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. 2010 Mar 9;20(5):435-40.
doi: 10.1016/j.cub.2009.12.062.

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity

Swati Suryavanshi  1 , Bernard Eddé, Laura A Fox, Stella Guerrero, Robert Hard, Todd Hennessey, Amrita Kabi, David Malison, David Pennock, Winfield S Sale, Dorota Wloga, Jacek Gaertig
Affiliations

Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity

Swati Suryavanshi et al. Curr Biol. .

Abstract

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.

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Figures

Figure 1
Figure 1. Deletion of Ttll6Ap and Ttll6Fp leads to a loss of tubulin glutamylation in cilia
(A) A neighbor-joining phylogenetic tree based on the catalytic domain of TTLL6 E-ligases [5]. Tt-Ttll4Ap was used as an outgroup. Abbreviations of species: Hs, Homo sapiens; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Tt, Tetrahymena thermophila. (B) A fluorogram of mammalian microtubule proteins (20 μg) separated by SDS-PAGE after in vitro glutamylation with partially purified GFP-Ttll6Ap and GFP-Ttll1p, ATP and 3H-glutamate. (C) A western blot of cilia proteins. The anti-SerH antigen antibodies were used as a loading control. (D–E″) Immunofluorescence images of pairs of wildtype and 6AF-KO cells imaged side by side. Wildtype cells were prefed with India Ink to reveal dark food vacuoles. Cells were labeled with 12G10 anti-α-tubulin mAb and polyE anti-polyglutamylation antibodies (D–D″) or with SG anti-total tubulin antibodies and GT335 anti-glutamylated tubulin mAb (E–E″). Arrowheads mark oral membranelles. Bar = 10 μm. Quantitative data are shown in Fig. S1A.
Figure 2
Figure 2. Cells lacking Ttll6Ap and Ttll6Fp display a loss of cilia-dependent functions
(A) A histogram shows the average linear cell motility rate during 5 sec for wild type, 6AF-KO and 6AF-KO cells rescued with a GFP-Ttll6Ap transgene (6AF-KOR) (n=40 for each strain). Bars represent standard deviations. *p<0.001. (B) Culture growth curves. (C–D) Images of a wild type (C) and 6AF-KO (D) cell exposed to India ink for 30 min. Bar = 20 μm. (E) The graph shows the percentage of paired cells following mixing of either two starved wild type strains (CU428 and B2086) or 6AF-KO cells with either of the two wild type strains. (F) The average ciliary beat frequency for wild type (n=27) and 6AF-KO cells (n=27, p<0.0001). Error bars represent standard deviations. (G–H′). Swimming responses to SDBS. Wild type (G–G′) or 6AF-KO (H–H′) cells were exposed to either a buffer alone or SDBS (20 μg/ml), and the paths of live cells were recorded for 1 sec. Bar = 1 mm.
Figure 3
Figure 3. Ttll6Ap and Ttl6Fp generate polyglutamylation primarily on B-tubule of outer microtubules
(A–B) Cross sections of wild type (A) and 6AF-KO (B) cilia of cells grown at 30°C. Bar = 100 nm (C) A graph that documents the average number of IDAs and ODAs per axoneme cross-section (wild type n= 27; 6AF-KO n= 27). Error bars represent standard errors. (D–E) Sections of cells expressing GFP-Ttll6Ap that were labeled with anti-GFP antibodies using immunogold TEM. Bar in D = 750 nm. Bar in E = 250 nm. (F) A graph that quantifies the localization of polyglutamylated tubulin epitopes in doublet microtubules labeled by whole mount immunogold microscopy (shown in Fig. 3G, H and Fig. S3) using polyE antibodies and anti-rabbit IgG 10 nm gold conjugates. Each gold particle was scored as associated more closely with either the A- or B-tubule, or the inter-tubule junction (A/B). Error bars represent standard deviations. *p=0.0001, **p<0.0001, ***p=0.0085. Twelve wild type and 12 6AF-KO axonemes were scored (G, H) Examples of isolated doublet microtubules of wild type (G) and 6AF-KO origin (H) analyzed by whole mount immunogold microscopy using polyE antibodies. Arrowheads mark the A-tubule covered with dynein arms. Additional images are shown in Fig. S3. Bar = 100 nm.
Figure 4
Figure 4. Tubulin glutamylation regulates the velocity of inner dynein arm driven microtubule sliding in axonemes in vitro
(A) TEM cross-sections of wildtype, 6AF-KO and 6AF-KO;oad1 axonemes grown for 12 hr at 38°C. Bar = 125 nm. The average numbers of dynein arms on scored axoneme cross-sections were as follows: 6AF-KO: 8.6 +/− 0.2 ODA, 6.4 +/− 0.6 IDA per section, n=15; 6AF-KO;oad1-1: 2.8 +/− 0.5 ODA, 6.5 +/− 0.2 IDA per section, n=15. (B) A graph shows the average sliding velocity of wild type (n=40) and 6AF-KO (n=40) axonemes obtained from cells grown at the standard temperature (30°C). Error bars represent standard deviations. Data were collected in 3 independent experiments. (C) A graph that documents the average sliding velocity of wild type (n=74), oad1-1 (n=41) 6AF-KO (n=73), and 6AF-KO;oad1-1 (n=46) axonemes obtained from cells grown at the 38°C, to induce the loss of ODAs in cells that are homozygous for the oad1-1 allele. Error bars represent standard deviations. *p<0.0001 for 6AF-KO;oad1-1 vs wildtype; **p<0.0001 for oad1-1 vs wildtype. Data were collected in 3 independent experiments.
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