Β-tubulin Mutant Research Articles

Computational analysis of drug resistance of taxanes bound to human β-tubulin mutant (D26E)

The single-point mutation D26E in human β-tubulin is associated with drug resistance seen with two anti-mitotic taxanes (paclitaxel and docetaxel) when used to treat cancers. The molecular mechanism of this resistance remains elusive. However, docetaxel and a third-generation taxane, cabazitaxel, are thought to overcome this resistance. Here, structural models of both the wildtype (WT) and D26E mutant (MT) human β-tubulin were constructed based on the crystal structure of pig β-tubulin in complex with docetaxel (PDB ID: 1TUB). The three taxanes were docked into the WT and MT β-tubulin, and the resulting complexes were submitted to three independent runs of 200 ns molecular dynamic simulations, which were then averaged. MM/GBSA calculations revealed the binding energy of paclitaxel with WT and MT β-Tubulin to be −101.5 ± 8.4 and −90.4 ± 8.9 kcal/mol, respectively. The binding energy of docetaxel was estimated to be −104.7 ± 7.0 kcal/mol with the WT and −103.8 ± 5.5 kcal/mol with the MT β-tubulin. Interestingly, cabazitaxel was found to have a binding energy of −122.8 ± 10.8 kcal/mol against the WT and −106.2 ± 7.0 kcal/mol against the MT β-tubulin. These results show that paclitaxel and docetaxel bound to the MT less strongly than the WT, suggesting possible drug resistance. Similarly, cabazitaxel displayed a greater binding propensity against WT and MT β-tubulin than the other two taxanes. Furthermore, the dynamic cross-correlation matrices (DCCM) analysis suggests that the single-point mutation D26E induces a subtle dynamical difference in the ligand-binding domain. Overall, the present study revealed how the single-point mutation D26E may reduce the binding affinity of the taxanes, however, the effect of the mutation does not significantly affect the binding of cabazitaxel.

Dual Impact of a Benzimidazole Resistant β-Tubulin on Microtubule Behavior in Fission Yeast.

The cytoskeleton microtubule consists of polymerized αβ-tubulin dimers and plays essential roles in many cellular events. Reagents that inhibit microtubule behaviors have been developed as antifungal, antiparasitic, and anticancer drugs. Benzimidazole compounds, including thiabendazole (TBZ), carbendazim (MBC), and nocodazole, are prevailing microtubule poisons that target β-tubulin and inhibit microtubule polymerization. The molecular basis, however, as to how the drug acts on β-tubulin remains controversial. Here, we characterize the S. pombe β-tubulin mutant nda3-TB101, which was previously isolated as a mutant resistance to benzimidazole. The mutation site tyrosine at position 50 is located in the interface of two lateral β-tubulin proteins and at the gate of a putative binging pocket for benzimidazole. Our observation revealed two properties of the mutant tubulin. First, the dynamics of cellular microtubules comprising the mutant β-tubulin were stabilized in the absence of benzimidazole. Second, the mutant protein reduced the affinity to benzimidazole in vitro. We therefore conclude that the mutant β-tubulin Nda3-TB101 exerts a dual effect on microtubule behaviors: the mutant β-tubulin stabilizes microtubules and is insensitive to benzimidazole drugs. This notion fine-tunes the current elusive molecular model regarding binding of benzimidazole to β-tubulin.

Abstract 2764A: Elucidating the pharmacological effects of covalent microtubule stabilizing taccalonolides through functional tagging

Abstract Microtubule stabilizing taxanes are highly effective in the treatment of solid tumors and continue to be used as single agents and in combination with targeted therapies and immunotherapies. However, limitations of taxane therapy include acquired and inherent drug resistance. The taccalonolides are a novel class of microtubule stabilizers that circumvent clinically relevant forms of drug resistance to the taxanes in vitro and in vivo. The taccalonolides are efficacious in taxane-resistant models due to their distinct mechanism of microtubule stabilization that involves covalent binding to β-tubulin. Pharmacological studies of the taccalonolides have been complicated by their irreversible binding to tubulin. A functional tagged taccalonolide would facilitate a greater understanding of the pharmacological, cellular, and physiological effects of this novel drug class. We have generated and characterized a series of fluorescently tagged taccalonolides that retain microtubule binding and stabilizing activities to serve as functional pharmacological taccalonolide probes. Biochemical tubulin polymerization, live cell fluorescence, immunofluorescence imaging, and the sulforhodamine B cytotoxicity assay were conducted to evaluate the microtubule binding and stabilizing effects of these taccalonolide probes. We identified stable taccalonolide probes that retain the biological activities of untagged taccalonolides and facilitate their detection in live cells and in cell lysates. The rates of taccalonolide uptake and tubulin binding in cells, tumors, and normal tissues were determined. These studies have also allowed for an evaluation of the anatomical distribution of the taccalonolides in vivo, which can inform on their potential for antitumor efficacy in distinct tumor sites. Furthermore, we have generated a cellular model to evaluate the kinetics of taccalonolide binding to β-tubulin mutants that are predicted to influence drug binding based on the crystal structure. These studies have confirmed the covalent interaction between the taccalonolides and D224 on β-tubulin and informed on other residues critical for drug binding. The generation of a functional tagged taccalonolide will continue to be a valuable tool in evaluating the pharmacokinetics of these agents and inform on drug targeting strategies that will facilitate their clinical development. Citation Format: Samantha S. Yee, Lin Du, April L. Risinger. Elucidating the pharmacological effects of covalent microtubule stabilizing taccalonolides through functional tagging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2764A.

Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips.

Disassembling microtubules can generate movement independently of motor enzymes, especially at kinetochores where they drive chromosome motility. A popular explanation is the 'conformational wave' model, in which protofilaments pull on the kinetochore as they curl outward from a disassembling tip. But whether protofilaments can work efficiently via this spring-like mechanism has been unclear. By modifying a previous assay to use recombinant tubulin and feedback-controlled laser trapping, we directly demonstrate the spring-like elasticity of curling protofilaments. Measuring their mechanical work output suggests they carry ~25% of the energy of GTP hydrolysis as bending strain, enabling them to drive movement with efficiency similar to conventional motors. Surprisingly, a β-tubulin mutant that dramatically slows disassembly has no effect on work output, indicating an uncoupling of disassembly speed from protofilament strain. These results show the wave mechanism can make a major contribution to kinetochore motility and establish a direct approach for measuring tubulin mechano-chemistry.

Contributions of β-tubulin to cellular morphology, sporulation and virulence in the insect-fungal pathogen, Metarhizium acridum

β-tubulin is an elementary subunit of microtubules that form the cytoskeleton, participating in a wide range of cellular processes. The contributions of the single β-tubulin gene in affecting cell morphology, sporulation and virulence were examined in the entomopathogenic fungus Metarhizium acridum. Targeted gene knockout of β-tubulin resulted in resistance to benomyl but impaired proper nuclear segregation, lipid droplet transport, and deposition of chitin to the cell wall. M. acridum β-tubulin mutants displayed wavy hyphal growth and densely packed, wrinkled colonies. Decreases in the rate of phialides formation and conidial yield were observed for the β-tubulin mutant, which was also impaired in virulence towards locust hosts as compared to wild type and complemented strains. Morphological analyses of infection structures revealed development of bifurcated germ tubes, with reduced appressoria formation seen in the β-tubulin mutant. M. acridum β-tubulin mutant appressoria were aberrant in morphology and displayed decreased turgor pressure. The ability of the M. acridum β-tubulin mutant to proliferate in the insect hemolymph both in vitro and in vivo was also significantly reduced. Our results indicate that in M. acridum, β-tubulin is not essential for survival but that it contributes to cellular transport of organelles and cell wall materials, impacting growth, appressorial differentiation, virulence, and sporulation.

Abstract 435: MT regulation of tumor-dependent microenvironment remodeling

Abstract Microtubules (MTs) represent one of the most effective targets in cancer chemotherapy. However, drugs that target MTs, like the taxanes, often fail in the metastatic setting. Most studies are performed in a two-dimensional monoculture thus, overlook the complexity of the tumor microenvironment. Therefore, to gain a better understanding of tumor MT biology within a physiological context we studied the role of MT dynamics in 3D co-cultures closer mimicking the tumor microenvironment. Since tumor cells need to remodel their ECM in order to metastasize we hypothesized that the interphase MT cytoskeleton, through the coordination of cellular signaling regulates this process. To test this hypothesis we first assessed the MT-dependence of tumor-mediated ECM remodeling, using a floating collagen gel contraction assay. In this assay, breast cancer cells were embedded into a floating collagen gel and monitored for their ability to contract the gel as a result of cell generated traction force. We observed that the MDA-MB-231 metastatic breast cancer cells exhibited greater contractility as compared with the MCF-7 non-metastatic breast cancer cells or the non-tumorigenic human breast epithelial cell line MCF10A. However upon disruption of MTs with paclitaxel (PTX), tumor cell contractility was severely inhibited at drug concentrations that did not affect cell number, suggesting that dynamic MTs are essential for cell traction force generation. In addition, we observed that human mammary fibroblasts when co-cultured with breast cancer cells in 3D exhibited increased motility as compared to 3D fibroblast monocultures. Interestingly, fibroblast activation was inhibited in co-cultures with breast cancer cells previously treated with a taxane, suggesting that tumor MT stabilization negatively affects fibroblast activation. To investigate the factors that mediated the tumor cell communication with surrounding fibroblasts we monitored fibroblast motility in 3D monocultures following incubation with conditioned medium collected from either untreated or Taxol pre-treated breast cancer cells. In agreement with our 3D co-culture results, fibroblast 3D motility was inhibited following exposure to conditioned medium from the Taxol-pretreated cells. These results suggest that the composition of the tumor secretome changes upon MT stabilization, which in turn affects the ability of tumor cells to communicate with stromal fibroblasts. Currently, we are performing proteomic analysis of the conditioned media to identify soluble factors whose secretion is dependent on dynamic microtubules. In addition, to determine whether the effects of taxane treatment is solely due to microtubule stabilization we are performing the 3D co-culture experiments using a Taxol-resistant β-tubulin mutant breast cancer cell line. These findings point to a novel role of interphase MTs in tumor biology and can possibly lead to new avenues for taxane clinical efficacy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 435. doi:10.1158/1538-7445.AM2011-435

Katanin cuts some, spares others

A microtubule-severing protein plays favorites, say Sharma et al. The slicer, called Katanin, builds up microtubules in cilia while it cuts down those in the cell body. Figure 1 Cells that lack katanin (KO) have more microtubules (green) in the cell body than do normal cells (WT). All microtubules are not created equal. In Tetrahymena, for example, the polymers come in many flavors, including ciliary extensions, an internal network, and cortical bundles. These subsets, the new results indicate, are differentially affected by katanin activity. After knocking out Tetrahymena katanin, the authors found that cilia were missing their central microtubules and had shorter outer doublets. Cortical bundles and internal microtubules, by contrast, were more abundant and unusually stable. These inner polymers were more heavily laden with posttranslational modifications, including acetylation, glutamylation, and glycylation. These modifications normally increase as microtubules age, so their accumulation might be a simple byproduct of the loss in severing activity, which may keep the polymer dynamic. The katanin mutants, however, resembled a β-tubulin mutant lacking the glutamate and glycyl projections. The similarity suggests that microtubule modifications might activate katanin, thereby focusing its activity on long, old filaments. Why cilial microtubules were shorter or missing is unclear. Cilia that were lopped off regrew to a similarly stunted length, suggesting that free tubulin subunits were available. Perhaps katanin cuts microtubules to a particular length that can be transported into or along the cilia, as has been suggested for axons. Reference: Sharma, N., et al. 2007. J. Cell Biol. 178:1065–1079. [PubMed]

Measuring Nanometer Scale Gradients in Spindle Microtubule Dynamics Using Model Convolution Microscopy

A computational model for the budding yeast mitotic spindle predicts a spatial gradient in tubulin turnover that is produced by kinetochore-attached microtubule (kMT) plus-end polymerization and depolymerization dynamics. However, kMTs in yeast are often much shorter than the resolution limit of the light microscope, making visualization of this gradient difficult. To overcome this limitation, we combined digital imaging of fluorescence redistribution after photobleaching (FRAP) with model convolution methods to compare computer simulations at nanometer scale resolution to microscopic data. We measured a gradient in microtubule dynamics in yeast spindles at approximately 65-nm spatial intervals. Tubulin turnover is greatest near kinetochores and lowest near the spindle poles. A beta-tubulin mutant with decreased plus-end dynamics preserves the spatial gradient in tubulin turnover at a slower time scale, increases average kinetochore microtubule length approximately 14%, and decreases tension at kinetochores. The beta-tubulin mutant cells have an increased frequency of chromosome loss, suggesting that the accuracy of chromosome segregation is linked to robust kMT plus-end dynamics.

Both Microtubule-Stabilizing and Microtubule-Destabilizing Drugs Inhibit Hypoxia-Inducible Factor-1α Accumulation and Activity by Disrupting Microtubule Function

We have recently identified a mechanistic link between disruption of the microtubule cytoskeleton and inhibition of tumor angiogenesis via the hypoxia-inducible factor-1 (HIF-1) pathway. Based on this model, we hypothesized that other microtubule-targeting drugs may have a similar effect on HIF-1alpha. To test that hypothesis, we studied the effects of different clinically relevant microtubule-disrupting agents, including taxotere, epothilone B, discodermolide, vincristine, 2-methoxyestradiol, and colchicine. In all cases, HIF-1alpha protein, but not mRNA, was down-regulated in a drug dose-dependent manner. In addition, HIF-1alpha transcriptional activity was also inhibited by all drugs tested. To further examine whether these effects were dependent on microtubule network disruption, we tested the ability of epothilone B to inhibit HIF-1alpha protein in the human ovarian cancer cell line 1A9 and its beta-tubulin mutant epothilone-resistant subclone 1A9/A8. Our data showed that epothilone B treatment down-regulated HIF-1alpha protein in the parental 1A9 cells but had no effect in the resistant 1A9/A8 cells. These observations were confirmed by confocal microscopy, which showed impaired nuclear accumulation of HIF-1alpha in parental 1A9 cells at epothilone B concentrations that induced extensive microtubule stabilization. In contrast, epothilone B treatment had no effect on either microtubules or HIF-1alpha nuclear accumulation in the resistant 1A9/A8 cells. Furthermore, epothilone B inhibited HIF-1 transcriptional activity in 1A9 cells, as evidenced by a hypoxia response element-luciferase reporter assay, but had no effect on HIF-1 activity in the resistant 1A9/A8 cells. These data directly link beta-tubulin drug binding with HIF-1alpha protein inhibition. Our results further provide a strong rationale for testing taxanes and epothilones in clinical trials targeting HIF-1 in cancer patients.

Paclitaxel resistance in cells with reduced β-tubulin

We previously described the isolation of colcemid resistant Chinese hamster ovary cell lines containing α- and β-tubulin mutations that increase microtubule assembly and stability. By analyzing colcemid sensitive revertants from one of the β-tubulin mutants, we now find that loss or inactivation of the mutant allele represents the most common mechanism of reversion. Consistent with this loss, the revertants have 35% less tubulin at steady state, no evidence for the presence of a mutant polypeptide, and a normal extent of tubulin polymerization. In addition to the loss of colcemid resistance, the revertant cells exhibit increased resistance to paclitaxel relative to wild-type cells. This paclitaxel resistance can be suppressed by transfecting the revertant cells with a cDNA for wild-type β-tubulin, indicating that the reduction in tubulin in the revertant cells is responsible for the resistance phenotype. We propose that reducing tubulin levels may represent a novel mechanism of paclitaxel resistance.

Mutations of Tubulin Glycylation Sites Reveal Cross-talk between the C Termini of α- and β-Tubulin and Affect the Ciliary Matrix in Tetrahymena

Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.

Survivin Deregulation in β-Tubulin Mutant Ovarian Cancer Cells Underlies Their Compromised Mitotic Response to Taxol

Taxol is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Taxol, the development of drug resistance hampers its clinical applicability. Herein we report that beta-tubulin mutant, Taxol-resistant ovarian cancer cells exhibit defective mitotic response to Taxol, even at high concentrations that are sufficient to trigger apoptosis. This mitotic response-defective phenotype is independent of p53 status. We have found that survivin, the mitosis regulator and inhibitor of apoptosis protein, is deregulated in these Taxol-resistant cancer cells; Taxol fails to induce survivin levels and survivin phosphorylation in these cells, in contrast to their parental drug-sensitive counterparts. Exogenous expression of wild-type survivin is able to restore the mitotic response of the resistant cells to Taxol treatment. On the other hand, exogenous expression of dominant-negative survivin abrogates the Taxol-induced mitotic response in drug-sensitive cancer cells. We have also found that overexpression of the mitotic kinase Cdk1, which phosphorylates survivin, is unable to restore the Taxol-induced mitotic response in the resistant cells. Our results show the importance of survivin for the mitotic response in the context of Taxol resistance and provide novel insights into the mechanisms of mitotic arrest and apoptosis induced by microtubule-targeting agents.

Transfer of amiprophosmethyl resistance from a Nicotiana plumbaginifolia mutant by somatic hybridisation

Transfer of resistance to the phosphorothioamidate herbicide, amiprophosmethyl (APM), from the β-tubulin mutant of Nicotiana plumbaginifolia to the interspecific N. plumbaginifolia (+)N. sylvestris and to the intertribal N. plumbaginifolia (+) Atropa belladonna somatic hybrids has been demonstrated. Transfer to the recipient species was accomplished by: (1) symmetric hybridisationand (2) asymmetric hybridisation using γ-irradiation of donor protoplasts. Cytogenetic analysis confirmed the hybrid origin of the hybrids obtained. It was established that most of them typically inherited no more than three donor chromosomes, although it was possible to obtain symmetric hybrids in the case of symmetric fusion. Immunofluorescent microscopy analysis has shown that protoplasts of the mutant, and of the N. plumbaginifolia (+) N. sylvestris and N. plumbaginifolia (+) A. belladonna hybrids, retained the normal structure of interphase microtubule (MT) arrays and mitotic figures after treatment with 5 µM APM, whereas MTs of protoplasts of the recipients were destroyed under these conditions. It was also shown that hybrid clones contained an altered β-tubulin isoform originating from the N. plumbaginifolia mutant. The selected hybrid clones were characterised by cross-resistance to trifluralin, a dinitroaniline herbicide with the same mode of anti-MT action. Some of the somatic hybrids which could flower were fertile. It was established that seeds of some fertile hybrids were able to germinate in the presence of 5 µM APM. The results obtained thus support the conclusion that the technique of somatic hybridisation, especially asymmetric fusion, can be used to transfer APM resistance from the N. plumbaginifolia mutant to different (related and remote) plant species of the Solanaceae, including important crops.

Effects of amino-acid substitutions in β tubulin on benomyl sensitivity and microtubule functions in Coprinus cinereus

The sensitivity of the homobasidiomyceteCoprinus cinereus to the benzimidazole fungicide benomyl allowed us to isolate β-tubulin mutants as strains resistant to benomyl. To understand the molecular basis for the interaction between benomyl and β tubulin and for cellular defects in the β-tubulin mutants, we first analyzed the wild-type β1-tubulin gene (benA) ofC. cinereus, revealing thatbenA contains eight introns and encodes a 445 amino-acid protein. We then characterized 16 β1-tubulin mutants. The 16 mutations involved 11 different amino-acid substitutions at 10 different residues in β1 tubulin. The mutated residues were widely distributed along the primary sequence of β1 tubulin, from residue 3 in the N-terminal domain to residue 350 in the intermediate domain, but half of them appeared to be close to the αβ intradimer interface in an atomic model determined by electron crystallography. The benomyl resistant strain BEN 193, which exhibits clear heat sensitivity for hyphal growth and defects in various cellular processes, had a novel mutation, i.e., the Leu to Phe substitution at residue 350. Benomyl resistance and the heat sensitivity in BEN 193 were suppressed by additional amino-acid substitutions at various residues in β1 tubulin, suggesting that conformational changes of β1 tubulin are involved in the alterations.

Newly-synthesized beta-tubulin demonstrates domain-specific interactions with the cytosolic chaperonin.

Tubulin folding requires two chaperone systems, i.e., the 900 kDa cytosolic chaperonin referred to as the TCP-1 complex or TRiC which facilitates folding of the alpha- and beta-tubulin subunits and a ca. 180 kDa complex which facilitates further assembly into heterodimer. beta-Tubulin mutants were expressed in rabbit reticulocyte lysates, and the effect of C-terminal, N-terminal, and internal deletions on the binding of beta-tubulin polypeptides to the 900 and 180 kDa complexes was ascertained. Proteolytic studies of chaperonin-bound beta-tubulin were also implemented. These studies support the concept of quasi-native chaperonin-bound intermediates [Tian et al. J. Biol. Chem. (1995) 270, 1-4]. Three "domains" similar in size to the domains in the native protein were implicated in facilitated folding: i.e., an internal or "M-domain" composed of residues approximately 140-260 which binds to TRiC; a "C-domain" composed of residues approximately 300-445 which interacts less strongly with TRiC and may contain regulatory sequences for tubulin release from the chaperonin; and an "N-domain" composed of residues approximately 1-140 which apparently does not interact with TRiC but does interact with the 180 kDa complex. The major TRiC-interacting region, residues approximately 150-350 (the "interactive core"), overlapped portions of the M- and C-domains and included a putative hydrophobic-rich interdomain segment which may be a preferential site of interaction with TRiC. This segment may also be important for microtubule assembly and/or tubulin dimer formation. Removal of two residues from the N-terminal end or ca. 27 residues from the C-terminal and caused the polypeptide to arrest on TRiC. It is proposed that N- and C-terminal regions of beta-tubulin structurally interact with TRiC-binding region approximately 150-350 to inhibit binding to TRiC.

The role of astral microtubules in conjugate division in the dikaryon of Coprinus cinereus

We used fluorescence microscopy to examine the positioning of the two nuclei, the configuration of the mitotic apparatus, and the site of septation during conjugate division in eight dikaryons homozygous for either one α-or one of the seven β-tubulin mutations as well as the parental wild-type dikaryon of Coprinus cinereus . In the wild-type dikaryon, more than 90% of the mitotic apparatus had distinct asters, whereas in the mutants the frequency of the apparatus with distinct asters was more or less lowered. In one of the β-tubulin mutants, BEN 193, the defect of astral microtubules was most remarkable and 75% of the mitotic apparatus lacked astral microtubules, although spindle formation and elongation occurred normally. In BEN 193, the two nuclei were positioned normally during conjugate division in the majority of hyphal cells examined, despite the defect of astral microtubules; the leading nucleus divided in the clamp and the second nucleus in the main hypha just beneath the clamp. This result provided evidence against a direct involvement of astral microtubules in the positioning of the two nuclei during conjugate division. In BEN 193, however, septation in the clamp was disturbed in spite of the fact that a mitotic nucleus was positioned normally in the clamp. In the mutant, the actin ring, which forms prior to septation, was also disturbed in its angle in the clamp. These results strongly suggested that astral microtubules control septation in the clamp.

The transition of cells of the fission yeast β-tubulin mutant nda3-311 as seen by freeze-substitution electron microscopy Requirement of functional tubulin for spindle pole body duplication

A previous fluorescence light-microscopic study showed that the fission yeast cold-sensitive beta-tubulin mutant nda3-311 was arrested with rod-like condensed chromosomes in a mitotic state at the restrictive temperature. Upon transfer to the permissive temperature, a spindle was formed and the nucleus was divided. In the present study, we employed freeze-substitution electron microscopy to examine the ultrastructure of arrested and released nda3-311 cells. In arrested cells, a single, displaced nucleus was seen with a single spindle pole body. Therefore, spindle pole body duplication seemed to require functional beta-tubulin. The nuclear membrane was highly deformed with a leaf-like profile in cross-section, possibly due to an interaction with the rod-like, condensed chromosomes. Upon transfer to the permissive temperature, the spindle pole duplicated and the daughter spindle pole bodies rapidly migrated to the opposite ends of the nucleus, accompanied by the formation of the mitotic spindle. Elongation of the nuclear envelope occurred with concomitant spindle extension, as in a wild-type mitosis. The deformed nuclear membrane became smooth and described a convex curve. The numerous vacuoles that are seen in the arrested cells decreased in number and increased in size. Septation was completed, leaving the two divided nuclei in one half of the cell. Hexagonally arranged microtubules, apparently forming the mitotic spindle, were observed in a cross-section of a cell after return to the permissive conditions.

Extragenic suppressor mutations of a β-tubulin mutation in the basidiomyceteCoprinus cinereus: Isolation and genetic and biochemical analyses

In total, 111 revertants were isolated from the oidia of a heat-sensitive, β-tubulin mutant BEN193 (benA193) of the basidiomyceteCoprinus cinereus after mutagenesis by ultraviolet. Of the 111 revertants, 48 were genetically analyzed. In 15 of the 48 revertants, reversion was due to mutations at loci unlinked tobenA, whereas in the remaining 33 revertants, reversion was due to mutations withinbenA or at loci closely linked tobenA. The 15 extragenic suppressor mutations comprised three groups in terms of genetic linkage; two of them were designated asmipA andmipB. Suppressor mutations in the third group were found to bebenC, one of the four loci we have previously identified as genes conferring benomyl resistance. Biochemical analysis revealed thatbenC + is a structural gene for a major α-tubulin in vegetative hyphae.

Use of α- and β-tubulin mutants for the study of spontaneous and induced chromosomal mis-distribution in Aspergillus nidulans

The effect of two different mutations, one involving an α-tubulin ( tubA) and the other a β-tubulin ( benA33) gene, on somatic segregation has been investigated in diploid strains of A. nidulans. Both mutations, particularly benA33, increase the level of spontaneous chromosomal mis-distribution (CMD) phenomena, without affecting the frequency of crossing-over. The employment of homozygous strains for each of the two mutations in sensitivity tests toward various chemicals, allowed the clear identification of those interfering with microtubule assembly-disassembly processes (i.e. chloral hydrate, diamide, aminocarb, N-ethyl-maleimide, p-chlormercuribenzoate. Such compounds turned out to be very efficient and specific inducers of CMD in a somatic segregation assay performed using the wild-type strain P1. The same assay, when carried out with some of these compounds but employing a tubA/tubA strain, revealed a marked proneness toward CMD to be associated with such mutation, which is known to confer microtubule hypostability.

Construction of a mini-chromosome by deletion and its mitotic and meiotic behaviour in fission yeast

A highly stable, partial aneuploid, HM248, of the fission yeast Schizosaccharomyces pombe was obtained from the unstable aneuploid disomic for chromosome III by γ-irradiation. It contained a 500 kb mini-chromosome (designated Ch16) that was separated as a single band by pulsed field gradient electrophoresis. Genetic analysis showed that Ch16 was deleted for most of chromosome III except for the pericentric region; three centromere-linked markers encompassing the centromere region remained. This was further substantiated by integrating the cloned fragments of Ch16 DNA extracted from the agarose gel; integrations took place in the pericentric region. A 400 kb derivative (Ch16D1) was constructed which appeared to lack a part of Ch16. A single haploid cell of S. pombe could stably maintain Ch16 and Ch16D1 in addition to the three regular chromosomes. Ch16 was visualized as a minute chromosomal body in the nucleus of a β-tubulin mutant under restrictive conditions. A single copy of Ch16 was highly stable and behaved like a natural chromosome in mitosis and meiosis. The frequency of chromosomal loss was 10-4. In meiosis, it segregated independently of the regular chromosome III. Segregation of two Ch16s per cell could be monitored by specifically marked Ch16s containing the Saccharomyces cerevisiae LEU2 gene (designated Ch16LE) of the fluorouracil resistance marker (Ch16FR). Two copies of Ch16 were mitotically unstable (chromosomal loss, 10-3) and frequently failed in meiotic segregation. The frequency of meiotic recombination between the two Ch16s was greatly reduced.