Tubulin Carboxypeptidase/Microtubules Association can be Detected in the Distal Region of Neural Processes

The association of tubulin carboxypeptidase with microtubules has been demonstrated in crude brain extracts and in living non-nervous cells. Here, we studied this phenomenon in cultured brain cells. To determine the association of the enzyme with neural microtubules we isolated the cytoskeletons (detergent-extraction under microtubule-stabilizing conditions) and measured the content of Tyr, Glu, and 2 tubulin as a function of the in vitro incubation time of the cytoskeletons. The carboxypeptidase was found associated with microtubules in 2 days-cultured cells but not in 7 days-cultured cells. Quantitative analysis of digitized images after immunofluorescent staining revealed that detyrosination during the incubation of the cytoskeletons occurred preferentially in the distal regions of the neural processes. Prolonged taxol-treatment of the cells promoted higher detyrosination but Tyr tubulin was not depleted suggesting the existence of a subset of microtubules that has not associated carboxypeptidase and therefore cannot be detyrosinated even after prolonged taxol-treatment. This hypothesis was supported, although not conclusively, by additional experiments.     

Postpolymerization detyrosination of alpha-tubulin: a mechanism for subcellular differentiation of microtubules

Tyrosinated (Tyr) and detyrosinated (Glu) alpha-tubulin, species interconverted by posttranslational modification, are largely segregated in separate populations of microtubules in interphase cultured cells. We sought to understand how distinct Tyr and Glu microtubules are generated in vivo, by examining time-dependent alterations in Tyr and Glu tubulin levels (by immunoblots probed with antibodies specific for each species) and distributions (by immunofluorescence) after microtubule regrowth and stabilization. When microtubules were allowed to regrow after complete depolymerization by microtubule antagonists, Glu microtubules reappeared with a delay of approximately 25 min after the complete array of Tyr microtubules had regrown. In these experiments, Tyr tubulin immunofluorescence first appeared as an aster of distinct microtubules, while Glu tubulin staining first appeared as a grainy pattern that was not altered by detergent extraction, suggesting that Glu microtubules were created by detyrosination of Tyr microtubules. Treatments with taxol, azide, or vinblastine, to stabilize polymeric tubulin, all resulted in time-dependent increases in polymeric Glu tubulin levels, further supporting the hypothesis of postpolymerization detyrosination. Analysis of monomer and polymer fractions during microtubule regrowth and in microtubule stabilization experiments were also consistent with postpolymerization detyrosination; in each case, Glu polymer levels increased in the absence of detectable Glu monomer. The low level of Glu monomer in untreated or nocodazole-treated cells (we estimate that Glu tubulin comprises less than 2% of the monomer pool) also suggested that Glu tubulin entering the monomer pool is efficiently retyrosinated. Taken together these results demonstrate that microtubules are polymerized from Tyr tubulin and are then rapidly converted to Glu microtubules. When Glu microtubules depolymerize, the resulting Glu monomer is retyrosinated. This cycle generates structurally, and perhaps functionally, distinct microtubules.     

Release of C-terminal tyrosine from tubulin and microtubules at steady state.

Microtubule protein preparations purified by cycles of assembly-disassembly contain the enzyme tubulinyltyrosine carboxypeptidase (TTCPase). Using these preparations, containing tubulinyl[14C]tyrosine, we studied the release of [14C]tyrosine from assembled and non-assembled tubulin under steady-state conditions. It was found that both states of aggregation were detyrosinated at similar rates by the action of the endogenous TTCPase. However, practically no release of [14C]tyrosine from the non-assembled tubulin pool was found when microtubules were previously eliminated from the incubation mixture. These results indicated that non-assembled tubulin requires to interact with microtubules to be detyrosinated. This interaction seems to occur through the incorporation of dimers into microtubules, since when the capability of tubulin to incorporate into microtubules was diminished by binding of colchicine a concomitant decrease in the rate of release of tyrosine was observed. When detyrosination was accelerated by increasing the concentration of TTCPase relative to the microtubule protein concentration, microtubules were found to be detyrosinated faster than was non-assembled tubulin. Using exogenous TTCPase in an incubation system in which the formation of microtubules was not allowed, tubulinyl[14C]tyrosine and tubulinyl[14C]tyrosine-colchicine complex were shown to have similar capabilities to act as substrates for this enzyme. Free colchicine was shown not to affect the activity of TTCPase.     

Motor-dependent microtubule disassembly driven by tubulin tyrosination

In cells, stable microtubules (MTs) are covalently modified by a carboxypeptidase, which removes the C-terminal Tyr residue of α-tubulin. The significance of this selective detyrosination of MTs is not understood. In this study, we report that tubulin detyrosination in fibroblasts inhibits MT disassembly. This inhibition is relieved by overexpression of the depolymerizing motor mitotic centromere-associated kinesin (MCAK). Conversely, suppression of MCAK expression prevents disassembly of normal tyrosinated MTs in fibroblasts. Detyrosination of MTs suppresses the activity of MCAK in vitro, apparently as the result of a decreased affinity of the adenosine diphosphate (ADP)–inorganic phosphate- and ADP-bound forms of MCAK for the MT lattice. Detyrosination also impairs MT disassembly in neurons and inhibits the activity of the neuronal depolymerizing motor KIF2A in vitro. These results indicate that MT depolymerizing motors are directly inhibited by the detyrosination of tubulin, resulting in the stabilization of cellular MTs. Detyrosination of transiently stabilized MTs may give rise to persistent subpopulations of disassembly-resistant polymers to sustain subcellular cytoskeletal differentiation.     

Stabilization of post-translational modification of microtubules during cellular morphogenesis

This review discusses the possible role of alpha-tubulin detyrosination, a reversible post-translational modification that occurs at the protein's C-terminus, in cellular morphogenesis. Higher eukaryotic cells possess a cyclic post-translational mechanism by which dynamic microtubules are differentiated from their more stable counterparts; a tubulin-specific carboxypeptidase detyrosinates tubulin protomers within microtubules, while the reverse reaction, tyrosination, is performed on the soluble protomer by a second tubulin-specific enzyme, tubulin tyrosine ligase. In general, the turnover of microtubules in undifferentiated, proliferating cells is so rapid that the microtubules accumulate very little detyrosinated tubulin; that is, they are enriched in tyrosinated tubulin. However, an early event common to at least three well-studied morphogenetic events--myogenesis, neuritogenesis, and directed cell motility--is the elaboration of a polarized array of stable microtubules that become enriched in detyrosinated tubulin. The formation of this specialized array of microtubules in specific locations in cells undergoing morphogenesis suggests that it plays an important role in generating cellular asymmetries.     

Detyrosination of tubulin is not correlated to cold-adaptation of microtubules in cultured cells from the Atlantic cod (Gadus morhua)

Summary Isolated cod brain microtubules from the cold-adapted Atlantic cod (Gadus morhua) have previously been shown to be highly detyrosinated, a post-translational modification of tubulin usually found in stable subsets of microtubules. In this study we found this was not restricted only to isolated brain microtubules. Microtubules in primary cultures of brain and skin cells were composed of both tyrosinated (Tyr)- and detyrosinated (Glu)-tubulin seen by immunocytochemistry. Immunoelectron microscopy of isolated microtubules showed that individual microtubules were composed of a mixture of Tyr- and Glu-tubulin. Leukocytes with extending lamellopodia contained only microtubules stained with the antibody against Tyr-tubulin, and isolated heart tubulin lacked both Tyr- and Glu-tubulin, suggesting that a relative high level of detyrosination is a characteristic of most, but not all, cod microtubules. Brain cell microtubules were more resistant to mitotic inhibitors than skin cell microtubules, but this was not correlated to a difference in detyrosination. Brain and skin cell microtubules were only partially disassembled when incubated at 0°C. Upon reassembly of microtubules at 12°C, microtubules were still made of mixtures of Tyr- and Glu-tubulin, indicating that detyrosination of assembled microtubules is rapid and/or that in cod cells, in contrast to mammalian cells, Glu-tubulin can reassemble to microtubules. Our data show that most cod microtubules are highly detyrosinated, but this is not the cause of their cold adaptation or drug stability.     

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.     

A rice tubulin tyrosine ligase‐like 12 protein affects the dynamic and orientation of microtubules

The detyrosination/retyrosination cycle is the most common post‐translational modification of α‐tubulin. Removal of the conserved C‐terminal tyrosine of α‐tubulin by a still elusive tubulin tyrosine carboxypeptidase, and religation of this tyrosine by a tubulin tyrosine ligase (TTL), are probably common to all eukaryotes. Interestingly, for plants, the only candidates qualifying as potential TTL homologs are the tubulin tyrosine ligase‐like 12 proteins. To get insight into the biological functions of these potential TTL homologs, we cloned the rice TTL‐like 12 protein (OsTTLL12) and generated overexpression OsTTLL12‐RFP lines in both rice and tobacco BY‐2 cells. We found, unexpectedly, that overexpression of this OsTTLL12‐RFP increased the relative abundance of detyrosinated α‐tubulin in both coleoptile and seminal root, correlated with more stable microtubules. This was independent of the respective orientation of cortical microtubule, and followed by correspondingly changing growth of coleoptiles and seminal roots. A perturbed organization of phragmoplast microtubules and disoriented cell walls were further characteristics of this phenotype. Thus, the elevated tubulin detyrosination in consequence of OsTTLL12 overexpression affects structural and dynamic features of microtubules, followed by changes in the axiality of cell plate deposition and, consequently, plant growth.     

Tyrosination State of Tubulin and the Activity of Tubulin:Tyrosine Ligase and Tubulin Carboxypeptidase in the Developing Retina of the Chick

The tyrosination state of tubulin and the enzymes involved in the tubulin tyrosination/detyrosination cycle--tubulin:tyrosine ligase and tubulin carboxypeptidase--were determined in chick retina during development. The amount of tyrosinable (tyrosinated plus detyrosinated) tubulin increased approximately 110% from embryonic day 7 to 14. Then it decreased, and by day 19 it was similar to the value on day 7. This result did not change after hatching, at least up to day 20. The proportion of tyrosinated and detyrosinated tubulin significantly changed with the development of the animal. At embryonic day 7, these tubulin species were at a proportion of 70 and 30%, respectively, and after hatching, the values inverted, to 30 and 70%, respectively. This change did not correlate with the activity of the ligase relative to that of the carboxypeptidase, as measured in vitro. This observation suggested that a change in the turnover rate of microtubules, in the proportion of assembled and nonassembled tubulin pools, or in both had occurred. Coincident with the last possibility, the proportion of assembled tubulin was found to increase during the development of the animal. This finding suggests that the tyrosination state of tubulin may be determined, at least in part, by the assembly state.     

Effect of polycations and polyanions on cell division of cultured mammalian cells

The polycations (H1 histone and polylysine) and polyanions (heparin and various RNA preparations) stimulate cell division of cultured mammalian cells. The mechanisms by which both polycations (H1 histone and polylysine) and polyanions (heparin and RNA) may increase the rate of cell division are discussed.     

Detyrosination of tubulin regulates the interaction of intermediate filaments with microtubules in vivo via a kinesin-dependent mechanism

Posttranslationally modified forms of tubulin accumulate in the subset of stabilized microtubules (MTs) in cells but are not themselves involved in generating MT stability. We showed previously that stabilized, detyrosinated (Glu) MTs function to localize vimentin intermediate filaments (IFs) in fibroblasts. To determine whether tubulin detyrosination or MT stability is the critical element in the preferential association of IFs with Glu MTs, we microinjected nonpolymerizable Glu tubulin into cells. If detyrosination is critical, then soluble Glu tubulin should be a competitive inhibitor of the IF-MT interaction. Before microinjection, Glu tubulin was rendered nonpolymerizable and nontyrosinatable by treatment with iodoacetamide (IAA). Microinjected IAA-Glu tubulin disrupted the interaction of IFs with MTs, as assayed by the collapse of IFs to a perinuclear location, and had no detectable effect on the array of Glu or tyrosinated MTs in cells. Conversely, neither IAA-tyrosinated tubulin nor untreated Glu tubulin, which assembled into MTs, caused collapse of IFs when microinjected. The epitope on Glu tubulin responsible for interfering with the Glu MT-IF interaction was mapped by microinjecting tubulin fragments of alpha-tubulin. The 14-kDa C-terminal fragment of Glu tubulin (alpha-C Glu) induced IF collapse, whereas the 36-kDa N-terminal fragment of alpha-tubulin did not alter the IF array. The epitope required more than the detyrosination site at the C terminus, because a short peptide (a 7-mer) mimicking the C terminus of Glu tubulin did not disrupt the IF distribution. We previously showed that kinesin may mediate the interaction of Glu MTs and IFs. In this study we found that kinesin binding to MTs in vitro was inhibited by the same reagents (i.e., IAA-Glu tubulin and alpha-C Glu) that disrupted the IF-Glu MT interaction in vivo. These results demonstrate for the first time that tubulin detyrosination functions as a signal for the recruitment of IFs to MTs via a mechanism that is likely to involve kinesin.     

Heparin binds with high affinity to voltage-dependent L-type Ca2+ channels. Evidence for an agonistic action

Heparin and related polyanions are a new class of compounds interacting with 1,4-dihydropyridine-sensitive L-type Ca2+ channels in a tissue-specific manner. Labeling of membrane-bound Ca2+ channels in rabbit skeletal muscle transverse tubules at the phenylalkylamine, benzothiazepine, and 1,4-dihydropyridine-selective domains was inhibited reversibly by a noncompetitive mechanism as shown by equilibrium saturation analysis and kinetic studies. (+)-cis-diltiazem but not (-)-cis-diltiazem reduced the inhibitory potency of heparin for 1,4-dihydropyridines. Antagonistic but not agonistic 1,4-dihydropyridines reversed heparin inhibition at the benzothiazepine site. Heparin forms a tight complex with the purified Ca2+ channel which is highly sensitive with respect to heparin inhibition (IC50 value: 0.05 microgram/ml) of 1,4-dihydropyridine binding. Reconstituted channel complexes have completely lost 1,4-dihydropyridine binding-inhibition by heparin and are not retained by lectin or heparin affinity columns. In whole cell patch clamp experiments with guinea-pig cardiac myocytes heparin increased the current through L-type Ca2+ channels when applied extracellulary. Synthetic peptides (representing putative heparin binding domains) which were derived from the rabbit skeletal muscle alpha 1-subunit reversed the inhibitory effects of heparin on 1,4-dihydropyridine receptors. Reversal for a peptide representing an extracellular domain occurred by an apparently competitive mechanism. It is suggested that heparin and related polyanions may interact with an evolutionary conserved cluster of basic amino acids in the large putative extracellular domain connecting the fifth and sixth putative transmembrane segment in the first motif of the ionic pore-forming alpha 1-subunit from skeletal muscle.     

Electrostatic interactions in the early events of VSV infection

The inportance of electrostatic interactions in the early phases of vesicular stomatitis virus (VSV) infection has been investigated in susceptible cells of different origin, human (HeLa) and avian (CER), by using some polyanions (heparin, polygalacturonic acid and mucin) and polycations (polymyxin B sulphate, poly-L-lysine, protamine, histone and polybrene). In HeLa cells, the attachment of VSV was enhanced by polymers having a positive charge and inhibited by those having a negative charge. In CER cells, all the polyanions tested reduced virus infection. Among the polycations, histone, polymyxin B sulphate and poly-L-lysine enhanced virus plaque forle protamine and polybrene reduced virus attachment. The effect of polyions on VSV particles and on cell membrane receptors has also been investigated. The analysis of the results obtained suggest that, although electrostatic interactions play an essential role in the binding of VSV to the cell membrane, more specific structural features appear to be required for viral attachment to occur.     

Increased uptake and desulphaton of heparin by mouse macrophages in the presence of polycations

Heparin uptake and desulphation by cultured macrophages were investigated. Histones, polyamino-acids, protamine and eosinophil-basic protein stimulated both heparin uptake and desulphation, processes found to be non-related. Poly-l-ornithine and poly-dl-lysine increased the heparin uptake by about 33-fold, and histone produced up to 7.5-fold increase in the desulphation. The same polycations inhibited heparin desulphation by macrophage extracts.     

Post-translational modifications of tubulin and the dynamics of microtubules in mouse oocytes and zygotes

The distribution of post-translationally modified forms of tubulin has been studied in mouse oocytes arrested in meiotic metaphase II and in interphase eggs after fertilisation. Tyrosinated and acetylated microtubules are present in the meiotic spindle but detyrosinated ones are not. Acetylation only occurs in the most stable subpopulation of microtubules in the spindles ("pole to kinetochore"). After fertilisation, many microtubules of the interphase array become acetylated, but detyrosination occurs only at a very low level.     

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the a-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75–78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.     

Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin.

The state of tubulin tyrosination in the fission yeast Schizosaccharomyces pombe was investigated using a combination of indirect immunofluorescence microscopy and Western blotting. Antibodies specific for the tyrosinated form of alpha-tubulin stained all microtubule arrays in wild type cells and recognised the two alpha-tubulin polypeptides in Western blots of cell extracts enriched for tubulin by DEAE-Sephadex chromatography. Antisera that specifically recognised the detyrosinated, glu, form, on the other hand, gave consistently negative results, both in cells undergoing rapid exponential growth and in those allowed to accumulate in stationary phase. Neither the "ageing" of microtubules, by arresting cells at different points (late G1 or G2/M) in the cell division cycle, nor stabilising them, using D2O, lead to any detectable tubulin detryrosination. These results suggest that S. pombe lacks the carboxypeptidase that carries out the tubulin detyrosination reaction. This is the first report of an organism that possesses the correct C-terminal alpha-tubulin sequence yet fails to carry out this post-translational modification. The implication of this novel finding for the biological role of these events is discussed.     

Lipids in the classification of Nocardioides: Reclassification of Arthrobacter simplex (Jensen) lochhead in the genus Nocardioides (Prauser) emend. O'Donnell et al. as Nocardioides simplex comb. nov.

The tubulin proteins of Blastocladiella emersonii have been characterized, and the pool sizes of soluble tubulins measured to evaluate turnover during early development. The axonemal tubulins and soluble tubulin dimers were typical of tubulin proteins from other eukaryotes.[³H]cholchicine binding assays were used to estimate the soluble tubulin pools of zoospores and during early development. The free colchicine-binding pool of tubulin in zoospores represents 1% of the soluble protein. It increases by 49% after encystment (at 30 min), decreases to 21% below the spore level by 50 min, and then increases slowly with growth. Neither deflagellation of zoospores prior to encystment, nor inhibition of axonemal disassembly, alter the postencystment pool increases. Disassembly of cytoskeletal microtubules occurs in either circumstance, but can account for only 54% of the pool increase. It was concluded that (1) the retracted axonemal tubulins are not returned to the soluble pool detected by cholchicine binding and are probably degraded; (2) new microtubules are supplied by the preexisting cytoplasmic pool that expands from disassembly of cytoplasmic microtubules; and (3) that the tubulins of the axonemes and soluble pools may be distinct.     

Purification and polyamine activation of a high-affinity 3',5'-cAMP phosphodiesterase from rabbit muscle

A high-affinity phosphodiesterase, termed PDE II, has been purified about 1400-fold from rabbit skeletal muscle. This enzyme is activated by treatment with proteases. It is also activated specifically by polyarginine and arginine-rich histones, but not by other polyanions. The activation is counteracted nonspecifically by polycations, such as heparin and chondroitin sulphate. When the enzyme is fully activated by polyarginine it is no longer susceptible to activation by proteases. A conformational or structural change must thus occur in the enzyme by the binding of the polyanions.     

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.