The AtMAP65-1 cross-bridge between microtubules is formed by one dimer

The microtubule-associated protein AtMAP65-1 from Arabidopsis thaliana dimerizes and forms 25 nm cross-bridges between microtubules, but the exact mechanism is unknown. Here, we used the predicted three-dimensional structure of AtMAP65-1 as a basis for analyzing the actual cross-bridging in detail. Fold-recognition predicts that AtMAP65-1 contains four coiled-coil domains and a flexible extended loop. The length of these coiled-coil domains is about 25 nm, suggesting that one molecule could span the gap, hence forming an antiparallel overlapping dimer instead of an end-to-end dimer. We then tested this model by using truncations of AtMAP65-1. EDC {[3-(dimethylamino) propyl] carbodiimide} cross-linking analysis indicated that the N-terminus of the rod domain of AtMAP65-1 (amino acids 1-339) binds to the C-terminus of the rod domain (amino acids 340-494) and also participates in connecting the two antiparallel proteins in the cross-bridge. Nevertheless, microtubules can still form bundles in the presence of AtMAP65-1 340-587 (amino acids 340-587) or AtMAP65-1 1-494 (amino acids 1-494). Comparing the cold stability of microtubule bundles induced by full-length AtMAP65-1 with that of AtMAP65-1 340-587 or AtMAP65-1 1-494, we conclude that AtMAP65-1 495-587 acts as a flexible extended loop, playing a crucial role in binding to and stabilizing microtubules in the AtMAP65-1 cross-bridge.     

Two microtubule-associated proteins of the Arabidopsis MAP65 family function differently on microtubules

The organization and dynamics of microtubules are regulated by microtubule-associated proteins, or MAPs. In Arabidopsis (Arabidopsis thaliana), nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might have distinct roles in the interaction with microtubules. In this study, two AtMAP65 proteins, AtMAP65-1 and AtMAP65-6, were chosen to test this hypothesis in vitro. Although both fusion proteins were able to cosediment with microtubules in vitro, different properties on tubulin polymerization and microtubule bundling were observed. AtMAP65-1 was able to promote tubulin polymerization, enhance microtubule nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large microtubule bundles by forming cross-bridges between microtubules evenly along the whole length of microtubules. In the presence of AtMAP65-1, microtubule bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, demonstrated no activity in promoting tubulin polymerization and stabilizing preformed microtubules. AtMAP65-6 induced microtubules to form a mesh-like network with individual microtubules. Cross-bridge-like interactions were only found at regional sites between microtubules. The microtubule network induced by AtMAP65-6 was more resistant to high concentration of NaCl than the bundles induced by AtMAP65-1. Purified monospecific anti-AtMAP65-6 antibodies revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis cells. It was concluded that these two MAP65 proteins were targeted to distinct sites, thus performing distinct functions in Arabidopsis cells.     

The Arabidopsis microtubule-associated protein AtMAP65-1: molecular analysis of its microtubule bundling activity

The 65-kD microtubule-associated protein (MAP65) family is a family of plant microtubule-bundling proteins. Functional analysis is complicated by the heterogeneity within this family: there are nine MAP65 genes in Arabidopsis thaliana, AtMAP65-1 to AtMAP65-9. To begin the functional dissection of the Arabidopsis MAP65 proteins, we have concentrated on a single isoform, AtMAP65-1, and examined its effect on the dynamics of mammalian microtubules. We show that recombinant AtMAP65-1 does not promote polymerization and does not stabilize microtubules against cold-induced microtubule depolymerization. However, we show that it does induce microtubule bundling in vitro and that this protein forms 25-nm cross-bridges between microtubules. We further demonstrate that the microtubule binding region resides in the C-terminal half of the protein and that Ala409 and Ala420 are essential for the interaction with microtubules. Ala420 is a conserved amino acid in the AtMAP65 family and is mutated to Val in the cytokinesis-defective mutant pleiade-4 of the AtMAP65-3/PLEIADE gene. We show that AtMAP65-1 can form dimers and that a region in the N terminus is responsible for this activity. Neither the microtubule binding region nor the dimerization region alone could induce microtubule bundling, strongly suggesting that dimerization is necessary to produce the microtubule cross-bridges. In vivo, AtMAP65-1 is ubiquitously expressed both during the cell cycle and in all plant organs and tissues with the exception of anthers and petals. Moreover, using an antiserum raised to AtMAP65-1, we show that AtMAP65-1 binds microtubules at specific stages of the cell cycle.     

Arabidopsis microtubule-associated protein AtMAP65-2 acts as a microtubule stabilizer

Nine genes that encode proteins of the MAP65 family have been identified in the Arabidopsis thaliana genome. In this study, we reported that AtMAP65-2, a member of the AtMAP65 family, could strongly stabilize microtubules (MTs). Bacterially-expressed AtMAP65-2 fusion proteins induced the formation of large MT bundles in vitro. Although AtMAP65-2 showed little effect on MT assembly or nucleation, AtMAP65-2 greatly stabilized MTs that were subjected to low-temperature treatment in vitro. Analyses of truncated versions of AtMAP65-2 indicated that the region that encompassed amino acids 495–578, which formed a flexible extended loop, played a crucial role in the stabilization of MTs. Analysis of suspension-cultured Arabidopsis cells that expressed the AtMAP65-2-GFP fusion protein showed that AtMAP65-2 co-localized with MTs throughout the cell cycle. Cortical MTs that were decorated with AtMAP65-2-GFP were more resistant to the MT-disrupting drug propyzamide and to ice treatment in vivo. The results of this study demonstrate that AtMAP65-2 strongly stabilizes MTs and is involved in the regulation of MT organization and dynamics. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. H. Li and X. Zeng have contributed equally to this paper and are considered as joint first authors.     

Microtubule nucleation from stable tubulin oligomers

Microtubule assembly from purified tubulin preparations involves both microtubule nucleation and elongation. Whereas elongation is well documented, microtubule nucleation remains poorly understood because of difficulties in isolating molecular intermediates between tubulin dimers and microtubules. Based on kinetic studies, we have previously proposed that the basic building blocks of microtubule nuclei are persistent tubulin oligomers, present at the onset of tubulin assembly. Here we have tested this model directly by isolating nucleation-competent cross-linked tubulin oligomers. We show that such oligomers are composed of 10-15 laterally associated tubulin dimers. In the presence of added free tubulin dimers, several oligomers combine to form microtubule nuclei competent for elongation. We provide evidence that these nuclei have heterogeneous structures, indicating unexpected flexibility in nucleation pathways. Our results suggest that microtubule nucleation in purified tubulin solution is mechanistically similar to that templated by gamma-tubulin ring complexes with the exception that in the absence of gamma-tubulin complexes the production of productive microtubule seeds from tubulin oligomers involves trial and error and a selection process.     

CRMP-2 binds to tubulin heterodimers to promote microtubule assembly

Regulated increase in the formation of microtubule arrays is thought to be important for axonal growth. Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33, mutations in which result in abnormal axon termination. We recently demonstrated that CRMP-2 is critical for axonal differentiation. Here, we identify two activities of CRMP-2: tubulin-heterodimer binding and the promotion of microtubule assembly. CRMP-2 bound tubulin dimers with higher affinity than it bound microtubules. Association of CRMP-2 with microtubules was enhanced by tubulin polymerization in the presence of CRMP-2. The binding property of CRMP-2 with tubulin was apparently distinct from that of Tau, which preferentially bound microtubules. In neurons, overexpression of CRMP-2 promoted axonal growth and branching. A mutant of CRMP-2, lacking the region responsible for microtubule assembly, inhibited axonal growth and branching in a dominant-negative manner. Taken together, our results suggest that CRMP-2 regulates axonal growth and branching as a partner of the tubulin heterodimer, in a different fashion from traditional MAPs.     

Taxol binds to polymerized tubulin in vitro

Taxol, a natural plant product that enhances the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells, was labeled with tritium by catalytic exchange with (3)H(2)O. The binding of [(3)H]taxol to microtubule protein was studied by a sedimentation assay. Microtubules assembled in the presence of [(3)H]taxol bind drug specifically with an apparent binding constant, K(app), of 8.7 x 19(-7) M and binding saturates with a calculated maximal binding ration, B(max), of 0.6 mol taxol bound/mol tubulin dimer. [(3)H]Taxol also binds and assembles phosphocellulose-purified tubulin, and we suggest that taxol stabilizes interactions between dimers that lead to microtubule polymer formation. With both microtubule protein and phosphocellulose- purified tubulin, binding saturation occurs at approximate stoichiometry with the tubulin dimmer concentration. Under assembly conditions, podophyllotoxin and vinblastine inhibit the binding of [(3)H]taxol to microtubule protein in a complex manner which we believe reflects a competition between these drugs, not for a single binding site, but for different forms (dimer and polymer) of tubulin. Steady-state microtubules assembled with GTP or with 5’-guanylyl-α,β-methylene diphosphonate (GPCPP), a GTP analog reported to inhibit microtubule treadmilling (I.V. Sandoval and K. Weber. 1980. J. Biol. Chem. 255:6966-6974), bind [(3)H]taxol with approximately the same stoichiometry as microtubules assembled in the presence of [(3)H]taxol. Such data indicate that a taxol binding site exists on the intact microtubule. Unlabeled taxol competitively displaces [(3)H]taxol from microtubules, while podophyllotoxin, vinblastine, and CaCl(2) do not. Podophyllotoxin and vinblastine, however, reduce the mass of sedimented taxol-stabilized microtubules, but the specific activity of bound [(3)H]taxol in the pellet remains constant. We conclude that taxol binds specifically and reversibly to a polymerized form of tubulin with a stoichiometry approaching unity.     

Determination of the net exchange rate of tubulin dimer in steady-state microtubules by fluorescence correlation spectroscopy

The microtubule cytoskeleton plays an important role in eukaryotic cells, e. g., in cell movement or morphogenesis. Microtubules, formed by assembly of tubulin dimers, are dynamic polymers changing randomly between periods of growing and shortening, a property known as dynamic instability. Another process characterizing the dynamic behaviour is the so-called treadmilling due to different binding constants of tubulin at both microtubule ends. In this study, we used tetramethylrhodamine (TMR)-labeled tubulin added to microtubule suspensions to determine the net exchange rate (NER) of tubulin dimers by fluorescence correlation spectroscopy (FCS) as a measure for microtubule dynamics. This approach, which seems to be suitable as a screening system to detect compounds influencing the NER of tubulin dimers into microtubules at steady-state, showed that taxol, nocodazole, colchicine, and vinblastine affect microtubule dynamics at concentrations as low as 10(-9)-10(-10) M.     

The C terminus of tubulin, a versatile partner for cationic molecules: binding of Tau, polyamines, and calcium

The C-terminal region of tubulin is involved in multiple aspects of the regulation of microtubule assembly. To elucidate the molecular mechanisms of this regulation, we study here, using different approaches, the interaction of Tau, spermine, and calcium, three representative partners of the tubulin C-terminal region, with a peptide composed of the last 42 residues of α1a-tubulin. The results show that their binding involves overlapping amino acid stretches in the C-terminal tubulin region: amino acid residues 421-441 for Tau, 430-432 and 444-451 for spermine, and 421-443 for calcium. Isothermal titration calorimetry, NMR, and cosedimentation experiments show that Tau and spermine have similar micromolar binding affinities, whereas their binding stoichiometry differs (C-terminal tubulin peptide/spermine stoichiometry 1:2, and C-terminal tubulin peptide/Tau stoichiometry 8:1). Interestingly, calcium, known as a negative regulator of microtubule assembly, can compete with the binding of Tau and spermine with the C-terminal domain of tubulin and with the positive effect of these two partners on microtubule assembly in vitro. This observation opens up the possibility that calcium may participate in the regulation of microtubule assembly in vivo through direct (still unknown) or indirect mechanism (displacement of microtubule partners). The functional importance of this part of tubulin was also underlined by the observation that an α-tubulin mutant deleted from the last 23 amino acid residues does not incorporate properly into the microtubule network of HeLa cells. Together, these results provide a structural basis for a better understanding of the complex interactions and putative competition of tubulin cationic partners with the C-terminal region of tubulin.     

植物微管结合蛋白AtMAP65-1在拟南芥气孔保卫细胞中的定位(简报)

植物细胞微管骨架的不同排列方式对细胞的生长分化及形态建成具有重要意义,微管的这种动态组织行为不仅需要自身的组成蛋白-微管蛋白(tubulin),还要有微管辅助蛋白MAPs(Microtubule-associated proteins)的参与[1,2]。即MAPs是一类能够与微管骨架特异结合并调节其动态装配过程及其结构、进而影响微管功能的蛋白大分子。其中,MAP65是最先在烟草悬浮细胞BY-2中纯化出来的、分子量约为65KDa的一个微管结合蛋白家族。     

Effect of tubulin self-association on GTP hydrolysis and nucleotide exchange reactions

We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. Both reactions are relevant for microtubule (MT) dynamics. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied by determining the concentrations of tubulin-free and tubulin-bound GTP and GDP. We observed no GTP hydrolysis below the critical conditions for MT assembly (either below the critical tubulin concentration and/or at low temperature), despite the assembly of tubulin 1D curved oligomers and single-rings, showing that their assembly did not involve GTP hydrolysis. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Cryo-TEM images showed that GTP-tubulin 1D oligomers were curved also at 36 °C. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to determine the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites. This conclusion may have implications on the dynamics at the tip of the MT plus end.     

The binding of Ruthenium red to tubulin

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.     

Microtubule assembly of isotypically purified tubulin and its mixtures

Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5′-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.     

Possible regulation of the in vitro assembly of bovine brain tubulin by the bovine thioredoxin system

Microtubule assembly in vitro and in vivo is highly sensitive to a variety of sulfhydryl-reactive reagents, raising the question of the possible existence of a physiological sulfhydryl-mediated system for regulating microtubule assembly. However, the specific reagents which have previously been used to inhibit microtubule assembly in vitro are either nonphysiological or, if physiological, effective only at concentrations much higher than their physiological ones. Because of reports of association in vivo between microtubules and the sulfhydryl-reactive proteins thioredoxin and thioredoxin reductase, we decided to examine the interaction in vitro between microtubules and the thioredoxin system, comprising thioredoxin, thioredoxin reductase and NADPH. At pH 6.8, both the mammalian and the Escherichia coli thioredoxin systems inhibited microtubule assembly by 4-35% (19 +/- 9%) by reducing one intra-subunit disulfide bond in the tubulin dimer. The thioredoxin-reducible disulfide of the tubulin dimer remains protected from thioredoxin in the assembled microtubules. Thioredoxin or thioredoxin reductase alone, or together in the absence of NADPH, were incapable of either reducing tubulin or inhibiting microtubule assembly. Microtubules formed from reduced tubulin were found to be stable and morphologically identical to those obtained from native tubulin dimers. Since the components of the thioredoxin system were used at concentrations similar to their physiological ones, our results suggest a potential role of the thioredoxin system in regulation of microtubule assembly in vivo.     

In vivo dynamics and differential microtubule-binding activities of MAP65 proteins

Plant cells produce different microtubule arrays that are essential for cell division and morphogenesis without equivalent in other eukaryotes. Microtubule-associated proteins influence the behavior of microtubules that is presumed to culminate into transitions from one array to another. We analyzed the microtubule-binding properties of three Arabidopsis (Arabidopsis thaliana) members, AtMAP65-1, AtMAP65-4, and AtMAP65-5, in live cells using laser scanning confocal microscopy. Depending on the overall organization of the cortical array, AtMAP65-1-GFP (green fluorescent protein) and AtMAP65-5-GFP associated with a subset of microtubules. In cells containing both coaligned and oblique microtubules, AtMAP65-1-GFP and AtMAP65-5-GFP tended to be associated with the coaligned microtubules. Cortical microtubules labeled with AtMAP65-1-GFP and AtMAP65-5-GFP appeared as thick bundles and showed more resistance to microtubule-destabilizing drugs. The polymerization rates of AtMAP65-1-GFP and AtMAP65-5-GFP microtubules were similar to those of tubulin-GFP marked microtubules but were different from AtEB1a-GFP, a microtubule plus-end-binding EB1-like protein that stimulated polymerization. By contrast, depolymerization rates of AtMAP65-1-GFP- and AtMAP65-5-GFP-labeled microtubules were reduced. AtMAP65-1-GFP associated with polymerizing microtubules within a bundle, and with fixed microtubule termini, suggesting that AtMAP65-1's function is to bundle and stabilize adjacent microtubules of the cortex. Polymerization within a bundle took place in either direction so that bundling occurred between parallel or antiparallel aligned microtubules. AtMAP65-4-GFP did not label cortical microtubules or the preprophase band, despite continuous expression driven by the 35S promoter, and its subcellular localization was restricted to microtubules that rearranged to form a spindle and the polar sides of the spindle proper. The expression of AtMAP65-4 peaked at mitosis, in agreement with a function related to spindle formation, whereas AtMAP65-1 and AtMAP65-5 were expressed throughout the cell cycle.     

Collapsin response mediator protein-2 regulates neurite formation by modulating tubulin GTPase activity

Collapsin response mediator protein-2 (CRMP-2) plays a key role in axonal development by regulating microtubule dynamics. However, the molecular mechanisms underlying this function have not been clearly elucidated. In this study, we demonstrated that hCRMP-2, specifically amino acid residues 480–509, is essential for stimulating tubulin GTPase activity. We also found that the GTPase-activating protein (GAP) activity of hCRMP-2 was important for microtubule assembly and neurite formation in differentiated PC12 pheochromocytoma cell lines. Mutant hCRMP-2, lacking arginine residues responsible for GAP activity, inhibited microtubule assembly and neurite formation. Interestingly, we found that the N-terminal region (amino acids150–299) of hCRMP-2 had an inhibitory role on GAP activity via a direct interaction with the C-terminal region (amino acids 480–509). Our results suggest that CRMP-2 as a tubulin direct binder may be a GAP of tubulin in neurite formation and that its GAP activity may be regulated by an intramolecular interaction with an N-terminal inhibitory region.     

Separation of active tubulin and microtubule-associated proteins by ultracentrifugation and isolation of a component causing the formation of microtubule bundles

A new method for separating microtubule-associated proteins (MAPs) and tubulin, appropriate for relatively large-scale preparations, was developed. Most of the active tubulin was separated from the MAPs by centrifugation after selective polymerization of the tubulin was induced with 1.6 M 2-(N-morpholino)ethanesulfonate (Mes) and GTP. The MAPs-enriched supernatant was concentrated and subsequently clarified by prolonged centrifugation. The supernatant (total soluble MAPs) contained almost no tubulin, most of the nucleosidediphosphate kinase activity of the microtubule protein, good activity in promoting microtubule assembly in 0.1 M Mes, and proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The pellet, inactive in supporting microtubule assembly, contained denatured tubulin, most of the ATPase activity of the microtubule protein, and significant amounts of protein with the electrophoretic mobility of MAP-2. Insoluble material at this and all previous stages, including the preparation of the microtubule protein, could be heat extracted to yield soluble protein active in promoting microtubule assembly and containing MAP-2 as a major constituent. The total soluble MAPs were further purified by DEAE-cellulose chromatography into bound and unbound components, both of which induced microtubule assembly. The bound component (DEAE-MAPs) contained proteins with the electrophoretic mobility of MAP-1, MAP-2, and tau factor. The polymerization reaction induced by the unbound component (flow-through MAPs) produced very high turbidity readings. This was caused by the formation of bundles of microtubules. Although the flow-through MAPs contained significantly more ATPase, tubulin-independent GTPase, and, especially, nucleosidediphosphate kinase activity than the DEAE-MAPs, preparation of a MAPs fraction without these enzymes required heat treatment.     

Detection of energy transfer between tryptophan residues in the tubulin molecule and bound bis(8-anilinonaphthalene-1-sulfonate), an inhibitor of microtubule assembly, that binds to a flexible region on tubulin

The fluorescent apolar probe bis(8-anilinonaphthalene-1-sulfonate) (Bis-ANS) is a potent inhibitor of microtubule assembly that binds to tubulin at a hitherto uncharacterized site distinct from those of the antimitotic drugs. We have found that energy transfer between tryptophan residues and bound Bis-ANS leads to quenching of the intrinsic tubulin fluorescence. The quenching is biphasic, implying two types of Bis-ANS binding sites. The estimated Kd values are 2.7 and 22.2 microM, consistent with reported values for the primary and secondary Bis-ANS binding sites. Preincubation of tubulin at 37 degrees C results in increased quenching of tryptophan fluorescence without any effect on the Kd values, suggesting localized structural change in the protein around the Bis-ANS binding sites. Concentration-dependent depolarization of Bis-ANS fluorescence was observed, suggesting energy transfer among bound Bis-ANS molecules. Such a concentration-dependent decrease in fluorescence polarization was not observed with 8-anilinonaphthalene-1-sulfonate (1,8-ANS), the monomeric form of Bis-ANS. Perrin-Weber plots were obtained for bound Bis-ANS and 1,8-ANS by varying the viscosity with sucrose. The rotational relaxation times calculated for Bis-ANS and 1,8-ANS are 18 and 96 ns, respectively. Comparison with the theoretical value (125 ns) suggests that Bis-ANS binds to a flexible region of tubulin. This, coupled with the fact that Bis-ANS, but not 1,8-ANS, inhibits microtubule assembly, suggests that the region in the tubulin molecule responsible for microtubule assembly is relatively flexible.     

Theory for modeling the copolymerization of tubulin and tubulin-colchicine complex

Substoichiometric concentrations of tubulin-colchicine complex (TC) inhibits microtubule assembly through a copolymerization reaction between tubulin and TC. We have determined the rates and extent of TC incorporation into bovine brain microtubules and developed a theory that models copolymerization. Our analysis suggests that while the apparent association rate constants for tubulin and TC are similar, the apparent dissociation rate constants for TC are a factor of five or more larger than those of tubulin. Copolymer composition showed only slight changes during assembly despite changes in the solution phase and showed little dependence at high TC upon the initial tubulin concentration. The theory was based on coupled Oosawa-Kasai equations that allow for the co-assembly of two components, tubulin and TC. An expression was derived that relates copolymer composition to reaction mixture composition and to the affinity of microtubule ends for tubulin and TC. This expression predicts copolymer composition at TC concentrations less than 10 microM and correlates composition with assembly inhibition. We perceive copolymerization as a facilitated incorporation of TC requiring the presence of tubulin. TC incorporation was dependent on the ratio of total tubulin to the dissociation constant for TC bound to microtubule ends. The copolymerization reaction is thus characterized by an interplay of two effects (a) where tubulin facilitates the incorporation of TC into the microtubule, and (b) where TC inhibits the assembly of tubulin into microtubules.     

Proteins associated with tubulin.

The distribution of proteins on SDS-urea polyacrylamide (7.5%) disc gel electrophoresis is studied from rat brain tubulin purified by three different procedures, including ammonium sulfate precipitation followed by DEAE cellulose chromotography, three cycles of polymerization-depolymerization and colchicinecontaining agarose affinity columns. Three tubulin-associated proteins other than the principal tubulin dimer are identified and characterized with respect to molecular weight, behavior on gel filtration chromatography and method of tubulin purification. One of these proteins (H₁) is released from the tubulin complex when colchicine is irreversibly bound to tubulin. These proteins may participate in processes related to microtubule assembly or function.