Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable Axonemal Microtubules in Paramecium

Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the α- and β- tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence of regulatory mechanisms for glycylation. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of this organism. These observations establish that polyglycylation is reversible and indicate that, in vivo, an equilibrium between glycylating and deglycylating enzymes might be responsible for the length of the oligoglycine side chains of tubulin.     

Tubulin polyglycylation: a morphogenetic marker in ciliates

The occurrence of the tubulin post-translational modification, polyglycylation, in stable microtubular structures was investigated during morphogenesis in two ciliates, Paramecium and Frontonia atra, belonging to the Epiplasmata group. This analysis was carried out by means of immunofluorescence and post-embedding immunoelectron microscopy using two monoclonal antibodies, TAP 952 and AXO 49, respectively recognizing mono- and polyglycylated sites in alpha- and beta-tubulin. In the course of cell division, the TAP 952 epitope is detected in all microtubular structures including the newly assembled ones, such as cortical and oral basal bodies and cilia. In contrast, the AXO 49 epitope is only present in 'old' microtubular structures such as parental cortical and oral basal bodies and cilia. Our observations show that, in ciliates: 1) this tubulin post-translational modification takes place early in the course of morphogenesis; and 2) the lengthening of the polyglycine chains occurs after a great delay following addition of the first glycine residues on the tubulin glycylation sites, and following microtubule assembly. Thus, a sequential mechanism of polyglycylation is shown to take place in the tubulin molecule and during morphogenesis in Paramecium and Frontonia atra. Accordingly, polyglycylation, through a time-dependent polyglycine chain elongation process, appears to be a morphogenetic marker in ciliates.     

Where and when is microtubule diversity generated inParamecium? Immunological properties of microtubular networks in the interphase and dividing cells

Summary Ciliates are highly differentiated cells which display extensive deployment of microtubular systems. Because genetic diversity of tubulin is extremely reduced in these cells, microtubule diversity is mostly generated at the post-translational level either through direct modification of tubulin or through the binding of associated proteins to microtubules. We have undertaken a systematic exploration of microtubule diversity in ciliates by way of production of monoclonal antibodies. Previously we reported the biochemical characterization of these antibodies. In addition to antibodies directed against primary sequences, we obtained antibodies directed against post-translational modifications. In this paper, we report a detailed analysis of the distribution of the various epitopes on the microtubular networks ofParamecium, both in interphase cells and during division morphogenesis. Each of these antibodies decorates a subset of microtubules. Acetylation, recognized by antibodies TEU 318 and TEU 348, is detected on stable microtubules early after microtubule assembly. Epitopes recognized by two other antibodies (TAP 952 and AXO 58) are found on a subset of stable microtubules; in addition, the TAP 952 antibody is also found on labile microtubules; both epitopes are detected as soon as microtubule assembly occurs. In contrast, the epitope of the antibody, AXO 49, is associated with only a restricted subset of stable microtubules in the interphase cell, and is detected a lag-time after microtubule assembly during division morphogenesis. These data show that microtubule diversity is generated through a time-dependent sequence and according to a definite spatial pattern.     

Structural characterization by tandem mass spectrometry of the posttranslational polyglycylation of tubulin.

Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.     

Isolation and characterization of libraries of monoclonal antibodies directed against various forms of tubulin in Paramecium

Summary— Ciliates are very good models for studying post-translationally generated tubulin heterogeneity because they exhibit highly differentiated microtubular networks in combination with reduced genetic diversity. We have approached the analysis of tubulin heterogeneity in Paramecium through extensive isolation and characterization of monoclonal antibodies using various antigens and several immunization protocols. Eight monoclonal antibodies and 10 hybridoma supernatants were characterized by: i) immunoblotting on ciliate and pig brain tubulins as well as on peptide maps of Paramecium axonemal tubulin; ii) immunoblotting on ciliate tubulin fusion peptides generated in E coli, a procedure which allows in principle to discriminate antibodies that are directed against tubulin sequence (reactive on fusion peptides) from those directed against a post-translational epitope (non-reactive); and iii) immunofluorescence on Paramecium, 3T3 and PtK2 cells. Twelve antibodies labeled all microtubules in Paramecium cells and were found to be directed against tubulin primary sequences (nine of them being located in the α N-terminal domain, one in the β C-terminal one, and two in α and β central stretches). The remaining ones decorated only a specific subset of microtubules within the cell and were presumably directed against post-translational modifications. Among these, three antibodies are directed against an N-terminal acetylated epitope of α-tubulin whereas the epitopes of three other ones (TAP 952°, AXO 58 and AXO 49°) apparently correspond to still unidentified post-translational modifications, located in the C-terminal domain of both α- and β-tubulins. The AXO 49° specificity is similar to that of a previously described polyclonal serum raised against Paramecium axonemal tubulin [2]. The results are discussed in terms of identification and accessibility of the epitopes and immunogenicity of ciliate tubulin with reference to mammalian and ciliate tubulin sequences.     

Mammalian cilia function is independent of the polymeric state of tubulin glycylation

Polyglycylation is a polymeric post-translational modification of tubulin that is ubiquitous and widely present in cilia and flagella. It consists of the addition of highly variable numbers of glycyl residues as side chains onto the gamma carboxyl group of specific glutamyl residues at the C-termini of alpha- and beta-tubulin. The function of polyglycylation is poorly understood, however, studies in Tetrahymena have shown that the mutation of polyglycylation sites in beta-tubulin resulted in axonemal abnormality or lethality. This suggests that polyglycylation is functionally essential in protists. We hypothesize that polyglycylation is also essential in mammalian cilia and that the extent of polyglycylation has functional significance. In this study, we examined polyglycylation states in ciliated tissues and in mouse tracheal epithelial cell cultures. We utilized two antibodies, TAP 952 and AXO 49, which recognize glutamyl sites possessing monomeric glycylation sites and glutamyl sites possessing polymeric glycylation sites, respectively. Monomeric glycylation sites were observed in cilia of all the ciliated tissues examined but were invariably excluded from the distal tips. In contrast, polymeric glycylation sites were rare, but when observed, they were localized at the bases of cilia. During ciliogenesis, in epithelial cell cultures, monomeric glycylation sites were observed, but the extent of polymeric glycylation sites were variable and were only observed during the early stages of the cultures. Our observations suggest that while monomeric glycylation sites are universal and likely essential in mammalian cilia, polymeric glycylation sites are not required for ciliary beating. Rather, our observations suggest that the number of added glycyl residues increases progressively from the tips of cilia toward their bases.     

Tubulin diversity in trophozoites of<Emphasis Type="Italic"> Giardia lamblia</Emphasis>

Giardia lamblia is a diplomonad that parasitizes the small intestine of vertebrates. The trophozoite is multiflagellar and its cytoskeleton presents a complex organization of microtubular structures. One of these, the adhesive disk, consists of a microtubular spiral. The median body, whose function is not yet determined, is also composed by microtubules. The cell has eight flagella and two microtubule sheets, known as the funis. In this study we used several antibodies and immunofluorescence microscopy to help in the characterization of these structures. We observed that Giardia tubulin reacts with antibodies raised against very distinct immunogens. The antibodies used were against: (1) alpha-tubulin from chicken embryo brain, Trypanosoma brucei, sea urchin sperm, Paramecium, acetylated alpha-tubulin from Paramecium, and tyrosinated alpha-tubulin, (2) beta-tubulin from chicken embryo brain and Physarum polycephalum, and (3) an antibody with specificity to beta-tubulin having as immunogen the FtsZ bacterial protein. Each cytoskeletal structure of Giardia presented a distinct pattern of labeling by the several antibodies used. These data indicate that even being considered one of the most ancient of organisms, Giardia shares similarities (at least in relation to tubulin) with other organisms. They also open some questions about the organization and composition of its microtubular structures.     

Accessory tubules and axonemal microtubules of Apis mellifera sperm flagellum differ in their tubulin isoform content

In the insect sperm flagellum, an extra set of nine additional microtubules, named accessory tubules, is present surrounding the axoneme. Using a sarcosyl/urea extraction, we were able to fractionate the microtubular cytoskeleton of the sperm flagellum of the insect Apis mellifera resulting in the dissociation of the axonemal microtubule protein components and the accessory tubules. This has allowed us to compare the tubulin isoform content of axonemal microtubules and accessory tubules by immunoelectron microscopy and immunoblotting using a panel of monoclonal antibodies directed against different tubulin post-translational modifications (PTMs). All the PTMs occurring in axonemal tubulin are also present in accessory tubules, which indicates the close relativeness of accessory tubules to axonemal rather than to cytoplasmic microtubules. However, our results demonstrate the presence of significant differences in the tubulin isoform content of axonemal microtubules and accessory tubules. First, the tubulin tyrosination extent of accessory tubules is far lower than that of axonemal microtubules, thus confirming at the molecular level their morphogenetic origin as outgrowths from the B-subtubule of each microtubular doublet. Second, although polyglycylation seems to occurr at the same extent in both microtubular systems, alpha-tubulin exhibits a larger amount of monoglycylated sites in axonemal microtubules than in accessory tubules. Third, a greater amount of beta-tubulin molecules is glutamylated in axonemal microtubules than in accessory tubules. Moreover, highly acidic isoforms, likely molecules with longer polyglutamate side chains, are present only in axonemal microtubules. Taken together, our data are indicative of a higher level of tubulin heterogeneity in axonemal microtubules than in accessory tubules. They also show a segregation of post-translationally modified isoforms between accessory tubules and axonemal microtubules and suggest the implication of PTMs in the functional specialization of the two microtubular systems.     

Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.     

Molecular mechanisms of microtubular organelle assembly in Tetrahymena

Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.     

Age-related changes in microtubules in the guinea pig organ of Corti

Biochemical and immunocytochemical analyses have been used to provide new insights into age-related changes in the sensory and supporting cells of the guinea pig organ of Corti. Quantitative densitometry of immunoblots showed that, while levels of alpha-tubulin remained relatively constant in guinea pigs from 3 weeks to 18 months old, there were progressive shifts in some tubulin isoforms. Levels of tyrosinated tubulin increased with age, nontyrosinatable tubulin (delta2-tubulin) showed a compensatory decrease, but detyrosinated tubulin did not change; acetylated, polyglutamylated, and glycylated tubulin levels also decreased. Immunolabeled tissue sections showed that cell type-specific distribution of tubulin seen in young guinea pigs (tyrosinated in the microtubules of the sensory cells, and post-translationally modified isoforms in the supporting cells) did not change as animals aged. However, there were age-related decreases in labeling for alpha-tubulin and all post-translationally modified isoforms. Biochemical and immunocytochemical results both support an age-related decrease in the number and/or length of microtubules as well as an increase in the pool of soluble tyrosinated and detyrosinated tubulin. They further suggest that microtubules containing nontyrosinatable tubulin from older animals are the sites for further modification of tubulin by acetylation, polyglutamylation, and glycylation. Changes in tubulin isoform levels and stability of microtubules in the organ of Corti may alter its micromechanical properties; the resulting changes in conduction of sound-induced vibration would provide one mechanism for age-related hearing loss.     

Glutamylated tubulin: diversity of expression and distribution of isoforms

Glutamylation of alpha and beta tubulin isotypes is a major posttranslational modification giving rise to diversified isoforms occurring mainly in neurotubules, centrioles, and axonemes. Monoglutamylated tubulin isoforms can be differentially recognized by two mAbs, B3 and GT335, which both recognize either polyglutamylated isoforms. In the present study, immunoelectron microscopy and immunofluorescence analyses were performed with these two mAbs to determine the expression and distribution of glutamylated tubulin isoforms in selected biological models whose tubulin isotypes are characterized. In mouse spermatozoa, microtubules of the flagellum contain polyglutamylated isoforms except in the tip where only monoglutamylated isoforms are detected. In spermatids, only a subset of manchette microtubules contain monoglutamylated tubulin isoforms. Cytoplasmic microtubules of Sertoli cells are monoglutamylated. Mitotic and meiotic spindles of germ cells are monoglutamylated whereas the HeLa cell mitotic spindle is polyglutamylated. Three models of axonemes are demonstrated as a function of the degree and extent of tubulin glutamylation. In lung ciliated cells, axonemes are uniformly polyglutamylated. In sea urchin sperm and Chlamydomonas, flagellar microtubules are polyglutamylated in their proximal part and monoglutamylated in their distal part. In Paramecium, cilia are bi- or monoglutamylated only at their base. In all cells, centrioles or basal bodies are polyglutamylated. These new data emphasize the importance of glutamylation in all types of microtubules and strengthen the hypothesis of its role in the regulation of the intracellular traffic and flagellar motility.     

Composition and organization of tubulin isoforms reveals a variety of axonemal models

In the flagellum of mammalian spermatozoa, glutamylated and glycylated tubulin isoforms are detected according to longitudinal gradients and preferentially in axonemal doublets 1-5-6 and 3-8, respectively. This suggested a role for these tubulin isoforms in the regulation of flagellar beating. In the present work, using antibodies directed against various tubulin isoforms and quantitative immunogold analysis, we aimed at investigating whether the particular accessibility of tubulin isoforms in the mammalian sperm flagellum is restricted to this model of axoneme surrounded with periaxonemal structures or is also displayed in naked axonemes. In rodent lung ciliated cells, all studied tubulin isoforms are uniformly distributed in all axonemal microtubules with a unique deficiency of glutamylated tubulin in the transitional region. A similar distribution of tubulin isoforms is observed in cilia of Paramecium, except for a decreasing gradient of glutamylated tubulin labeling in the proximal part of axonemal microtubules. In the sea urchin sperm flagellum, predominant labeling of tyrosinated and detyrosinated tubulin in 1-5-6 and 3-8 doublets, respectively, were observed together with decreasing proximo-distal gradients of glutamylated and polyglycylated tubulin labeling and an increasing gradient of monoglycylated tubulin labeling. In flagella of Chlamydomonas, the glutamylated and glycylated tubulin isoforms are detected at low levels. Our results show a specific composition and organization of tubulin isoforms in different models of cilia and flagella, suggesting various models of functional organization and beating regulation of the axoneme.     

Polyglycylation domain of beta-tubulin maintains axonemal architecture and affects cytokinesis in Tetrahymena

Polyglycylation occurs through the post-translational addition of a polyglycine peptide to the gamma-carboxyl group of glutamic acids near the C terminus of alpha- and beta-tubulin, and has been found only in cells with axonemes, from protists to humans. In Tetrahymena thermophila, multiple sites of polyglycylation on alpha-tubulin are dispensable. By contrast, mutating similar sites on beta-tubulin has site-specific effects, affecting cell motility and cytokinesis, or resulting in cell death. Here, we address the lethality of a polyglycylation deficiency in T. thermophila using heterokaryons. Cells with a lethal mutation in the polyglycylation domain of beta-tubulin assembled axonemes that lack the central pair, B-subfibres and the transitional zone of outer microtubules (MTs). Furthermore, an arrest in cytokinesis occurred, and was associated with incomplete severing of cortical MTs positioned near the cleavage furrow. Thus, tubulin polyglycylation is required for the maintenance of some stable microtubular organelles that are all known to be polyglycylated in vivo, but its effects on MTs appear to be organelle-specific.     

Visualization of the funis of Giardia lamblia by high-resolution field emission scanning electron microscopy--new insights

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a cytoskeleton made of several microtubular structures-an adhesive disc, four pairs of flagella, median body, and funis. This protozoan displays different types of movements, including a lateral and dorso-ventral dislocation of its posterior region, which has not been completely elucidated. In the present study, high-resolution field emission scanning electron microscopy was used to analyze the funis structure of G. lamblia trophozoites. It was shown that the funis is made of short arrays of microtubules emanating from the axonemes of the caudal flagella, which are anchored to dense rods that run parallel to the posterior-lateral flagella. After emergence of the posterior-lateral flagella, funis microtubules are anchored to the epiplasm, a fibrous layer that underlies the portion of membrane that presents tail contractility. Based on these observations a model for the tail flexion of G. lamblia is proposed.     

Giardia lamblia: evaluation of the in vitro effects of nocodazole and colchicine on trophozoites

Giardia lamblia is the most commonly detected parasite in the intestinal tract of humans and other mammals causing giardiasis. Giardia presents several cytoskeletal structures with microtubules as major components such as the ventral adhesive disk, eight flagella axonemes, the median body and funis. Many drugs have already been tested as antigiardial agents, such as albendazole and mebendazole, which act by specifically inhibiting tubulin polymerization and hence microtubule assembly. In the present work, we used the microtubule inhibitors nocodazole and colchicine in order to investigate their direct and indirect effects on Giardia ultrastructure and attachment to the glass surface, respectively. Axenically grown G. lamblia trophozoites were treated with nocodazole or colchicine for different time intervals and analyzed by light and electron microscopy. It was observed that trophozoites became completely misshapen, detached from the glass surface and failed to complete cell division. The main alterations observed included disc fragmentation, presence of large vacuoles, and appearance of electrondense deposits made of tubulin. The cytokinesis was blocked, but not the karyokinesis, and membrane blebs were observed. These findings show that Giardia behavior and cytoskeleton are clearly affected by the commonly used microtubule targetting agents colchicine and nozodazole.     

The microtubular system and posttranslationally modified tubulin during spermatogenesis in a parasitic nematode with amoeboid and aflagellate spermatozoa

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis. Mol. Reprod. Dev. 49:150–167, 1998. © 1998 Wiley-Liss, Inc.     

Centrin in Giardia lamblia - ultrastructural localization

Giardia lamblia is a multiflagellar parasite and one of the earliest diverging eukaryotic cells. It possesses a complex cytoskeleton based on different groups of microtubular structures - a ventral adhesive disc, four pairs of flagella, a median body and funis. Centrin is an important member of the EF-hand family of calcium-binding proteins, and it is known to show calcium-sensitive contractile behaviour. In the present study, we performed an ultrastructural localization of centrin in G. lamblia using several monoclonal antibodies to centrin. Microtubular structures such as the basal bodies, all the flagella axonemes, the adhesive disc, funis, and the median bodies presented positive labelling to centrin. In addition, the dense rods also demonstrated positive labelling. These results show that centrin is located in key positions related to microtubules. The role of centrin in these dynamic regions is discussed.     

Cytoskeleton: functions for tubulin modifications at last

New studies show that three types of evolutionarily conserved post translational tubulin modification, polyglutamylation, polyglycylation and detyrosination, play important roles in vivo. These modifications appear to act by modulating the binding of molecular motors to the external surface of microtubules.