Pure Tubulin Research Articles

Dynamic instability of microtubules assembled from microtubule-associated protein-free tubulin: neither variability of growth and shortening rates nor "rescue" requires microtubule-associated proteins.

The growth and shortening of microtubules in dynamic instability is known to be modulated by microtubule-associated proteins (MAPs). A full understanding of the mechanism of dynamic instability requires that one distinguish which of its aspects are mediated by microtubule-associated proteins (even in small residual concentrations) and which are intrinsic properties of the tubulin lattice itself. This paper addresses two of those aspects: whether MAPs cause the rescue events of dynamic instability (i.e., the transitions from shortening to growth) and whether MAPs are responsible for the marked variability of the rates at which microtubules grow and shorten. Very pure tubulin was prepared by sequential chromatographies on phosphocellulose and DEAE-Sephadex. Analysis by electrophoresis and immunoblotting showed it to be essentially MAP-free; it contained fewer than one MAP molecule per 10000 tubulin dimers. When its dynamic instability was studied by video-DIC microscopy, rescues were found to occur at a mean frequency of one per 4 microns of shortening. Variability of rates of growth and shortening, which is observed on the length scale of a few micrometers, was not changed by removal of MAPs. Because the mean distance between bound MAP molecules was calculated to be greater than 14 microns in these experiments, it is concluded that they cannot cause either rescue or variability of rates.

Ethacrynic acid disrupts steady state microtubules in vitro.

Ethacrynic acid (ECA) has been shown to increase facility of aqueous outflow in whole eyes and perfused anterior segments, to open up spaces between cells in the trabecular meshwork and inner wall of Schlemm's canal, and to cause separation and retraction of trabecular meshwork and endothelial cells in culture. One mechanism by which ECA has been proposed to act in cells is via disruption of microtubules, leading to cell retraction. Although it is known that ECA can inhibit de novo assembly of microtubules from tubulin subunits in vitro, we wanted to determine, as a better correlate to the proposed effect of ECA in cells, whether ECA could disrupt microtubule polymers that had reached steady state. We also wanted to determine whether calcium ion could enhance this process. We therefore assembled purified and crude porcine brain tubulin to steady state at 37 degrees C and then added ECA and/or calcium. Reaction kinetics were followed spectrophotometrically. We found that ECA effectively disrupted assembled microtubules in vitro. Although 0.8-1.0 mM ECA was required to produce a half-maximal effect in pure tubulin microtubules and 0.2-0.3 mM ECA was necessary with crude microtubule protein, significant disassembly also occurred in the 0.01-0.2 mM range. Calcium had a greater maximal effect than ECA, and was more potent on a molar basis, showing half maximal effect between 2 and 12 microM free calcium ion. Combination experiments showed that ECA did not act synergistically with calcium to increase microtubule disassembly. Our results are consistent with the proposed disruptive action of ECA on the assembled microtubules of outflow pathway cells, but do not support a rise in intracellular calcium as being an added factor.

Domains of tau protein, differential phosphorylation, and dynamic instability of microtubules.

The dynamic instability of microtubules is thought to be regulated by MAPs and phosphorylation. Here we describe the effect of the neuronal microtubule-associated protein tau by observing the dynamics of single microtubules by video microscopy. We used recombinant tau isoforms and tau mutants, and we phosphorylated tau by the neuronal kinases MARK (affecting the KXGS motifs within tau's repeat domain) and cdk5 (phosphorylating Ser-Pro motifs in the regions flanking the repeats). The variants of tau can be broadly classified into three categories, depending on their potency to affect microtubule dynamics. "Strong" tau variants have four repeats and both flanking regions. "Medium" variants have one to three repeats and both flanking regions. "Weak" variants lack one or both of the flanking regions, or have no repeats; with such constructs, microtubule dynamics is not significantly different from that of pure tubulin. N- or C-terminal tails of tau have no influence on dynamic instability. The two ends of microtubules (plus and minus) showed different activities but analogous behavior. These results are consistent with the "jaws" model of tau where the flanking regions are considered as targeting domains whereas the addition of repeats makes them catalytically active in terms of microtubule stabilization. The dominant changes in the parameters of dynamic instability induced by tau are those in the dissociation rate and in the catastrophe rate (up to 30-fold). Other rates change only moderately or not at all (association rate increased up to twofold, rates of rescue or rapid shrinkage decreased up to approximately twofold). The order of repeats has little influence on microtubule dynamics (i.e., repeats can be re-arranged or interchanged), arguing in favor of the "distributed weak binding" model proposed by Butner and Kirschner (1991); however, we confirmed the presence of a "hotspot" of binding potential involving Lys274 and Lys281 observed by Goode and Feinstein, 1994. Phosphorylation of Ser-Pro motifs by cdk5 (mainly Ser 202, 235, and 404) in the flanking regions had a moderate effect on microtubule dynamics while phosphorylation at the "Alzheimer"-site Ser262 MARK eliminated tau's interactions with microtubules. In both cases the predominant effects of phosphorylation are on the rates of tubulin dissociation and catastrophe whereas the effects on the rates of association or rescue are comparatively small.

Stabilization and bundling of subtilisin-treated microtubules induced by microtubule associated proteins

The acidic carboxy-terminal regions of alpha- and beta-tubulin subunits are currently thought to be centrally involved in microtubule stability and in microtubule association with a variety of proteins (MAPs) such as MAP2 and tau proteins. Here, pure tubulin microtubules were exposed to subtilisin to produce polymers composed of cleaved tubulin subunits lacking carboxy termini. Polymer exposure to subtilisin was achieved in buffer conditions compatible with further tests of microtubule stability. Microtubules composed of normal alpha-tubulin and cleaved beta-tubulin were indistinguishable from control microtubules with regard to resistance to dilution-induced disassembly, to cold temperature-induced disassembly and to Ca(2+)-induced disassembly. Microtubules composed of cleaved alpha- and beta-tubulins showed normal sensitivity to dilution-induced disassembly and to low temperature-induced disassembly, but marked resistance to Ca(2+)-induced disassembly. Polymers composed of normal alpha-tubulin and cleaved beta-tubulin or of cleaved alpha- and beta-tubulins were stabilized in the presence of added MAP2, myelin basic protein and histone H1. Cleavage of tubulin carboxy termini greatly potentiated microtubule stabilization by tau proteins. We show that this potentiation of polymer stabilization can be ascribed to tau-induced microtubule bundling. In our working conditions, such bundling upon association with tau proteins occurred only in the case of microtubules composed of cleaved alpha- and beta-tubulins and triggered apparent microtubule cross-stabilization among the bundled polymers. These results, as well as immunofluorescence analysis, which directly showed interactions between subtilisin-treated microtubules and MAPs, suggest that the carboxy termini of alpha- and beta-tubulins are not primarily involved in the binding of MAPs onto microtubules. However, interactions between tubulin carboxy termini and MAPs remain possible and might be involved in the regulation of MAP-induced microtubule bundling.

The minimum GTP cap required to stabilize microtubules

Background: Microtubules polymerized from pure tubulin show the unusual property of dynamic instability, in which both growing and shrinking polymers coexist at steady state. Shortly after its addition to a microtubule end, a tubulin subunit hydrolyzes its bound GTP. Studies with non-hydrolyzable analogs have shown that GTP hydrolysis is not required for microtubule assembly, but is essential for generating a dynamic polymer, in which the subunits at the growing tip have bound GTP and those in the bulk of the polymer have bound GDP. It has been suggested that loss of the ‘GTP cap’ through dissociation or hydrolysis exposes the unstable GDP core, leading to rapid depolymerization. However, evidence for a stabilizing cap has been very difficult to obtain.Results We developed an assay to determine the minimum GTP cap necessary to stabilize a microtubule from shrinking. Assembly of a small number of subunits containing a slowly hydrolyzed GTP analog (GMPCPP) onto the end of dynamic microtubules stabilized the polymer to dilution. By labeling the subunits with rhodamine, we measured the size of the cap and found that as few as 40 subunits were sufficient to stabilize a microtubule.Conclusion On the basis of statistical arguments, in which the proportion of stabilized microtubules is compared to the probability that when 40 GMPCPP-tubulin subunits have polymerized onto a microtubule end, all protofilaments have added at least one GMPCPP-tubulin subunit, our measurements of cap size support a model in which a single GTP subunit at the end of each of the 13 protofilaments of a microtubule is sufficient for stabilization. Depolymerization of a microtubule may be initiated by an exposed tubulin–GDP subunit at even a single position. These results have implications for the structure of microtubules and their means of regulation.

Centrosome assembly in vitro: role of gamma-tubulin recruitment in Xenopus sperm aster formation.

Centrioles organize microtubules in two ways: either microtubules elongate from the centriole cylinder itself, forming a flagellum or a cilium ("template elongation"), or pericentriolar material assembles and nucleates a microtubule aster ("astral nucleation"). During spermatogenesis in most species, a motile flagellum elongates from one of the sperm centrioles, whereas after fertilization a large aster of microtubules forms around the sperm centrioles in the egg cytoplasm. Using Xenopus egg extracts we have developed an in vitro system to study this change in microtubule-organizing activity. An aster of microtubules forms around the centrioles of permeabilized frog sperm in egg extracts, but not in pure tubulin. However, when the sperm heads are incubated in the egg extract in the presence of nocodazole, they are able to nucleate a microtubule aster after isolation and incubation with pure calf brain tubulin. This provides a two-step assay that distinguishes between centrosome assembly and subsequent microtubule nucleation. We have studied several centrosomal antigens during centrosome assembly. The CTR2611 antigen is present in the sperm head in the peri-centriolar region. gamma-tubulin and certain phosphorylated epitopes appear in the centrosome only after incubation in the egg extract. gamma-tubulin is recruited from the egg extract and associated with electron-dense patches dispersed in a wide area around the centrioles. Immunodepletion of gamma-tubulin and associated molecules from the egg extract before sperm head incubation prevents the change in microtubule-organizing activity of the sperm heads. This suggests that gamma-tubulin and/or associated molecules play a key role in centrosome formation and activity.

In vitro analysis of microtubule assembly of isotypically pure tubulin dimers. Intrinsic differences in the assembly properties of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers in the absence of microtubule-associated proteins.

Microtubule assembly of different beta tubulin isotypes in the presence of 4 M glycerol and 6 mM magnesium ion demonstrates significantly different characteristics. alpha beta II and alpha beta IV assembled faster and to a greater extent than did unfractionated phosphocellulose-purified tubulin (PC-tubulin). Microtubule assembly from alpha beta III showed a distinctive delay in nucleation, proceeded at a slower rate than those of the other beta tubulin isotypes, and had the highest critical concentration. However, treatment of beta tubulin isotypes with subtilisin to remove the C-terminal domain of the tubulin dimer abolished these differences in microtubule assembly pattern and enhanced self-assembly. The kinetic analysis of microtubule elongation of different beta tubulin isotypes also showed significant differences. Elongation of alpha beta III from microtubule seeds had a lower apparent K alpha and a lower apparent Kd than did alpha beta II and alpha beta IV. The dynamic behaviors of different beta tubulin isotypes were qualitatively similar to each other and fit the dynamic instability model. However, microtubules formed from alpha beta III appeared to be less dynamic than microtubules formed from other beta tubulin isotypes. Our results suggest that the beta III isotype might have a different conformation than do the other beta tubulin isotypes. The distinctive nucleation and elongation behaviors of the alpha beta III dimers demonstrated in vitro may have a significant influence on microtubule functions in vivo.

Microtubule-associated protein 2 alters the dynamic properties of microtubule assembly and disassembly

The influence of microtubule-associated protein 2 (MAP2) on the dynamics of microtubule assembly and disassembly from axonemal fragments was characterized in vitro in solutions of pure tubulin and varying concentrations of MAP2. A mechanistic description of interactions between MAP2 and individual microtubules was developed from analysis of recorded images obtained by video-enhanced differential-interference-contrast light microscopy. MAP2 decreased the rates and lengths of shortening events and decreased the frequency of transitions between growth and shortening over a wide range of concentrations, thereby producing the increases in net microtubule growth previously described by light-scattering techniques. Increases in rates and lengths of elongation phases, as well as rescue frequencies (i.e. transition from shortening to growth), were observed under conditions in which microtubules are expected to be saturated with MAP2. During early stages of nucleated assembly, MAP2 greatly increased the number of microtubules growing from a given axoneme and caused elongation of "curved" structures which may be sheet-like microtubules.

Effects of the fluorescence dye DAPI on microtuble structure in vitro: Formation of novel types of tubulin assembly products

It has been found that the DNA fluorescence dye 4',6-diamidino-2-phenylindole (DAPI) is able to stain also microtubules. However, electron microscopy revealed that DAPI changed microtubule structure and induced the formation of a broad spectrum of polymorphic tubulin assembly products. Upon addition of DAPI to microtubules assembled from 10 to 15 mumol tubulin (molar DAPI/tubulin ratios of 10 to 40) in the presence of microtubule-associated proteins, most of the microtubules were decorated with additional protofilaments usually running parallel to the protofilaments of the microtubule wall (microtubule-protofilament complexes). When DAPI was already present during assembly, curved C- and S-shaped protofilament ribbons and microtubule-ribbon complexes with 6-shaped profiles were the most prominent products, beside microtubules. Additionally, protofilament bundles, some flat sheets, and hoops occurred. Electrophoresis revealed that DAPI lowered the amount of associated proteins, especially of tau-proteins, bound to the assembly products. Nevertheless, DAPI stimulated the assembly, enabled pure tubulin to assemble even at concentrations as low as 10 mumol, and stabilized the assembly products against cold. The microtubule-protofilament complexes, observed for the first time, are interpreted as the result of DAPI-induced protofilament linking as well as of activation of an additional tubulin-tubulin binding site which is possibly identical to that involved in the formation of microtubule doublets.

Brain microtubule-associated proteins modulate microtubule dynamic instability in vitro. Real-time observations using video microscopy.

We used video assays to study the dynamic instability behavior of individual microtubules assembled in vitro with purified tau, purified MAP2 or a preparation of unfractionated heat-stable MAPs. Axoneme-nucleated microtubules were assembled from pure tubulin at concentrations between 4 and 9 microM in the presence of MAPs, and observed by video-differential interference contrast microscopy. Microtubules co-assembled with each MAP preparation exhibited the elongation and rapid shortening phases and the abrupt transitions (catastrophe and rescue) characteristic of dynamic instability. Each MAP preparation increased the microtubule elongation rate above that for purified tubulin alone by decreasing the tubulin subunit dissociation rate during elongation. The brain MAPs used in this study reduced the rate of microtubule rapid shortening, but allowed significant loss of polymer during the shortening phase. Purified tau and MAP2 decreased the frequency of catastrophe and increased the frequency of rescue, while the heat-stable MAPs suppressed catastrophe at all but the lowest tubulin concentrations. Thus, each of these MAPs modulates, but does not abolish, dynamic instability behavior of microtubules. We propose a model to explain how MAP2 and tau bind to the microtubule lattice at sites along protofilaments so that the MAPs promote polymerization, but do not significantly block the mechanism of rapid shortening inherent in the tubulin lattice. Rapid shortening, when it occurs, proceeds primarily by the dissociation of short fragments of protofilaments, which contain the bound MAPs.

X-ray solution scattering studies on vinblastine-induced polymers of microtubule protein: structural characterisation and effects of temperature

We report here on X-ray solution scattering and electron microscopy studies of microtubule protein in the presence of the antimitotic drug, vinblastine. In buffer conditions used for microtubule assembly, vinblastine caused the formation of coil-like structures. The coils appeared to be made up of two protofilaments. Details of the structure and behaviour of coils in solution were obtained from interpretation of their solution scattering patterns. Upon increasing temperature from 4 to 37°C the pitch of the coils increased from 25.92 to 26.96 nm. However, little change was observed in their mean diameters (38.46 and 38.45 nm, respectively). Increasing the temperature also favoured increased formation and/or elongation of the coils. The effect of temperature on the pitch was fully reversible. Vinblastine-induced assembly of pure tubulin also showed the formation of coils. However, these coils appeared to consist of only one proto-filament. Their mean diameters (38.35 nm) were similar to those of the coils formed from microtubule protein.

Aging of tubulin at neutral pH: Stabilization by colchicine and its analogues

The effect of colchicine and its analogues, allocolchicine, 2,3,4-trimethoxy-4′-carbomethoxy-1, 1′biphenyl, 2,3,4,4′-tetramethoxy-1,1′-biphenyl, 2,3,4-trimethoxy-4′-acetyl-1, 1′-biphenyl, and tropolone methyl ether, on the aging process of tubulin has been examined. In contrast to the vinca alkaloid drugs which accelerate the formation of the paucidisperse 9 S polymers by a factor of 3.5, the colchicine class of ligands stabilize α,β-tubulin. Less than 10% of the protein is transformed into the aggregates after 50 h of incubation in the presence of 1 × 10 −3 m colchicine, as compared to nearly 70–75% transformation in its absence. These results are supported by fluorescence examination of the retention of colchicine binding ability, as well as circular dichroism spectroscopy. In the presence of colchicine, the rate determining step is a conformational change, just as in its absence. The colchicine analogues which bind to tubulin in a rapidly reversible equilibrium were almost as effective in tubulin stabilization. Addition of vincristine to the system reduced the stability of the tubulin-colchicine complex. Furthermore, vincristine was found to have the same effects on the fresh complex as it does on pure tubulin; i.e., it induced the isodesmic linear polymerization and inhibited assembly into the microtubule-mimicking large polymers. This inhibition, however, was stoichiometric, whereas it is substoichiometric in the case of microtubules.

Microtubules grow and shorten at intrinsically variable rates.

Microtubules were assembled from pure tubulin with axonemal pieces serving as nuclei. They were observed by video-enhanced differential-interference-contrast light microscopy. Their lengths were measured from videotaped images at frequent intervals (0.13-5 s). Error analysis indicated that the uncertainty in measuring a single length was quite small; the 95% confidence limit approximated the microscope's limit of resolution. Rates of growth and shortening of the dynamically unstable microtubules, obtained from the length-versus-time data, were found to be highly variable. The variability was far too large to be attributed to known random error of measurement and must be a property of the microtubules. Further experiments were aimed at finding its structural cause. The variability of rates exhibited by a single microtubule was as great as that of the whole population. The locations at which a growing microtubule changed its rate of growth were not related to the locations at which rates changed during its subsequent shortening. The cause of the variability must therefore be both small relative to the size of a microtubule and transient relative to its lifetime. Fluctuations in configuration of the microtubule's cap appear to be the likeliest source.

Regulation of the microtubule nucleating activity of centrosomes in Xenopus egg extracts: role of cyclin A-associated protein kinase.

Isolated centrosomes nucleate microtubules when incubated in pure tubulin solutions well below the critical concentration for spontaneous polymer assembly (approximately 15 microM instead of 60 microM). Treatment with urea (2-3 M) does not severely damage the centriole cylinders but inactivates their ability to nucleate microtubules even at high tubulin concentrations. Here we show that centrosomes inactivated by urea are functionally complemented in frog egg extracts. Centrosomes can then be reisolated on sucrose gradients and assayed in different concentrations of pure tubulin to quantify their nucleating activity. We show that the material that complements centrosomes is stored in a soluble form in the egg. Each frog egg contains enough material to complement greater than 6,000 urea-inactivated centrosomes. The material is heat inactivated above 56 degrees C. One can use this in vitro system to study how the microtubule nucleating activity of centrosomes is regulated. Native centrosomes require approximately 15 microM tubulin to begin nucleating microtubules, whereas centrosomes complemented in interphase extracts begin nucleating microtubules around 7-8 microM tubulin. Therefore, the critical tubulin concentrations for polymer assembly off native centrosomes is higher than that observed for the centrosomes first denatured and then complemented in egg extracts. In vivo, the microtubule nucleating activity of centrosomes seems to be regulated by phosphorylation at the onset of mitosis (Centonze, V. E., and G. G. Borisy. 1990. J. Cell Sci. 95:405-411). Since cyclins are major regulators of mitosis, we tested the effect of adding bacterially produced cyclins to interphase egg extracts. Both cyclin A and B activate an H1 kinase in the extracts. Cyclin A-associated kinase causes an increase in the microtubule nucleating activity of centrosomes complemented in the extract but cyclin B does not. The critical tubulin concentration for polymer assembly off centrosomes complemented in cyclin A-treated extracts is similar to that observed for centrosomes complemented in interphase extracts. However, centrosomes complemented in cyclin A treated extracts nucleate much more microtubules at high tubulin concentration. We define this as the "capacity" of centrosomes to nucleate microtubules. It seems that the microtubule nucleating activity of centrosomes can be defined by two distinct parameters: (a) the critical tubulin concentration at which they begin to nucleate microtubules and (b) their capacity to nucleate microtubules at high tubulin concentrations, the latter being modulated by phosphorylation.

Preparation of a monoclonal antibody specific for the class IV isotype of beta-tubulin. Purification and assembly of alpha beta II, alpha beta III, and alpha beta IV tubulin dimers from bovine brain.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.

Microtubule assembly protects the region 28-38 of the beta-tubulin subunit.

Polyclonal antibodies have been raised against the peptide 28-38 of the beta-subunit of the tubulin heterodimer in order to study the accessibility of this region in the tubulin heterodimer and in various tubulin assemblies. These antibodies were specific for all beta-tubulin subunits, except for beta'-tubulin isotypes, and did not recognize the alpha-tubulin subunit. The 28-38 region does not play a role in the interaction between the alpha- and beta-subunits since it was accessible to the antibodies on the native heterodimer. The accessibility of the antibodies was not modified by several microtubular poisons. In contrast, in all tubulin assemblies obtained in the presence of microtubular associated proteins, the region 28-38 was not available to the antibodies. These antibodies did not react with microtubules or tubulin spirals assembled either from microtubule proteins or from pure tubulin when these tubulin assemblies were probed in the absence of free tubulin after centrifugation on glass coverslips. In addition, antibodies failed to interact with the microtubule cytoskeleton in cultured Ptk2 cells indicating that the 28-38 region of beta-tubulin is also protected in cellular structures. These observations suggest that the 28-38 region of the beta-tubulin subunit is either located in a zone of interaction between two successive tubulin dimers within a protofilament or hidden by an allosteric conformational change which occurs during tubulin assembly.

Kinetics of the spontaneous organization of microtubules in solution

Optically anisotropic zones occur spontaneously in solutions of microtubules. These tactoids, in which microtubules are arranged in parallel arrays, can be visualized by their birefringence. With microtubules assembled in the presence of associated proteins (MAPs), birefringence appears immediately after nucleation of polymerization, even at relatively low protein concentrations. It is not dependent on whether the assembly is initiated by temperature jump or by isothermal addition of GTP. With pure tubulin, assembled in buffers containing 25% glycerol or 4% dimethylsulfoxide and/or taxol, birefringence appears within a few hours, but it can be speeded up by gentle agitation. With tubulin assembled in the presence of MAPs, spontaneous orientation occurs simultaneously with polymerization. This may be due to the existence of more pronounced repulsive forces between microtubules when they are covered with MAPs. A simple calculation of the covolume, suggests that tactoid formation is expected for microtubules of lengths of 5 to 10 microns at protein concentrations in the range 1 to 3 mg/ml (as observed), and that repulsive forces will promote tactoid formation at even lower protein concentrations.

Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex.

Complete replacement of the nucleotide on the exchangeable binding site of purified calf brain tubulin by the non-hydrolyzable GTP-analogue guanylyl-(beta,gamma-methylene)diphosphonate (GMPPCP) has been achieved by treatment of tubulin-GDP with phosphodiesterase-free alkaline phosphatase. GMPPCP binds to tubulin with a low affinity relative to GTP or GDP. Binding of the analogue is linked to magnesium ion concentration and, like the binding of other guanine nucleotides, is promoted by high concentrations of glycerol. The complex of pure tubulin and GMPPCP readily assembles at 37 degrees C into microtubules or curled ribbons of protofilaments, depending on buffer composition. Assemblies are cold-reversible at 0-2 degrees C, and multiple reversible assemblies can be observed during repeated heating/cooling cycles.

Characterization of microtubule protofilament numbers: How does the surface lattice accommodate?

Frozen-hydrated specimens of microtubules assembled in vitro were observed by cryo-electron microscopy. Specimens were of both pure tubulin, and of microtubule protein isolated by three cycles of assembly and disassembly. It is shown that the characteristic image contrast of individual microtubules allows the microtubule protofilament number to be determined unambiguously. Microtubules with 13, 14 and 15 protofilaments are observed to coexist in specimens prepared under various assembly conditions. Confirmation of these results is obtained by observations of thin sections of pelleted samples fixed and stained using the glutaraldehyde/tannic acid technique. Images of individual microtubules show both characteristic contrast profiles across their width and typical variations of these profiles along their length. The profiles across the images indicate the protofilament number of the microtubule. The lengthwise variations indicate how the protofilaments are aligned with respect to the microtubule axis giving what has previously been called a supertwist. In 13 protofilament microtubules the protofilaments are paraxial. In 14 and 15 protofilament microtubules, the protofilaments are skewed with respect to the microtubule axis. The skew is greater for the 15 protofilament case than for 14 protofilaments. The skew allows the extra protofilaments to be accommodated by the surface lattice. These results should also be relevant to situations in vivo.

The effect of Podophyllotoxin on Microtubule Dynamics

We have investigated the effects of podophyllotoxin on the dynamic properties of microtubules assembled from pure tubulin dimer. Excess podophyllotoxin causes the complete disassembly of microtubules, through formation of a tubulin-GTP-podophyllotoxin ternary complex with a dissociation rate constant of 160 s-1 at 37 degrees C, similar to that found upon extensive isothermal dilution in this buffer system. Addition of substoichiometric concentrations of podophyllotoxin causes partial disassembly of microtubules through production of an equivalent amount of the ternary complex. Microtubule length measurements and incorporation of [3H]GTP-tubulin dimer show that podophyllotoxin can suppress the dynamic instability of tubulin dimer microtubules and that it acts substoichiometrically in so doing. We interpret the action of substoichiometric podophyllotoxin on microtubule ends in terms of effects on interconversion of growing and shrinking microtubules in a dynamic system in which tubulin-GTP-podophyllotoxin is kinetically analogous to tubulin-GTP in addition and to tubulin-GDP in dissociation. The ability to suppress dynamic instability may be one way in which drugs such as podophyllotoxin, acting at relatively low concentrations, are able to arrest cell growth and development in a selective way, without necessarily affecting the integrity of the major part of the cytoskeletal microtubule network.