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• W2054442207 abstract "Tubulin 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 [1Janke C. Rogowski K. van Dijk J. Polyglutamylation: A fine-regulator of protein function? ‘Protein modifications: Beyond the usual suspects’ review series.EMBO Rep. 2008; 9: 636-641Crossref PubMed Scopus (73) Google Scholar, 2Gaertig J. Wloga D. Ciliary tubulin and its post-translational modifications.Curr. Top. Dev. Biol. 2008; 85: 83-113Crossref PubMed Scopus (47) Google Scholar]. 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 [3van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar], 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. Tubulin 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 [1Janke C. Rogowski K. van Dijk J. Polyglutamylation: A fine-regulator of protein function? ‘Protein modifications: Beyond the usual suspects’ review series.EMBO Rep. 2008; 9: 636-641Crossref PubMed Scopus (73) Google Scholar, 2Gaertig J. Wloga D. Ciliary tubulin and its post-translational modifications.Curr. Top. Dev. Biol. 2008; 85: 83-113Crossref PubMed Scopus (47) Google Scholar]. 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 [3van Dijk J. Rogowski K. Miro J. Lacroix B. Eddé B. Janke C. A targeted multienzyme mechanism for selective microtubule polyglutamylation.Mol. Cell. 2007; 26: 437-448Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar], 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 Chlamydomonas mutant, tpg1, lacking α-tubulin polyglutamate ligase was isolated The tpg1 mutation displayed slightly lowered flagellar motility This mutation caused complete loss of motility in flagella lacking outer-arm dynein The paralyzed axoneme underwent faster microtubule sliding than motile axonemes We isolated a novel Chlamydomonas mutant, tpg1, by screening ultraviolet irradiation-mutagenized cells for low motility [4Kamiya R. Mutations at twelve independent loci result in absence of outer dynein arms in Chylamydomonas reinhardtii.J. Cell Biol. 1988; 107: 2253-2258Crossref PubMed Scopus (180) Google Scholar]. Amplified fragment length polymorphism analysis after genetic cross with the S1-D2 strain [5Kathir P. LaVoie M. Brazelton W.J. Haas N.A. Lefebvre P.A. Silflow C.D. Molecular map of the Chlamydomonas reinhardtii nuclear genome.Eukaryot. Cell. 2003; 2: 362-379Crossref PubMed Scopus (95) Google Scholar] mapped this mutation to a region on linkage group XIX. This region contained three genes coding for proteins FAP59, RPL24, and FAP267, registered in the flagellar proteome database [6Pazour G.J. Agrin N. Leszyk J. Witman G.B. Proteomic analysis of a eukaryotic cilium.J. Cell Biol. 2005; 170: 103-113Crossref PubMed Scopus (729) Google Scholar]. Sequence analysis of genomic DNA revealed that the isolate has a 502 bp deletion that covers the entire exon 7 sequence of the gene encoding FAP267, a homolog of TTLL9 of mammals (Figures 1A and 1B ; see also Figures S1A and S1B available online). TTLL9 is one of tubulin tyrosine ligase-like proteins (TTLLs) with tubulin glutamic acid ligase activity on α-tubulin [7Janke C. Rogowski K. Wloga D. Regnard C. Kajava A.V. Strub J.M. Temurak N. van Dijk J. Boucher D. van Dorsselaer A. et al.Tubulin polyglutamylase enzymes are members of the TTL domain protein family.Science. 2005; 308: 1758-1762Crossref PubMed Scopus (210) Google Scholar, 8Wloga D. Rogowski K. Sharma N. Van Dijk J. Janke C. Eddé B. Bré M.H. Levilliers N. Redeker V. Duan J. et al.Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.Eukaryot. Cell. 2008; 7: 1362-1372Crossref PubMed Scopus (69) Google Scholar]. The tpg1 mutation was found to have a deletion in the C-terminal third of this protein (CrTTLL9) where the ATP binding site is located (Figure 1A). Hence, the product of the mutated gene, if any, should lack the enzymatic activity. Immunoblot with the polyE antibody, which recognizes tubulin with a side chain of three or more glutamates [8Wloga D. Rogowski K. Sharma N. Van Dijk J. Janke C. Eddé B. Bré M.H. Levilliers N. Redeker V. Duan J. et al.Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.Eukaryot. Cell. 2008; 7: 1362-1372Crossref PubMed Scopus (69) Google Scholar], detected a significantly weaker band in the tpg1 axoneme than in the wild-type axoneme (Figure 1C). In wild-type axonemes, this antibody detected only a weak band of polyglutamylated β-tubulin compared with that of polyglutamylated α-tubulin (Figure S1C). In two-dimensional SDS polyacrylamide gel electrophoresis patterns, α-tubulin separated into at least seven discrete spots in the wild-type axoneme, whereas only about four spots were detected in tpg1. In addition, upon close examination, both α- and β-tubulin spots were associated with faint long smears on the acidic side in the wild-type, but not in the tpg1, axoneme sample (Figure 1D). However, the β-tubulin spot did not separate into discrete spots. These results indicate that the tpg1 mutation causes loss of long-chain polyglutamylation mostly in α-tubulin. It is likely that CrTTLL9 catalyzes the elongation of polyglutamylate side chains as shown for the TTLL9 ortholog in Tetrahymena thermophila [8Wloga D. Rogowski K. Sharma N. Van Dijk J. Janke C. Eddé B. Bré M.H. Levilliers N. Redeker V. Duan J. et al.Glutamylation on alpha-tubulin is not essential but affects the assembly and functions of a subset of microtubules in Tetrahymena thermophila.Eukaryot. Cell. 2008; 7: 1362-1372Crossref PubMed Scopus (69) Google Scholar]. Initiation of polyglutamylation is probably carried out by some TTLL protein(s) other than CrTTLL9; in other organisms, different TTLL proteins have been assigned to the initiation and elongation of polyglutamylation [1Janke C. Rogowski K. van Dijk J. Polyglutamylation: A fine-regulator of protein function? ‘Protein modifications: Beyond the usual suspects’ review series.EMBO Rep. 2008; 9: 636-641Crossref PubMed Scopus (73) Google Scholar]. Database search indicates that Chlamydomonas genome contains ∼10 TTLL proteins. Indirect fluorescent microscopy with the polyE antibody showed staining of the wild-type axoneme over the entire length (Figure 2A ). In extensively frayed axonemes, staining occurred on single or bundled microtubules except for the central pair, which can be distinguished from the outer doublet by its strongly curved shape [9Kamiya R. Nagai R. Nakamura S. Rotation of the central-pair microtubules in Chlamydomonas flagella.in: Sakai H. Mohri H. Borisy G. Biological Functions of Microtubules and Related Structures. Academic Press, New York1982: 189-198Crossref Google Scholar] (Figure S2). Because we encountered no outer doublet that lacked polyE staining, it is likely that all of the nine outer doublets are polyglutamylated. The staining intensity of doublet bundles in frayed axonemes did not show a gradient, unlike the staining pattern of the whole axoneme (Figure 2A). Thus, the origin of the graded staining pattern in the wild-type axoneme is not clear. In a striking contrast with the wild-type axoneme, the tpg1 axoneme was stained only in the proximal 1–2 μm region of the 10–11 μm flagellum (Figures 2A and 2B). This observation is consistent with the immunoblot results showing that long polyglutamate side chains are greatly decreased but not completely eliminated in tpg1. The polyglutamylation at the proximal portion may well be carried out by other TTLL protein(s). Immunoelectron microscopy with colloid gold indicated that long-chain polyglutamylation takes place predominantly on the B-tubule of outer doublet in the wild-type axoneme (Figure 2C). These observations are consistent with the results of a previous study [10Lechtreck K.F. Geimer S. Distribution of polyglutamylated tubulin in the flagellar apparatus of green flagellates.Cell Motil. Cytoskeleton. 2000; 47: 219-235Crossref PubMed Scopus (58) Google Scholar]. In contrast, tpg1 axonemes showed very low levels of staining (Figure 2C). We raised polyclonal antibodies against the total length of CrTTLL9 expressed in E. coli. Western blot of detergent-extracted wild-type flagella showed that this protein is present mostly in the axoneme fraction and is extractable with 0.6 M KCl (Figures 3A and 3B ). In tpg1, this antibody did not detect bands corresponding to CrTTLL9 or truncated products in either axoneme or detergent-soluble fractions. Because the expression level of the CrTTLL9 gene (the FAP267-encoding gene) has been shown to increase upon deflagellation [6Pazour G.J. Agrin N. Leszyk J. Witman G.B. Proteomic analysis of a eukaryotic cilium.J. Cell Biol. 2005; 170: 103-113Crossref PubMed Scopus (729) Google Scholar], we examined the change in the amount of the flagellar CrTTLL9 protein after deflagellation in the wild-type axoneme. As shown in Figures 3C and 3D, the amount of CrTTLL9 in a constant weight of axoneme increased after deflagellation by pH shock and decreased to the basic level following flagellar regeneration. The relative CrTTLL9 amount per flagellum can be estimated by multiplying the protein amount by the average flagellar length. This value also showed an increase immediately after deflagellation and a decrease following the flagellar growth, indicating that excess CrTTLL9 protein is removed as flagella grow (Figure 3D). With the same antibodies, we were unable to detect CrTTLL9 in the wild-type axonemes via immunofluorescence. To examine whether the loss of CrTTLL9 causes any defects in axonemal structure, we examined isolated axonemes by electron microscopy (data not shown), analyzed dynein composition by ion-exchange chromatography (Figures S3A and S3B), and measured the average length (Figure S3C) and the time course of flagellar growth after deflagellation (Figure S3D). None of these assays detected any differences between wild-type and mutant axonemes. Therefore, we conclude that loss of long-chain tubulin polyglutamylation does not interfere with the formation of axoneme and assembly of dyneins. The tpg1 mutant swam at a velocity that was 70%–80% as fast as the wild-type velocity (Figure 4A ). The flagellar beat frequency in tpg1 was reduced to a similar extent, indicating that the tpg1 and wild-type cells move for a similar distance per flagellar beat (Figure 4B). Thus, the flagellar beat pattern does not appear to greatly differ between the two strains (Movies S1 and S2), although direct waveform analysis must be performed to conclude it. Strikingly, however, the double mutant of tpg1 and the mutant oda2 lacking outer-arm dynein completely lacked motility, whereas oda2 swam at about 30% of the wild-type velocity (Figure 4A; Movies S3 and S4). In contrast to the difference in motility, axonemal ATPase activities were almost the same between wild-type and tpg1 and between oda2 and oda2tpg1 (Table S1). To explore the function of polyglutamylation on axonemal motility, we next examined the microtubule sliding in disintegrating axonemes induced by treatment with protease and ATP [11Summers K.E. Gibbons I.R. Adenosine triphosphate-induced sliding of tubules in trypsin-treated flagella of sea-urchin sperm.Proc. Natl. Acad. Sci. USA. 1971; 68: 3092-3096Crossref PubMed Scopus (494) Google Scholar, 12Okagaki T. Kamiya R. Microtubule sliding in mutant Chlamydomonas axonemes devoid of outer or inner dynein arms.J. Cell Biol. 1986; 103: 1895-1902Crossref PubMed Scopus (64) Google Scholar] (Figure 4C). The tpg1 axoneme underwent sliding at almost the same velocity as the wild-type axoneme. Unexpectedly, however, the axoneme of paralyzed oda2tpg1 displayed much faster sliding than the axoneme of motile oda2. Thus, long-chain tubulin polyglutamylation in oda2 axonemes apparently functions to suppress microtubule sliding produced by inner-arm dyneins under low-load conditions wherein no axonemal bending takes place. The presence or absence of long polyglutamate chains must be severely affecting the function of inner-arm dyneins, but not so much that of outer-arm dynein. Chlamydomonas has seven major species of inner-arm dyneins called dyneins a–g [13Kagami O. Kamiya R. Translocation and rotation of microtubules caused by multiple species of Chlamydomonas inner-arm dynein.J. Cell Sci. 1992; 103: 653-664Crossref Google Scholar, 14Yagi T. Uematsu K. Liu Z. Kamiya R. Identification of dyneins that localize exclusively to the proximal portion of Chlamydomonas flagella.J. Cell Sci. 2009; 122: 1306-1314Crossref PubMed Scopus (92) Google Scholar]. Of these, dynein f (also called I1) is the only two-headed species containing two heavy chains, and all others are one-headed species containing a single heavy chain. The subunit composition of dynein f/I1 is totally different from other inner-arm dyneins. To examine which type of inner-arm dynein species is most strongly affected by tubulin polyglutamylation, we next examined the effect of the tpg1 mutation on the motility of two inner-arm-deficient mutants, ida1 and ida5. The mutant ida1 lacks dynein species f/I1 because of a mutation in one of its heavy chains [15Myster S.H. Knott J.A. O'Toole E. Porter M.E. The Chlamydomonas Dhc1 gene encodes a dynein heavy chain subunit required for assembly of the I1 inner arm complex.Mol. Biol. Cell. 1997; 8: 607-620Crossref PubMed Scopus (84) Google Scholar], whereas ida5 lacks species a, c, d, and e because of the loss of actin [16Kato-Minoura T. Hirono M. Kamiya R. Chlamydomonas inner-arm dynein mutant, ida5, has a mutation in an actin-encoding gene.J. Cell Biol. 1997; 137: 649-656Crossref PubMed Scopus (83) Google Scholar], a common subunit of one-headed inner-arm dyneins. We found that the double mutant ida1tpg1 swam 5–6 times slower than ida1 (Figure 4A), with flagella beating at about 70% frequency of ida1 (Figure 4B). The reduction in swimming velocity is apparently caused by a significant decrease in flagellar bend angle, as is evident from the extremely small amplitude of the envelope of beating flagella (data not shown). The other double mutant, ida5tpg1, displayed a distinct motility phenotype; like ida5 cells, the ida5tpg1 cells swam slowly, but unlike ida5 mutants alone, double mutants tended to stick to the glass surface a few seconds after the onset of observation under the microscope. A fraction (∼40%) of cells remained motile when observation was performed under red (>630 nm) light. In this case, the average velocity of swimming cells was ∼50 μm/s, i.e., ∼70% of the ida5 swimming velocity (Figure 4A). Thus, in this mutant, flagellar beating did not appear to be so greatly impaired as in ida1tpg1 as long as the cells did not stick to the surface in response to light. The reason for this light sensitivity is not understood. These observations suggest that polyglutamylation must be affecting the dynein species remaining in ida1 (outer-arm dynein and one-headed inner-arm dynein species a–e and g) more severely than those remaining in ida5 (outer-arm dynein, two-headed inner-arm dynein species f, and one-headed species b and g). From the comparison of the dynein species involved, we suppose that polyglutamylation affects one-headed dyneins more strongly than two-headed dynein. However, these observations do not rule out the possibility that polyglutamylation also affects two-headed inner-arm dyneins to some extent, because ida5tpg1 displayed weaker motility than ida5. Microtubule sliding velocity in disintegrating axonemes also showed a difference between the two inner-arm mutants; the sliding velocity in ida1 slightly increased in the background of tpg1 mutation, whereas no change occurred in ida5 (Figure 4C). Thus, in this experiment also, the tpg1 mutation affects the ida1 axoneme more significantly than the ida5 axoneme. How the tpg1 mutation lowers flagellar motility while increasing microtubule sliding velocity in some mutant axonemes remains to be studied. It is conceivable that lack of long, negatively charged polyglutamate side chains from the microtubule causes a significant effect on the strength of dynein-microtubule interaction. Electrostatic interaction between dynein and the C-terminal portion of tubulin has been suggested to be critical for the processive movements in inner-arm dynein species c [17Sakakibara H. Kojima H. Sakai Y. Katayama E. Oiwa K. Inner-arm dynein c of Chlamydomonas flagella is a single-headed processive motor.Nature. 1999; 400: 586-590Crossref PubMed Scopus (152) Google Scholar], as well as in cytoplasmic dynein [18Wang Z. Sheetz M.P. The C-terminus of tubulin increases cytoplasmic dynein and kinesin processivity.Biophys. J. 2000; 78: 1955-1964Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar]. The function of other dyneins may well also critically depend on the surface charge of the microtubule. Interestingly, the microtubule binding site of dynein, the stalk tip, is positively charged in all kinds of inner-arm dyneins, and the stalk tip of a one-headed species, dynein e, has the highest pI among all dyneins (T.Y., unpublished data). In the background of the tpg1 mutation, electrostatic dynein-microtubule interaction should decrease. We speculate that the decreased interaction may increase sliding velocity by preventing some slow-moving dynein from entering into a strong binding state or by decreasing interdoublet friction that acts as a drag against sliding. At the same time, a strong interaction of inner-arm dyneins with microtubules may be prerequisite for axonemal beating, which requires stronger force generation than simple microtubule sliding. However, the exact mechanism by which the tpg1 mutation accelerates microtubule sliding and inhibits flagellar beating must await further studies. In summary, the novel Chlamydomonas mutant tpg1 lacking a tubulin polyglutamylating enzyme revealed that lack of long polyglutamate side chains in doublet microtubules specifically interferes with motility. In particular, the tpg1 mutation causes complete loss of motility in oda2 lacking outer-arm dynein and an extremely slow swimming velocity in ida1 lacking two-headed inner-arm dyneins. Curiously, the mutation increased microtubule sliding velocity in the background of oda2 or ida1. These findings support the idea that CrTTLL9 tubulin glutamic acid ligase most significantly affects the function of one-headed inner-arm dyneins. In accordance with this conclusion, a recent study in Tetrahymena concluded that tubulin polyglutamylation mediated by TTLL6, a chain elongase for β-tubulin, regulates ciliary motility by restraining the activity of inner-arm dynein (Suryavanshi et al. [19Suryavanshi S. Edde B. Fox L.A. Guerrero S. Hard R. Hennessey T. Kabi A. Malison D. Pennock D. Winfield S.S. et al.Tubulin glutamylation regulates ciliary motility by altering inner dynein arm activity.Curr. Biol. 2009; (in press. Published online February 25, 2010)https://doi.org/10.1016/j.cub.2009.12.062Abstract Full Text Full Text PDF Scopus (87) Google Scholar], this issue of Current Biology). Different sensitivities to polyglutamylation among different dyneins may be due to the difference in intrinsic properties of dyneins. Alternatively, it may be due to a biased localization of polyglutamylated tubulin in the B-tubule. How tubulin polyglutamylation affects dynein function, as well as how it takes place specifically on the B-tubule, must await further studies. We thank M.A. Gorovsky (University of Rochester) for providing polyE antibody and J. Gaertig (University of Georgia) and W. Sale (Emory University) for discussion and critical reading of the manuscript. This study has been supported by a grant from the Japan Society for the Promotion of Science (JSPS). Download .pdf (1.22 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Three Figures, and One Table Download .avi (.53 MB) Help with avi files Movie S1. Movement of Wild-Type Cells, Related to Figure 4Flagellar movements in wild-type cells. Taken at 500 frames/s. Beat frequency is ∼60 Hz. Download .avi (.38 MB) Help with avi files Movie S2. Movement of tpg1 Cells, Related to Figure 4Flagellar movements in tpg1 cells. Taken at 500 frames/s. Beat frequency is ∼50 Hz. Download .avi (1.09 MB) Help with avi files Movie S3. Movement of oda2, Related to Figure 4Cells of oda2 with flagella beating at ∼30 Hz. Taken at 30 frames/s. Download .avi (.96 MB) Help with avi files Movie S4. Movement of oda2tpg1, Related to Figure 4Cells of oda2tpg1 with paralyzed flagella. Taken at 30 frames/s. Tubulin Glutamylation Regulates Ciliary Motility by Altering Inner Dynein Arm ActivitySuryavanshi et al.Current BiologyMarch 4, 2010In BriefHow 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]. Full-Text PDF Open Archive" @default.
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• W2054442207 title "Tubulin Polyglutamylation Regulates Axonemal Motility by Modulating Activities of Inner-Arm Dyneins" @default.
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