Effect of estramustine phosphate on the assembly of trypsin-treated microtubules and microtubules reconstituted from purified tubulin with either tau, MAP2, or the tubulin-binding fragment of MAP2

Estramustine phosphate, an estradiol nitrogen-mustard derivative is a microtubule-associated protein (MAP)-binding microtubule inhibitor, used in the therapy of prostatic carcinoma. It was found to inhibit assembly and to induce disassembly of microtubules reconstituted from phosphocellulose-purified tubulin with either tau, microtubule-associated protein 2, or chymotrypsin-digested microtubule-associated protein 2. Estramustine phosphate also inhibited assembly of trypsin-treated microtubules, completely depleted of high-molecular-weight microtubule-associated proteins, but with their microtubule-binding fragment present. In all cases estramustine phosphate induced disassembly to about 50%, at a concentration of approximately 100 microM, at similar protein concentrations. However, estramustine phosphate did not affect dimethyl sulfoxide-induced assembly of phosphocellulose-purified tubulin. Estramustine phosphate is a reversible inhibitor, as the nonionic detergent Triton X-100 was found to counteract the inhibition in a concentration-dependent manner. The reversibility was nondisruptive, as Triton X-100 itself did not affect microtubule assembly, microtubule protein composition, or morphology. This new reversible MAPs-dependent inhibitor estramustine phosphate affects the tubulin assembly, induced by tau, as well as by the small tubulin-binding part of MAP2 with the same concentration dependency. This indicates that tau and the tubulin-binding part of MAP2, in addition to their assembly promoting functions also have binding site(s) for estramustine phosphate in common.     

Estramustine-phosphate binds to a tubulin binding domain on microtubule-associated proteins MAP-2 and tau.

Estramustine-phosphate (EMP), a phosphorylated conjugate of estradiol and nor-nitrogen mustard binds to microtubule-associated proteins MAP-2 and tau. It was shown that this estramustine derivative inhibits the binding of the C-terminal tubulin peptide beta-(422-434) to both MAP-2 and tau. This tubulin segment constitutes a main binding domain for these microtubule-associated proteins. Interestingly, estramustine-phosphate interacted with the synthetic tau peptides V187-G204 and V218-G235, representing two major repeats within the conserved microtubule-binding domain on tau and also on MAP-2. This observation was corroborated by the inhibitory effects of estramustine-phosphate on the tau peptide-induced tubulin assembly into microtubules. On the other hand, the nonphosphorylated drug estramustine failed to block the MAP peptide-induced assembly, indicating that the negatively charged phosphate moiety of estramustine-phosphate is of importance for its inhibitory effect. These findings suggest that the molecular sites for the action of estramustine-phosphate are located within the microtubule binding domains on tau and MAP-2.     

Stoichiometry of estramustine phosphate binding to MAP2 measured by the disassembly of chick brain MAP2:tubulin microtubules

The concentration of estramustine phosphate required to inhibit the assembly or to induce the disassembly of chick brain MAP2:tubulin microtubules is markedly dependent upon the microtubule protein concentration. Analysis of this relationship shows that estramustine phosphate and tubulin compete for common MAP2 sites, that MAP2 can bind 5-6 moles.mole-1 estramustine phosphate, and that the Kd of these sites is congruent to 20 microM estramustine phosphate. It is proposed that two molecules of estramustine phosphate interact with each of the three tubulin-binding sites of MAP2 and inhibit the MAP2:tubulin interaction by neutralising two highly conserved basic residues.     

Molecular weight dependency of heparin inhibition of microtubule assembly in vitro

Low molar ratios of heparin inhibited in vitro assembly of bovine brain microtubule proteins and disassembled preformed microtubules. Addition of purified microtubule-associated proteins counteracted the assembly inhibition by heparin. Our results suggest that the polyanion heparin affects microtubule assembly by binding to the microtubule-associated proteins. This complex can not support nucleation or stabilize the microtubule structure although it still can associate with the tubulin polymer. In the presence of heparin, the critical concentration needed for microtubule assembly was increased. Furthermore, the absolute assembly difference induced by heparin, the delta A350, was only dependent on the concentration and the molecular weight of heparin, not of the total microtubule protein concentration, or the addition of microtubule-associated proteins. Commercial, standard heparin (Mr 6000-25 000) had an I50 of about 0.1/tubulin dimer. The heparin fraction(s) with a high molecular weight had a stronger effect than those with lower molecular weight. Substoichiometric amounts of taxol completely relieved the inhibition of assembly by heparin, although aberrant forms were present. These microtubules had a reduced amount of coassembled microtubule-associated proteins, and furthermore contained heparin.     

Cysteine oxidation of tau and microtubule-associated protein-2 by peroxynitrite: modulation of microtubule assembly kinetics by the thioredoxin reductase system

Alterations in the redox status of proteins have been implicated in the pathology of several neurodegenerative conditions including Alzheimer and Parkinson diseases. We report that peroxynitrite- and hydrogen peroxide-induced disulfides in the neuron-specific microtubule-associated proteins tau and microtubule-associated protein-2 are substrates for the ubiquitous thioredoxin reductase system composed of thioredoxin reductase, human or Escherichia coli thioredoxin, and NADPH. Tau and microtubule-associated protein-2 cysteine oxidation and reduction were quantitated by monitoring the incorporation of 5-iodoacetamidofluorescein, a thiol-specific labeling reagent. Cysteine oxidation of tau and microtubule-associated protein-2 to disulfides altered the ability of the proteins to promote the assembly of microtubules from purified porcine tubulin. Treatment of tau and microtubule-associated protein-2 with either the thioredoxin reductase system or small molecule reductants fully restores the ability of the MAPs to promote microtubule assembly. Thus changes in the redox state of microtubule-associated proteins may regulate microtubule polymerization in vivo.     

Dependency of microtubule-associated proteins (MAPS) for tubulin stability and assembly; use of estramustine phosphate in the study of microtubules

Summary Microtubule-associated proteins (MAPS) were separated from tubulin with several different methods. The ability of the isolated MAPs to reinduce assembly of phosphocellulose purified tubulin differed markedly between the different methods. MAPs isolated by addition of 0.35 M NaCl to taxol-stabilized microtubules stimulated tubulin assembly most effectively, while addition of 0.6M NaCl produced MAPs with a substantially lower ability to stimulate tubulin assembly. The second best preparation was achieved with phosphocellulose chromatographic separation of MAPs with 0.6 M NaCl elution.The addition of estramustine phosphate to microtubules reconstituted of MAPS prepared by 0.35 M NaCl or phosphocellulose chromatography, induced less disassembly than for microtubules assembled from unseparated proteins, and was almost without effect on microtubules reconstituted from MAPs prepared by taxol and 0.6 M NaCl. Estramustine phosphate binds to the tubulin binding part of the MAPs, and the results do therefore indicate that the MAPs are altered by the separation methods. Since the MAPs are regarded as highly stable molecules, one probable alteration could be aggregation of the MAPs, as also indicated by the results. The purified tubulin itself seemed not to be affected by the phosphocellulose purification, since the microtubule proteins were unchanged by the low buffer strenght used during the cromatography. However, the assembly competence after a prolonged incubation of the microtubule proteins at 4° C was dependent on intact bindings between the tubulin and MAPs.Abbreviations Pipes 1,4-Piperazinediethanesulfonic acid - EDTA Ethylenedinitrilo Tetraacetic Acid - MAPs Microtubule-Associated Proteins - SDS-PAGE SDS-Polyacrylamide Gel Electrophoresis     

Taxol assembles tubulin in the absence of exogenous guanosine 5'-triphosphate or microtubule-associated proteins

Taxol increases the rate and extent of microtubule assembly in vitro and stabilizes microtubules in vitro and in cells [Schiff, P. B., Fant, J., & Horwitz, S. B. (1979) Nature (London) 277, 665-667; Schiff, P. B., & Horwitz, S. B. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 1561-1565]. We report herein that taxol has the ability to promote microtubule assembly in the absence of microtubule-associated proteins, rings, and added guanosine 5'-triphosphate (GTP or organic buffer. The drug enhances additional microtubule assembly when added to microtubules at apparent steady state. This additional assembly can be attributed to both elongation of existing microtubules and spontaneous nucleation of new microtubules. Taxol-treated microtubules have depressed dissociation reactions as determined by dilution experiments. The drug does not inhibit the binding of GTP or the hydrolysis of GTP or guanosine 5'-diphosphate (GDP) in our microtubule protein preparations. Taxol does not competitively inhibit the binding of colchicine to tubulin.     

Incorporation of GDP-tubulin during elongation of microtubules in vitro

Removal of GDP from tubulin E-site is not obligatory for the in vitro assembly of microtubule protein in 0.5 mM GMPPCP. This assembly, which is significantly enhanced by glycerol, produces microtubules of normal morphology and with normal composition of microtubule-associated proteins (MAPs). [3H]-GDP initially present at the E-site is shown to be incorporated directly into microtubules during assembly; this incorporation, maximally 60% of the assembled polymer, is dependent upon MAPs. These results are consistent with oligomeric species composed principally of GDP-tubulin plus MAPs, being incorporated directly into microtubules. The finding that stoichiometric GTP-tubulin formation is not an essential prerequisite for microtubule assembly may have important implications for the energetics of microtubule formation.     

The benzophenanthridine alkaloid sanguinarine perturbs microtubule assembly dynamics through tubulin binding. A possible mechanism for its antiproliferative activity

Sanguinarine has been shown to inhibit proliferation of several types of human cancer cell including multidrug-resistant cells, whereas it has minimal cytotoxicity against normal cells such as neutrophils and keratinocytes. By analyzing the antiproliferative activity of sanguinarine in relation to its effects on mitosis and microtubule assembly, we found that it inhibits cancer cell proliferation by a novel mechanism. It inhibited HeLa cell proliferation with a half-maximal inhibitory concentration of 1.6 +/- 0.1 microM. In its lower effective inhibitory concentration range, sanguinarine depolymerized microtubules of both interphase and mitotic cells and perturbed chromosome organization in mitotic HeLa cells. At concentrations of 2 microM, it induced bundling of interphase microtubules and formation of granular tubulin aggregates. A brief exposure of HeLa cells to sanguinarine caused irreversible depolymerization of the microtubules, inhibited cell proliferation, and induced cell death. However, in contrast with several other microtubule-depolymerizing agents, sanguinarine did not arrest cell cycle progression at mitosis. In vitro, low concentrations of sanguinarine inhibited microtubule assembly. At higher concentrations (> 40 microM), it altered polymer morphology. Further, it induced aggregation of tubulin in the presence of microtubule-associated proteins. The binding of sanguinarine to tubulin induces conformational changes in tubulin. Together, the results suggest that sanguinarine inhibits cell proliferation at least in part by perturbing microtubule assembly dynamics.     

Vinblastine-induced aggregation of brine shrimp (Artemia) tubulin

Tubulin from the brine shrimp Artemia readily assembles in vitro in the absence of microtubule-associated proteins under conditions which do not permit assembly of tubulin from brain. Heated microtubule-associated protein preparations from bovine brain do, however, interact with Artemia tubulin, resulting in stimulation of tubulin assembly and formation of morphologically normal cold-sensitive microtubules. Addition of vinblastine to mixtures containing microtubules assembled in the presence of neural microtubule-associated proteins caused a drop and then a rise in turbidity of the solution. The turbidity changes were accompanied by the appearance of coils, presumably derived from the microtubules which disappeared upon addition of vinblastine. Coils also resulted when microtubule-associated proteins and vinblastine were added to tubulin before polymerization was initiated. Vinblastine prevented normal assembly and caused disruption of Artemia microtubules polymerized in the absence of microtubule-associated proteins. Under these conditions clumped or compact coils, different in appearance from those formed in the presence of the microtubule-associated proteins, were observed. The data confirm that tubulin from Artemia, an organism that is phylogenetically far removed from mammals, has retained binding sites for vinblastine and microtubule-associated proteins and that the interrelationship of these sites has been at least partially preserved. The incomplete depolymerization of Artemia microtubules in response to vinblastine when microtubule-associated proteins are absent suggests that the longitudinal tubulin-tubulin interactions involved in microtubule formation are more stable for Artemia than for neural tubulin.     

The Balance Between τ Protein's Microtubule Growth and Nucleation Activities: Implications for the Formation of Axonal Microtubules

Abstract: The microtubule-associated protein τ is found primarily in neuronal tissues and is highly enriched in the axon. It promotes microtubule assembly in vitro and stabilizes microtubules in cells. To study how τ protein might be involved in the unique features of axonal microtubules, we have analyzed the effect of E. coli -synthesized τ protein using an in vitro centrosome-mediated microtubule regrowth assay over a wide range of τ/tubulin ratios. We report that microtubule assembly promoted by τ protein exhibits characteristic changes dependent on the τ/tubulin ratio. Above a threshold level, nucleation of new microtubules is favored over growth of existing ones, τ isoform variation does not change this phase transition in microtubule assembly. We discuss how τ might participate in the elaboration of axonal morphology based on our results and present evidence that the phase transition from microtubule growth to nucleation is critical for axonal development.     

The effects of estramustine on metaphase and anaphase in DU 145 prostatic carcinoma cells

The chemotherapeutic drug, estramustine, has been shown to cause the disassembly of microtubules via binding to microtubule-associated proteins. In this report, estramustine is shown to be a potent inhibitor of mitotic progression in the human prostatic carcinoma cell line, DU 145. Examination of individual living cells via video-enhanced differential interference contrast (DIC) optics shows that the drug delays the onset of anaphase, reduces anaphase spindle-pole elongation (anaphase B), and delays cytokinesis. In addition, immunofluorescent studies demonstrate that estramustine causes a rapid disorganization of the mitotic apparatus at significantly lower concentrations than those reported previously. Electron microscopic studies show that microtubule bundles are present in drug-treated mitotic cells in association with kinetochores and centrioles.     

The secretion of urokinase-like plasminogen activator is inhibited by microtubule-interacting drugs

The secretion of proteinases into the extracellular matrix is one of the main features of tumour cells, as related to their invasive behaviour. Considering the role of the microtubule cytoskeleton, and particularly the action of microtubule-associated protein (MAPs) in mediating protein secretion, the effects of the anti-microtubule drugs estramustine and taxol, on the secretion of urokinase-type plasminogen activator (u-PA) and the 72 kDa gelatinase were investigated. Treatment of 5637 bladder carcinoma cells with estramustine and taxol inhibited u-PA secretion into the conditioned medium in a drug concentration-dependent fashion. This inhibition was confirmed by determinations of u-PA enzymatic activities and by measurements of the levels of immunoreactive activator. Studies using gelatin zymograms also showed an inhibition of another tumoural proteinase namely the 72 kDa gelatinase. Time-course uptake experiments showed that estramustine was incorporated into the cells, a process which depended on temperature. On the other hand, immunofluorescence studies indicated that the microtubule network was affected by taxol with the formation of bundles of microtubules at different cell domains. Minor effects were visualized after treatment of the cells with estramustine-phosphate, a drug that blocks primarily the action of microtubule-associated proteins. The studies provide a way to analyse the relationships between u-PA secretion and the integrity of the cytoskeletal network.     

Changes in the hydrodynamic properties of microtubules induced by taxol

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.     

Phosphatidylinositol inhibits microtubule assembly by binding to microtubule-associated protein 2 at a single, specific, high-affinity site.

The effects of various anionic phospholipids on the in vitro assembly of MAP2/tubulin microtubules has been examined. We show that the potency to inhibit is related to the polarity of the phospholipids and that this is consistent with a mode of action involving the sequencing of microtubule-associated proteins (MAPs) by nonspecific electrostatic interactions. The inhibitory potency of phosphatidylinositol (PI) is, however, considerably larger than predicted by this model. The effects of PI on MAP2/tubulin microtubule assembly have therefore been examined in greater detail by preparing phosphatidylcholine (PC) liposomes doped with increasing amounts of PI. We show that when the PI is sufficiently dispersed by dilution with PC, it inhibits microtubule assembly by binding to MAP2 with an apparent stoichiometry, after correction for the bilamellar nature of the liposomes, of 1:1 mol.mol-1 PI:MAP2. Furthermore, we show that the Kd of this interaction is in the submicromolar range.     

Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules

Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.     

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.     

Nerve growth factor-induced neurite outgrowth in PC12 cells involves the coordinate induction of microtubule assembly and assembly-promoting factors

Nerve growth factor (NGF) regulates the microtubule-dependent extension and maintenance of axons by some peripheral neurons. We show here that one effect of NGF is to promote microtubule assembly during neurite outgrowth in PC12 cells. Though NGF causes an increase in total tubulin levels, the formation of neurites and the assembly of microtubules follow a time course completely distinct from that of the tubulin induction. The increases in microtubule mass and neurite extension closely parallel 10- and 20-fold inductions of tau and MAP1, proteins shown previously to promote microtubule assembly in vitro. When NGF is removed from PC12 cells, neurites disappear, microtubule mass decreases, and both microtubule-associated proteins return to undifferentiated levels. These data suggest that the induction of tau and MAP1 in response to NGF promotes microtubule assembly and that these factors are therefore key regulators of neurite outgrowth.     

Microtubule-associated proteins from Antarctic fishes

Microtubules and presumptive microtubule-associated proteins (MAPs) were isolated from the brain tissues of four Antarctic fishes (Notothenia gibberifrons, N. coriiceps neglecta, Chaenocephalus aceratus, and a Chionodraco sp.) by means of a taxol-dependent, microtubule-affinity procedure (cf. Vallee: Journal of Cell Biology 92:435-442, 1982). MAPs from these fishes were similar to each other in electrophoretic pattern. Prominent in each preparation were proteins in the molecular weight ranges 410,000-430,000, 220,000-280,000, 140,000-155,000, 85,000-95,000, 40,000-45,000, and 32,000-34,000. The surfaces of MAP-rich microtubules were decorated by numerous filamentous projections. Exposure to elevated ionic strength released the MAPs from the microtubules and also removed the filamentous projections. Addition of fish MAPs to subcritical concentrations of fish tubulins at 0-5 degrees C induced the assembly of microtubules. Both the rate and the extent of this assembly increased with increasing concentrations of the MAPs. Sedimentation revealed that approximately six proteins, with apparent molecular weights between 60,000 and 300,000, became incorporated into the microtubule polymer. Bovine MAPs promoted microtubule formation by fish tubulin at 2-5 degrees C, and proteins corresponding to MAPs 1 and 2 co-sedimented with the polymer. MAPs from C. aceratus also enhanced the polymerization of bovine tubulin at 33 degrees C, but the microtubules depolymerized at 0 degrees C. We conclude that MAPs are part of the microtubules of Antarctic fishes, that these proteins promote microtubule assembly in much the same way as mammalian MAPs, and that they do not possess special capacities to promote microtubule assembly at low temperatures or to prevent cold-induced microtubule depolymerization.     

Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization.

Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of [14C]NAD+ and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the alpha and beta chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight mirotubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated [14C]ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD+ resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.