Bidirectional role of microtubule dynamics in the acquisition and maintenance of temporal information in dorsolateral striatum.

Accurate and precise timing is crucial for complex and purposeful behaviors, such as foraging for food or playing a musical instrument. The brain is capable of processing temporal information in a coordinated manner, as if it contains an 'internal clock'. Similar to the need for the brain to orient itself in space in order to understand its surroundings, temporal orientation and tracking is an essential component of cognition as well. While there have been multiple models explaining the neural correlates of timing, independent lines of research appear to converge on the conclusion that populations of neurons in the dorsal striatum encode information relating to where a subject is in time relative to an anticipated goal. Similar to other learning processes, acquisition and maintenance of this temporal information is dependent on synaptic plasticity. Microtubules are cytoskeletal proteins that have been implicated in synaptic plasticity mechanisms and therefore are considered key elements in learning and memory. In this study, we investigated the role of microtubule dynamics in temporal learning by local infusions of microtubule stabilizing and destabilizing agents into the dorsolateral striatum. Our results suggested a bidirectional role for microtubules in timing, such that microtubule stabilization improves the maintenance of learned target durations, but impairs the acquisition of a novel duration. On the other hand, microtubule destabilization enhances the acquisition of novel target durations, while compromising the maintenance of previously learned durations. These findings suggest that microtubule dynamics plays an important role in synaptic plasticity mechanisms in the dorsolateral striatum, which in turn modulates temporal learning and time perception.

Pocket similarity identifies selective estrogen receptor modulators as microtubule modulators at the taxane site.

Taxanes are a family of natural products with a broad spectrum of anticancer activity. This activity is mediated by interaction with the taxane site of beta-tubulin, leading to microtubule stabilization and cell death. Although widely used in the treatment of breast cancer and other malignancies, existing taxane-based therapies including paclitaxel and the second-generation docetaxel are currently limited by severe adverse effects and dose-limiting toxicity. To discover taxane site modulators, we employ a computational binding site similarity screen of > 14,000 drug-like pockets from PDB, revealing an unexpected similarity between the estrogen receptor and the beta-tubulin taxane binding pocket. Evaluation of nine selective estrogen receptor modulators (SERMs) via cellular and biochemical assays confirms taxane site interaction, microtubule stabilization, and cell proliferation inhibition. Our study demonstrates that SERMs can modulate microtubule assembly and raises the possibility of an estrogen receptor-independent mechanism for inhibiting cell proliferation.

Effect of Aluminum, Iron, and Zinc Ions on the Assembly of Microtubules from Brain Microtubule Proteins.

Al(3+), Fe(3+), and Zn(2+) ions can disturb microtubule assembly from tubulin and microtubuleassociated proteins in rat brain. The main structural forms of these microtubules are rings and tangled bundles. These structures are formed only in the presence of Al(3+) and Fe(3+) ions. Therefore, Zn(2+) ions can be excluded from possible causes of structural abnormalities in microtubules during Alzheimer's disease. Al(3+) ions are the most probable etiological cause of Alzheimer's disease. The concentration of Al(3+) ions affecting the structure of microtubules is one order of magnitude lower than that of Fe(3+) ions (10 and 100 µM, respectively), which corresponds to their brain concentration reported in Alzheimer's disease.

Anti-microtubule activity of tubeimoside I and its colchicine binding site of tubulin.

BACKGROUND: Tubeimoside I (TBMS1) was isolated from the tubers of Bolbostemma paniculatum (Maxim.) Franquet. TBMS1 shows potent anti-tumor activity. The present study was conducted to investigate the anti-microtubule role of TBMS1 and its binding site of tubulin. METHODS: Cell growth inhibition was measured by MTT after treatment with TBMS1. Uptake kinetics of TBMS1 by human nasopharyngeal carcinoma CNE-2Z cell line (CNE-2Z) was assayed by HPLC. Microtubule protein (MTP) was prepared from porcine brain through two cycles of polymerization-depolymerization in a high molarity buffer. Inhibition of MTP polymerization induced by TBMS1 was determined by a turbidity measurement and a sedimentation assay; the interactions of TBMS1 with tubulin within CNE-2Z cells were investigated by immunofluorescence microscopy and immunoblotting. TBMS1 was tested for its ability to inhibit binding of known tubulin ligands through competitive binding assay. RESULTS: TBMS1 displayed growth inhibitory activity against CNE-2Z cells with IC(50) value of 16.7 microM for 72 h. HPLC analysis of TBMS1 uptake by CNE-2Z cells displayed the initial slow TBMS1 uptake and then gradually reaching an maximum uptake near 18 h. CNE-2Z cells treated with TBMS1 (25 microM, 3 h) were sufficient to cause the microtubular network disruption. Immunoblot analysis showed that the proportion of cytosolic tubulin of cells treated with TBMS1 increased in a time- and concentration-dependent manner. TBMS1 did not inhibit the binding of vinblastine to tubulin. Colchicine binding to tubulin was inhibited in the presence of TBMS1. CONCLUSIONS: TBMS1 is an anti-microtubule agent, and its binding site of tubulin is the colchicine binding site of tubulin.

Reduced tubulin tyrosination as an early marker of mercury toxicity in differentiating N2a cells.

The aims of this work were to compare the effects of methyl mercury chloride and thimerosal on neurite/process outgrowth and microtubule proteins in differentiating mouse N2a neuroblastoma and rat C6 glioma cells. Exposure for 4h to sublethal concentrations of both compounds inhibited neurite outgrowth to a similar extent in both cells lines compared to controls. In the case of N2a cells, this inhibitory effect by both compounds was associated with a fall in the reactivity of western blots of cell extracts with monoclonal antibody T1A2, which recognises C-terminally tyrosinated alpha-tubulin. By contrast, reactivity with monoclonal antibody B512 (which recognises total alpha-tubulin) was unaffected at the same time point. These findings suggest that decreased tubulin tyrosination represents a neuron-specific early marker of mercury toxicity associated with impaired neurite outgrowth.

Curcuminoids form reactive glucuronides in vitro.

Curcumin is of current interest because of its putative anti-inflammatory, anticarcinogenic, and anti-Alzheimer's activity, but its pharmacokinetic and metabolic fate is poorly understood. The present in vitro study has therefore been conducted on the glucuronidation of curcumin and its major phase I metabolite, hexahydro-curcumin, as well as of various natural and artificial analogs. The predominant glucuronide generated by rat and human liver microsomes from curcumin, hexahydro-curcumin, and other analogs with a phenolic hydroxyl group was a phenolic glucuronide according to LC-MS/MS analysis. However, a second glucuronide carrying the glucuronic acid moiety at the alcoholic hydroxyl group was formed from the same curcuminoids, but not hexahydro-curcuminoids, by human microsomes. Curcuminoids without a phenolic hydroxyl group gave rise to the aliphatic glucuronide only. The phenolic glucuronides of curcuminoids, but not of hexahydro-curcuminoids, were rather lipophilic and, in part, unstable in aqueous solution, their stability depending strongly on the type of aromatic substitution. The phenolic glucuronide of curcumin and of its natural congeners, but not the parent compounds, clearly inhibited the assembly of microtubule proteins under cell-free conditions, implying chemical reactivity of the glucuronides. These novel properties of the major phase II metabolites of curcuminoids deserve further investigation.

PUMA overexpression induces reactive oxygen species generation and proteasome-mediated stathmin degradation in colorectal cancer cells.

Increased amounts of reactive oxygen species (ROS) induce apoptosis in mammalian cells. PUMA (P53 up-regulated modulator of apoptosis), a mitochondrial proapoptotic BH3-only protein, induces rapid apoptosis through a Bax- and mitochondria-dependent pathway. However, the molecular basis of PUMA-induced apoptosis is largely not understood. Using a combination of biophysical and biochemical methods and PUMA-inducible colorectal cells, DLD-1.PUMA, we showed that (a) PUMA-induced apoptosis is dose and time dependent; (b) PUMA-induced apoptosis is directly associated with ROS generation; (c) diphenyleneiodonium chloride, a ROS blocker, or BAX-inhibiting peptide, a suppressor of BAX translocation, decreased ROS generation and apoptosis in DLD-1.PUMA cells; (d) overexpression of PUMA induced up-regulation (>1.34-fold) of peroxiredoxin 1 and down-regulation (by 25%) of stathmin through proteasome-mediated degradation; and (e) hydrogen peroxide down-regulated stathmin and disrupted the cellular microtubule network. Our findings indicate that PUMA induces apoptosis, in part, through the BAX-dependent generation of superoxide and hydrogen peroxide. ROS overproduction and oxidative stress induce proteome-wise alterations, such as stathmin degradation and disorganization of the cell microtubule network, in apoptotic cells.

A sesquiterpene lactone, costunolide, interacts with microtubule protein and inhibits the growth of MCF-7 cells.

Costunolide is an active sesquiterpene lactone of medicinal herbs with anti-inflammatory and potential anti-cancer activity. Nevertheless, the pharmacological pathways of costunolide have not yet been fully elucidated. In this study we showed that costunolide exerts a dose-dependent antiproliferative activity in the human breast cancer MCF-7 cells. In addition, light microscopy observations indicated that costunolide affected nuclear organization and reorganized microtubule architecture. The antiproliferative and antimicrotubular effects of costunolide were not influenced by paclitaxel, well-known microtubule-stabilizing anticancer agent. The microtubule-interacting activity of costunolide was confirmed by in vitro studies on purified microtubular protein. In fact, costunolide demonstrated polymerizing ability, by inducing the formation of well organized microtubule polymers. Our data suggest an interaction of costunolide with microtubules, which may represent a new intracellular target for this drug.

Mutations in alpha-tubulin promote basal body maturation and flagellar assembly in the absence of delta-tubulin.

We have isolated suppressors of the deletion allele of delta-tubulin, uni3-1, in the biflagellate green alga Chlamydomonas reinhardtii. The deletion of delta-tubulin produces cells that assemble zero, one or two flagella and have basal bodies composed primarily of doublet rather than triplet microtubules. Flagellar number is completely restored in the suppressed strains. Most of the uni3-1 suppressors map to the TUA2 locus, which encodes alpha2-tubulin. Twelve independent tua2 mutations were sequenced. Amino acids D205 or A208, which are nearly invariant residues in alpha-tubulin, were altered. The tua2 mutations on their own have a second phenotype - they make the cells colchicine supersensitive. Colchicine supersensitivity itself is not needed for suppression and colchicine cannot phenocopy the suppression. The suppressors partially restore the assembly of triplet microtubules. These results suggest that the delta-tubulin plays two roles: it is needed for extension or stability of the triplet microtubule and also for early maturation of basal bodies. We suggest that the mutant alpha-tubulin promotes the early maturation of the basal body in the absence of delta-tubulin, perhaps through interactions with other partners, and this allows assembly of the flagella.

Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification.

Microtubules (MTs) polymerized with GMPCPP, a slowly hydrolyzable GTP analogue, are stable in buffer but are rapidly depolymerized in Xenopus egg extracts. This depolymerization is independent of three previously identified MT destabilizers (Op18, katanin, and XKCM1/KinI). We purified the factor responsible for this novel depolymerizing activity using biochemical fractionation and a visual activity assay and identified it as XMAP215, previously identified as a prominent MT growth-promoting protein in Xenopus extracts. Consistent with the purification results, we find that XMAP215 is necessary for GMPCPP-MT destabilization in extracts and that recombinant full-length XMAP215 as well as an NH2-terminal fragment have depolymerizing activity in vitro. Stimulation of depolymerization is specific for the MT plus end. These results provide evidence for a robust MT-destabilizing activity intrinsic to this microtubule-associated protein and suggest that destabilization may be part of its essential biochemical functions. We propose that the substrate in our assay, GMPCPP-stabilized MTs, serves as a model for the pause state of MT ends and that the multiple activities of XMAP215 are unified by a mechanism of antagonizing MT pauses.

Theonezolide A, a novel marine macrolide, induces drastic shape change in rabbit platelets by reorganization of microtubules.

Theonezolide A, a marine macrolide, and thrombin caused a shape change followed by an aggregation in the rabbit platelets. Theonezolide A-induced platelet shape change, estimated by a decrease in light transmission, appeared to a greater extent than thrombin-induced one. Morphological studies using an electron microscope showed that theonezolide A changed platelet shape with various numbers of long pseudopods, loosing their discoid shape. Theonezolide A-induced shape change was inhibited by a microtubule-stabilizing agent, taxol, but not by an actin-depolymerizing agent, cytochalasin B. In contrast, thrombin-induced shape change was inhibited by cytochalasin B but not by taxol. Confocal fluorescence microscopy showed that circumferential microtubule bundle disappeared in the platelets treated with theonezolide A. Theonezolide A had no direct effect on polymerization of microtubules isolated from bovine brain, indicating that it indirectly causes microtubule reorganization. These results suggest that theonezolide A induces drastic shape change through reorganization of microtubules in rabbit platelets. Thus, theonezolide A is a useful drug to examine microtubule reorganization in the cells.

Celogentins A-C, new antimitotic bicyclic peptides from the seeds of Celosia argentea.

Three new bicyclic peptides, celogentins A (1), B (2), and C (3), have been isolated together with a known-related peptide, moroidin (4), from the seeds of Celosia argentea, and their structures including absolute stereochemistry were determined by using extensive NMR methods and chemical means. Celogentins A (1), B (2), and C (3) inhibited the polymerization of tubulin, and celogentin C (3) was four times more potent than moroidin (4) in the inhibitory activity. Structure-activity relationship study using moroidin derivatives 5-7 and analogue 8 as well as celogentins A-C (1-3) and moroidin (4) indicates that the bicyclic ring system including unusual non-peptide connections among beta(s)-Leu, Trp, and His residues characteristic of celogentins and moroidin, with ring size and conformations suitable for interaction with tubulin would be important for their biological activity.

Additivity of dilantin and vinblastine inhibitory effects on microtubule assembly.

Dilantin (phenytoin) is a commonly used antiepileptic agent that is known to decrease conductance of sodium and calcium ions and delay outward potassium currents. Separate from its antiseizure activity, dilantin interferes with microtubule protein polymerization. It induces metaphase arrest and potentiates the effects of the antimitotics vincristine and vinblastine in cell culture. We show here by fluorescence binding studies that dilantin interacts directly with tubulin at a low affinity site [Ka = 3.5 (+/- 2.5) x 10(3) M(-1); Kd = 286 microM]. We quantitatively examined the effect of dilantin on bulk microtubule formation and found that the drug raises the critical concentration for microtubule polymerization in 2 M glycerol identically in the presence or absence of vinblastine. The change in free energy for microtubule polymerization attributable to 400 microM dilantin [deltadelta G = 117 (+/- 28) cal/mol] is additive with vinblastine effects. Under the same conditions, mean microtubule lengths are 7.7 +/- 4.3 microm (n = 558) and 7.4 +/- 4.0 microm (n = 477) in the presence or absence of dilantin, respectively. Dilantin has no effect on vinblastine-induced tubulin spiral formation, as measured by sedimentation velocity. Our data suggest that the mechanism for the antimicrotubule effects of dilantin involves sequestration of tubulin heterodimers in 1:1 drug:tubulin complexes that do not participate in tubulin polymerization. The dilantin binding site is distinct from the Vinca binding site, and these independent binding modes account for the additive effects in vitro. The sequestration of tubulin heterodimers could explain the combined drug synergy in cell cultures if it disrupted interactions with proteins that regulate microtubule dynamics and/or cell cycle events.

Kinesin accumulation in chick spinal axonal swellings with beta,beta'-iminodipropionitrile (IDPN) intoxication.

Kinesin is a major molecular motor responsible for anterograde axonal transport. Chicks were injected with beta,beta'-iminodipropionitrile (IDPN) to induce axonal swellings in spinal motor neurons and spinal sensory ganglion neurons. Cylindrical swollen axons were found in the anterior horn and anterior funiculus of the spinal cord, anterior root, and spinal ganglia. All of the axonal swellings were heavily stained with two anti-kinesin monoclonal antibodies. The swellings were mildly stained with an anti-cytoplasmic dynein and anti-tubulin antibodies, and weakly stained with an anti-tau antibody. These suggest the isolated disturbance of kinesin transport with neurofilament accumulation in IDPN intoxication.

Tubulin assembly in the presence of calcium ions and taxol: microtubule bundling and formation of macrotubule-ring complexes.

It has been confirmed that taxol is able to prevent Ca(2+)-induced inhibition of microtubule formation from tubulin in the presence of microtubule-associated proteins. However, by means of electron microscopy and scanning force microscopy it could be demonstrated that assembly in the presence of Ca2+ and taxol leads to structural aberrations. The kind of aberration depends on the order of addition of taxol and Ca2+ to tubulin. When taxol was added first, microtubules were formed preferentially. But, these microtubules typically associated with each other by close wall-to-wall alignments or they formed complexes with some C-shaped protofilament ribbons, resulting in microtubule bundles or doublet- and triplet-like microtubule structures, respectively. When Ca2+ was added first, macrotubules, rings, and ring crystals were the dominant assembly products. Mostly, the macrotubules were also bundled or they enclosed rings in their lumen. The findings clearly demonstrate the potency of Ca2+ to induce different polymorphic assemblies with additional protofilament associations, not realized in microtubules.

Interaction of p-benzoquinone and p-biphenoquinone with microtubule proteins in vitro.

p-benzoquinone (BQ) and p-biphenoquinone (BPQ) are metabolites of the human myelotoxin and leukemogen benzene, which has been reported to induce aneuploidy in mammalian cells. Because a possible mechanism for the aneuploidogenic effect of benzene may be the disruption of the mitotic spindle by covalent binding of BQ and BPQ to microtubule proteins (MTP), we have studied the reaction of these quinones with MTP and its consequences for microtubule (MT) formation under cell-free conditions. Both BQ and BPQ inhibit the assembly of MTP to MT in a concentration-dependent manner. This interaction is accompanied by a spectral change of the quinones and loss of free sulfhydryl groups of MTP. With 40 microM BQ or BPQ, 50% inhibition of MT assembly was observed and associated with the loss of 1.3 thiol groups per tubulin dimer. Further analysis showed that native MTP form monoadducts, but no diadducts nor disulfide bonds with both BQ and BPQ. The formation of covalent quinone/MTP monoadducts was unequivocally demonstrated by GC/MS analysis of the respective thioanisols liberated by alkaline permethylation. Denatured MTP or glutathione or 2-mercaptoethanol gave rise to the virtually exclusive formation of monoadducts with BQ but led to a high proportion of disulfide bonds with BPQ. Therefore, BQ and BPQ react differently with thiol compounds capable of disulfide bond formation. The fact that both quinones form only monoadducts with native MTP can be explained by the assumption that the thiol groups of native tubulin are not prone to oxidative disulfide bond formation. This proposition was supported by the lack of native MTP to form disulfide bridges upon treatment with hydrogen peroxide/horseradish peroxidase under conditions leading to a complete oxidation of glutathione. The covalent binding of the benzene metabolites BQ and BPQ to critical thiol groups of tubulin inhibits MT formation under cell-free conditions and may also interfere with the formation of a functional spindle apparatus in the mitotic cell, thus leading to the abnormal chromosome segregation and aneuploidy induction reported for benzene.

Promotion of microtubule assembly in vitro by a novel 35-kDa protein purified from human term placenta.

Microtubule assembly promoting-protein (taxol-like protein, TALP) was purified by combination of high salt extraction, phosphocellulose chromatography and hydroxyapatite chromatography from human term placenta. Molecular weight of purified protein was identified as 35kDa on SDS-polyacrylamide gel electrophoresis. In vitro, TALP promoted microtubule assembly in dose-dependent manner in spite of the absence of GTP. Both TALP (0.5 microM) and taxol (10 microM) gave hyperbolic kinetics and shortened the lag time for microtubule polymerization. TALP-stabilized microtubules were resistant to depolymerization by cold (4 degrees C) and CaCl2 (4 mM) like taxol-stabilized microtubules. TALP showed its direct binding on the microtubule in cosedimentation assay. These results suggest that the action mechanism of TALP on the microtubule assembly is similar to taxol in vitro and the binding site of TALP is available on the intact microtubule.

Isolation and structures of an antifungal antibiotic, fusarielin A, and related compounds produced by a Fusarium sp.

A new antifungal antibiotic, fusarielin A, and three related compounds, fusarielins B, C and D, were obtained from a culture of a Fusarium sp. The skeletal structure and the relative stereochemistry of fusarielin A were determined mainly on the basis of its NMR data, and the absolute structure was elucidated by using the exciton chirality method and the modified Mosher method. The structures of the other homologues were determined by comparison of their spectral data with those of fusarielin A.

Ustiloxins, antimitotic cyclic peptides from false smut balls on rice panicles caused by Ustilaginoidea virens.

Ustiloxins A (1a), B (1b), C (1c), D (1d) and E (1e), antimitotic peptides, have been isolated from the water extract of false smut balls caused on the panicles of rice plant by a fungus Ustilaginoidea virens. The structure of 1b was assigned from its spectral property and its amino acid analysis in relation to 1a whose structure was determined previously by a combination of X-ray crystallographic and amino acid analyses. Structures of 1c and 1d were elucidated by their spectroscopic data, specially based on their 1H and 13C NMR spectra. Bioactivities of these compounds against microtubule assembly as well as mammal, plant and fungal cells have been studied.

Inhibition of microtubules and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its chiral isomer, NSC 613863.

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7 microM and 1.6-2.1 microM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 microM colchicine. Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.