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• W2767908258 abstract "•TTLL-11 and CCPP-1 fine-tune ciliary microtubule glutamylation•Velocity of OSM-3/KIF17 and KLP-6/KIF28 motors is sensitive to glutamylation defects•TTLL-11 and CCPP-1 are required for ciliary extracellular vesicle release•TTLL-11 and CCPP-1 and microtubule glutamylation specialize ciliary ultrastructure Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a “tubulin code” that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia. Ciliary microtubules (MTs) are extensively decorated with post-translational modifications (PTMs), such as glutamylation of tubulin tails. PTMs and tubulin isotype diversity act as a “tubulin code” that regulates cytoskeletal stability and the activity of MT-associated proteins such as kinesins. We previously showed that, in C. elegans cilia, the deglutamylase CCPP-1 affects ciliary ultrastructure, localization of the TRP channel PKD-2 and the kinesin-3 KLP-6, and velocity of the kinesin-2 OSM-3/KIF17, whereas a cell-specific α-tubulin isotype regulates ciliary ultrastructure, intraflagellar transport, and ciliary functions of extracellular vesicle (EV)-releasing neurons. Here we examine the role of PTMs and the tubulin code in the ciliary specialization of EV-releasing neurons using genetics, fluorescence microscopy, kymography, electron microscopy, and sensory behavioral assays. Although the C. elegans genome encodes five tubulin tyrosine ligase-like (TTLL) glutamylases, only ttll-11 specifically regulates PKD-2 localization in EV-releasing neurons. In EV-releasing cephalic male (CEM) cilia, TTLL-11 and the deglutamylase CCPP-1 regulate remodeling of 9+0 MT doublets into 18 singlet MTs. Balanced TTLL-11 and CCPP-1 activity fine-tunes glutamylation to control the velocity of the kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 without affecting the intraflagellar transport (IFT) kinesin-II. TTLL-11 is transported by ciliary motors. TTLL-11 and CCPP-1 are also required for the ciliary function of releasing bioactive EVs, and TTLL-11 is itself a novel EV cargo. Therefore, MT glutamylation, as part of the tubulin code, controls ciliary specialization, ciliary motor-based transport, and ciliary EV release in a living animal. We suggest that cell-specific control of MT glutamylation may be a conserved mechanism to specialize the form and function of cilia. Cilia and flagella are antenna-like organelles that protrude from most eukaryotic cells and serve sensory and motility functions that are important for development, physiology, and behavior. Cilia have a conserved structural core called an axoneme, composed of microtubules (MTs) that typically form a ring of nine outer A-B doublet MTs surrounding two or zero inner singlets—the so-called “9+2” or “9+0” formations in motile or primary/sensory cilia, respectively [1Fisch C. Dupuis-Williams P. Ultrastructure of cilia and flagella—back to the future!.Biol. Cell. 2011; 103: 249-270Crossref PubMed Scopus (119) Google Scholar]. Although virtually all cilia are built by a conserved intraflagellar transport (IFT) process and share a similar architecture, cilia and flagella adopt morphological specializations and serve diverse functions [1Fisch C. Dupuis-Williams P. Ultrastructure of cilia and flagella—back to the future!.Biol. Cell. 2011; 103: 249-270Crossref PubMed Scopus (119) Google Scholar]. For example, the rods and cones of the retina are elaborately shaped cilia [2Falk N. Lösl M. Schröder N. Gießl A. Specialized cilia in mammalian sensory systems.Cells. 2015; 4: 500-519Crossref PubMed Scopus (62) Google Scholar], whereas sperm have simple whip-like flagella that are variable in length and axoneme structure [3Konno A. Ikegami K. Konishi Y. Yang H.J. Abe M. Yamazaki M. Sakimura K. Yao I. Shiba K. Inaba K. Setou M. Ttll9−/− mice sperm flagella show shortening of doublet 7, reduction of doublet 5 polyglutamylation and a stall in beating.J. Cell Sci. 2016; 129: 2757-2766Crossref PubMed Scopus (27) Google Scholar]. C. elegans amphid channel cilia, mammalian olfactory cilia, and mammalian renal primary cilia possess a proximal doublet region followed by a distal A tubule singlet region [1Fisch C. Dupuis-Williams P. Ultrastructure of cilia and flagella—back to the future!.Biol. Cell. 2011; 103: 249-270Crossref PubMed Scopus (119) Google Scholar, 2Falk N. Lösl M. Schröder N. Gießl A. Specialized cilia in mammalian sensory systems.Cells. 2015; 4: 500-519Crossref PubMed Scopus (62) Google Scholar, 4Perkins L.A. Hedgecock E.M. Thomson J.N. Culotti J.G. Mutant sensory cilia in the nematode Caenorhabditis elegans.Dev. Biol. 1986; 117: 456-487Crossref PubMed Scopus (674) Google Scholar, 5Tsuji T. Matsuo K. Nakahari T. Marunaka Y. Yokoyama T. Structural basis of the Inv compartment and ciliary abnormalities in Inv/nphp2 mutant mice.Cytoskeleton (Hoboken). 2016; 73: 45-56Crossref PubMed Scopus (10) Google Scholar]. Another ciliary specialization is the ability to produce extracellular vesicles (EVs) called ectosomes [6Wood C.R. Huang K. Diener D.R. Rosenbaum J.L. The cilium secretes bioactive ectosomes.Curr. Biol. 2013; 23: 906-911Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 7Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Wang J. Kaletsky R. Silva M. Williams A. Haas L.A. Androwski R.J. Landis J.N. Patrick C. Rashid A. Santiago-Martinez D. et al.Cell-specific transcriptional profiling of ciliated sensory neurons reveals regulators of behavior and extracellular vesicle biogenesis.Curr. Biol. 2015; 25: 3232-3238Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 9Long H. Zhang F. Xu N. Liu G. Diener D.R. Rosenbaum J.L. Huang K. Comparative analysis of ciliary membranes and ectosomes.Curr. Biol. 2016; 26: 3327-3335Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 10Cao M. Ning J. Hernandez-Lara C.I. Belzile O. Wang Q. Dutcher S.K. Liu Y. Snell W.J. Uni-directional ciliary membrane protein trafficking by a cytoplasmic retrograde IFT motor and ciliary ectosome shedding.eLife. 2015; 4: e05242Crossref Scopus (80) Google Scholar, 11Salinas R.Y. Pearring J.N. Ding J.D. Spencer W.J. Hao Y. Arshavsky V.Y. Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release.J. Cell Biol. 2017; 216: 1489-1499Crossref PubMed Scopus (76) Google Scholar]. The molecular underpinnings and functions of these specializations are only beginning to be appreciated. Regulation of the function of conserved ciliogenesis proteins by post-translational modifications (PTMs) of MTs could provide a mechanism for generating structural and functional diversity of cilia. Ciliary MTs are marked by diverse PTMs that have been proposed to act as a “tubulin code” to regulate particular motors, MT-binding proteins, and MAPs (MT-associated proteins) [12Verhey K.J. Gaertig J. The tubulin code.Cell Cycle. 2007; 6: 2152-2160Crossref PubMed Scopus (394) Google Scholar, 13Janke C. The tubulin code: molecular components, readout mechanisms, and functions.J. Cell Biol. 2014; 206: 461-472Crossref PubMed Scopus (356) Google Scholar, 14Sirajuddin M. Rice L.M. Vale R.D. Regulation of microtubule motors by tubulin isotypes and post-translational modifications.Nat. Cell Biol. 2014; 16: 335-344Crossref PubMed Scopus (349) Google Scholar]. The tubulin tyrosine ligase-like (TTLL) family of proteins includes glutamylases, which act as writers of the tubulin code by adding or elongating glutamate side chains on MTs [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar]. Carboxypeptidases of the M14D deglutamylase subfamily act as erasers of the tubulin code that remove or reduce the length of glutamate side chains on tubulins [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar, 16Rogowski K. van Dijk J. Magiera M.M. Bosc C. Deloulme J.C. Bosson A. Peris L. Gold N.D. Lacroix B. Bosch Grau M. et al.A family of protein-deglutamylating enzymes associated with neurodegeneration.Cell. 2010; 143: 564-578Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar]. Hence, MT glutamylation is a reversible modification, and a balance of glutamylase and deglutamylase activity may fine-tune the extent or pattern of glutamylation in tubulin C-terminal tails [13Janke C. The tubulin code: molecular components, readout mechanisms, and functions.J. Cell Biol. 2014; 206: 461-472Crossref PubMed Scopus (356) Google Scholar]. Ciliary MTs are heavily glutamylated [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar]. Defects in glutamylation are implicated in human ciliopathies. Joubert syndrome [17Lee J.E. Silhavy J.L. Zaki M.S. Schroth J. Bielas S.L. Marsh S.E. Olvera J. Brancati F. Iannicelli M. Ikegami K. et al.CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium.Nat. Genet. 2012; 44: 193-199Crossref PubMed Scopus (129) Google Scholar], blindness [18Sergouniotis P.I. Chakarova C. Murphy C. Becker M. Lenassi E. Arno G. Lek M. MacArthur D.G. Bhattacharya S.S. Moore A.T. et al.UCL-Exomes ConsortiumBiallelic variants in TTLL5, encoding a tubulin glutamylase, cause retinal dystrophy.Am. J. Hum. Genet. 2014; 94: 760-769Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar], and schizophrenia [19Fullston T. Gabb B. Callen D. Ullmann R. Woollatt E. Bain S. Ropers H.H. Cooper M. Chandler D. Carter K. et al.Inherited balanced translocation t(9;17)(q33.2;q25.3) concomitant with a 16p13.1 duplication in a patient with schizophrenia.Am. J. Med. Genet. B. Neuropsychiatr. Genet. 2011; 156: 204-214Crossref PubMed Scopus (14) Google Scholar] are associated with defects in TTLL glutamylases. Defects in the Ccp1 deglutamylase cause neuronal degeneration in mice [20Fernandez-Gonzalez A. La Spada A.R. Treadaway J. Higdon J.C. Harris B.S. Sidman R.L. Morgan J.I. Zuo J. Purkinje cell degeneration (pcd) phenotypes caused by mutations in the axotomy-induced gene.Nna1. Science. 2002; 295: 1904-1906Crossref PubMed Scopus (185) Google Scholar]. How dysregulated glutamylation might contribute to human disease is largely unknown. C. elegans possess variant 9+0 cilia whose ultrastructures can be simultaneously analyzed using transmission electron microscopy (TEM) and electron tomography [4Perkins L.A. Hedgecock E.M. Thomson J.N. Culotti J.G. Mutant sensory cilia in the nematode Caenorhabditis elegans.Dev. Biol. 1986; 117: 456-487Crossref PubMed Scopus (674) Google Scholar, 21Doroquez D.B. Berciu C. Anderson J.R. Sengupta P. Nicastro D. A high-resolution morphological and ultrastructural map of anterior sensory cilia and glia in Caenorhabditis elegans.eLife. 2014; 3: e01948Crossref PubMed Scopus (116) Google Scholar]. Variant 9+0 cilia are not nematode-specific oddities. Variations from the “typical” 9+2 and 9+0 doublet structures may be more common than appreciated, largely due to technical difficulty of serial-section TEM of mammalian cilia. Of the 302 neurons in the C. elegans hermaphrodite, 60 have dendritic endings that terminate in cilia [22Inglis P.N. Ou G. Leroux M.R. Scholey J.M. The sensory cilia of Caenorhabditis elegans.WormBook. 2007; : 1-22PubMed Google Scholar]. In addition to the shared ciliated nervous system (common between hermaphrodites and males), the C. elegans male possesses 48 ciliated neurons of 385 total neurons [22Inglis P.N. Ou G. Leroux M.R. Scholey J.M. The sensory cilia of Caenorhabditis elegans.WormBook. 2007; : 1-22PubMed Google Scholar]. Specialized male-specific cilia shed and release bioactive EVs that contain the polycystin receptor LOV-1 and TRP channel PKD-2 [7Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar]. The diversity of C. elegans sensory cilia enables study of ciliary specialization and the role of the tubulin code in this process in a living animal. CCPP-1, a homolog of the mammalian deglutamylase Ccp1, is required in C. elegans sensory neuronal cilia to regulate MT stability [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. In nematodes lacking CCPP-1, EV-releasing cephalic male-specific CEM cilia contain fewer MTs [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. In CEM cilia, CCPP-1 regulates localization of the ciliary TRP channel PKD-2 and the kinesin-3 KLP-6, and the velocity of homodimeric kinesin-2 OSM-3/KIF17 without affecting the anterograde heterotrimeric kinesin-II motor [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. These pleiotropic defects are likely to result from MT hyperglutamylation. CEM cilia display an ultrastructural specialization in which nine MT doublets splay into nine A tubule and nine B tubule singlets in middle regions of the axoneme but remain joined in distal and proximal regions [24Silva M. Morsci N. Nguyen K.C.Q. Rizvi A. Rongo C. Hall D.H. Barr M.M. Cell-specific α-tubulin isotype regulates ciliary microtubule ultrastructure, intraflagellar transport, and extracellular vesicle biology.Curr. Biol. 2017; 27: 968-980Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The α-tubulin isotype TBA-6 is essential for B tubule singlet formation, and hence the tubulin code is implicated in generating this specialized EV-releasing cilium [24Silva M. Morsci N. Nguyen K.C.Q. Rizvi A. Rongo C. Hall D.H. Barr M.M. Cell-specific α-tubulin isotype regulates ciliary microtubule ultrastructure, intraflagellar transport, and extracellular vesicle biology.Curr. Biol. 2017; 27: 968-980Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar]. The mammalian and C. elegans genomes encode nine and five ttll glutamylases, respectively [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar, 25Kimura Y. Kurabe N. Ikegami K. Tsutsumi K. Konishi Y. Kaplan O.I. Kunitomo H. Iino Y. Blacque O.E. Setou M. Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).J. Biol. Chem. 2010; 285: 22936-22941Crossref PubMed Scopus (78) Google Scholar]. TTLL glutamylases are biochemically distinguished by their preferences for the C-terminal tails of α- or β-tubulin and whether they are initiases (adding the first E) or elongases (extending chains of polyE) [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar]. However, the true physiological function of each TTLL enzyme is not known. Here we focus on how hypoglutamylation resulting from genetic ablation of the TTLL glutamylases affects the cilia of a set of C. elegans male-specific EV-releasing neurons (EVNs) that express PKD-2 [26Barr M.M. Sternberg P.W. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.Nature. 1999; 401: 386-389Crossref PubMed Scopus (408) Google Scholar]—specifically, the four CEM neurons in the head, the HOB (hook B type) neuron in the tail, and 16 RnB (ray B type, where n = 1–9, excluding ray 6) neurons that innervate the copulatory fan structure of the male tail. We show that CCPP-1 deglutamylase and TTLL-11 glutamylase act in concert to sculpt the CEM axoneme and regulate ciliary kinesin-2 OSM-3/KIF17 and kinesin-3 KLP-6 motors. CEM cilia are functionally specialized to shed and release bioactive EVs. We find that CCPP-1 and TTLL-11 are required for environmental release of EVs from ciliated neurons in C. elegans males, and that TTLL-11 itself is a novel EV cargo. Our results suggest that CCPP-1 and TTLL-11 fine-tune glutamylation to regulate ciliary transport, EVs, and axonemal structure in cilia. ccpp-1 is widely expressed in ciliated sensory neurons in hermaphrodites and males [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. Here we focus on the role of glutamylation in ciliary specialization of the male-specific EVNs, where CCPP-1-mediated regulation of MT glutamylation is important for appropriate localization of the ciliary TRP channel PKD-2::GFP. PKD-2::GFP abnormally accumulates in ccpp-1 mutant cilia and distal dendrites (Figure 1A) [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar]. To identify the TTLL glutamylase that opposes CCPP-1 in EVNs, we hypothesized that loss of a TTLL glutamylase might suppress the ccpp-1 PKD-2::GFP ciliary (Cil) defective phenotype caused by hyperglutamylation. The C. elegans genome encodes five TTLL family members: TTLL-4, TTLL-5, TTLL-9, TTLL-11, and TTLL-15 (http://www.wormbase.com). (We exclude TTLL-12 for two reasons: it is an ortholog to mammalian TTLL12, which lacks glutamylase and glycylase activity [15Yu I. Garnham C.P. Roll-Mecak A. Writing and reading the tubulin code.J. Biol. Chem. 2015; 290: 17163-17172Crossref PubMed Scopus (126) Google Scholar], and is not neuronally expressed [http://www.wormbase.com].) Mutant alleles that delete portions of coding regions were available for each of the five ttll genes: ttll-4(tm3310); ttll-5(tm3360); ttll-9(tm3389); ttll-11(tm4059); ttll-11(gk482); and ttll-15(tm3871). We examined ttll mutants for the ability to suppress the ccpp-1 PKD-2::GFP Cil phenotype. None of the tested TTLL glutamylase deletion mutations suppressed the ccpp-1 PKD-2::GFP localization defect. However, the ttll-11(tm4059) and ttll-11(gk482) deletion mutants displayed a PKD-2::GFP Cil phenotype similar to ccpp-1 mutants, with PKD-2::GFP abnormally accumulating in ciliary bases and distal dendrites (Figures 1A and S1). These results suggest that regulated MT glutamylation is essential for normal ciliary localization and abundance of PKD-2. We conclude that different TTLL enzymes may act in a cell-specific manner and may possess different enzymatic activities in vivo. The C. elegans ttll-11 locus encodes two isoforms: the long TTLL-11B and short TTLL-11A proteins [25Kimura Y. Kurabe N. Ikegami K. Tsutsumi K. Konishi Y. Kaplan O.I. Kunitomo H. Iino Y. Blacque O.E. Setou M. Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).J. Biol. Chem. 2010; 285: 22936-22941Crossref PubMed Scopus (78) Google Scholar] (http://www.wormbase.com). A BLAST search of the human TTLL11 long isoform (UniProtKB: Q8NHH1) against the C. elegans WS259 protein database identified both TTLL-11B and TTLL-11A as top hits [28Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. Basic local alignment search tool.J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70322) Google Scholar], and alignments showed that amino acids 213–742 from the human TTLL11 long isoform are 35% identical and 58% similar to C. elegans TTLL-11B amino acids 119–642 and TTLL-11A amino acids 19–542. TTLL-11A lacks the first 100 amino acids of TTLL-11B but is otherwise identical (Figure 1B) [25Kimura Y. Kurabe N. Ikegami K. Tsutsumi K. Konishi Y. Kaplan O.I. Kunitomo H. Iino Y. Blacque O.E. Setou M. Identification of tubulin deglutamylase among Caenorhabditis elegans and mammalian cytosolic carboxypeptidases (CCPs).J. Biol. Chem. 2010; 285: 22936-22941Crossref PubMed Scopus (78) Google Scholar] (http://www.wormbase.com). TTLL-11B contains a putative myristoylation sequence at its N terminus [27Eisenhaber F. Eisenhaber B. Kubina W. Maurer-Stroh S. Neuberger G. Schneider G. Wildpaner M. Prediction of lipid posttranslational modifications and localization signals from protein sequences: big-Pi, NMT and PTS1.Nucleic Acids Res. 2003; 31: 3631-3634Crossref PubMed Scopus (71) Google Scholar]. We previously showed that myristoylation is necessary for targeting and function of the EV regulator and EV cargo CIL-7 [29Maguire J.E. Silva M. Nguyen K.C. Hellen E. Kern A.D. Hall D.H. Barr M.M. Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis.Mol. Biol. Cell. 2015; 26: 2823-2832Crossref PubMed Scopus (38) Google Scholar]. The ttll-11(tm4059) deletion allele is predicted to produce an early stop codon after 90 amino acids in TTLL-11A and 190 amino acids in TTLL-11B (Figure 1B; http://www.wormbase.com), removing all except seven amino acids of the predicted ATP-grasp_4 domain [30Marchler-Bauer A. Derbyshire M.K. Gonzales N.R. Lu S. Chitsaz F. Geer L.Y. Geer R.C. He J. Gwadz M. Hurwitz D.I. et al.CDD: NCBI’s conserved domain database.Nucleic Acids Res. 2015; 43: D222-D226Crossref PubMed Scopus (2256) Google Scholar]. The gk482 deletion allele affects the coding region of only the TTLL-11B long isoform (Figure 1B) (http://www.wormbase.com). Both the tm4059 and the gk482 alleles produced a PKD-2::GFP Cil phenotype (Figures 1A and S1). Therefore, at least the TTLL-11B isoform is required for normal localization of PKD-2. Hereafter, unless specifically noted as the ttll-11b(gk482) allele, reference to mutation of ttll-11 indicates the tm4059 allele, which affects both isoforms. To examine where the ttll isoforms function, we created transcriptional reporters. The ttll-11b reporter was exclusively expressed in ciliated EVNs. Expression of GFP [31Chalfie M. Tu Y. Euskirchen G. Ward W.W. Prasher D.C. Green fluorescent protein as a marker for gene expression.Science. 1994; 263: 802-805Crossref PubMed Scopus (5487) Google Scholar] driven by the ttll-11b promoter was observed in the inner labial type 2 (IL2) ciliated sensory neurons in both males and hermaphrodites, as well as the male-specific PKD-2-expressing neurons (CEMs in the head; HOB and RnBs in the tail; Figures 1C and 1D). The IL2, CEM, HOB, and RnB neurons comprise the EVNs, which release bioactive EVs to the environment [7Wang J. Silva M. Haas L.A. Morsci N.S. Nguyen K.C. Hall D.H. Barr M.M. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication.Curr. Biol. 2014; 24: 519-525Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 8Wang J. Kaletsky R. Silva M. Williams A. Haas L.A. Androwski R.J. Landis J.N. Patrick C. Rashid A. Santiago-Martinez D. et al.Cell-specific transcriptional profiling of ciliated sensory neurons reveals regulators of behavior and extracellular vesicle biogenesis.Curr. Biol. 2015; 25: 3232-3238Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar]. The ttll-11a promoter drove GFP expression in a distinct and non-overlapping set of ciliated sensory neurons in the head, including the IL1s, outer labial quadrant neurons (OLQs), cephalic neurons (CEPs), and amphids, but not in the EVNs (Figure 1E). In the male tail, expression was seen in the hook A type neuron (HOA), ray type A neurons (RnAs), and phasmids but not the HOB and RnB neurons (Figure 1F). Expression was also seen in phasmid neurons in the hermaphrodite tail (data not shown). These expression patterns suggest that TTLL-11B functions in the EVNs and is essential for normal localization of PKD-2, whereas TTLL-11A functions in other ciliated sensory neuronal types. The polycystin PKD-2 and the male-specific EVNs mediate male mating behaviors [26Barr M.M. Sternberg P.W. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.Nature. 1999; 401: 386-389Crossref PubMed Scopus (408) Google Scholar]. Because ttll-11b expressed in these neurons and the ttll-11 mutant displayed abnormal accumulation of PKD-2::GFP, we examined the mating behaviors of ttll-11 mutant males (Figures 1G and 1H). ttll-11 mutant males were “Lov” defective (i.e., abnormal location of the vulva substep of mating behavior), but not “Rsp” defective (i.e., the response substep of mating behavior was normal). These results suggest that abnormal glutamylation impairs the function of these male-specific EVNs, and that hyperglutamylation caused greater impairment than hypoglutamylation. The RnBs play a role in response behavior, whereas the HOB functions in the location of vulva behavior [26Barr M.M. Sternberg P.W. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.Nature. 1999; 401: 386-389Crossref PubMed Scopus (408) Google Scholar]. The fact that ttll-11 mutants are Lov defective, but not Rsp defective, suggests that requirements for MT glutamylation may not be identical even among the neurons that express ttll-11 and ccpp-1 and mediate mating behaviors. Alternatively, location of vulva behavior may be more sensitive to abnormal glutamylation, because a single pair of neurons (HOB and HOA) senses the vulva, whereas multiple ray neurons redundantly sense the hermaphrodite for response behavior [26Barr M.M. Sternberg P.W. A polycystic kidney-disease gene homologue required for male mating behaviour in C. elegans.Nature. 1999; 401: 386-389Crossref PubMed Scopus (408) Google Scholar]. ttll-11 encodes a glutamylase and ttll-11 mutations are predicted to reduce glutamylation. To determine whether TTLL-11 regulates glutamate side-chain length, we analyzed glutamylation state by immunodetection with a polyclonal polyglutamylation (polyE) antibody (IN105) that recognizes chains of 3 or more glutamates [32van 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 (182) Google Scholar]. Glutamylation in cephalic CEM and CEP cilia was undetectable by polyE staining in ttll-11(tm4059) mutant males, which lack both TTLL-11B and TTLL-11A (Figure S2). However, in the absence of TTLL-11B only, some ciliary polyE staining remained in cephalic cilia. To determine whether TTLL-11 is required for glutamylation branchpoint initiation, we stained animals with the monoclonal antibody GT335, which detects the branchpoint of glutamylation side chains on tubulin C-terminal tails [23O’Hagan R. Piasecki B.P. Silva M. Phirke P. Nguyen K.C. Hall D.H. Swoboda P. Barr M.M. The tubulin deglutamylase CCPP-1 regulates the function and stability of sensory cilia in C. elegans.Curr. Biol. 2011; 21: 1685-1694Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 33Wolff A. de Néchaud B. Chillet D. Mazarguil H. Desbruyères E. Audebert S. Eddé B. Gros F. Denoulet P. Distribution of glutamylated α and β-tubulin in mouse tissues using a specific monoclonal antibody, GT335.Eur. J. Cell Biol. 1992; 59: 425-432PubMed Google Scholar]. In ttll-11(tm4059) mutants, glutamylation was undetectable by GT335 staining in all cilia, including those in male-specific EVNs (Figure 2). In ttll-11b(gk482) mutant males, GT335 still stained ci" @default.
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• W2767908258 date "2017-11-01" @default.
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• W2767908258 title "Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia" @default.
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