Assembly of unfertilized sea urchin egg tubulin at physiological temperatures

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by DEAE-column chromatography and cycles of temperature-dependent assembly and disassembly. Tubulin-containing column fractions self-assemble into intact microtubules in the absence of high molecular weight microtubule-associated proteins. Egg microtubules assembled during the third cycle of assembly following DEAE-chromatography are composed of 2 or 3 alpha tubulins and 2 beta tubulins as assayed by isoelectric focusing and two-dimensional electrophoresis. The critical protein concentrations necessary for assembly of egg tubulin at 37 and 25 degrees C are 0.15-0.24 and 0.24-0.28 mg/ml, respectively. At physiological temperatures, the critical protein concentrations are 0.81 mg/ml at 15 degrees C and 0.70-0.79 mg/ml at 18 degrees C. At 18 degrees C, bovine brain microtubule-associated proteins stoichiometrically stimulate the initial rate and final extent of egg tubulin assembly. These hybrid microtubules assemble at 18 degrees C at a critical protein concentration of 4-20 micrograms/ml.     

Gamma-tubulin can both nucleate microtubule assembly and self-assemble into novel tubular structures in mammalian cells

alpha-, beta-, and gamma-tubulins are evolutionarily highly conserved members of the tubulin gene superfamily. While the abundant members, alpha- and beta-tubulins, constitute the building blocks of cellular microtubule polymers, gamma-tubulin is a low abundance protein which localized to the pericentriolar material and may play a role in microtubule assembly. To test whether gamma-tubulin mediates the nucleation of microtubule assembly in vivo, and co-assembles with alpha- and beta-tubulins into microtubules or self-assembles into macro- molecular structures, we experimentally elevated the expression of gamma-tubulin in the cell cytoplasm. In most cells, overexpression of gamma-tubulin causes a dramatic reorganization of the cellular microtubule network. Furthermore, we show that when overexpressed, gamma-tubulin causes ectopic nucleation of microtubules which are not associated with the centrosome. In a fraction of cells, gamma-tubulin self-assembles into novel tubular structures with a diameter of approximately 50 nm (named gamma-tubules). Furthermore, unlike microtubules, gamma-tubules are resistant to cold or drug induced depolymerization. These data provide evidence that gamma-tubulin can cause nucleation of microtubule assembly and can self-assemble into novel tubular structures.     

Purification of tau,a microtubule-associated protein that induces assembly of microtubules from purified tubulin

Tau, a microtubule-associated protein which copurifies with tubulin through successive cycles of polymerization and depolymerization, has been isolated from tubulin by phosphocellulose chromatography and purified to near homogeneity. The purified protein is seen to migrate during electrophoresis on acrylamide gels as four closely spaced bands of apparent molecular weights between 55,000 and 62,000. Specific activity for induction of microtubule formation from purified tubulin has been assayed by quantitative electron microscopy and is seen to be enhanced three- to fourfold in the purified tau when compared with the unfractionated microtubule-associated proteins. Nearly 90% of available tubulin at 1 mg/ml is found to be polymerizable into microtubules with elevated levels of tau. Moreover, the critical concentration for polymerization of the reconstituted tau + tubulin system is seen to be a function of tau concentration and may be lowered to as little as 30 μg of tubulin per ml. Under depolymerizing conditions, 50% of the tubulin at only 1 mg/ml may be driven into ring structures. A separate purification procedure for isolation of tau directly from cell extracts has been developed and data from this purification suggest that tau is present in the extract in roughly the same proportion to tubulin as is found in microtubules purified by cycles of assembly and disassembly. Tau is sufficient for both nucleation and elongation of microtubules from purified tubulin and hence the reconstituted tau + tubulin system defines a complete microtubule assembly system under standard buffer conditions. In an accompanying paper (Cleveland et al., 1977) the physical and chemical properties of tau are discussed and a model by which tau may function in microtubule assembly is presented.     

Towards an understanding of microtubule function and cell organization: an overview.

Microtubules exhibit dynamic instability, converting abruptly between assembly and disassembly with continued growth dependent on the presence of a tubulin-GTP cap at the plus end of the organelle. Tubulin, the main structural protein of microtubules, is a heterodimer composed of related polypeptides termed alpha-tubulin and beta-tubulin. Most eukaryotic cells possess several isoforms of the alpha- and beta-tubulins, as well as gamma-tubulin, an isoform restricted to the centrosome. The isoforms of tubulin arise either as the products of different genes or by posttranslational processes and their synthesis is subject to regulation. Tubulin isoforms coassemble with one another and isoform composition does not appear to determine whether a microtubule is able to carry out one particular activity or another. However, the posttranslational modification of polymerized tubulin may provide chemical signals which designate microtubules for a certain function. Microtubules interact with proteins called microtubule-associated proteins (MAPs) and they can be divided into two groups. The structural MAPs stimulate tubulin assembly, enhance microtubule stability, and influence the spatial distribution of microtubules within cells. The dynamic MAPs take advantage of microtubule polarity and organization to vectorially translocate cellular components. The interactions between microtubules and MAPs contribute to the structural-functional integration that characterizes eukaryotic cells.     

Temperature-dependent reversible assembly of taxol-treated microtubules

Taxol is a plant alkaloid that binds to and strongly stabilizes microtubules. Taxol-treated microtubules resist depolymerization under a variety of conditions that readily disassemble untreated microtubules. We report here that taxol-treated microtubules can be induced to disassemble by a combination of depolymerizating conditions. Reversible cycles of disassembly and reassembly were carried out using taxol-containing microtubules from calf brain and sea urchin eggs by shifting temperature in the presence of millimolar levels of Ca2+. Microtubules depolymerized completely, yielding dimers and ring-shaped oligomers as revealed by negative stain electron microscopy and Bio-Gel A-15m chromatography, and reassembled into well-formed microtubule polymer structures. Microtubule-associated proteins (MAPs), including species previously identified only by taxol-based purification such as MAP 1B and kinesin, were found to copurify with tubulin through reversible assembly cycles. To determine whether taxol remained bound to tubulin subunits, we subjected depolymerized taxol-treated microtubule protein to Sephadex G-25 chromatography, and the fractions were assayed for taxol content by reverse-phase HPLC. Taxol was found to be dissociated from the depolymerized microtubules. Protein treated in this way was found to be competent to reassemble, but now required conditions comparable with those for protein that had never been exposed to taxol. Thus, the binding of taxol to tubulin can be reversed. This has implications for the mechanism of taxol action and for the purification of microtubules from a wide variety of sources for use in self-assembly experiments.     

Polymerization of Antarctic fish tubulins at low temperatures: energetic aspects

Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.     

Purification and characterization of tubulin from the catfishHeteropneustes fossilis

Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.     

Developmental and Biochemical Analysis of Chick Brain Tubulin Heterogeneity

Tubulin, isolated from brain tissue of chicks at different stages during late embryonic and early post-hatched development by ion-exchange chromatography and by in vitro microtubule reassembly, was analyzed by high-resolution isoelectric focusing and by two-dimensional polyacrylamide gel electrophoresis. Similar results were obtained with tubulins purified by the two methods. Sixteen isoelectric species of tubulin that differ in apparent net charge under denaturing conditions were detected by isoelectric focusing. By two-dimensional polyacrylamide gel electrophoresis, the chick brain tubulins were resolved into at least seven forms of alpha and 10 forms of beta tubulin. The number and relative proportions of the multiple brain tubulins were modulated during development. Since there are only four alpha tubulin and four beta tubulin genes in chickens, posttranslational modification of the tubulins must play a prominent role in the heterogeneity. Analysis of isotubulin distributions through cycles of microtubule assembly and disassembly indicated that the tubulins differ very little, if at all, in their capacity to assemble into microtubules. Therefore, the chemical differences that distinguish the multiple tubulins have very little structural impact on the protein surface areas involved in microtubule formation. Partial fractionation of the multiple tubulins during ion-exchange chromatography was observed, suggesting that it may be possible to isolate individual native tubulin variants for biochemical studies.     

A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end

We have isolated a protein factor from Xenopus eggs that promotes microtubule assembly in vitro. Assembly promotion was associated with a 215-kD protein after a 1,000-3,000-fold enrichment of activity. The 215-kD protein, termed Xenopus microtubule assembly protein (XMAP), binds to microtubules with a stoichiometry of 0.06 mol/mol tubulin dimer. XMAP is immunologically distinct from the Xenopus homologues to mammalian brain microtubule-associated proteins; however, protein species immunologically related to XMAP with different molecular masses are found in Xenopus neuronal tissues and testis. XMAP is unusual in that it specifically promotes microtubule assembly at the plus-end. At a molar ratio of 0.01 mol XMAP/mol tubulin the assembly rate of the microtubule plus-end is accelerated 8-fold while the assembly rate of the minus-end is increased only 1.8-fold. Under these conditions XMAP promotes a 10-fold increase in the on-rate constant (from 1.4 s-1.microM-1 for microtubules assembled from pure tubulin to 15 s-1.microM-1), and a 10-fold decrease in off-rate constant (from 340 to 34 s-1). Given its stoichiometry in vivo, XMAP must be the major microtubule assembly factor in the Xenopus egg. XMAP is phosphorylated during M-phase of both meiotic and mitotic cycles, suggesting that its activity may be regulated during the cell cycle.     

Identification of betaIII- and betaIV-tubulin isotypes in cold-adapted microtubules from Atlantic cod (Gadus morhua): antibody mapping and cDNA sequencing

Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.     

Assembly of nonneural microtubules in the absence of glycerol and microtubule-associated proteins.

Microtubule protein from Ehrlich ascites tumor cells purified by an in vitro polymerization process in the absence of glycerol and calcium chelators contains several accessory proteins but lacks the high molecular weight proteins which are present in neurotubulin. DEAE-Sephadex chromatography of two-times cycled tubulin removes these nontubulin proteins, resulting in pure tubulin, as critically examined by sodium dodecyl sulfate gel electrophoresis. This tubulin can readily assemble into microtubules in assembly buffer, at low magnesium concentrations, without glycerol and at tubulin concentrations above 0.8 mg/mL. Electron microscopy shows that the tubules are identical with normal microtubules. When the purified tubulin fraction was reduced and carboxymethylated, a significant minor protein component could be observed electrophoretically, migrating between alpha- and beta-tubulin. At present, the identity and function of this protein are not known. The results demonstrate that the in vitro assembly of tubulin from Ehrlich ascites tumor cells does not require high molecular weight proteins or gamma-like factor(s) as has been proposed for the neurotubulin system.     

Localization and Characterization of Tubulin-Like Proteins Associated with Brain Mitochondria: The Presence of a Membrane-Specific Isoform

A mitochondrial fraction, purified from pig brain, was found to contain associated polypeptides with the same electrophoretic migration and isoelectric points as the alpha- and beta-tubulin subunits present in brain microtubules. When analyzed by Western blotting these polypeptides reacted specifically with purified tubulin antibodies. The tubulin-like proteins were then visualized in mitochondrial membranes by protein A-gold complexes after the incubation of purified mitochondria with tubulin antibodies. When membrane and microtubule proteins were compared by isoelectric focussing and two-dimensional gel electrophoresis, differences were observed in the patterns of tubulin isoforms. An additional polypeptide, with the electrophoretic migration of beta-tubulin but the isoelectric point of alpha-tubulin, was found to be enriched in the mitochondrial fraction. This peptide had several Staphylococcus aureus V8 protease peptides in common with alpha-tubulin and may result from a posttranslational modification of that subunit.     

Properties of microtubules assembled from mammalian tubulin synthesized in Escherichia coli

When isolated from tissues, the alpha beta-dimeric protein tubulin consists of multiple isoforms which originate from the expression and subsequent posttranslational modification of multiple polypeptide sequences. Microtubules studied in vitro consist of mixtures of these isoforms. It is therefore not known whether dimers composed of single sequences of alpha- and beta-tubulin can polymerize to form microtubules, or whether posttranslational modifications may be necessary for microtubule assembly. To initiate investigation of these questions, rabbit reticulocyte lysate, which contains the cytoplasmic chaperonin CCT and its cofactors, was employed to prepare substantial quantities (tens of micrograms) of active tubulin by in vitro folding of mouse alpha- and beta-tubulins recombinantly synthesized in E. coli. This recombinant tubulin is composed of only a single alpha-chain and a single beta-chain. When analyzed after folding by isoelectric focusing, each chain yielded only one band, indicating that neither was detectably posttranslationally modified in the course of the folding reaction. When subjected to assembly-promoting conditions, this tubulin formed microtubules without the addition of any exogenous protein. Electron microscopy showed them to be of normal morphology. Analysis of their protein composition showed that they are composed nearly entirely of recombinant tubulin. These results demonstrate that the naturally occurring mixtures of isoforms are not strictly required for the formation of microtubules. They also open a route to other studies, both biomedical and structural, of fully defined tubulin in vitro.     

In vitro assembly of microtubule proteins from myxamoebae of Physarum polycephalum

Microtubule protein of >95% purity has been isolated by self-assembly from concentrated cell extracts of myxamoebae of Physarum polycephalum. Ninety-eight percent of the amoebal microtubule protein was tubulin. Both a and β subunits of amoebal tubulin were different from neurotubulin α and β subunits, but very similar to those of Tetrahymena ciliary tubulin. The non-tubulin components, which co-purified with tubulin through three assembly cycles, were essential to microtubule formation and contained several polypeptides including some of apparent molecular weights 49000, 57000 and 59000. Purified amoebal microtubule protein formed microtubules on warming in the absence of glycerol which were cold- and Ca²⁺-labile. In vitro, microtubule assembly was inhibited by vinblastine, benzimidazole derivatives and griseofulvin, but not by 10^?4 M colchicine. Amoebal tubulin had a much lower affinity than neurotubulin for colchicine.     

A novel tubulin from Mimosa pudica. Purification and characterization

From Mimosa pudica fresh leaves and pulvinar callus cells, we have purified tubulin protein using an anion-exchange resin, DEAE-Sephadex A-50, followed by ammonium sulfate fractionation and Sephadex G-200 gel filtration. The purified protein consisted of alpha and beta subunits and trace quantities of other proteins. When analysed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis at pH 8.8, both alpha and beta subunits of tubulin almost comigrated with their counterparts from goat brain. Two-dimensional electrophoresis revealed that this tubulin contains one major alpha-tubulin having a pI value of 7.1 and three beta species having pI values of 6.70, 6.46 and 6.40. Morphologically normal microtubules were observed by electron microscopy; self-polymerization in vitro, even in the cold, can also be achieved. Radioimmunoassays, and also immunoblotting with the antibodies raised against alpha- and beta-tubulins of this plant, showed that the nature of alpha-tubulin is different from that obtained from other sources. This is an example of plant tubulin where strong colchicine binding at 1 microM was observed. This protein constitutes 5-6% of the total extractable protein in the leaves. We propose that movement of the leaves of this plant may be regulated by the presence of a high amount of this protein.     

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

"Buttonin," a unique button-shaped microtubule-associated protein (75 kD) that decorates spindle microtubule surface hexagonally

A 75-kD protein was purified from sea urchin egg microtubule proteins through gel filtration. It enhanced the polymerization of porcine brain tubulin, but was not heat-stable and did not bind to calmodulin in the presence of calcium as demonstrated by calmodulin affinity column chromatography. Rotary shadowing of the freeze-etched 75-kD protein adsorbed on mica revealed the protein to be a spherical molecule (approximately 9 nm in diameter). Quick-freeze deep-etch electron microscopy revealed that the surface of microtubules polymerized with 75-kD protein was entirely covered with hexagonally packed, round, button-like structures that were quite uniform in shape and size (approximately 9 nm) and similar to the buttons observed on microtubules of mitotic spindles in vivo or microtubules isolated from mitotic spindles. Judging from calibration studies of molecular mass by gel filtration, the 75-kD protein probably exists in a dimeric form (approximately 150 kD) in its native condition. The stoichiometry of tubulin (dimer) versus 75-kD protein (dimer) in the polymerized pellet was 3-3.4:1. Hence, we concluded that the 75-kD protein was a unique microtubule-associated protein that formed the microtubule button in vivo and in vitro. We propose to name this protein "buttonin".     

Effect of tau on the vinblastine-induced aggregation of tubulin

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.     

Tubulin carbamoylation. Functional amino groups in microtubule assembly.

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