The effects of various GTP analogues on microtubule assembly.

We synthesized 27 GTP analogues with modification or substitution at positions C2, C6, C8 and ribose moiety to investigate their effect on microtubule (Mt) assembly. It was found that C2 and C6 are both functional for the analogues supporting Mt assembly. It was surprising to find that 2-amino- ATP (n2ATP) substantially supports assembly, and that the appearance of the assembled Mts was indistinguishable from those assembled in the standard GTP assembly buffer solution. Furthermore, 2-amino dATP and dGTP are even more potent than GTP in supporting assembly. The substitution of oxo group at C6 with reactive thiol largely reduced the activity of the analogue to support assembly. When free rotation of the glycosidic linkage of GTP was blocked by the introduction of sulfur atom between C8 and C2' of ribose moiety, it resulted in total suppression of assembly. Purine nucleoside triphosphate was found to support assembly better than GTP, and even more efficient was 2-amino purine nucleoside triphosphate. Interestingly, their deoxy-type analogues were totally inhibitory. Although 2-amino 8-hydroxy ATP and other analogues supported assembly much better than did GTP, their diphosphate analogues were totally incapable of supporting assembly. Finally, bulky fluorescent probes were introduced at C3' of ribose moiety (Mant-8-Br-GTP or Mant-GTP) to visualize the fluorescent signal in assembled Mts. Even in this case, the number of most protofilaments was found to be 14, consistent with that found in Mts assembled in GTP standard buffer solution.     

An oscillatory mode for microtubule assembly.

Depending upon the conditions under which polymerization takes place, pure tubulin can assemble into microtubules following either the usual monotonic kinetics or a more complex oscillatory mechanism. When present, these oscillations involve large cyclic changes in the extent of polymer formed before a steady-state is reached. Analysis of the microtubules formed at different times shows that these oscillations involve marked redistribution in both the length and number of microtubules. No significant difference is found between two populations of microtubules corresponding to the same level of assembly, one for which the extent of polymerization will remain stable with time and one for which it will decrease by as much as 90% in the next oscillation. The amplitude of these oscillations is sensitive to changes in the concentrations of protein, nucleotide (GTP, GDP or GMPpNp), magnesium ion or GTP regenerating system. A complete shift from an oscillatory to a monotonic polymerization can be induced by a minor increase in the concentration of free nucleotide, GTP or GDP.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

Cytoplasmic microtubule assembly is altered in cytoplasts.

Nucleation of microtubule (MT) organization of the cytoplasmic microtubule complex (CMTC) from the microtubule organizing centres (MTOC) was studied in enucleated cytoplasts of human diploid fibroblast (MRC-5) and mouse peritoneal macrophages in culture. Cytoplasts of both cell types could not organize the complete CMTC. Aberrant MT patterns were seen in MRC-5 cells while mouse macrophages showed occurrence of few short MT. The studies suggest that nucleus may have a role in determining CMTC.     

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.     

Colchicine inhibition of microtubule assembly via copolymer formation.

Colchicine.tubulin complex (CD) inhibits microtubule assembly. We examined this inhibition under conditions where spontaneous nucleation was suppressed and assembly was restricted to an elongation polymerization. We found that CD inhibited assembly by a mechanism which preserved the ability of microtubule ends to add tubulin. This observation is inconsistent with the end-poisoning model which recently was proposed as a general mechanism for assembly inhibition by CD. Our data are consistent with the following model: (a) microtubules formed in the presence of CD are CD-tubulin copolymers; (b) these copolymers can have appreciable numbers of incorporated CDs which are, most likely, randomly distributed in the copolymers; (c) CD-tubulin copolymers have assembly-competent ends with association and dissociation rate constants which decrease as the CD/tubulin ratio in the copolymers, (CD/T)MT, increases; and (d) the critical tubulin concentrations required for microtubule assembly increase in the presence of CD, indicating that copolymer affinity for tubulin decreases as (CD/T)MT increases.     

Ethacrynic acid inhibition of microtubule assembly in vitro.

Ethacrynic acid (ECA) is a sulfhydryl reactive diuretic drug. Recent studies show that ocular administration of ECA may have potential efficacy for treatment of glaucoma. ECA affects cell shape in cultured cells from the eye outflow pathway and the microtubule system is disrupted. We have studied the effect of ECA on microtubule protein (MTP) (tubulin and microtubule-associated proteins) and purified tubulin assembly. Fifty percent inhibition of MTP (1.8 mg/ml) assembly was found at 70 microM ECA in buffer and 410 microM ECA in 30% glycerol in buffer. If all sulfhydryl groups were attributed to tubulin, then approximately two sulfhydryls were blocked at 50% inhibition. Tubulin (2 mg/ml) assembly showed 50% inhibition at 175 microM ECA and approximately 2 sulfhydryl groups were lost. Increasing ECA preincubation times (0-60 min) with tubulin showed that the longer the preincubation time, the longer the lag time, and the slower the rate of assembly and that the percentage of inhibition was proportional to the ECA preincubation time. The number of blocked sulfhydryls also increased with preincubation time. Approximately two sulfhydryls were blocked at 50% inhibition of assembly. The critical concentration for assembly increased twofold when tubulin was preincubated with 0.1 mM ECA, suggesting a loss of active tubulin. Fifty percent inhibition of taxol-induced MTP and tubulin assembly occurred at 190 and 280 microM ECA, respectively, with 3.6 to 3.8 sulfhydryls blocked, respectively. Taxol protects microtubules from disassembly by ECA, suggesting that the ECA binding key sulfhydryls are blocked in the microtubule. These results suggest that ECA reacts slowly with tubulin and blocks sulfhydryl groups important for assembly. Microtubule-associated proteins and glycerol protect the sulfhydryls and so more ECA is necessary to inhibit assembly. Since the number of blocked sulfhydryls is greater at 50% inhibition for taxol-induced microtubules, sulfhydryl blocked tubulin incompetent to assemble under normal conditions may be induced to do so with taxol.     

Dephosphorylation of tubulin-bound guanosine triphosphate during microtubule assembly.

1. Tubulin purified from porcine brain in the presence of GTP contained 0.16 mole of GDP and 0.73 mole of GTP per 60,000 g of protein. 2. Microtubules reconstituted from the purified tubulin contained 0.43 mole of GDP and 0.41 mole of GTP per 60,000 g of protein. Guanine nucleotide bound to the exchangeable site of tubulin was converted to GDP during microtubule assembly, while GTP at the non-exchangeable site remained intact. 3. Guanine nucleotide which had been bound to the exchangeable site of tubulin before microtubule assembly was also exchangeable during disassembly.     

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.     

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Two non-hydrolyzable analogs of GTP, guanylyl-β,γ-methylene diphosphonate and guanylyl imidodiphosphate, have been found to induce rapid and efficient microtubule assembly in vitro by binding at the exchangeable site (E-site) on tubulin. Characterization of microtubule polymerization by several criteria, including polymerization kinetics, nucleotide binding to depolymerized and polymerized microtubules, and microtubule stability, reveals strong similarities between microtubule assembly induced by GTP and non-hydrolyzable GTP analogs. Nucleoside triphosphates which bind weakly or not at all to tubulin, such as ATP, UTP and CTP, are shown to induce microtubule assembly by means of a nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6.) activity which is not intrinsic to tubulin. The NDP-kinase mediates microtubule polymerization by phosphorylating tubulin-bound GDP in situ at the E-site. Although hydrolysis of exchangeably bound GTP occurs, it is found to be uncoupled from the polymerization reaction. The non-exchangeable nucleotide binding site on tubulin (N-site) is not directly involved in microtubule assembly in vitro. The N-site is shown to contain almost exclusively GTP which is not hydrolyzed during microtubule assembly. A scheme is presented in which GTP acts as an allosteric effector at the E-site during microtubule assembly in vitro.     

Tubulin carbamoylation. Functional amino groups in microtubule assembly.

The characteristics of the carbamoylation of pig brain tubulin were examined by using the modification conditions with cyanate described previously [Mellado, Slebe + Maccioni (1980) Biochem. Int. I, 584--590]. The carbamoylation reaction resulted in an inhibition of microtubule assembly, which was dependent on the concentration of the modifying agent. This tubulin modification appears to inhibit the growth of microtubules. The presence of GTP did not protect tubulin against this inhibition. Electron microscopy showed a marked decrease in the number of tubules after carbamoylation, but no alterations were observed in the microtubule morphology. The incorporation of KN14CO into alpha- and beta-subunits with similar kinetics was also shown, and the carbamoylated residues were identified as epsilon-N-carbamoyl-lysine residues.     

Actin assembly by lithium ions.

The ability of Li+ to promote the assembly of actin has been compared with the more common cations used in actin assembly assays, K+, Mg2+, and Ca2+. The principal assay of actin assembly utilized was fluorescence photobleaching recovery (FPR), from which it is possible to determine the fraction of actin protomers incorporated into filaments and the average diffusion coefficients of the filaments. In addition, critical concentrations of actin over a range of concentrations of all of these cations have been determined using an assay that involves sonication and dilution of assembled actin filaments containing trace amounts of pyrene-labeled actin. The results demonstrate that Li+ is a more potent promoter of actin assembly than is K+. The more rapid assembly of actin in the presence of Li+ is attributable to an increased rate of filament elongation. Filaments assembled in equivalent concentrations of Li+ or K+ have the same diffusion coefficients, and thus presumably the same average lengths. The critical concentration of actin is about three times less in the presence of Li+ than in the presence of an equal concentration of K+. Cytochalasin D accelerates the rate of Li+-promoted actin assembly and reduces slightly the total fraction of actin assembly. However, cytochalasin D causes less shortening of filaments in the presence of Li+ than in the presence of K+ or Mg2+. By the criteria of assembly kinetics and critical concentration, Li+ is much less potent as a promoter of actin assembly than either Mg2+ or Ca2+. These results are discussed in terms of the role of electrostatic forces in the actin assembly mechanism and in terms of possible relationships to therapeutic and toxicity mechanisms for Li+.     

Identification of the major 68,000-dalton protein of microtubule preparations as a 10-nm filament protein and its effects on microtubule assembly in vitro.

The major 68,000-dalton protein present in cycled microtubule preparations from bovine brain can be isolated in a rapidly sedimenting fraction consisting of filaments 10 nm in diameter. This 68,000-dalton protein remains in the filament fraction after gel filtration, phosphocellulose chromatography, or salt extraction of microtubule protein. Microtubule protein devoid of 10-nm filaments contains ring structures under depolymerizing conditions, and it polymerizes into microtubules with a characteristically low critical concentration, although all of the 68,000-dalton protein has been removed from it. When cycled microtubule protein is subjected to chromatography on phosphocellulose, the tubulin fraction (PC-tubulin) assembles into microtubules only at concentrations greater than 2 mg/mL. The other fraction, eluted from phosphocellulose at high ionic strength, contains the major 68,000-dalton protein and can be further resolved into two components by centrifugation. The supernatant, which consists mainly of high molecular weight microtubule-associated proteins, stimulates low concentrations of PC-tubulin to assemble. The pellet contains all of the 68,000-dalton protein, consists of 10-nm filaments, and does not stimulate assembly of PC-tublin. Boiling of purified filaments, however, releases several proteins, including the 68,000-dalton protein, and these released proteins stimulate the assembly of PC-tubulin. The morphology and protein composition of the filaments isolated from microtubule preparations by these techniques are very similar to those of mammalian neurofilaments. These results suggest that the major 68,000-dalton protein in cycled microtubule preparations, which may correspond to tubulin assembly protein [Lockwood, A.H. (1978) Cell 13, 613--627], is a constituent of neurofilaments.