Tubulin Molecule Research Articles

Serotonin type-3 receptor antagonists selectively kill melanoma cells through classical apoptosis, microtubule depolymerisation, ERK activation, and NF-κB downregulation.

Malignant melanoma is a highly metastatic tumour, resistant to treatment. Serotonin type-3 (5-HT3) receptor antagonists, such as tropisetron and ondansetron, are well-tolerated antiemetic drugs commonly used to prevent nausea caused by chemotherapy or radiotherapy. We investigated the anticancer effects of these drugs on melanoma cancer cell lines WM-266-4 and B16F10 with or without paclitaxel. We constructed IC50 curves and performed Chou-Talalay analysis, using data obtained with the MTT assay. Flow cytometry and fluorescent microscopy were used to examine characteristics of the cell cycle, cell death and cytoskeleton changes. Protein levels and activation were analysed by western blotting and molecular docking studies carried out. Data were analysed by one way ANOVA and post hoc testing. Ondansetron and tropisetron showed selective concentration-dependent cytotoxicity in melanoma cell lines WM-266-4 and B16F10. The effect in combination with paclitaxel was synergistic. The drugs did not cause cell cycle arrest but did promote characteristics of classical apoptosis, including accumulation of subG1 DNA, cleaved caspase-3, mitochondrial membrane permeability and phosphatidylserine exposure. As well, the cytosolic calcium level in the melanoma cells was enhanced, phosphorylated ERK1/2 induced and NF-κB inhibited. Finally, the formation of microtubules was shown to be impaired in melanoma cells treated with ondansetron or tropisetron. Docking studies were used to predict that these drugs could bind to the colchicine binding site on the tubulin molecule. Antiemetic drugs, already given in combination with chemotherapy, may enhance the cytotoxic effect of chemotherapy, following successful delivery to the tumour site.

Quantitative detection of optical anisotropy of single microtubules by polarization-sensitive interferometric scattering microscopy

Microtubules (MTs) are ubiquitous cytoskeletal biopolymers essential for diverse cellular processes. MTs consist of strictly ordered tubulin dimers arranged into hollow cylindrical filaments and are known to be optically anisotropic, which enables their direct observation in microscopes based on polarization contrast. However, there are no experimental data to quantify the relation between the momentary optical anisotropy of the MT and the immediate arrangement of proteins in the MT structure. In this work, we introduce polarization-sensitive microscopy based on interferometric detection of scattering to quantify the scattering anisotropy of single unlabeled MTs with high precision. Our data explain the structural origin of MT anisotropy with a marginal contribution of the intrinsic asymmetry of a single tubulin molecule. We monitor changes in the MT scattering resulting from the binding of tau proteins to single MTs with a resolution of several proteins per diffraction-limited spot. We associate the changes in the contrast as well as in the scattering anisotropy with the formation of a shell around the MT formed by densely packed tau proteins. Our experimental results match closely with the theoretical model of the MT and include quantitative details about the polarization-dependent interaction of light with biological matter.

The emerging role of the tubulin code: From the tubulin molecule to neuronal function and disease.

Across different cell types and tissues, microtubules are assembled from highly conserved dimers of α- and β-tubulin. Despite their highly similar structures, microtubules have functional heterogeneity, generated either by the expression of different tubulin genes, encoding distinct isotypes, or by posttranslational modifications of tubulin. This genetically encoded and posttranslational generated heterogeneity of tubulin-the "tubulin code"-has the potential to modulate microtubule structure, dynamics, and interactions with associated proteins. The tubulin code is therefore believed to regulate microtubule functions on a cellular and sub-cellular level. This review highlights the importance of the tubulin code for tubulin structure, as well as on microtubule dynamics and functions in neurons. It further summarizes recent developments in the understanding of mutations in tubulin genes, and how they are linked to neurodegenerative and neurodevelopmental disorders. The current advances in the knowledge of the tubulin code on the molecular and the functional level will certainly lead to a better understanding of how complex signaling events control microtubule functions, especially in cells of the nervous system. © 2016 Wiley Periodicals, Inc.

High-affinity accumulation of a maytansinoid in cells via weak tubulin interaction.

The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4–6 × 107 copies). Efflux of 3 [H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of 3 [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.

Molecular insight of isotypes specific β-tubulin interaction of tubulin heterodimer with noscapinoids.

Noscapine and its derivatives bind stoichiometrically to tubulin, alter its dynamic instability and thus effectively inhibit the cellular proliferation of a wide variety of cancer cells including many drug-resistant variants. The tubulin molecule is composed of α- and β-tubulin, which exist as various isotypes whose distribution and drug-binding properties are significantly different. Although the noscapinoids bind to a site overlapping with colchicine, their interaction is more biased towards β-tubulin. In fact, their precise interaction and binding affinity with specific isotypes of β-tubulin in the αβ-heterodimer has never been addressed. In this study, the binding affinity of a panel of noscapinoids with each type of tubulin was investigated computationally. We found that the binding score of a specific noscapinoid with each type of tubulin isotype is different. Specifically, amino-noscapine has the highest binding score of -6.4, -7.2, -7.4 and -7.3kcal/mol with αβI, αβII, αβIII and αβIV isotypes, respectively. Similarly 10 showed higher binding affinity of -6.8kcal/mol with αβV, whereas 8 had the highest binding affinity of -7.2, -7.1 and -7.2kcal/mol, respectively with αβVI, αβVII and αβVIII isotypes. More importantly, both amino-noscapine and its clinical derivative, bromo-noscapine have the highest binding affinity of -46.2 and -38.1kcal/mol against αβIII (overexpression of αβIII has been associated with resistance to a wide range of chemotherapeutic drugs for several human malignancies) as measured using MM-PBSA. Knowledge of the isotype specificity of the noscapinoids may allow for development of novel therapeutic agents based on this class of drugs.

Abstract 4493: Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors.

Abstract Eribulin (E7389) is an antimitotic drug that inhibits microtubule assembly in vitro and in vivo by binding to tubulin. Several studies have been performed on eribulin mechanism of action; however, the mode of interaction with soluble tubulin (αβ-heterodimer) and binding site differences with vinca alkaloids (vinblastine and vincristine) and paclitaxel have not been well characterized. To understand binding differences, we performed an in vitro conformational change assays and established a surface plasmon resonance (SPR)-based direct binding assay using chemical probes and soluble tubulin. Unlike vinca alkaloids and paclitaxel, binding of eribulin with tubulin has little or no effect on conformational change of tubulin which is consistent with previous observations in the following three independent assays: 1) limited proteolysis pattern, 2) intrinsic tryptophan fluorescence and 3) exposure of hydrophobic area on the tubulin molecule. We therefore examined whether eribulin binds to the vinca or paclitaxel site of tubulin in competitive inhibition assay on SPR. Vinca alkaloids and paclitaxel failed to inhibit the binding of tubulin to immobilized eribulin chemical probe. Similarly, eribulin and paclitaxel did not inhibit the binding of tubulin to immobilized vinblastine chemical probe. Thus, the eribulin binding site on tubulin is different from that of vinca alkaloids and paclitaxel. In addition, we further investigated the binding affinities and characteristics of interaction of eribulin with GTP- or GDP-forms of tubulin, respectively. Eribulin chemical probe binds both forms of tubulin specifically with significantly higher affinity (∼40 nM) as compared with vinblastine chemical probe (∼1 μM). Interestingly, we found that eribulin chemical probe preferentially recognizes GTP- over GDP-tubulin and dissociation of GTP-form from eribulin chemical probe was slower than that of GDP-form of tubulin. In contrast, vinblastine chemical probe did not show significant difference in binding to the two nucleotide states of tubulin. This indicates that eribulin distinguishes different nucleotide state of tubulin and binds to GTP-tubulin more tightly. These characteristic features of eribulin differ from those of vinca alkaloids and paclitaxel. Overall, our results suggest that eribulin exerts its unique antimitotic activity by inhibiting microtubule assembly by binding with high affinity at a novel tubulin site at the plus-end of the growing microtubule. This may potentially relate to the mechanism of eribulin clinical efficacy and diminished neuronal toxicity. Citation Format: Kishan Lal Agarwala, Kenji Kubara, Koji Sagane, Boris M. Seletsky, Bruce A. Littlefield. Eribulin recognizes the nucleotide states of soluble tubulin and binds with high-affinity on a site distinct from known tubulin inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4493. doi:10.1158/1538-7445.AM2013-4493

Atomic water channel controlling remarkable properties of a single brain microtubule: Correlating single protein to its supramolecular assembly

Microtubule nanotubes are found in every living eukaryotic cells; these are formed by reversible polymerization of the tubulin protein, and their hollow fibers are filled with uniquely arranged water molecules. Here we measure single tubulin molecule and single brain-neuron extracted microtubule nanowire with and without water channel inside to unravel their unique electronic and optical properties for the first time. We demonstrate that the energy levels of a single tubulin protein and single microtubule made of 40,000 tubulin dimers are identical unlike conventional materials. Moreover, the transmitted ac power and the transient fluorescence decay (single photon count) are independent of the microtubule length. Even more remarkable is the fact that the microtubule nanowire is more conducting than a single protein molecule that constitutes the nanowire. Microtubule's vibrational peaks condense to a single mode that controls the emergence of size independent electronic/optical properties, and automated noise alleviation, which disappear when the atomic water core is released from the inner cylinder. We have carried out several tricky state-of-the-art experiments and identified the electromagnetic resonance peaks of single microtubule reliably. The resonant vibrations established that the condensation of energy levels and periodic oscillation of unique energy fringes on the microtubule surface, emerge as the atomic water core resonantly integrates all proteins around it such that the nanotube irrespective of its size functions like a single protein molecule. Thus, a monomolecular water channel residing inside the protein-cylinder displays an unprecedented control in governing the tantalizing electronic and optical properties of microtubule.

Molecular dynamics simulation study of tubulin dimer interaction with cytostatics

Colchicine, podophylotoxin, and indibulin are natural cytostatics that are used in the treatment of neoplasms. However, application of the compounds is restricted due to their high toxicity and low specificity. Computational experiments modeling tubulin interactions with the cytostatics seem a promising approach to design new analogues of the above-mentioned drugs with higher cytostatic activity and lower toxicity. Therefore, the CHARMM software was used to examine the macromolecules using molecular dynamics and mechanics methods. Particularly, a procedure was applied according to which molecules of each studied cytostatics were placed at several various random positions around the predicted binding site on tubulin. As a result, cytostatic binding regions were identified on the tubulin molecule. It was shown that, during the interaction, structural alterations occurred in these regions that may be responsible for tubulin polymerization. Thus, alterations have been revealed for the first time in the structure of tubulin in the regions of cytostatic binding that can substantially affect its function.

A phase 1 trial of E7974 administered on day 1 of a 21‐day cycle in patients with advanced solid tumors

E7974, a synthetic analog of hemiasterlin, interacts with the tubulin molecule and overcomes resistance to other antitubulin drugs (taxanes and vinca alkaloids). In a phase 1 study, E7974 was given intravenously over a 2- to 5-minute infusion on day 1 of every 21-day cycle. Adult patients with advanced refractory solid tumors who had adequate organ function and Eastern Cooperative Oncology Group performance status 0 to 2 were eligible for this study. A modified Fibonacci schema was used. The maximal tolerated dose (MTD) was the dose where <2 of 6 patients developed a dose-limiting toxicity (DLT). Twenty-eight patients (19 men and 9 women; median age, 64 years) treated at different cohort dose levels (0.18 mg/m(2) , 0.27 mg/m(2) , 0.36 mg/m(2) , 0.45 mg/m(2) , and 0.56 mg/m(2) ) received a total of 66 courses of E7974. The MTD was established at 0.45 mg/m(2) , where 1 of 6 patients experienced DLT (grade 4 febrile neutropenia). Of the 17 refractory colon cancer patients with a median of 3 prior treatments, stable disease was seen in 7 patients (41%). There were no tumor responses. Median progression-free survival was 1.2 months, and median overall survival was 6.7 months. In pharmacokinetic analysis, E7974 was characterized by a fast and moderately large distribution (37.95-147.93 L), slow clearance (2.23-7.15 L/h), and moderate to slow elimination (time to half-life, 10.4-30.5 hours). This study shows that E7974 once every 21-day cycle shows antitumor activity in patients with refractory solid tumors. The recommended phase 2 dose is 0.45 mg/m(2).

2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone Inhibits Skin Cancer M21 Cell Proliferation through Aberrant Expression of Microtubules and the Cell Cycle

Microtubules are a proven target for anticancer drug development because they are critical for mitotic spindle formation and the separation of chromosomes at mitosis. 2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (HL66) induced cell death with the large cells and multiple micronuclei in M21 skin cancer cells. We demonstrated that HL66-induced cell death is caspase-independent and accompanied by the failure of cell cycle progression. Therefore, HL66-induced cell death may be a mitotic catastrophe. HL66 inhibits the dephosphorylation on Thr14 or Tyr15 of cyclin-dependent kinase (Cdk) 1 and the formation of Cdk1/cyclin B1 complex, which might be associated with cell cycle arrest at the S and G(2)/M phases. HL66 is an antimicrotubule agent by molecular modeling on the basis of ligand binding to tubulin molecule. Furthermore, we also demonstrated that HL66, like vinblastine, is a tubulin-destabilizing agent via microtubule disruption in M21 cells. These results describe a novel pharmacological property of HL66 as a microtubule inhibitor, which may make it an attractive new agent for the treatment of skin cancer.

Tau phosphorylation in hippocampus results in toxic gain-of-function

The MAP (microtubule-associated protein) tau binds to tubulin, the main component of MTs (microtubules), which results in the stabilization of MT polymers. Tau binds to the C-terminal of tubulin, like other MAPs (including motor proteins such as kinesin) and it therefore may compete with these proteins for the same binding site in the tubulin molecule. In pathological conditions, tau is the main component of aberrant protein aggregates found in neurodegenerative disorders known as tauopathies where tau is present in its hyperphosphorylated form. GSK3 (glycogen synthase kinase 3, also known as tau kinase I) has been described as one of the main kinases involved in tau modifications. We have analysed the role of phospho-tau as a neurotoxic agent. We have analysed a transgenic mouse model which overexpresses GSK3beta. In this transgenic mouse, a clear degeneration of the dentate gyrus, which increases with age, was found. In a double transgenic mouse, which overexpresses GSK3 and tau at the same time, dentate gyrus degeneration was dramatically increased. This result may suggest that phospho-tau may be toxic inside neurons of the dentate gyrus. Once neuronal degeneration takes place, intracellular tau is secreted to the extracellular space. The present review discusses the toxicity of this extracellular tau for surrounding neurons.

Structure and Evolution of Tubulin C-terminal Tails

Both α- and β-tubulin have a highly charged, acidic, unstructured, C-terminal “tail” peptide (CTT) that extends from the outer surface of the microtubule (MT).These peptides, ∼10 residues for α and ∼20 for β, do not appear in crystal structures or in cryo-EM reconstructions but are critical to the interaction of other proteins with tubulin in dimer or MT form. The CTT are the locus of isotype-defining sequence differences as well as the site of most posttranslational modifications (PTM) of tubulin. Molecular modeling shows that the CTT are flexible and mobile compared to the body of the protein, explaining why they don't diffract. MT are covered with a sparse negatvely charged brush composed of these peptides: their length is comparable to their grafting distance. CTT mediate interactions with many proteins, including the dimer with the mitochondrial outer membrane protein VDAC. Modeling shows that the tails fit into the channel, but not with branch-producing PTMs (polyglutamylation or polyglycylation). Thus PTMs could alter (prevent) tubulin from acting as a regulator of VDAC, and hence mitochondrial, function. Evolutionarily the tails are highly conserved, but not in exact sequence. Rather the residue length and charge of each CTT are closely maintained. This is found throughout the eukaryotic world, except in organisms that lack mitochondria, in which case the tail can be much shorter and/or less charged. Bacterial tubulin (αβ-tubuin, not FtsZ), arriving from an unknown eukaryote by horizontal gene transfer, shows great divergence in tail length (from total loss to much increased length) and charge (including some with many positive charges). We conclude that the interactions of the tail peptides with other proteins, including VDAC on mitochondria, provide a major selective constraint on this critical part of the tubulin molecule.

Identification and Characterization of an Intermediate Taxol Binding Site Within Microtubule Nanopores and a Mechanism for Tubulin Isotype Binding Selectivity

Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.

Evolution of host resistance in a toxin-producing bacterial–fungal alliance

The rice seedling blight fungus Rhizopus microsporus harbors endosymbiotic Burkholderia sp. for the production of the virulence factor, the antimitotic agent rhizoxin. Since the toxin highly efficiently blocks mitosis in most eukaryotes, it remained elusive how self-resistance emerged in the fungal host. In this study, rhizoxin sensitivity was systematically correlated with the nature of beta-tubulin sequences in the kingdom Fungi. A total of 49 new beta-tubulin sequences were generated for representative species of Ascomycota, Basidiomycota and Zygomycota. Rhizoxin sensitivity assays revealed two further amino acids at position 100 (Ser-100 and Ala-100), in addition to the known Ile-100 and Val-100, which convey rhizoxin resistance. All sensitive strains feature Asn-100. This hot spot was verified by modeling studies, which support the finding that rhizoxin preferentially interacts with the tubulin molecule in a cavity near position 100. Ancestral character state reconstructions conducted in a Bayesian framework suggest that rhizoxin sensitivity represents the ancestral character state in fungi, and that evolution of rhizoxin resistance took place in the ancestor of extant resistant Zygomycota. These findings support a model according to which endosymbiosis became possible through a parasitism--mutualism shift in insensitive fungi.

Anticancer effects of diallyl trisulfide derived from garlic.

Alk(en)yl sulfides are characteristic flavor components of garlic. Several lines of epidemiological study indicate that the risk of a certain cancer can be prevented by consumption of garlic. In this manuscript, we examined the anticancer property of garlic-derived alk(en)yl sulfides, and the molecular basis especially for diallyl trisulfide which is a major constituent of the garlic oil. Alk(en)yl sulfides with different numbers of sulfur atom (i.e., mono-, di-, and trisulfide) were synthesized and purified (>99%). The anticancer activity of the alk(en)yl sulfides was primarily examined using human colon cancer cells HCT-15 and DLD-1. The growth of the cells was significantly suppressed by diallyl trisulfide, but neither diallyl monosulfide nor diallyl disulfide showed such an effect. The number of cells arrested at G2/M phase, the cells with a sub-G1 DNA content, and the cells with caspase-3 activity were dramatically increased by diallyl trisulfide treatment. Diallyl trisulfide disrupted microtubule network formation of the cells, and microtubule fragments could be seen at the interphase. There was a specific oxidative modification of cysteine residues Cys12 beta and Cys354 beta, forming S-allylmercaptocysteines in the tubulin molecule. These results suggest that diallyl trisulfide is responsible, at least in part, for the epidemiologically proven anticancer effect for garlic eaters.

Ixabepilone and the Narrow Path to Developing New Cytotoxic Drugs

During the last decade, the focus of attention in the field of new drug development has drifted away from the uninterrupted quest of new and better cytotoxic drugs to the newer and more glamorous grounds of targeted therapy, proof of principle and proof of concept, and sophisticated pharmacodynamic investigations. After years of relentless work behind the scenes, ixabepilone is emerging from the somewhat neglected list of cytotoxic compounds to stand on stage as something more than an apprentice, as reported in this issue of the Journal of Clinical Oncology (JCO). There are reports of five phase II studies of the drug, four in metastatic breast cancer (MBC) and one in non–small-cell lung cancer (NSCLC). Ixabepilone (BMS-247550) is an analog of epothilone B and a reference compound of a novel class of tubulin-active drugs that blocks tubulin polymerization in a way similar to that of the taxanes. Ixabepilone binds to a different site of the tubulin molecule than the taxanes; such a feature may be relevant to the reported lack of complete cross resistance with paclitaxel and docetaxel in preclinical models. The data presented in this issue deserve attention for multiple reasons, forcing us to reconsider the role of the development of old-fashioned chemotherapy to complement the current armamentarium of drugs for cancer treatment in the era of targeted therapies. Ixabepilone enters with the almost mandatory ambition of challenging the still undisputed role of the taxanes. The data presented in this issue of JCO, as summarized in Table 1, support such ambition: ixabepilone worked well as first-line therapy in women with MBC, and had clear antitumor activity in tumors resistant to or even refractory to multiple other classes of cytotoxic drugs and—most important—to paclitaxel or docetaxel. For NSCLC, the activity was observed despite progression within 4 months from treatment with cisplatin or carboplatin. Such therapeutic activity is an encouraging start to widening the range of therapeutic options after failure of the taxanes, prompting the testing of ixabepilone in early stages of disease. But ixabepilone treatment comes at a price: neurotoxicity. Tubulin-active drugs almost invariably cause neurologic adverse effects. Ixabepilone, which was well tolerated regarding bone marrow and other organ toxicities, is not an exception, causing a paclitaxel-like neuropathy that was primarily sensory in nature, cumulative, and slowly reversible in the majority of patients. The neuropathy associated with ixabepilone had much to do with the uneven and slower than expected clinical development of the drug. Table 1 reveals a situation that points to the differences of dose and schedule of ixabepilone in the different trials reported in this issue of JCO. The difference is indeed larger than it appears from the table, due to the extent and severity of the neuropathy initially observed in four of the studies that forced a lowering of the dose from 50 to 40 or even to 32 mg/m per cycle to make treatment feasible with the schedule of administration once every 3 weeks. The only exception to the remedy of amending the dose was the study in women who never received taxanes before ixabepilone, in which daily administration for 5 consecutive days was adopted. One may be tempted to conclude that the daily schedule was less neurotoxic. However, after dose titration to feasibility of the once-per-cycle dosing, there was no obvious pattern of different

Ultrastructural analysis of the aberrant axoneme morphogenesis in thrips (Thysanoptera, Insecta)

Thrips spermiogenesis is characterized by unusual features in the differentiating spermatid cells. Three centrioles from which three individual short flagella are initially assembled, make the early spermatid a tri-flagellated cell. Successively, during spermatid maturation, the three basal bodies maintain a position close to the most anterior end of the elongating nucleus, so that the three axonemes are progressively incorporated in the spermatid cytoplasm, where they run in parallel to the main nuclear axis. Finally, the three axonemes amalgamate to form a microtubular bundle. The process starts with the formation of rifts at three specific points in each axonemal circumference, corresponding to sites 1,3,7 and leads to the formation of 9 microtubular rows of different length, i.e. 3 "dyads", 3 "triads" and 3 "tetrads". In the spermatozoon, the nucleus, the mitochondrion and the bundle of microtubules are arranged in a helicoidal pattern. The elongation of the spermatozoon is allowed by the deep anchorage of the spermatid to the cyst cell through a dense mass of material which, at the end of spermiogenesis, becomes a long anterior cylindrical structure. This bizarre "axoneme" does not show any trace of progressive movement but it is able to beat. According to the presence of dynein arms, sliding can take place only within each row and not between the rows. The possible molecular basis underlying the peculiar instability of thrips axonemes is discussed in light of the present knowledge on the organization of the axoneme in mutant organisms carrying alterations of the tubulin molecule.

Expression of beta-tubulin epitope in human sperm with pathological spermiogram

To determine the location of the corresponding epitope on the tubulin molecule and to find any differences in its exposition in human sperm with normal and pathological spermiograms. The mature spermatozoon exhibits extraordinary structural compartmentalization that is related to the presence of cytoskeletal proteins and has a functional role in connection with fertilization and motility. Previously, we have shown that anti-beta-tubulin antibody TU-12 provided an unexpectedly strong reaction in human and boar sperm head. A retrospective study. Academic research laboratories and private IVF center. One hundred thirteen men participating in the IVF program. Sperm were divided into five categories: normozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia, and asthenoteratozoospermia. Well-characterized monoclonal antibodies were applied for monitoring tubulin epitope distributions in pathological spermatozoa. Qualitative and quantitative detection of tubulin. TU-12 epitope was located in the beta-tubulin region beta 426-435. Immunoblotting revealed differences in the amount of tubulin among men with normozoospermia and pathological spermiogram. Striking differences were observed in the exposition of TU-12 epitope in heads of normal and pathological spermatozoa. The results suggest that tubulin epitopes could be useful biomarkers of the pathological sperm state.

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.

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