Absence Of Microtubule-associated Proteins Research Articles

Reconstitution of Neuronal Motor Traffic on Septin-Associated Microtubules.

Reconstitution of intracellular transport in cell-free in vitro assays enables the understanding and dissection of the molecular mechanisms that underlie membrane traffic. Using total internal reflection fluorescence (TIRF) microscopy and microtubules, which are immobilized to a functionalized glass surface, the kinetic properties of single kinesin molecules can be imaged and analyzed in the presence or absence of microtubule-associated proteins. Here, we describe methods for the in vitro reconstitution of the motility of the neuronal kinesin motor KIF1A on microtubules associated with heteromeric septin (SEPT2/6/7) complexes. This method can be adapted for various neuronal septin complexes and kinesin motors, leading to new insights into the spatial regulation of neuronal membrane traffic by microtubule-associated septins.

Ceratamines, Structurally Simple Microtubule-Stabilizing Antimitotic Agents with Unusual Cellular Effects

Ceratamine A and ceratamine B are heterocyclic alkaloids recently identified in a screen for compounds that arrest cells in mitosis. Treatment of breast carcinoma MCF-7 cells causes a concentration-dependent block of cell cycle progression exclusively at mitosis. In vitro studies with purified tubulin indicate that the ceratamines directly stimulate microtubule polymerization in the absence of microtubule-associated proteins. Cells treated with ceratamines show a dense perinuclear microtubule network in interphase and multiple pillar-like tubulin structures in mitotic cells. The ceratamines do not compete with paclitaxel for binding to microtubules in vitro. Unlike other microtubule-stabilizing agents, the ceratamines have simple structures with no chiral centers, making them attractive drug leads.

Cytoplasmic dynein mediates adenovirus binding to microtubules.

During infection, adenovirus (Ad) capsids undergo microtubule-dependent retrograde transport as part of a program of vectorial transport of the viral genome to the nucleus. The microtubule-associated molecular motor, cytoplasmic dynein, has been implicated in the retrograde movement of Ad. We hypothesized that cytoplasmic dynein constituted the primary mode of association of Ad with microtubules. To evaluate this hypothesis, an Ad-microtubule binding assay was established in which microtubules were polymerized with taxol, combined with Ad in the presence or absence of microtubule-associated proteins (MAPs), and centrifuged through a glycerol cushion. The addition of purified bovine brain MAPs increased the fraction of Ad in the microtubule pellet from 17.3% +/- 3.5% to 80.7% +/- 3.8% (P < 0.01). In the absence of tubulin polymerization or in the presence of high salt, no Ad was found in the pellet. Ad binding to microtubules was not enhanced by bovine brain MAPs enriched for tau protein or by the addition of bovine serum albumin. Enhanced Ad-microtubule binding was also observed by using a fraction of MAPs purified from lung A549 epithelial cell lysate which contained cytoplasmic dynein. Ad-microtubule interaction was sensitive to the addition of ATP, a hallmark of cytoplasmic dynein-dependent microtubule interactions. Immunodepletion of cytoplasmic dynein from the A549 cell lysate abolished the MAP-enhanced Ad-microtubule binding. The interaction of Ad with both dynein and dynactin complexes was demonstrated by coimmunoprecipitation. Partially uncoated capsids isolated from cells 40 min after infection also exhibited microtubule binding. In summary, the primary mode of Ad attachment to microtubules occurs though cytoplasmic dynein-mediated binding.

755. Cytoplasmic Dynein-Dependent Adeno-Associated Virus Interaction with Microtubules

The importance of adeno-associated virus (AAV)-based gene therapy vectors as therapeutic vehicles is derived in part from efficient cell membrane penetration and translocation of the adeno-associated virus capsid to the nucleus where its genetic cargo is delivered. Other viruses have enhanced intracellular trafficking to the nucleus through interaction with the microtubule cytoskeleton with subsequent energy-dependent translocation in a retrograde manner towards the nucleus. The retrograde molecular motor, cytoplasmic dynein, participates in the microtubule-based nuclear-directed translocation of numerous viruses. Based on the hypothesis that the cytoplasmic dynein motor complex mediates a biochemical linkage of the AAV capsid with microtubules, a microtubule binding assay was developed in which binding of fluorophore-conjugated AAV capsids to microtubules was evaluated by mixing fluorophore-conjugated AAV capsid with polymerized microtubules, centrifuging to pellet the microtubules, and measuring the presence of the AAV-associated fluorescence in either the microtubule-free supernatant or microtubule-containing pellet. SDS-polyacrylamide gel electrophoresis of supernatant and pellet fractions showed that fluorophore-labeled AAV capsid was located predominantly in the supernatant when AAV was incubated with taxol-stabilized microtubules. However, when purified bovine microtubule-associated proteins were combined with microtubules, the majority of AAV was found in the pellet after centrifugation (69 ± 4 % vs 23 ± 4% in the absence of microtubule-associated proteins). The interaction of microtubule-associated proteins and AAV with microtubules did not occur in the presence of 500 mM NaCl. Using freshly isolated A549 lung epithelial cell lysate, the direct involvement of cytoplasmic dynein was evaluated. A549 cell lysate was depleted of endogenous cytoplasmic dynein by immunoprecipitation with a monoclonal antibody against the 74.1 kDa intermediate chain of cytoplasmic dynein. Microtubule-associated proteins isolated from whole A549 cell lysate versus cytoplasmic dynein-depleted A549 cell lysate were compared for the ability to support AAV-microtubule interaction. Whereas AAV capsid pelleted with microtubules when incubated with microtubule-associated proteins from whole A549 cell lysate, AAV capsid was found predominantly in the supernatant when cytoplasmic dynein-depleted cell lysate was used. No effect was observed when an irrelevant primary antibody was used for immunprecipitation. To confirm the involvement of cytoplasmic dynein in AAV-microtubule interaction, the nucleotide-sensitive binding property of cytoplasmic dynein was utilized. Under conditions that are known to disrupt cytoplasmic dynein interaction with microtubules (10 mM ATP), the efficiency of the AAV-microtubule interaction was reduced. However, the presence of an identical concentration of AMP-PNP, a non-hydrolyzable analog of ATP, had no effect on AAV-microtubule interactions. The results of this study suggest that the AAV capsid interacts with microtubules in a cytoplasmic dynein-dependent manner, providing a potential mechanism for the efficient translocation of the AAV capsid to the nucleus during infection.

Individual expression of recombinant alpha- and beta-tubulin from Haemonchus contortus: polymerization and drug effects.

Three tubulin isotypes from the parasitic nematode Haemonchus contortus were individually expressed in Escherichia coli, purified, and induced to polymerize into microtubules in the absence of microtubule-associated proteins. The effect of different conditions on the rate of polymerization of pure tubulin was assessed. This is the first time that recombinant alpha-tubulin has been shown to be capable of polymerization into microtubule-like structures when incubated with recombinant beta-tubulin. In addition, the present study has shown that: (1) microtubule-associated proteins are not required for tubulin polymerization; and (2) pure beta-tubulin isotype, beta12-16, alone was capable of forming microtubule-like structures in the absence of alpha-tubulin. Polymerization of the recombinant invertebrate tubulin, as measured by a spectrophotometric assay, was found to be enhanced by a concentration of tubulin >0.25 mg/mL; temperature > or =20 degrees C; 2 mM GTP; glycerol; EGTA; and Mg(2+). Polymerization was inhibited by GTP (>2 mM) and albendazole. Calcium ions and a pH range of 6 to 8.5 had no measurable effect on polymerization. Individual isotypes of tubulin polymerized to approximately the same extent as an alpha-/beta-tubulin mixture. Samples of tubulin assembled under the above conditions for 60 min were also examined under a transmission electron microscope. Although the spectrophotometric assay indicated polymerization, it did not predict the structure of the polymer. In many cases tubulin sheets, folded sheets, and rings were observed in addition to, or instead of, microtubule-like structures.

Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals

Vinblastine, vincristine, vindesine, and vinorelbine, the four vinca alkaloids used in cancer therapy, differ in their antitumoral spectra and toxicities, but not in their inhibitory effects on microtubule assembly in vitro. At higher drug concentrations, vinca alkaloids induce the assembly of spiral filaments of tubulin, which, in turn, can interact laterally and form paracrystals. Using methods that distinguish spiral filaments and paracrystals (aggregated spirals), we found that spiral filament formation was largely independent of the incubation temperature, of the alkaloid used, and of the presence or absence of microtubule-associated proteins (MAPs). In contrast, the formation of aggregated spirals was markedly dependent on the alkaloid used, on the incubation temperature, and on the absence or presence of MAPs. Aggregated spirals failed to assemble in the presence of high concentrations of MAP-1A or MAP-1B, whereas they assembled readily with tau and MAP-2. Differences in patterns of turbidity development using pure tubulin allowed the classification of thirteen cytotoxic vinca alkaloids into five distinct groups, with centrifugal recovery of aggregated spirals in close agreement with the various turbidity patterns. With microtubule protein, i.e. tubulin preparations containing MAPs, only four groups were defined by turbidity patterns, and centrifugal protein recovery was more divergent. Vinblastine, vincristine, vindesine, and vinorelbine fell into distinct groups under both reaction conditions, and thus they appear to have qualitatively distinguishable in vitro interactions with tubulin. These differential effects on spiral filament and aggregated spiral assembly revealed that the four drugs induce different constraints on the tubulin molecule.

Differential Assembly Kinetics of α-Tubulin Isoforms in the Presence of Paclitaxel

The antitumor drug paclitaxel (PTX) inhibits cell growth by binding to microtubules, the eukaryotic structures consisting of α- and β-tubulin. PTX also promotes the assembly of tubulin in the absence of microtubule-associated proteins. Although recent studies have implicated β-tubulin as the site of PTX binding, no information is available that relates α-tubulin to the binding site. In an effort to understand whether the α-tubulin is involved in the drug binding, we have studied the assembly of α-tubulin isoforms in the presence of PTX. The assembly results in the presence of 10 μM paclitaxel (PTX) show that the isoforms assemble at differential rates. The rate of assembly for tyrosinated Mα1/2 is about three-fold higher than that of the nontyrosinated Mα1/2 isoform. Such a strikingly different assembly behavior of the α-tubulin isoforms indicates that α-tubulin may be involved in the interaction of PTX with microtubules.

Coassembly of bovine and cod microtubule proteins: the ratio of the different tubulins within hybrid microtubules determines the ability to assemble at low temperatures, MAPs dependency and effects of Ca2+.

Cod and bovine microtubule proteins (MTP) differ from each other in many respects, e.g., tubulin isoforms and microtubule-associated proteins (MAPs) but only cod MTP are cold-adapted. We used these differences to determine how tubulin isoform composition affects microtubule properties. Mixtures of cod and bovine MTP coassembled at 30 degrees C as shown by light scattering and immunoelectron microscopy, with no apparent preference for one set of MAPs over the other. Bovine tubulin was, in contrast to cod tubulin, unable to assemble in the absence of MAPs, while 50%/50% mixtures of bovine and cod tubulin, respectively, coassembled readily without exclusion of cod or bovine tubulin isoforms in the hybrids, as shown by two-dimensional gel electrophoresis. Alteration in MAPs dependency was also confirmed by the use of the MAPs-binding microtubule inhibitor estramustine phosphate. Addition of 10 mM Ca2+ to microtubules induced formation of spirals or rings depending on the ratio of the cod and bovine MTP, respectively. Bovine MTP were unable to assemble at low temperatures, while cod MTP are cold-adapted and assembled efficiently at 14 degrees C in the presence of MAPs. Amounts of cod MTP as low as 33% were enough to induce assembly of bovine/cod MTP hybrids. The critical concentration for assembly of a 50%/50% mixture was similar to that of 100% cod MTP. Taken together, the results show that the divergent cod and bovine MTP can coassemble, and that alterations in tubulin isotype/isoform composition above certain thresholds significantly modulate microtubule properties such as MAPs dependency, effects of Ca2+, and ability to assemble at low temperatures.

(+)-Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest

Background: The marine natural product (+)-discodermolide has potent immunosuppressive activity. It inhibits proliferation of a wide range of human and murine cells, induces cell cycle arrest in the G2 or M phase and was recently shown to stabilize microtubules. Total synthesis of discodermolide has made it possible to generate variants of the compound to study its intracellular function in detail. Results: We have determined that (+)-discodermolide arrests MG63 cells at M phase, and has a stabilizing effect on microtubules. In vitro studies show that discodermolide induces polymerization of purified tubulin in the absence of microtubule-associated proteins, and that it binds to tubulin dimers in microtubules at 1:1 stoichiometry. Discodermolide binds taxol-polymerized microtubules at near stoichiometric level, whereas taxol binds discodermolide-induced microtubules poorly. Competition data show that the binding of microtubules by discodermolide and taxol are mutually exclusive; discodermolide binds with higher affinity than taxol. The results of binding assays carried out in vivo or in cell lysates also suggest that the microtubule network is discodermolide's cellular target. Condusions: (+)-Discodermolide causes cell cycle arrest at the metaphase-anaphase transition in mitosis, presumably due to its stabilizing effect on microtubules. In vitro, discodermolide polymerizes purified tubulin potently in the absence of MAPs. It binds microtubules at one molecule per tubulin dimer with a higher affinity than taxol, and the binding of microtubules by discodermolide and taxol are mutually exclusive. In total cell lysates discodermolide displays binding activity that is consistent with its effects on microtubules.

Phosphorylation of beta III-tubulin.

There is considerable evidence that mammalian beta-tubulin is phosphorylated. Specifically, of the seven beta isotypes, the phosphorylated one is beta III, the isotype found almost entirely in neurons. The phosphate is added at a serine and perhaps a tyrosine near the C-terminus. All the evidence to date has been gathered by growth of cells and tissues in the presence of radioactive inorganic phosphate followed by tubulin isolation and determination of the labeled tubulin; thus, the actual extent of phosphorylation of beta III is unknown. Nor is it known if alpha-tubulin and the other beta isotypes are phosphorylated by a mechanism which would not be revealed by previous experiments. In addition, the role of tubulin phosphorylation is unknown. We have purified the alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers from bovine brain and have determined their phosphate content chemically. We have found that alpha-tubulin is not phosphorylated and neither are the beta II or beta IV isotypes. However, beta III is phosphorylated with a stoichiometry of about 1.52 mol/mol. We have found that the phosphate on beta III is resistant to a wide variety of phosphatases except for human erythrocyte phosphatase 2A and that removal of the phosphate inhibits microtubule assembly in vitro stimulated by microtubule-associated protein 2 (MAP 2). However such an inhibition was not evident when microtubule assembly was induced in the absence of microtubule-associated proteins. Our results suggest the possibility that beta III phosphorylation may play a role in regulating microtubule assembly in vivo.

Microtubule-stabilizing activity of microtubule-associated proteins (MAPs) is due to increase in frequency of rescue in dynamic instability: shortening length decreases with binding of MAPs onto microtubules.

The role of microtubule associated proteins (MAPs) on the dynamic instability of microtubules was examined under a dark-field microscope using bovine brain tubulin purified by DEAE-Sepharose column chromatography. In the absence of MAPs, the transition from the shortening phase to the growing phase (the rescue) occurred rarely both in self-assembled microtubules and seeded ones, especially at the plus end. Even under the conditions unfavorable to stabilize microtubule, the addition of a small amount of crude MAPs or purified microtubule associated protein 2 (MAPs) to the microtubules allowed them to undergo the rescue. At increased concentrations of MAPs or MAP2, both the length change required for a rescue during shortening phase ("shortening length") and for a catastrophe (transition from the growing to the shortening phase) ("growth length") decreased. Under these conditions, the rescue often occurred at the same site where previous rescues occurred. Distribution of immunofluorescent MAP2 antibodies along individual microtubules showed that MAP2 molecules bound onto microtubules by forming discrete clusters. The number of MAP2 molecules per cluster was estimated to be between 25 and 60. Because both the "shortening length" and the distance between MAP2 clusters in a microtubule decreased with increased MAPs concentration, we suggest that the MAP2 clusters may form the specific site at which the shortening of the microtubule readily stops. MAP2 possibly regulates the dynamic instability by stopping the shortening, which is a prerequisite for the rescue.

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.

Comparative Effects of Cryosolvents on Tubulin Association, Thermal Stability, and Binding of Microtubule-Associated Proteins

Organic cryosolvents essential for cryopreservation of living cells have a colligative effect on water properties, but also affect cellular structures such as the membrane, actin, or tubulin cytoskeleton. The effects of cryosolvents on actin and its binding proteins are starting to be well investigated. In parallel, tubulin assembly characteristics were investigated comparatively, with 0-30% 1,2-propanediol, dimethyl sulfoxide, or glycerol, and with or without microtubule-associated proteins, at 37 or 4°C. Tubulin association was monitored by spectrometry and sedimentation, providing the concentration in free protein, cold-depolymerizable microtubules, and cold-resistant associations. At 37°C, 1,2-propanediol and dimethyl sulfoxide induce a similar association level and cold stability of the assemblies. Glycerol yields a lower level of tubulin association. Cold stability of the assemblies requires the presence of solvent, the amount of which is modulated by microtubule-associated proteins (MAPs): 15% 1,2-propanediol or dimethyl sulfoxide, decreasing down to 10% with MAPs\\, or 10% glycerol with MAPs only. At 4°C, some cold-stable association is promoted by 1.2-propanediol or dimethyl sulfoxide above 10-15%, in the presence or absence of MAPs, but not with glycerol. In addition, protein content of the various fractions obtained with MAPs and 30% solvent was examined by densitometry of electrophoresis gels. Cold-labile associations obtained at 37°C with 1,2-propanediol or dimethyl sulfoxide are lacking in tubulin and enriched in τ proteins relative to control or glycerol. Associations formed at 37°C and stable to subsequent cold treatment, or at 4°C, regardless of the solvent, present a large tubulin content, as well as few τ proteins and high-molecular-weight MAPs.

Conformational properties of the .beta.(400-436) and .beta.(400-445) C-terminal peptides of porcine brain tubulin

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.

Substoichiometric inhibition of microtubule formation by acetaldehyde-tubulin adducts

We have shown previously that acetaldehyde forms stable covalent adducts with tubulin, resulting in impaired microtubule formation. The present study explored the mechanism responsible for impaired microtubule formation caused by the substoichiometric stable binding of acetaldehyde to tubulin. The free tubulin dimer was much more reactive with acetaldehyde than microtubules, binding more than twice as much aldehyde. The dimer also formed nearly twice as many stable adducts on its α-chain as on its β-chain, whereas microtubules exhibited an equal distribution of adducts between the two subunits. These data confirm that the α-chain of free tubulin, but not microtubules, has an accessible highly reactive lysine (HRL) residue that is a preferential target of acetaldehyde binding. Adduct formation with the HRL residue also correlated with impaired tubulin polymerization, and only 0.08 moles of acetaldehyde bound per mole of HRL was required for complete inhibition; however, adducts with other lysine residues (bulk adducts) did not affect assembly. Adducts to microtubule-associated proteins (MAPs) also impaired the assembly of tubulin, but were much less effective than HRL adducts. In a copolymerization assay, HRL-adducted tubulin, in addition to being itself assembly incompetent, also interfered with polymerization of normal (unadducted) tubulin. Bulk adducts did not alter assembly and were incorporated normally into the growing polymer. When tubulin was cleaved by the proteolytic enzyme, subtilisin, microtubule formation could readily take place in the absence of MAPs. In this polymerization system, HRL adducts, but not bulk adducts, still markedly inhibited assembly. When low concentrations of acetaldehyde (50 μM) were used to generate HRL adducts, an adduct on only 1 out of 20 tubulin molecules was sufficient to totally block polymerization. These findings indicate that substoichiometric amounts of acetaldehyde bound to HRL of tubulin can markedly inhibit microtubule formation via direct interference of dimer-dimer interactions, and further suggest that low concentrations of acetaldehyde could generate sufficient amounts of HRL adducts in cellular systems to alter microtubule formation and function.

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua.

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.

Peptides corresponding to the second repeated sequence in MAP-2 inhibit binding of microtubule-associated proteins to microtubules

Bovine brain high molecular weight microtubule-associated proteins (MAPs) can be displaced from assembled tubules by peptides corresponding to the second of three nonidentical repeated sequences in mouse MAP-2. The octadecapeptide m2 (VTSKCGSLKNIRHRPGGG) can release MAP-1b from MAP-containing microtubules, and the extended second-sequence peptide m2' (VTSKCGSLKNIRHRPGGGRVK) displaces MAP-1a and MAP-1b as well as MAP-2a and MAP-2b. Peptides m2 and m2' stimulate tubulin polymerization in the absence of MAPs or microtubule-stabilizing agents, and m2' acts as a competitive inhibitor of radiolabeled MAP-2 binding. The dissociation constant for MAP-2 binding to taxol-stabilized tubules was 3.4 microM in the absence of m2' and 14 microM in the presence of 1.5 mM of the m2' peptide. We estimate that the inhibition constant for peptide m2' is about 0.5 mM, about 100 times lower than for the Km of MAP-2. These observations suggest that the second repeated sequence in MAP-2 may represent an important recognition site for MAP binding to microtubules and that other structural features within MAP-2 may reinforce the strength of MAP-microtubule interactions.

Effect of meso-hexestrol, a synthetic estrogen, on S-tubulin.

We have reported that meso-hexestrol, a synthetic estrogen, inhibits microtubule assembly and induces microtubule proteins into twisted ribbon structures. On the other hand, Serrano et al. proved that S-tubulin, which lacks the C-terminal moiety of tubulin subunits, assembles into sheet structures in the absence of microtubule-associated proteins (MAPs). In the present investigation, we attempted to clarify whether meso-hexestrol could induce the ribbon structure from S-tubulin. meso-Hexestrol delayed the initiation of polymerization of S-tubulin into sheet structures in a dose-dependent manner below 50 microM. But the effect of meso-hexestrol on S-tubulin was reduced in the presence of either tau or microtubule-associated protein 2 (MAP2) in a MAPs-concentration-dependent manner. At concentrations higher than 100 microM, meso-hexestrol inhibited the polymerization of S-tubulin into sheet structures, without forming ribbon structures. The present results may indicate that moso-hexestrol interacts with S-tubulin, and its interaction is affected by MAPs.

Microtubule solutions display nematic liquid crystalline structure.

We report a study of the spontaneous formation of ordered arrays of microtubules in solution. Form birefringence and anisotropic light-scattering appear rapidly and spontaneously when tubulin, initially present in homogeneous solution, self-assembles into microtubules. This phenomenon is reversible and occurs at protein concentrations of a few milligrams per ml, in the presence or absence of microtubule-associated proteins. Light and electron microscopic examination reveals that extensive regions of these birefringent solutions consist of nearly parallel microtubules. Measurement of the order parameter, S, yields a value of 0.81 +/- 0.05, indicating a high degree of alignment. Comparison of these observations to qualitative predictions developed from the theory of Onsager ((1949) Ann. N.Y. Acad. Sci. 51, 627-659) leads to the conclusion that microtubules form a nematic liquid crystalline phase in vitro under ordinary conditions. Simultaneous spectrophotometric observation of turbidity (a measure of microtubule assembly) and birefringence shows that the parallel ordering lags only slightly behind assembly, thus demonstrating that much microtubule growth must occur by addition of tubulin to the ends of microtubules that are already aligned. These observations of anisotropy are important to the understanding of microtubule dynamics in vitro.

Dynamic Instability of Sheared Microtubules Observed by Quasi-Elastic Light Scattering

The kinetics of microtubule reassembly was studied in vitro by quasi-elastic light scattering (QELS). When microtubules assembled in the absence of microtubule-associated proteins (MAPs) were sheared, they rapidly depolymerized, recovered, and reassembled. The mean length of the recovered microtubules was the same as that observed just before shearing, implying that on average one fragment per original microtubule survived the fragmentation and recovery. When microtubules that contained 25 percent brain MAP were sheared, the fragments did not depolymerize extensively and the average length of the fragments decreased by a factor of 3 relative to the unsheared sample. The results support the dynamic instability model, which predicts that cellular microtubules are latently unstable structures protected on their ends by stabilizing caps.