Effect of tau on the vinblastine-induced aggregation of tubulin.

Two microtubule-associated proteins, tau and the high molecular weight microtubule-associated protein 2 (MAP 2), were purified from rat brain microtubules. Addition of either protein to pure tubulin caused microtubule assembly. In the presence of tau and 10 microM vinblastine, tubulin aggregated into spiral structures. If tau was absent, or replaced by MAP 2, little aggregation occurred in the presence of vinblastine. Thus, vinblastine may be a useful probe in elucidating the individual roles of tau and MAP 2 in microtubule assembly.

Effects of Tau and MAP2 on the interaction of maytansine with tubulin: inhibitory effect of maytansine on vinblastine-induced aggregation of tubulin.

Maytansine, a potent inhibitor of mitosis and in vitro microtubule assembly, was used to demonstrate a striking difference in the mechanism by which two of the main groups of brain microtubule-associated proteins, Tau and MAP2, interact with tubulin. At the low concentrations of 0.5 to 2 microM, maytansine inhibited Tau-catalyzed tubulin assembly more effectively than it did MAP2-catalyzed assembly. This effect differed markedly from that of vinblastine, although both drugs bind competitively to tubulin. At the same low concentrations, vinblastine almost completely inhibited Tau- and MAP2-mediated tubulin assembly. At higher concentrations of 10 to 40 microM, a more striking difference was observed between the actions of the two drugs. Maytansine very effectively inhibited tubulin assembly promoted by either Tau or MAP2. Vinblastine also had this effect on MAP2-mediated tubulin assembly but in the presence of Tau induced extensive tubulin aggregation into spirals. In addition maytansine strongly inhibited vinblastine-induced Tau-dependent tubulin aggregation into spiral polymers. Even at very low concentrations, maytansine completely inhibited the effect of very high concentrations of vinblastine. These results very strongly suggest that the binding sites of maytansine and vinblastine on the tubulin molecule overlap and that the changes that they probably induce in the conformation of this molecule are markedly different, at least in the presence of microtubule-associated proteins.

SIAH-1 interacts with alpha-tubulin and degrades the kinesin Kid by the proteasome pathway during mitosis.

SIAH-1, a human homologue of the Drosophila seven in absentia (Sina), has been implicated in ubiquitin-mediated proteolysis of different target proteins through its N-terminal RING finger domain. SIAH-1 is also induced during p53-mediated apoptosis. Furthermore, SIAH-1-transfected breast cancer cell line MCF-7 exhibits an altered mitotic process resulting in multinucleated giant cells. Now, using the two-hybrid system, we identified two new SIAH interacting proteins: Kid (kinesin like DNA binding protein) and alpha-tubulin. We demonstrate that SIAH is involved in the degradation of Kid via the ubiquitin-proteasome pathway. Our results suggest that SIAH-1 but not its N-terminal deletion mutant, affects the mitosis by an enhanced reduction of kinesin levels. Our results imply, for the first time, SIAH-1 in regulating the degradation of proteins directly implicated in the mitotic process.

Intracellular localization of MAP2-related protein (O-map) in prophase I and metaphase II oocytes of Xenopus.

An antibody directed against rat brain microtubule-associated protein 2 (MAP2) immunoprecipitated a protein of 240 kDa from a Xenopus oocyte extract. In contrast, in Xenopus brain extract, this antibody recognizes a protein of 280 kDa. The oocyte MAP2-related protein (called O-MAP) is present in both prophase I and metaphase II-blocked oocytes as demonstrated by immunoblotting experiments; it is in vivo phosphorylated. Immunocytochemical studies using the anti-rat brain MAP2 antibody demonstrated that the O-MAP colocalizes within the cortical microtubular array present in both prophase I and metaphase II oocytes. However, O-MAP is not associated with the microtubular structures which are organized during the oocyte prophase-metaphase transition, i.e., a giant cytoplasmic network and both the meiotic spindles. O-MAP therefore appears as a microtubule-associated protein oocyte specific and may play a role in the regulation of microtubule stability and the organization of the oocyte cytoskeleton.

Association of p34cdc2 kinase and MAP kinase with microtubules during the meiotic maturation of Xenopus oocytes.

p34cdc2 protein is found in prophase, metaphase and activated Xenopus oocytes at a similar level whereas its kinase activity oscillates within meiosis. Using an anti-PSTAIRE antibody that recognizes Xenopus p34cdc2, it was demonstrated that the major part of p34cdc2 was associated with microtubules isolated in vitro from Xenopus oocytes. Conversely, tubulin was recovered in association with p34cdc2 in p13-Sepharose pellets. The abundance of the fraction of p34cdc2 which was associated with microtubules did not oscillate during the meiotic maturation and the activation process. By contrast, the histone H1 kinase activity of p34cdc2 estimated in microtubular oocyte pellets was much higher in metaphase than in prophase oocytes. Cyclin B, which is associated in vivo with p34cdc2 in prophase and metaphase oocytes, was also present in the microtubular fractions. However, cyclin was not necessary for the binding of p34cdc2 to microtubules since p34cdc2 from activated eggs, where cyclin was missing, still copurified with microtubules. Purified MAP2, but not tubulin, was able to bind to p34cdc2, demonstrating that the association between p34cdc2 and microtubules was mediated by microtubule-associated proteins. During the meiotic maturation of Xenopus oocytes, several protein kinases were activated, among them MAP kinase. MAP kinase also associated with microtubules. It was demonstrated that both p34cdc2 kinase and MAP kinase purified from Xenopus oocytes were able to phosphorylate in vitro rat brain MAP2. However both protein kinases phosphorylated different domains of MAP2, suggesting that they might regulate microtubules in different ways.

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments.

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.

Developmental differentiation of MAP2 expression in the central versus the peripheral and efferent projections of the inner ear.

The goal of this study was to extend our knowledge of MAP2 localization in the peripheral nervous system of mammals, since most results on MAP2 distribution are obtained in the central nervous system (CNS). This study shows the presence of microtubule-associated protein 2b (MAP2b) and MAP2c in the inner ear and describes the immunocytochemical distribution of MAP in adult and developing spiral ganglion of the rat by using a well-characterized antibody for MAP2a and MAP2b. (This antibody does not recognize the immature MAP2c). MAP2 labeling is already present in spiral ganglion neurons at 16 days of gestation. From this stage and up to the first postnatal week, MAP2 labeling was strong in all spiral ganglion neurons and their central processes. Double immunostaining at the 16-day stage with anti-MAP2 and anti-neurofilament (NF) antibodies mainly showed NF labeling in central branches that corresponded to anatomically and functionally described axons of spiral neurons. The peripheral branches lacked MAP2 labeling. In neonatal and postnatal stages, MAP2 reactivity was located in spiral ganglion perikarya and their neurites. The intensity of adult labeling was, however, lower than in younger animals. The antibody used in this study did not label axons originating in the CNS as seen by a negative response in efferent fibers from the intraganglionic spiral bundle of the cochlea. Our results suggest that during ontogenesis, MAP2 is highly expressed in the central projection of spiral ganglion neurons, and then is reduced to lower quantities in the central branch after the first postnatal week and persists into adulthood.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ analysis of the action of Navelbine on various types of microtubules using immunofluorescence.

Preliminary clinical studies demonstrated that 5' nor-anhydro-vinblastine, Navelbine (NVB) has a broader antitumor activity and fewer neurotoxic effects than vinblastine or vincristine. The tectal plate anlage of mouse embryos at the earliest stages of neuronal differentiation were used to analyze and compare the effect of NVB, vincristine and vinblastine on axonal and mitotic microtubules after culture of post-implantation embryos in a medium containing the agent. All drugs are active on mitotic microtubules at the same concentration (0.1 mumol/L), inducing a depolymerization of microtubules and a blockade of cells at metaphase. At higher concentrations. NVB is the only one of the three drugs that induces a blockade of the cells at prophase. A depolymerization of axonal microtubules occurs at higher concentrations with NVB than with the two other vinca alkaloids. These results demonstrate that NVB is as active on mitotic microtubules and less active on axonal microtubules than vincristine and vinblastine. These findings can be related to the potent antitumor effect of the drug with minor neurotoxicity.

Biochemical effects of Navelbine on tubulin and associated proteins.

Navelbine (NVB) or 5' nor-anhydro-vinblastine was shown to present a broader antitumor activity and to induce fewer side effects than vinblastine (VBL) or vincristine (VCR). The possible mechanisms of these differences were analyzed with in vitro methods. At substoichiometric concentrations, the three drugs inhibit microtubule assembly. NVB, in comparison with VCR and VBL, is shown to have a lower inhibitory effect. At stoichiometric concentrations, the three drugs are able to induce tubulin aggregation into spirals and paracrystals. This process involves a microtubule-associated protein (MAPs) family referred to as Tau and is inhibited by another MAPs family referred to as MAP2. However, dramatic quantitative and qualitative differences are observed between NVB and VLB or VCR in TAU-induced aggregation of tubulin. The rate and extent of NVB-induced tubulin aggregation is much lower. With NVB, only certain TAU isoforms are able to induce paracrystals, while all TAU isoforms may contribute to VCR-induced or VBL-induced paracrystals. The TAU isoforms that are not able to induce crystallization with NVB, at least in a certain range of concentrations, are probably involved in mitotic microtubules--the hypothetical antitumoral target of vinca alkaloids (VAS). The present work shows for the first time that an anticancer drug is able to discriminate between the various types of microtubules. A next step will be to investigate whether this property is limited to a modulating effect of the various TAU isoforms on the affinity of VAS for tubulin. These biochemical investigations will be extended to tubulins extracted from tumor cell lines in order to further discriminate NVB from the other VAS.

Association of estramustine resistance in human prostatic carcinoma cells with modified patterns of tubulin expression.

Estramustine (EM) is an antimicrotubule drug used in the treatment of hormone refractory advanced prostate cancer. To investigate the mechanism of resistance to EM, we compared its effects on human prostate cancer cells (DU145) and an estramustine-resistant derived cell line (E4). Immunofluorescence demonstrated that EM caused depolymerization of microtubules and blocked cells in mitosis in DU145 cells, with less effect in E4 cells. Using tubulin isotype-specific antibodies, a threefold increase in betaIII and approximately twofold increase in betaI + II isotype in E4 cells compared to DU145 cells were observed. A most interesting observation concerned an increase in the posttranslational modification of alpha-tubulin of both polyglutamylation and acetylation in the E4 cells. Significant to this observation, using direct EM photoaffinity labeling of tubulin, drug binding to the most acidic posttranslationally modified forms of alpha-tubulin was shown to be minimal. Taken together, these results indicate that the modification of the tubulin expression pattern may be responsible for estramustine resistance by both lowering the amount of drug bound to microtubules and inducing more stable microtubules.

Tau microheterogeneity: an immunological approach with monoclonal antibodies.

The family of tau polypeptides purified from mammalian brain exhibit both extensive heterogeneity and large similarities in their chemical, physical, and functional properties. All the tau isoforms generated at a transcriptional or posttranscriptional level share the property of interacting with tubulin dimers in a specific manner. They strengthen longitudinal interactions between tubulin dimers and thus may stabilize microtubules once they are formed. Mild proteolysis or phosphorylation does not remove but only modulates the tau specific function that is probably related to the conserved sequences of the molecules. Monoclonal antibodies raised against tau were found to recognize epitopes conserved not only between species but also in different tissues. Using indirect immunofluorescence, a specific staining pattern was observed on rat neuronal cells and also on human skin fibroblasts. The same antibodies did not recognize glial cells, suggesting that these cells either do not contain detectable levels of tau or contain tau molecules different from the neuronal ones. These data suggest that tau protein is widely distributed, highly conserved, and may be preferentially associated with special subclasses of microtubules.

Different microtubule network alterations induced by pachymatismin, a new marine glycoprotein, on two prostatic cell lines.

Pachymatismin is a new cytostatic factor extracted from the marine sponge Pachymatisma johnstonii Bowerbank. To investigate the mechanism of action of pachymatismin, we studied its effects on two human prostate cell lines (DU145 and E4) of tumor origin. Immunocytochemistry demonstrated that the drug caused depolymerization of microtubules in DU145 cells, this effect being similar to that of estramustine, known to be a microtubule-depolymerizing agent. E4 cells, described to be resistant to the microtubule-depolymerizing agent estramustine, were also found resistant to pachymatismin. Pachymatismin at the same dose that destroys microtubule organization in DU145 cells is not able to induce microtubule depolymerization in E4 cells. Compared to the estramustine- and pachymatismin-sensitive DU145 cells, E4 cells revealed an increase of betaI+II, betaIII, betaIV isotypes as well as post-translational modifications of tubulin, such as polyglutamylation and acetylation. In addition, the level of tau protein was also enhanced in E4 cells compared to DU145 cells. The effects of pachymatismin were tested in vitro using calf brain microtubules. It was shown that the drug lowers the capacity of microtubules to reassemble in vitro. Interestingly, pachymatismin has been found to inhibit microtubule assembly less efficiently when the ratio of tau to tubulin is increased. Taken together, pachymastismin has been shown to induce in vivo microtubule depolymerization following binding to microtubule proteins. Changes in microtubule components such as tubulin isoforms or tau may be involved in a decrease of sensitivity to pachymatismin.

Mouse brain Tau proteins: microheterogeneity and phosphorylation.

Tau proteins are involved in polymerization of tubulin into microtubules. They comprise a heterogeneous group of proteins that can be resolved by two-dimensional gel electrophoresis using a non-equilibrium pH gradient in the first dimension. Developmental studies show that mouse brain Tau proteins are more heterogeneous in 15-day old mice than in newborn pups or adults. Tau phosphorylation is also more heterogeneous at this stage.

Taxol resistance.

BACKGROUND: Taxol has gained considerable attention in cancer therapy in recent years and is successfully used in treating a variety of tumors, including those of the breast, ovary and lung. Despite its preclinical and clinical success, the ability of tumors to develop an acquired resistance to drugs used for treatment remains a major obstacle to cancer cure. A better understanding of the various mechanisms of Taxol resistance may have important implications for strategies designed for cancer treatment. METHOD: This review summarizes the information available to date concerning the effects of Taxol and the development of drug resistance, focusing particularly on alterations in microtubules. RESULTS AND DISCUSSION: Acquired resistance to Taxol is a function of multiple adaptations. Although the cellular transport of Taxol appears to be an important mechanism of resistance to Taxol, altered transcription and posttranslational modification of microtubular changes may be involved in the mechanism of drug resistance.

Estramustine resistance.

Estramustine (EM), a conjugate of nornitrogen mustard and estradiol, is a antimicrotubule drug currently in use for the treatment of advanced prostatic carcinoma. Experimental data are accumulating concerning the antitumor effect of EM in other malignancies, and clinical studies in other malignancies are ongoing. This review summarizes the information available to date concerning the effects of EM and the development of drug resistance. EM depolymerizes microtubules by binding to microtubule-associated proteins (MAPs) as well as tubulin. Because of the radiosensitizing effect of this drug there has been a recent increase in interest concerning estramustine and its clinical use. Recently, it was proposed that EM induces an apoptotic cell death in glioma cells in vitro and in a rat model. EM resistance is distinct from MDR phenotype; it has been used in combination with antimitotic agents which are part of the MDR phenotype. Observations made with estramustine-resistant cell lines show the acquisition of estramustine resistance is a function of multiple adaptation by changes at tubulin expression pattern, and is also associated with changes in tau expression and phosphorylation.