tRNA glycylation system from Thermus thermophilus. tRNAGly identity and functional interrelation with the glycylation systems from other phylae.

The systems of tRNA glycylation belong to the most complex aminoacylation systems since neither the oligomeric structure of glycyl-tRNA synthetases (GlyRS) nor the discriminator bases in tRNAGly are conserved in the phylae. To better understand the structure-function relationship in glycylation systems of various origins and the functional peculiarities related to their structural divergences, the elements in tRNA conferring its glycine identity in Thermus thermophilus were characterized and compared to those of other systems. Thermophilic identity is conferred by the G1-C72, C2-G71, G3-C70, and C50-G64 pairs together with the G10, U16, C35, and C36 single residues. In contrast to most other aminoacylation systems, the discriminator base is not directly involved in identity. Transplantation of these elements in tRNAAsp and tRNAPhe converts specificity toward glycine albeit conservation of nucleotide 73. Analysis of the functional interrelation of the identity elements shows coupling in synthetase recognition of the elements from anticodon and G10 whereas those from acceptor arm are recognized independently. Despite nondirect implication in identity, the discriminator base contributes cooperatively with C36 in specificity of glycylation. The link between the structural heterogeneity and the functional divergence of the glycylation systems and the phylogenic interrelation of these systems were approached by comparing the ability of GlyRSs of various phylae to glycylate heterologous tRNAGly. Dimeric GlyRSs from mammalian and archaebacteria acylate efficiently only eukaryotic and archaebacterial tRNAGly with a discriminatory A73, whereas tetrameric Escherichia coli GlyRS acylates only eubacterial tRNAGly with a discriminatory U73. In contrast, dimeric yeast GlyRS acylates efficiently both eukaryotic and archaebacterial tRNAGly as well as peculiar prokaryotic isoacceptors. Species specificity is lost with the dimeric GlyRS from Thermus thermophilus that acylates efficiently eubacterial, archaebacterial, and eukaryotic tRNAGly. These features are discussed in the context of the evolution of the glycylation systems and the phylogenic interrelation of the organisms.     

Regulatory aspects of the glutamylation of methotrexate in cultured hepatoma cells

The glutamylation of methotrexate has been evaluated in H35 hepatoma cells in vitro as a function of the conditions of culture. Glutamylation yields methotrexate polyglutamate with two to five additional glutamate residues and is a saturable process. The rate of glutamylation increases little above 10 microM extracellular methotrexate which corresponds to an intracellular concentration of approximately 4 microM. The rate of glutamylation measured over a 6-h period was stimulated by a reduction in cellular folates and prior incubation of the cells with insulin. Glutamylation was also more rapid in dividing cultures than in confluent cells. The combination of insulin inclusion and folate reduction, which was additive, caused approximately a fourfold increase in the rate of glutamylation over control cells under the conditions tested. The maximal rate of methotrexate glutamylation, which was 100 nmol/g/h, occurred in folate-depleted, insulin-supplemented cells. Supplementing folate-depleted cells with reduced folate coenzymes caused the glutamylation to be reduced by more than 90%. The turnover of methotrexate polyglutamates in cells saturated with these derivatives occurred at approximately one-half the rate of net synthesis and was stimulated to nearly the same extent by folate depletion and insulin. In addition to showing that folates can modify the rates of methotrexate polyglutamate formation, data are presented suggesting that methotrexate polyglutamates can regulate their own synthesis. The consequences of the formation of these retained forms of methotrexate in H35 hepatoma cells (M. Balinska, J. Galivan, and J.K. Coward (1981) Cancer Res. 41,2751-2756) and the effects of potential regulators of this process are discussed in terms of the glutamylation of folates in the cells and the chemotherapeutic effects of antifolates.     

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.     

Tubulin transport in neurons

A question of broad importance in cellular neurobiology has been, how is microtubule cytoskeleton of the axon organized? It is of particular interest because of the history of conflicting results concerning the form in which tubulin is transported in the axon. While many studies indicate a stationary nature of axonal microtubules, a recent series of experiments reports that microtubules are recruited into axons of neurons grown in the presence of a microtubule-inhibitor, vinblastine (Baas, P.W., and F.J. Ahmad. 1993.J. Cell Biol. 120:1427-1437: Ahmad F.J., and P.W. Baas. 1995. J. Cell Sci, 108:2761-2769; Sharp, D.J., W. Yu, and P.W. Baas. 1995. J. Cell Biol, 130:93-103; Yu, W., and P.W. Baas. 1995. J. Neurosci. 15:6827-6833.). Since vinblastine stabilizes bulk microtubule-dynamics in vitro, it was concluded that preformed microtubules moved into newly grown axons. By visualizing the polymerization of injected fluorescent tubulin, we show that substantial microtubule polymerization occurs in neurons grown at reported vinblastine concentrations. Vinblastine inhibits, in a concentration-dependent manner, both neurite outgrowth and microtubule assembly. More importantly, the neuron growth conditions of low vinblastine concentration allowed us to visualize the footprints of the tubulin wave as it polymerized and depolymerized during its slow axonal transport. In contrast, depolymerization resistant fluorescent microtubules did not move when injected in neurons. We show that tubulin subunits, not microtubules, are the primary form of tubulin transport in neurons.     

Structural insights into microtubule doublet interactions in axonemes

Coordinated sliding of microtubule doublets, driven by dynein motors, produces periodic beating of eukaryotic cilia and flagella. Recent structural studies of the axoneme, which forms the core of cilia and flagella, have used cryo-electron tomography to reveal new details of the interactions between some of the multitude of proteins that form the axoneme and regulate its movement. Connections between the several types of dyneins, in particular, suggest ways in which their action might be coordinated. Study of the molecular architecture of isolated doublets has provided a structural basis for understanding mechanical properties related to the bending of the axoneme, and has also offered insight into the potential role of doublets in the mechanism of dynein activity regulation.     

Tubulin Conformation in Zinc-Induced Sheets and Macrotubes

The protein tubulin is the main constituent of microtubules. Previous studies have shown that zinc ions induce the formation of crystalline sheets and macrotubes of tubulin. Both crystal types are suitable for structural studies by electron crystallography. However, crystallographic structural analysis of tubulin has been hampered by limited crystal size and quality and the inability to control crystal polymorphism. We can obtain well-ordered crystals which are grown upon prolonged incubations (up to 24 hr). The presence of NaCl delays the degradation of the crystals, and addition of the protease inhibitor pepstatin improves crystal quality. The crystal form (sheet or macrotube) can be controlled with incubation conditions. The size of the crystals can reach up to 2 μm in width for the sheets and up to 0.5 μm in diameter for the macrotubes. Both crystal types can reach several micrometers in length. Comparison of the projection maps of the two crystal structures shows that adjacent protofilaments in the macrotubes are shifted by about 6 Å relative to their positions in the sheets. Observable changes of to monomer shape appear to allow close interprotofilament contacts to be maintained in both crystal forms. Images of glucose-embedded specimens obtained under these conditions give structural information beyond 4 Å resolution. Merging of high- and low-resolution data allows for unambiguous assignment of monomer boundaries to high-resolution features.     

Tubulin protofilaments and kinesin-dependent motility

Microtubules are built of tubulin subunits assembled into hollow cylinders which consist of parallel protofilaments. Thus, motor molecules interacting with a microtubule could do so either with one or several tubulin subunits. This makes it difficult to determine the structural requirements for the interaction. One way to approach the problem is to alter the surface lattice. This can be done in several ways. Proto-filaments can be exposed on their inside (C-tubules or "sheets"), they can be made antiparallel (zinc sheets), or they can be rolled up (duplex tubules). We have exploited this polymorphism to study how the motor protein kinesin attached to a glass surface interacts and moves the various tubulin assemblies. Microtubules glide over the surface along straight paths and with uniform velocities. In the case of C-tubules, approximately 40% glide similarly to microtubules, but a major fraction do not glide at all. This indicates (a) that a full cylindrical closure is not necessary for movement, and (b) that the inside surface of microtubules does not support gliding. With zinc sheets, up to 70% of the polymers move, but the movement is discontinuous, has a reduced speed, and follows along a curved path. Since zinc sheets have protofilaments alternating in orientation and polarity, this result suggests that in principle a single protofilament can produce movement, even when its neighbors cannot. Duplex microtubules do not move because they are covered with protofilaments coiled inside out, thus preventing the interaction with kinesin. The data can be explained by assuming that the outside of one protofilament represents the minimal track for kinesin, but smooth gliding requires several parallel protofilaments. Finally, we followed the motion of kinesin-coated microbeads on sea-urchin sperm flagella, from the flagellar outer doublet microtubules to the singlet microtubule tips extending from the A-tubules. No change in behavior was detected during the transition. This indicates that even if these microtubules differ in surface lattice, this does not affect the motility.     

Tubulin structure and biochemistry.

In the past year, much has been learned about structure-function correlations in the tubulin molecule, and specifically about the nature and roles of post-translational modifications and tubulin isotypes. The interactions between tubulin and its ligands--both microtubule-associated proteins and anti-mitotic drugs--are becoming clearer at the molecular level.     

In vitro phosphorylation of Paramecium axonemes and permeabilized cells

This study seeks to identify phosphoproteins in axonemes from Paramecium tetraurelia whose phosphorylation responses to adenosine 3', 5'-cyclic monophosphate (cAMP) and Ca2+ parallel responses induced by these agents in ciliary behavior in this cell. In purified axonemes, over 15 bands ranging from Mr greater than 300 kDa to 19 kDa on SDS-PAGE incorporate 32P from adenosine 5'-gamma-[32P]triphosphate (gamma-32P-ATP) at pCa 7 in the absence of cAMP. A major band whose label turns over rapidly was identified at Mr 43 kDa. In the presence of 5 microM cAMP, more than eight bands, but not the Mr 43 kDa band, were labeled additionally or enhanced their labeling. These phosphoproteins and their kinases are structural components of the axoneme. Overall, some of the same major bands are labeled in the presence of cAMP in Triton X-100-permeabilized paramecia that retain their behavioral responses and in axonemes mechanically isolated from these cells. In particular, two major bands have been identified whose phosphorylation is greatly enhanced by cAMP at low concentrations: 1) a 29 kDa polypeptide whose cAMP-dependent phosphorylation is diminished at pCa 4 compared with pCa 7 and 2) a 65 kDa polypeptide whose phosphorylation is pCa insensitive. These polypeptides meet minimal criteria for signal-sensitive regulators of motility parameters in the Paramecium axoneme.     

Tubulin superfamily genes in Tribolium castaneum and the use of a Tubulin promoter to drive transgene expression

The use of native promoters to drive transgene expression has facilitated overexpression studies in Drosophila and other insects. We identified 12 Tubulin family members from the genome sequence of the red flour beetle, Tribolium castaneum, and used the promoter from one of these to drive constitutive expression of a transgene. The activity of the T. castaneum alpha-Tubulin1 (TcalphaTub1) putative promoter was pre-tested in conjunction with an eye-color gene, T. castaneum vermilion (Tcv), by transient expression in Tcv-deficient embryos. Such embryos showed complete rescue of larval eyespot pigmentation. We also examined the TcalphaTub1 expression pattern in germline transformants using the enhanced green fluorescent protein (EGFP) reporter. Beetles transformed with this piggyBac-based reporter ubiquitously expressed EGFP at all stages.     

Direct measurement of inter-doublet elasticity in flagellar axonemes.

The outer doublet microtubules in ciliary and flagellar axonemes are presumed to be connected with each other by elastic links called the inter-doublet links or the nexin links, but it is not known whether there actually are such elastic links. In this study, to detect the elasticity of the putative inter-doublet links, shear force was applied to Chlamydomonas axonemes with a fine glass needle and the longitudinal elasticity was determined from the deflection of the needle. Wild-type axonemes underwent a high-frequency, nanometer-scale vibration in the presence of ATP. When longitudinal shear force was applied, the average position of the needle tip attached to the axoneme moved linearly with the force applied, yielding an estimate of spring constant of 2.0 (S.D.: 0.8) pN/nm for 1 microm of axoneme. This value did not change in the presence of vanadate, i.e., when dynein does not form strong cross bridges. In contrast, it was at least five times larger when ATP was absent, i.e., when dynein forms strong cross bridges. The measured elasticity did not significantly differ in various mutant axonemes lacking the central-pair microtubules, a subset of inner-arm dynein, outer-arm dynein, or the radial spokes, although it was somewhat smaller in the latter two mutants. It was also observed that the shear displacement in an axoneme in the presence of ATP often took place in a stepwise manner. This suggests that the inter-doublet links can reversibly detach from and reattach to the outer doublets in a cooperative manner. This study thus provides the first direct measure of the elasticity of inter-doublet links and also demonstrates its dynamic nature.     

Cytoplasmic carboxypeptidase 5 regulates tubulin glutamylation and zebrafish cilia formation and function

Glutamylation is a functionally important tubulin posttranslational modification enriched on stable microtubules of neuronal axons, mitotic spindles, centrioles, and cilia. In vertebrates, balanced activities of tubulin glutamyl ligase and cytoplasmic carboxypeptidase deglutamylase enzymes maintain organelle- and cell type–specific tubulin glutamylation patterns. Tubulin glutamylation in cilia is regulated via restricted subcellular localization or expression of tubulin glutamyl ligases (ttlls) and nonenzymatic proteins, including the zebrafish TPR repeat protein Fleer/Ift70. Here we analyze the expression patterns of ccp deglutamylase genes during zebrafish development and the effects of ccp gene knockdown on cilia formation, morphology, and tubulin glutamylation. The deglutamylases ccp2, ccp5, and ccp6 are expressed in ciliated cells, whereas ccp1 expression is restricted to the nervous system. Only ccp5 knockdown increases cilia tubulin glutamylation, induces ciliopathy phenotypes, including axis curvature, hydrocephalus, and pronephric cysts, and disrupts multicilia motility, suggesting that Ccp5 is the principal tubulin deglutamylase that maintains functional levels of cilia tubulin glutamylation. The ability of ccp5 knockdown to restore cilia tubulin glutamylation in fleer/ift70 mutants and rescue pronephric multicilia formation in both fleer- and ift88-deficient zebrafish indicates that tubulin glutamylation is a key driver of ciliogenesis.     

Writing and Reading the Tubulin Code

Microtubules give rise to intracellular structures with diverse morphologies and dynamics that are crucial for cell division, motility, and differentiation. They are decorated with abundant and chemically diverse posttranslational modifications that modulate their stability and interactions with cellular regulators. These modifications are important for the biogenesis and maintenance of complex microtubule arrays such as those found in spindles, cilia, neuronal processes, and platelets. Here we discuss the nature and subcellular distribution of these posttranslational marks whose patterns have been proposed to constitute a tubulin code that is interpreted by cellular effectors. We review the enzymes responsible for writing the tubulin code, explore their functional consequences, and identify outstanding challenges in deciphering the tubulin code.     

Structural and functional reconstitution of inner dynein arms in Chlamydomonas flagellar axonemes

The inner row of dynein arms contains three dynein subforms. Each is distinct in composition and location in flagellar axonemes. To begin investigating the specificity of inner dynein arm assembly, we assessed the capability of isolated inner arm dynein subforms to rebind to their appropriate positions on axonemal doublet microtubules by recombining them with either mutant or extracted axonemes missing some or all dyneins. Densitometry of Coomassie blue-stained polyacrylamide gels revealed that for each inner dynein arm subform, binding to axonemes was saturable and stoichiometric. Using structural markers of position and polarity, electron microscopy confirmed that subforms bound to the correct inner arm position. Inner arms did not bind to outer arm or inappropriate inner arm positions despite the availability of sites. These and previous observations implicate specialized tubulin isoforms or nontubulin proteins in designation of specific inner dynein arm binding sites. Further, microtubule sliding velocities were restored to dynein-depleted axonemes upon rebinding of the missing inner arm subtypes as evaluated by an ATP-induced microtubule sliding disintegration assay. Therefore, not only were the inner arm dynein subforms able to identify and bind to the correct location on doublet microtubules but they bound in a functionally active conformation.     

A physical model of microtubule sliding in ciliary axonemes.

Ciliary movement is caused by coordinated sliding interactions between the peripheral doublet microtubules of the axoneme. In demembranated organelles treated with trypsin and ATP, this sliding can be visualized during progressive disintegration. In this paper, microtubule sliding behavior resulting from various patterns of dynein arm activity and elastic link breakage is determined using a simplified model of the axoneme. The model consists of a cylindrical array of microtubules joined, initially, by elastic links, with the possibility of dynein arm interaction between microtubules. If no elastic links are broken, sliding can produce stable distortion of the model, which finds application to straight sections of a motile cilium. If some elastic links break, the model predicts a variety of sliding patterns, some of which match, qualitatively, the observed disintegration behavior of real axonemes. Splitting of the axoneme is most likely to occur between two doublets N and N + 1 when either the arms on doublet N + 1 are active and arms on doublet N are inactive or arms on doublet N - 1 are active while arms on doublet N are inactive. The analysis suggests further experimental studies which, in conjunction with the model, will lead to a more detailed understanding of the sliding mechanism, and will allow the mechanical properties of some axonemal components to be evaluated.     

Tubulin dimer dissociation and proteolytic accessibility

The alpha and beta subunits of the tubulin dimer each possess a distal C-terminal subtilisin cleavage site which, when cleaved, releases an acidic, small peptide. In addition, each possesses an internal site, cleaved by trypsin in alpha and chymotrypsin in beta, which connects the amino and carboxyl structural domains. A model of the dimer is presented which suggests that the beta C-terminal subtilisin site may be more accessible in the monomer than in the dimer. Kinetics of cleavage at this site on the dimer yield straight-line plots of log (undigested fraction) versus time, from which pseudo-first-order rate constants are obtained. Temperature effects on the rate constant are due to changes in the activity of subtilisin, not to temperature-induced unfolding around this site. The rate constant is proportional to the subtilisin/tubulin ratio, whether this is varied by changing the concentration of subtilisin or of tubulin. However, if the rate constant increases due to decreasing tubulin concentration, the extrapolated zero time intercept decreases. The decrease in zero time intercept is interpreted as being due to the appearance of a rapidly digested fraction upon dilution of tubulin. The increase observed in this fast fraction with dilution of tubulin is fully reversible upon reconcentration. It is suggested that this fast fraction represents monomeric beta-tubulin and the concentration dependence of this fast fraction indicates a dissociation constant of about 1.5 X 10(-7) M.     

Tubulin and microtubule-like structures in mammalian acrosomes.

Immunofluorescence and electron microscopy were used to demonstrate the presence of tubulin and microtubule-like structures in rabbit and rhesus monkey sperm acrosomes. The distribution of tubulin within the acrosome appeared similar to the distribution of proteolytic activity, suggesting an association of proteinase with these structures. Electron microscopy of rhesus monkey sperm incubated in vitro demonstrated a progressive condensation of the acrosomal matrix into microtubule-like structure. A system of this type could explain the linear patterns of silver-proteinate staining in the pentration tunnel through the zona pellucida.