On the mechanism of calmodulin-induced inhibition of microtubule assembly in vitro

Binding of calmodulin to microtubule-associated proteins (MAPs) was analyzed by the equilibrium gel filtration method. The apparent dissociation constant (Kd) of calmodulin binding was found to be 2 microM for tau, and 5 microM for MAP2. These Kd values were similar to the Kd previously determined for calmodulin binding to tubulin. The inhibitory effect of increasing concentrations of calmodulin on the kinetics of microtubule assembly from tau and tubulin was not mimicked by decreasing the concentration of tau alone or tubulin alone. These results suggest that calmodulin inhibits microtubule assembly by its binding to both MAPs and tubulin.     

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.     

Griseofulvin: Association with tubulin and inhibition of in vitro microtubule assembly

Griseofulvin—shown previously to disrupt the mitotic apparatus $\text{in vivo}$—inhibited the $\text{in vitro}$ microtubule assembly reaction completely at 8 × 10^?4M griseofulvin. In a gel filtration assay, randomly tritiated griseofulvin associated stoichiometrically with purified tubulin, as determined by chromatography on Sephadex G-25. No detectable drug binding was observed when bovine serum albumin was used as a control in an identical column assay. Both gel filtration chromatography and a kinetic analysis of the inhibition of assembly by griseofulvin suggest that the drug interacts directly and stoichimetrically with the tubulin dimer, and that the interaction is both rapid and independent of temperature.     

Colchicine inhibition of microtubule assembly via copolymer formation.

Colchicine.tubulin complex (CD) inhibits microtubule assembly. We examined this inhibition under conditions where spontaneous nucleation was suppressed and assembly was restricted to an elongation polymerization. We found that CD inhibited assembly by a mechanism which preserved the ability of microtubule ends to add tubulin. This observation is inconsistent with the end-poisoning model which recently was proposed as a general mechanism for assembly inhibition by CD. Our data are consistent with the following model: (a) microtubules formed in the presence of CD are CD-tubulin copolymers; (b) these copolymers can have appreciable numbers of incorporated CDs which are, most likely, randomly distributed in the copolymers; (c) CD-tubulin copolymers have assembly-competent ends with association and dissociation rate constants which decrease as the CD/tubulin ratio in the copolymers, (CD/T)MT, increases; and (d) the critical tubulin concentrations required for microtubule assembly increase in the presence of CD, indicating that copolymer affinity for tubulin decreases as (CD/T)MT increases.     

Nucleotide specificity in microtubule assembly in vitro

A procedure is described for removing most of the GDP bound at the exchangeable GTP binding site (E site) of tubulin. Microtubule protein containing substoichiometric amounts of GDP at the E site is found to polymerize in response to: (a) two nonhydrolyzable ATP analogues, adenylyl imidodiphosphate (AMP-PNP) and adenylyl beta, gamma-methylenediphosphonate (AMP-PCP); and (b) substoichiometric levels of GTP or dGTP. The results are interpreted as suggesting that: (1) when GDP is removed from tubulin, the E site shows broad specificity for nucleoside triphosphates: (2) microtubule assembly can be induced by the binding of substoichiometric amounts of nucleoside triphosphate to the E site.     

Erythrocyte microtubule assembly in vitro. Tubulin oligomers limit the rate of microtubule self-assembly

Chicken erythrocyte tubulin containing a unique beta tubulin variant polymerizes with greater efficiency (lower critical concentration) but at a slower rate than chicken brain tubulin. In a previous study we demonstrated that the low net rate of assembly is partly due to the presence of large oligomers and rings which reduce the initial rate of subunit elongation on microtubule seeds (Murphy, D.B., and Wallis, K.T. (1985) J. Biol. Chem. 260, 12293-12301). In this study we show that erythrocyte tubulin oligomers also retard the rate of microtubule nucleation and the net rate of self-assembly. The inhibitory effect is most likely to be due to the increased stability of erythrocyte tubulin oligomers, including a novel polymer of coiled rings that forms during the rapid phase of microtubule polymerization. The slow rate of dissociation of rings and coils into dimers and small oligomers appears to limit both the nucleation and elongation steps in the self-assembly of erythrocyte microtubules.     

Nucleotide binding and phosphorylation in microtubule assembly in vitro.

Two non-hydrolyzable analogs of GTP, guanylyl-β,γ-methylene diphosphonate and guanylyl imidodiphosphate, have been found to induce rapid and efficient microtubule assembly in vitro by binding at the exchangeable site (E-site) on tubulin. Characterization of microtubule polymerization by several criteria, including polymerization kinetics, nucleotide binding to depolymerized and polymerized microtubules, and microtubule stability, reveals strong similarities between microtubule assembly induced by GTP and non-hydrolyzable GTP analogs. Nucleoside triphosphates which bind weakly or not at all to tubulin, such as ATP, UTP and CTP, are shown to induce microtubule assembly by means of a nucleoside diphosphate kinase (NDP-kinase, EC 2.7.4.6.) activity which is not intrinsic to tubulin. The NDP-kinase mediates microtubule polymerization by phosphorylating tubulin-bound GDP in situ at the E-site. Although hydrolysis of exchangeably bound GTP occurs, it is found to be uncoupled from the polymerization reaction. The non-exchangeable nucleotide binding site on tubulin (N-site) is not directly involved in microtubule assembly in vitro. The N-site is shown to contain almost exclusively GTP which is not hydrolyzed during microtubule assembly. A scheme is presented in which GTP acts as an allosteric effector at the E-site during microtubule assembly in vitro.     

Cep70 promotes microtubule assembly in vitro by increasing microtubule elongation

Microtubules are dynamic cytoskeletal polymers present in all eukaryotic cells. In animal cells, they are organized by the centrosome, the major microtubule-organizing center. Many centrosomal proteins act coordinately to modulate microtubule assembly and organization. Our previous work has shown that Cep70, a novel centrosomal protein regulates microtubule assembly and organization in mammalian cells. However, the molecular details remain to be investigated. In this study, we investigated the molecular mechanism of how Cep70 regulates microtubule assembly using purified proteins. Our data showed that Cep70 increased the microtubule length without affecting the microtubule number in the purified system. These results demonstrate that Cep70 could directly regulate microtubule assembly by promoting microtubule elongation instead of microtubule nucleation.     

Rotenone inhibition of spindle microtubule assembly in mammalian cells

Rotenone, a potent inhibitor of mitochondrial respiration is also an effective antimitotic agent. The addition of either rotenone or Colcemid to exponentially growing Chinese hamster ovary cells resulted in a dramatic increase in mitotic index after 90 min. When the cultures were washed free of the drugs, mitosis was completed and the cells progressed into G 1 at approximately the same rate. Further similarity of rotenone-arrested cells to Colcemid-induced mitotic inhibition was apparent at the ultrastructural level. Mitotic cells treated by either drug contained monopolar spindles with chromosomes grouped around centriole pairs near the cell center. Occasional microtubules were seen near the kinetochore and centrioles. These observations, along with the fact that rotenone inhibited the binding of ³H-colchicine to isolated bovine brain tubulin, suggested that rotenone inhibited mitosis by binding directly to tubulin to prevent microtubule assembly.     

Effect of gamma-irradiation on microtubule assembly in vitro

Microtubular protein was exposed to gamma-radiation from 500 to 1000 Gy, Within that dose range its polymerization ability was decreased by 20-60 per cent when samples were irradiated in assembled state, and by 40-75 per cent when irradiated in unassembled state. Microtubules assembled from irradiated subunits were shorter and of more uniform lengths than control microtubules. For the dose of 1000 Gy the mean length and its standard deviation were reduced to about one-half of the values of the control.     

Ethacrynic acid induced inotropism.

Ethacrynic acid (ECA), a sulfhydryl group inhibiting diuretic was examined for positive inotropic effects. These were found to be present in isolated guinea pig left atria studied in 0.9 and 1.8 mM Ca bathing solutions and were partially dependent upon adrenergic mechanisms (presumably secondary to norepinephrine release from sympathetic nerve endings) and partly independent of such mechanisms as demonstrated by propranolol induced beta-blockade and reserpine-induced catecholamine depletion. The mechanism of the non-beta adrenergic inotropism is unclear but may relate to the ability of ECA to inhibit the sarcolemmal Na-K-Mg-dependent ATPase. ECA-induced premature contractile failure occurred in all atria as well as a late increase in diastolic tension, the latter being comparable to that described for toxic doses of cardiac glycosides in similar preparations.     

Inhibition of microtubule assembly in vitro by anti-tubulin monoclonal antibodies

Five monoclonal antibodies against N-terminal domains of alpha- or beta-tubulin were tested for their ability to interfere with the in vitro formation of microtubules. Although all the antibodies exhibited similar association constants for immobilized tubulin, they differed in their inhibitory effect on microtubule assembly. For the most potent antibody, TU-13, the antibody/tubulin molar ratio of about 1:320 was sufficient for a 50% inhibition. The data indicate that the surface regions of N-terminal domains of tubulin are involved in the formation of microtubules.     

Double and single exponential kinetics of microtubule assembly in vitro

The kinetics of the microtubule protein assembly were studied in Mes buffer, pH 6.6, at 28 degrees C. The assembly under above conditions follow a kinetic expression containing two exponential terms. The observed two rate constants depend on protein concentration, and are on the order of 10(-2) sec-1 and 10(-3) sec-1. When CaCl2 is added to the system in low concentration, the kinetic expression becomes single exponential. The observed rate constant is independent of protein concentration and its value is 5 X 10(-3) sec-1. It is concluded that the double exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics correspond to favorable assembly conditions, probably to a high extent of nucleation, whereas the single exponential kinetics is a slower process which occur under hindered assembly conditions.     

The assembly of microtubule protein in vitro. The kinetic role in microtubule elongation of oligomeric fragments containing microtubule-associated proteins.

The kinetics of assembly were studied for bovine and pig microtubule protein in vitro over a range of conditions of pH, temperature, nucleotide and protein concentration. The kinetics are in general biphasic with two major processes of similar amplitude but separated in rate by one order of magnitude. Rates and amplitudes are complex functions of solution conditions. The rates of the fast phase and the slow phase attain limiting values as a function of increasing protein concentration, and are more stringently limited at pH 6.5 than pH 6.95. Such behaviour indicates that mechanisms other than the condensation polymerization of tubulin dimer become rate-limiting at higher protein concentration. The constancy of the wavelength-dependence of light-scattering and ultrastructural criteria indicate that microtubules of normal morphology are formed in both phases of the assembly process. Electrophoretic analysis of assembling microtubule protein shows that MAP- (microtubule-associated-protein-)rich microtubules are formed during the fast phase. The rate of dissociation of oligomeric species on dilution of microtubule protein closely parallels the fast-phase rate in magnitude and temperature-dependence. We propose that the rate of this process constitutes an upper limit to the rate of the fast phase of assembly. The kinetics of redistribution of MAPs from MAP-rich microtubules may be a factor limiting the slow-phase rate. A working model is derived for the self-assembly of microtubule protein incorporating the dissociation and redistribution mechanisms that impose upper limits to the rates of assembly attainable by bimolecular addition reactions. Key roles are assigned to MAP-containing fragments in both phases of microtubule elongation. Variations in kinetic behaviour with solution conditions are inferred to derive from the nature and properties of fragments formed from oligomeric species after the rapid temperature jump. The model accounts for the limiting rate behaviour and indicates experimental criteria to be applied in evaluating the relative contributions of alternative pathways.     

In vitro microtubule assembly on centrioles from mammalian spermatids

In the present study we have shown that the centriolar structures, which form the neck region of the spermatid tail, can act as microtubule-organizing centers.     

Copolymerization of two distinct tubulin isotypes during microtubule assembly in vitro

Cells contain multiple tubulin isotypes that are the products of different genes and posttranslational modifications. It has been proposed that tubulin isotypes become segregated into different classes of microtubules each adapted to specific activities and functions. To determine if mixtures of tubulin isotypes segregate into different classes of polymers in vitro, we used immunoelectron microscopy to examine the composition of microtubule copolymers that assembled from mixtures of purified tubulin subunits from chicken brain and erythrocytes, each of which has been shown to exhibit distinct assembly properties in vitro. We observed that (a) the two isotypes coassemble rapidly and efficiently despite the fact that each isotype exhibits its own unique biochemical and assembly properties; (b) at low monomer concentrations the ratio of tubulin isotypes changes along the lengths of elongating copolymers resulting in gradients in immuno-gold labeling; (c) two distinct classes of copolymers each containing a distinct ratio of isotypes assemble simultaneously in the same subunit mixture; and (d) subunits and polymers of different isotypes associate nearly equally well with each other, there being only a slight bias favoring interactions among subunits and polymers of the same isotype. The observations agree with previous studies on the homogeneous distribution of multiple isotypes within cells and suggest that if segregation of isotypes does occur in vivo, it is most likely directed by cell-specific microtubule-associated proteins (MAPs) or specialized intracellular conditions.     

Calcium/calmodulin-dependent inhibition of microtubule assembly by brain synaptic junction

The effect of synaptic junction (SJ) on microtubule assembly was examined. After preincubation with ATP at 37°C, rat SJ decreased the initial velocity and the extent of the porcine brain microtubule assembly (initiated by the addition of GTP) in a Ca²⁺/calmodulin (CaM)-dependent manner. The degree of the inhibition reached 35% of the control assembly (0-min preincubation) after 20-min preincubation with ATP. There was no inhibition either with heat-treated SJ, at 0°C, or in the presence of EGTA or W-7 (CaM antagonist). The inhibition was due neither to protease(s) nor CaM contaminating the preparations. Free Ca²⁺ concentration level required for the inhibition of microtubule assembly was 10^–6 M. Phosphorylation of microtubule proteins was inhibited by SJ in a Ca²⁺/CaM-dependent manner, and the inhibition occurred in a physiological increase range of intracellular Ca²⁺ concentration (10^–6M) The heat-treated SJ caused no inhibition. The result suggested that the microtubule assembly in the postsynaptic region was regulated by a Ca²⁺/CaM-dependent protein kinase associated with SJ; i. e., major postsynaptic density protein.Abbreviations used CaM calmodulin - DTT dithiothreitol - MAPs microtubule-associated proteins - MES 2-(N-morphorino)ethanesulfonic acid - mPSDp major postsynaptic density protein - PSD postsynaptic density - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride