The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro

The yeast Saccharomyces cerevisiae has two genes for α-tubulin, TUB1 and TUB3, and one β-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents ~10% of α-tubulin in the cell. We determined the effects of the two α-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only α-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only α-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.     

The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus.

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 +/- 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 +/- 0.13 microm. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.     

Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.

The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.     

Association of ribosomes with in vitro assembled microtubules

Microtubules were purified from unfertilized eggs of the sea urchins Arbacia punctulata, Lytechinus pictus, Lytechinus variegatus, and Strongylocentrotus purpuratus. Numerous densely stained particles (24 x 26 nm) are associated with microtubules isolated from each of these sea urchins. The most striking aspect of this structure is an extended, slightly curved arm that appears to attach the particles to the microtubule. Morphologically similar particles are associated with microtubules of the isolated first cleavage mitotic apparatus. The particles are attached to the microtubules by ionic interactions and contain large amounts of extractable RNA. Based upon their size and density, RNA and protein composition, and sedimentation in sucrose gradients, the microtubule-associated particles are identified as ribosomes.     

Alkaline pH favors microtubule self-assembly in surf clam, Spisula solidissima, oocyte extracts

Microtubule assembly in surf clam oocytes is dependent upon events that occur during fertilization. Prior to fertilization there are few, if any microtubules, but within minutes after fertilization microtubules assemble to form the meiotic apparatus. This study demonstrates that the assembly of microtubules after fertilization may be dependent on the fertilization-induced pH change of the cytoplasm. Since the magnitude of the intracellular pH (pHi) change in Spisula oocytes has not been determined, surf clam microtubule assembly was examined at pH values that reflect the pHi change that occurs during sea urchin fertilization. The results indicate that microtubule assembly in crude oocyte extracts is favored at alkaline pH. In contrast, purified surf clam tubulin assembles to a greater extent at pH 6.6 than at pH 7.2. These results reveal that the tubulin in unfertilized oocytes can assemble into microtubules at pH 6.6 but that they are prevented from doing so by pH-dependent cytoplasmic regulatory factors in the oocyte.     

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.     

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.     

Unidirectional microtubule assembly in cell-free extracts of Spisula solidissima oocytes is regulated by subtle changes in pH

Most, if not all, microtubules in vivo grow unidirectionally from a nucleation site such as the centrosome. This organized growth of microtubules can generate and maintain the radially symmetrical array of interphase microtubules as well as the bipolar mitotic apparatus. To investigate the regulation of polarized microtubule growth, we have prepared a cell-free extract from surf clam oocytes that exhibits unidirectional microtubule assembly. Immunofluorescence microscopy was used to visualize the net assembly of microtubules onto the fast (plus)- and slow (minus)- growing ends of isolated ciliary axonemes. All detectable microtubule growth in these cytoplasmic extracts occurred at the plus (+) ends and the extent of (+) end growth was regulated by subtle changes in pH. Microtubule assembly in these crude extracts was highly favored at pH 7.3, the pH of the post-fertilization cytoplasm. In contrast, when tubulin was purified from these oocyte extracts, integral components were lost, and microtubule growth became predominantly bidirectional and was favored at acidic pH. These results indicate that cytoplasmic factors may inhibit bidirectional growth in vivo and that temporal or local changes in cytoplasmic pH may influence microtubule assembly during the cell cycle.     

beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast

Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.