Molecular mechanisms of microtubular organelle assembly in Tetrahymena

Thanks to recent technological advances, the ciliate Tetrahymena thermophila has emerged as an attractive model organism for studies on the assembly of microtubular organelles in a single cell. Tetrahymena assembles 17 types of distinct microtubules, which are localized in cilia, cell cortex, nuclei, and the endoplasm. These diverse microtubules have distinct morphologies, stabilities, and associations with specific Microtubule-Associated Proteins. For example, kinesin-111, a microtubular motor protein, is required for assembly of cilia and is preferentially targeted to microtubules of actively assembled, immature cilia. It is unlikely that the unique properties of individual microtubules are derived from the utilization of diverse tubulin genes, because Tetrahymena expresses only a single isotype of alpha- and two isotypes of 1-tubulin. However, Tetrahymena tubulins are modified secondarily by a host of posttranslational mechanisms. Each microtubule organelle type displays a unique set of secondary tubulin modifications. The results of systematic in vivo mutational analyses of modification sites indicate a divergence in significance among post-translational mechanisms affecting either alpha- or beta-tubulin. Both acetylation and polyglycylation of alpha-tubulin are not essential and their complete elimination does not change the cell's phenotype in an appreciable way. However, the multiple polyglycylation sites on 1-tubulin are essential for survival, and their partial elimination dramatically affects cell motility, growth and morphology. Thus, both high-precision targeting of molecular motors to individual organelles as well as organelle-specific tubulin modifications contribute to the creation of diverse microtubules in a single cytoplasm of Tetrahymena.     

Resistance to Microtubule-Stabilizing Drugs Involves Two Eventse: β-Tubulin Mutation in One Allele Followed by Loss of the Second Allele

Resistance to paclitaxel (PTX) or the epothilones (Epo) occurs via the acquisition of point mutations in β-tubulin residues important for drug-tubulin binding. We have isolated four drug-resistant clones selected with PTX or Epo A which harbor distinct β-tubulin mutations. During the development of a stable drug-resistant phenotype, early clones expressing both wild-type (wt) and mutant β-tubulin sequences exhibited a 10-fold drug resistance, while more advanced clones expressing only the mutant β-tubulin sequence exhibited 30 to 50-fold drug resistance. The drug-sensitive parental 1A9 ovarian carcinoma cell line and the drug resistant clones (1A9-A8, 1A9-PTX10 and 1A9-PTX22) were evaluated for loss of heterozygosity (LOH) for β-tubulin (6p25) by single nucleotide polymorphism (SNP) and fluorescent in situ hybridization (FISH) analyses. Functional assays such as drug-induced tubulin polymerization, cell cycle analysis by FACS, DNA sequencing for β-tubulin and mitotic index by immunofluorescence were performed to correlate the β-tubulin LOH status with drug response in the early- and late-step drug-resistant clones. Late-step drug resistant clones revealed LOH in one allele for wt b-tubulin in addition to a β-tubulin mutation in the other allele leading to increased levels of drug resistance, while the early-step clones that contained both a wt and a mutant b-tubulin allele were considerably less drug resistant. The LOH and functional assays revealed cell response that was proportional to the tubulin gene and heterozygosity status. Acquired tubulin mutations in conjunction with LOH for the wt tubulin resulted in a highly resistant phenotype, revealing a new mechanism for taxane resistance.     

Acetylated alpha-tubulin in Physarum: immunological characterization of the isotype and its usage in particular microtubular organelles

We have used monoclonal antibodies specific for acetylated and unacetylated alpha-tubulin to characterize the acetylated alpha-tubulin isotype of Physarum polycephalum, its expression in the life cycle, and its localization in particular microtubular organelles. We have used the monoclonal antibody 6-11B-1 (Piperno, G., and M. T. Fuller, 1985, J. Cell Biol., 101:2085-2094) as the probe for acetylated alpha-tubulin and have provided a biochemical characterization of the monoclonal antibody KMP-1 as a probe for unacetylated tubulin in Physarum. Concomitant use of these two probes has allowed us to characterize the acetylated alpha-tubulin of Physarum as the alpha 3 isotype. We have detected this acetylated alpha 3 tubulin isotype in both the flagellate and in the myxameba, but not in the plasmodium. In the flagellate, acetylated tubulin is present in both the flagellar axonemes and in an extensive array of cytoplasmic microtubules. The extensive arrangement of acetylated cytoplasmic microtubules and the flagellar axonemes are elaborated during the myxameba-flagellate transformation. In the myxameba, acetylated tubulin is not present in the cytoplasmic microtubules nor in the mitotic spindle microtubules, but is associated with the two centrioles of this cell. These findings, taken together with the apparent absence of acetylated alpha-tubulin in the ephemeral microtubules of the plasmodium suggest a natural correspondence between the presence of acetylated alpha-tubulin and microtubule organelles that are intrinsically stable or cross-linked.     

Multiple forms of tubulin in the cytoskeletal and flagellar microtubules of polytomella

The alga polytomella contains several organelles composed of microtubules, including four flagella and hundreds of cytoskeletal microtubules. Brown and co-workers have shown (1976. J. Cell Biol. 69:6-125; 1978, Exp. Cell Res. 117: 313-324) that the flagella could be removed and the cytoskeletans dissociated, and that both structures could partially regenerate in the absence of protein synthesis. Because of this, and because both the flagella and the cytoskeletons can be isolated intact, this organism is particularly suitable for studying tubulin heterogeneity and the incorporation of specific tubulins into different microtubule-containing organelles in the same cell. In order to define the different species of tubulin in polytonella cytoplasm, a (35)S- labeled cytoplasmic fraction was subjected to two cycles of assembly and disassembly in the presence of unlabeled brain tubulin. Comparison of the labeled polytomella cytoplasmic tubulin obtained by this procedure with the tubulin of isolated polytomella flagella by two-dimensional gel electrophoresis showed that, whereas the β-tubulin from both cytoplasmic and flagellar tubulin samples comigrated, the two α-tubulins had distinctly different isoelectic points. As a second method of isolating tubulin from the cytoplasm, cells were gently lysed with detergent and intact cytoskeletons obtained. When these cytoskeletons were exposed to cold temperature, the proteins that were released were found to be highly enriched in tubulin; this tubulin, by itself, could be assembled into microtubules in vitro. The predominant α-tubulin of this in vitro- assembled cytoskeletal tubulin corresponded to the major cytoplasmic α-tubulin obtained by coassembly of labeled polytomella cytoplasmic extract with brain tubulin and was quite distinct from the α-tubulin of purified flagella. These results clearly show that two different microtubule-containing organelles from the same cell are composed of distinct tubulins.     

Brain t-complex polypeptide 1 (TCP- 1) related to its natural substrate beta1 tubulin is decreased in Alzheimer's disease.

The t-complex polypeptide 1 is a selective molecular chaperone in tubulin biogenesis, by that nascent tubulin subunits are bound to t-complex polypeptide 1 and released in assembly competent forms. In neurodegenerative diseases with Alzheimer pathology cytoskeletal proteins are deficient and aggregated. Therefore we examined t-complex polypeptide 1 as represented by the zeta subunit and its specific substrate beta 1 tubulin represented by a truncated product in six brain regions of nine patients with Alzheimer's disease, nine patients with Down syndrome and nine controls. We used 2 dimensional electrophoresis with in-gel-digestion and matrix-assisted laser desorption/ ionization- mass spectrometry for the separation and identification of human brain t-complex polypeptide 1 and beta 1 tubulin. When t-complex polypeptide I was related to its natural and specific substrate beta 1 tubulin, the ratio was significantly decreased in the temporal, frontal, parietal cortex and in thalamus of patients with Alzheimer's disease. In Down syndrome the t-complex polypeptide 1/beta 1 tubulin ratio was significantly increased in frontal and parietal cortex suggesting a different mechanism for aggregation of microfilament proteins e.g. beta 1 tubulin. Relatively decreased molecular chaperoning of beta 1 tubulin by t-complex polypeptide 1 may lead to misfolded tubulin aggregating and accumulating in plaques and tangles, a hallmark of Alzheimer's disease. Our contribution provides first clues for a mechanism of microtubular accumulation in Alzheimer's disease and challenges further studies on different chaperones and chaperonins in the brain of patients with neurodegenerative diseases.     

During Drosophila spermatogenesis beta 1, beta 2 and beta 3 tubulin isotypes are cell-type specifically expressed but have the potential to coassemble into the axoneme of transgenic flies

alpha and beta Tubulins exist in a number of different isotypes with distinct expression patterns during development. We have shown by immunofluorescent staining that beta 1, beta 2 and beta 3 tubulins are distributed very specifically in the testes of Drosophila. beta 3 Tubulin is present exclusively in cytoplasmic microtubules of cells somatic in origin, while the beta 1 isotype is localized in the somatic cells and in early germ cells of both the microtubules of the cytoskeleton as well as in the mitotic spindle. In contrast, beta 2 tubulin is present in all microtubular arrays (cytoskeleton, meiotic spindles, axoneme) of germ cells from meiotic prophase onward, though not detectable in somatic cells. Thus, a switch of beta tubulin isotypes from beta 1 to beta 2 occurs during male germ cell differentiation. This switch is also observed in the distantly related species Drosophila hydei. By fusing beta 1 or beta 3 amino acid coding regions to the control region of the beta 2 tubulin gene and performing germ line transformation experiments, we have examined the copolymerization properties of the different tubulin isotypes. Neither beta 1 nor beta 3 are detectable in the axoneme in the wild-type situation. Analysis of transgenic flies carrying beta 2-beta 1 fusion genes or beta 2-beta 3 fusion genes revealed that both beta 1 and beta 3 tubulin isotypes have the potential to co-incorporate with beta 2 tubulin into microtubules of the sperm axoneme. Male flies homozygous for the fusion genes (beta 2-beta 1 or beta 2-beta 3) remain fertile, despite the mixture of beta tubulin isotypes in the axoneme.     

Androgen-induced up-regulation of tubulin isoforms in neuroblastoma cells

Androgens regulate the physiology of motor neurones both during development and in adult life. In particular, androgens increase the rate of axonal regeneration after axotomy, an effect correlated with the up-regulation of tubulin. In order to determine whether this was the result of a direct hormone action on neurones, we examined the effect of testosterone on microtubular proteins in human neuroblastoma SH-SY5Y cells. Treatment of proliferating SH-SY5Y cells with testosterone resulted in an up-regulation of alpha- and beta-tubulin. By contrast, no change in tubulin was observed either in cells differentiated into a neuronal phenotype by retinoic acid or in adrenal SW13 cells. We also show that an up-regulation of the ubiquitous beta(II)-tubulin and of the neurone-specific beta(III)-tubulin isoforms contributes to the overall increase in tubulin in response to androgen treatment. The increase in tubulin levels following testosterone treatment was abolished by co-incubation with antiandrogens, indicating that this effect is mediated through a classical mechanism of steroid action. The two microtubule-associated proteins, tau and MAP2b, remained unchanged following testosterone exposure. Thus, these results demonstrate that tubulin is a direct neuronal target of androgen regulation and suggest that dysregulation of tubulin expression may contribute to the pathogenesis of some motor neuronopathies.     

Bacterial tubulin distinct loop sequences and primitive assembly properties support its origin from a eukaryotic tubulin ancestor

The structure of the unique bacterial tubulin BtubA/B from Prosthecobacter is very similar to eukaryotic αβ-tubulin but, strikingly, BtubA/B fold without eukaryotic chaperones. Our sequence comparisons indicate that BtubA and BtubB do not really correspond to either α- or β-tubulin but have mosaic sequences with intertwining features from both. Their nucleotide-binding loops are more conserved, and their more divergent sequences correspond to discrete surface zones of tubulin involved in microtubule assembly and binding to eukaryotic cytosolic chaperonin, which is absent from the Prosthecobacter dejongeii draft genome. BtubA/B cooperatively assembles over a wider range of conditions than αβ-tubulin, forming pairs of protofilaments that coalesce into bundles instead of microtubules, and it lacks the ability to differentially interact with divalent cations and bind typical tubulin drugs. Assembled BtubA/B contain close to one bound GTP and GDP. Both BtubA and BtubB subunits hydrolyze GTP, leading to disassembly. The mutant BtubA/B-S144G in the tubulin signature motif GGG(T/S)G(S/T)G has strongly inhibited GTPase, but BtubA-T147G/B does not, suggesting that BtubB is a more active GTPase, like β-tubulin. BtubA/B chimera bearing the β-tubulin loops M, H1-S2, and S9-S10 in BtubB fold, assemble, and have reduced GTPase activity. However, introduction of the α-tubulin loop S9-S10 with its unique eight-residue insertion impaired folding. From the sequence analyses, its primitive assembly features, and the properties of the chimeras, we propose that BtubA/B were acquired shortly after duplication of a spontaneously folding α- and β-tubulin ancestor, possibly by horizontal gene transfer from a primitive eukaryotic cell, followed by divergent evolution.     

A variant beta-tubulin isoform of Drosophila melanogaster (beta 3) is expressed primarily in tissues of mesodermal origin in embryos and pupae, and is utilized in populations of transient microtubules

The beta 3-tubulin gene of Drosophila melanogaster codes for a variant tubulin isoform which is expressed at two distinct times during development: (1) during midembryogenesis from 8-16 hr postfertilization, and (2) during the 4 days of pupal development. We have determined the spatial pattern of beta 3-tubulin expression by localizing the beta 3 mRNA in paraffin sections using a 3' message-specific RNA probe and by localizing the beta 3 protein using a polyclonal antibody specific for Drosophila beta 3-tubulin. During embryogenesis beta 3 is restricted to and is expressed in all of the developing muscles. During pupal development beta 3 is also expressed at high levels in developing adult muscles. In addition, early in pupal development beta 3 is expressed in the imaginal discs, while at later times beta 3 is expressed in the epidermal cells of the wing blade, the optic lobe, the ovaries, and the testes. The expression of beta 3 tubulin ceases by the end of pupal development in all of these tissues except the ovaries and testes where expression persists into the adult. In both developing muscles and wings our results indicate that beta 3-tubulin is utilized in populations of specialized but transient cytoskeletal microtubules which are involved in establishing the final form of the tissue.     

A direct test of the hypothesis that increased microtubule network density contributes to contractile dysfunction of the hypertrophied heart

Contractile dysfunction in pressure overload-hypertrophied myocardium has been attributed in part to the increased density of a stabilized cardiocyte microtubule network. The present study, the first to employ wild-type and mutant tubulin transgenes in a living animal, directly addresses this microtubule hypothesis by defining the contractile mechanics of the normal and hypertrophied left ventricle (LV) and its constituent cardiocytes from transgenic mice having cardiac-restricted replacement of native beta(4)-tubulin with beta(1)-tubulin mutants that had been selected for their effects on microtubule stability and thus microtubule network density. In each case, the replacement of cardiac beta(4)-tubulin with mutant hemagglutinin-tagged beta(1)-tubulin was well tolerated in vivo. When LVs in intact mice and cardiocytes from these same LVs were examined in terms of contractile mechanics, baseline function was reduced in mice with genetically hyperstabilized microtubules, and hypertrophy-related contractile dysfunction was exacerbated. However, in mice with genetically hypostabilized cardiac microtubules, hypertrophy-related contractile dysfunction was ameliorated. Thus, in direct support of the microtubule hypothesis, we show here that cardiocyte microtubule network density, as an isolated variable, is inversely related to contractile function in vivo and in vitro, and microtubule instability rescues most of the contractile dysfunction seen in pressure overload-hypertrophied myocardium.     

Tubulin isotypes optimize distinct spindle positioning mechanisms during yeast mitosis

Microtubules are dynamic cytoskeleton filaments that are essential for a wide range of cellular processes. They are polymerized from tubulin, a heterodimer of α- and β-subunits. Most eukaryotic organisms express multiple isotypes of α- and β-tubulin, yet their functional relevance in any organism remains largely obscure. The two α-tubulin isotypes in budding yeast, Tub1 and Tub3, are proposed to be functionally interchangeable, yet their individual functions have not been rigorously interrogated. Here, we develop otherwise isogenic yeast strains expressing single tubulin isotypes at levels comparable to total tubulin in WT cells. Using genome-wide screening, we uncover unique interactions between the isotypes and the two major mitotic spindle positioning mechanisms. We further exploit these cells to demonstrate that Tub1 and Tub3 optimize spindle positioning by differentially recruiting key components of the Dyn1- and Kar9-dependent mechanisms, respectively. Our results provide novel mechanistic insights into how tubulin isotypes allow highly conserved microtubules to function in diverse cellular processes.     

A lineage-restricted and divergent beta-tubulin isoform is essential for the biogenesis, structure and function of blood platelets

BACKGROUND: Mammalian megakaryocytes release blood platelets through a remarkable process of cytoplasmic fragmentation and de novo assembly of a marginal microtubule band. Cell-specific components of this process include the divergent beta-tubulin isoform beta1 that is expressed exclusively, and is the predominant isoform, in platelets and megakaryocytes. The functional significance of this restricted expression, and indeed of the surprisingly large repertoire of metazoan tubulin genes, is unclear. Fungal tubulin isoforms appear to be functionally redundant, and all mammalian beta-tubulins can assemble in a variety of microtubules, whereas selected fly and worm beta-tubulins are essential in spermatogenesis and neurogenesis. To address the essential role of beta1-tubulin in its natural context, we generated mice with targeted gene disruption. RESULTS: beta1-tubulin(-/-) mice have thrombocytopenia resulting from a defect in generating proplatelets, the immediate precursors of blood platelets. Circulating platelets lack the characteristic discoid shape and have defective marginal bands with reduced microtubule coilings. beta1-tubulin(-/-) mice also have a prolonged bleeding time, and their platelets show an attenuated response to thrombin. Two alternative tubulin isoforms, beta2 and beta5, are overexpressed, and the total beta-tubulin content of beta1-tubulin(-/-) megakaryocytes is normal. However, these isoforms assemble much less efficiently into platelet microtubules and are thus unable to compensate completely for the absence of beta1-tubulin. CONCLUSIONS: This is the first genetic study to address the essential functions of a mammalian tubulin isoform in vivo. The results establish a specialized role for beta1-tubulin in platelet synthesis, structure, and function.     

Involvement of a particular species of beta-tubulin (beta 3) in conidial development in Aspergillus nidulans

Strains of Aspergillus containing the benA22 mutation are resistant to benomyl for vegetative growth but do not produce conidia. To test whether conidiation involved an additional benomyl-sensitive tubulin (i.e., was mediated by a tubulin other than the tubulins coded for by the benA locus), a collection of mutants was produced that formed conidia in the presence of benomyl, i.e., were conidiation-resistant (CR-) mutants. We analyzed the tubulins of these CR- mutants using two-dimensional gel electrophoresis and found that the mutants lacked one species of beta-tubulin (designated beta 3). We have examined two of these mutants in detail. In crosses with strains containing wild-type tubulins, we found that the absence of the beta 3-tubulin co-segregated perfectly with the CR- phenotype. In diploids containing both the benA22 and CR- mutations, we found that the CR- phenotype was recessive and that beta 3-tubulin was present on two-dimensional gels of tubulins prepared from these diploids. In another set of crosses, these two CR- strains and seven others were first made auxotrophic for uridine and then crossed against strains that had homologously integrated a plasmid containing an incomplete internal fragment of the beta 3-tubulin gene and the pyr4 gene of Neurospora crassa (which confers uridine prototrophy on transformants). If the CR- phenotype were produced by a mutation in a gene distinct from the structural gene for beta 3-tubulin (designated the tubC gene), then crossing over should have produced some CR+ segregants among the uridine auxotrophic progeny of the second cross. All of the uridine auxotrophs from this type of cross, however, showed the CR- phenotype, suggesting that the mutation in these strains is at or closely linked to the tubC locus. The most obvious explanation of these results is that beta 3-tubulin is ordinarily used during conidiation and the presence of this species of beta-tubulin renders conidiation sensitive to benomyl. In the CR- mutants, beta 3-tubulin is absent, and in the presence of the benA22 mutation the benomyl-resistant beta 1-and/or beta 2-tubulin substitutes for beta 3 to make conidiation benomyl resistant. We discuss these results and give two models to explain the interactions between these beta-tubulin species.     

Occurrence of nuclear beta(II)-tubulin in cultured cells

Microtubules are cylindrical organelles that play critical roles in cell division. Their subunit protein, tubulin, is a target for various antitumor drugs. Tubulin exists as various forms, known as isotypes. In most normal cells, tubulin occurs only in the cytosol and not in the nucleus. However, we have recently reported the finding of the beta(II) isotype of tubulin in the nuclei of cultured rat kidney mesangial cells. Mesangial cells, unlike most normal cell lines, have the ability to proliferate rapidly in culture. In efforts to determine whether nuclear beta(II)-tubulin occurred in other cell lines, we examined the distribution of the beta(I), beta(II), and beta(IV) mammalian tubulin isotypes in a variety of normal and cancer human cell lines by immunofluorescence microscopy. We have found that, in the normal cell lines, all three isotypes are present only in the cytoplasm. However, the beta(II) isotype of tubulin is located not only in the cytoplasm, but also in the nuclei of the following cell lines: LNCaP prostate carcinoma, MCF-7, MDA-MB-231, MDA-MB-435, and Calc18 breast carcinoma, C6 and T98G glioma, and HeLa cells. In contrast, the beta(I) and beta(IV) isotypes, which are also synthesized in cancer cells, are not localized to the nucleus but are restricted to the cytoplasm. We have also seen beta(II) in breast cancer excisions. In most of these cells, beta(II) appears to be concentrated in the nucleoli. These results suggest that transformation may lead to localization of beta(II)-tubulin in cell nuclei, serving an as yet unknown function, and that nuclear beta(II) may be a useful marker for detection of tumor cells.     

Microtubule regulation in mitosis: tubulin phosphorylation by the cyclin-dependent kinase Cdk1

The activation of the cyclin-dependent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates beta-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of beta-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-beta3-tubulin(S172D/E) mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to beta-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.     

Presence of a tubulin pool in the resting cyst of the hypotrich ciliate, Histriculus muscorum

Although microtubular organelles have not been observed in the resting cyst of the ciliate, Histriculus muscorum, the cyst was immunocytochemically stained with anti-tubulin antiserum, and one of its polypeptide bands reacted to the same antiserum after the immunoblotting test. Both these findings demonstrate the presence of tubulin in the resting cyst. The reacted band appeared in the 100,000 g supernatant of the cyst but not in the pellet, suggesting a depolymerized state of the tubulin in the resting cyst.     

A tubulin identified by a monoclonal antibody is not present in mitotic spindles

A monoclonal antibody, G8, which recognizes a form of tubulin (G8-tubulin) with a novel distribution in Rat-1 cells and Potorous tridactylis kidney (Ptk-2) cells was isolated. G8 labeled the interphase cytoskeleton of Rat-1 fibroblasts but not mitotic spindles or midbodies. G8 also stained a fiber network in some but not all Ptk-2 interphase cells but did not label mitotic spindles or midbodies in these cells. G8-tubulin is the only identified tubulin known to be absent from these structures. This distribution may indicate that G8-tubulin possesses functional specificity.