Collagen-induced exposure of anionic phospholipid in platelets and platelet-derived microparticles.

We have shown recently that the calcium-dependent phospholipid-binding protein annexin V (placental anticoagulant protein I) can be used to study the exposure of anionic phospholipid after platelet activation. In this study we have further examined the mechanism of this process. Collagen-induced exposure of annexin V binding sites correlated directly with increased ability to support activity of the reconstituted prothrombinase complex. The potency of annexin V as an inhibitor of platelet prothrombinase was the same as its Kd for platelets. Prior incubation of platelets with 5'-p-fluorosulfonylbenzoyladenosine or p-chloromercuribenzenesulfonate had no significant effect on annexin V binding. Similarly, inhibition of platelet cyclic endoperoxide synthesis by acetylsalicylic acid or indomethacin did not inhibit annexin V binding. Staurosporine inhibited collagen-induced, but not A23187-induced, annexin V binding. Agents that increase intraplatelet cyclic nucleotides partially inhibited collagen-induced annexin V binding. Thus, collagen-induced exposure of anionic phospholipid appears to depend primarily on increases in intraplatelet free calcium and may be independent of ADP- or endoperoxide-mediated pathways. Binding sites for annexin V on microparticles derived from collagen-stimulated platelets were demonstrated by flow cytometry and gel filtration. In addition, prior incubation of platelets with 100 nM annexin V inhibited factor Va binding to both platelets and platelet-derived microparticles. These results support the concept that the procoagulant effect of platelets and platelet-derived microparticles is mediated by calcium-induced exposure of anionic phospholipids.     

Microtubule-associated proteins-dependent colchicine stability of acetylated cold-labile brain microtubules from the Atlantic cod, Gadus morhua

Assembly of brain microtubule proteins isolated from the Atlantic cod, Gadus morhua, was found to be much less sensitive to colchicine than assembly of bovine brain microtubules, which was completely inhibited by low colchicine concentrations (10 microM). The degree of disassembly by colchicine was also less for cod microtubules. The lack of colchicine effect was not caused by a lower affinity of colchicine to cod tubulin, as colchicine bound to cod tubulin with a dissociation constant, Kd, and a binding ratio close to that of bovine tubulin. Cod brain tubulin was highly acetylated and mainly detyrosinated, as opposed to bovine tubulin. When cod tubulin, purified by means of phosphocellulose chromatography, was assembled by addition of DMSO in the absence of microtubule-associated proteins (MAPs), the microtubules became sensitive to low concentrations of colchicine. They were, however, slightly more stable to disassembly, indicating that posttranslational modifications induce a somewhat increased stability to colchicine. The stability was mainly MAPs dependent, as it increased markedly in the presence of MAPs. The stability was not caused by an extremely large amount of cod MAPs, since there were slightly less MAPs in cod than in bovine microtubules. When "hybrid" microtubules were assembled from cod tubulin and bovine MAPs, these microtubules became less sensitive to colchicine. This was not a general effect of MAPs, since bovine MAPs did not induce a colchicine stability of microtubules assembled from bovine tubulin. We can therefore conclude that MAPs can induce colchicine stability of colchicine labile acetylated tubulin.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of microtubules in platelet secretory release

The role of microtubules in platelet aggregation and secretion has been analyzed using platelets permeabilized with digitonin and monoclonal antibodies to alpha (DM1A) and beta (DM1B) subunits of tubulin. Permeabilized platelets were able to undergo aggregation and secretory release. However, threshold doses of agonists capable of eliciting a second wave of aggregation and the platelet release reaction were higher than in control platelets exposed to dimethyl sulfoxide, the solvent for digitonin. Both antibodies to alpha and beta tubulin caused a further increase in the threshold concentration of agonists and inhibited the secretory release of permeabilized platelets, but were ineffective using intact platelets. Neither monoclonal antibody inhibited polymerization or depolymerization of platelet tubulin in vitro. Antibodies to platelet actin and myosin also exhibited an inhibitory activity on platelet aggregation albeit less severe than that observed with the antibodies to alpha and beta tubulin. There was evidence of an interaction between DM1A and DM1B and the antibodies to actin and myosin. The interaction of platelet tubulin and myosin was investigated by two different methods. (1) Coprecipitation of the proteins at low ionic strength at which tubulin by itself did not precipitate and (2) affinity chromatography on columns of immobilized myosin. Tubulin freed of its associated proteins (MAPs) by phosphocellulose chromatography bound to myosin in a molar ratio which approached 2. Platelet actin competed with tubulin for 1 binding site on the myosin molecule. MAPs also reduced the binding stoichiometry of tubulin/myosin. Treatment of microtubule protein with p-chloromercuribenzoate or colchicine did not influence its binding to myosin. DM1A and DM1B inhibited the interaction of tubulin and myosin. This effect could also be demonstrated by reaction of electrophoretic transblots of extracted platelet tubulin with the respective proteins. We interpret these results as evidence for an interference of the two monoclonal antibodies to the tubulin subunits (DM1A and DM1B) with the translocation of microtubule protein from its submembranous site to a more central one during the activation process.     

Mechanism of inhibition of microtubule polymerization by colchicine: inhibitory potencies of unliganded colchicine and tubulin-colchicine complexes.

The tubulin-colchicine binding reaction appears to involve a number of intermediate steps beginning with rapid formation of a transient preequilibrium complex that is followed by one or more slow steps in which conformational changes in tubulin and colchicine lead to formation of a poorly reversible final-state complex. In the present study, we investigated the relative ability of unliganded colchicine and preformed final-stage tubulin-colchicine complex to incorporate at microtubule ends and to inhibit addition of tubulin at the net assembly ends of bovine brain microtubules in vitro. Addition of 0.1 microM final-stage tubulin-colchicine complex to suspensions of microtubules at polymer-mass steady-state resulted in rapid incorporation of one to two molecules of tubulin-colchicine complex per microtubule net assembly end concomitant with approximately 50-60% inhibition of tubulin addition. Incorporation of colchicine-tubulin complex continued slowly with time, without significant additional change in the rate of tubulin addition. In contrast, addition of unliganded colchicine to microtubule suspensions resulted in incorporation of small numbers of colchicine molecules at microtubule ends and inhibition of tubulin addition only after periods of time that varied from several minutes to approximately 20 min depending upon the concentration of colchicine. Inhibition of tubulin addition beginning with unliganded colchicine increased slowly with time, concomitant with increases in the concentration of final-state tubulin-colchicine complex and the amount of colchicine bound per microtubule end. The results indicate that inhibition of tubulin incorporation at microtubule ends is caused by colchicine-liganded tubulin in the form of a final-state complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Microtubules and the calcium-dependent regulation of rat granulosa cell steroidogenesis

The possible relationship between calcium and microtubules in the regulation of granulosa cell steroidogenesis was assessed by using agents known to alter microtubule-tubulin equilibrium together with the ionophore A23187, an antibiotic that facilitates the movement of calcium across plasma membranes. Using immunofluorescence and morphometric analysis, we determined alterations in microtubule organization and overall cell shape, respectively, in response to ionophore-stimulated production of progesterone and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-progesterone) during 24 h of culture. In addition, the influences of colchicine and nocodazole, two agents known to induce microtubule depolymerization, and of taxol, an agent that stabilizes tubulin polymers, on calcium-dependent regulation of granulosa cell progestin production in vitro were examined. Cells cultured as controls were flattened, highly irregular in outline, and associated with a complexly organized, well-spread cytoplasmic network of microtubules. In contrast, those maintained in the presence of increasing concentrations of ionophore were progressively more circular and smooth in outline, occupied less area on the growth surface, and contained cytoplasmic arrays of microtubules considerably less extensive than those of the controls and occupying areas defined by the more regular cellular perimeters. While progestin production in the absence or presence of a submaximally stimulatory concentration of A23187 was increased by both colchicine and nocodazole, the microtubule-depolymerizing agents had little to no effect on the production of the steroids by granulosa cells maximally stimulated by the ionophore. However, both basal and ionophore-induced progestin production were unaltered by taxol except at a concentration of 10 microM in the presence of 0.25 micrograms/ml A23187.(ABSTRACT TRUNCATED AT 250 WORDS)     

Colchicine inhibits ionophore-induced formation of leukotriene B4 by human neutrophils: the role of microtubules

Neutrophils which ingest particles (serum-treated zymosan, monosodium urate crystals) or are exposed to calcium ionophore A23187 generate leukotriene B4 (LTB4). Earlier work has shown that cells exposed to colchicine before exposure to monosodium urate crystals produce less LTB4; the formation of 5-HETE is unaffected. To determine whether inhibition by colchicine of LTB4 generation was stimulus-specific and was mediated by microtubule integrity, the effects of colchicine (10 microM, 60 min) on the release of lipoxygenase products from neutrophils exposed to ionophore A23187 (10 microM, 5 min) were examined. In the presence of exogenous arachidonic acid (100 microM, 15 min), colchicine decreased LTB4 to 48% +/- 11.7 of control and 5-HETE to 60.5% +/- 5.7 of control (mean +/- SEM); 15-HETE was also decreased to 61% +/- 10.3 of control. In the absence of exogenous arachidonate, LTB4 was decreased to 22.2% +/- 11.7 of control and 5-HETE to 13% +/- 4.8 of control. Lumicolchicine did not significantly affect formation of 5-HETE or LTB4. However, vinblastine sulfate (20 microM, 60 min), another microtubule-disruptive agent, decreased the formation of both 5-lipoxygenase products. The effects of colchicine and vinblastine were not due to impairment of cell viability because the release of cytoplasmic lactic dehydrogenase was unaffected. Ultrastructural analysis of centriolar microtubules showed that decrements in microtubule numbers of colchicine- and vinblastine-treated cells paralleled decrements in 5-lipoxygenase products. These pharmacologic manipulations suggested that functional microtubules might be required for optimal lipoxygenase activity. Consequently, we prepared neutrophil-derived cytoplasts, devoid of an intact microtubule system. No significant decreases in the 5- or 15-lipoxygenase products were found when cytoplasts were exposed to colchicine in the presence of exogenous arachidonate and A23187. The data show that colchicine inhibits the formation of lipoxygenase products from neutrophils stimulated with A23187, most likely via its effect on microtubules, the integrity of which appears necessary for full expression of 5- and 15-lipoxygenases.     

The natural naphthoquinone plumbagin exhibits antiproliferative activity and disrupts the microtubule network through tubulin binding

Plumbagin (5-hydroxy-2-methyl-1,4-naphthoquinone), a naphthoquinone isolated from the roots of Plumbaginaceae plants, has potential antiproliferative activity against several tumor types. We have examined the effects of plumbagin on cellular microtubules ex vivo as well as its binding with purified tubulin and microtubules in vitro. Cell viability experiments using human non-small lung epithelium carcinoma cells (A549) indicated that the IC 50 value for plumbagin is 14.6 microM. Immunofluorescence studies using an antitubulin FITC conjugated antibody showed a significant perturbation of the interphase microtubule network in a dose dependent manner. In vitro polymerization of purified tubulin into microtubules is inhibited by plumbagin with an IC 50 value of 38 +/- 0.5 microM. Its binding to tubulin quenches protein tryptophan fluorescence in a time and concentration dependent manner. Binding of plumbagin to tubulin is slow, taking 60 min for equilibration at 25 degrees C. The association reaction kinetics is biphasic in nature, and the association rate constants for fast and slow phases are 235.12 +/- 36 M (-1) s (-1) and 11.63 +/- 11 M (-1) s (-1) at 25 degrees C respectively. The stoichiometry of plumbagin binding to tubulin is 1:1 (mole:mole) with a dissociation constant of 0.936 +/- 0.71 microM at 25 degrees C. Plumbagin competes for the colchicine binding site with a K i of 7.5 microM as determined from a modified Dixon plot. Based on these data we conclude that plumbagin recognizes the colchicine binding site to tubulin. Further study is necessary to locate the pharmacophoric point of attachment of the inhibitor to the colchicine binding site of tubulin.     

Perturbation of microtubule polymerization by quercetin through tubulin binding: a novel mechanism of its antiproliferative activity

The dietary flavonoid quercetin has a broad range of biological activities, including potent antitumor activity against several types of tumors. Recently, it has been shown that quercetin inhibits cancer cells proliferation by depleting cellular microtubules and perturbing cellular microtubule functions. However, the direct interactions of quercetin with tubulin and microtubules have not been examined so far. Here, we found that quercetin inhibited polymerization of microtubules and depolymerized microtubules made from purified tubulin in vitro. The binding of quercetin with tubulin was studied using quercetin fluorescence and intrinsic tryptophan fluorescence of tubulin. Quercetin bound to tubulin at a single site with a dissociation constant of 5-7 microM, and it specifically inhibited colchicine binding to tubulin but did not bind at the vinblastine site. In addition, quercetin perturbed the secondary structure of tubulin, and the binding of quercetin stimulated the intrinsic GTPase activity of soluble tubulin. Further, quercetin stabilized tubulin against decay and protected two cysteine residues of tubulin toward chemical modification by 5,5'-dithiobis-2-nitrobenzoic acid. Our data demonstrated that the binding of quercetin to tubulin induces conformational changes in tubulin and a mechanism through which quercetin could perturb microtubule polymerization dynamics has been proposed. The data suggest that quercetin inhibits cancer cells proliferation at least in part by perturbing microtubule functions through tubulin binding.     

Interplay of cyclic AMP and microtubules in modulating the initiation of DNA synthesis in 3T3 cells

The results presented here show that disruption of the microtubule network acts synergistically with cAMP-elevating agents to stimulate the entry into DNA synthesis of 3T3 cells. Antimicrotubule agents and increased cAMP levels require an additional growth-promoting factor for inducing initiation of DNA synthesis; such requirement can be furnished by insulin, vasopressin, epidermal growth factor, platelet-derived growth factor, or fibroblast-derived growth factor. The involvement of the microtubules is indicated by the fact that enhancement of the DNA synthetic response was demonstrated with the chemically diverse agents colchicine, nocodazole, vinblastine, or demecolcine, all of which elicited the response in a dose-dependent manner. We verified that colchicine and nocodazole, at the doses used in this study, induced microtubule disassembly in the absence as well as in the presence of cAMP-elevating agents as judged by measurement of [3H]colchicine binding of total and pelletable tubulin. The involvement of cAMP was revealed by increasing its endogenous production by cholera toxin or by treatment with 8BrcAMP. The enhancing effects of antimicrotubule drugs and cAMP-elevating agents could be demonstrated by incorporation of [3H]thymidine into acid-insoluble material, autoradiography of labeled nuclei, or flow cytofluorometric analysis. The addition of antimicrotubule drugs does not increase the intracellular level of cAMP nor does addition of cAMP-elevating agents promote disassembly of microtubules (as judged by measuring [3H]colchicine binding of total and pelletable tubulin) in 3T3 cells. In view of these findings and the striking synergistic effects between these agents in stimulating DNA synthesis in the presence of a peptide growth factor, we conclude that increased cAMP levels and a disrupted microtubule network regulate independent pathways involved in proliferative response.     

Microtubules and protein secretion in rat lacrimal glands. Inhibitory effect of the tubulin . colchicine complex isolated from lacrimal glands upon brain tubulin polymerization. Identification of the complex by gel electrophoresis.

The specific inhibitory effect of colchicine upon protein secretion by lacrimal glands could be related to the formation of a complex between colchicine and tubulin from the soluble fraction of the gland. By gel electrophoresis under nondissociating conditions, it is shown that this complex is similar to the colchicine . tubulin complex from brain. The complex isolated from lacrimal glands is highly inhibitory upon brain tubulin assembly since as low as 0.07 microM complex impedes the polymerization of 8 microM tubulin by 50%, compared to 3 microM for free colchicine. Therefore, a small percentage of complexed tubulin (0.9%) is enough for polymerization to be blocked. In lacrimal glands the complex might prevent the polymerization of tubulin, and colchicine shift the tubulin in equilibrium microtubules equilibrium to microtubules disassembly. The disorganization of the labile microtubular system could lead to a modification of the transport of the secretory granules and to a perturbation of secretion.     

The binding of isocolchicine to tubulin. Mechanisms of ligand association with tubulin

Isocolchicine is a structurally related isomer of colchicine altered in the methoxytropone C ring. In spite of virtual structural homology of colchicine and isocolchicine, isocolchicine is commonly believed to be inactive in binding to tubulin and inhibiting microtubule assembly. We have found that isocolchicine does indeed bind to the colchicine site on tubulin, as demonstrated by its ability to competitively inhibit [3H]colchicine binding to tubulin with a KI approximately 400 microM. Isocolchicine inhibits tubulin assembly into microtubules with an I50 of about 1 mM, but the affinity of isocolchicine for the colchicine receptor site, 5.5 +/- 0.9 x 10(3) M-1 at 23 degrees C, is much less (approximately 500-fold) than that of colchicine. Unlike colchicine, isocolchicine binds rapidly, and the absorption and fluorescence properties of the complex are only modestly altered compared to free ligand. It is proposed that the binding of isocolchicine to tubulin may be rationalized either in terms of conformational states of colchicinoids when liganded to tubulin or by the structural requirements for C-10 substituents for high affinity binding to the colchicine receptor.     

A novel microtubule protein in the marginal band of human blood platelets

A characterization is reported of the major cytoskeletal protein, called IEF (isoelectric focusing)-51K, of marginal band microtubule coils from human blood platelets (Kenney, D. M. and Linck, R. W. (1985) J. Cell Sci. 78, 1-22). IEF-51K is a unique biochemical species which is distinguishable from platelet and mammalian neuronal alpha-tubulin and beta-tubulin by 1) its faster mobility on discontinuous sodium dodecyl sulfate electrophoresis corresponding to an apparent Mr 51,000; 2) its more alkaline relative isoelectric point at pH 5.7 compared with that of alpha- and beta-tubulin at pH 5.3 and 5.5, respectively; 3) lack of coincidence in peptide maps prepared with chymotrypsin or Staphylococcus aureus V8 protease; and 4) lack of immunochemical cross-reactivity of polyclonal anti-IEF-51K with alpha- and beta-tubulin and of monoclonal anti-alpha-tubulin and anti-beta-tubulin with IEF-51K. In contrast to its chemical uniqueness, IEF-51K is tubulin-like in some of its properties. IEF-51K is localized in the marginal band of intact platelets by immunofluorescence; it undergoes cycles of microtubule disassembly/reassembly both in vitro and in vivo. Furthermore, IEF-51K was not extracted from isolated Taxol-stabilized marginal band microtubules by elevated NaCl concentrations (to 0.45 M), conditions that do not disrupt the polymeric structure of alpha- and beta-tubulin. These results indicate that IEF-51K together with alpha-tubulin and beta-tubulin are the major structural polypeptides of platelet marginal band microtubules. The unusual subunit composition of the platelet marginal band microtubule may be related to specialization(s) of microtubule structure and function in the marginal band coil of platelets.     

Antimitotic antifungal compound benomyl inhibits brain microtubule polymerization and dynamics and cancer cell proliferation at mitosis, by binding to a novel site in tubulin

The antifungal agent benomyl [methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate] is used throughout the world against a wide range of agricultural fungal diseases. In this paper, we investigated the interaction of benomyl with mammalian brain tubulin and microtubules. Using the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonic acid, benomyl was found to bind to brain tubulin with a dissociation constant of 11.9 +/- 1.2 microM. Further, benomyl bound to at a novel site, distinct from the well-characterized colchicine and vinblastine binding sites. Benomyl altered the far-UV circular dichroism spectrum of tubulin and reduced the accessibility of its cysteine residues to modification by 5,5'-dithiobis-2-nitrobenzoic acid, indicating that benomyl binding to tubulin induces a conformational change in the tubulin. Benomyl inhibited the polymerization of brain tubulin into microtubules, with 50% inhibition occurring at a concentration of 70-75 microM. Furthermore, it strongly suppressed the dynamic instability behavior of individual brain microtubules in vitro as determined by video microscopy. It reduced the growing and shortening rates of the microtubules but did not alter the catastrophe or rescue frequencies. The unexpected potency of benomyl against mammalian microtubule polymerization and dynamics prompted us to investigate the effects of benomyl on HeLa cell proliferation and mitosis. Benomyl inhibited proliferation of the cells with an IC(50) of 5 microM, and it blocked mitotic spindle function by perturbing microtubule and chromosome organization. The greater than expected actions of benomyl on mammalian microtubules and mitosis together with its relatively low toxicity suggest that it might be useful as an adjuvant in cancer chemotherapy.     

Characterization and in vitro polymerization of Tetrahymena tubulin

Tetrahymena tubulin was purified from the cell extract using DEAE-Sephadex A-50 ion-exchanger and ammonium sulfate precipitation. About 2.2% of the total protein in the 20,000 X g supernatant was recovered as DEAE-Sephadex-purified tubulin fraction. Applying the temperature-dependent polymerization-depolymerization method to this fraction in the presence of Tetrahymena outer fibers as a seed, almost pure tubulin was obtained. Tetrahymena tubulin dimer showed different behavior on SDS-polyacrylamide gels from porcine brain tubulin, and showed very low affinity for colchicine, amounting to about one-twentieth of the binding to porcine brain tubulin. The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragment induced polymerization as demonstrated by viscometric measurements, but the reconstituted microtubules were very unstable in the absence of glycerol. Microtubule-depolymerizing agents such as Ca2+ ions, low temperature, or colchicine all inhibited in vitro polymerization. Although Tetrahymena tubulin purified by the polymerization-depolymerization method could copolymerize with porcine brain microtubules, the DEAE-Sephadex-purified tubulin fraction suppressed the initial rate of porcine brain microtubule assembly in vitro. There seemed to be no differences between cytoplasmic tubulin and outer fiber tubulin in colchicine binding activity or SDS-gel electrophoretic behavior, or between the fine structure of both reconstituted microtubules observed by electron microscopy.     

4',6-Diamidino-2-phenylindole, a fluorescent probe for tubulin and microtubules

A new fluorophor for tubulin which has permitted the monitoring of microtubule assembly in vitro is reported. DAPI (4',6-diamidino-2-phenylindole), a fluorophor already known as a DNA intercalator, was shown to bind specifically to a unique tubulin site as a dimer (KD(app) = 43 +/- 5 microM at 37 degrees C) or to tubulin associated in microtubules (KD(app) = 6 +/- 2 microM at 37 degrees C) with the same maximum enhancement in fluorescence. When tubulin polymerization was induced with GTP, the change in DAPI affinity for tubulin resulted in an enhancement of DAPI binding and, consequently, of fluorescence intensity. DAPI, whose binding site is different from that of colchicine, vinblastine, or taxol, did not interfere greatly with microtubule polymerization. It induced a slight diminution of the critical concentration for tubulin assembly due to a decrease in the depolymerizing rate constant. Moreover, DAPI did not interfere with GTP hydrolysis correlated with tubulin polymerization, but it decreased the GTPase activity at the steady state of tubulin assembly. Even at substoichiometric levels DAPI can be used to follow the kinetics of microtubule assembly.     

Microtubule structure at improved resolution.

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.     

Interaction of captan with mammalian microtubules

Using turbidometry, electron microscopy and immunofluorescent microscopy experiments we studied the effect of captan, a widely used pesticide on mammalian microtubules and microfilaments. Turbidometry at 350 nm showed a dose-dependent inhibition of tubulin assembly incubated with captan. The pesticide, given at equimolar concentration with tubulin (30 microM), caused the total inhibition of microtubule formation, while at lower concentrations (5-20 microM) the inhibition of tubulin polymerization was less extensive. At the same concentration range (5-30 microM), captan also promoted the disassembly of performed microtubules. The results of the in vitro effects of captan with microtubules were confirmed in parallel by electron microscopic studies. In vivo, captan caused also depolymerization of microtubules in cultured mouse fibroblasts as shown by indirect immunofluorescent staining of tubulin. The extent of microtubules disassembly was concentration- and time-dependent. While incubation of the cells with 10 microM captan for 3 h disturbs totally the microtubular structures, incubation with 5 microM captan needs 12 h for the same effect. Recovery of microtubules was observed, when preincubated cells were extensively washed. No interaction of this drug with equimolar concentration of G- or F-actin could be observed in vitro, as shown by polymerization experiments. In line with this, the fluorescent actin pattern in mouse fibroblasts incubated with 10 mM captan for up to 12 h did not seem to be altered. From these results it is concluded that captan interacts in equimolar concentrations with tubulin affecting the assembly and disassembly of microtubules in vitro and in cultures of mammalian cells.     

Traces of brain microtubule-associated proteins affect dynamic properties of microtubules

A method is described for measuring the quantities of stable and dynamic microtubules in a population in vitro. The method exploits the tendency of dynamic microtubules to depolymerize rapidly after being sheared. Stable microtubules, such as those protected by microtubule-associated proteins (MAPs), are broken to a smaller size by shearing, but do not depolymerize into subunits. The usual difficulty with this procedure is that the tubulin released from the dynamic microtubules rapidly repolymerizes before the end point of depolymerization can be measured. This has been overcome by including a small quantity of tubulin-colchicine complex in the mixture to block the repolymerization. For a total of 24 microM tubulin in a polymerization mixture, 10 microM of the sample polymerized originally under the conditions used. When 1.05 microM tubulin-colchicine complex was added at the time of shearing, the dynamic microtubules depolymerized, but the tubulin was released was unable to repolymerize and a small fraction of stable microtubules that resisted shear-induced depolymerization could then be detected. When traces of MAPs (0.23-2.8% by mass) were included in the tubulin mixture, the fraction of stable microtubules increased from 5% in the absence of added MAPs to 41% in the presence of 2.8% MAPs. All the MAPs in the mixture were found in the stable fraction and this stable fraction forms early during microtubule assembly. Calculations on the extent of enrichment of MAPs in the stable fraction indicated that as little as 4% MAPs in a microtubule protected it from shear-induced disassembly. The results suggest that low levels of MAPs may distribute nonrandomly in the microtubule population.     

The binding of Ruthenium red to tubulin

The inhibition of microtubule assembly by Ruthenium red (Deinum, J., Wallin, M., Kanje, M. and Lagercrantz, C. (1981) Biochim. Biophys. Acta 675, 209-213) could be counteracted by either taxol or dimethyl sulfoxide. Ruthenium red remained bound to the assembled microtubules. Microtubules assembled in the presence of Ruthenium red and taxol showed the typical taxol-dependent stability. The dimethyl sulfoxide-induced microtubules showed normal assembly characteristics, e.g., were GTP dependent, could be disassembled by cold, colchicine and Ca2+ and had no alterations in ultrastructure. The absolute disassembly induced by Ca2+ in the presence of dimethyl sulfoxide and Ruthenium red was dependent on the microtubule protein concentration, but independent in the absence of Ruthenium red. Ruthenium red was strongly bound to purified tubulin also in the presence of 8% (v/v) dimethyl sulfoxide. The dimethyl sulfoxide-induced assembly of purified tubulin in the presence of Ruthenium red was slightly stimulated, although the critical protein concentration was the same. It was found by resonance Raman spectroscopy with a flow technique that Ruthenium red did not bind to a specific calcium binding site on tubulin, although binding to a GTP binding site cannot be excluded. The wavenumbers of the lines in the region 375-500 cm-1 differ from those found for Ruthenium red bound to typical calcium-binding proteins such as calmodulin. Although Ruthenium red binds to serum albumin as well, the spectrum with albumin resembled that of the free dye.