Yeast Tubulin Research Articles

Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.

Stu2, the Budding Yeast XMAP215/Dis1 Homolog, Promotes Assembly of Yeast Microtubules by Increasing Growth Rate and Decreasing Catastrophe Frequency

Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells.

Chapter 22 - Structure–Function Analysis of Yeast Tubulin

Microtubules play essential roles in a wide variety of cellular processes including cell division, motility, and vesicular transport. Microtubule function depends on the polymerization dynamics of tubulin and specific interactions between tubulin and diverse microtubule-associated proteins. To date, investigation of the structural and functional properties of tubulin and tubulin mutants has been limited by the inability to obtain functional protein from overexpression systems, and by the heterogeneous mixture of tubulin isotypes typically isolated from higher eukaryotes. The budding yeast, Saccharomyces cerevisiae, has emerged as a leading system for tubulin structure-function analysis. Yeast cells encode a single beta-tubulin gene and can be engineered to express just one of two alpha isotypes. Moreover, yeast allows site-directed modification of tubulin genes at the endogenous loci expressed under the native promoter and regulatory elements. These advantageous features provide a homogeneous and controlled environment for analysis of the functional consequences of specific mutations. Here, we present the techniques to generate site-specific tubulin mutations in diploid and haploid cells, assess the ability of the mutated protein to support cell viability, measure overall microtubule stability, and define changes in the specific parameters of microtubule dynamic instability. We also outline strategies to determine whether mutations disrupt interactions with microtubule-associated proteins. Microtubule-based functions in yeast are well defined, which allows the observed changes in microtubule properties to be related to the role of microtubules in specific cellular processes.

Rationalization of paclitaxel insensitivity of yeast β-tubulin and human βIII-tubulin isotype using principal component analysis

BackgroundThe chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among β-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin, within a common theoretical framework, we have performed structural principal component analyses of β-tubulin sequences across eukaryotes.ResultsThe paclitaxel-resistance of human βIII tubulin isotype and yeast β-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of β-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in βIII isotype was shown to be crucial for paclitaxel-resistance.ConclusionsThe present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human βIII tubulin isotype, through two common collective sequence vectors.

Chemical genetic profiling of the microtubule-targeting agent peloruside A in budding yeast Saccharomyces cerevisiae

Peloruside A, a microtubule-stabilising agent from a New Zealand marine sponge, inhibits mammalian cell division by a similar mechanism to that of the anticancer drug paclitaxel. Wild type budding yeast Saccharomyces cerevisiae (haploid strain BY4741) showed growth sensitivity to peloruside A with an IC50 of 35μM. Sensitivity was increased in a mad2Δ (Mitotic Arrest Deficient 2) deletion mutant (IC50=19μM). Mad2 is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. Haploid mad2Δ cells were much less sensitive to paclitaxel than to peloruside A, possibly because the peloruside binding site on yeast tubulin is more similar to mammalian tubulin than the taxoid site where paclitaxel binds. In order to obtain information on the primary and secondary targets of peloruside A in yeast, a microarray analysis of yeast heterozygous and homozygous deletion mutant sets was carried out. Haploinsufficiency profiling (HIP) failed to provide hits that could be validated, but homozygous profiling (HOP) generated twelve validated genes that interact with peloruside A in cells. Five of these were particularly significant: RTS1, SAC1, MAD1, MAD2, and LSM1. In addition to its known target tubulin, based on these microarray ‘hits’, peloruside A was seen to interact genetically with other cell proteins involved in the cell cycle, mitosis, RNA splicing, and membrane trafficking.

Binding mod of microtubule stabilizing agents to yeast tubulin

binding mod of microtubule stabilizing agents to yeast tubulin

Human TUBB3 Mutations Perturb Microtubule Dynamics, Kinesin Interactions, and Axon Guidance

We report that eight heterozygous missense mutations in TUBB3, encoding the neuron-specific beta-tubulin isotype III, result in a spectrum of human nervous system disorders that we now call the TUBB3 syndromes. Each mutation causes the ocular motility disorder CFEOM3, whereas some also result in intellectual and behavioral impairments, facial paralysis, and/or later-onset axonal sensorimotor polyneuropathy. Neuroimaging reveals a spectrum of abnormalities including hypoplasia of oculomotor nerves and dysgenesis of the corpus callosum, anterior commissure, and corticospinal tracts. A knock-in disease mouse model reveals axon guidance defects without evidence of cortical cell migration abnormalities. We show that the disease-associated mutations can impair tubulin heterodimer formation in vitro, although folded mutant heterodimers can still polymerize into microtubules. Modeling each mutation in yeast tubulin demonstrates that all alter dynamic instability whereas a subset disrupts the interaction of microtubules with kinesin motors. These findings demonstrate that normal TUBB3 is required for axon guidance and maintenance in mammals.

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Differences in Paclitaxel and Docetaxel Interactions with Tubulin Detected by Mutagenesis of Yeast Tubulin

Immunocytochemistry of tubulin at meiosis in Tradescantia by a protein-A gold method

CLAPHAM, D.H. and OSTERGREN, G. 1984. Immunocytochemistry of tubulin at meiosis in Tradescantia by a protein-A gold method.–Hereditas 101:137–142. Lund, Sweden. ISSN 0018–0661. A method for the immunocytochemical demonstration of microtubules in pollen mother cells of Tradescantia paludosa is described. Cells were fixed in paraformaldehyde and attached to slides; and cell-walls were partially degraded with enzymes. Then, in the first stage of the indirect staining procedure, monoclonal antibodies to yeast tubulin were applied to the preparations, followed in the second stage by protein-A coupled to colloidal gold. At first metaphase, separately organized cone-shaped bands of microtubules extend from the polar region to each half-bivalent, with the broad end of each cone located near the kinetochore. Most of the microtubules lie between the kinetochores and the nearer pole. A few fibres curve round the half-bivalents and: occasionally cross the equator, but rarely if ever extend into the opposite half-spindle. It appears that kinetochore microtubules are arranged in a manner that does not permit them to interact with fibres from the opposite half-spindle at first metaphase. Further, to judge from the gradient in the density of staining along each cone of microtubules, many or most of the fibres that begin at the kinetochore region do not reach the pole, which reduces the opportunity for kinetochore microtubules to react with polar microtubules in the same half-spindle.

Understanding tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin.

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

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.

Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae.

We describe a method for isolating milligram quantities of assembly-competent tubulin from the budding yeast Saccharomyces cerevisiae. The tubulin is > 95% purified and free of contaminating enzyme activities. As a result, it has been possible to determine the yeast tubulin equilibrium-binding constant for Mg-GTP and the tubulin GTPase activity under nonassembling and assembling conditions. We also determined the critical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identical conditions. Similarly, the dynamic properties both of individual yeast microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule-associated proteins. The data suggest that the properties of the yeast tubulin may reflect the particular functional requirements of the yeast cell. With this method, it is now possible to introduce any desired tubulin gene mutation into the budding yeast and correlate the phenotypic effects of the mutation in cells with the effects of the mutation on the biochemical and polymerization properties of the tubulin.

Purification of assembly-competent tubulin from Saccharomyces cerevisiae.

We have developed a straightforward, two-step procedure to isolate highly purified yeast tubulin that reproducibly assembles into microtubules. The starting extracts are obtained from cells genetically engineered to overproduce both the alpha and beta subunits of tubulin, under control of the galactose promoter, to approximately 10-times wild-type levels. The first step of purification is carried out with the high-speed supernatant of lysed cells loaded onto a DEAE-Sephadex column; after this step the tubulin preparation is approximately 30% pure. In the second step, the tubulin fractions are loaded onto an immunoaffinity column prepared by coupling the anti-(alpha-tubulin) monoclonal antibody YL 1/2 to Sepharose-4B. Following elution with 0.8 M KCl, the tubulin present in the peak is 90% pure. Upon addition of porcine brain microtubule-associated proteins or DEAE-dextran, this tubulin preparation is functionally active for assembly into microtubules, as visualized by electron microscopy on negatively stained samples. Virtually identical microtubule structures are produced in parallel experiments on the assembly of yeast or porcine brain tubulin, with differences observed only at acidic pH values. Overall, this relatively simple procedure provides a useful tool for the production of functional tubulin suitable both for structural studies and for investigations of the assembly process.

Yeast proteins associated with microtubules in vitro and in vivo.

Conditions were established for the self-assembly of milligram amounts of purified Saccharomyces cerevisiae tubulin. Microtubules assembled with pure yeast tubulin were not stabilized by taxol; hybrid microtubules containing substoichiometric amounts of bovine tubulin were stabilized. Yeast microtubule-associated proteins (MAPs) were identified on affinity matrices made from hybrid and all-bovine microtubules. About 25 yeast MAPs were isolated. The amino-terminal sequences of several of these were determined: three were known metabolic enzymes, two were GTP-binding proteins (including the product of the SAR1 gene), and three were novel proteins not found in sequence databases. Affinity-purified antisera were generated against synthetic peptides corresponding to two of the apparently novel proteins (38 and 50 kDa). Immunofluorescence microscopy showed that both these proteins colocalize with intra- and extranuclear microtubules in vivo.

Changes in the level of tubulin subunits during development of cotton (Gossypium hirsutum) fiber

The levels of tubulin protein in developing cotton (Gossypium hirsutum L. cv. Stoneville 825) fibers were measured from 8 to 28 days post‐anthesis using commercially available monoclonal antibodies against alpha‐ and beta‐tubulin. As the monoclonal antibodies against alpha‐ and beta‐tubulin were prepared from yeast tubulin and chick brain tubulin, respectively, indirect immunofluorescence microscopy was used to establish that the two monoclonal antibodies recognized microtubule structures in cotton fibers. Western blots of electrophoretically separated proteins in crude extracts of cotton roots and fibers showed that single polypeptides with the expected apparent molecular weight for tubulin subunits were recognized by the antisera. An enzyme‐linked immunosorbent assay was used to quantify tubulin levels. From 10 to 20 days post‐anthesis the level of tubulin protein increases approximately three‐fold. After 20 days post‐anthesis, the amount of tubulin relative to total fiber protein reaches a plateau or decreases slightly. The rapid rise in tubulin is correlated with the elongation of the fiber and an increase in cellulose synthesis.

Microtubule cycle in Chlamydomonas reinhardtii: An Immunofluorescence study

AbstractMonoclonal antibodies to yeast tubulin have been used to investigate the distribution of microtubules throughout the cell cycle of the green alga Chlamydomonas reinhardtii. By indirect immunofluorescence microscopy we detect all the classes of microtubule‐containing structures previously described from ultrastructural investigations but we can now demonstrate the complex spatial and temporal relationships between the different microtubule arrays. During interphase a cortical cytoplasmic microtubule system emanates from the base of the flagellar microtubules. These microtubules become reorganised on entry into mitosis, being largely disassembled and replaced by the spindle and “metaphase band” microtubules. The “metaphase band” is shown to be not one array but two distinct sets of microtubules, each linking a spindle pole with a region of the cell cortex near the spindle equator. It persists until late telophase, when it is replaced by cortical and cleavage furrow microtubules.

Calcium ions initiate the selective depolymerization of the pellicular microtubules in bloodstream forms of Trypanosoma brucei.

Calcium ions (100 microM) were found to initiate the selective and complete depolymerization of the pellicular microtubules of Trypanosoma brucei. The Ca2+-dependent release of tubulin was found to occur without the detectable mediation of calmodulin. The released, depolymerized, pellicular tubulin from T. brucei cross-reacted with a monoclonal antibody raised against yeast tubulin. The pellicular tubulin was found to be composed of two alpha isotypes (apparently equal amounts) and one beta isotype. No other proteins were released from the plasma membrane-microtubule complexes during treatment with Ca2+. The released pellicular tubulin was capable of reassembly into microtubules with normal ultrastructure. The observations reported here suggest that a special process may be required to accommodate the cleavage furrow during cytokinesis. This process would either be the Ca2+-dependent depolymerization of at least two of the cross-linked pellicular microtubules or the detachment of the cross-bridges between two pairs of pellicular microtubules on opposite sides of the cell.

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.

Immunofluorescence microscopy of microtubules in intact cell lineages of the moss, Physcomitrella patens. I. Normal and CIPC-treated tip cells.

Monoclonal antibodies to yeast tubulin have been used to visualize the distribution of microtubules in the intact filamentous protonemata of the moss Physcomitrella patens. Protonemata were prepared for immunofluorescence by fixation in formaldehyde and cells were made permeable with Driselase. Extensive cell files were preserved by 'blotting' the moss onto glutaraldehyde-derivatized coverslips. Problems due to fluorescence from chloroplasts were obviated by extraction with dimethyl sulphoxide and the non-ionic detergent, Nonidet NP40. These improvements allowed us to determine that microtubules were present throughout the cell cycle in the apical dome of caulonemal tip cells, that was a pronounced association of microtubules with the nucleus, that 'astral' microtubules were associated with the mitotic spindle and during anaphase may be involved in reorientation of the spindle before an oblique cytokinesis in caulonemata and that the cytokinetic phragmoplast appeared identical to the structure described for higher plants. Microtubules appeared to converge at the very tip of apical caulonemal cells and this was studied further by treating cells with CIPC--a drug that is known to produce multiple microtubule-organizing centres--and which here produces multiple foci for microtubules at the tip. These observations emphasize the involvement of microtubules in tip growth, alignment of the cell plate and nuclear migration--processes that are fundamental to the morphogenesis of filamentous organisms.

The mitotic spindle of Chinese hamster ovary cells isolated in taxol-containing medium.

Mitotic spindles from Chinese hamster ovary (CHO) cells were isolated and purified by a one-step procedure in an isolation medium containing the microtubule-stabilizing drug, taxol. Released mitotic spindles were examined by phase-contrast, polarizing and differential-interference microscopy. They were also stained with monoclonal antibody raised against yeast tubulin and examined by epifluorescence microscopy. The spindles were free from visible cytoplasmic contaminants and the chromosomes were generally lost from the preparations. Electron microscopy showed that microtubules were the dominant structural component and sodium dodecyl sulphate/gel electrophoresis showed that tubulin was the major molecular species present, although a number of minor components, possibly representing microtubule-associated proteins (MAPs), were present. The taxol procedure was also useful in obtaining other microtubule-containing structures such as the midbody or the cytoplasmic microtubule complex in interphase cells. The taxol procedure was also used to isolate mitotic spindles from HeLa cells. The HeLa spindles stained positively with an antibody specific for the 210 X 10(3) Mr microtubule-associated protein, indicating that the MAP was retained by the taxol procedure. The taxol procedure appears to be of great advantage in large-scale preparations of spindles for biochemical analysis.