# Patent

## Title
Biological system and assay for identifying modulators of tubulin ligases

## Abstract

Tetrahymena is used as a host cell in a biological assay for identification of modulators of tubulin ligases.

## Background
This application is a continuation-in-part of U.S. application Ser. No. 11/479,098, filed Jun. 30, 2006, which claims the benefit of U.S. Provisional Application Ser. No. 60/695,776, filed Jun. 30, 2005, each of which is incorporated herein by reference in its entirety.


This invention was made with government support under grants from the National Science Foundation, Grant Nos. MBC-0235826 and MBC-033965, and under a grant from the National Institutes of Health, Grant No. R37GM051173. The U.S. Government has certain rights in this invention.


BACKGROUND OF THE INVENTION
Microtubules are fibers made of α-tubulin and β-tubulin dimers. Microtubules form cytoplasmic networks and serve as frameworks of important organelles, including the mitotic spindle, centrioles, cilia and bundles inside neurites. The biogenesis of microtubules involves the synthesis of tubulin polypeptides, chaperonin-assisted folding and dimerization of α-tubulin and β-tubulin, transport to the sites of assembly, nucleation, polymerization, deposition of post-translational modifications (PTMs), and binding of diverse microtubule-associated proteins (MAPs).
During mitosis, the microtubule undergoes dramatic changes to transition from an interphase monopolar organization to the bipolar spindle. Thus, tubulins are a major target of anti-cancer drugs which act by disrupting the dynamic properties of MTs during mitosis and in some cases inducing apoptosis. However currently available widely used microtubule-targeting compounds (such as vinblastine or paclitaxel) suffer from a major limitation—they target microtubules indiscriminately. Paclitaxel, for example, a compound that hyperstabilizes microtubules and blocks cells in mitosis, is currently the most widely used drug to treat ovarian, breast, lung cancers and AIDS-related Kaposi's sarcoma (Ring et al. (2005) Cancer Treat Rev 31, 618-627; Cheung et al., (2005) Oncologist 10, 412-426). However, paclitaxel produces strong side effects by affecting non-mitotic microtubules, in particular in nerve cells (Hennenfent et al. (2006). Ann Oncol. 17, 735-749). Paclitaxel also affects the bone marrow leading to hematopoietic deficiencies in  ̃90% of patients (Hagiwara et al. (2004) Breast Cancer 11, 82-85). Ideally, future anti-microtubule compounds should affect as few cell types as possible besides intended targets. Humans have several isotypes of α-tubulin and β-tubulin, some of which are expressed in a restricted fashion. However, tubulins are highly conserved and differ mainly in the small portion of their primary sequence near the C-terminal end (Luduena (1998) Int. Review Cytol. 178, 207-274). Thus, developing isotype-specific inhibitors for tubulin primary polypeptides is likely to be difficult.
Post-translational modifications of microtubules are ubiquitously present in eukaryotes and their physiological importance is increasingly well documented (Rosenbaum (2000) Current Biol. 10, R801-R803, Westermann et al. (2003) Nat Rev Mol Cell Biol 4, 938-947). The most studied post-translational modifications include acetylation of α-tubulin, detyrosination of α-tubulin, palmitoylation of α-tubulin, and phosphorylation, glutamylation, and glycylation of α-tubulin and β-tubulin.
Post-translational modifications are believed to function in regulating interactions of microtubules with MAPs (such as dynein and kinesin motors) (Rosenbaum (2000) Current Biol. 10, R801-R803), Westermann et al. (2003) Nat Rev Mol Cell Biol 4, 938-947). Most post-translational modifications are located on the C-terminal tails of tubulins, highly flexible acidic domains present on the surface of microtubules (Nogales et al. (1999) Cell 96, 79-88). The tails are also the major sites of interactions with kinesin and dynein motors, structural MAPs (MAP2, Tau), microtubule-severing protein katanin, and plus end-depolymerizer (MCAK) (Skiniotis et al. (2004) Embo J 23, 989-999, Ovechkina et al. (2002) J Cell Biol 159, 557-562, Lu et al. (2004) Mol Biol Cell 15, 142-150). By regulating the activity of tubulin modifying enzymes, cells can mark microtubules in specific subcellular areas to regulate binding and activity of MAPs in a localized fashion. Detyrosination of α-tubulin promotes transport of vimentin intermediate filaments mediated by kinesin-1 (Kreitzer et al. (1999) Mol. Biol. Cell 10, 1105-1118). Another post-translational modification, polyglycylation, appears to acts as a mark to regulate assembly of cilia and severing of stable cortical microtubules in Tetrahymena (Thazhath et al. (2002) Nature Cell Biol. 4, 256-259; Thazhath et al. (2004) Mol Biol Cell 15, 4136-4147). The basic principle of specific post-translational modifications acting alone or in combination to regulate binding of a variety of microtubule interactors is likely to be general. By analogy with the epigenetic “histone code”, eukaryotic cells appear to utilize a “microtubule code” to coordinate MAPs.
Glutamylation, a conserved post-translational modification, occurs by addition of a variable number of glutamate (also known as glu or “E”) residues onto specific glutamate residues of the C-terminal tail domain of α-tubulin or β-tubulin (Eddé et al. (1990) Science 247, 83-85), Weber et al. (1996) FEBS Lett. 393, 27-30). Besides tubulins only the nucleosome assembly proteins, NAP-1 and NAP-2 are known to undergo glutamylation (Regnard et al. (2000) J. Biol. Chem. 275, 15969-15976). The added glutamates form a peptide side chain using the gamma-carboxyl group of the glutamate in the primary sequence. Due to its negative charge and bulky nature, glutamate side chains have a strong structural impact on microtubules. Because the number of added glutamates changes as the function of age of microtubules and cell cycle stage, this post-translational modification is probably not a simple on/off signal. Rather, glutamylation may act like a rheostat to fine tune the function of microtubules. Tubulin glutamylation is particularly abundant on microtubules inside cellular projections including neuritis (Wolff et al. (1992) Eur. J. Cell Biol. 59, 425-432) and cilia (Bré et al. (1994) Cell Motility and the Cytoskeleton 27, 337-349). Glutamylation accumulates on microtubules of centrioles and in the central part of the mitotic spindle (Bobinnec et al. (1998) Cell Motil. Cytoskeleton 39, 223-232). Tubulin glutamylation is important in vivo. Injection of antibodies specific to glutamylated tubulin caused disassembly of centrioles in mammalian cells (Bobinnec et al. (1998) J. Cell Biol. 143, 1575-1589), suggesting that inhibitors of glutamylation could have anti-mitotic properties. A mutation of a subunit associated with the TTLL1 glutamylase in the mouse blocked assembly of sperm axonemes and affected behavior (Campbell et al. (2002) Genetics 162, 307-320). In vitro, glutamylation of microtubules strongly affects binding of kinesins and structural MAPs, MAP2 and Tau (Bonnet et al. (2000) J. Biol. Chem. 276, 12839-12848, Boucher et al. (1994) Biochemistry 33, 12471-12477).
Inhibitors of a forward post-translational modification enzyme are not known in the art. Furthermore, inhibitors are known for only one of the reverse post-translational modification enzymes, tubulin deacetylase HDAC6 (related to histone deacetylases). Overexpression of HDAC6 decreased acetylation and increased chemotactic motility of mammalian cells (Hubbert et al. (2002) Nature 417, 455-458). HDAC6 can be inhibited with trichostatin A (a broad inhibitor of deacetylases) (Matsuyama et al. (2002) Embo J 21, 6820-6831), and tubacin (a specific inhibitor) (Haggarty et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4389-4394). Chemically blocking HDAC6 increased the level of acetylation on microtubules and decreased cell motility as well as disrupted localization of the p58 MAP (a Golgi-microtubule linker) in vivo (Haggarty et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 4389-4394). Inhibitors of HDAC6 has helped to uncover potential new functions for α-tubulin acetylation, including its role in the immune synapse formation (Serrador et al. (2004) Immunity 20, 417-428), and during infection of cells by HIV (Valenzuela-Fernandez et al. (2005) Mol Biol Cell 16, 5445-5454). HDAC6 is upregulated in the acute myeloid leukemia cells (Bradbury et al. (2005) Leukemia 19, 1751-1759), and is one of the estrogen-responsive genes in breast carcinoma. Blocking HDAC6 with tubacin inhibited estradiol-induced cell migration of breast carcinoma cells (Saji et al. (2005) Oncogene 24, 4531-4539). Tubacin also increased anti-cancer effects of other compounds, including a proteasome inhibitor, bortezomid (Hideshima et al. (2005) Proc Natl Acad Sci USA 102, 8567-8572). Although HDAC6 effects could be mediated by at least two different substrates (α-tubulin and HSP90, (Kovacs et al. (2005) Mol Cell 18, 601-607), it is very likely that the effects on cell motility are microtubule-mediated. Importantly, HDAC6 is required for the synergistic inhibitory action of paclitaxel and lonafarmib (an inhibitor of farnesyltransferase) on cancer cells (Marcus et al. (2005). Cancer Res 65, 3883-3893). However, these targeting efforts are limited to one post-translational modification and specifically, to one enzyme, HDAC6 deacetylase. A general strategy for identifying post-translational modification drugs and in particular inhibitors of forward enzymes responsible for deposition of PTMs is needed.
SUMMARY OF THE INVENTION
The invention provides a biological system and assay for identification of inhibitors of enzymes responsible tubulin glutamylation.
In one aspect, the invention is directed to a biological system that includes a host protozoan, preferably a ciliate such as Tetrahymena, that overexpresses a tubulin glutamylase, preferably Tetrahymena Ttll6Ap, Ttll6Bp, Ttll6Cp, Ttll6Dp, Ttll6Ep or Ttll6Fp. In one embodiment, the Tetrahymena overexpresses a wild-type tubulin glutamylase. In another embodiment, the Tetrahymena expresses a modified tubulin glutamylase, for example a truncated form of a Tetrahymena tubulin glutamylase that is missing the C-terminal region responsible for targeting the glutamylase to the cilia. A modified tubulin glutamylase can include, for example, a subunit of a tubulin glutamylase or a mutated tubulin glutamylase having mutations at one or more sites, or a fusion construct that includes all or part of a tubulin glutamylase. A preferred truncated tubulin glutamylase is Tetrahymena Ttll6Ap-Δ710. Preferably, the tubulin glutamylase is under the control of a metallothionein promoter, more preferably an MTT1 promoter. MTT1 is conveniently inducible by cadmium.
The invention should be understood as including a Tetrahymena cell or cell line that overexpresses a wild-type or modified tubulin glutamylase as described above. In a particularly preferred embodiment, the Tetrahymena cell or cell line contains polynucleotide sequence encoding a wild-type or modified tubulin glutamylase that is integrated into the β-tubulin 1 (BTU1) locus. A preferred construct for use in transforming the Tetrahymena includes an MTT1 promoter region upstream of a region that encodes a fusion protein. The fusion protein optionally includes a marker protein, such as green fluorescent protein (GFP), followed by a tubulin glutamylase, or vice versa. Preferably, the construct encodes the marker protein followed by an in-frame coding region of a genomic sequence of the tubulin glutamylase gene, preferably the Tetrahymena TTLL6A gene (locus number 25.m00404 at Tetrahymena genome database; The Institute for Genomic Research website at www.tigr.org), or a truncated version thereof, TTLL6A-Δ710. A particularly preferred Tetrahymena is one that expresses Ttll6Ap-GFP or Ttll6Ap-Δ710-GFP.
In another aspect, the invention is directed to a Tetrahymena tubulin glutamylase, as well as a nucleic acid encoding a Tetrahymena tubulin glutamylase. The invention encompasses amino acid sequences having at least about 90%, preferably at least about 95%, and more preferably at least about 98% identity to the amino acid sequences described herein.
In another aspect, the invention is directed toward a high throughput in vivo assay for identification, validation and/or analysis of selective inhibitors of tubulin glutamylases. The assay is performed using a protozoan, preferably the ciliate Tetrahymena, a model protist with a sophisticated microtubular cytoskeleton. The Tetrahymena used in the in vivo assay overexpresses a tubulin glutamylase, as described herein. This assay is a phenotypic screen based on rescue from growth arrest caused by overexpression of a tubulin glutamylase enzyme in Tetrahymena. 
In another aspect, the invention is directed toward an in vitro assay for identification, validation and/or analysis of selective inhibitors of tubulin glutamylases. This assay makes use of a glutamylation reaction using a purified Tetrahymena enzyme and microtubules. A variant of this assay, useful as a primary screen, employs luciferase activity as a readout signal.
Inhibitors of tubulin glutamylases are expected to be therapeutically useful to treat or prevent many diseases including cancer and disorders of central nervous system including mental diseases. They may also find utility as a male contraceptive.
Accordingly, the present invention is directed to a Tetrahymena cell that overexpresses a tubulin glutamylase. Preferably the Tetrahymena includes a polynucleotide operably encoding a tubulin glutamylase, wherein the polynucleotide is integrated into a BTU1 locus. The polynucleotide preferably includes an MTT1 promoter region upstream of a region encoding a fusion protein, which fusion protein incorporates a marker protein and a tubulin glutamylase. The marker protein is preferably an optically detectable protein; more preferably it is a fluorescent protein such as green, red or blue fluorescent protein. The tubulin glutamylase may be a wild-type or a modified tubulin glutamylase; preferably the tubulin glutamylase is Tetrahymena Ttll6Ap glutamylase or Ttll6Ap-Δ710 glutamylase. A preferred Tetrahymena cell is one that overexpresses Tetrahymena TTLL6A gene (locus number 25.m00404 at Tetrahymena genome database), or its truncated version, Tetrahymena TTLL6A-Δ710.
The invention further provides a method for identifying an inhibitor of tubulin glutamylase that includes contacting a Tetrahymena cell of the invention with a candidate inhibitor compound; and detecting an increase in cell growth and/or motility; wherein an increase in cell growth or motility is indicative of tubulin glutamylase inhibition.
In another aspect, the invention provides a genetically engineered Tetrahymena cell that expresses tubulin exhibiting reduced polymodification compared to a wild-type Tetrahymena cell. Polymodification can take the form of, for example, polyglutamylation or polyglycylation.
In one embodiment of the genetically engineered Tetrahymena cell that exhibits reduced polyglutamylation, the tubulin present in the Tetrahymena cell has shortened glutamyl side chains compared to wild-type Tetrahymena tubulin. In a preferred embodiment, at least a portion of the shortened glutamyl side chains contain less than 6 glutamic acid residues, and the density, or proportion, of the glutamyl side chains containing less than 6 glutamic acid residues is increased relative to wild-type Tetrahymena tubulin. More preferably, at least portion of the shortened glutamyl side chains contain only one glutamic acid residue, and the density, or proportion, of the glutamyl side chains containing only one glutamic acid residue is increased relative to wild-type Tetrahymena tubulin.
In one embodiment of the genetically engineered Tetrahymena cell that exhibits reduced polyglycylation, tubulin present in the Tetrahymena cell lacks glycyl side chains that are present in wild-type tubulin. In a preferred embodiment, the tubulin glycylation level is less than 80%, more preferably less than 90%, and even more preferably less than 95%, of the tubulin glycylation level in wild-type Tetrahymena tubulin.
In another preferred embodiment of the Tetrahymena cell, a plurality of the tubulin glutamic acid ligase genes, preferably TTLL6 genes, or a plurality of the tubulin glycine ligase genes, preferably TTLL3 genes, of the Tetrahymena cell have been disrupted.
A preferred genetically engineered Tetrahymena cell has reduced function of at least two paralogs of TTLL6 or TTLL3. Paralogs of TTLL6 may be selected from the group consisting of TTLL6A, TTLL6B, TTLL6C, TTLL6D, TTLL6E, and TTLL6F. Paralogs of TTLL3 may be selected from the group consisting of TTLL3A, TTLL3B, TTLL3C, TTLL3D, TTLL3E, and TTLL3F. Particularly preferred genetically engineered Tetrahymena cells are knockout cells, and include a TTLL6A/TTLL6F knockout cell, a TTLL6A/TTLL6B/TTLL6D/TTLL6F knockout cell, a TTLL6A/TTLL6B/TTLL6D/TTLL6E/TTLL6F knock out cell, a TTLL3A/TTLL3B knockout cell, a TTLL3A/TTLL3B/TTLL3C/TTLL3D knockout cell, and a TTLL3A/TTLL3B/TTLL3C/TTLL3D/TTLL3E/TTLL3F knockout cell.
Cellular components which, in wild-type Tetrahymena cells, are polymodified with glutamic acid side chains and/or glycine side chains, but which, in the genetically engineered Tetrahymena cells of the invention, exhibit shortened or absent glutamic acid or glycine side chains, can be isolated from genetically engineered Tetrahymena cells of the invention and employed in in vitro assays to identify inhibitors or enhancers of glutamic acid ligases or glycine ligases. Thus, in another aspect, the invention provides an in vitro method for identifying an inhibitor or enhancer of a glutamic acid ligase or a glycine ligase, which method utilizes cellular components isolated from the genetically engineered Tetrahymena of the invention. Preferably, the cellular components used in the assay methods of the invention are tubulin-containing components such as axonemes, microtubules, and tubulin.
In one embodiment, the method is a method for identifying an inhibitor of a glutamic acid ligase that includes contacting a cellular component, such as an axoneme, a microtubule, a stabilized microtubule or tubulin, isolated from the Tetrahymena cell of the invention, with a candidate inhibitor compound and a glutamic acid ligase; and detecting a change in glutamylation of the cellular component, wherein the absence of additional glutamylation, or a slower rate of glutamylation compared to the rate observed in the absence of the candidate inhibitor compound, is indicative of a compound that inhibits glutamic acid ligase.
In another embodiment, the is a method for method for identifying an inhibitor of a glycine ligase that includes contacting a cellular component, such as an axoneme, a microtubule, a stabilized microtubule or tubulin, isolated from the Tetrahymena cell of the invention, with a candidate inhibitor compound and a glycine ligase; detecting a change in glycylation of the cellular component, wherein the absence of additional glycylation, or a slower rate of glycylation compared to the rate observed in the absence of the candidate inhibitor compound, is indicative of a compound that inhibits glycine ligase.
Unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows ciliary localization of Ttll6Ap-GFP (left panel, A), immuno-fluorescence with the anti-glutamylation antibody of cells overproducing Ttll6Ap (middle panel, E) and controls (right panel, H). Abbreviations: sc, subcortical MTs; ic, intracytoplasmic MTs; oc, oral cilia; oa, oral apparatus.
FIG. 2 shows that hyperglutamylation by Ttll6Ap-ΔCT-GFP stabilizes cytoplasmic MTs. Cells overproducing truncated Ttll6Ap-GFP or control cells were treated with 40 μM nocodazole for 30 minutes and labeled by immunofluorescence using anti-α-tubulin antibodies. Note the presence of drug-stable wavy and thick MTs in the cell body of Ttll6Ap-ΔCT cells.
FIG. 3 shows exemplary in vivo and in vitro screens for inhibitors of glutamylases.
FIG. 4 shows fluorescence intensity as a function of Tetrahymena cell concentration; cells with MTT1-driven GFP, 100 μl/well, 96 well black plastic optical bottom plates, 485/530 nm.
FIG. 5 shows characterization of the neuronal glutamylase. (A) A Coomassie Brilliant Blue stained NEPHGE 2D gel of a purified glutamylase fraction, immunoprecipitated from 200 3-day old mouse brains using mAb 206. All numbered protein spots were identified by nano-LC-MS-MS (Table I). “HC” and “LC” mark the positions of heavy and light chains of mAb 206. (B) Immunoprecipitations with mAb 206, L83 (anti-p32/PGs1; Regnard et al., J Cell Sci 116, 4181 (2003)), L80 (anti-p79) and DM1A (Mab anti-α-tubulin) used as a control. Equal proportions of input and supernatants were assayed for glutamylase activity. (C) Input, supernatants and beads (equal proportions) were analyzed by western blotting. The glutamylase activity, and all 5 glutamylase complex subunits were strongly depleted from the supernatants. The proteins were quantitatively recovered with the beads of mAb 206, L83 and L80, but not of DM1A. Arp1 did not quantitatively co-purify with the enzyme.
FIG. 6 shows a structural model of TTLL 1. (A) Crystal structure of glutathione synthetase from E. coli (pdb code 1GSA) in a complex with ADP, glutathione, Mg2+ and sulphate (Hara et al., Biochemistry 35, 11967 (1996)). (B) A model of mouse TTLL1 (PGs3) in a complex with ATP, Mg2+ and a protein substrate shown in ball-and-stick representation. The regions of TTLL1 that could not be modeled are drawn as thin lines. (C) A close-up view of the active center of TTLL 1. The protein substrate is a three-residue peptide with a central modified glutamate. The flanking amino acids are drawn only with Cβ atoms for clarity of the picture. The glutamate side chain contains two additional glutamate residues (Glu 1, Glu 2). The putative site for the next glutamate residue to be added to the side chain is indicated (Glu 3 site). All residues of the active site that are conserved with other ATP-dependent carboxylate-amine ligases are shown in dark green (see FIG. 11). Some positively charged amino acid residues (K 13, K 142 and K215 in light grey) that are specific to TTLL1 and close to the active site might be important for substrate binding. The proximity of the carboxyl group of Glu 2 and the phosphate of ATP could catalyze the formation of an acylphosphate intermediate (broken line).
FIG. 7 shows that Ttll1p of Tetrahymena is associated with tubulin glutamylation. (A) A cell expressing Ttll1p-GFP, labeled by immunofluorescence using anti-GFP antibodies. (B-D) Glutamylated MTs labeled by ID5 antibody in a cell overproducing Ttll1p-GFP (B), a wild type cell (C) and a TTLL1-null cell (D). Note a strong reduction in the labeling of rows of basal bodies in D as compared to C. (E) Immunoblotting studies on total Tetrahymena cell extracts before (−) and after (+) 3 hrs cadmium induction of Ttll1p-GFP, analyzed with anti-GFP (GFP), anti-glutamylation GT335 and polyE antibodies (arrowhead points to Ttll1p-GFP; * marks the comigrating α- and β-tubulin bands). (F) The soluble (S) and cytoskeletal (C) fractions from cells overproducing Ttll1p-GFP (+) or non-induced controls (−) were analyzed for tubulin glutamylase activity in vitro. (G, H) Soluble fractions of cells overproducing Ttll1p-GFP were subjected to immunoprecipitation with anti-GFP antibodies. The input (In), unbound (Un) and bound (Bo) fractions were analyzed by immunoblotting with anti-GFP antibodies (G; arrowhead TG points to Ttll1p-GFP, HC to the anti-GFP heavy chain) and assayed for glutamylase activity in vitro (H). Equal volumes of input, unbound and bead fractions were analyzed by immunoblotting, while 20 times more of bead fraction was assayed for glutamylase activity. Abbreviations: oa, oral apparatus; noa, new oral apparatus prior to cell division; df, deep fiber; bb, basal bodies.
FIG. 8 shows an evolutionary tree of TTL domain proteins, based on the Neighbor-Joining method. Numbers correspond to bootstrap values for 100 repeats. Dots represent predicted TTL domain sequences in several genomes analyzed. The grey lines denote clades discussed in the paper. The yeast sequence was used as an outgroup. See FIGS. 12 and 13 for complete data.
FIG. 9 shows that Ttll6p-GFP is associated with strong tubulin glutamylation in vivo and in vitro. (A-D) GFP fluorescence (A, B) and immunofluorescence images using ID5 anti-glutamylation antibody (C, D) of cells overproducing Ttll6Ap-GFP (A, C) or Ttll6ApΔ710-GFP (B, D). The staining detected in A, B co-localized with MTs visualized by anti-α-tubulin antibodies (results not shown). Note appearance of strong glutamylation in the cell body in cells overproducing forms of Ttll6Ap, while glutamylation is restricted to cilia and basal bodies in control cells (compare with the wild type cell shown in FIG. 3C). Abbreviations: c, cilia; oc, oral cilia; oa, oral apparatus; bb, basal bodies; sc, subcortical MTs, ic, intracytoplasmic MTs. (E) Tubulin glutamylase assays using soluble (S) and cytoskeletal (C) fractions from Ttll6ApΔ710-GFP cells grown in the absence (−) and presence (+) of cadmium for 3 hrs. (F) Immunoblots of total cells (anti-GFP) or cytoskeletons of the following strains following a 3 hr cadmium treatment: wild type (WT), cells overproducing GFP (GFP), Ttll6ApΔ710-E422G-GFP (Δ710-G), Ttll6ApΔ710-GFP (Δ710), and Ttll6Ap-GFP (Ttll6p). The antibodies used were: anti-GFP (GFP), anti-α-tubulin (12G10), anti-glutamylation (ID5), anti-polyglycylation (polyG). (G-I) Cells overproducing an enzymatically active Ttll6Ap and not an ATPase-deficient form fail to multiply and undergo ciliary paralysis. Growth curves on SPP medium for strains expressing Ttll6Ap-GFP (G), Ttll6ApΔ710-GFP or Ttll6ApΔ710-E422G-GFP (H) with or without cadmium induction. Note that only the ATPase-capable proteins reduce the growth rate. (I) The percentage of motile cells in several overproducing strains. After 3 hrs of cadmium induction 100-200 cells were scored for vigorous motility. Either extremely sluggish (showing rotations but no directional movement) or completely paralyzed cells were counted as non-motile. The data shown in all panels represent mean values from three independent experiments.
FIG. 10 shows co-purification of the glutamylase subunits and enzymatic activity.
FIG. 11 shows a sequence profile alignment of 1GSA (SEQ ID NO:6) and mTTLL1 (SEQ ID NO:7) for TTLL1 modeling.
FIG. 12 shows a phylogenetic analysis of predicted protein gene sequences containing a TTL domain.
FIG. 13 shows a multiple sequence alignment of TTL domains (SEQ ID NOs:8-65) of TTL and TTLL proteins.
FIG. 14 shows glutamylation activity of Ttll6Ap in vitro.
FIG. 15 shows overproduction of GFP-Ttll6Ap causes elongation of glutamyl side chains on tubulin in vivo. (A) Western blots of total proteins from GFP-Ttll6Ap-expressing cells that are either uninduced (0 h) or induced for 1, 2, or 4 h with either 1 or 2.5 μg of CdCl2/ml. (B and C) 2D silver-stained protein gels containing cytoskeletons of uninduced (B) or induced (C) GFP-Ttll6Ap cells. The arrows point at strings of highly acidic protein isoforms that appear at the mass level of the main spots of α- and β-tubulin upon induction of GFP-Ttll6Ap. (D to E) Confocal fluorescence images of the GFP signal in growing (D) and cilia regenerating (E) GFP-Ttll6Ap cells induced with 2.5 μg of CdCl2/ml for 3 h. Note the increase of the signal of GFP-Ttll6Ap in short growing cilia (arrowheads) and on subcortical microtubules (arrows). Bar, 10 μm.
FIG. 16 shows Imaging of Tetrahymena cells expressing either full length or truncated GFP-Ttll6Ap. (A, B) Fluorescence confocal images of GFP-Ttll6Ap overexpressed in a growing cell induced for 2 hr with 2.5 μg/ml CdCl2 (A) or in a starved cell induced for 2 hr with 0.05 μg/ml CdCl2 (B). Note predominant localization of GFP-Ttll6Ap to short assembling cilia (arrows) and weaker signal in the full length cilia. (C-J) Fluorescence confocal images of GFP in cells overproducing truncated variants of GFP-Ttll6Ap after 3 hr of 2.5 μg/ml CdCl2 induction. Bar=10 μm. Note that the variant lacking the 240 N-terminal amino acids (Ttll6Ap M241-L1217) showed increased ciliary signal, and accumulated at the tips of cilia (C). An enrichment at the ciliary tips was also observed for a fragment with a larger N-terminal deletion that lacked the TTL-like catalytic domain, R712-L1217 (H). Furthermore, shortening of the M241-L1217 protein from the C-terminal end (M241-V929 and M241-Q828) resulted in a localization pattern that was similar to the localization pattern of the full length protein (D, E compare to FIG. 15D) however note that the majority of GFP-Ttll6Ap-M241-Q828 is associated with cytoplasmic microtubules and only weak signal is observed in growing oral cilia (arrowhead). Thus, the N-terminal 240 (M1-E240) and the C-terminal 288 (K930-L1217) amino acids appear to contain determinants that promote and inhibit uniform ciliary localization of Ttll6Ap, respectively. Note also that further deletions of the C-terminal, microtubule targeting fragment from either protein end created fragments (R712-V929 (I) and R924-L1217 (J)) that failed to localize to cilia. Thus, either multiple parts of the R712-L1217 region are required for ciliary localization or deletions at both ends of this fragment affect its conformation in the context of ciliary targeting. (K) A scheme illustrating the organization of amino acid sequences of expressed variants. The arrows pointing up mark enzymatically active proteins. The “+” or “−” mark presence or absence of the Ttll6Ap variant in the cilia.
FIG. 17 shows overproduced GFP-Ttll6Ap increases the levels of tubulin polyglutamylation on ciliary and cell-body microtubules in a time- and overexpression strength-dependent manner. (A to D) Confocal fluorescence images of GFP-Ttll6Ap cells that are untreated (A) or induced with 2.5 μg of CdCl2/ml for 1 (B), 2 (C), or 3 h (D) and subjected to immunofluorescence with a MAb that recognized polyglutamylation (ID5). We reduced the gain levels in B to D to avoid overexposure. Arrows point to short hyperglutamylated cilia, arrowheads points to hyperglutamylated cell body microtubules. Bar, 10 μm. (E to G) Graphs that document the distribution of cells with distinct pattern of tubulin hyperglutamylation as shown in panels A to D, as a function of either cadmium concentration or duration of induction with cadmium.
FIG. 18 shows overproduction of GFP-Ttll6Ap results in formation of short, hyperglutamylated axonemes and hypeglutamylation of cytoplasmic, subcortical and spindle microtubules. Microtubules in wild-type (A, A1-A3, C, D, H, I) and GFP-Ttll6Ap overproducing cells (B, B1-B3, E-G, J-L) induced for 3 hr with 2.5 μg/ml CdCl2 and stained with anti-α-tubulin antibodies (A1, B1, C, E, H, J) and polyE antibodies (A2, B2, D, F, I, K). A, A3, B, B3, G, L represent merged images. Note short hyperglutamylated cilia in GFP-Ttll6A cells (B1-B3, arrows), hyperglutamylated spindle in the micronucleus (F, arrow), intranuclear microtubules in the dividing macronucleus (K, arrows) and bundles of hyperglutamylated subcortical and cytoplasmic microtubules. Bar, 10 μm.
FIG. 19 shows mapping the minimal domain of Ttll6Ap that is required for E-ligase activity. (A-F) Immunofluorescence images of cells labeled for polyglutamylation with ID5 mAb that are either wild-type (A) or express specific truncated versions of GFP-Ttll6Ap (B-F) as indicated in the panels. By comparing the signal of ID5 mAb, it is apparent that the only proteins expressed in cells shown in panels B-D are enzymatically active. All images were obtained using the same gain level and consequently the images in shown in B-D are overexposed. Bar=10 μm. (G) Growth rate of wild type cells (+Cd) and cells overexpressing truncated GFP-Ttll6Ap as indicated in the legend.
FIG. 20 shows tubulin hyperglutamylation stabilizes cell body microtubules. (A to I′) Confocal microscopic images of cells stained with anti-α-tubulin MAb 12G10. (A and B) Wild-type cells grown without (A) or with (B) 40 μM paclitaxel. Note the appearance of thick bundles of subcortical microtubules in the drug-treated cells. (C to G). Cells expressing GFP-Ttll6Ap or its truncated variant M241-V929 that are uninduced (C) and induced with cadmium (D to G). Note the appearance of bundles of microtubules in the cell body and macronuclei (arrow) in the overproducing cell. (H to I′) Overexpression of GFP-Ttll6Ap protects subcortical and cytoplasmic microtubules against oryzalin-induced depolymerization. Wild-type (H and H′) and GFP-Ttll6Ap-overexpressing (I and I′) cells grown for 4 h in the presence of 2.5 μg of CdCl2/ml were treated for 30 min with 10 μM oryzalin. Panels H′ and I′ show the internal sections of cells shown in panels H and I, respectively. Note the nearly complete depolymerization of cell body microtubules in the wild-type cells but not in Ttll6Ap-overproducing cells. Bar, 10 μm. (J) A graph documents the percentages of wild-type and GFP-Ttll6Ap cells with cytoplasmic microtubules during oryzalin treatment.
FIG. 21 shows hyperelongation of glutamyl side chains in the cell body causes accumulation of α-tubulin K40 acetylation. (A) Wild-type and GFP-Ttll6Ap cells (indicated by the asterisk) were grown for 4 h in the presence of 2.5 μg of CdCl2/ml and labeled side-by-side with antiacetylated tubulin antibodies (6-11 B-1). (A) Projection image of z-section from the top half of the cell; (A′) section showing the middle part of the cell. Bar, 10 μm.
FIG. 22 shows Overproduction of GFP-Ttll6Ap caused decrease in the levels of tubulin monoglycylation. (A-A′′) levels of tubulin monoglycylation detected by TAP952 antibodies in wild-type (arrow) and GFP-Ttll6Ap cells induced with 2.5 μg/ml CdCl2 for 4 hr. (A′, A′′) Inserts showing higher magnification of cilia in wild-type (A′) and GFP-Ttll6Ap cells (A′′), both regions marked in A with white frame. Note that growing cilia in wild-type cell has higher levels of tubulin monoglycylation than full length cilia (A′, arrow) while short cilia in GFP-Ttll6Ap cells have similar levels of tubulin monoglycylation as full length cilia (A′′, arrow). Bar=10 μm. (B) Quantitative immunofluorescence analysis showing average intensity of tubulin monoglycylation detected by TAP952 antibodies in cilia in wild-type and GFP-Ttll6Ap overproducing cells. Bars represent standard deviation.
FIG. 23 shows hyperelongation of glutamyl side chains delays cilium regeneration. (A to F) Confocal immunofluorescence images of wild-type (A and B) and GFP-Ttll6Ap (C and D), GFP-Ttll6Ap-M241-V929-E662G (E)-, and GFP-Ttll6Ap-M241-V929 (F)-overexpressing cells that regenerated cilia for 1 (A and C), 2 (E and F), or 4 h (B and D) after deciliation. Cells were costained with anti-α-tubulin 12G10 MAb and the anti-polyglutamylation antibody poly(E). Note the shorter size of cilia of GFP-Ttll6Ap cells. Insets show cilia at higher magnifications from the areas indicated by a bracket. (G) A graph shows the average lengths of cilia as a function of time of cilium regeneration. Bar, 10 μM.
FIG. 24 shows tetrahymena cells overexpressing M241-Q828 GFP-Ttll6A regenerate cilia with the rate similar to wild-type cells. Confocal immunofluorescence images of wild-type (A) and GFPTtll6Ap M241-Q828 overexpressing cells (B) that regenerated cilia for 2 hr in culture medium supplied with 2.5 μg/ml cadmium chloride. Cells were co-stained with anti-α-tubulin 12G10 mAb and anti-polyglutamylation antibodies polyE. Note the similar length of cilia of wild-type and GFP-Ttll6Ap M241-Q828 overexpressing cells. Bar, 10 μm.
FIG. 25 shows hyperglutamylation of ciliary microtubules results in structural defects in axonemes. TEM cross-sections (A, B, and D) and longitudinal sections (C, E, and F) of cilia in wild-type (D) and GFP-Ttll6Ap-overproducing (A to C, E, and F) cells. Note that short cilia (A to C) lack a central pair (90, arrows) and that broken microtubules are visible (E and F, arrows). Bar, 200 nm.
FIG. 26 shows paclitaxel partly rescues the destabilizing effect of hyperglutamylation on elongation of assembling cilia in vegetatively growing Tetrahymena. Confocal immunofluorescence images of wild-type (A and B) and GFP-Ttll6Ap-overexpressing (D and E) cells grown without (A and D) or with (B and E) 40 μM paclitaxel. Cells were costained with anti-α-tubulin antibodies and anti-poly(E) antibodies. The arrows in panel E point to hyperglutamylated distal segments. Bar, 10 μm. (C and F) A graph documents the distribution of cilium length in wild-type (C) and GFP-Ttll6Ap-overexpressing (F) cells. Black diamonds indicate the cilium lengths in cells grown without drug, and open triangles represent the cilium lengths in cells grown in the presence of paclitaxel.
FIG. 27 shows Levels of tubulin glutamylation in cilia in wild-type cells treated with paclitaxel. (A) Confocal microscope image of mixed population of wild-type cells (cell on the left) and wildtype cells grown for 4 hr in the presence of 40 μM paclitaxel (cell on the right, indicated by arrow) showing α-tubulin (12G10) in green and levels of tubulin polyglutamylation with polyE in red; (A1-A6) Magnified images of cilia from area indicated by brackets in wild-type cells grown without paclitaxel (A1-A3) or wild-type cells grown in the presence of paclitaxel (A4-A6). Bar, 10 μm.
FIG. 28 shows 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 (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372). 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. 29A.
FIG. 29 shows loss of Ttll6Ap and Ttll6Fp leads to a loss of elongated glutamyl side chains in axonemes. (A). A histogram that documents the average intensity of fluorescence signals detected for antibodies against tubulin or glutamylated tubulin using quantitative immunofluorescence. Wild type (labeled with India ink) and 6AF-KO cells were mixed and processed for double immunofluorescence for total tubulin and glutamylated tubulin (using either GT335 or polyE antibodies). Confocal images were obtained of pairs of wild type and mutant cells located near each other. Signal intensities were measured for individual axonemes using ImageJ. A total of 4 images (8 cells) were analyzed, Between 73 and 196 axonemes were measured for antibody. For any given pair of cells, the mutant signal average was calculated as a percentage of the adjacent wild type cell signal. The histogram contains an average of each image value. Error bars represent standard errors. (B) Images of portions of tubulin regains of 2D gels of axonemal proteins. The arrowhead marks a region that contains a string of highly acidic protein forms migrating near the main spots of tubulin in the wild type that are missing in the 6AF-KO cilia. Note that on SDS-PAGE, ciliate tubulin migrates in an inverted order as compared to most other species (β-tubulin migrates more slowly).
FIG. 30 shows 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.
FIG. 31 shows 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 G, H and FIG. 32) 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. 32. Bar, 100 nm.
FIG. 32 shows Ttll6Ap and Fp primarily affect the polyglutamylation levels on the B-tubule or outer doublets. Images of negatively stained immunogold-labeled (using polyE antibodies) wildtype and 6AF-KO axonemes. Bar, 100 nm.
FIG. 33. 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.
FIG. 34 shows deletion of multiple paralogs genes of TTLL6 progressively reduces the rate of growth, phagocytosis and cell motility.
FIG. 35 shows deletion of multiple TTLL6 paralog genes leads to a progressive loss of tubulin polyglutamylation.
FIG. 36 shows deletion of multiple TTLL6 paralog genes leads to progressive shortening of cilia.
FIG. 37A shows a phylogenetic tree of TTLLs calculated using the conserved catalytic TTL domain by a neighbor-joining method and HsTTL as an outgroup. All known human proteins that have a TTL catalytic domain are included. From other organisms, sequences that were best matches for HsTTLL3 and identified as TTLL3 type sequence in a reverse BLASTp were included. TTLL sequences were obtained from National Center for Biotechnology Information (NCBI), Tetrahymena genome database (available on the worldwide web at ciliate.org/), Ciona intestinalis and Chlamydomonas reinhardtii from DOE Joint Genome Institute (available on the worldwide web at genome.jgipsf.org/ciona4/ciona4.home.html). Abbreviations and accession numbers: Ci, Ciona intestinalis; Cr, Chlamydomonas reinhardtii (C—540087); Dm, Drosophila melanogaster (CG11323 and CG11201); Dr, Danio rerio (AAI17657.1); Gl, Giardia lamblia (XP—001708169.1); Hs, Homo sapiens (TTL, NP—714923.1; TTLL1, CAG30485.1; TTLL2, AAK20169.1; TTLL3, NP—056459.3; TTLL4, Q14679.2; TTLL5, Q6EMB2.1; TTLL6, BAC05032.1; TTLL7, NP—078962.4; TTLL8, XP—943304.2; TTLL9, EAW76406.1; TTLL10, Q6ZVT0.2; TTLL11, NP—919228.2; TTL12, NP—055955.1; TTLL13, NP—001025135.2); Nv, Nematostella vectensis (XP—001636414.1); Sp, Strongylocentrotus purpuratus (X—784103.1); Tt, Tetrahymena thermophila (Ttll3Ap, TTHERM—00666600; Ttll3Bp, TTHERM—00125600; Ttll3Cp, TTHERM—00378750; Ttll3Dp, TTHERM—00196050; Ttll3Ep, TTHERM—00770730; Ttll3Fp, TTHERM—00316230). FIG. 37B-G shows fluorescence confocal images of GFP in cells overproducing GFP-tagged Ttll3Ap-A414-875 (a fragment lacking the catalytic domain and C-terminal part) (B), GFP-Ttll3Bp (C), GFPTtll3Cp (D), GFP-Ttll3Dp, (E) GFP-Ttll3Ep (F) and GFP-Ttll3Fp (G) after 3 hrs of 2.5 μg/ml cadmium chloride induction. Abbreviations: bb, basal body; c, cilium; oa, oral apparatus. Bar, 10 μm.
FIG. 38 shows TTLL3 enzymes increase tubulin monoglycylation in Tetrahymena. FIGS. 38A and B show a cell expressing GFP-Ttll3Ap before (A) and after (B) induction with 2.5 μg/ml CdCl2 for 5 hr, labeled by TAP952. FIG. 38C shows GFP fluorescence in GFP-Ttll3Ap-expressing cells. Scale bar, 10 μm. c, cilia; oc, oral cilia. FIG. 38D shows a fluorogram of an SDS-PAGE gel with brain microtubule proteins after in vitro glycylation with fractions enriched in overexpressed TTLL3 proteins of Tetrahymena. FIG. 38E shows a graph showing levels of incorporation of 3H-glycine (in disintegrations per min) into brain α- or β tubulin in in vitro glycylation assays. FIG. 38F shows Western blots of ciliary proteins from wild-type and cells overproducing either GFP-Ttll3Ap or GFP-DN-Ttll3Ap. FIG. 38G shows a Western blot of cells expressing either GFPTtll3Ap or GFP-DN-Ttll3Ap before (−) and after (+) cadmium induction, probed either with anti-GFP or anti-α-tubulin antibodies.
FIG. 39 shows overproduction of GFP-Ttll3Ap increases tubulin monoglycylation and reduces tubulin glutamylation. FIG. 39A-D′ shows wild type and GFP-Ttll3Ap overproducing cells (marked by arrows) were induced by 2.5 μg/ml cadmium chloride for 5 hrs, mixed and processed side-by side for quantitative immunofluorescence with the following antibodies: TAP952 anti-monoglycylated tubulin (A′); AXO49 anti-polyglycylated tubulin (B′); polyG, anti-polyglycylation side chain (C′) and ID5, anti-polyglutamylated tubulin (D′). Corresponding merged images with the GFP signal are shown in A-D (in A, in the wildtype control, the faint green signal corresponds to autofluorescence of food vacuoles loaded with Ink). FIG. 39A′′-D′′ shows graphs representing the corresponding quantitative immunofluorescence analyses of the average pixel intensity of axoneme sections. N—number of analyzed cilia, P—total number of analyzed pixel. Error bars show standard deviations. Note that there is an increase in the levels of tubulin monoglycylation but not polyglycylation and decrease in the level of tubulin polyglutamylation in cilia of GFP-Ttll3Ap overproducing cells. These data are consistent with Ttll3Ap having a glycine ligase chain initiating activity but lacking chain elongating activity and negatively inhibiting tubulin glutamylation. Bar, 10 μm.
FIG. 40 shows disruption of TTLL3 genes reduces tubulin glycylation and increases tubulin glutamylation. FIG. 40A-D shows WT (fed with India ink) and 3AB-KO cells (arrows) imaged side by side. (A) and (C) show immunofluorescence with TAP952 and AXO49 respectively, while (B) and (D) show the corresponding phase contrast images. Insets show higher magnifications (2.53) of boxed areas of cilia. FIG. 40E-E′ shows growth curves of WT and TTLL3 knockout strains grown without (E) or with (E′) 35 μM paclitaxel. Note that in (E), WT and 3AB-KO curves completely overlap. FIG. 40F shows Western blots of ciliary proteins from WT (loaded at multiple dilutions) and TTLL3 knockout strains. FIG. 40G shows a graph representing the length of cilia in WT and TTLL3 knockout cells. Error bars represent SEM. The differences between WT and 3AB-KO, 3ABCD-KO, and 3ABCDEF-KO cells were statistically significant (p<0.0001). FIG. 40G′-G′′ shows immunofluorescence images of cilia in WT and sextuple knockout cells labeled with anti-tubulin antibodies. Images of the entire cells are shown in FIGS. 41K and L. FIG. 40H-K shows immunofluorescence with anti-total tubulin antibodies of Tetrahymena cells that are either WT (H and J) or 3AB-KO (I and K), and were either untreated (H-I) or treated with 25 μM paclitaxel for 4 hr (J and K).
FIG. 41 shows knockouts of TTLL3 genes reduce the levels of tubulin glycylation and increase the levels of tubulin glutamylation. FIG. 41A-F shows 3AB-KO (A-C) and 3ABCDEF-KO (D-F) cells were mixed with wild type cells (indicated by arrows, earlier fed with India ink for identification) and stained with the following antibodies: TAP952, tubulin monoglycylation (A, D); AXO49, tubulin polyglycylation (B, E) and ID5, tubulin polyglutamylation (C, F). FIG. 41A′-F′ shows graphs contain the corresponding quantitative immunofluorescence data based on the average pixel intensity of axonemes. N—number of analyzed axonemes, P—number of analyzed pixels. Error bars show standard deviations. Note the dramatic reduction in the level of tubulin monoglycylation in both double and sextuple knockouts (A,A′ D,D′) and polyglycylation in the sextuple knockouts (E,E′) and an increase in the level of tubulin polyglutamylation (C,C′, F,F′) in TTLL3 knockout cells. FIG. 41G-J shows knockouts of TTLL3 genes reduce the levels of tubulin polyglycylation in cilia. All cells were analyzed at the same gain level to visualize polyglycylated tubulin in wild type (G) and TTLL3 double (H), quadruple (I) and sextuple (J) knockout cells. Note that wild type and double knockout cells are overexposed. FIG. 41K and L shows immunofluorescence with a mixture of 12G10 anti-α-tubulin and SG anti-total tubulin antibodies of Tetrahymena wild type (K) and 3ABCDEF-KO cells (L). Note the reduction in the length of cilia in TTLL3 knockout cells. The boxed areas are shown at higher magnification in FIG. 40G-G′. Bar, 10 μm.
FIG. 42 shows a knockout of TTLL1 and TTLL9 genes that encode α-tubulin preferring glutamic acid ligases (Wloga et al. 2008. Eukaryot. Cell 7, 1362-1372) reduces the signal of tubulin glutamylation and increases the signal of tubulin monoglycylation in cilia. TTLL1 and TTLL9 knockout cells were mixed with wild type cells (earlier fed with India ink) and stained with TAP952 antibodies to detect monoglycylation (A). A graph representing the quantitative immunofluorescence analysis of the intensity of cilia staining in WT and TTLL1/9 knockout cells; the intensity of staining was measured in the part of cilia indicated by parenthesis (A′). N—number of analyzed cilia, P—number of analyzed pixels. Error bars show standard deviations. Bar, 10 μm.
FIG. 43 shows TTLL3 proteins and tubulin glycylation are restricted to outer microtubules and dominant-negative TTLL3 disrupts the axoneme structure. FIG. 43A-G shows TEM cross-sections of axonemes of WT (A) and GFP-DN-Ttll3Ap-overproducing cells (B-G). The arrow in (G) marks an abnormal triplet. Scale bar, 0.2 μm. FIG. 43H-J shows immunogold localization of either GFPTtll3Ap (H) or GFP-DN-Ttlll3Ap (I and J) with anti-GFP antibodies. Scale bar, 0.2 μm. FIG. 43K-L shows longitudinal TEM sections of cilia of GFPDN-Ttll3Ap overproducing cells. Scale bar, 0.2 μm. FIG. 43M and N shows TEM cross-sections of axonemes of 3AB-KO (M) and 3ABCDEF-KO (N) cells. Scale bar, 0.2 μm. FIG. 430 and P shows immunogold localization of monoglycylated (O) or polyglycylated tubulin (P) in WT cells using TAP952 and AXO49 mAb, respectively. Scale bar, 0.2 μm.
FIG. 44 shows a PCR analysis of quadruple and sextuple knockout cells. To confirm deletion of part of the TTLL3 genes in each locus, wild type, quadruple and sextuple knockout cells were analyzed by PCR with primers that amplify junctions between the targeted regions and non-targeted flanking sequences. The absence of the product indicates that a specific region has been entirely deleted in the tested strain. (A) TTLL3A locus, (B) TTLL3B locus, (C) TTLL3C locus, (D) TTLL3F locus, (E) TTLL3D locus, (F) positive control for PCR amplification of a non-targeted NRK9 locus, (G) TTLL3E locus.
FIG. 45 shows expression of a dominant-negative TTLL3 affects ciliary length. FIG. 45A-C shows cells expressing GFP-DN-Ttll3Ap induced with 2.5 mg/ml CdCl2 for 6 hr. The GFP fluorescence is shown in (A), AXO49 immunofluorescence in (B), and a merged image in (C). GFPDN-Ttlll3Ap is enriched in short, newly formed cilia (arrowheads) and in the distal segments of long preexisting cilia (arrows). FIG. 45D-F shows WT (D), GFP-Ttll3Ap-(E), and GFP-DNTfi13Ap (F)-overexpressing cells were labeled with 12G10 anti-α-tubulin and polyG antibodies. Arrows point at short, new cilia, while arrowheads mark long, pre-existing cilia. FIG. 45G-I′ shows WT (G and G′) and either GFP-Ttll3Ap- (H and H′) or GFP-DN-Ttll3Ap (I and I′)-overproducing cells were double labeled with ID5 anti-polyglutamylated tubulin and polyG antibodies. Note that short cilia and distal segments of elongated cilia in GFP-DN-Ttll3Ap cells are hypoglycylated and hyperglutamylated. Scale bar, 10 μm. FIG. 45J-L) Graphs representing the distribution of axoneme lengths in cells overexpressing either GFP-Ttll3Ap (J) or GFP-DN-Ttll3Ap (K and L) that were uninduced (black diamonds) or induced with CdCl2 (white circles) for 6 (K) or 11 hr (J and L). Each data point represents a single measured axoneme.
FIG. 46 shows overproduction of GFP-DN-Ttll3Ap reduces tubulin glycylation. Wild-type (A-C and G-I) and GFP-DN-Ttll3Ap (D-F) cells were induced with 2.5 μg/ml CdCl2 for 6 hr and imaged at the same gain. Images of GFP or background (A and D), monoglycylation (B and E) and merged signals (C and F) are shown. The GFP-DN-Ttll3Ap cells have lower levels of tubulin monoglycylation and polyglycylation even without transgene induction probably due to leaky expression of the MTT1 promoter. In addition, after the transgene induction, the signal of monoglycylation is nearly non-detectable in locations that accumulate DN-Ttll3Ap (D-F). Bar, 10 μm.
FIG. 47 shows overproduction of GFP-DN-Ttll3Ap inhibits elongation of axonemes during cilia regeneration. Uninduced (A1-A5 and C1-C5) and induced (4.5 hr, 2 μg/ml CdCl2) cells (B1-B5 and D1-D5) expressing either GFP-Ttll3Ap (A1-B5) or GFP-DN-Ttll3Ap (C1-D5), were deciliated, allowed to regenerate cilia in SPP medium (Al-AS and C1-C5) or SPP with 2.5 μg/ml CdCl2 (B1-B5 and D1-D5) and analyzed by immunofluorescence using 12G10 and SG anti-tubulin antibodies within the following 15-120 min. Bar, 10 μm. E. Graphs that document the length of cilia in regenerating cells. Note that induction of DN-Ttll3Ap and not Ttll3Ap inhibits elongation of cilia.
FIG. 48 shows TTLL3 is required for tubulin glycylation and cilia assembly in zebrafish. FIG. 48A-F shows images of live control and morphant embryos at 24 (A and B), 48 (E and F), and 55 hpf. FIGS. 48C and D shows hydrocephaly is apparent in TTLL3-ATG morphant embryos, as indicated by the enlarged vesicles in the hindbrain (B and F, arrows). FIG. 48G shows T=the head of a WT embryo stained by 6-11 B1 antibodies at 72 hpf (the olfactory placode is marked with an arrow). FIG. 48H-I′′ shows in olfactory cilia, TTLL3-ATG MOs reduce the levels of tubulin monoglycylation detected by TAP952 mAb at 72 hpf (compare [H] and [I]), but not the levels of tubulin polyglutamylation revealed by polyE antibodies (H′ and I′′). FIG. 48J and K shows the olfactory cilia stained with AXO49 anti-polyglycylated tubulin mAb in a control (J) and a TTLL3-ATG morphant (K) at 72 hpf. FIG. 48L and M) Olfactory cilia stained with 6-11 B-1 mAb in a control (L) and a TTLL3-ATG morphant (M) at 72 hpf. FIG. 48N-R shows SEM images of the zebrafish olfactory placode at 72 hpf in control (N and O) and a TTLL3-ATG morphant (P and R); (O) and (R) are higher magnifications of areas of placodes shown in (N) and (P). FIG. 48S shows TEM image of a fragment of the olfactory placode in the TTLL3-ATG embryo.
FIG. 49 shows ttll3 MOs reduce tubulin monoglycylation in zebrafish cilia. FIG. 49A-B′ shows ear in a control (A and A′) and a morphant fish (B and B′) labeled with TAP952 mAb. (A′) and (B′) show higher magnifications (5×) of areas boxed in (A) and (B). FIG. 49C-F shows the pronephric duct area in control (C and E) and morphant fish (D and F) labeled with TAP952 anti-monoglycylated tubulin mAb (C and D) and GT335 anti-glutamylated tubulin mAb (E and F). Note lack of cilia staining in morphants in corresponding area (all indicated by arrows). FIG. 49G-J shows the spinal cord and hypochord area in control (G and I) and morphant fish (H and J) labeled with TAP952 (G and H) and GT335 mAb (I and J). Arrows indicate cilia in spinal cord, while arrowheads point to cilia in hypochord in control embryos (G and I). Note lack of cilia staining in morphants in corresponding regions, as indicated by arrows and arrowheads (H and J).
FIG. 50A-C shows a knockdown of TTLL3 expression in zebrafish changes fish morphology and affects cilia in multiple organs. Morphology of control embryos (A) and TTLL3-ATG morphants (B-C) at 48 hpf that were released manually from the chorion after 24 hpf (B) or at 72 hpf released manually from the chorion after 48 hpf (C). FIG. 50D shows results of an RT-PCR assay for TTLL3 mRNA using total RNA from control and morphants injected with 3 or 5 ng of TTLL3-SP MO. FIGS. 50E and F shows depletion of TTLL3 reduces the TAP952 signal of tubulin monoglycylation in pronephron cilia; a control embryo (E) and a TTLL3-ATG morphant (F). FIG. 50G-N shows TTLL3 depletion shortens cilia and reduces the TAP952-positive tubulin monoglycylation. G and H are images of neuromasts stained with 6-11 B-1 anti-acetylated α-tubulin antibodies in control (G) and a TTLL3-ATG morphant (H). FIG. 50I-N shows double labeling of control embryos (I-K) and TTLL3-ATG morphants (L-N) with TAP952 (I and L) and polyE anti-polyglutamylation antibodies (J and M) and merged images (K and N). Note a decrease in the level of tubulin monoglycylation and increase in the level of tubulin polyglutamylation in morphants.
FIG. 51 shows injection of random sequence morpholinos into zebrafish embryos does not affect the levels of monoglycylation detected by TAP952. Uninjected embryos (A-D) and embryos injected with 1 ng of random sequence morpholinos (A′-D′), were fixed at 72 hpf, stained with TAP952 antibodies and the levels of tubulin monoglycylation were analyzed by immunofluoresce in cilia in the otic vesicle (arrowheads) and neuromasts (arrows) (A-A′), spinal cord (B-B′) and in middle (C-C′) and distal (D-D′) part of the pronephric duct. Scale 100 μm.
FIG. 52 shows depletion of ttll3 expression produces defects in the LR asymmetry in zebrafish. Ventral views of 22 hpf embryos stained for expression of lefty1 (A-D), or 33 hpf embryos stained for expression of cmlc2 (E-H). Insets in (A)-(D) depict 43 magnifications of the heads of embryos. In all cases, the left side of the embryo is to the left of the panel. lefty1 is normally expressed on the left side of the epiphysis (A). ttll3 morphant embryos express lefty1 on the left body side (B), or display bilateral (C) or right-sided expression (D). Arrows mark the epiphysis. In untreated embryos, cmlc2 (E) is expressed in the myocardium, which is normally located in the left lateral plate. The position of the heart field is unaltered in some TTLL3 morphants (F). Other morphants display midline (G) or right-side expression (H). Arrows in E-H mark the heart field. (I and J) Cilia in KV stained with 6-11 B-1 anti-acetylated K40 α-tubulin mAb in a control (I) and a TTLL3-ATG morphant (J) at 8-10 somite stage at 14 hpf. (K) A graph represents the average length of cilia in KV in control (n=3) and TTLL3-ATG morphants (n=3). Data are presented as means±SEM.
FIG. 53 shows TEM study of cilia in the olfactory placode in a control (A-D, I) and in a morphant (E-H, J-K). Note that in a control placode, there are axoneme cross-sections that have both inner and outer (A) or only inner (B) dynein arms and some cross-sections lack the central pair (C). The distal ends of control cilia were contain the A-subfiber extensions (D). Ultrastructure of the remaining few morphant cilia was normal except that longitudinal sections show that some cilia have swollen tips (J).



DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention is directed to a new group of enzymes which regulate the assembly and dynamics of microtubules, called tubulin ligases. Two types of tubulin PTMs, known as polymodifications are glutamylation and glycylation. Glutamylation and glycylation are the addition and/or elongation of peptide branches made of either glutamyl or glycyl residues, respectively, that are attached to tubulin. The enzymes responsible for post-translational glutamylation, in which a side branch of one or more glutamic acid is added to the tubulin protein, are tubulin glutamylases, also known as and referred to herein as tubulin glutamic acid ligases or “E-ligases,” wherein the “E” refers to the amino acid abbreviation for glutamic acid. The enzymes responsible for post-translational glycylation, in which a side branch of one or more glycine is added to the tubulin protein, are tubulin glycine ligases, also known as and referred to herein as tubulin glycylases or “G-ligases,” wherein the “G” refers to the amino acid abbreviation for glycine. The invention is further directed to cells having reduced levels of polymodification as well as methods of using said cells.
In the following examples, overproduction of one of these enzymes in Tetrahymena, Ttll6Ap, using MTT1 promoter is shown to lead to cell multiplication arrest of transgenic Tetrahymena. Advantageously, the lethal condition brought about by overproduction of the enzyme can be used to screen for novel compounds which inhibit the enzyme, based on a rescue of the lethal phenotype associated with overproduction. Thus, the invention includes screening methods to identify inhibitors of a tubulin glutamylase, as well as compounds identified using the screening method and methods of using them.
Tetrahymena as a Model System
Tetrahymena is a free-living ciliate and a model eukaryote with a sequenced genome. Tetrahymena has been used in research that led to some key achievements, including the Nobel award winning discovery of self-splicing RNA, and identification of telomeres and telomerase, histone acetyltransferase, dynein, and siRNAs that guide DNA rearrangement (Turkewitz et al. (2002) Trends in Genetics 18, 35-40). The Tetrahymena system is well equipped for reverse genetics (by homologous DNA recombination), and biochemical studies (Collins et al. (2005) Curr Biol 15, R317-318).
Tetrahymena assembles a diverse set of microtubules including those forming the cell body networks, cilia, centriole-like basal bodies, and the mitotic spindle. Due to the importance of their diverse microtubules, ciliates are sensitive to perturbations in microtubule-dependent functions which result in characteristic (potentially screenable) phenotypes (Janke et al. (2005) Science 308, 1758-1762 (see Example I), Thazhath, et al. (2002) Nature Cell Biol. 4, 256-259, Fujiu et al. (2000) Cell Motil Cytoskeleton 46, 17-27).
Tetrahymena posttranslationally modifies its microtubules, using highly conserved post-translational modifications, including extensive glutamylation of microtubules in the basal bodies and cilia (Gaertig (2000) Microbiol. 47, 185-190). Tetrahymena has been in use as a model to dissect the function of post-translational modifications. The use of Tetrahymena led to the first report a mutant phenotype caused by lack of specific sites of post-translational modifications on tubulins (Thazhath et al. (2002) Nature Cell Biol. 4, 256-259), and the discovery of the first post-translational modification forward enzyme (Janke et al. (2005) Science 308, 1758-1762 (see Example I)).
Tetrahymena is attractive for high throughput manipulations due to its: 1) rapid growth (generation time of 3 hrs), 2) low cost of culture, 3) nearly transparent culture medium, 4) ability to grow on defined medium without animal products (lowers the cost of culture and reduced the strigency of required biosafety procedures, 5) lack of pathogenicity, 6) routine culture on 96-well plates, 7) growth to high density in microdrops (compatibility with 384-1536 well plates), 8) lack of autofluorescence (no cell wall or plastids), 9) rapid cell motility (promotes mixing of assay components), 10) sensitivity to established inhibitors (e.g. cycloheximide, paclitaxel) within the concentration range similar to animal cells, 11) methods for introduction of transgenes and protein tagging, 12) targeted mutation approaches that allow for exploration of loss-of-function phenotypes, 13) inducible-repressible promoters that allow for generation of gain-of-function phenotypes, 14) large cell size—highly amenable for HT cytological profiling at low microscopic magnification, 15) advanced cellular functions shared with animal cells including sophisticated microtubule-based organelles, regulated secretion, nuclear apoptosis, DNA rearrangements, phagocytosis, and chemotactic cell motility.
Tubulin Ligases
Microtubules are filamentous structures that have a variety of important functions in living cells, e.g. in cell division, nerve cell differentiation or transport inside the cells. Microtubular networks are involved in a variety of important functions in living cells, e.g. in cell division, nerve cell differentiation or transport inside the cells. In most proliferating mammalian cells, interphase microtubules are glutamylated at a very low level, and only during mitosis.
How microtubule-associated motor proteins are regulated is not well understood. A potential mechanism for spatial regulation of motor proteins is provided by post-translational modifications of tubulin subunits that form patterns on microtubules. Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of α- and β-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (Verhey and Gaertig. 2007. Cell Cycle 6:2152-2160). Two types of polymeric posttranslational modifications of α/β-tubulin occur widely in cilia and flagella. These two PTMs will be referred as “polymodifications.” Polymodifications generate peptide branches of variable lengths that are attached to several acceptor sites in the C-terminal tails of α- and β-tubulin (Redeker et al., (1998) Biochemistry 37, 14838-14844; Schneider et al., (1998) FEBS Lett. 429, 399-402; Vinh et al., (1999) Biochemistry 38, 3133-3139). These modifications, conserved throughout evolution, are thought to act individually or in combination to control specific microtubule-based functions. However, their cellular functions are poorly understood. Polymodifications substantially increase the heterogeneity of the α/β-tubulin heterodimer. These tubulin polymodifications, referred to as glutamylation and glycylation, correspond to the addition of a peptide polymer consisting of several glutamates (Eddé et al., (1990) Science 247, 83-85) or glycines (Redeker et al., (1994) Science 266, 1688-1691) onto the γ-carboxyl group of a glutamate of the primary sequence of tubulin. The C-terminal tail domains of tubulin are also the major binding sites of many microtubule-associated proteins (MAPs) and MT-based molecular motors (kinesins and dyneins). It is thus highly probable that both modifications regulate the interactions between microtubules and their partners.
It appears that microtubule populations can be distinguished between different cell types as well as inside a single cell by specific polymodification patterns. The flexible parameters that could specify those patterns on microtubules are (i) the density of the modification (fraction of modified tubulins within a microtubule), (ii) the choice of the tubulin subunit (α- or β-tubulin), (iii) the length of the side chains and (iv) the modification sites occupied on the tubulin molecule.
In ciliates, both polymodification types are not only present in cilia and basal bodies (Péchart et al., (1999) Biol. Cell 91, 685-697; Adoutte et al., (1991) Biol. Cell 71, 227-245; Bré et al., (1994) Cell Motil. Cytoskeleton 27, 337-49; Iftode et al., (2000) Biol. Cell 92, 615-628), but also occur on the more dynamic intracytoplasmic microtubules (Bré et al., (1994) Cell Motil. Cytoskeleton 27, 337-49; Fleury et al., (1995) Protoplasma 189, 37-60; Bré et al., (1998) Mol. Biol. Cell 9, 2655-2665). α- and β-tubulins of ciliates are biochemically heterogeneous (Bré et al., (1994) Cell Motil. Cytoskeleton 27, 337-49; Bré et al., (1998) Mol. Biol. Cell 9, 2655-2665; Suprenant et al., (1985) Proc. Natl. Acad. Sci. USA 82, 6908-6912), suggesting that structural differences among tubulin isoforms are important in generating functionally distinct types of microtubules in a single cell. However, genetic and biochemical studies showed that in ciliates only one or two gene isotypes of α- and β-tubulin form the bulk of microtubules (Redeker et al., (1994) Science 266, 1688-1691; Dupuis, (1992) EMBO J. 11, 3713-3719; Gaertig et al., (1993) Cell Motil. Cytoskeleton 25, 243-53; McGrath et al., (1994) Cell Motil. Cytoskeleton 27, 272-283; Dupuis-Williams et al., (1996) Biol. Cell 87, 83-93). Therefore, the large number of tubulin isoforms present in ciliates is mainly the result of PTMs (see Refs. 6, 29). Thus, ciliates provide a favorable model for assessing the function of PTMs.
The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases) and tubulin glycine ligases (tubulin G-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (Janke et al., 2005. Science 308:1758-1762; van Dijk et al., 2007. Mol. Cell 26:437-448; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Tubulin ligases may be either chain initiating ligases or chain elongation ligases.
The TTL-like proteins (TTLLs) are members of a large family of conserved eukaryotic proteins that are likely involved in PTMs such as polymodifications. Phylogenetic analyses showed that TTLLs of diverse eukaryotes belong to several conserved subtypes (example I, FIGS. 8, 12 and 13). In Tetrahymena, the TTLL family has over 50 genes, including a number paralogs (genes related by duplication within a genome that may evolve new functions, even if these are related to the original one). The unusually large number of TTLL genes in Tetrahymena and the lack of a detectable loss of function phenotype for TTLL1 (example I) suggests functional redundancy. The phylogenetic association of TTLL1, TTLL9, TTLL4, TTLL6, TTLL5 and TTLL15 protein types (86% bootstrap value; FIG. 8) suggests that these protein types are all involved in glutamylation of tubulin or possibly other proteins such as NAPs (Regnard et al., J Biol Chem 275, 15969 (2000)). The phylogenetic association of TTLL3, TTLL10, and TTLL14 suggests that these protein types are all involved in glycylation of tubulin or possibly other MAPs or NAPs.
Studies on several glutamic acid ligases indicate that tubulin glutamylation is important for assembly of axonemes. Hyperelongation of glutamyl side chains stabilizes cell body and destabilizes axonemal microtubules in the same cell (example IV). Further, deletion of two paralogs of a tubulin E-ligase caused severe deficiency in ciliary motility and increased the velocity of microtubule sliding in axonemes (example V).
The role of tubulin glycylation remains unknown, mainly because the modifying enzymes are yet to be discovered. In vivo, glutamic acid and glycine ligases oppose each other. Reduction in tubulin glycylation led to hyperglutamylation, likely by competing for shared modification sites on tubulin (example VII). Thus, it is likely that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation. Thus, glycylation likely acts by competing with glutamylation for shared modification sites on tubulin.
Tubulin glutamic Acid Ligases
Until now the enzymes which deposit post-translational modifications on microtubules remained unidentified due to, among other things, difficulties in their purification. As a result of synergistic research that explored advantages of the mouse model (in collaboration with Dr. Bernard Eddé and Carsten Janke at CRBM, Montpelier, France) and Tetrahymena, our laboratories identified a family of glutamylases differing in substrate preferences (α-tubulin vs. β-tubulin) (Janke et al. (2005) Science 308, 1758-1762 (see Example I)).
The catalytic subunit of the major neural glutamylase is the product of the TTLL1 gene that encodes a protein with a tubulin-tyrosine ligase (TTL)-like domain. TTL is a well known reverse enzyme for another post-translational modification, tubulin detyrosination (Ersfeld et al. (1993) J. Cell Biol. 120, 725-732). TTLL1 and TTL belong to a larger family of conserved proteins with a common catalytic domain. We named these proteins “TTL-like” (TTLLs). Phylogenetic analyses identified conserved subtypes of TTLLs, that may all be involved in ligation of specific amino acids to tubulins (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). Members of this novel family are structurally related to other proteins designated as ADP-forming enzymes, that display an ATP hydrolysis-dependent carboxylate-amine ligase activity. TTLL1 type proteins are associated with glutamylase activity on α-tubulin. TTLL1-mediated glutamylation is required for neurite extension in the mouse (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). A mutation in the noncatalytic subunit of TTLL1 complex, PGs1, led to disruption of assembly of axonemal microtubules in murine sperm and interestingly, caused reduction in aggressive behavior in males (Campbell et al. (2002) Genetics 162, 307-320). In Tetrahymena, a gene knockout of Ttll1p reduced the level of α-tubulin glutamylation, which in turn was associated with slow growth, defects in specific types of microtubules including basal bodies (centriole-like structures that template cilia), and slow ciliary beating (Janke et al. (2005) Science 308, 1758-1762 (see Example I)).
Another TTLL, TTLL6, was found to be a highly active glutamylase for β-tubulin (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). A TTLL6 type protein of Tetrahymena, Ttll6Ap, localizes mainly to cilia. Tetrahymena cells overproducing Ttll6Ap-GFP showed a dramatic increase in the level of tubulin glutamylation on cilia and some cell body microtubules (compare FIG. 1, middle and right panels, with FIG. 9C). A reliable in vitro assay for glutamylation that includes tritiated glutamic acid, taxol-stabilized microtubules and ATP is available (Regnard et al. (1998) Biochemistry 37, 8395-8404, Regnard et al. (1999) J. Cell Sci 112, 4281-4289). Remarkably, crude extracts of Tetrahymena cells overproducing Ttll6Ap under a cadmium-inducible promoter had 100 times more of activity in vitro (as compared to non-induced cells) (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). Ttll6Ap-GFP with a mutation in the predicted ATP-binding site lacked any detectable glutamylase activity. Thus, Ttll6Ap expresses well in Tetrahymena and large quantities of this enzyme can be produced cheaply.
Because there are several expressed paralogs, it has not yet been possible to determine what the loss of function for the Ttll6Ap-type activity is in Tetrahymena. A single knockout of TTLL6A gene did not produce a mutant phenotype.
Hyperglutamylation of β-Tubulin by Ttll6Ap Stabilizes Microtubules, Blocks Cells Proliferation and Inhibits Ciliary Motility in Tetrahymena. 
A full-length Ttll6Ap localizes primarily to cilia in Tetrahymena (FIG. 1, left panel; Example I and FIG. 9A). When the full length Ttll6Ap-GFP was overproduced, cells stopped dividing in a few hours (Example I) and become paralyzed due to lack of ciliary beating (Example I).
A truncated but enzymatically active form of Ttll6Ap lacking 288 amino acids of its C-terminus (Ttll6Ap-Δ710) failed to localize to cilia and accumulated in the cell body. As Ttll6Ap-Δ710 accumulated on cell body microtubules, their appearance changed dramatically—they acquired extensive glutamylation and become thick and wavy, which indicated that these microtubules were hyperstable. Indeed we found that these microtubules were resistant to nocodazole (FIG. 2).
An inactive (ATPase-dead) variant of the truncated protein (Ttll6Ap-Δ710-G422) accumulated to the same level as the enzymatically active protein, but failed to hyperglutamylate and hyperstabilize cell body microtubules. The ATPase-dead variant did not have any effect on cell growth and motility (Example I). This experiment indicates that the ATP-binding site within the TTL homology domain of the glutamylases is one of the preferred target region for development of specific inhibitors.
The above observations argue that the observed phenotypic effects of active Ttll6tAp are mediated by its glutamylation activity and not by the mere binding to microtubules. Importantly, excessive glutamylation on β-tubulin strongly inhibits cell multiplication and ciliary motility. The biological assay of the invention takes advantage of these effects, because a glutamylase inhibitor will rescue the host organism from a lethal condition, restoring growth and, where a full length glutamylase is used such that it localizes to the cilia, cell motility. Of course, glutamylation on b-tubulin may be required for cell viability. Therefore, the inhibitor would need to be applied in a range of concentrations to find its concentration that titrates out the overproduced enzyme but does not deplete the total activity below the endogenous level. It should be noted that cells overproducing Ttll6Ap-ΔcT also ceased dividing, but the effect on ciliary motility was relatively mild because this isoform cannot be targeted to cilia (Example I). This is advantageous because the truncated Ttll6Ap variant is especially useful in the cell-based, biological assay to avoid sedimentation of paralyzed cells to the well bottom that could disturb the uniformity of signal strength across the well.
Tubulin Glycine Ligases
An analysis of ciliary tubulin from the protist Paramecium led to the discovery of polyglycylation, which consisted of an additional lateral chain of up to 34 glycine units on both axonemal tubulin subunits (Redeker et al., Science. 1994; 266:1688-1691). Since then, studies on polyglycylation, using either mass spectrometry (Rüdiger et al., FEBS Lett. 1995; 364:147-151; Mary et al., J Biol Chem. 1996; 271:9928-9933; Multigner et al., Biochemistry. 1996; 35:10862-10871; Weber et al., FEBS Lett. 1996; 393:27-30) or two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49 (Bré et al., J Cell Sci. 1996; 109:727-738), have involved principally axonemes of various cell types. mAbs, TAP 952 and AXO 49, serve as complementary tools for detection of mono- and polyglycylated tubulin (Bre et al., 1998 Mol. Biol. Cell 9(9):2655-2665). Taken together, the data suggest that axonemal tubulin could be the preferred substrate for polyglycylation. This would contrast with the broad occurrence of polyglutamylation in both cytoplasmic (Eddé et al., Science. 1990; 247:83-85; Alexander et al., Proc Natl Acad Sci USA. 1991; 88:4685-4689; Redeker et al., FEBS Lett. 1992; 313:185-192; Rüdiger et al., FEBS Lett. 1992; 308:101-105; Wolff et al., Eur J Cell Biol. 1992; 59:425-432; Mary et al., FEBS Lett. 1994; 353:89-94) and axonemal tubulin (Bré et al., Cell Motil Cytoskeleton. 1994; 27:337-349; Fouquet et al., Cell Motil Cytoskeleton. 1994; 27:49-58; Mary et al., J Biol Chem. 1996; 271:9928-9933; Schneider et al., J Cell Sci. 1997; 110:431-437). Therefore, polyglycylation is likely a selective marker of the most stable microtubules.
The role of tubulin glycylation remains largely unknown, mainly because the modifying enzymes are yet to be discovered. TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. Tetrahymena TTLL3 is a tubulin glycine ligase (G-ligase) with a chain-initiating activity. Tetrahymena cells lacking all TTLL3 genes have shortened axonemes that are resistant to paclitaxel, indicating that tubulin glycylation changes the lattice properties of axonemal microtubules. Deletion of TTLL3 in zebrafish development by morpholino (MO)-based depletion studies indicate that TTLL3 is required for either elongation or stability of axonemes. In both organisms, a reduction in tubulin glycylation led to hyperglutamylation (example VII). Thus, glycylation could act by competing with glutamylation for shared modification sites on tubulin.
Cells
Cells useful in the method of the invention include but not limited to animal, plant, yeast, protozoan, and bacterial cells. Unicellular eukaryotic cells, such as protozoan cells, are preferred. Even more preferably, the cell of the present invention is a ciliated cell. Examples of ciliated cells include, without limitation, Tetrahymena, Paramecium, Plasmodium, and Chlamydomonas. An example of a particularly preferred cell for use in the method of the invention is Tetrahymena. Also included in the present invention are cellular components, such as axonemes, microtubules or tublin, derived from the cells.
Cells useful in the present invention have been engineered to result in reduced levels of polymodification. Preferred types of polymodification in the present invention include, without limitation, polyglutamylation and polyglycylation.
Preferably, the reduced level of polymodification in the cell of the present invention is a result of the disruption of a tubulin ligase gene (e.g., via a “knockout” of a gene), such that the modification is reduced when compared to a wild type cell (i.e., a cell that does not have a disruption in a tubulin ligase gene). Methods of disrupting or altering levels of polymodification can be effected at any level of gene expression (e.g., DNA replication, transcription, translation, or post-translationally) of a tubulin ligase and are routine and well known in the art. For example, enzymatic function can be inhibited when the enzyme is targeted by a molecular inhibitor, such as an antibody or a small molecule inhibitor. Translation of an RNA message into an enzyme can be disrupted, for example, by introducing a small interfering RNA, a short-hairpin RNA, a morpholino, or a hybridization probe into the cell. Transcription of a gene encoding an enzyme can be disrupted, for example, by targeting the gene with a molecular inhibitor or physically altering the gene to prevent or confound gene replication or transcription. Cells can be engineered through the introduction of polynucleotides, as well as the directed mutagenesis of coding regions. Common gene disruption techniques include mutagenesis, gene deletion or knock-out, and heterologous gene transformation. Such methods are well known in the art; see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989), and Methods for General and Molecular Bacteriology, (eds. Gerhardt et al.) American Society for Microbiology, chapters 13-14 and 16-18 (1994). Optionally, the cell may have multiple gene disruptions. The multiplicity of disruptions may occur from engineering more than one disruption in a single cell or from engineering the disruptions in separate cells and crossing the cells to obtain progeny harboring multiple disruptions.
A particularly useful cell having reduced levels of polymodification is a cell that has been engineered to “knock out” a gene encoding a tubulin ligase. In one embodiment, at least two paralogs of a tubulin glutamic acid ligase gene are disrupted. Non-limiting examples of tubulin glutamic acid ligase genes are TTLL1, TTLL9, TTLL4, TTLL6, TTLL5 and TTLL15. Preferably, the knocked out tubulin glutamic acid ligase gene is the TTLL6 gene. Preferred cells of the present invention include, without limitation, cells having a disruption of the TTLL6A and F paralogs, such as a TTLL6A/TTLL6F knockout cell (e.g, cell line “6AF-KO” as described in Example V), cells having a disruption of the TTLL6A, B, D, and F paralogs, such as a TTLL6A/TTLL6B/TTLL6D/TTLL6F knockout cell (e.g., cell line “6ABDF-KO” as described in Example VI), or cells having a disruption of the TTLL6A, B, D, E, and F paralogs, such as a TTLL6A/TTLL6B/TTLL6C/TTLL6D/TTLL6E/TTLL6F knockout cell (e.g., cell line “6ABDEF-KO” as described in Example VI).
Deletion of TTLL6A and TTLL6F genes from Tetrahymena produces double knockout (KO) cells (termed herein 6AF-KO cells) that have cilia with microtubule axonemes that have greatly shortened side chains. The shortened glutamyl side chains may be, but are not necessarily, as short as a single glutamic acid residue. Axonemes isolated from 6AF-KO cells, as well as microtubule preparations and tubulin preparations derived therefrom, are well-suited for use as substrates for in vitro glutamylation reactions using purified or crude enzymes that have glutamyl side chain elongase activity, such as Ttll6Ap and similar enzymes.
It should be noted that Tetrahymena and other organisms have a large number of TTLL genes ( ̃50), including TTLL1, TTLL9, that potentially could encode tubulin glutamic acid ligases and be functionally redundant with TTLL6. It was thus entirely unexpected that the deletion of just the TTLL6A and 6F genes, from among the many TTLL genes present in Tetrahymena, would lead such a significant loss of tubulin glutamylation in cilia.
Advantageously, due to its high growth rate and inexpensive methods of culture, Tetrahymena with deletions of TTLL6A and 6F is an excellent source of axonemes and other tubulin-containing structures for in vitro assay for tubulin glutamylation at an industrial scale for the purpose of screening for compounds that either enhance or inhibit the reaction. Moreover, the deletion of five TTLL genes simultaneously (TTTL6A, B, D, E and F, to produce a strain termed herein 6ABDEF-KO), was found to produce Tetrahymena cells evidencing further reduction in the levels of tubulin polyglutamylation, and thus could provide an even lower background signal in axonemes and other tubulin-containing structures used for in vitro assays.
In another embodiment, the cell of the invention has at least two paralogs of a tubulin glycine ligase gene disrupted. Non-limiting examples of tubulin glycine ligases are TTLL3, TTLL10, and TTLL14. Preferably, the knocked out tubulin glycine ligase gene is a TTLL3 gene. Preferred cells include, without limitation, cells having a disruption of the TTLL3A and B paralogs, such as a TTLL3A/TTLL3B knockout (e.g., cell line “3AB-KO” as described in Example VII), cells having a disruption of the TTLL3A, B, C, and D paralogs, such as a TTLL3A/TTLLL3B/TTLL3C/TTLL3D knockout cell (e.g., cell line “3ABCD-KO” as described in Example VII), or cells having a disruption of the TTLL3A, B, C, D, E, and F paralogs, such a TTLL3A/TTLL3B/TTLL3C/TTLL3D/TTLL3E/TTLL3F knockout cell (e.g., cell line “3ABCDEF-KO” as described in Example VII).
Antibodies that bind to axonemes, microtubules and tubulin isolated from cells of the invention, which have utility as diagnostic reagents and have potential therapeutic uses as well, are also included in the invention.
Antibody-based Screening Assays
The invention further includes screening assays based on anti-glutamylation antibodies, which give an extremely strong signal reflecting Ttll6Ap activity (see Example I; FIG. 9C,D). Examples of antibody-based assays include cytoblot for an in vivo assay, or ELISA-type assay with plates coated with microtubules for in vitro assay.
Assays
The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Due to its high growth rate and inexpensive methods of culture, Tetrahymena is an excellent model for in vivo assays as well as an ideal source for axonemes, microtubules and tubulin for use in in vitro assays on an industrial scale. The cells and methods of the present invention provide a system in which to perform high-throughput screens of the methods as described below.
The present invention thus includes both in vivo and in vitro methods for identifying compounds that enhance or inhibit a tubulin ligase reaction. In vitro methods advantageously utilize cellular components such as axonemes, microtubules or tubulins, that are isolated from the genetically engineered Tetrahymena cells of the invention. The cellular components useful in the in vitro assays are components that are, in a wild-type Tetrahymena cell, polymodified with glutamic acid and/or glycine side chains. Also included in the present invention are the enhancers and inhibitors identified using the method of the invention.
A inhibitor or enhancer compound identified by the method of the present invention may be, without limitation, a protein, a peptide, a peptide fragment, an antibody, a small molecule, a nucleic acid, a chemical compound, or any other molecule capable of inhibiting or enhancing the ligase reaction. A compound of identified by the method of the present invention may be a natural or a synthetic compound. Also included in the present invention are compositions including an inhibitor or enhancer molecule identified by the method of the present invention. Such compositions typically include a pharmaceutically acceptable carrier. As used herein “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
In vivo assays may be performed using the cells of the present invention and may include biological assays or molecular assays. For example, a biological assay may involve phenotypic studies. Phenotypic studies typically used to examine microtubules include, but are not limited to, cell growth, cell motility, phagocytosis, and ciliary beat frequency. In one embodiment, an in vivo assay may be used as a primary screen and an in vitro assay may be used as a secondary screen, or vice versa.
In vitro assays may utilize isolated cellular components of the cells of the present invention. An “isolated” component, such as an axoneme, microtubule or tubulin, is one that has been removed from the cell. Preferably, the cellular component is purified, i.e., essentially free from any other cellular products or impurities. In vitro assays may include biological or molecular assays. For example, a molecular assay typically used to examine microtubules may include, without limitation, biochemical studies such as glutamylation assays or glycylation assays and microtubule sliding assays. For example, an in vitro assay with purified enzyme will determine whether the compound directly inhibits or enhances the ligase activity. Further assays, both in vitro and/or in vivo, can be used to determine whether the enzyme is a broad or selective modulator and whether it can act on mammalian ligases. These assays are commonly used in the field and would be known to a skilled artisan.
Assays for detecting glutamylation or glycylation are also well known in the art and are further described in the Examples, below. Glutamylation assays may include, for example, contacting the experimental sample with a suitable substrate, preferably a stabilized microtubule, under conditions that allow for glutamylation, and detecting the level of glutamylation on the substrate. Detection of glutamylation level can be accomplished using any convenient method, for example using antibody detection. Likewise, a glycylation assays may include, for example, contacting the experimental sample with a suitable substrate, preferably a stabilized microtubule, under conditions that allow for glycylation, and detecting the level of glycylation on the substrate. Optionally, the tubulin ligase assay may further include, prior to contacting the sample with the substrate, lysing the cell of the present invention and/or isolating cellular components such as the axonemes, microtubules or tubulin.
Cellular components such as axonemes, microtubules or tubulin, which have been isolated from a Tetrahymena cell of the invention that exhibits reduced tubulin polyglutamylation, are useful in in vitro assays for identifying inhibitors or enhancers of tubulin glutamylation. For example, a substrate, such as an as axoneme, microtubule or tubulin, can be contacted with a candidate glutamic acid ligase-inhibitor compound and a known glutamic acid ligase, preferably a tubulin glutamic acid ligase. A change in the glutamylation level of the substrate, dependent upon the presence or absence of the inhibitor compound, can be detected. The absence of additional glutamylation, or a slower rate of glutamylation in the presence of the candidate compound compared to the glutamylation rate in the absence of the candidate compound, indicates that the compound functions as a glutamic acid ligase inhibitor. Likewise, to identify an enhancer compound, an increase in glutamylation amount or rate, compared to the glutamylation rate in the absence of the candidate compound, is indicative of a compound that enhances or stimulates glutamic acid ligase activity. Optionally, the inhibitory nature of an inhibitory compound can be confirmed or further characterized or studied using in vivo assays. Advantageously, such in vivo assays can be performed in wild-type Tetrahymena or a Tetrahymena cell that has been genetically engineered to overexpress a tubulin glutamic acid ligase, such as TTLL6A.
Alternatively, a primary in vivo assay can be used to identify candidate glutamic acid ligase inhibitor compounds. In this embodiment, a Tetrahymena cell exhibiting reduced glycine ligase activity (and hence, hyperglutamylation) as described herein can be contacted with a candidate glutamic acid ligase-inhibitor compound, and a change in at least one of cell growth or motility can be detected. A decrease in cell growth or motility is indicative of tubulin glutamic acid inhibition. The method may further include detecting the amount of glutamylation. A decrease in glutamylation relative to an untreated cell is indicative of tubulin glutamic acid ligase inhibition.
In analogous methods, cellular components such as axonemes, microtubules or tubulin, which have been isolated from a Tetrahymena cell of the invention that exhibits reduced tubulin polyglycylation, are useful in in vitro assays for identifying inhibitors or enhancers of tubulin glycine ligase. For example, the method may include contacting a substrate isolated from a Tetrahymena cell having reduced glycine ligase activity, with a candidate glycine ligase-inhibitor compound and a known glycine ligase, preferably a tubulin glycine ligase, and detecting a change in glycylation of the substrate. A change in the glycylation level of the substrate, dependent upon the presence or absence of the inhibitor compound, can be detected. The absence of additional glycylation, or a slower rate of glycylation in the presence of the candidate compound compared to the glycylation rate in the absence of the candidate compound, indicates that the compound functions as a glycine ligase inhibitor. Likewise, to identify an enhancer compound, an increase in glycylation amount or rate, compared to the glycylation rate in the absence of the candidate compound, is indicative of a compound that enhances or stimulates glycine ligase activity. Optionally, the inhibitory nature of an inhibitor compound can be confirmed or further characterized or studied using in vivo assays. Advantageously, such in vivo assays can be performed in wild-type Tetrahymena or a Tetrahymena cell that has been genetically engineered to overexpress a tubulin glycine ligase, such as TTLL3A.
Scientific and Medical Applications
It is expected that an inhibitor of glutamylase will be of great value in assessing the function of glutamylation in diverse cell types, including those that are difficult to grow in vitro or manipulate genetically and are known to have highly glutamylated microtubules (such as ciliated epithelial cells (Million et al. (1999)). There is evidence that glutamylation affects assembly of certain organelles such as flagella (Campbell et al. (2002) Genetics 162, 307-320), centrioles (Bobinnec et al. (1998) J. Cell Biol. 143, 1575-1589), and neural bundles (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). Thus, genetic means alone may not adequately assess the function of glutamylation in already assembled organelles.
Additionally, glutamylase inhibitors could find innovative uses in medicine. As noted earlier, tubulins are a major target of anti-cancer drugs. However, tubulins are highly conserved. Thus, developing isotype-specific inhibitors for tubulin primary polypeptides is likely to be difficult. The present invention provides an alternative strategy that involves targeting the post-translational modification enzymes rather then tubulins per se. Tubulin glutamylases identified herein are suitable objects for highly focused targeting strategies. Mammals have nine glutamylase enzymes (Janke et al. (2005) Science 308, 1758-1762 (see Example I)), many of which display restricted patterns of expression (see Unigene entries: Hs.91930, Hs.445826, Hs.567737 Mm.276780). Thus, a restricted chemical targeting of microtubules may be possible by inhibition of specific tubulin glutamylases.
Furthermore, ciliates are evolutionary relatives of important apicomplexan parasites (e.g., malaria-causing Plasmodium and Toxoplasma), known to extensively glutamylate pellicular microtubules (Plessmann et al. (2004) Parasitol Res 94, 386-389). Thus narrow specificity inhibitors developed against a Tetrahymena enzyme may be useful as anti-parasite compounds. For example, the eukaryotic parasite Toxoplasma gondii has highly glutamylated pellicular microtubules (Plessmann et al., (2004) Parasitol. Res. 94, 386-389) and Toxoplasma has several glutamylase genes. An inhibitor of a glutamylase can inhibit the growth of, or kill, for example, ciliated protozoans.
An inhibitor of glutamylase can be used in a number of diverse and medically relevant ways. For example, two glutamylases (TTLL6 and TTLL13) in humans are highly expressed in the testis and only at low level or are non-detectable in other tissues (NCBI/Unigene entry Hs.632164; Hs.91930). A mutation in the noncatalytic subunit of one glutamylase, PGs1, blocked assembly of sperm axonemes in the mouse (Campbell et al. (2002). Genetics 162, 307-320). Thus, a glutamylase inhibitor could be used as a male contraceptive, as it can be expected to interfere with the function of sperm flagellum. As shown in the following examples, one of the enzymes in Tetrahymena localizes to cilia, which are structures similar to flagella. Another potential application is in cancer. It is known that glutamylases modify microtubules of spindles and centrioles in dividing cells (Bobinnec et al. (1998) Cell Motility Cytoskeleton 39, 223-232). Thus, an inhibitor of a glutamylase is expected to inhibit cell proliferation.
Another potential application is as a psychiatric drug. A mutation in the non-catalytic subunit of the glutamylase was found to reduce male-male aggression in the mouse model likely based on its effect on the brain (Campbell et al. (2002). Genetics 162, 307-320). Thus, an inhibitor of glutamylation could be useful as a psychiatric drug.
EXAMPLES
The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.
Example I
Tubulin Glutamylase Enzymes are Members of the TTL Domain Protein Family
Summary
Glutamylation of tubulin has been implicated in several functions of microtubules but the identification of the responsible enzyme(s) has been challenging. We show here that the neuronal glutamylase is a protein complex containing a tubulin tyrosine ligase-like (TTLL) protein, TTLL1. TTLL1 is a member of a large family of proteins with a TTL domain, whose members could catalyze ligations of diverse amino acids to tubulins or other substrates. In the model protist Tetrahymena thermophila two conserved types of glutamylases were characterized, which differ in substrate preference and subcellular localization. Janke et al. (2005) Science 308, 1758-1762.
Introduction
Polyglutamylation is an uncommon type of posttranslational modification that adds multiple glutamic acids to a γ-carboxyl group of a glutamate residue of target proteins, including tubulin and nucleosome assembly proteins NAP1 and NAP2 (Edde et al., Science 247, 83 (1990); Rüdiger et al., FEBS Lett 308, 101 (1992); Redeker et al. FEBS Lett 313, 185 (1992); Regnard et al., J Biol Chem 275, 15969 (2000)). The resulting glutamate side chains are of variable length, allowing for a graded regulation of protein-protein interactions. Glutamylation regulates the binding of neuronal microtubule (MT) associated proteins as a function of the length of the glutamate chain, suggesting that the modification is important for the organization of the neuronal MT network (Bonnet et al., J Biol Chem 276, 12839 (2001)). Tubulin glutamylation may also play a role in centriole maintenance (Bobinnec et al., J Cell Biol 143, 1575 (1998)) axoneme motility (Gagnon et al., J Cell Sci 109, 1545 (1996); Million et al., J Cell Sci 112, 4357 (1999)) and mitosis (Bobinnec et al., Cell Motil Cytoskeleton 39, 223 (1998); Regnard et al., J Cell Sci 112, 4281 (1999)).
Identification of mouse brain glutamylase. Monoclonal antibody (mAb 206), raised against a partially purified brain tubulin glutamylase fraction, immunoprecipitates the enzyme complex, including the PGs1 protein (Regnard et al., J Cell Sci 116, 4181 (2003)). Here we used gel electrophoresis followed by nano-LC-MS-MS (Liquid Chromatography-Mass Spectrometry) to identify 4 additional protein components of the same complex: p24, p33, p49 and p79 (FIG. 5A; Table I). Polyclonal antibodies were raised against recombinant proteins or peptides of p24, p32/PGs1, p33, p49 and p79 and used for co-immunoprecipitation analyses. mAb 206, anti-p32/PGs1 antibodies (L83) and anti-p79 antibodies (L80) precipitated≧80% of the glutamylase activity (FIG. 5B) along with all 5 proteins (FIG. 5C). These 5 proteins also consistently copurified with the glutamylase activity during several purification steps (FIG. 10) and were named Glutamylase subunits PGs5, PGs1, PGs2, PGs3 and PGs4 (for p24, p32, p33, p49 and p79, respectively). Additional proteins were found in the mAb 206-immunoprecipitated fraction, including Arp1 and CF Im25 (Table I), but these did not consistently copurify with the enzyme activity.
The apparent size of the neuronal glutamylase complex (360 kDa; Regnard et al., Biochemistry 37, 8395 (1998)) is greater than the theoretical sum of the predicted molecular masses of all 5 subunits (217 kDa), suggesting that the complex contains multiples of one or more subunits. Some of the components appear on 2D gels as multiple spots at different isoelectric points, suggesting that they are themselves subject to charge-altering post-translational modifications (FIG. 5A). Indeed, PGs1 was phosphorylated on Ser 279. The 3 more acidic spots of PGs1/p32 contain the phosphorylated peptide 277-284 (RPSVPMAR (SEQ ID NO:5)) (FIG. 5A: spots 5-7). Phosphorylation site prediction (available on the world wide web at expasy.org/) indicates that 5279 can be phosphorylated by cAMP- or cGMP-dependent protein kinases. The 3 more acidic spots of PGs1/p32 contain the phosphorylated peptide 277-284 (RPSVPMAR; SEQ ID NO:5) (FIG. 5A: spots 5-7). Phosphorylation site prediction (available on the world wide web at expasy.org/) indicates that S279 can be phosphorylated by cAMP- or cGMP-dependent protein kinases.
PGs1 (a product of the mouse gene GTRGEO22) is required for sperm axoneme assembly and normal animal behavior (Campbell et al., Genetics 162, 307 (2002)) and may act in the intracellular targeting of the glutamylase complex (Regnard et al., J Cell Sci 116, 4181 (2003)). PGs3 is an ortholog of the human TTLL1 protein (Trichet et al. Gene 257, 109 (2000)). The amino acid sequence of TTLL1 exhibits 17% identity to tubulin tyrosine ligase (TTL), which catalyses the addition of tyrosine to the C-terminal glutamate of detyrosinated α-tubulin (Ersfeld et al., J Cell Biol 120, 725 (1993)). Despite obvious differences, both glutamylation and tyrosination reactions involve an amino acid addition to a glutamate residue through the formation of an amide bond. Thus, we examined the possibility that TTLL1 is the catalytic subunit of neural tubulin glutamylase.
A structural model for TTLL1. The amino acid sequence of TTLL1 (PGs3) contains three conserved motifs which correspond to the ATP/Mg2+-binding site typical of enzymes with a carboxylate-amine/thiol ligase activity, such as glutathione synthetase (Galperin et al., Protein Sci 6, 2639 (1997)). Although the overall sequence similarity between TTLL1 and the known carboxylate-amine/thiol ligase enzymes is low, we could align the ATP-binding regions, and also all major parts of TTLL1. A structural model of TTLL1 was obtained by homology-based modeling using glutathione synthetase from E. coli as a template (FIG. 6A, B). Docking of ATP and Mg2+ into the model supports the localization of the ATP/Mg2+-binding site (FIGS. 6B, C and FIG. 11). We were also able to fit a peptide with a glutamate side chain into the active site, which located a putative binding site for free glutamate (FIG. 6C). Thus, it is very likely that TTLL1 protein is the catalytic subunit of neural tubulin glutamylase.
Ttll1p is associated with α-tubulin glutamylase activity in vivo. When expressed in bacteria or in various cell lines as well as in several heterologous systems, the murine TTLL1 had a strong tendency to precipitate and did not show glutamylase activity in vitro. We used a homologous protein expression system based on the ciliated protist Tetrahymena thermophila to assess the role of TTLL1-related proteins in glutamylation. Tetrahymena has a complex cytoskeleton with a large number of distinct types of MTs (Gaertig, J Eukaryot Microbiol 47, 185 (2000)). Most types of MTs in Tetrahymena are monoglutamylated, while a small subset, including MTs of the basal bodies (BBs), cilia, contractile vacuole pore (CVP) and oral deep fiber (DF), have side chains composed of two or more glutamates. Using the recently sequenced macronuclear genome of Tetrahymena, we identified the likely TTLL1 ortholog, Ttll1p (54% of amino acid sequence identity to TTLL1). Ttll1p with an N-terminal GFP was strongly overexpressed using a cadmium-inducible gene promoter (Shang et al., Proc Natl Acad Sci USA 99, 3734 (2002)). Ttll1p-GFP localized to a subset of glutamylated MT organelles: including BBs, CVPs and DF (FIG. 3A), but no increase in the level of MT glutamylation over normal level was detected (FIG. 3B, C, E), and the phenotype appeared normal. Similar results were obtained for the HA epitope C-terminally tagged protein. No change in in vitro tubulin glutamylation activity was detected in cell extracts despite the strong accumulation of Ttll1p-GFP (FIG. 7F). However, a glutamylase activity directed mostly toward α-tubulin was co-immunoprecipitated with anti-GFP antibodies from extracts of overexpressing cells (FIG. 7G, H). The overexpressed protein apparently can replace a part of the endogenous Ttll1p but may not function alone. Based on the data obtained for the murine homolog, it is likely that Ttll1p also acts in a complex and that other subunits are limiting. We also constructed cells completely lacking the TTLL1 gene, using gene disruption. The TTLL1-null cells had a normal phenotype, but showed a strong reduction in tubulin glutamylation in the BBs (FIG. 7C, D), confirming that Ttll1p is involved in glutamylation but also suggesting that there are additional glutamylase activities in this organism that do not require Ttll1p.
The TTLL family. TTLL1 and TTL are members of a large family of conserved eukaryotic proteins with a TTL homology domain, raising the possibility that other members of this family are also involved in glutamylation or other types of posttranslational amino acid ligations. Phylogenetic analyses showed that TTL-like proteins (TTLLs) of diverse eukaryotes belong to several conserved subtypes (FIGS. 8, 12 and 13). We used the HsTTLL1 sequence (Trichet et al., Gene 257, 109 (2000)) as a template for tBLASTn searches to identify all TTLL loci of Tetrahymena and several other model eukaryotes. Phylogenetic analyses revealed 10 clades of TTLLs, 8 of which contain mammalian proteins. Tetrahymena has between 1-7 sequences in most groups, and one clade of 20 ciliate-specific TTLLs. Among the genomes surveyed, only Trypanosoma has a close homolog of the mammalian TTL, which may be why an enzymatic activity of TTL was not detected in invertebrates (Preston et al., J Mol Evol 13, 233 (1979)) and Tetrahymena (Raybin et al., J Cell Biol 73, 492 (1977)).
Tetrahymena Ttll6Ap is a β-tubulin-preferring glutamylase. The Tetrahymena TTLL6A sequence belongs to a clade related to TTLL1 type (FIGS. 8 and 13). Overproduced Ttll6Ap-GFP localized mainly to cilia, with a small amount associated with BBs and cell body MTs (FIG. 9A). Overproduction of Ttll6Ap-GFP led to a strong increase in glutamylation in cilia, and on cell body MTs (FIG. 9C; compare FIG. 7C). A strong increase in tubulin glutamylation (but not in polyglycylation) was also detected in whole cells using immunoblotting (FIG. 9F).
A truncated variant lacking the 286 C-terminal amino acids, Ttll6ApΔ710-GFP localized predominantly to the cell body with strong preference for subcortical (SC) MTs that extend from the apical end of the cell and run below the BBs (FIG. 9B). Consistently, overexpression of Ttll6ApΔ710-GFP led to a strong increase in glutamylation on MTs in the cell body and to a much lesser extent in cilia (FIG. 9D). Thus, the 286 C-terminal amino acids of Ttll6Ap are required for preferential targeting to cilia.
We used the truncated version of Ttll6Ap for biochemical studies, due to the increased presence of this variant in the soluble/cytosolic pool, as compared to the full-length protein. Extracts of cells overexpressing Ttll6ApΔ710-GFP showed a 100-fold increase in in vitro glutamylase activity for β-tubulin and a 10-fold increase for α-tubulin compared to non-induced cells (FIG. 9E). This activity co-purified with Ttll6ApΔ710-GFP protein under all conditions tested (FIG. 14A-C). No in vitro activity toward NAP proteins was detected. Thus, Ttll6Ap is a tubulin glutamylase displaying a strong preference for the β-tubulin subunit.
Increased glutamylation affects cell growth and motility. Tetrahymena cells overexpressing Ttll6Ap-GFP ceased to multiply in a few hours following cadmium-induction (FIG. 9G) and most had paralyzed cilia (FIG. 9I), indicating that excessive glutamylation inhibits cell proliferation and ciliary dynein-based motility. Cells overexpressing the truncated protein also ceased to proliferate but the effect on ciliary motility was much weaker, in accordance with the altered protein localization pattern (FIG. 9H, I). These effects did not occur when a mutation in the predicted ATP binding site (E422G) of the TTL homology domain was introduced (FIG. 11). Compared to the ATPase-active protein, the inactive variant, Ttll6ApΔ710-E422G-GFP, was expressed at a similar level (FIG. 9F) and localized to the same types of MT organelles. However, neither increase in tubulin glutamylation (FIG. 9F), nor alteration of cell growth or motility were observed (FIG. 9H, I), confirming that excessive glutamylation was responsible for the observed effects of Ttll6Ap overproduction.
Conclusion. The simplest interpretation of all our data is that TTLL1/Ttll1p and Ttll6Ap are two types of tubulin glutamylase catalytic components with distinct tubulin subunit preferences. The neuronal TTLL1 as well as Ttll1p have a preference for α-tubulin, while Ttll6Ap preferentially glutamylates β-tubulin. Unlike Ttll6Ap, Ttll1p (and its murine ortholog) did not increase glutamylation in vitro and in vivo upon overproduction. However, TTLL1 exists in a protein complex and additional subunits may be required for its activity and could be limiting in vivo. Indeed, immunoprecipitation of Ttll1p-GFP from overproducing Tetrahymena cells led to a recovery of glutamylase activity.
Ttll6Ap is a much larger protein (116 kDa) compared to TTLL1 (49 kDa) and Ttll1p (42 kDa) and may contain all properties required for autonomous glutamylase activity. The four non-catalytic subunits identified in the neuronal TTLL1 complex may be involved in tubulin substrate recognition, regulation of the enzymatic activity, or subcellular localization as suggested for PGs 1 (Regnard et al., J Cell Sci 116, 4181 (2003)). It is likely that Ttll1p is also in a complex, as is the murine homolog. Except for PGs4, we could not identify homologs of the other subunits of the neural complex (PGs1, PGs2, PGs5) outside of vertebrates, including Tetrahymena, indicating that variations in composition of non-catalytic subunits occur across phyla.
The unusually large number of TTLL genes in Tetrahymena and the lack of a detectable loss of function phenotype for TTLL1 suggests functional redundancy. In contrast, a mutation in the PGs1 component of the murine TTLL 1-complex lead to defective sperm axonemes and changes in animal behavior (Campbell et al., Genetics 162, 307 (2002)). In C. elegans, RNAi depletion of C55A6.2 (a TTLL5 type) causes embryonic lethality and sterility (Maeda et al., Curr Biol 11, 171 (2001)). Depletion of TTLL1 mRNA in PC12-E2 cells inhibited neurite outgrowth, suggesting an essential function in neurogenesis. The phylogenetic association of TTLL1, TTLL9, TTLL4, TTLL6, TTLL5 and TTLL15 protein types (86% bootstrap value; FIG. 8) suggests that these protein types are all involved in glutamylation of tubulin or possibly other proteins such as NAPs (Regnard et al., J Biol Chem 275, 15969 (2000)). Other members of the TTLL family may catalyze different types of posttranslational addition of an amino acid, such as polyglycylation.
Materials and Methods
Purification and Identification of Mouse Brain Glutamylase
Glutamylase was partially purified (400×) from 200 3 day-old mouse brains and immunoprecipitated with mAb 206 linked to protein G magnetic beads as described Regnard et al. (J Cell Sci 116, 4181 (2003)). The precipitated proteins were analyzed by two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). For the first dimension, non-equilibrium pH gradient electrophoresis (NEPHGE; O'Farrell et al. (1977) Cell 12, 1133) and for the second PAGE dimension, an acrylamide gradient (8-13%) were applied. Proteins were stained with Coomassie brilliant blue (Serva) and all visible protein spots (apart from two large spots corresponding to the heavy and light chain of mAb 206) were excised and submitted to in-gel digestion by trypsin. Extracted peptides were analyzed by nano-LC-MS-MS on a Q-TOF 2 mass spectrometer (Micromass Ltd., Manchester, UK) and identified using the Mascot software (Fraering et al. (2004) Biochemistry 43, 9774).
Tetrahymena Cell Extracts
To prepare extracts for glutamylase activity assays, cells were lysed in the T buffer (Tris-HCl 50 mM pH 8.0, NaCl 0.2 M, EGTA 1 mM, MgCl2 1 mM, NP-40 0.5%) containing protease inhibitors (leupeptin 0.5 E-64 10 μg/ml, chymostatin 10 μg/ml, antipain 12.5 μg/ml, according to Chilcoat et al. (J Cell Biol 135, 1775 (1996)). Total extracts were either directly analyzed or centrifuged at 50,000×g. The supernatant constituted the soluble fraction. The cytoskeletal fraction was obtained by resuspending and sonicating the pellet in T buffer.
To prepare whole cytoskeletons for western blots, 107 Tetrahymena cells were spun down and processed according to Williams et al. (Methods Cell Biol 47, 301 (1995)) with some modifications. Cells were resuspended in 2 ml of 10 mM Tris-HCl, pH 7.5 with protease inhibitors and incubated on ice for 5 min. An equal volume of 2× lysis buffer with protease inhibitors was added (1% Triton-X100 final concentration) and after 1 min on ice, cytoskeletons were collected by centrifugation (10 min, 16,000 g at 4° C.). Pellets were resuspended in a phosphate buffer and protein concentration was determined using the BCA TM Protein Assay (Pierce). Eight μg of protein was loaded per lane, separated on a 12% SDS-PAGE gel and processed for western blotting.
Size Exclusion Chromatography
A soluble extract of Ttll6ApΔ710-GFP producing cells was fractionated on an HPLC
TSK G300 SWXL, 7.8×300 mm column (TosoHaas, PA, USA) in MES 50 mM pH 6.9, EGTA 1 mM, MgCl2 1 mM, NaCl 0.35 M, Triton-X100 0.1%, at a flow rate of 0.8 ml/min. Fractions of 0.4 ml were collected. Protein standards used for calibration were thyroglobulin (669 kDa), aldolase (158 kDa), transferrin (81 kDa), hemoglobin (32 kDa) and cytochrome C (12.4 kDa).
Glutamylase Assay
The tubulin glutamylase activity was measured as described Regnard et al. (Biochemistry 37, 8395 (1998)). Briefly, reaction mixtures (20 μl) containing 50 mM Tris-HCl pH 9.0, 2 mM ATP (equilibrated to pH 7.0 with NaOH), 12 mM MgCl2, 2.5 mM DDT, 10 μM taxotere, l-[3H]-glutamate (45-55 Ci/mmol, Amersham, UK) and 0.1 mg/ml taxotere-stabilized MTs, were incubated at 30° C. for 40 min. In some cases, to increase the radioactive signal, l-[3H]-glutamate was concentrated by centrifugation under vacuum and added at a final concentration of up to 25 μM. Protein fractions to be tested were added at a maximal final concentration of 0.5 mg/ml. The taxotere-stabilized mouse brain MTs were prepared as described Regnard et al. (J Cell Sci 112, 4281 (1999)). The salt concentration was maintained at ≦20 mM to avoid inhibition of the enzyme activity (Regnard et al. (1998) Biochemistry 37, 8395). Quantifications were done by scintillation counting of the α- and β-tubulin bands after SDS-PAGE and electro-transfer onto nitrocellulose, as described Regnard et al. (Biochemistry 37, 8395 (1998)). The specificity of glutamylation to tubulins was verified by submitting the reaction mixtures to SDS-PAGE and fluorography. Exposures were performed at −80° C. using Kodak (Rochester, N.Y.) XAR-5 films after enhancement with Amplify (Amersham). As noted before (Regnard et al. (2003) J Cell Sci 116, 4181), bead fractions obtained after immunoprecipitation have a disproportionally low activity compared to the input or unbound fractions, presumably because the beads hinder proper interactions between the glutamylase and the MT substrate. Therefore, 10-20 times more of the bead material was used compared to the equivalent amount of unbound and input material, but the levels of activity remained relatively low. Thus, the glutamylase assays of bead fractions cannot be considered as quantitative; they only provide qualitative information about the type of activity present. However, relevant quantitative information can be deduced from the comparison of the activities in the input and unbound material.
Antibodies
Mouse monoclonal antibodies (mAbs) used were mAb 206 (Regnard et al. (2003) J Cell Sci 116, 4181), GT335 (anti-glutamylation; Wolff et al., Eur J Cell Biol 59, 425 (1992)), ID5 (anti-glutamylation; Rüdiger et al., Eur J Cell Biol 78, 15 (1999)), 12G10 (anti-α-tubulin, from J. Frankel).
Polyclonal rabbit Abs were anti-Arp1 (courtesy of R. Melki), anti-CF Im25 (courtesy of S. Dettwiler and W. Keller), anti-GFP (Torrey Pines Biolabs, Houston, Tex.), polyE anti-glutamylation (serum 2303) and polyG anti-polyglycylation (serum 2301), the latter two courtesies of M. Gorovsky.
Laboratory-made rabbit polyclonal Abs were L26 (anti-p24/PGs5), L83 (anti-p32/PGs1; Regnard et al. (2003) J Cell Sci 116, 4181), L91 (anti-p33/PGs2 anti-peptide Ab), L90 (anti-p49/PGs3) and L80 (anti-p79/PGs4 anti-peptide Ab).
Secondary antibodies used were: HRP-labeled donkey anti-rabbit Ig 1:10,000, HRP-labeled protein A 1:5,000 (Amersham Pharmacia Biotech), goat-anti-mouse FITC and goat-anti-rabbit FITC (Zymed).
Immunoprecipitation
For immunoprecipitation, antibodies (FIGS. 5, 7, 14) were pre-incubated with protein G magnetic beads (Dynal Biotech), washed in PBS with 0.05% Tween-20 and incubated with the input material for 3 hrs at 4° C. The beads were extensively washed with Tris-HCl 50 mM pH 8.0, NaCl 0.5 M, NP-40 0.1% before analysis.
Western Blotting
For western blots shown in FIG. 5, antibodies were used at 0.5 μg/ml. For FIGS. 7 and 14, anti-GFP, GT335 and anti-polyE antibodies were diluted at 1:5,000, 1:20,000 and 1:3,000, respectively. For western blots presented in FIG. 9 antibodies were used at the following dilutions: ID5 (1:100), polyG 2301 (1:2,000), 12G10 (1:100), anti-GFP (1:1,000). Proteins were visualized with HRP-labeled donkey anti-rabbit Ig 1:10,000 or HRP-labeled protein A 1:5,000, followed by detection with chemo luminescence (Western Lightning Chemo-luminescence Reagent Plus, Perkin Elmer).
Modeling of the Structure of TTLL1
TTLL1 (PGs3) contains an ATP/Mg2+-binding site typical of enzymes with an ATP-dependent carboxylate-amine/thiol ligase activity (Galperin et al. (1997) Protein Sci 6, 2639; Dideberg et al. (1998) Trends Biochem Sci 23, 57). These so-called ADP-forming enzymes catalyze ATP-dependent ligations of carboxyl group carbon atoms of the first substrate to an amino (or imino) acid group nitrogen atoms of the second substrate via the formation of acylphosphate intermediates, as glutamylation reaction is predicted to involve. Previous structural alignments of ADP-forming enzymes have identified three conserved sequence motifs that correspond to the ATP-binding cleft (Artymiuk et al. (1996) Nat Struct Biol 3, 128), which are also well conserved in TTLL1. In enzymes with a known structure, these conserved regions are located at the ATP/Mg2+-binding sites. Nevertheless, about 85% of the sequence of the ATP/Mg2+-binding regions of the structurally characterized enzymes differs from TTLL1. To test our hypothesis about the enzymatic function of TTLL1, we applied a combination of a structure-based alignment, sequence profile search and molecular modeling. Several structures of ADP-forming enzymes (pdb codes: 1GSA, 1BNC, 2DLN, 1MOW, 1EHI, 1JDB, 1EYZ) were superposed with the Insightll program (Dayring et al. (1986) J Mol Graph 4, 82). The initial CLUSTAL alignment of the enzyme sequences was manually improved according to structural superposition. The resulting sequence alignments served for the generation of sequences profiles with the generalized sequence profile method and the pftools package (Bucher et al. (1996) Comput Chem 20, 3). The probability of random alignment was calculated by analyzing the score distribution obtained from a profile search versus a regionally randomized version of the protein database, assuming an extreme value distribution (Hofmann et al. (1995) Trends Biochem Sci 20, 347). Apart from the central ATP-binding site, the structures of all analyzed enzymes did not match. This changed when the enzyme structures were divided into four sub-structures (N-terminal substrate-binding domain, part 1 of ATP-binding domain, part 2 of ATP-binding domain and C-terminal region; FIG. 11) and separately superimposed with each other, yielding four structure-based alignments with corresponding sequence profiles. These sequence profiles (Bucher et al. (1996) Comput Chem 20, 3) were then applied to the sequence of TTLL1.
The initial structure of TTLL1 was constructed using the HOMOLOGY module of InsightII program (Dayring et al. (1986) J Mol Graph 4, 82) based on the alignment of TTLL1 to the known 3D-structures of ADP-forming enzymes, but mainly to glutathione synthetase (pdb code: 1GSA; Hara et al., Biochemistry 35, 11967 (1996)). The resulting model was subjected to 300 steps of minimization based on the steepest descent algorithm with the backbone atoms of α-helical segments restrained to their starting positions with the force constant K=100. The next 500 steps of the refinement were performed without any restrictions, using conjugate gradients algorithm. The CHARMM force field (Brooks et al. (1983) J Comp Chem 4, 187) and the distance dependent dielectric constant were used for the energy calculations. The program PROCHECK (Laskowski et al. (1993) J Appl Crystallog 26, 282) was used to verify the quality of the modeled structure. The figures were generated with the programs Molscript and Raster3D (FIG. 6) (Kraulis et al. (1991) J Appl Crystallog 24, 946; Bacon et al. (1988) J Mol Graph 6, 219). As a result, we obtained an alignment of TTLL 1 not only in the previously mentioned ATP-binding regions, but also in all major parts of the protein (FIG. 11) with a structure highly similar to glutathione synthetase. ATP, Mg2+ and a peptide with a glutamate side chain were docked into the model taking into consideration an analogous molecular co-crystallization with know ATP-dependent carboxylate-amine ligases (FIG. 6). For simplification of the model, we have fitted an A-E-A peptide modified on the central E into the structure, however, we have before shown that the flanking A residues could be replaced by others without changes for the fitting.
Searching of Genomes and Phylogenetic Analyses
The Arabidopsis thaliana, yeast, human and Giardia lamblia predicted TTL domain protein sequences were identified by BLAST searches of the NCBI databases (available on the world wide web at ncbi.nlm.nih.gov/BLAST/). The Drosophila melanogaster sequences were identified by BLAST searches at the FlyBase (available on the world wide web at flybase.bio.indiana.edu/). The Caenorhabditis elegans, and Trypanosoma brucei sequences were identified using The Sangers Institute (available on the world wide web at sanger.ac.uk/DataSearch/) and The Institute for Genomic Research (TIGR) databases for respective species (available on the world wide web at tigr.org). The Tetrahymena thermophila TTL domain sequences were identified by tBLASTn searches of the recently assembled macronuclear genome at the TIGR Tetrahymena blast server (available on the world wide web at tigrblast.tigr.org/er-blast/index.cgi?project=ttg) using genomic scaffolds of the November 2003 assembly. Using HsTTLL1 we first identified the Tetrahymena TTLL1 homolog gene and exhaustive searches of the Tetrahymena genome were continued using TTLL1 and several other TTL domain sequences. Starting from the region of homology to TTL domains of other organisms, the probable coding region was reconstructed based on the higher G/C content. The intron junctions were tentatively identified based on the known consensus splice sites (Wuitschick et al. (1999) J Eukaryot Microbiol 46, 239). As we have completed the manual prediction of the coding regions, the Tetrahymena Genome Project at TIGR has published the preliminary gene predictions based on gene finder programs. In general there was an agreement between the manual and software-based predictions. We verified each putative TTLL sequence by a reverse BLASTp of the NCBI databases. A cDNA sequence of TTLL4A was kindly provided by Drs. Kathleen Clark and Martin Gorovsky (University of Rochester, N.Y.). The cDNA plasmid of TTLL14F was kindly provided by Dr. Aaron Turkewitz (University of Chicago, Ill.).
The ends of the transcribed regions of TTLL1 and TTLL6A genes were identified by PCR of cDNAs pools or cDNA libraries from Tetrahymena cells grown under several different conditions. Total RNA was isolated using the modified TRI Reagent procedure (MRC; Chomczynski et al. (1995) Biotechniques 19, 942). cDNA was synthesized using primers of the SMART cDNA library construction kit and the PowerScript Reverse Transcriptase (Clontech). One pg of total RNA was used for the first strand cDNA synthesis (1.5-2 hrs at 42° C.). Next, 1 μl of the first strand cDNA reaction mix was used for a PCR reaction with the same primers (extension step at 68° C. for 8 min). A cDNA from cilia-regenerating cells was used to construct a small cDNA library (43,000 clones) by cloning into a plasmid vector according to the Clontech kit instructions. The 5′ end of TTLL1 UTR was determined by 5′RACE PCR of the cDNA library using a gene specific primer anchored inside the conserved region and the SMART IV primer (Clontech). The 3′ end of the TTLL1 cDNA was identified by 3′ RACE PCR using a gene-specific primer and the CDS III/3′ primer (Clontech). The same 5′ and 3′ RACE strategy with the cDNA library as a template was used to obtain terminal fragments of cDNAs for TTLL6. The fragment of cDNA initially amplified for the 3′ end of the TTLL6 UTR lacked a stop codon in the translational frame, suggesting that the cDNA template was truncated. The protein prediction TTLL6A produced by the TIGR gene finder software indicated that the coding region extends for another 286 codons. Using a primer walking strategy with a pool of cDNA we subsequently mapped the end of TTLL6A transcribed region at a more downstream position, consistent with the TIGR prediction.
The TTL domains of each predicted protein sequence were identified manually using highly conserved peptide motifs and a preliminary alignment was done using Clustal X 1.82 program (Jeanmougin et al. (1998) Trends Biochem Sci 23, 403) followed by extensive manual adjustment using the Seaview program (Galtier et al., (1996) Comput Appl Biosci 12, 543). Phylogenetic analyses were performed using the Phylip version 3.6 package (Felsenstein, (1997) Syst Biol 46, 101) using the following programs. One hundred replicates of the sequence set were created using SEQBOOT. The distances were calculated using PROTDIST, trees were reconstructed using NEIGHBOR. The Jones-Taylor-Thorton (JTT) substitution model was used. A consensus tree was obtained using CONSENSE and the tree was plotted using DRAWGRAM.
Gene Knockout in Tetrahymena 
A 1.9 kb 5′ untranslated region immediately upstream of the TTLL1 coding region was amplified using the KOS 1 (5′-ATTTTATGAGCTCCACCATCTTTTATTTTGCTTT-3′) (SEQ ID NO:1) and KOA1 (5′-TAAATAAGGATCCACACAAAATAGATAAAAAGGAG-3′) (SEQ ID NO:2) primers. SacI and BamHI restriction sites were introduced at the ends of the above primer sequences, respectively. The PCR product was digested with BamHI and SacI and cloned into the p4T2-1 plasmid on one side of the neo2 cassette. A 1.6 kb fragment of the 3′ UTR of TTLL1 containing a part of TTLL1 coding region was amplified using the TTL-K02-F (5′-ATTTTATATCGATTTGTT AAACCAGCATCACGA-3′) (SEQ ID NO:3) and TTL-KO2-R (5′-TAAATAACTCGAGAAAATTAAATGTCTGGCTGGAT-3′) (SEQ ID NO:4) primers. The ClaI and XhoI restriction sites were introduced at the ends of the primers and used for subcloning into the other side of the neo2 cassette of p4T21. The resulting gene knockout targeting plasmid named pTTLL1-KO was digested with SacI and XhoI to release the disruption fragment of TTLL1::neo2 from the plasmid backbone. The germline disruption of the TTLL1 gene was done by biolistic bombardment and the knockout heterokaryons were constructed as described in Cassidy-Hanley et al. (Genetics 146, 135 (1997)). Cells homozygous for the TTLL1 deletion were obtained by a cross of two TTLL1 knockout heterokaryons (Hai et al. (2000) Methods Cell Biol 62, 513).
Expression and Localization of GFP Fusion Proteins in Tetrahymena 
The pMTT1-GFP-N vector suitable for expression of coding regions with an N-terminal GFP tag under control of the MTT1 promoter was constructed on the basis of the pMTT1-IFT52-GFP plasmid (Brown et al. (2003) Mol Biol Cell 14, 3192). The coding region of IFT52 of this plasmid was replaced by the coding region of GFP (Haddad et al. (1997) Proc Natl Acad Sci USA 94, 10675) using a forward primer with a HindIII site and a reverse primer with MluI-BamHI sites (and a TGA stop codon between these two sites). Subcloning a coding region between the HindIII and BamHI sites of the resulting pMTT1-GFP-N provides a fragment which can be integrated into the BTU1 locus of Tetrahymena and the coding region can be expressed using the MTT1 promoter fragment in response to cadmium (Shang et al. (2002) Proc Natl Acad Sci USA 99, 3734). To overexpress TTLL1, the entire predicted coding region of TTLL1 gene was amplified from a total genomic DNA with addition of an MluI site to the forward primer and a BamHI site to the reverse primer and subcloned into pMTT1-GFP-N to create pMTT1-GFP-TTLL1. The same strategy was used to amplify the predicted coding region of TTLL6A to create pMTT1-GFP-TTLL6L plasmid, except that the reverse primer was designed to anchor at the position of 390 by downstream of the predicted TGA stop codon. Thus, after integration into the Tetrahymena genome, using the pMTT1-GFP-TTLL6L fragment, the TTLL6A coding region is probably expressed using the native 3′ nontranscribed region. To create a plasmid for expression of the truncated version of Ttll6Ap gene product (Ttll6ApΔ710-GFP), the coding region encompassing the amino acid codons 1-710 was amplified with MluI and BamHI sites added at the ends of the primers and subcloned into pMTT1-GFP-N to create pMTT1-GFP-TTLL6S. To produce a plasmid for expression of the ATPase deficient form of Ttll6ApΔ710-GFP, we changed the predicted codon 422 from glutamic acid to glycine using the QuickChange site-directed mutagenesis kit (Stratagene) and obtained the plasmid named pTTLL6Δ-GFP.
For expression of GFP fusion proteins in Tetrahymena, we used the negative selection method based on targeting to the BTU1 locus (Gaertig et al. (1999) Nat Biotechnol 17, 462). All plasmids used for expression of GFP fusions had fragments of the BTU1 locus flanking sequences for homologous targeting. Following biolistic bombardment of starved CU522 cells, transformants with integrations into the BTU1 locus were selected with 20 μM paclitaxel and the presence of a transgene was confirmed by PCR using total genomic DNA. The expression of the transgene was induced by adding 2.5 μg/ml cadmium chloride to the medium and cells were analyzed microscopically or biochemically 2-4 hrs later.
Immunofluorescence and Microscopy
For immunofluorescence, cells were processed as described in Thazhath et al. (Nat Cell Biol 4, 256 (2002)) with modifications. Briefly, we hand-picked 50-100 cells using a pipette, washed cells by releasing into a drop of 10 mM Tris-HCl, pH 7.5 buffer on a cover slip. Cells were permeabilized on cover slips for 45-60 sec by exposure to 10 μl of 0.5% Triton-X100 in the PHEM buffer (Gaertig et al. (1992) Protoplasma 167, 74), followed by addition of 15 μl of 2% paraformaldehyde in the PHEM buffer. The fixed cells were air-dried and coverslips incubated for 10 min in the blocking solution (3% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) with 0.1% Tween-20) and incubated for a few hours at room temperature (or overnight at 4° C.) in primary antibodies in PBS with 3% BSA and 10% normal goat serum. The following primary antibodies were used at the indicated dilutions: anti-GFP (1:100), and ID5 (1:10). The cover slips were washed 3 times for 5 min by immersion in PBS using small staining jars followed by incubation in the secondary antibodies diluted 1:100 for 1 hr at room temperature. After 3 washes cover slips were mounted as described in Gaertig et al. (Protoplasma 167, 74 (1992)). To visualize only Ttll6Ap-GFP fluorescence, cells were fixed as described above and analyzed directly. Images were collected using a Leica TCS SP2 Spectral Confocal Microscope with Coherent Ti: sapphire multiphoton laser (Mira Optima 900-F). Usually a series of optical sections was obtained and the top half of the sections was assembled into a composite image.
FIG. 10 describes co-purification of the glutamylase subunits and enzymatic activity. Fractions of the two main purification steps of glutamylase from brain extract, phospho cellulose chromatography (A, B) and sucrose gradient centrifugation (C, D), are analyzed for glutamylase activity (A, C) and by western blot for the proteins p24, p32, p33, p49, p′79, Arp1 and CF Im25 (B, D). The five subunits p24 to p79 all eluted together with the peaks of enzymatic activity (A, C). CF Im25 and Arp1 show partial co-elution with glutamylase in the phospho cellulose step, but were mostly recovered in other fractions of the sucrose gradient. Note that L80 cross-reacts on western blots with another protein band of slightly higher apparent mass than p79, which is not immunoprecipitated (* the arrow points to the specific protein band for p79).
FIG. 11 shows a sequence profile alignment for TTLL1 modeling. The sequences of glutathione synthetase from E. coli (1GSA; SEQ ID NO:6) and TTLL1 from mouse (mTTLL1; SEQ ID NO:7) have been aligned. The elements of the secondary structure, α-helices (turquoise arrows) and β-sheets (violet boxes), are shown above the alignment. Amino acids in lower case (mTTLL1) stand for regions that were not modeled. Red numbers indicate the beginning of four sub-domains: (1) N-terminal substrate-binding domain, (2) part 1 and (3) part 2 of the ATP-binding domain and (4) the C-terminal region. Residues that interact with ATP and Mg2+ are in bold letters and the corresponding domains are outlined in yellow. All residues of the active site that are conserved with other ATP-dependent carboxylate-amine ligases are written in dark green (see FIG. 6C). Residues located in the proximity of the substrates are outlined in green. The E326 residue (mTTLL1) indicated by an arrowhead corresponds to the matched E422 residue of Ttll6Ap that has been mutated in Ttll6ApΔ710-E422G.
FIG. 12 presents a phylogenetic analysis of predicted protein gene sequences containing a TTL domain. The TTL domains of each predicted protein sequence were aligned was using Clustal X 1.82 followed by manual adjustment in SeaView. Phylogenetic analyses were performed using the Phylip version 3.6 package. One hundred replicates of the sequence set were created using SEQBOOT. The distances were calculated using PROTDIST, and trees were constructed using NEIGHBOR. The Jones-Taylor-Thorton (JTT) substitution model was used. A single consensus tree was obtained using CONSENSE. The yeast sequence was used as an outgroup. Tetrahymena has an extraordinarily large number of predicted TTL domain genes (50). Only one paralog of Tetrahymena is shown in each orthologous group and the number of additional paralogs is indicated in parentheses. The following are GenBank accession numbers of predicted protein sequences used in the analysis. Some sequences are not yet deposited in GenBank including some of the Trypanosoma brucei genes (whose sequences were derived from TIGR annotations) and most Tetrahymena sequences (also derived from the genomic sequences identified using the TIGR Tetrahymena blast server. Homo sapiens: TTL (Q8NG68), TTLL1 (NP—036395), TTLL2 (AAH30650), TTLL3 (T12515), TTLL4 (NP—055455), TTLL5 (NP—05587), TTLL6 (BACO5032), TTLL7 (AAH60878), TTLL8 (XP—104657), TTLL9 (XP—092778), TTLL10 (BAC85781), TTLL11 (AAM81328), TTLL12 (AAH01070), TTLL13 (XP—496092). Drosophila melanogaster: CG32238 (NP—729025), CG31108 (NP—733081), CG16833 (NP—723643), CG16716 (NP—725916), CG11201 (NP—609068), CG11323 (NP—609069), CG8918 (NP—573197), CG5987 (NP—651549), CG4089 (NP—650021), CG3964 (NP—722946), CG1550 (NP—610325), Saccharomyces cerevisiae: Ybr094wp (NP—009652.1). Arabidopsis thaliana: Atlg77550 (NP—177879.2). Trypanosoma brucei: Tb927.1.1550 (CAB95431), Tb927.2.5250 (XM—340623) Tb05.6E7.820 (TIGR annotation 314.m00409), Tb06.2N9.120 (TIGR 315.m00360) Tb03.5L5.580 (TIGR 312.m00548), Tb09.211.1170 (TIGR 320.m00798) Tb11.02.4640 (322.m00560), Tb10.61.3050 (TIGR 319.m01288) Tb11.02.0020 (322.m00126). Caenorhabditis elegans: F25C8 (part of the F25C8 cosmid, Sanger Institute, UK), C55A6.2 (NP—505918), ZK1128.6 (CAA87425), I-123L24.3 (AAL06035), K07C5.7 (CAA94900), D2013.9 (CAA87783). Giardia lamblia: GLP—43—54366—55577 (EAA40434), GLP—43—15991—17301 (EAA40412.1), GLP—618—12970—14472 (EAA38012.1), GLP—205—13412—15397 (EAA38969.1), GLP—223—2566—4779 (EAA37058.1), GLP—251—3885—7112 (EAA38326.1), GLP—203—36475—34136 (EAA39544.1).
The following are scaffold designations for the Tetrahymena TTLL gene sequences using the November 2003 assembly of the Tetrahymena macronuclear genome by TIGR: TTLL1 (8254582), TTLL2 (8254617), TTLL3A (8254579), TTLL3B (8254645), TTLL3C (8254670), TTLL3D (8254548), TTLL3E (8254652), TTLL4A (8254630), TTLL4B (8254717), TTLL4C (8254496), TTLL4D (8254359), TTLL4E (8254495), TTLL6A (8254600), TTLL6B (8254747), TTL6C (8254577), TTLL6D (8254607), TTLL6E (8254650), TTLL6F (8254010), TTLL9 (8254449), TTLL10A (8254580), TTLL10B (8254751), TTLL10C (8254034), TTLL10D (8254666), TTLL10E (8254504), TTLL10F (8254563), TTLL12A (8254576), TTLL12B (8254820), TTLL14A (8254487), TTLL14B (8254479), TTLL14C (8254589), TTLL14D (8254379), TTL14E (8254555), TTLL14F (8254814), TTLL14G (8253864), TTLL14H (8254469), TTLL14I (8254688), TTLL14J (8254798), TTLL14K (8254403), TTLL14L (8254823), TTLL14M (8254557), TTLL14N (8254748), TTLL14O (8254609), TTLL14P (8254819), TTL14Q (8254545), TTLL14R (8254737), TTLL14S (8254811), TTLL14T (8254373), TTLL15A (8254385), TTLL15B (8254460), TTLL15C (8254459).
FIG. 13 shows a multiple sequence alignment of TTL domains (SEQ ID NOs:8-65) of TTL and TTLL proteins. This alignment was used to construct the tree shown in FIGS. 8 and 12. Regions with low homology were not included in the alignment. Shaded regions indicate either amino acid identity or amino acid similarity (Clustal X alignment was colored with BoxShade and is available on the world wide web at ch.embnet.org/software/BOX_form.html). At: Arabidopsis thaliana, Ce: Caenorhabditis elegans, Dm: Drosophila melanogaster, Gl: Giardia lamblia, Hs: Homo sapiens, Sc: Saccharomyces cerevisiae, Tb: Trypanosoma brucei, Tt: Tetrahymena thermophila. TTL domains represented in FIG. 13 are Homo sapiens: TTL (SEQ ID NO:31), TTLL1 (SEQ ID NO:10), TTLL2 (SEQ ID NO:44), TTLL3 (SEQ ID NO:33), TTLL4 (SEQ ID NO:25), TTLL5 (SEQ ID NO:43), TTLL6 (SEQ ID NO:22), TTLL7 (SEQ ID NO:45), TTLL8 (SEQ ID NO:32), TTLL9 (SEQ ID NO:15), TTLL10 (SEQ ID NO:57), TTLL11 (SEQ ID NO:53), TTLL12 (SEQ ID NO:37), TTLL13 (SEQ ID NO:63); Drosophila melanogaster: CG32238 (SEQ ID NO:9), CG31108 (SEQ ID NO:41), CG16833 (SEQ ID NO:26), CG16716 (SEQ ID NO:20), CG11201 (SEQ ID NO:49), CG11323 (SEQ ID NO:39), CG8918 (SEQ ID NO:11), CG5987 (SEQ ID NO:21), CG4089 (SEQ ID NO:56), CG3964 (SEQ ID NO:27), CG1550 (SEQ ID NO:36); Saccharomyces cerevisiae: Ybr094wp (SEQ ID NO:62); Arabidopsis thaliana: Atlg77550 (SEQ ID NO:64); Trypanosoma brucei: Tb927.1.1550 (SEQ ID NO:50), Tb927.2.5250 (SEQ ID NO:30), Tb05.6E7.820 (SEQ ID NO:13), Tb06.2N9.120 (SEQ ID NO:51), Tb03.5L5.580 (SEQ ID NO:48), Tb09.211.1170 (SEQ ID NO:58), Tb11.02.4640 (SEQ ID NO:59), Tb10.61.3050 (SEQ ID NO:60), Tb11.02.0020 (SEQ ID NO:61); Caenorhabditis elegans: F25C8 (SEQ ID NO:16), C55A6.2 (SEQ ID NO:42), ZK1128.6 (SEQ ID NO:24), H23L24.3 (SEQ ID NO:52), K07C5.7 (SEQ ID NO:65), D2013.9 (SEQ ID NO:38); Giardia lamblia: EAA40434 (SEQ ID NO:12), EAA40412.1 (SEQ ID NO:17), EAA38012.1 (SEQ ID NO:19), EAA38969.1 (SEQ ID NO:54); Tetrahymena: TTLL1 (SEQ ID NO:8), TTLL2 (SEQ ID NO:46), TTLL3B (SEQ ID NO:55), TTLL4A (SEQ ID NO:23), TTLL6A (SEQ ID NO:18), TTLL6E (SEQ ID NO:40), TTLL9 (SEQ ID NO:14), TTLL10A (SEQ ID NO:34), TTLL12A (SEQ ID NO:35), TTLL14A (SEQ ID NO:28), TTLL14H (SEQ ID NO:29), TTLL1 SC (SEQ ID NO:47). Amino acid sequences within a conserved region located within the ATP-binding region and in close proximity to a substrate include subsequences NHFPGMFSLARK (SEQ ID NO:66) from T. thermophila Ttll6Ap and NHFPNHYELTRK (SEQ ID NO:67) from T. thermophila Ttll1 at amino acid positions 58-69 in FIG. 13A. A generic subsequence showing highly conserved amino acids, based on SEQ ID NO:67 from TtTtll1 as the reference subsequence, is NHFPXXXXLXRK (SEQ ID NO:68).
FIG. 14 shows glutamylation activity of Ttll6Ap in vitro. Glutamylase activity co-purified with Ttll6ApΔ710-GFP protein under all conditions tested, including immunoprecipitation (A, B), phosphocellulose (results not shown) and size exclusion chromatography (C). A-B. Soluble extracts of Ttll6ApΔ710-GFP expressing cells were submitted to immunoprecipitation with anti-GFP antibodies or unrelated rabbit antibodies (mock). The input (In) as well as the unbound (Un) and bound (Bo) fractions were analyzed by western blot with anti-GFP antibody (A) and assayed for tubulin glutamylase activity (B). Equal proportions of input, unbound and bead fractions were analyzed by western blotting, while 10 times more bead fraction was assayed for glutamylase activity. C. Size exclusion chromatography using a soluble fraction of cells expressing Ttll6ApΔ710-GFP. A soluble extract (4 mg of protein) of induced Ttll6ApΔ710-GFP expressing cells was fractionated on an HPLC TSK column. Each fraction was analyzed by western blotting with anti-GFP antibodies (lower panel) and assayed for tubulin glutamylase activity using adult brain MTs as substrate (radioactive glutamate concentration 5 μM). The results are expressed as dpm incorporated into α- (open circles) and β-tubulin (filled circle). The absorbance profile at 280 nm is shown (dashed line). Arrowheads indicated the elution peak position of standard proteins used for calibration. The enzymatic activity and Ttll6ApΔ710-GFP co-eluted in a peak of 180 kDa, while the expected size of Ttll6ApΔ710-GFP is 116 kDa. It is possible that the Ttll6ApΔ710-GFP is a homodimer, especially because the N-terminal region of the predicted peptide sequence contains a coiled-coil region.







TABLE I







Identification of all proteins precipitated with mAb 206 (see FIG. 5A).












spot

Genebank
recovered
MASCOT



NoA)
identified proteinB)
accession NoB)
sequenceB)
scoreB)
protein identity















p79C)
D430025H09Rik protein
16741525
26%
622
p79/PGs4


1
nicolin 1D)
13384852
42%
315
p24/PGs5


2
nicolin 1D)
13384852
48%
404
p24/PGs5


3
cleavage and polyadenylation specific factor 5,
13386106
23%
190
CF Im25



25 kD subunit



(Ruegsegger et al.,







Mol Cell 1, 243







(1998))


3
complement component 1, q subcomponent,
6671652
33%
236
non-specific



gamma polypeptide


4
chain A, IgG2a Fab fragment
640171
15%
198
non-specific


5
gene trap ROSA b-geo 22
22507341
32%
496
p32/PGs1


6
gene trap ROSA b-geo 22
22507341
34%
496
p32/PGs1


7
gene trap ROSA b-geo 22
22507341
57%
607
p32/PGs1


8
gene trap ROSA b-geo 22
22507341
38%
572
p32/PGs1


9
RIKEN cDNA 5730494M16
25029762
43%
384
p33/PGs2


10
ARP1 actin-related protein 1 homolog A,
5031569
20%
287
Arp1 (Holleran et al.,



centractin alpha



J Cell Biol 135, 1815







(1996); Schafer et al.,







J Cell Biol 126, 403







(1994))


11
tubulin tyrosine ligase-like 1
30725861
18%
321
p49/PGs3


12
tubulin tyrosine ligase-like 1
30725861
30%
428
p49/PGs3


13
tubulin tyrosine ligase-like 1
30725862
45%
531
p49/PGs3


14
similar to Ig gamma-2B chain C region secreted
28523035
35%
156
non-specific



form


14
fibrinogen, B beta polypeptide
20872398
44%
665
non-specific


15
immunoglobulin heavy chain constant region
14091948
21%
198
non-specific


15
fibrinogen, B beta polypeptide
20872398
30%
552
non-specific


16
chaperonin subunit 8 (theta)
6753328
43%
1110
TRIC component







(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))


17
chaperonin containing TCP-1 theta subunit
5295992
23%
531
TRIC component







(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))


18
chaperonin subunit 5 (epsilon)
6671702
10%
225
TRIC component







(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))


19
keratin 1
17318569
25%
620
contamination


20
matricin
631730
30%
640
TRIC component







(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))


21
chaperonin subunit 6a (zeta); chaperonin
6753324
25%
678
TRIC component



containing TCP-1



(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))


22
chaperonin subunit 6a (zeta); chaperonin
6753324
27%
594
TRIC component



containing TCP-1



(Lopez-Fanarraga et







al., J Struct Biol 135,







219 (2001); Leroux et







al., Curr Biol 10,







R260 (2000))





A)numbering according to excised spots from 2D-gel, FIG. 5A.


B)obtained with: MASCOT Matrix science, available on the world wide web at matrixscience.com.


C)identified from one-dimensional SDS-PAGE as shown in FIG. 6A (C. Regnard et al., J Cell Sci\ 116, 4181 (2003)).


D)protein of unknown function named nicolin (Backofen et al., Eur J Biochem 269, 5240 (2002)).






Example II
Biological Assay for Glutamylase Inhibition
The invention provides a phenotype-based assay for inhibitors of tubulin glutamylation. The assay is a biological assay; that is, it is performed in a host cell, preferably Tetrahymena. It is also referred to herein as an “in vivo” assay or “Assay I.”
Overproduction of the truncated Ttll6Ap-GFP under cadmium-inducible promoter (at 2.5 μg/ml CdCl2) caused a tight arrest in cell multiplication of Tetrahymena. Decreasing the concentration of cadmium allowed for more growth while increasing it two-fold was lethal within 18 hours. Thus, the extent of growth inhibition by Ttll6Ap is dependent on its intracellular concentration (which in turn is dependent on cadmium concentration). The presence of a compound which inhibits Ttll6Ap activity should partly or completely rescue the cell growth arrest. A preferred embodiment of the in vivo assay is outlined in FIG. 3 (left panel). The assay involves adding CdCl2 to Tetrahymena cells in which Ttll6Ap expression is controlled by the cadmium-dependent microtubule T1 promoter, distributing cells into wells with compounds, incubating for a time required for uninhibited cells to grow, and reading a fluorescent signal that reflects cell density. Previous studies have established that there is an approximately linear response in the level of protein production using the microtubuleT1 promoter within the range of 0.25-5 μg/ml of CdCl2 (Shang et al. (2002). Proc Natl Acad Sci 99, 3734-3739). A range of cadmium concentrations is used to create a spectrum of target/inhibitor ratios.
The in vivo assay can be optimized as follows:
1) A strain with a red fluorescent protein (RFP) reporter is constructed so that cell density can be determined instantly using plate reader (e.g., BMG Fluorostar Optima reader). Fluorescence is beneficial over alternative readout types because there is no need to add detection substrates. In a pilot experiment and without any optimization we were able to detect a signal in living highly mobile GFP-expressing Tetrahymena cells above 10,000 cells/ml (FIG. 4). A simultaneous quantification of RFP and Ttll6Ap-GFP will help in sorting the primary hits. First, a strain that has RFP coding region stably integrated under a strong promoter whose activity is known to reflect cell proliferation (nonessential histone locus HHT1) is constructed. Next, a microtubuleT1-driven Ttll6Ap-GFP transgene is introduced into the nonessential BTU1 locus using a negative selection method (Gaertig et al. (1999) Nat. Biotech. 17, 462-465; U.S. Pat. No. 6,846,481, issued Jan. 25, 2005). As a control, a similar strain containing a version of Ttll6Ap that is inactive due to a mutation in the ATP-binding site (RFP, Ttll6Ap-G422-GFP) is prepared. The simultaneous use of this strain is valuable as a counterscreen for unrelated growth stimulators and compounds that are highly toxic. Our data indicate that Ttll6Ap-overproducing cells upon induction with 2.5 μg/ml CdCl2 at density of  ̃104 cells/ml divide only once and do not proliferate for at least 12 hrs while controls undergo between 4-5 doublings (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). Thus, the signal to noise ratio could theoretically be  ̃20 but the signal could be amplified by RFP fluorescence.
2) Assay conditions can be established for the following parameters:
a) Cadmium concentration and exposure time. An important aspect is the titration of potential inhibitory compounds. As discussed, the presence of a compound at a concentration that depletes the overproduced enzyme should promote cell growth. However, a relevant compound is likely to inhibit cell growth at its higher concentration that also depletes the endogenous glutamylases. This will especially be the case of potential broad specificity inhibitors that affect both TTLL6 and TTLL1 type glutamylases β-tubulin and α-tubulin preferring, respectively). The issue of titration could be dealt with by using compounds in several different concentrations as it is done routinely in HTS. Alternatively, Ttll6Ap can be induced at several levels by varying the cadmium concentration. This could lower the cost of screened compounds especially if the assay volume can also be reduced (see below). Using 96-well plates, the most optimal range of CdCl2 concentration to produce the desired signal/noise ratio can be determined. Three concentrations can be chosen: 1) the lowest, 2) mid-range and 3) highest concentration of CdCl2 that inhibit the multiplication of RFP, Ttll6Ap-GFP cells for a period sufficient to give a signal/noise ratio above 5. If needed, the exposure time can be increased to allow uninhibited cells to produce stronger readout signal.
b) Cell concentration. The lower the initial cell concentration, the lower the background but potentially longer time of incubation may be needed to produce a sufficiently strong signal.
c) Assay volume. The smaller the volume, the smaller the required quantities of screened chemicals. Furthermore, Tetrahymena cells grow faster and to higher densities in smaller drops due to increase aeration, which may increase the signal/noise ratio.
d) DMSO. DMSO is commonly used as a solvent for compounds. Published data indicate that Tetrahymena tolerates well DMSO at up to 2.5% (Nilsson et al. (1974) J Cell Sci 16, 39-47.
e) Assay sensitivity. Assay sensitivity can be evaluated by running assays on plates and including control wells with a series of CdCl2 concentrations that are progressively lower. Lowering the cadmium concentration will mimic the Ttll6Ap activity-depleting action of an inhibitory compound. The actual effect of lowering CdCl2 on the level of
Ttll6Ap-GFP can be evaluated quantitatively by western blotting (using purified Ttll6Ap as a standard). Thus, we can determine the minimal depletion in the Ttll6Ap activity that can be detected using our assay.
If this assay is used as a primary screen, two secondary screens for hit validation can be used: 1) immunofluorescence and western blotting of compound-treated cells with anti-glutamylation antibodies will determine whether the compound affects the level and pattern of tubulin glutamylation; microscopic observations of treated cells will also determine whether the compound's action gives phenotypic changes that are consistent with deficiency in tubulin, and 2) an in vitro assay with purified enzyme will determine whether the compound directly inhibits the glutamylase enzyme activity. Further assays can be used to determine whether the enzyme is a broad or selective inhibitor and whether it can act on mammalian enzymes.
Example III
Cell-Free Assay for Glutamylase Inhibition
The assay is an “in vitro” assay; that is, it is performed in a cell-free environment. The in vitro assay is referred to herein as “Assay II.” An in vitro assay is useful for hit validation (secondary screen) or possibly also as a primary high throughput screen. Assay for the purpose of a secondary screen is described first. The in vivo screen described (Assay I) could identify a large number of compounds. Several mechanisms could give a positive readout: 1) direct inhibition of glutamylases, 2) inhibition of an activator of glutamylases, 3) inhibition of an effector of microtubule glutamylation, 4) stimulation of a tubulin deglutamylase (there is evidence that reverse enzymes exist (Audebert et al. (1993) Mol. Biol. Cell 4, 615-626), 5) inhibition of expression by the MTT1 promoter, 6) growth stimulation by unrelated mechanisms, 7) intrinsic fluorescence of a compound. If we simultaneously read the RFP and GFP signals, we should eliminate compounds which affect the MTT1-driven expression. Compounds that promote growth non-specifically will show an increase in RFP signal in the counterscreen with cells expression inactive glutamylase. Compounds with intrinsic fluorescence will increase signal in the counterscreen as well. Compounds of type 1) (direct inhibitors) can be identified using an in vitro reaction with a purified Ttll6Ap. A reliable in vitro assay has been established for Ttll6Ap using a crude or purified Ttll6Ap, taxol-stabilized brain microtubules, ATP, and tritiated glutamic acid (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). Following the reaction in vitro, products are separated by 1D SDS-PAGE, blotted onto nitrocellulose and the level of incorporation of tritiated E into α- and β-tubulin is determined for excised bands using liquid scintillation counting. A fairly large number of compounds can be readily screened with a crude cytosolic assay. While this will eliminate some compounds, a relatively pure enzyme will be needed to identify compounds that are direct inhibitors (although compounds that are in categories 2-4 are also of great interest in long-term). Ttll6Ap has been partially purified from cytosol of overproducing cells by a phosphocellulose chromatography and gel filtration (Janke et al. (2005) Science 308, 1758-1762 (see Example I)). An alternative method for purifying the enzyme is possible using a modification of the tandem affinity purification (TAP) method (O'Connor et al. (2005) J Biol Chem 280, 17533-17539, Prathapam et al. (2005) Nat Struct Mol Biol 12, 252-257). The batch-purified enzyme can be used for validation of primary hit compounds using our established (radioactive) assay. The assay is time-consuming (1 day) but highly reliable and suitable as a validation screen.
Because TTLL6Ap is an ATPase, another assay is possible based on the production of ATP-dependent luminescence by luciferase with luciferin substrate. This assay measures the amount of ATP left after the reaction with an ATP consuming-enzyme and the level of luminescence is inversely proportional to the measured enzyme activity. The assay is widely used with kinases and has been used with other ATPases such as myosin (Cheung et al. (2002) Nat Cell Biol 4, 83-88). The assay is shown in in FIG. 3 (right panel). The ATP concentration is first optimized using a standard assay with radiolabelled glutamate. Next, a relatively low ATP concentration (micromolar range) is used, under which the reaction is still robust, to determine the timing of the reaction under which most of ATP is depleted by the activity of the TTLL6Ap. Once these conditions are established, the reaction is performed at 95% of ATP hydrolysis with luciferin and luciferase, and the luminescence signal is measured using the Lmax luminescence plate reader. The reliability and sensitivity of this assay can be determined by running test reactions in the presence of tritiated glutamic acid and analyzing products by both luminescence and liquid scintillation counting. Many parameters of the assay can be optimized, including the enzyme and substrates concentration, pH, Mg2+ concentration. One modification which could substantially lower the cost of high throughput screening is the potential use of ciliary axonemes as a microtubule substrate, in place of standard taxol-stabilized brain microtubules. Tetrahymena microtubules are certainly a formidable substrate in vivo (FIG. 2) and ciliary microtubules can be easily purified at extremely low cost. The assay sensitivity to inhibitors can be determined by using different ratios of ATP and nonhydrolyzable analog, AMP-PNP.
If the in vitro assay (Assay II) is used as a primary screen, the following secondary screens can be used for validation: 1) an in vitro activity assay with purified enzyme and tritiated glutamate; 2) immunofluorescence and western blotting of treated cells with anti-glutamylation antibodies to determine whether the compound affects the level of tubulin glutamylation in vivo (and causes anticipated types of phenotypic changes) which would indicate that it can penetrate the plasma membrane. Finally, using an overexpression in vivo assay (Assay I) we can determine whether the compound inhibition can be relieved by mild overproduction of Ttll6Ap.
Example IV
Hyperglutamylation of Tubulin Can either Stabilize or Destabilize Microtubules in the Same Cell
In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. In this example, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.
Microtubules are dynamic elements of the cytoskeleton that are assembled from heterodimers of α- and β-tubulin. Once assembled, tubulin subunits undergo several conserved posttranslational modifications (PTMs) that diversify the external and luminal surfaces of microtubules (Verhey and Gaertig. 2007. Cell Cycle 6:2152-2160). Two tubulin PTMs, glycylation and glutamylation, collectively known as polymodifications, form peptide side chains that are attached to the γ-carboxyl groups of glutamic acids in the primary sequence of the C-terminal tails (CTTs) of α- and β-tubulin (Eddé et al., 1990. Science 247:83-85; Redeker et al., 1994. Science 266:1688-1691). Glutamylated microtubules are abundant in projections of neurons (Eddé et al., 1990. Science 247:83-85), axonemes (Bre ́ et al., 1994. Cell Motil. Cytoskeleton 27:337-349; Fouquet et al., 1994. Cell Motil. Cytoskeleton 27:49-58; Gagnon et al., 1996. J. Cell Sci. 109(Pt. 6):1545-1553), and centrioles/basal bodies (Bobinnec et al., 1998. Cell Motil. Cytoskeleton 39:223-232; Million et al., 1999. J. Cell Sci. 112(Pt. 23):4357-4366) and are detectable in the mitotic spindle and on a subset of cytoplasmic network microtubules (Abal et al., 2005. Biol. Cell 97:425-434; Bobinnec et al., 1998. Cell Motil. Cytoskeleton 39:223-232). The modifying enzymes, tubulin glutamic acid ligases (tubulin E-ligases), belong to the family of proteins related to the tubulin tyrosine ligase (TTL), known as TTL-like (TTLL) proteins (Janke et al., 2005. Science 308:1758-1762; van Dijk et al., 2007. Mol. Cell 26:437-448; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Tubulin glutamylation appears to be important in vivo. A knockdown of the TTLL7 E-ligase mRNA in cultured neurons inhibits the outgrowth of neurites (Ikegami et al., 2006. J. Biol. Chem. 281:30707-30716). A loss of PGs1, a protein associated with TTLL1 E-ligase (Janke et al., 2005. Science 308:1758-1762; Regnard et al., 2003. J. Cell Sci. 116:4181-4190), disorganizes sperm axonemes in the mouse (Campbell et al., 2002. Genetics 162:307-320), and a morpholino knockdown of TTLL6 E-ligase expression in zebrafish inhibits the assembly of olfactory cilia (Pathak et al., 2007. Mol. Biol. Cell 18:4353-4364). The biochemical consequences of tubulin glutamylation in vivo are poorly understood, but the emerging model is that this PTM regulates interactions between microtubules and microtubule-associated proteins (MAPs) (Bonnet et al., 2001. J. Biol. Chem. 276:12839-12848; Boucher et al., 1994. Biochemistry 33:12471-12477; Ikegami et al., 2007. Proc. Natl. Acad. Sci. U. S. A. 104:3213-3218; Larcher et al., 1996. J. Biol. Chem. 271:22117-22124).
The ciliate Tetrahymena thermophila has 18 types of diverse microtubules that are all assembled in a single cell. Although most, if not all, of these microtubules are glutamylated, the length of glutamyl side chains is spatially regulated (Bré et al., 1994. Cell Motil. Cytoskeleton 27:337-349; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Minimal side chains composed of a single glutamic acid (monoglutamylation) are present on the cytoplasmic and nuclear microtubules, whereas elongated side chains are present on the basal bodies and axonemes (Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). In Tetrahymena, Ttll6Ap is a β-tubulin-preferring E-ligase (Janke et al., 2005. Science 308:1758-1762), with a strong if not exclusive, side chain elongating activity (van Dijk et al., 2007. Mol. Cell 26:437-448). Here, by overproducing Ttll6Ap in vivo, we explore the consequences of glutamyl side chain hyper-elongation. Unexpectedly, we show that in the same cells, hyperelongation of glutamyl side chains stabilizes cell body and destabilizes axonemal microtubules. The simplest explanation of these data is that, in vivo, the cellular outcomes of tubulin glutamylation are mediated by diverse microtubule type-specific MAPs. To our knowledge, we are first to report that excessive tubulin glutamylation can either stabilize or destabilize microtubules in the same cell.
Materials and Methods
Strains, culture, and green fluorescent protein (GFP) tagging. Tetrahymena cells were grown in the SPP medium (Gorovsky et al., 1975. Methods Cell Biol. 9:311-327) supplied with the antibiotic-antimycotic mix (Invitrogen, Carlsbad, Calif.). To overexpress Ttll6Ap variants tagged at the N terminus with GFP, fragments of the coding region of TTLL6A were amplified with addition of MluI and BamHI sites at the 5′ and 3′ ends, respectively, and cloned into pMTT1-GFP plasmid (Wloga et al., 2006. Mol. Biol. Cell 17:2799-2810). The primers are listed in Table 2. The transgenic strains were constructed and induced as described previously (Janke et al., 2005. Science 308:1758-1762; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372).







TABLE 2







Primers used for overexpression plasmids.









Name
Sequence
Seq ID





Tt116A-forward
5′ AATAAACGCGTCATGTCATAGAAAGATATATAT 3′
69


 


Tt116A-M241-forward
5′ AATAAACGCGTCATGGATAATAAATAAAGTGATATA 3′
70


 


Tt116A-A327-forward
5′ ATATACGCGTCGCCTAGTTGTTAAAAAAAAAG 3′
71


 


Tt116A-E337-forward
5′ AATAAACGCGTCGAGTAGGCAAAGAAGCAATAAG 3′
72


 


Tt116A-R712-forward
5′ AATTACGCGTCAGAGTTTTGACTGGTAAAAAAGTG 3′
73


 


Tt116A-1781-forward
5′ TTTAAACGCGTCATTAGAAGAAATACAAAAAAGGTC 3′
74


 


Tt116A-R924-forward
5′ AATTACGCGTCAGAGTAGCAATAGGAGTCAAAAG 3′
75


 


Tt116A-reverse
5′ ATATAGGATCCATACATACATACATCCATTCA 3′
76


 


Tt116A-K1116-reverse
5′ TTTATGGATCCTCACTTTATTGTCACTCGTTTATC 3′
77


 


Tt116A-V929-reverse
5′ TTATTGGATCCTTGACTCCTATTGCTACTCTG 3′
78


 


Tt116A-Q828-reverse
5′ AATTGGATCCTCAAGTCTAAGAATTAGGCTT 3′
79


 


Tt116A-E725-reverse
5′ TTTATGGATCCTCATTCTTCAGGTGTGTACTTC 3′
80









Immunofluorescence and electron microscopy. GFP-Ttll6Ap-expressing cells were grown in SPP with 0.5 to 2.5 μg of CdCl2/ml for 2 to 4 h. For GFP-Ttll6Ap localization, a 10-μl drop of cells was placed on a coverslip, followed by the addition of 20 μl of 2% paraformaldehyde in PHEM buffer (Schliwa and van Blerkom. 1981. J. Cell Biol. 90:222-235) and, after 20 s, the addition of 10 μl of 0.5% Triton X-100 in PHEM. The cells were subjected to immunofluorescence (Janke et al., 2005. Science 308:1758-1762) with the following primary antibodies: 12G10, an anti-α-tubulin monoclonal antibody (MAb) (Jerka-Dziadosz et al., 1995. Dev. Biol. 169:644-661) at 1:50; MAb ID5 (40), which in Tetrahymena is specific to polyglutamylated tubulin (Wloga et al., 2008. Eukaryot. Cell 7:1362-1372), at 1:50; poly(E) antipolyglutamic acid antibodies (1:100) (Shang et al., 2002. J. Cell Biol. 158:1195-1206); TAP952, an anti-monoglycylated tubulin MAb (1:5,000) (Bré et al., 1998. Mol. Biol. Cell 9:2655-2665; Callen et al., 1994. Biol. Cell 81:95-119); and 6-11 B-1, an anti-acetyl-K40 α-tubulin MAb (1:200) (LeDizet and Piperno. 1991. Methods Enzymol. 196:264-274). The secondary goat antibodies were as follows: anti-mouse-FITC, anti-mouse-Cy3, anti-rabbit-Cy3, and anti-rabbit-Cy5 (Zymed) at 1:200 dilutions. Cells were viewed under either Leica TCS SP or Zeiss LSM 510 VIS/META confocal microscopes. To measure the length of the cilia, the cells were labeled with MAb 12G10, [and, in some experiments, double labeled with poly(E) antibodies]. Confocal images were recorded with a 0.8-μm distance between z-sections, and sets of two to four z-sections were merged. The lengths of the cilia were measured by using ImageJ 1.37. For transmission electron microscopy (TEM), the cells were prepared as described previously (Jerka-Dziadosz et al., 2001. Protist 152:53-67).
Western blots and 2D gels. For a two-dimensional (2D) separation of tubulin isoforms, cytoskeletons were prepared as described previously (Janke et al., 2005. Science 308:1758-1762) from uninduced or induced GFP-Ttll6Ap-expressing cells and washed with the lysis buffer without Triton X-100. A 100-μg portion of the cytoskeletons (15 μl) was separated by isoelectric focusing on 18-cm Immobiline dry strips (4.5-5.5), followed by SDS-PAGE (10%) and silver staining. For Western blots, total extracts from 5 x 103 cells were separated by SDS-8% PAGE (Janke et al., 2005. Science 308:1758-1762). The primary antibodies were used as follows at the indicated concentrations: 12G10 (1:10,000), poly(E) (1:2,000), GT335 anti-glutamylation MAb (1:1,000) (55), TAP952 (1:10,000), and 6-11 B-1 (1:10,000).
Results
Overproduction of Ttll6Ap hyperelongates glutamyl side chains on axonemal and cell body microtubules. Ttll6Ap of Tetrahymena is a potent E-ligase (Janke et al., 2005. Science 308:1758-1762) with strong side chain-elongating activity on β-tubulin (van Dijk et al., 2007. Mol. Cell 26:437-448). Here, we explore the consequences of deregulation of the glutamyl side chain length by overexpression of Ttll6Ap in Tetrahymena. We overexpressed Ttll6Ap as an N-terminal GFP fusion using the strong cadmium-dependent MTT1 promoter (Janke et al., 2005. Science 308:1758-1762; Shang et al., 2002. Proc. Natl. Acad. Sci. U. S. A. 99:3734-3739). A Western blot with the anti-polyglutamic acid antibody, poly(E), which recognizes elongated glutamyl side chains (>=3E), showed that overexpression of GFP-Ttll6Ap increased the levels of polyglutamylation of proteins in the tubulin size range, whereas the levels of other proteins recognized by the same antibody (likely glutamylated nontubulin proteins), remained unchanged (FIG. 15A). 2D SDS-PAGE showed that overproduction of GFPTtll6Ap led to appearance of strings of highly acidic isoforms migrating near the main spots of α- and β-tubulin (FIGS. 15B and C, arrows). The levels of tubulin glutamylation detected by GT335, an antibody that recognizes a glutamyl side chain of any length (Wolff et al., 1992. Eur. J. Cell Biol. 59:425-432), were unchanged or slightly lower in overexpressing cells (FIG. 15A). Thus, in vivo, overexpressed Ttll6Ap appears to have primarily a side chain-elongating activity on tubulin.
GFP-Ttll6Ap localized mainly to a subset of short, most likely assembling cilia (see FIGS. 16A and B [arrows]). Consistently, when GFP-Ttll6Ap was overproduced in cilium-regenerating cells, the transgenic protein was targeted to most if not all cilia (FIG. 15E). Overproduction of increased duration (or increased strength) resulted in colocalization of GFP-Ttll6Ap to subcortical and cytoplasmic microtubules (FIG. 15D, arrows), in addition to cilia (FIG. 15D, arrowheads) (Janke et al., 2005. Science 308:1758-1762).
Overproduction of GFP-Ttll6Ap strongly increased the levels of tubulin polyglutamylation on ciliary, cell body, and nuclear microtubules (FIGS. 17 and 18). The pattern of accumulation of tubulin polyglutamylation was dependent on the time and strength of overexpression of GFP-Ttll6Ap (FIGS. 15A and 17). A 1-h induction of GFP-Ttll6Ap with 2.5 μg of CdCl2/ml or induction for a longer time by a lower cadmium concentration increased the level of tubulin polyglutamylation mainly in assembling axonemes (FIGS. 17A and B). This indicates that cilia are the primary site of the native Ttll6Ap activity. Functional studies agree with this hypothesis: deletion of TTLL6A and a closely related TTLL6F led to a loss of ciliary motility and deletion of additional paralogs (TTLL6B and 6D) led to shortening of cilia (Examples V and VI). The hyperglutamylated axonemes in GFP-Ttll6Ap cells were shorter than axonemes in untreated cells (FIG. 17B and FIG. 18B1 to B3). With longer induction period or increased strength of induction (cadmium concentration), tubulin polyglutamylation accumulated on cortical and cytoplasmic microtubules (FIGS. 17C and D [arrowheads] and FIG. 17E to G).
To determine which parts of Tll6Ap are required for ciliary localization and enzymatic activity, we overexpressed truncated variants of Ttll6Ap as GFP fusions (see FIGS. 16 and 19). The predicted Ttll6Ap is composed of 1,217 amino acids with the conserved TTL-like domain located between V395 and N703 (based on SMART prediction [Letunic et al., 2009. Nucleic Acids Res. 37:D229-D232]). Truncations of the C-terminal portion of the protein beyond Q828 residue resulted in increased retention of the fusion protein in the cell body (see FIG. 16). On the other hand, the fragment R712-L1217 lacking the TTL-like domain was sufficient to target GFP to cilia (see FIG. 16H). Thus, the R712-L1217 region contains determinants involved in targeting of Ttl6Ap to cilia.
Truncation of 240 amino acids on the N-terminal side and 390 amino acids on the C-terminal side of the TTL-like domain (GFP-Ttll6Ap-M241-Q828 variant) had an E-ligase activity in vivo (see FIG. 19A to D). Further deletions on either the N- or C-terminal side (resulting in fragments E337-Q828, M241-E725, and A326-V929) abolished the E-ligase activity in vivo (see FIG. 19E). Therefore, among the tested variants, the M241-Q828 fragment is the smallest enzymatically active protein. The TTL homology domain is contained between V395 and N703. Thus, less conserved amino acids adjacent to the TTL-like homology domain contribute to the enzymatic activity, as seen earlier for mammalian E-ligases (van Dijk et al., 2007. Mol. Cell 26:437-448). The majority of GFP-Ttll6Ap-M241-Q828 was associated with cytoplasmic microtubules, and only weak signal was observed in growing cilia during the formation of new oral apparatus prior to cell division (see FIG. 16E [arrowhead]). In vegetatively growing GFP-Ttll6Ap-M241-Q828-overexpressing cells, short hyperglutamylated cilia were rarely observed but bundles of hyperglutamylated cell body microtubules were abundant (see FIG. 19D). In contrast to GFP-Ttll6Ap and GFPTtll6Ap-M241-V929-overexpressing cells that gradually loose motility (Janke et al., 2005. Science 308:1758-1762), GFP-Ttll6Ap-M241-Q828-overexpressing cells remained motile. Thus, GFP-Ttll6Ap M241-Q828 acts primarily in the cell body. We used truncated variants of GFP-Ttll6Ap to selectively drive tubulin hyperglutamylation in the cell body (see below). The growth rate of cells overexpressing either full-length or enzymatically active fragments of GFP-Ttll6Ap was reduced compared to wild-type cells treated with the same cadmium concentration (see FIG. 19G), suggesting that hyperglutamylation of cell body microtubules is deleterious for cell growth.
Tubulin hyperglutamylation increases the abundance and stability of cell body microtubules. In wild-type Tetrahymena cells, the length of the glutamyl side chains on tubulin is spatially regulated. The cell body microtubules (cytoplasmic, subcortical, and nuclear) have tubulin subunits with side chains limited to a single E (monoglutamylated) except for the postoral fiber microtubules that carry biglutamylated side chains (Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Within the cell cortex, axonemes and basal bodies contain mono- and polyglutamylated microtubules (with biglutamylated or longer side chains), whereas cortical bundles have side chains limited to a single E (Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Overproduction of GFPTtll6Ap resulted in the polyglutamylation of diverse microtubules in the cell body, which in wild-type cells are only monoglutamylated, including cytoplasmic network and nuclear microtubules (see FIG. 18C to). Moreover, in GFP-Ttll6Ap-overexpressing cells, anti-α-tubulin antibodies revealed abnormally thick bundles of subcortical and nuclear microtubules (FIG. 20C to G and see FIG. 18C to L). In dividing cells, abnormally thick bundles of intramacronuclear microtubules were present around and within the cytoplasmic bridge connecting the future daughter cells (FIG. 20G, arrow). Bundling of nuclear microtubules was especially apparent in the GFP-Ttll6Ap-M241-V929 cells, and this was likely due to the increased presence of this variant in the cell body (see FIG. 16D; FIGS. 20E and F, arrows). The increased abundance, bundling, and curvature of microtubules indicated that hyperglutamylated microtubules are excessively stable. For example, similar curved bundles of microtubules appear in Tetrahymena cells treated with the microtubule stabilizing drug, paclitaxel (Fujiu and Numata. 2000. Cell Motil. Cytoskeleton 46:17-27) (FIGS. 20A and B) and in cells with a K350M mutation in β-tubulin that confers paclitaxel sensitivity (Smith et al., 2004. Eukaryot. Cell 3:1217-1226). To probe the stability of cell body microtubules, we treated the wild-type and GFP-Ttll6Ap-overexpressing cells with the microtubule-destabilizing compounds, nocodazole (40 μM) and oryzalin (10 μM). Although these drugs caused rapid depolymerization of cytoplasmic microtubules in wild-type cells (FIGS. 20H, H′, and J), similarly treated GFPTtll6Ap-overexpressing cells retained abundant cell body microtubules (FIGS. 201, I′, and J). Next, we investigated the levels of α-tubulin K40 acetylation in GFP-Ttll6Ap-overproducing cells, since this PTM accumulates on long-lived microtubules (Piperno et al., 1987. J. Cell Biol. 104:289-302). Although wild-type cells had a strong K40 acetylation signal that was limited to the stable microtubules of cell cortex and cilia, the GFP-Ttll6Ap-overproducing cells showed abundant K40 acetylation on cytoplasmic microtubules, a finding consistent with increased stability of these microtubules (FIG. 21). Cells overexpressing a variant of GFP-Ttll6Ap that lacks enzymatic activity due to a mutation in the catalytic domain (Janke et al., 2005. Science 308:1758-1762) had a wild-type organization of cell body and cortical microtubules and a normal the pattern of K40 acetylation. Thus, hyperelongation of glutamyl side chains stabilizes at least a subset of cell body microtubules.
GFP-Ttll6Ap-overproducing cells also had reduced levels of tubulin monoglycylation on stabilized cytoplasmic and cortical microtubules (FIG. 15A and see FIG. 22). Similar observations were made for tubulin polyglycylation. We have previously documented that the two tubulin polymodifications, glutamylation and glycylation, inhibit each other, and we suggested that this mutual inhibition can be explained either by competition for the same modification sites or steric inhibition of adjacent sites (Wloga et al., 2009. Dev. Cell 16:867-876). Overproduction of GFP-Ttll6Ap does not lead to an increase in the total number of glutamyl side chains on tubulin (based on Western blots with GT335 MAb that recognizes a side chain of any length [FIG. 15A]). Thus, it is more likely that elongation of glutamyl side chains sterically inhibits the activity of tubulin G-ligases (TTLL3 [Ikegami and Setou. 2009. FEBS Lett. 583:1957-1963; Rogowski et al., 2009. Cell 137:1076-1087; Wloga et al., 2009. Dev. Cell 16:867-876]) on adjacent polymodification sites.
Hyperglutamylation destabilizes microtubules in axonemes. GFP-Ttll6Ap-overexpressing cells contained both excessively short hyperglutamylated cilia (in which GFP-Ttll6Ap accumulates) and unaffected cilia (with a low GFP-Ttll6Ap signal; FIG. 15D, FIGS. 16A and B, and FIG. 18B). The short, hyperglutamylated cilia observed within 1 to 4 h after induction of GFP-Ttll6Ap overexpression could be assembling cilia that had failed to elongate or preexisting cilia that had undergone shortening (or both). Since mildly overproduced GFP-Ttll6Ap is preferentially targeted to assembling cilia (FIG. 15E), hyperglutamylation could primarily affect axonemes during their assembly. To test this hypothesis, we deciliated wild-type and GFP-Ttll6Ap-overproducing cells and examined the lengths of the cilia during regeneration. While at 1 h after deciliation wild-type cells had regenerated cilia to 85% of the original length (4.32±0.57 μm, n=30), during the same period the GFP-Ttll6Ap-overexpressing cells regenerated significantly shorter cilia (1.07±0.35 μm, n=50 [FIGS. 23A and C]). The GFP-Ttll6Ap-overexpressing cells maintained short hyperglutamylated cilia even 4 h after deciliation (3.88±0.89 μm, n=82; wild-type cells 5.43±0.59 μm, n=60 [FIGS. 23B, D, and G]).
The failed elongation of regenerating cilia in cells overproducing GFP-Ttll6Ap could be caused either by the physical presence of GFP-Ttll6Ap or by tubulin hyperglutamylation. To distinguish between these two effects, we compared the lengths of cilia in Tetrahymena cells overexpressing either an enzymatically active and cilium-targeted enzyme (GFP-Ttll6Ap-M241-V929) or an inactive enzyme with an amino acid substitution in the ATP-binding site (GFP-Ttll6Ap-M241-V929-E662G [Janke et al., 2005. Science 308:1758-1762]). Whereas cells overproducing an active enzyme regenerated excessively short axonemes (1.44±0.49, n=62, after 2 h [FIGS. 23F and G]), cells overexpressing an inactive enzyme assembled normal length axonemes (5.05±0.59 μm, n=40, after 2 h [FIGS. 23E and G]) despite the fact that the inactive enzyme is targeted to cilia (Janke et al., 2005. Science 308:1758-1762). Cells overexpressing GFP-M241-Q828 that localizes mainly to cell bodies, regenerated cilia at the rate similar to that of wild-type cells (see FIG. 24). Thus, most likely, the inhibitory effect of GFPTtll6Ap on the elongation of axonemes is mediated by hyperelongation of glutamyl side chains on axonemal tubulin.
The TEM analysis of vegetatively growing cells overproducing GFP-Ttll6Ap (FIG. 25) revealed two types of axonemes: unaffected 92 axonemes and structurally defective mostly 90 axonemes. The intact 92 axonemes are likely present in the nonassembling cilia that do not accumulate GFP-Ttll6Ap. The defective axonemes frequently lacked a central pair (FIG. 25A to C) and had broken outer doublets (FIGS. 25E and F). These observations indicate that, in GFP-Ttll6Ap-overproducing cells, assembling axonemes are unstable.
In an attempt to probe how the dynamic properties of axonemal microtubules change as a function of glutamyl side chain hyper-elongation, we treated control and GFP-Ttll6Ap-overproducing cells with 40 μM paclitaxel for 3 h. As previously described in wild-type cells (Wloga et al., 2009. Dev. Cell 16:867-876), paclitaxel increased the average axoneme length by 20% (length before treatment, 5.02±0.52 μm, n=129; length after treatment, 6.05±1.12 μm, n=173 [FIGS. 26A to C]). In GFP-overproducing cells, theassembling cilia were 44% treated GFP-Ttll6Ap-overproducing cells (no drug, 1.87±0.36 μm; paclitaxel treated, 2.7±0.6 μm [FIGS. 26D to F]). Furthermore, the assembling cilia in GFPTtll6Ap-overproducing, paclitaxel-treated cells had elevated levels of tubulin polyglutamylation comparable to those in axonemes of the untreated GFP-Ttll6Ap-overproducing cells. The simplest explanation of these observations is that hyperelongation of glutamyl side chains on the tubulin of assembling axonemal microtubules makes them unstable and that this effect can be counteracted by paclitaxel. Interestingly, in GFPTtll6Ap-overexpressing paclitaxel-treated cells, preexisting cilia underwent elongation by 22% by forming a hyperglutamy-lated distal segment (before treatment, 5.25±0.5 μm; paclitaxel treated, 6.39±0.82 μm [FIGS. 26E and F, arrows]). In wild-type cells treated with paclitaxel the distal segments of preexisting elongated axonemes had lower levels of tubulin polyglutamylation compared to the preexisting proximal axoneme segment (see FIG. 27). This argues again that the destabilizing effects of hyperglutamylation in the axoneme are counteracted by paclitaxel.
Discussion
The polymeric character of glycylation and glutamylation, as well as the fact that these PTMs affect multiple modification sites within the tubulin CTTs and can coexist on the same tubulin proteins, generates an exceptionally large number of tubulin isoforms. The polymodification sites on β-tubulin are required for axoneme assembly (Thazhath et al., 2002. Nat. Cell Biol. 4:256-259). Functional studies on the E- and G-ligases indicate that both glutamylation and glycylation on tubulin are important and contribute to either the assembly or the stability of microtubules, including those present in neural extensions (Ikegami et al., 2006. J. Biol. Chem. 281:30707-30716), and axonemes (Pathak et al., 2007. Mol. Biol. Cell 18:4353-4364; Rogowski et al., 2009. Cell 137:1076-1087; Wloga et al., 2009. Dev. Cell 16:867-876). However, it is not known what the structural consequences of tubulin polymodifications on microtubules are.
The length of the polymodification side chain is spatially regulated and dependent on the microtubule type and possibly on the position of tubulin subunits within the microtubule (Bré et al., 1994. Cell Motil. Cytoskeleton 27:337-349; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Moreover, in multicellular organisms, the glutamyl side chain length changes during organismal development (Audebert et al., 1994. J. Cell Sci. 107(Pt. 8):2313-2322; Ikegami et al., 2006. J. Biol. Chem. 281:30707-30716). Among the mechanisms that regulate the side chain length could be (i) temporal and spatial regulation of the initiation and elongation steps performed by E-ligases and (ii) selective shortening of glutamyl side chain by deglutamylases (Audebert et al., 1994. J. Cell Sci. 107(Pt. 8):2313-2322). Whereas some E-ligases, such as the murine TTLL7, can both initiate and elongate the side chains (Mukai et al., 2009. Biochemistry 48:1084-1093), the majority of studied E-ligases have a bias for either chain elongation or initiation (Janke et al., 2005. Science 308:1758-1762; van Dijk et al., 2007. Mol. Cell 26:437-448; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). Ttll6Ap is a strong elongate for β-tubulin (van Dijk et al., 2007. Mol. Cell 26:437-448; this example). We have studied here the consequences of hyperelongation of glutamyl side chain in vivo by overexpressing Ttll6Ap. Although the activity mediated by GFP-Ttll6Ap on the nonciliary microtubules could be nonphysiological, this ectopic activity gave us a tool to investigate the importance of the side chain length regulation on multiple types of microtubules within the same cell.
We show that the consequences of overexpression of Ttll6Ap depend on the cellular context. Hyperelongation of glutamyl side chains on cell body microtubules increased the density and bundling of microtubules, resistance to depolymerizing drugs, and α-tubulin K40 acetylation. These effects are consistent with an increased stability of hyperglutamylated cell body microtubules. In wild-type Tetrahymena cells, the most dynamic microtubules (e.g., the cytoplasmic network, micronuclear spindle, and macronuclear and longitudinal cortical microtubules) have tubulin subunits with side chains limited to monoglutamylation (Bré et al., 1994. Cell Motil. Cytoskeleton 27:337-349; Wloga et al., 2008. Eukaryot. Cell 7:1362-1372). In contrast, basal bodies and axonemes have elongated glutamyl side chains, and these microtubules are known to be extremely stable (turnover slowly and resist standard depolymerizing treatments) (Thazhath et al., 2004. Mol. Biol. Cell 15:4136-4147). We speculate that the physiological elongation of glutamyl side chains on tubulin of basal body and axonemal microtubules contributes to their increased stability. Indeed, deletion of some TTLL6 genes led to shortening of axonemes (Example VI). In GFP-Ttll6Ap-overproducing cells, hyperelongation of the cell body microtubules could lead to capture of axoneme-stabilizing MAPs that are in transit to cilia. A model that elongation of the glutamyl side chains stabilizes microtubules by recruiting MAPs likely applies to other contexts. In neurons, the accumulation of tubulin polyglutamylation during differentiation correlates with increased stability of microtubules and the accumulation of MAP2 in dendrites and the cell body (Ikegami et al., 2006. J. Biol. Chem. 281:30707-30716). Moreover, knockdown of TTLL7 E-ligase inhibited the formation of MAP2-positive neurites in PC-12 cells (Ikegami et al., 2006. J. Biol. Chem. 281:30707-30716). In vitro studies show that the levels of tubulin glutamylation affect the binding of certain structural MAPs and motor proteins to microtubules (Bonnet et al., 2001. J. Biol. Chem. 276:12839-12848; Boucher et al., 1994. Biochemistry 33:12471-12477; Ikegami et al., 2007. Proc. Natl. Acad. Sci. U. S. A. 104:3213-3218; Larcher et al., 1996. J. Biol. Chem. 271:22117-22124). Future studies in vitro based on microtubule polyglutamylation with purified E-ligases should shed light on the mechanism of polyglutamylation-induced microtubule stabilization and, in particular, should reveal whether polyglutamylation has a direct effect on the microtubule dynamics or acts via MAPs.
Given the apparent stabilizing effect of the hyperelongation of glutamyl side chains on the cell body microtubules and the fact that native axonemes have relatively long glutamyl side chains, it was surprising that we observed a seemingly opposite effect of hyperglutamylation on axonemes. It appears that the destabilizing effects of overexpressed Ttll6Ap on axonemes are largely autonomous and cannot be explained by the retention of stabilizing axoneme-destined MAPs in the cell body. The shortening of axonemes could be explained by inhibition of the intraflagellar transport (IFT) pathway, a motility mechanism that moves precursors required for cilia assembly along growing outer doublet microtubules (Kozminski et al., 1993. Proc. Natl. Acad. Sci. U. S. A. 90:5519-5523). However, the defect in the elongation of axonemes in GFP-Ttll6Ap cells is partly rescuable by paclitaxel, suggesting that the elongation of glutamyl side chains destabilizes axonemal microtubules. Moreover, paclitaxel failed to rescue an axoneme assembly defect caused by a loss of function of IFT in the DYF1 knockout strain of Tetrahymena (Dave et al., 2009. Eukaryot. Cell 8:1397-1406).
Hyperelongation of glutamyl side chains on tubulin could affect the dynamics of microtubules. This model agrees with the observation that the removal of CTTs by proteolysis with subtilisin increases the resistance of microtubules to depolymerization by high salt and cold (Bhattacharyya et al., 1985. J. Biol. Chem. 260:10208-10216; Sackett et al., 1985. J. Biol. Chem. 260:43-45), although other studies disagree with this conclusion (Knipling et al, 1999. Cell Motil. Cytoskeleton 43:63-71; Saoudi et al., 1995. J. Cell Sci. 108(Pt. 1):357-367). CTTs are highly negatively charged and could interact with the positively charged surface of the tubulin dimer (Priel et al., 2005. Eur.Biophys. J. 35:40-52). Glutamylation further increases the negative charge of CTTs. The bonds between the dimers could be weakened by charge repulsion, especially if CTTs of neighboring tubulin subunits can interact with each other. However, we would need to assume that in GFPTtll6Ap-overproducing cells, and specifically in the cell body, the lattice-weakening effects of glutamylation are counteracted by stabilizing MAPs that preferentially bind to hyperglutamylated microtubules.
Alternatively, the restriction of the destabilizing effect of hyperglutamylation to the axoneme could result from differential utilization of tubulin subunits. In vitro studies showed that Ttll6Ap prefers β-tubulin (Janke et al., 2005. Science 308:1758-1762), but the same enzyme, when overexpressed in vivo, modified both α- and β-tubulin (FIG. 15C). Thus, some differences in the consequences of excessive activity of Ttll6Ap could result from the differential utilization of α- and β-tubulin subunits in different microtubules, which in turn could be caused by competing MAPs that selectively hinder one of the two tubulin subunits.
However, another explanation of the restriction of destabilizing influence of hyperglutamylation to axonemes is that this effect is mediated by axoneme-restricted factors that are regulated by polyglutamylation. Specifically, in assembling axoneme, hyperelongation of glutamyl side chains could increase the activity of factors that promote microtubule depolymerization. A microtubule-severing protein, katanin, plays a prominent role in the axoneme assembly. Katanin localizes to cilia in Tetrahymena and Chlamydomonas (Dymek et al., 2004. Eukaryot. Cell 3:870-879; Sharma et al., 2007. J. Cell Biol. 178:1065-1079). The presence of CTTs is required for the katanin-mediated microtubule-severing activity in vitro (McNally and Vale. 1993. Cell 75:419-429). Knockouts of katanin subunit genes in Tetrahymena phenocopy the substitutions of glutamic acids that undergo polymodifications in the CTT of β-tubulin (Sharma et al., 2007. J. Cell Biol. 178:1065-1079). The activity of spastin, another microtubule-severing protein, is blocked by an antibody that recognized a terminal glutamic acid, a finding consistent with a requirement of either detyrosination or polyglutamylation (or both) for severing activity (Roll-Mecak and Vale. 2008. Nature 451:363-367). In the axoneme, tubulin hyperglutamylation could cause an excessive activity of severing factors such as katanin, specifically during axoneme assembly, and this could prevent axoneme elongation and assembly of central microtubules. Interestingly, the levels of tubulin glutamylation appear to change during axoneme assembly in wild-type cells. The short assembling cilia label more strongly with antibodies that recognize elongated glutamyl side chains. The signal of polyglutamylation decreases as cilia mature, while the levels of polyglycylation increase in these cilia (Sharma et al., 2007. J. Cell Biol. 178:1065-1079). Thus, axonemal microtubules undergo remodeling of the PTM composition as part of the polymer maturation. It is possible that some glutamyl side chains are trimmed down or completely removed by deglutamylating enzymes (Audebert et al., 1993. Mol. Biol. Cell 4:615-626) and are replaced by glycyl side chains. Thus, tubulin glutamylation could play distinct roles during and after assembly of the axoneme. In a growing axoneme, tubulin polyglutamylation could promote polymer turnover, whereas in the mature axoneme the modification could contribute to increased stability of the polymer.
To summarize, this example shows that the effects of tubulin hyperglutamylation are subcellular context specific. In the same cells, hyperglutamylation stabilizes cell body microtubules and destabilizes axonemes. We propose that the differential effects of hyperglutamylation are mediated by nonuniformly distributed MAPs.
Example V
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 post-translational modifications of tubulin subunits that form patterns on microtubules. Glutamylation is a conserved tubulin modification (Edde et al., 1990. Science 247, 83-85) that is enriched in axonemes. The enzymes responsible for this PTM, glutamic acid ligases (E-ligases), belong to a family of proteins with a tubulin tyrosine ligase (TTL) homology domain (TTL-like or TTLL proteins) (Janke et al. 2005. Science 308, 1758-1762). 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.
Results and Discussion
TTLL6 enzymes generate tubulin polyglutamylation in cilia. The genome of Tetrahymena contains 6 genes encoding TTLL6 paralogs, namely, Ttll6Ap through Ttll6Fp (FIG. 28A). Ttll6Ap is targeted to cilia (Janke et al. 2005. Science 308, 1758-1762; Wloga et al., 2010. Eukaryot Cell. 9,184-193). To characterize the enzymatic properties of Ttll6Ap, we overexpressed GFP-Ttll6Ap in Tetrahymena, partially purified and assayed the enzyme for glutamylation of microtubules in vitro. Glutamylation involves two distinct steps: initiation and elongation. To distinguish between the two reactions, we used microtubules with varying levels of pre-existing glutamylation: high (adult murine brain tubulin), intermediate (young murine brain tubulin) and low (HeLa tubulin) (Regnard et al., 2003. J Cell Sci 116, 4181-4190). Enriched GFP-Ttll6Ap strongly modified microtubules made of adult brain tubulin, less efficiently microtubules made of young brain tubulin, and failed to detectably modify microtubules made of HeLa tubulin (FIG. 28B). The activity was primarily on β-tubulin as seen earlier (Janke et al. 2005. Science 308, 1758-1762). Moreover, enriched GFP-Ttll6Ap did not modify unpolymerized adult brain tubulin (FIG. 28B). As a control, we used another partially purified E-ligase, Ttll1p, (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372) with the same microtubule substrates, and detected a distinct enzymatic profile (FIG. 28B). These and earlier studies (Wloga et al., 2010. Eukaryot Cell. 9,184-193; van Dijk et al., 2007. Mol Cell 26, 437-448) are consistent with Ttll6Ap acting primarily as a glutamyl side chain elongase for β-tubulin in microtubules.
In Tetrahymena, glutamylation occurs on most types of microtubules, but the length of the glutamyl side chain is spatially regulated (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372), presumably by localized activities of elongases such as Ttll6Ap. While the modification is detectable on most if not all microtubules, only microtubules of cilia and basal bodies are labeled by the polyE antibodies that recognize elongated (poly)glutamyl side chains ((Wloga et al. 2008. Eukaryot Cell 7, 1362-1372) and FIG. 28D′). Knocking out the TTLL6A gene by DNA homologous recombination neither changed the levels of tubulin glutamylation (FIG. 28C) nor affected the gross phenotype. TTLL6F encodes a close paralog (FIG. 28A). Cells with a deletion of TTLL6F showed no reduction in the levels of tubulin glutamylation (FIG. 28C). However, a double knockout strain, 6AF-KO, had strongly reduced levels of elongated side chains recognized by polyE antibodies (FIG. 28C), indicating that Ttll6Ap and Ttl1Fp act synergistically. Consistent with this result, 2D gel electrophoresis of axonemal proteins showed a prominent reduction in the abundance of protein isoforms migrating as a smear on the more acidic side of the major (3-tubulin spots in 6AF-KO cilia (FIG. 29B). Immunofluorescence with polyE antibodies showed a decrease in tubulin polyglutamylation signal in cilia and basal bodies of 6AF-KO cells imaged side by side with wild type cells (FIG. 28D-D′′). The levels of tubulin glutamylation recognized by the GT335 antibody that detects an epitope at the base of the glutamyl side chain and probably recognizes side chains of any length (Wolff et al., 1992. Eur J Cell Biol 59, 425-432) appeared unchanged in 6AF-KO cilia based on immunofluorescence (FIG. 28E-E′′, FIG. 29A) and western blotting (FIG. 28C). These data indicate that, the absence of Ttll6Ap and Ttll6Fp leads to shortening but not complete loss of glutamyl side chains, which agrees with the enzymatic profile of Ttll6Ap obtained in vitro. In cilia of 6AF-KO cells, the levels of tubulin acetylation and glycylation appeared nearly normal (FIG. 28C). Thus, Ttll6Ap and Ttll6Fp together contribute to β-tubulin glutamylation in cilia and are responsible primarily if not exclusively for the chain elongation in vivo.
Ttll6Ap and Ttll6Fp affect ciliary motility. The 6AF-KO cells have a normal density of cilia that appear only slightly shorter than wild type cilia (wild type 5.6±0.8 μm, n=150; 6AF-KO, 5.0±0.5 μm, n=150). However, the 6AF-KO cells moved at only 1⁄5 of wild type rate (FIG. 30A). In ciliates, phagocytosis requires the motility of ciliary membranelles that sweep food particles into the oral cavity. Although, the 6AF-KO cells assemble oral membranelles (arrowheads, FIG. 28D,E), they exhibited a greatly reduced rate of formation of food vacuoles (FIG. 30C,D), consistent with malfunction of oral cilia. 6AF-KO cells also showed a reduced rate of multiplication (FIG. 30B). Tetrahymena cells require motile cilia for conjugation (our unpublished data). When starved 6AF-KO cells (earlier grown for over 100 generations to reach sexual maturity) were mixed with wild type cells, few pairs formed and these pairs dissociated quickly (FIG. 30E). Thus, all functions dependent on normal ciliary motility appear to be severely affected in 6AF-KO cells.
Biolistic bombardment of 6AF-KO cells with a GFP-Ttll6Ap transgene (targeted to an unrelated locus) resulted in the appearance of cells with vigorous motility (at the frequency of 0.014%), and no such cells were found in the mock-transformed population (n=107). The rescued cells had a GFP signal in cilia and basal bodies (results not shown) and recovered a nearly normal rate of motility, multiplication (FIG. 30A,B), and phagocytosis (Table 3). Thus, the dramatic loss of ciliary functions seen in 6AF-KO cells is caused by the loss of TTLL6 protein activity.







TABLE 3







Phagocytic activity is restored by re-introduction


of GFP-Ttll6Ap transgene into 6AF-KO cells.











Wild Type
6AF-KO
GFP-TTLL6AR














Average number of
42.8
0
36.3


labeled food


vacuoles/cell


Standard deviation
3.7
—
3.5


N =
25
25
25





Food vacuoles were labeled with India ink for 30 min, cells were fixed and the average number of food vacuoles per cell was determined.






High-speed video microscopy showed that in wild type cells, locomotory cilia had an asymmetric waveform and rows of cilia were engaged in metachronal. In contrast, in 6AF-KO cells, many cilia appeared straight and some were seen rotating around a central pivot point, often colliding with each other in an uncoordinated motion. Furthermore, immunofluorescence images indicate that 6AF-KO cilia are more straight than wild type (FIG. 28D,E). In some 6AF-KO cultures grown for over 480 generations, the waveform was partly restored to normal. In these “adapted” 6AF-KO cells (6AF-KOA), the beat frequency could be measured, and was found to be  ̃60% of wild type (FIG. 30F).
Exposure of wild type Tetrahymena cells to 20 μg/ml of sodium dodecyl benzene sulfonate (SDBS), causes rapid avoidance reaction associated with backward motility, likely by depolarizing the ciliary plasma membrane. While wild type cells showed rapid SDBS-induced avoidance responses (based on deviations from the linearity of swimming paths), the 6AF-KO cells failed to swim backwards, and instead slightly increased the rate of forward motility (FIG. 30G,H). The responses of 6AF-KO cells to other plasma membrane-depolarizing treatments (1 mM Ba++, 20 mM Ca++) were similar to SDBS. At the time of addition of SDBS, some 6AF-KO cells showed a slight turn, indicating that the signal detection pathways that regulate motility are at least partly functional. These data suggests that the response to signals, that requires proper modulation of activity of dynein arms, is affected. However, ultrastructural studies revealed that 6AF-KO axoneme cross-sections have a normal morphology (FIG. 31A,B, n=209). No difference was found in the frequency of outer (ODA) and inner dynein arms (IDA) on wild type and 6AF-KO axoneme cross-sections (FIG. 31C). Thus, we considered that TTLL6 enzymes, via tubulin glutamylation, could be affecting the activity ciliary dyneins.
Ttll6Ap and Ttll6Fp primarily modify the B-tubule of outer microtubules. Immunogold TEM studies showed that overexpressed GFP-Ttll6Ap localized to the outer doublets and not to central microtubules in all of cross-sections examined (FIG. 31D,E n=38). We evaluated the distribution of polyglutamylated (polyE) epitopes on isolated doublet microtubules that were extruded from the axoneme with 40 μM ATP (see below). In negatively stained doublet microtubules viewed on edge, the A-tubule can be identified based on its increased width (as compared to the B-tubule), and dynein arms projecting from its surface (arrowhead, FIG. 31G,H). In wild type doublets, the majority of gold particles were detected in proximity of the B-tubule (FIG. 31F,G, FIG. 32) and the signal was dramatically reduced in 6AF-KO axonemes (FIG. 31F,H, FIG. 32). Thus, Ttll6Ap and Ttl1Fp primarily generate glutamylation on the B-tubule. Our observations are consistent with earlier microscopic and biochemical studies showing enrichment of tubulin glutamylation on the B-tubule (Kann et al., 1995. Tissue Cell 27, 323-329; Lechtreck and Geimer, 2000. Cell Motil Cytoskeleton 47, 219-235; Multigner et al., 1996. Biochemistry 35, 10862-10871). Since the B-tubule serves as a track for ciliary dynein, the primary role of tubulin glutamylation in cilia could be to regulate the motor activity of ciliary dynein.
Tubulin glutamylation strongly affects the inner dynein arm-driven microtubule sliding in isolated axonemes in vitro. When isolated Tetrahymena axonemes are exposed to ATP, outer doublet microtubules undergo unconstrained dynein-driven microtubule sliding (rather than reactivated bending) and the velocity of ATP-induced axonemal microtubule sliding can be used to assay the activity of ciliary dynein in situ (Summers Gibbons, 1971. Proc Natl Acad Sci U S A 68, 3092-3096; Okagaki and Kamiya, 1986. J Cell Biol 103, 1895-1902; Sale and Satir, 1977. Proc Natl Acad Sci U S A 74, 2045-2049). With 1 mMATP, microtubules underwent sliding at similar rates in wildtype and 6AF-KO axonemes (FIG. 33B). In wild type axonemes, the microtubule sliding velocity is believed to be primarily determined by the activity of ODAs (reviewed in Kamiya, 2002. Int Rev Cytol 219, 115-155). To test whether the TTLL6-mediated tubulin glutamylation affects the microtubule sliding generated specifically by IDAs, we constructed a strain that lacks TTLL6A and TTLL6F and is homozygous for the temperature-sensitive oadl-1 allele. When oadl-1 mutants are grown at restrictive temperature (38° C.), ODAs fail to assemble and cells are nearly paralyzed (Ludmann, 1993. J Eukaryot Microbiol 40, 650-660; Attwell et al., 1992. J Protozool 39, 261-266). We assessed microtubule sliding in axonemes isolated from wildtype, oadl-1, 6AF-KO and 6AF-KO;oadl-1 triple mutant cells, all grown for 12 hr at 38° C. We confirmed that 6AF-KO;oadl-1 cells had fewer ODAs as compared to 6AF-KO cells grown at the same temperature (FIG. 33A). As reported (Seetharam and Satir, 2005. Cell Motil Cytoskeleton 60, 96-103), oadl-1 axonemes showed a decreased rate of microtubule sliding as compared to wild type (FIG. 33C) while wild type and 6AF-KO axonemes showed nearly the same microtubule sliding velocity, as seen earlier for axonemes from cells grown at the standard temperature (compare FIG. 33B,C). Unexpectedly, the 6AF-KO;oadl-1 axonemes showed a nearly two-fold increase in the rate of microtubule sliding as compared to oadl-1 axonemes (FIG. 33C). These data indicate that tubulin glutamylation generated on the B-tubule by Ttll6Ap and Ttll6Fp has a restraining effect on the microtubule sliding velocity generated by the net activity of IDAs.
To conclude, we report that tubulin polyglutamylation generated by TTLL6 enzymes plays a major role in ciliary motility, and that the mechanism appears to involve regulation of inner dynein arm activity. Earlier studies have implicated tubulin glutamylation in axoneme assembly (Campbell et al. 2002. Genetics 162, 307-320; Pathak et al., 2007. Mol Biol Cell 18, 4353-4364). Deletion of additional TTLL6 paralogs in Tetrahymena led to major shortening of the axoneme (Example VI). Thus, tubulin glutamylation affects both axoneme assembly and motility and the latter function may require a higher dose of TTLL6 activity. A recent study of a Chlamydomonas mutant defective in TTLL9, an α-tubulin-preferring E-ligase, also revealed that tubulin polyglutamylation controls ciliary motility by affecting inner dynein arm activity (Kubo et al., 2009. Tubulin polyglutamylation regulates axonemal motility by changing activities of inner arm dyneins. Submitted). Thus, both studies link tubulin glutamylation, mediated by two conserved E-ligases on either α- or β-tubulin, to regulation of IDA activity. The abnormal waveform and lack of ciliary reversals that we observed in the 6AF-KO cells are also consistent with malfunctioning IDAs in Tetrahymena (Wood et al., 2007. J Cell Sci 120, 3075-3085; Hennessey et al., 2002. Cell Motil Cytoskeleton 53, 281-288). The mechanochemical properties of ODAs and IDAs are distinct but the underlying structural basis is not well understood (reviewed in Kamiya, 2002. Int Rev Cytol 219, 115-155). For example, in Tetrahymena, the 22S dynein fraction from the salt extract of axonemes (mainly ODAs) produces a linear gliding of microtubules at the rate of 8 μm/s, while the 14S dynein fraction (presumably IDAs) produces a motility at the rate of 4 μm/s associated with microtubule rotation (Vale and Toyoshima, 1988. Cell 52, 459-469). Importantly, two IDA subtypes that were studied in Chlamydomonas (dynein c and f/I1) display processive movements along microtubules (Sakakibara et al., 1999. Nature 400, 586-590; Kotani et al., 2007. Biophys J 93, 886-894). Kotani and colleagues proposed that in the bending cilium, processive IDAs, acting simultaneously with faster ODAs, impose a drag on sliding microtubules and could increase the axoneme curvature (Kotani et al., 2007. Biophys J 93, 886-894). We speculate that tubulin glutamylation is important for the processive motility of IDAs. One unusual feature of dynein motor domain is that it contacts the microtubule track by a conserved stalk domain (Gee et al., 1997. Nature 390, 636-639). Recent studies indicate that the stalk acts as a tether that allows for pulling of parts of the dynein molecule toward the microtubule during the power stroke (Ueno et al., 2008. Proc Natl Acad Sci U S A 105, 19702-19707; Carter et al., 2008. Science 322, 1691-1695). Thus, tubulin glutamylation could regulate the affinity of the stalk in inner dynein arms to the B-tubule. The patterns of glutamylation vary between axonemes of different species (Hoyle et al., 2008. Cell Motil Cytoskeleton 65, 295-313). Thus, specific beating patterns could be dependent on diverse patterns of tubulin glutamylation.
Experimental Procedures
Phylogenetic analyses. Sequences of TTLL6 protein homologs were obtained from NCBI databases. Conserved catalytic domains were aligned and neighbor-joining trees constructed as previously described (Edde et al., 1990. Science 247, 83-85).
Germ line-based targeting. The gene targeting plasmids were made based on the macronuclear genome sequence of Tetrahymena thermophila, available at the Tetrahymena Genome Database (Janke et al. 2005. Science 308, 1758-1762). Two fragments of macronuclear DNA were amplified for each locus. The fragments were designed to flank the genomic sequence that encodes the catalytic domain of TTLL6







TABLE 4







Primers used for construction of targeting fragments.















Restriction








sites used


TTLL





to release


catalytic





the
Size of

domain





targeted
targeting
Selectable
region














Gene
Primers sequence (5′-3′)
fragment
fragment
cassette
replaced
Seq. ID





TTLL6A
Forward
ApaI/SacI
1.5 kb
Neo3
926 bp
81



ATATTGGGCCCGAGGAAGATGATGATGAGA








Reverse:




82



ATAAACCCGGGGCTAAAGAAAACATACCAG








Forward

1.45 kb


83



AATTTACTAGTAGCCATGGGTTTTAGAAGT








Reverse:




84



ATTATGAGCTCCTTTTGGAAGTAATGICAG







 


TTLL6F
Forward:
ApaI/SacII
1.47 kb
mtt1-rp129
1.6kb
85



ATATTGGGCCCGAGCTAATCAAACATACGA








Reverse:




86



ATTATATCGATTTCCTAGCTATTCTGGTTA








Forward:

1.44 kb


87



ATATTCCCGGGAAAAAGCCTGATGTTGAAG








Reverse:




88



ATATTCCGCGGGGCTACAAATAAAGTCCAT









(Table 4). Fragments were subcloned on either side of a selectable drug resistance cassette (neo3 (Wloga et al., 2010. Eukaryot Cell. 9,184-193) or mtt1-rp129. The cassette was embedded in a reverse transcriptional orientation. The targeting fragments were separated from the rest of the plasmid using restriction enzymes and transformed biolistically into mating CU428 and B2086 strains (Regnard et al., 2003. J Cell Sci 116, 4181-4190) at 3, 3.5 and 4 hr after mixing. After shooting, cells were incubated in SPP and 2.5 μg/ml CdCl2 for 3 hr, and transformants were selected with appropriate drugs (neo3, paramomycin-100 μg/ml with CdCl2 2.5 μg/ml; mtt1-rp129, cycloheximide 15 μg/ml with CdCl2 2.5 μg/ml). Putative germline transformants were identified by co-resistance to 15 μg/ml 6-methylpurine. Knockout heterokaryons were generated by allowing the disrupted alleles to assort from the macronucleus via phenotypic assortment and by making the micronucleus fully homozygous using a star cross (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372). Double knockout strains were made by multiple rounds of standard crosses. Total homozygotes were created by crossing appropriate heterokaryons and isolating progeny. The absence of the targeted genomic regions in the homozygote was confirmed by PCR of genomic DNA with primers designed to amplify junctions between selectable cassettes and the nontargeted gene specific regions (Table 5).








TABLE 5







Diagnostic primers used for verification of


gene disruptions (amplify deleted regions)









Type

Seq.


of gene

ID





TTLL6A
Forward 5′-TATCTTTTGGACTGATAATGCT-3′
89



Reverse 5′-CTCTTAATATCTTTCCACAG-3′
90


 


TTLL6F
Forward 5′-AGATCTCTAAAGGAAAATGC-3′
91



Reverse 5′-TTCATGTAGTTATCTGGTTG-3′
92









To construct a triple mutant strain, 6AF-KO;oadl-1, a 6AF-KO heterokaryon was crossed to an oadl-1 germline mutant strain (van Dijk et al., 2007. Mol Cell 26, 437-448), triple heterozygotes were matured and drug resistance was lost from the macronucleus by phenotypic assortment. Strain with homozygous micronuclei were made by a cross to A*III. Specific heterokaryons that were homozygous for both gene disruptions and the oadl-1 allele were identified by an outcross to an oadl-1 homozygote strain, and identification their progeny that was resistant to paromomycin, cycloheksimide in the presence of cadmium and displayed ciliary paralysis after incubation at 38° C.
For rescue, vegetatively growing 6AF-KO cells were biolistically transformed with a fragment encoding MTT1-GFP-TTLL6A targeted to BTU1 (Janke et al. 2005. Science 308, 1758-1762). A strain lacking TTLL6A and TTLL6F and homozygous the oadl-1 mutation (Attwell et al., 1992. J Protozool 39, 261-266) was constructed by standard crosses.
Phenotypic studies. The multiplication, cell motility and phagocytosis rates were measured as described (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372). To assay swimming behavior, 2 ml of cells (2×105 cells/ml) were added to 100 ml of wash buffer (10 mM Tris, 50 μM CaCl2, MOPS pH 7.2), centrifuged at 1000×g for 2 min and suspended in 2 ml. After 30 min of adaptation, cells were assayed in 70 μl drop on a two-ring slide, with or without SDBS (20 μg/ml) and video recordings were done under a dissecting microscope with a Moticam 480 digital camera. To measure the beat frequency, cells (2 x 105 cells/ml) were recorded at 500 frames/sec by a Photronics 1280 PCI FastCam on a Nikon Eclipse E600 microscope.
Biochemical studies. Partial purifications of Ttll6Ap and Ttlllp from overproducing Tetrahymena strains and in vitro glutamylation assays with taxotere—stabilized microtubules made of the murine brain and HeLa tubulin were performed as described (Wloga et al. 2008. Eukaryot Cell 7, 1362-1372).
To purify cilia, Tetrahymena cells were grown to a density of 3×105 cells/ml in 500 ml of SPP, washed with 10 mM Tris pH 7.5 and suspended in 40 ml of 10 mM Tris, 50 mM sucrose, 10 mM CaCl2 with protease inhibitors (Complete, Roche). Deciliation was initiated by adding 600 μl of 0.5 M acetic acid, after 2 min followed by 550 μl of 0.6M KOH. Cell bodies were removed (1860×g, 5 min) and cilia were collected (23,300×g, 15 mM, 4° C.) and suspended in 500μ of the axoneme buffer (20 mM potassium acetate, 5 mM MgSO4, 0.5 mM EDTA, 20 mM HEPES, pH 7.6).
Microtubule sliding in isolated axonemes. Cilia were suspended at 0.1 mg protein/ml in the axoneme buffer (without protease inhibitors). For experiments on axonemes purified from strains carrying the oadl-1 mutation, all strains were grown for 12 hr at 38° C. with inocula adjusted according to individual growth rates to collect cells at 3×105 cells/ml. Cilia were suspended at 0.1 mg protein/ml in 500 μl of the motility buffer (1 mM DTT, 50 mM potassium acetate, 5 mM MgSO4, 1 mM EGTA, 30 mM HEPES, PEG 1%, pH 7.6). To demembranate, 10 μl of 1% NP-40 in motility buffer was added to 50 μl of diluted cilia. The axoneme suspension was pipetted into a perfusion chamber constructed with a glass slide and cover slip separated by double stick tape. The perfusion chamber was washed with 50 μl of axoneme buffer followed by perfusion with 50 μl of 1 mM of ATP, in the motility buffer. The sliding of microtubules was recorded on a Zeiss Axiovert 35 microscope equipped with dark field optics (40×PlanApo), on a silicon-intensified camera (VE-1000, Dage-MTI, Michigan City, Ind.). The video images were converted to a digital format using Labview 7.1 software (National Instruments, Austin, Tex.). The sliding velocity was determined manually by measuring microtubule end displacement, as a function of time, on tracings calibrated with a micrometer (Okagaki and Kamiya, 1986. J Cell Biol 103, 1895-1902).
Immunofluorescence and western blotting. The cells were stained by immunofluorescence as described in (Wolff et al., 1992. Eur J Cell Biol 59, 425-432) with the following primary antibodies: 12G10 (1:50), GT335 (1:100), SG polyclonal anti-total tubulin (1:600) and polyE (1:100).
For western blotting, 5 μg of cilia protein per lane were separated on a 10% SDS-PAGE and western blots were done as described (Kann et al., 1995. Tissue Cell 27, 323-329), with the primary antibodies at the following dilutions: GT335 anti-glutamylated tubulin mAb (1:1,000) (Lechtreck and Geimer, 2000. Cell Motil Cytoskeleton 47, 219-235), 12G10 anti-α-tubulin mAb (1:10,000) (Developmental Studies Hybridoma Bank), polyE anti-polyglutamic acid antibodies (1:1,000) (Wloga et al., 2010. Eukaryot Cell. 9,184-193), polyG, anti-polyglycine antibodies (Multigner et al., 1996. Biochemistry 35, 10862-10871), 6-11 B-1 anti-acetyl-K40 on α-tubulin (Summers Gibbons, 1971. Proc Natl Acad Sci U S A 68, 3092-3096), and anti-SerH surface antigen antibodies (Okagaki and Kamiya, 1986. J Cell Biol 103, 1895-1902).
Electron microscopy. For standard transmission electron microscopy (TEM), cells were fixed as described (Sale and Satir, 1977. Proc Natl Acad Sci U S A 74, 2045-2049). For immunogold TEM, cells carrying an MTT1-GFP-TTLL6A transgene were grown in 5 ml of SPP medium (2×105 cells/ml) and induced with 2.5 μg/ml CdCl2 for 3 hr, gently spun down briefly, fixed in an Eppendorf tube and stained as described above for immunofluorescence with anti-GFP antibodies (Abcam, 1:10,000) overnight followed by anti-rabbit IgG-10 nm gold (1:60) (Amersham Pharmacia) for 2 hr at room temperature. Cells were concentrated by centrifugation to 100 μl and post-fixed with 1 ml of 2% glutaraldehyde (in 0.1M sodium cacodylate buffer, p117.2) on ice for 1 h. Cells were washed 5 times in cold sodium cacodylate buffer (10 min each on ice) and fixed with 1 ml of 1% osmium tetroxide for 1 hour on ice. The pellet was washed 5 times in water, followed by dehydration in ethanol concentration/water series and embedding in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and analyzed on JEOL 1200 EX transmission electron microscope. For immunogold whole mount TEM, cilia were purified from 50 ml of cells (3×105 cells/ml) and demembranated in 500 μl of 1% NP40 in the axoneme buffer. After 5 min on ice, axonemes were collected at 23,000 g (15 min, 4° C.), washed with 1 ml of axoneme buffer and, centrifuged and suspended in the axoneme buffer at 10 mg tight pellet/ml of. To initiate sliding of axonemal microtubules, 0.5 μl of 2 mM ATP was added to 50 μl of axonemal suspension (final concentration 40 μM). After incubation for 3 min at room temperature, 10 μl of suspension was placed onto a formvar-coated grid for 1 min and the excess was absorbed by filter paper. Grids were processed for immunogold TEM (Kamiya, 2002. Int Rev Cytol 219, 115-155) as described below and stained using uranyl acetate.
For immunogold whole mount TEM, axonemes were purified and reactivated with ATP as described in the main text, and labeled with antibodies according to (Kamiya, 2002. Int Rev Cytol 219, 115-155) with minor modifications. Ten μl of the ATP-reactivated axonemes were placed on top of a formvar-coated copper EM grid, allowed to settle for 1 min, the excess of liquid was drained with a filter paper and the material was immediately fixed by floating the grid on top of a 50 μl drop of 2% paraformaldehyde in PHEM buffer. The grids were washed by dipping in 10 mM HEPES (pH 7.4) about 15 times and blocked by covering with 10 μl of 3% BSA, 0.01% Tween20 in PBS for 15 min. The grids were incubated in 50 μl drops of primary antibodies (1:100 polyE in the blocking buffer). The grids were washed by dipping in PBS about 15 times, incubated in 50 μl drops of the secondary antibody, anti-rabbit IgG-10 nm gold (GE Healthcare) (1:60 in the blocking buffer), washed 15 times in PBS, and negatively stained with 2% uranyl acetate.
Example VI
Tetrahymena Strains Lacking Paralogs of the TTLL6 Gene Demonstrate Reduced Glutamylation
Methods described in detail in Example V were used to obtain strains of Tetrahymena thermophila lacking 4 or 5 paralogs of TTLL6 gene family. Deletion of additional TTLL6 paralogs (on top of TTLL6A and TTLL6F genes that were deleted earlier and are described in Example V) led to further reduction in the levels of tubulin polyglutamylation but the reduction in the levels of total glutamylation is less pronounced. As a result, axonemes of cilia of deletion mutants have shortened glutamyl side chains. Such axonemes can potentially be used for an in vitro tubulin glutamylation assay with Ttll6Ap or similar enzymes that act mainly as glutamyl ligases with chain elongation activity.
Deletion of multiple paralogs genes of TTLL6 progressively reduces the rate of growth, phagocytosis and cell motility. In all panels of FIG. 34, WT designates a wild type strain while 6AF-, 6BD-, 6ABD-F, and 6ABDEF- designate strains lacking from 2 to 5 paralogous genes in the TTLL6 family (TTLL6A, TTLL6B, TTLL6D, TTLL6E, TTLL6F). FIG. 34A shows motility rates. FIG. 34B shows culture growth rates. FIG. 34C-D shows images of cells fed with India ink to evaluate the ability to perform phagocytosis. Note that ink-filled food vacuoles are present only in WT cells.
Deletion of multiple TTLL6 paralog genes leads to a progressive loss of tubulin polyglutamylation. In all panels of FIG. 35, WT designates a wild type strain while 6AF-, 6BD-, 6ABD-F, and 6ABDEF- designate strains lacking from 2 to 5 paralogous genes in the TTLL6 family (TTLL6A, TTLL6B, TTLL6D, TTLL6E, TTLL6F). FIG. 35A shows immunofluorescence images of a pair of cells imaged side by side. One of the cells is WT and another lacks two or more TTLL6 paralog genes. The left row contains images of cells labeled by an anti-tubulin antibody, 12G10. The middle row contains the same cells labeled by antibodies against polyglutamylation (polyE). The right row contains images of merged signals shown in the other two rows. Note a progressive loss of tubulin polyglutamylation along with the deletion of TTLL6 paralog genes. FIG. 35B shows immunofluorescence images of a pair of cells images side by side. One of the cells is WT and another lacks two or more TTLL6 paralog genes. The left row contains images of cells labeled by an anti-tubulin antibody, 12G10. The middle row contains the same cells labeled by antibodies against tubulin glutamylation (GT335). The right row contains images of merged signals shown in the other two rows. Note that the signals are similar in WT and TTLL6 knockout cells. Thus, the loss of TTLL6 paralogs leads to polyglutamylation side chain shortening and not its complete loss. FIG. 35C shows a western blot of total ciliary proteins isolated from WT and TTLL6 knockout strains. The following antibodies were used: polyE (anti-polyglutamylation), GT335 (tubulin glutamylation), polyG (anti-polyglycylation), 6-11 B-1 (anti-acetyl-K40 of α-tubulin), 12G10 (anti-α-tubulin). FIG. 35D shows a Coomassie Blue stained SDS-PAGE gel of protein samples used in FIG. 35C as a control for equal loading.
Deletion of multiple TTLL6 paralog genes leads to progressive shortening of cilia. FIG. 36A shows immunofluorescence images of cells labeled by 12G10 anti-α-tubulin antibodies. FIG. 36B shows a histogram that documents the distribution of ciliary length obtained by measurements of cilia of cells labeled by immunofluorescence. FIG. 36C-D shows electron micrographs of axoneme cross-sections of a WT and knockout cell which demonstrate that the axoneme structure is normal despite its shortening. FIG. 36E shows immunofluorescence images (with anti-α-tubulin antibodies) of isolated cilia.
Example VII
TTLL3 is a Tubulin Glycine Ligase that Regulates the Assembly of Cilia
In most ciliated cell types, tubulin is modified by glycylation, a posttranslational modification of unknown function. This example shows that the TTLL3 proteins act as tubulin glycine ligases with chain-initiating activity. In Tetrahymena, deletion of TTLL3 shortened axonemes and increased their resistance to paclitaxel-mediated microtubule stabilization. In zebrafish, depletion of TTLL3 led to either shortening or loss of cilia in several organs, including the Kupffer's vesicle and olfactory placode. This example also shows that, in vivo, glutamic acid and glycine ligases oppose each other, likely by competing for shared modification sites on tubulin.We propose that tubulin glycylation regulates the assembly and dynamics of axonemal microtubules and acts either directly or indirectly by inhibiting tubulin glutamylation. Wloga et al., “TTLL3 is a Tubulin Glycine Ligase that Regulates the Assembly of Cilia,” Jun. 16, 2009, Developmental Cell 16:867-876.
In a typical motile cilium, its microtubular scaffold, the axoneme, is built around a framework of nine outer and two central microtubules. The axoneme is composed of several hundred different proteins (Pazour et al., 2005. J. Cell Biol. 170, 103-113; Smith et al., 2005. J. Proteome Res. 4, 909-919). In most cell types, ciliogenesis involves the delivery of precursors to the tips of growing axonemes by the intraflagellar transport (IFT) pathway (Kozminski et al., 1995. J. Cell Biol. 131, 1517-1527). However, how the axoneme assembles once the precursors are delivered to cilia is poorly understood.
Ciliary tubulin is marked by conserved posttranslational modifications (PTMs), that could play a role in assembly of the axoneme (Verhey and Gaertig 2007. Cell Cycle 6, 2152-2160). Two types of tubulin PTMs, known as polymodifications—glutamylation (Eddé et al., 1990. Science 247, 83-85) and glycylation (Redeker et al., 1994. Science 266, 1688-1691)—are peptide branches made of either glutamyl or glycyl residues that are attached to the g-carboxyl group of glutamic acids of the C-terminal tail domains (CTTs) of α- and β-tubulin. Recently, we have identified the tubulin glutamic acid ligase enzymes as proteins related to tubulin tyrosine ligase (Ersfeld et al., 1993. J. Cell Biol. 120, 725-732), known as TTL-like (TTLL) proteins (Janke et al., 2005. Science 308, 1758-1762). Studies on several glutamic acid ligases (E-ligases) indicate that tubulin glutamylation is important for assembly of axonemes. The loss of the noncatalytic subunit of TTLL1 E-ligase led to defective sperm axonemes and male sterility in mice (Campbell et al., 2002. Genetics 162, 307-320), and depletion of TTLL6 inhibited assembly of olfactory cilia in zebrafish (Pathak et al., 2007. Mol. Biol. Cell 18, 4353-4364).
The role of tubulin glycylation remains unknown, mainly because the modifying enzymes are yet to be discovered. Here we use Tetrahymena to identify TTLL3 as a tubulin glycine ligase (G-ligase) with a chain-initiating activity. Tetrahymena cells lacking all TTLL3 genes have shortened axonemes that are resistant to paclitaxel, indicating that tubulin glycylation changes the lattice properties of axonemal microtubules. Next, we use zebrafish to study the significance of TTLL3 during vertebrate development. Morpholino (MO)-based depletion studies indicate that TTLL3 is required for either elongation or stability of axonemes. In both organisms, a reduction in tubulin glycylation led to hyperglutamylation. Thus, glycylation could act by competing with glutamylation for shared modification sites on tubulin.
Results
TTLL3 Homologs of Tetrahymena are Required for Initiation of Glycyl Side
Chains on Tubulin. The TTLL superfamily includes proteins that mediate the ligation of amino acids to proteins, including enzymes that catalyze tubulin tyrosination (Ersfeld et al., 1993. J. Cell Biol. 120, 725-732) and glutamylation (Janke et al., 2005. Science 308, 1758-1762). The murine TTLL10 glycylates NAP1 (Ikegami et al., 2008. FEBS Lett. 582, 1129-1134), but it is not known whether any TTLL glycylates tubulin. TTLL3 sequences (Janke et al., 2005. Science 308,1758-1762) are present in organisms that have tubulin glycylation (Bré et al., 1996. J. Cell Sci. 109, 727-738; Bressac et al., 1995. Eur. J. Cell Biol. 67, 346-355), but are absent in Trypanosoma and Plasmodium that lack this PTM (Fennell et al., 2008. Int. J. Parasitol. 38, 527-539; Schneider et al., 1997. J. Cell Sci. 110, 431-437). Thus, TTLL3 could be a G-ligase for tubulin.
Tetrahymena thermophila has four genes encoding members of the conserved TTLL3 clade: TTLL3A through TTLL3D, and two genes encoding divergent TTLL3-like proteins, TTLL3E and TTLL3F (see FIG. 37). All these proteins have peptide signatures in the ATPase domain that are diagnostic of the TTLL3 clade. Ectopic GFP-Ttll3Ap, Bp, Cp, and Ep localized mainly to cilia, with GFP-Ttll3Ap and Ep present mainly in locomotory and oral cilia (FIG. 37F and FIG. 38C), Bp in oral cilia, and Cp in assembling locomotory cilia (FIGS. 37C-D). The N-terminal noncatalytic domain of Ttll3Ap was sufficient for ciliary localization (FIG. 37B). GFPTtll3Dp remained in the cell body, while 3Fp was enriched in the basal bodies (FIGS. 37E-G).
We measured the tubulin glycylation activity in cytoplasmic fractions of Tetrahymena overproducing specific paralogs. A GFP-Ttll3Ap-enriched extract showed a strong increase in glycylation activity on α- and β-tubulin, while the GFP-Ttll3Bp-enriched extract had increased activity on α-tubulin (FIGS. 38D-E). The TAP952 monoclonal antibody (mAb) recognizes monoglycylated sites on tubulin, while the AXO49 mAb (Bré et al., 1998. Mol. Biol. Cell 9, 2655-2665) and polyG antibodies (Duan and Gorovsky, 2002. Curr. Biol. 12, 313-316) recognize elongated side chains (polyglycylation). Expression of GFP-Ttll3Ap increased the levels of tubulin monoglycylation, but not polyglycylation in cilia based on immunofluorescence (FIGS. 38A-B and FIGS. 39A-C′′) and Western blots (FIG. 38F). Thus, Ttll3Ap mediates glycyl side chain initiation activity.
Next, using homologous DNA recombination, we disrupted TTLL3A and TTLL3B genes that encode ciliary paralogs with a significant activity in vitro. In 3AB-KO cells, the levels of monoglycylated tubulin were strongly reduced (FIGS. 40A-B, and 40F; FIGS. 41A-A′) and the levels of tubulin polyglycylation were moderately reduced (FIGS. 40C-D, F; FIGS. 41B, B′, and H). Strikingly, the levels of tubulin glutamylation in cilia were increased by  ̃3-fold (FIG. 40F; FIGS. 41C-C′). Additional experiments indicate that, under a variety of conditions, the levels of tubulin glycylation and glutamylation are inversely correlated. For example, overexpression of GFP-Ttll3Ap decreased tubulin glutamylation in cilia (FIG. 39D-D′′), and conversely, axonemes from a strain lacking two E-ligases—Ttll1p and Ttll9p (Wloga et al. 2008. Eukaryot. Cell 7, 1362-1372)—have increased levels of tubulin glycylation (FIG. 42). Thus, the two polymodifications negatively regulate each other.
A transmission electron microscopy (TEM) analysis did not reveal any defects in 3AB-KO axonemes (FIG. 43M). The 3AB-KO cells grew at a normal rate on standard medium (FIG. 40E), but multiplied faster than wild-type in the presence of the microtubule stabilizing drug, paclitaxel (FIG. 40E′). In wild-type cells, paclitaxel induced a remarkable elongation of axonemes, but this effect was not observed in 3AB-KO cells (FIGS. 40H-K). Thus, 3AB-KO cells and specifically axonemes are resistant to paclitaxel.
Next, we constructed strains lacking either four conserved (3ABCD-KO), or all six TTLL3 genes (3ABCDEF-KO; FIG. 44). The quadruple and sextuple knockout strains showed further reduction in the levels of tubulin glycylation in cilia, including a strong reduction in polyglycylation (FIG. 40F; FIGS. 41D-E′ and G-J). Thus, in contrast to Ttll3Ap and Bp, the activity of the remaining TTLL3 paralogs is closely coupled to chain elongation. However, even cells lacking all TTLL3 genes (3ABCDEF-KO) had a residual glycylation signal in the tubulin region of a Western blot of ciliary proteins (estimated as  ̃8% of the wild-type level for mono- and <1% for polyglycylation [FIG. 40F]) and in axonemes of fixed cells (FIGS. 41D-E′ and J). Thus, it appears that another enzyme(s) distinct from TTLL3 also generates tubulin glycylation. Based on immunofluorescence, 3ABCDEF-KO cells had higher levels of tubulin polyglutamylation than the 3AB-KO cells (compare FIGS. 41F-F′ to FIGS. 41C-C′). The quadruple and sextuple KO strains grew more slowly as compared with wild-type and 3AB-KO cells (FIG. 40E), and the level of paclitaxel resistance in the 3ABCDEF-KO strain was higher as compared with the 3AB-KO strain (FIG. 40E′). Thus, there is correlation between the loss of TTLL3 function and the levels of hyperglutamylation and paclitaxel resistance.
The increased paclitaxel resistance suggests that hypoglycylated microtubules are more dynamic. Surprisingly, axonemes in TTLL3 knockout strains consistently had moderately elevated levels of K40 acetylation on α-tubulin, a marker of long-lived microtubules (FIG. 40F) (LeDizet and Piperno 1991. Meth. Enzymol. 196, 264-274). This suggests that glycylation promotes subunit turnover of axonemal microtubules. The 3ABCDEF-KO axonemes were normal at the ultrastructural level (FIG. 43N). However, the loss of TTLL3 function led to a progressive shortening of the axonemes; in 3ABCDEF-KO cells, axonemes are 15% shorter than wild-type (FIGS. 40G-G′′; FIGS. 41K and L). Thus, in Tetrahymena, TTLL3 is required for the vast majority of tubulin glycylation and promotes axoneme elongation.
A Dominant-Negative Mutation of TTLL3 Causes Severe Defects in Axoneme Assembly in Tetrahymena. If TTLL3 is a G-ligase, its activity should be blocked by a mutation that affects the ATPase domain. Using a cadmium-dependent promoter (MTT1), we overexpressed an active GFP-Ttll3Ap and an inactive GFP-Ttll3Ap, with an E818G mutation (GFPDN-Ttll3Ap) that was predicted to block the ATPase activity (Janke et al., 2005. Science 308, 1758-1762). Both proteins accumulated to similar levels (FIG. 38G) and localized to cilia (FIG. 38C and FIG. 45A). In cells overproducing GFP-Ttll3Ap, there was a moderate reduction in the length of cilia (from 5.2±0.7 to 4.9±0.9 mm [FIGS. 45D, E, and J]). In GFP-DN-Ttll3Ap cells, at 6 hr, most cilia were extremely short (2.1±0.8 mm), while some cilia were exceptionally long (7±0.9 mm [FIGS. 45C, F, and K]), and the proportion of short cilia increased after 11 hr (FIG. 45L). GFPDN-Ttll3Ap was enriched in short cilia and in the distal segments of long cilia (FIGS. 45A-C). The levels of mono- and polyglycylation were moderately reduced in GFP-DN-Ttll3Ap, even without induction of transgene expression, due to the basal expression of the MTT1. Moreover, in induced cells, segments of cilia with that accumulated GFP-DN-Ttll3Ap had extremely low levels of tubulin monoglycylation (FIG. 46) and polyglycylation (FIGS. 45A-C and F). As expected, the hypoglycylated segments of axonemes enriched in GFP-DNTtll3Ap had strongly increased levels of glutamylation (FIGS. 45G-I′).
It is most likely that short cilia are those that have assembled during induced GFP-DN-Ttll3Ap expression, while the long cilia arose by distal extension of pre-existing units. Indeed, GFPDN-Ttll3Ap-overexpressing cells that were subjected to complete deciliation could regenerate only excessively short cilia (FIG. 47).
TEM analysis showed that cells expressing GFP-Ttll3Ap had no apparent defects in the axonemes (FIG. 43H). In GFP-DN-Ttll3Ap cells, 60% of axoneme cross-sections lacked the central microtubules (FIGS. 43B, D, F, G, and I; and Table 6). Some peripheral doublets were rotated (FIGS. 43B and F) or absent (FIG. 43E). Rarely, abnormal peripheral triplet microtubules were present in the axoneme (FIG. 43G). Many axoneme cross-sections had ectopic singlet microtubules (FIGS. 43B, C, and E). Since such microtubules were not present in axonemes of cilia-regenerating cells (Table 6), we conclude that ectopic singlets form at the distal ends of the pre-existing cilia. Indeed, many longitudinal cilia sections in vegetatively growing GFPDN-Ttll3Ap cells had ectopic, disordered singlet microtubules, primarily in the distal regions (FIGS. 43K and L). These data suggest that the hypoglycylated/ hyperglutamylated segments added to ends of pre-existing motile axonemes are unstable.







TABLE 6







Axoneme cross-sections of wild-type and GFP-Ttll3Ap-E818G Tetrahymena cells that are either growing vegetatively or regenerating cilia.






















Two







9 + 2 with

9 + 0 with
singlet
axoneme




twisted

twisted
ectopic
profiles//
Peripheral
Fewer



9 + 2
doublets
9 + 0
doublets
MTs
cilium
triplets
Doublets
n
























Growing WT
99.57%
(229)
0%
(0)
0.43%
(1)
0%
(0)
0% (0)
0% (0)
0% (0)
0% (0)
230


somatic cilia


Growing WT
100%
(158)
0%
(0)
0%
(0)
0%
(0)
0% (0)
0% (0)
0% (0)
0% (0)
158


oral cilia


Growing
26.19
(33)
1.59%
(2)
42.87%
(54)
9.52%
(12)
5.55% (7)  
11.9% (15)  
0% (0)
2.38% (3)  
126


Ttll3Ap-E818G


(somatic cilia)


Growing
60.65%
(259)
0%
(0)
37.87%
(159)
0%
(0)
0.7% (3)  
0% (0)
1.41% (6)  
0% (0)
427


Ttll3Ap-E818G


(oral cilia)


Cilia
95.82%
(275)
0%
(0)
3.14%
(9)
1.04%
(3)
0% (0)
0% (0)
0% (0)
0% (0)
287


regenerating WT


(30 min)


Cilia
39%
(78)
0.5%
(1)
55%
(110)
5.5%
(11)
0% (0)
0% (0)
0% (0)
0% (0)
200


regenerating


Ttll3Ap-E818G


(30 min)


Cilia
41.81%
92
0.46%
1
53.64%
(118)
4.09
(9)
0% (0)
0% (0)
0% (0)
0% (0)
220


regenerating


Ttll3Ap-E818G


(60 min)









Immunogold TEM shows that both active and DN forms of the enzyme localized to the peripheral doublets (FIGS. 43H-J). Moreover, in wild-type cells, mono- and polyglycylation were detected exclusively on outer doublets in 98% (FIG. 30; n=93) and 100% (FIG. 43P; n=100) cross-sections, respectively. Thus, TTLL3 proteins act primarily by modifying tubulin of the outer microtubules, and the effects of DN-Ttll3Ap on the central pair assembly are probably indirect.
Knockdown of Ttll3 in Zebrafish Affects Ciliary Assembly and Cilia in Developing Embryos. Next, we addressed the significance of TTLL3 in a vertebrate. Zebrafish has a single TTLL3 gene, ttll3 (gi:113671247) (FIG. 37A). Immuno-fluorescence of zebrafish embryos (n=57) using TAP952 revealed monoglycylated tubulin in cilia of the olfactory placode (FIG. 48H), otic vesicle (FIGS. 49A and A′), spinal cord, hypochord (FIG. 49G), pronephric ducts (FIG. 49C and FIG. 50E), and neuromast hair cells (FIG. 50I). Cilia in the nasal epithelium (FIG. 48J), neuromasts, and the medial section of the pronephros stained with AXO49, indicating the presence of polyglycylation. Thus, the pattern of tubulin glycylation is tissue dependent.
To reduce ttll3 function, we used antisense MOs to block either the translation initiation (TTLL3-ATG) or splicing at the intron 10/exon 11 junction (TTLL3-SP) of ttll3 mRNA. Injection of 1 ng of TTLL3-ATG MO greatly reduced tubulin monoglycylation in multiple types of cilia at 72 hpf (compare FIG. 48H with FIG. 481; FIGS. 49A-D, G, H; FIGS. 50E, F, I, and L). In TTLL3-ATG MO embryos, the TAP952 signal was not detected in the cilia of the spinal cord (in 89% of morphants), the medial (61%), and the distal part of pronephric duct (98%) (n=46). TTLL3-SP MOs (3 ng) reduced tubulin monoglycylation in cilia of the olfactory placode, otic vesicle, spinal cord, and hypochord in all analyzed embryos (n=8). In morphants, in the olfactory placode, the levels of polyglycylation (using AXO49) were also reduced (FIGS. 48J and K). No change in the levels of tubulin glycylation was detected in multiple types of cilia in embryos injected with control (random sequence) MOs (n=5; FIG. 51). Since the two ttll3 MOs contain no overlapping sequences, yet produce similar effects on tubulin glycylation, and similar phenotypic defects (see below), we conclude that the reduction in glycylation in the morphants is due to a specific reduction in ttll3 function.
In control embryos, the GT335 antibody detected glutamylated tubulin in all types of cilia that also had glycylated tubulin (n=14). Despite the reduction of monoglycylation in ttll3 morphants, most cilia types maintained a strong GT335 signal (FIGS. 49I and J). Moreover, the polyE antibody that recognizes polyglutamylation (Shang et al., 2002. J. Cell Biol. 158, 1195-1206) detected an increased signal in cilia of the olfactory placode and neuromasts in morphants (FIGS. 48H′-I′′; FIGS. 50J, K, M, and N). Thus, in some zebrafish tissues, tubulin glycylation may inhibit tubulin glutamylation as it does in Tetrahymena. However, cilia in the distal parts of pronephric duct were either GT335 negative (n=7) or had very short GT335-positive segments (n=5) (FIGS. 49E and F). It is thus possible that depletion of ttll3 inhibits the assembly of a subset of pronephric cilia.
The majority of morphant embryos had a “curly tail” (FIGS. 48A-D; FIGS. 50A-C; TTLL3-ATG: 76%; n=128; TTLL3-SP: 64.3%; n=84). For unclear reasons, embryos that were manually released from the chorion at 24 hpf had curled-up tails, whereas those released at 48 hr had curled-down tails (FIGS. 50A-C). The curly tail phenotype is often associated with ciliary deficiencies (Sun et al., 2004. Development 131, 4085-4093).
Defective cilia disrupt fluid flow in the kidney and brain, resulting in severe distensions of these organs that manifest as hydrocephaly and cystic kidneys (Kramer-Zucker et al., 2005. Development 132, 1907-1921). Some ttll3 morphants were hydrocephalic (81% of TTLL3-SP, n=37; 10% of TTLL3-ATG, n=30) (FIGS. 48E and F). We did not observe kidney cysts, despite the dramatic reduction of glycylation in cilia of the pronephric duct (FIGS. 49C and D; FIGS. 50E and F).







TABLE 7







Heart position in zebrafish control and TTLL3 morphant


embryos obtained by scoring either live embryos or those


subjected to in situ hybridization using clm1 probe.












left
middle
right
N

















control
live
58 (93.6%)
4 (6.4%)
0
(0%)
62



hybridization
13 (92.9%)
1 (7.1%)
0
(0%)
14


control MO
live
76 (93.8%)
5 (6.2%)
0
(0%)
81


TTLL3-ATG-MO
live
56 (50%)  
33 (29.5%)
23
(20.5%)
112



hybridization
21 (50%)  
 7 (16.7%)
14
(33.3%)
42


TTLL3-SP-MO
live
21 (64%)  
8 (24%) 
4
(12%)
33



hybridization
23 (57.5%)
6 (15%) 
11
(27.5%)
40









In vertebrate embryos, motile cilia are required to establish the left-right (LR) axis at the end of gastrulation (Burdine and Schier, 2000. Genes Dev. 14, 763-776). In zebrafish, the first cilia form in a tissue called the Kupffer's vesicle (KV), which is analogous to the mouse node (Essner et al., 2005. Development 132, 1247-1260). Disruptions of the KV cilia result in a complete reversal of the LR axis, known as situs inversus, or in a randomized LR axis, called heterotaxia (Burdine and Schier, 2000. Genes Dev. 14, 763-776). During normal development, the developing heart is located on the left side of the embryo, as revealed by expression of cardiac myosin light chain 2 (cmlc2) (FIG. 52E) (Yelon, 2001. Dev. Dyn. 222, 552-563). In ttll3 morphants, by contrast, hearts were often located on the right side of the embryo, or along the midline (Table 7). Consistent with this, cmlc2 (FIGS. 52F-H; Table 7) and lefty2 (Table 8) were often abnormally expressed on the right side, along the midline, or bilaterally. In zebrafish, asymmetry in the brain is revealed by leftyl, which is expressed on the left side of the epiphysis, a structure in the diencephalon that gives rise to the pineal gland (FIG. 52A [Liang et al., 2000]). In ttll3 morphants; by contrast, lefty1 mRNA was often present on the left side, bilaterally, or on the right side of the epiphysis, indicating that asymmetry in the brain was also disrupted (FIGS. 52B-D). The nodal-related gene, southpaw (spaw), is the earliest marker of LR asymmetry and is normally expressed in the left lateral plate mesoderm (Long et al., 2003). In ttll3 morphants, spaw was often expressed bilaterally or in the right lateral plate mesoderm (Table 8). Injection of control random sequence MOs did not lead to patterning defects (Table 7). Injection of MOs designed to deplete non-TTLL3 TTLLs (TTLL10 and -12) have produced a distinct combination of defects, and none these MOs affected the LR asymmetry. These results suggest that tubulin glycylation is needed either for assembly or function of KV cilia. Indeed, we found that morphants had fewer and shorter KV cilia as compared with control embryos, as revealed by staining with the 6-11 B-1 antibody, which recognizes α-tubulin with acetyl-K40 (LeDizet and Piperno 1991. Meth. Enzymol. 196, 264-274; FIGS. 52I-K).
In addition to KV, the assembly of cilia in other tissues is also affected by Ttll3 depletion. Based on the staining with 6-11 B-1 mAb, cilia in the neuromasts cells were shorter in ttll3 morphants (FIGS. 50G and H). In the wild-type nose, the 6-11 B-1-positive cilia were densely packed around the edge of the olfactory placode (FIGS. 48G







TABLE 8







Expression patterns of lefty2 and southpaw in


zebrafish control and TTLL3 morphant embryos.









Pattern of Expression















No


Bilatera






specific


or













signal
left
midline
right
N











lefty2
















Control
1
(7%)
13
(93%)
0
(0%)
0
(0%)
14


TTLL3-
8
(19%)
15
(35.7%)
5
(11.9%)
14
(33.4%)
42


ATG-MO


TTLL3-
16
(38.1%)
21
(50%)
2
(4.8%)
3
(7.1%)
42


SP-MO







southpaw
















Control
3
(8.8%)
31
(91.2%)
0
(0%)
0
(0%)
34


TTLL3-
19
(24.3%)
14
(17.9%)
34
(43.7%)
11
(14.1%)
78


ATG-MO


TTLL3-
6
(14.6%)
12
(29.3%)
22
(53.7%)
1
(2.4%)
41


SP-MO









and L). In the placodes of morphants, there were large gaps lacking 6-11 B 1-positive cilia (FIG. 48M). To rule out the possibility that K40 α-tubulin acetylation is reduced in the morphants without a loss of the axoneme, we examined the olfactory cilia by SEM. In wild-type, the olfactory placode is an oval structure filled with cilia (FIGS. 48N and O; n=7). In all ttll3 morphants examined, the density of cilia was reduced, and many of the remaining cilia were truncated (FIGS. 48P and R; n=7). TEM analysis of the olfactory placode in the wildtype embryo revealed the presence of axonemes with either 9+0 or 9+2 organization (the latter were usually close to the placode rim [Hansen and Zeiske, 1993. J. Comp. Neurol. 333, 289-300]) (FIGS. 53A-D and I). Most 9+2 axoneme cross-sections had dynein arms, but some axonemes lacked dynein arms (FIG. 53B). The distal ends of axonemes contained singlet A-subfiber extensions lacking projections (FIG. 53D). Morphant cilia appeared normal (FIGS. 53E-H and K), but longitudinal sections showed that many morphant cilia were excessively short and some had swollen tips (FIG. 48S and FIG. 53J). Thus, the main effect of Ttll3 depletion in zebrafish is shortening and loss of axonemes.

Discussion

We show that TTLL3 proteins are required for tubulin glycylation in a protist and in a vertebrate. Overproduction of TTLL3 in Tetrahymena led to a strong increase in tubulin monoglycylation, but not polyglycylation, consistent with chain-initiating activity. A mutation that inactivates the ATPase domain of TTLL3 inhibited its G-ligase activity. In a separate study, Rogowski et al. show that the mammalian and Drosophila TTLL3 proteins mediate G-ligase/initiase activity (Rogowski et al., 2009. Cell, 137, 1076-1087). The simplest explanation of all these data is that TTLL3 is a conserved tubulin G-ligase with a chain-initiating activity.
Loss of TTLL3 proteins led to dramatic reduction in the levels of tubulin glycylation in cilia of Tetrahymena and zebrafish. However, Tetrahymena mutants lacking all TTLL3 genes have residual tubulin glycylation signal in cilia. The mammalian TTLL10 mediates glycine side chain elongation activity on tubulin (Rogowski et al., 2009. Cell, 137, 1076-1087). However, TTLL10 can directly glycylate a recombinant NAP1, indicating an ability to initiate (Ikegami et al., 2008. FEBS Lett. 582, 1129-1134). Thus in Tetrahymena, TTLL10 enzymes could initiate side chains on tubulin, perhaps only in the absence of TTLL3. Alternatively, yet-uncharacterized TTLL types (e.g., TTLL14 or TTLL15) could act as G-ligases.
Tetrahymena cells lacking TTLL3 assembled shorter cilia. In zebrafish, lack of
TTLL3 activity leads to dramatic shortening or complete loss of cilia. Thus, the zebrafish and Tetrahymena phenotypes are consistent, although cilia are less affected in Tetrahymena, possibly due to action of non-TTLL3 G-ligases. In Tetrahymena, besides tubulin, the only immunologically detectable glycylated protein is Pgplp, which is targeted to the endoplasmic reticulum (Xie et al., 2007. Eukaryot. Cell 6, 388-397). Thus, the effects of TTLL3 deficiencies on cilia assembly and function are likely mediated by tubulin glycylation. In Drosophila, several nontubulin proteins undergo TTLL3-dependent glycylation (Rogowski et al., 2009. Cell, 137, 1076-1087). Drosophila may be unusual, because this species lacks TTLL10, and thus all protein glycylation may be dependent on TTLL3. We can not exclude, however, the possibility that a protein distinct from tubulin undergoes glycylation mediated by TTLL3 and contributes to ciliogenesis in Tetrahymena and zebrafish.
Overproduction of an ATPase-deficient form of Ttll3Ap in Tetrahymena led to shortening of the axoneme and structural defects, including lack of assembly of central microtubules. These effects can be explained either by competitive inhibition of TTLL3 and non-TTLL3 G-ligases that act on tubulin, or by disturbance of another function of CTT, such as glutamylation. Consistent with the latter hypothesis, strong overexpression of a ciliary E-ligase, Ttll6Ap resulted in a phenotype that strongly resembles that of cells overexpressing DN-Ttll3Ap. Interestingly, the expression of GFPDN-Ttll3Ap inhibited the assembly of central microtubules, while the enzyme itself and tubulin glycylation appear restricted to outer microtubules. This observation indicates that the assembly of central microtubules is dependent on the properties of outer doublets.
This example shows that TTLL3, most likely by modifying tubulin, is of critical importance for assembly of cilia. It remains to be determined how tubulin glycylation affects ciliary microtubules at the molecular level. This PTM is present primarily on the outer axonemal microtubules, and therefore its absence could inhibit IFT (Kozminski et al., 1995. J. Cell Biol. 131, 1517-1527). A reduction in the rate of IFT motors could explain the shortening of cilia caused by deficiencies in TTLL3 in both Tetrahymena and zebrafish. Indeed, a mutation of a subset of polymodification sites on the CTT of β-tubulin in Tetrahymena led to accumulation of electron-dense materials near outer microtubules in the cilia (Redeker et al., 2005. J. Biol. Chem. 280, 596-606). However, the sperm axoneme in Drosophila is strongly glycylated (Bressac et al., 1995. Eur. J. Cell Biol. 67, 346-355), but assembles without IFT (Han et al., 2003. Curr. Biol. 13,1679-1686). Thus, the evolutionary conservation of tubulin glycylation is probably based on a function distinct from IFT.
TTLL3-null cells (and specifically axonemes) showed increased resistance to paclitaxel, a microtubule-stabilizing agent. While paclitaxel binds inside the microtubule lumen (Nogales et al., 1998. Nature 391, 199-202), there are interactions between the microtubule lumen and its surface (Raff et al., 2008. Curr. Biol. 18, 911-914). Thus glycylation of CTTs could indirectly affect the paclitaxel binding site in the lumen. However, TTLL3-deficient axonemes are excessively short, suggesting that glycylation affects turnover rate or stability of axonemal microtubules. In mammalian cells, paclitaxel resistance is associated with increased dynamics of cytoplasmic microtubules (Barlow et al., 2002. J. Cell Sci. 115, 3469-3478). The axonemal microtubules exchange subunits even when fully assembled (Wloga and Gaertig 2008. Ciliary tubulin and its post-translational modifications. In Ciliary Function in Mammalian Development, Yoder (ed.), San Diego: Academic Press, pp. 83-109). Unexpectedly, axonemes deficient in glycylation show a moderate increase in the K40 acetylation, a marker of long-lived microtubules (LeDizet and Piperno 1991. Meth. Enzymol. 196, 264-274), arguing that lack of glycylation increases, rather than decreases, the residence time for tubulin subunits. To reconcile these data, we can consider that lack of tubulin glycylation makes axonemal microtubules less dynamic and, at the same time, limits the access of paclitaxel to its luminal binding sites. Paclitaxel diffuses across the microtubule walls via small fenestrations (Nogales et al., 1998. Nature 391, 199-202; Ross and Fygenson 2003. Biophys. J. 84, 3959-3967). The lack of tubulin glycylation on CTTs could reduce the size of these fenestrations. As the axonemal microtubules are coated with protein complexes and their plus ends are capped (Dentler, 1980. J. Cell Sci. 42, 207-220), diffusion across the microtubule wall could be rate limiting for buildup of paclitaxel concentration inside the lumen. A tighter microtubule lattice could also reduce the level of axoneme dynamics, and promote accumulation of K40 acetylation. A lack of proper turnover of axonemal microtubules could, in turn, lead to shorter size of cilia. While counterintuitive, there is already evidence that katanin, a factor that promotes turnover of microtubules by severing, is required for assembly of axonemes, and interacts with posttranslationally modified tubulin (Dymeket al., 2004. Eukaryot. Cell 3,870-879; Sharma et al., 2007. J. Cell Biol. 178, 1065-1079).
Further evidence that the status of polymodifications affects the axonemal lattice organization is provided by the results of overexpression of GFP-DN-Ttll3Ap. In these cells, distal segments of pre-existing cilia that elongated using hypoglycylated tubulin were apparently unstable. Since the pre-existing cilia were initially motile, their movement could lead to breakage of the newly assembled distal hypoglycylated/hyperglutamylated segments. Thus, axonemes with an abnormal composition of polymodifications could be more fragile, possibly due to reduced flexibility. The mechanical properties of the lattice could be important to both cilia with intrinsic motility and primary cilia that bend passively in response to flow of fluid in the extracellular space in mammals. In support of this idea, tubulin glycylation has been detected in both motile (Redeker et al., 1994. Science 266, 1688-1691) and primary cilia (Davenport et al., 2007. Curr. Biol. 17, 1586-1594). Some protists, like Trypanosoma, are endowed with motile cilia and 9+2 axonemes and lack tubulin glycylation (Schneider et al., 1997. J. Cell Sci. 110, 431-437), but these organisms assemble a paraflagellar rod, which could affect the mechanical properties of the flagellum.
This example shows that G- and E-ligases oppose each other on tubulin. A reduction of tubulin glycylation increased the levels of tubulin glutamylation and, conversely, a reduction of tubulin glutamylation increased the levels of tubulin glycylation. Moreover, overexpression of TTLL3 reduced the levels of tubulin glutamylation (this example) and overexpression of the TTLL6 E-ligase reduced the levels of tubulin glycylation. Both polymodifications occur on the glutamic acids of the CTTs and can coexist on the same tubulin CTTs (Redeker et al., 2005. J. Biol. Chem. 280, 596-606). In ciliates, glycylation occurs on several adjacent glutamic acids on the CTT of b-tubulin (Vinh et al., 1999. Biochemistry 38, 3133-3139), but the sites of glutamylation have not been mapped. In other organisms, glutamylation sites are located at glutamic acids adjacent to or overlapping with the glycylation sites on the CTT (Popodi et al., 2005. Cell Motil. Cytoskeleton 62, 48-64). It is thus likely that G- and E-ligases compete for the same sites or influence each other by sterically blocking adjacent sites. In contrast to tubulin glutamylation, which may be present in all organisms with axonemes, a few lineages (e.g., Trypanosoma and Plasmodium) lack glycylation despite the presence of axonemes (Fennell et al., 2008. Int. J. Parasitol. 38, 527-539; Schneider et al., 1997. J. Cell Sci. 110, 431-437). It is tempting to speculate that tubulin glutamylation is directly involved in the axoneme properties, while tubulin glycylation acts as competitive inhibitor of tubulin glutamylation. The few lineages that lack tubulin glycylation could use other means to decrease the levels of tubulin glutamylation, including unidentified enzymes that trim the glutamyl side chains (Audebert et al., 1993. Mol. Biol. Cell 4, 615-626).
Experimental Procedures
Cultures. Tetrahymena thermophila cells were grown in SPP medium. To determine the growth rate, cells were diluted to 104 cells/ml and grown in 10 ml of SPP in 150 ml flasks. In some experiments, paclitaxel (LC Laboratories, Woburn, Mass.) was added to the SPP medium. WT in Kalkutta and Tupfel longfin (commonly known as “WIK” and “TL”, respectively) zebrafish were raised at 28° C. at 14 hr light/10 hr dark cycle. The embryos were cultured in egg water at 28° C.
GFP Tagging and Gene Disruptions in Tetrahymena. The coding regions (based on the Tetrahymena Gene Database) of TTLL3 paralogs were amplified from genomic DNA with primers listed in Table 9, and ligated into pMTT1-GFP (Wloga et al., 2006. Mol. Biol. Cell 17,2799-2810). A plasmid for expression of GFP-DN-Ttll3Ap was made by changing the E codon GAA 818 to a G codon GGT using PCR with overlapping primers. Plasmids were digested with Apal and SacII, and 20 mg was used to transform CU522 cells. Transformants were subjected to phenotypic assortment to increase the copy number by culturing with 20 mM paclitaxel (Gaertig et al., 1999. Nat. Biotechnol. 17, 462-465).







TABLE 9







Primers used for amplification of the predicted


coding regions of TTLL3 homologs in Tetrahymena.












Restriction
Seq.


TTLL
Primers
sites added
ID













TTLL3A
5′ ATATACGCGTCATGTTCTAATAGAATTAGCAG 3′
MluI
93



5′ ATAATGGATCCTCATTTTTATTCTCTATAGAT 3′
BamHI
94


 


TTLL3A
5′ ATATACGCGTCATGTTCTAATAGAATTAGCAG 3′
MluI
95


tail
5′ TATTGGATCCTCATTCTTTTAAGTAATTCTTGAG 3′
BamHI
96


 


TTLL3B
5′ TTTATACGCGTCATGTTTAGCATCGATATTTAAGG 3′
MluI
97



5′ TTATTCTCGAGTCATTTTTTGTAAGAAGCTAGTAC 3′
XhoI
98


 


TTLL3C
5′ ATTATACGCGTCATGAGTTCTTTAGATGAAGGITTA 3′
MluI
99



5′ TTTTTGGATCCTCATGAGAAATTAGGTTTTAGATT 3′
BamHI
100


 


TTLL3D
5′ ATATACGCGTCATGGATAAAAGTTACAATATA 3′
MluI
101



5′ ATAATGGATCCTCACAATTTTTTACTACTTTAAGT 3′
BamHI
102


 


TTLL3E
5′ ATATACGCGTCATGATATTGCCITTTGAATACTATTTT 3′
MluI
103



5′ ATTTAGGATCCAACTGATTTATCGTTGATTGAGTG 3′
BamHI
104


 


TTLL3F
5′ ATATACGCGTCATGAGTGATCGAATATATCAT 3′
MluI
105



5′ ATAATGGATCCTCATATTTTTTAATTTTCATTTTTC 3′
BamHI
106









For gene disruptions, two fragments of a TTLL3 gene were amplified (Table 10) and cloned into a plasmid with a drug resistance cassette. The fragments were designed to flank the region encoding the catalytic domain. CU428 and B2086 mating cells were biolistically transformed (Cassidy-Hanley et al., 1997. Genetics 146, 135-147). The multiple knockout cells were obtained by crosses (Sharma et al., 2007. J. Cell Biol. 178, 1065-1079). Because multiple genes were disrupted using the same selectable marker, heterokaryons with a desired combination of alleles were identified by backcrosses and screening of progeny using PCR with primers that amplify specific disrupted loci.







TABLE 10







Primers and Resistance Cassettes Used to Prepare


Plasmids for Gene Knockouts in Tetrahymena.












Disruption
Seq.


Gene
Primers
cassette
ID





TTLL3A
F1-5′ AAATAGGGCCCCTTAAACCAGCAGCAACAGAC 3′
Neo3
107



R1-5′ AAATACCCGGGCAAGACTCTCTGGATTAGGAT 3 ′

108



F2-5′ AAATTATCGATTGATGTTTATCACAGGCTAAG 3′

109



R2-5′ AAATTGAGCTCTGCTTTAGTTATGTTTAGAAC 3′

110


 


TTLL3B
F1-5′ AAATAGGGCCCATCATAGTAGAGTAACAGCCT 3′
MTT1-RPL29
111



R1-5′ AAATAATCGATCTGGTGAATGTTTGGTTGTTG 3′

112



F2-5′ AAAATCCCGGGTACCTTGTGTAAGTCCAGAAG 3′

113



R2-5′ ATATTCCGCGGTCATTTTTTGTAAGAAGCTAGTAC 3′

114


 


TTLL3C
F-5′ TAAACCGCGGGATAGCCTGTCTTCTCCTCCA 3′
Neo3
115



R-5′ AATAGGGCCCGATACTTTAATATCAAATACTTGC 3′

116


 


TTLL3D
F1-5′ AAATGGGCCCTAGATGACAGTATTGTTGATGC 3′
Neo3
117



R1-5′ AATTCCCGGGTTAGGTCTGCATTGTCAAACC 3′

118



F2-5′ TTAAATCGATAGTTGAGATAATTGATCATGC 3′

119



R2-5′ TTAAGAGCTCCTCATAAATAATCTACCACAC 3′

120


 


TTLL3E
F1-5′ ATTTGGGCCCTTAGGGAGTTCTGCTGCTAAGAGT 3′
MTT1-RPL29
121



R1-5′ AATTAATCGATACTTGTAGTCATCATTTGAATC 3′

122



F2-5′ ATTAACCCGGGCCATCTGCTAATTCAAGAGGA 3′

123



R2-5′ ATTAAGAGCTCACTGATTTATCGTTGATTGAGTG 3′

124


 


TTLL3F
F1-5′ AAAAAGGGCCCAACAACCATCCATATGACAGC 3′
Neo3
125



R1-5′ AAATTCCCGGGCCATCCTAGCTCAATTAGAAC 3′

126



F2-5′ TATAATCGATAAGGCTTGCAGATAATGAGTG 3′

127



R2-5′ TATAGAGCTCGGGAATTTGAACTTAACACAG 3′

128









Immunofluorescence and Electron Microscopy of Tetrahymena. Immuno-fluorescence was done as previously described (Wloga et al., 2006. Mol. Biol. Cell 17, 2799-2810) and quantified using mixed mutant and WT cells (fed with India ink) imaged side by side. The average pixel intensity was determined for sections of axonemes from the base to below the tip using ImageJ. TEM was performed as previously described (Jerka-Dziadosz et al., 2001. Protist 152, 53-67). For immunoelectron TEM, cells were fixed as for immunofluorescence, reacted with anti-GFP antibodies (1:1000; Abcam) in 3% bovine serum albumin (BSA) in the PHEM buffer with 0.1% Tween-20 for 2 hr, followed by anti-rabbit IgG-10 nm gold antibodies (GE Healthcare) at 1:60 in 3% BSA in PBS for 1.5 hr. Cells were postfixed in 2.5% glutaraldehyde in PBS for 1 hr, followed by 1% osmium tetroxide in PBS for 1 hr. After washing, cells were dehydrated in an ethanol series, embedded in Epon, sectioned, and analyzed in a JEM 1210 electron microscope.
Western Blots. Total protein from 4 3 103 cells, or 4 mg of cilia, were used per lane and blots were prepared as previously described (Wloga et al. 2008. Eukaryot. Cell 7, 1362-1372) with the following mAbs: 12G10 anti-a-tubulin (1:20,000); 6-11 B-1 anti-acetylated α-tubulin (1:10,000) (LeDizet and Piperno 1991. Meth. Enzymol. 196, 264-274); TAP952 anti-monoglycylated tubulin (1:5000) (Bré et al., 1998. Mol. Biol. Cell 9, 2655-2665; Callen et al., 1994. Biol. Cell 81, 95-119); AXO49 anti-polyglycylated tubulin (1:10,000) (Bré et al., 1998. Mol. Biol. Cell 9, 2655-2665); and polyclonal antibodies polyE (1:1000) (Shang et al., 2002. J. Cell Biol. 158, 1195-1206), polyG (1:5000) (Duan and Gorovsky, 2002. Cum Biol. 12, 313-316), and anti-GFP (1:6,000) (Torrey Pines Biolabs).
In Vitro G-Ligase Assay. Tetrahymena strains expressing a TTLL3 homolog were grown to 2×105 cells/ml, induced with 2.5 mg/ml of CdCl2 for 4 hr. The cells were lysed in the enzyme reaction buffer, ERB (50 mM Tris HCl [pH 8.0], 10% glycerol, 5 mM MgCl2, 1 mM EGTA) with 0.2% NP-40 (2 ml/g of cell pellet), centrifuged at 100,000 3 g, and the supernatant was assayed. The assay was performed in 50 ml of ERB with 2 mM ATP, 250 pmol [3H]-glycine (5 ml of 20.0 Ci/mmol; GE Healthcare), and 10 ml of cell extract. Taxotere (20 mg)-stabilized brain microtubules were added and incubated at 30° C. for 4 hr. The reaction was stopped by the addition of the SDS sample buffer and boiling for 5 min at 95° C. The samples were subjected to 10% SDS-PAGE and electrotransferred onto nitrocellulose. The tubulin bands were visualized by Ponceau S, excised, and subjected to liquid scintillation counting. For fluorography, SDS-PAGE gels were stained with Coomassie blue, incubated in Amplify (GE Healthcare), dried, and subjected to autoradiography.
MO Knockdowns in Zebrafish Embryos. TTLL3-ATG MO (5′ GTGTTGGTGCATGTTTGAGTTAACC 3′; SEQ ID NO:129) and TTLL3-SP MO (5′ GATGTACAGAGCTGAGATTGAAAAG 3; SEQ ID NO:130) were obtained from Open Biosystems (Huntsville, AL). TTLL3-ATG targets the translation initiation site of ttll3 mRNA. TTLL3-SP is predicted to prevent splicing of exon 11 and cause a frame shift in exon 12. This would eliminate the coding sequence downstream of exon 10, which includes a conserved catalytic domain-encoding region. To evaluate the effect of TTLL3-SP on ttll3 mRNA, total RNA was isolated with TRIzol reagent (Invitrogen, Inc., Carlsbad, Calif.) from embryos at 7 hpf: 50 uninjected control embryos, 40 embryos injected with 3 ng TTLL3-SP MO, or 30 embryos injected with 5 ng TTLL3-SP MO. Total RNA (1 mg) was used as a template for cDNA synthesis with random hexamers and oligo-dT primers and the iScript TM cDNA synthesis kit (Bio-Rad Laboratories, Hercules, Calif.). cDNA (2 ml) was amplified with the following primers spanning exon 11: TTLL3-splice F, 5′-CTTACTCCACTCATACGCTG-3′ (exon 10; SEQ ID NO:131); TTLL3-splice R, 5′-GTCGAATGTTGATCACCTCC-3′ (exon 12; SEQ ID NO:132). As expected, primers flanking exon 11 produced a shorter product in RT-PCR from embryos injected with TTLL3-SP than in controls (FIG. 50D). There was a dramatic reduction in the levels of endogenous TTLL3 mRNA in embryos injected with TTLL3-SP-MO, suggesting that this MO affects mRNA stability. As a negative control, we injected 25 base MOs with a random base mixture at each position (Gene Tools, Inc., Philomath, Oreg.).
Microscopy of Zebrafish. For whole-mount, in situ hybridization, embryos were fixed as previously described (Hagos and Dougan, 2007), stored in 100% methanol at −20° C., and stained with spaw, lefty1, lefty2, and cmlc2 probes (Yelon, 2001). Embryos were photographed under differential interference contrast with a Zeiss Axioplan 2 microscope with Slidebook (Intelligent Imaging Innovations, Denver, Colo.). For immunofluorescence, the embryos were manually dechorionated and fixed with 0.5% Triton X-100 and 4% paraformaldehyde in PBT (0.1% Tween-20 in PBS) at 4° C. overnight. After washing with PBT, embryos were dehydrated in methanol/PBT series and incubated overnight in 100% methanol at −20° C. After rehydration, embryos were blocked for 1-2 hr in 2% BSA, 0.5% normal goat serum,l%DMSO, 0.5% Triton X-100 in PBS, and incubated overnight at 4° C. with one of the following antibodies: 6-11 B-1 (1:1000); TAP952 (1:2000); AXO49 (1:2000); and GT335 (1:1000); polyE (1:500). After washing 3×10 min with 0.5% Triton X-100 in PBS, embryos were incubated overnight at 4° C. in anti-mouse-IgG-FITC or anti-rabbit-IgG-Cy3 (1:500; Zymed), washed (3×10 min), mounted in 100 mg/ml of DABCO (Sigma-Aldrich) in PBS, and examined on a Leica TCS SP confocal microscope.
For electron microscopy, the dechorionated embryos were fixed at 4° C. overnight in 2.5% glutaraldehyde in 0.1Mcacodylate buffer (pH 7.2), postfixed for 1 hr on ice in 1% osmium tetroxide in 0.1 M cacodylate buffer, and dehydrated through ethanol series. For SEM, embryos were dried at a critical point using CO2, sputter coated with gold, and observed in an SEM Leo982 microscope. For TEM, fixed embryos were dehydrated in 100% acetone, incubated in a mixture of acetone and Epon 812 (3:1, 1:1, and 1:3, 2 hr each) followed by overnight infiltration in 100% Epon 812. After transfer to fresh Epon 812, blocks were polymerized at 60° C.
The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for example, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

## Claims

1. A genetically engineered Tetrahymena cell comprising a deletion of at least two paralogs of TTLL6 or TTLL3; wherein said cell expresses tubulin exhibiting reduced polymodification compared to a wild-type Tetrahymena cell.

2. The Tetrahymena cell of claim 1 wherein the tubulin comprises shortened glutamyl side chains compared to wild-type Tetrahymena tubulin.

3. The Tetrahymena cell of claim 2 wherein at least a portion of the shortened glutamyl side chains contain less than 6 glutamic acid residues, and wherein the density of the glutamyl side chains containing less than 6 glutamic acid residues is increased relative to wild-type Tetrahymena tubulin.

4. The Tetrahymena cell of claim 2 wherein at least a portion of the shortened glutamyl side chains contain only one glutamic acid residue, and wherein the density of the glutamyl side chains containing only one glutamic acid residue is increased relative to wild-type Tetrahymena tubulin.

5. The Tetrahymena cell of claim 1 wherein the tubulin lacks glycyl side chains that are present in wild-type tubulin.

6. The Tetrahymena cell of claim 5 wherein the tubulin glycylation level is less than 90% of the tubulin glycylation level in wild-type Tetrahymena tubulin.

7. The Tetrahymena cell of claim 1 which is a TTLL6A/TTLL6F knockout cell.

8. The Tetrahymena cell of claim 1 wherein the two paralogs are selected from the group consisting of TTLL6A, TTLL6B, TTLL6C, TTLL6D, TTLL6E, and TTLL6F.

9. The Tetrahymena cell of claim 1 wherein the two paralogs are selected from the group consisting of TTLL3A, TTLL3B, TTLL3C, TTLL3D, TTLL3E, and TTLL3F.

10. The Tetrahymena cell of claim 1 which is selected from the group consisting of a TTLL6A/TTLL6F knockout cell, a TTLL6A/TTLL6B/TTLL6D/TTLL6F knockout cell, and a TTLL6A/TTLL6B/TTLL6D/TTLL6E/TTLL6F knock out cell.

11. The Tetrahymena cell of claim 1 which is selected from the group consisting of a TTLL3A/TTLL3B knockout cell, a TTLL3A/TTLL3B/TTLL3C/TTLL3D knockout cell, and a TTLL3A/TTLL3B/TTLL3C/TTLL3D/TTLL3E/TTLL3F knockout cell.

12. A method for identifying an inhibitor of a glutamic acid ligase, the method comprising:
contacting a cellular component isolated from the Tetrahymena cell of claim 1 with a candidate inhibitor compound and a glutamic acid ligase; and
detecting a change in glutamylation of the cellular component, wherein the absence of additional glutamylation is indicative of a compound that inhibits glutamic acid ligase.

13. The method of claim 12 wherein the cellular component is selected from the group consisting of an axoneme, a microtubule, a stabilized microtubule and a tubulin.

14. A method for identifying an inhibitor of a glycine ligase, the method comprising:
contacting a cellular component isolated from the Tetrahymena cell of claim 1 with a candidate inhibitor compound and a glycine ligase; and
detecting a change in glycylation of the cellular component, wherein the absence of additional glycylation is indicative of a compound that inhibits glycine ligase.

15. The method of claim 14 wherein the cellular component is selected from the group consisting of an axoneme, a microtubule, a stabilized microtubule and a tubulin.

16. A genetically engineered Tetrahymena cell comprising a disruption of at least two paralogs of TTLL6 or TTLL3; wherein the disruption is the result of homologous recombination, and wherein said cell expresses tubulin exhibiting reduced polymodification compared to a wild-type Tetrahymena cell.