acid ligase enzyme [3], we found that the lack of a long polyglutamate side chain in α-tubulin moderately weakens flagellar motility without noticeably impairing the axonemal structure. Furthermore, the double mutant of tpg1 with oda2, a mutation that leads to loss of outer-arm dynein, completely lacks motility. More surprisingly, when treated with protease and ATP, the axoneme of this paralyzed double mutant displayed faster microtubule sliding than the motile oda2 axoneme. These and other results suggest that polyglutamylation directly regulates microtubule-dynein interaction mainly by modulating the function of inner-arm dyneins

A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase-like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility

Correction for \u3cem\u3eIC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding\u3c/em\u3e

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 α- and β-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

A Tektin Homologue Is Decreased in Chlamydomonas Mutants Lacking an Axonemal Inner-Arm Dynein

In ciliary and flagellar axonemes, various discrete structures such as inner and outer dynein arms are regularly arranged on the outer doublet microtubules. Little is known about the basis for their regular arrangement. In this study, proteins involved in the attachment of inner-arm dyneins were searched by a microtubule overlay assay on Chlamydomonas mutant axonemes. A 58-kDa protein (p58) was found ∼80% diminished in the mutants ida6 and pf3, both lacking one (species e) of the seven inner-arm species (a–g). Analysis of its cDNA indicated that p58 is homologous to tektin, a protein that was originally found in sea urchin and thought to be crucial for the longitudinal periodicity of the doublet microtubule. Unlike sea urchin tektin, which is a component of protofilament ribbons that occur after Sarkosyl treatment of axonemes, p58 was not contained in similar Sarkosyl-resistant ribbons from Chlamydomonas axonemes. Immunofluorescence microscopy showed that p58 was localized uniformly along the axoneme and on the basal body. The p58 signal was reduced in ida6 and pf3. These results suggest that a reduced amount of p58 is sufficient for the production of outer doublets, whereas an additional amount of it is involved in inner-arm dynein attachment

Novel 44-Kilodalton Subunit of Axonemal Dynein Conserved from Chlamydomonas to Mammals▿

Cilia and flagella have multiple dyneins in their inner and outer arms. Chlamydomonas inner-arm dynein contains at least seven major subspecies (dynein a to dynein g), of which all but dynein f (also called dynein I1) are the single-headed type that are composed of a single heavy chain, actin, and either centrin or a 28-kDa protein (p28). Dynein d was found to associate with two additional proteins of 38 kDa (p38) and 44 kDa (p44). Following the characterization of the p38 protein (R. Yamamoto, H. A. Yanagisawa, T. Yagi, and R. Kamiya, FEBS Lett. 580:6357-6360, 2006), we have identified p44 as a novel component of dynein d by using an immunoprecipitation approach. p44 is present along the length of the axonemes and is diminished, but not absent, in the ida4 and ida5 mutants, both lacking this dynein. In the ida5 axoneme, p44 and p38 appear to form a complex, suggesting that they constitute the docking site of dynein d on the outer doublet. p44 has potential homologues in other ciliated organisms. For example, the mouse homologue of p44, NYD-SP14, was found to be strongly expressed in tissues with motile cilia and flagella. These results suggest that inner-arm dynein d and its subunit organization are widely conserved

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii

Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes

FAP20 is an inner junction protein of doublet microtubules essential for both the planar asymmetrical waveform and stability of flagella in Chlamydomonas.

The axoneme-the conserved core of eukaryotic cilia and flagella-contains highly specialized doublet microtubules (DMTs). A long-standing question is what protein(s) compose the junctions between two tubules in DMT. Here we identify a highly conserved flagellar-associated protein (FAP), FAP20, as an inner junction (IJ) component. The flagella of Chlamydomonas FAP20 mutants have normal length but beat with an abnormal symmetrical three-dimensional pattern. In addition, the mutant axonemes are liable to disintegrate during beating, implying that interdoublet connections may be weakened. Conventional electron microscopy shows that the mutant axonemes lack the IJ, and cryo-electron tomography combined with a structural labeling method reveals that the labeled FAP20 localizes at the IJ. The mutant axonemes also lack doublet-specific beak structures, which are localized in the proximal portion of the axoneme and may be involved in planar asymmetric flagellar bending. FAP20 itself, however, may not be a beak component, because uniform localization of FAP20 along the entire length of all nine DMTs is inconsistent with the beaks localization. FAP20 is the first confirmed component of the IJ. Our data also suggest that the IJ is important for both stabilizing the axoneme and scaffolding intra-B-tubular substructures required for a planar asymmetrical waveform

Inner lumen proteins stabilize doublet microtubules in cilia and flagella

Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes