Nucleotide specificity in microtubule assembly in vitro

A procedure is described for removing most of the GDP bound at the exchangeable GTP binding site (E site) of tubulin. Microtubule protein containing substoichiometric amounts of GDP at the E site is found to polymerize in response to: (a) two nonhydrolyzable ATP analogues, adenylyl imidodiphosphate (AMP-PNP) and adenylyl beta, gamma-methylenediphosphonate (AMP-PCP); and (b) substoichiometric levels of GTP or dGTP. The results are interpreted as suggesting that: (1) when GDP is removed from tubulin, the E site shows broad specificity for nucleoside triphosphates: (2) microtubule assembly can be induced by the binding of substoichiometric amounts of nucleoside triphosphate to the E site.     

Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

Effect of gamma-irradiation on microtubule assembly in vitro

Microtubular protein was exposed to gamma-radiation from 500 to 1000 Gy, Within that dose range its polymerization ability was decreased by 20-60 per cent when samples were irradiated in assembled state, and by 40-75 per cent when irradiated in unassembled state. Microtubules assembled from irradiated subunits were shorter and of more uniform lengths than control microtubules. For the dose of 1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.     

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.     

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.     

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.     

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.     

Long-time stretched exponential kinetics in single DNA duplex dissociation

We probe DNA hybridization kinetics by measuring the lifetime distribution of single 16-bp duplexes under thermal dissociation. Our unique approach, based on two DNA-coated microspheres in an extended optical tweezer, allows the study of single duplex DNA molecules under negligible molecular tension. In contrast to earlier experiments, we find a stretched exponential lifetime distribution, which is likely due to dissociation proceeding via a number of competing pathways between highly force-sensitive intermediate states. Similar measurements of microspheres linked by multiple DNA bridges find they have unexpected short bound lifetimes, also consistent with force sensitivity.     

On the mechanism of calmodulin-induced inhibition of microtubule assembly in vitro

Binding of calmodulin to microtubule-associated proteins (MAPs) was analyzed by the equilibrium gel filtration method. The apparent dissociation constant (Kd) of calmodulin binding was found to be 2 microM for tau, and 5 microM for MAP2. These Kd values were similar to the Kd previously determined for calmodulin binding to tubulin. The inhibitory effect of increasing concentrations of calmodulin on the kinetics of microtubule assembly from tau and tubulin was not mimicked by decreasing the concentration of tau alone or tubulin alone. These results suggest that calmodulin inhibits microtubule assembly by its binding to both MAPs and tubulin.     

A reassessment of the factors affecting microtubule assembly and disassembly in vitro

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

In vitro microtubule assembly on centrioles from mammalian spermatids

In the present study we have shown that the centriolar structures, which form the neck region of the spermatid tail, can act as microtubule-organizing centers.     

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.     

Soluble immune response suppressor (SIRS) inhibits microtubule function in vivo and microtubule assembly in vitro

Soluble immune response suppressor (SIRS) is a product of concanavalin A-stimulated murine T cells that, when activated or oxidized by macrophages or H2O2 (SIRSox), suppresses in vitro immune responses and inhibits cell division by normal and neoplastic cells. SIRSox is inactivated by a variety of electron donors, which suggests that SIRSox may be an oxidizing agent. Incubation of lymphocytes with SIRSox, but not with SIRS, partially reversed concanavalin A-mediated inhibition of capping of membrane immunoglobulin on B cells, and disrupted the cytoplasmic array of microtubules visualized by fluorescence microscopy. SIRSox also inhibited microtubule assembly in vitro in a concentration-dependent manner. Inactivation of SIRSox by dithiothreitol prevented SIRSox-mediated reversal of inhibition of capping and inhibition of microtubule assembly. These results reveal a pattern of SIRSox activity similar to sulfhydryl-dependent cytoskeletal disrupting agents (e.g., N-ethylmaleimide, cytochalasin A, p-benzoquinone), and suggest that SIRSox-mediated suppression of proliferation may involve interference with sulfhydryl-dependent cytoskeletal events critical for cell division.     

Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.     

The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism

A model describing the nucleation and assembly of purified tubulin has been developed. The novel feature of this model is a two stage nucleation process to allow the explicit inclusion of the two-dimensional nature of the early stages of microtubule assembly. In actin assembly the small starting nucleus has only one site for subunit addition as the two-stranded helix is formed. In contrast, microtubule assembly begins with the formation of a small two-dimensional section of microtubule wall. The model we propose is a modification of the work of Wegner and Engel (Wegner, A., and Engel, J. (1975) Biophys. Chem. 3, 215-225) wherein we add a second stage of nucleation to directly account for lateral growth, i.e. the addition of a small number of subunits to the side of an existing sheet structure. Subsequent elongation of the sheets is treated in the usual way. The experimental system used to test this model was the Mg2+/glycerol induced assembly of purified tubulin. The computer simulation of the polymerization time courses gave a fairly good fit to experimental kinetics for our model, where the primary nucleus comprises two protofilaments, of four and three subunits, and lateral growth requires a three-subunit nucleus to initiate a new protofilament.     

Putative involvement of a 49 kDa protein in microtubule assembly in vitro

In higher plant cells, thus far only a few molecules have been inferred to be involved in microtubule organizing centers (MTOCs). Examination of a 49 kDa tobacco protein, homologous to a 51 kDa protein involved in sea urchin MTOCs, showed that it also accumulated at the putative MTOC sites in tobacco BY-2 cells. In this report, we show that the 49 kDa protein is likely to play a significant role in microtubule organization in vitro. We have established a system prepared from BY-2 cells, capable of organizing microtubules in vitro. The fraction, which was partially purified from homogenized miniprotoplasts (evacuolated protoplasts) by salt extraction and subsequent ion exchange chromatography, contained many particles of diameters about 1 micron after desalting by dialysis. When this fraction was incubated with purified porcine brain tubulin, microtubules were elongated radially from the particles and organized into structures similar to the asters observed in animal cells, and therefore also termed "asters" here. Since we could hardly detect BY-2 tubulin molecules in this fraction, the microtubules in "asters" seemed to be solely composed of the added porcine tubulin. Tubulin molecules were newly polymerized at the ends of the microtubules distal to the particles, and the elongation rate of microtubules was more similar to the reported rate of the plus-ends than that of the minus-ends in vitro. By fluorescence microscopy, the 49 kDa protein was shown to be located at the particles. Thus, its location at the centers of the "asters" suggests that the protein plays a role in microtubule organization in vitro.