Effects of ATP and cyclic AMP on the in vitro assembly and stability of mammalian brain microtubules

The relevance of protein phosphorylation, transphosphorylation and binding phenomena in the kinetics of the ATP-induced assembly of cycle-purified microtubule protein from mammalian brain were studied. ATP was able to induce the polymerization of microtubules of normal appearance. However, the assembled structures, were unstable and microtubules depolymerized after achievement of a transitory maximum. Cyclic AMP reduced the amplitude of the polymerization maximum in a concentration-dependent manner, correlating with the stimulation of the endogenous phosphorylation reaction. When microtubule assembly was induced by GTP, in the presence of various concentrations of ATP, the slope of the depolymerization phase was found to depend on the concentration of ATP. Fluoride ion inhibited the endogenous phosphorylation reaction and reduced the disassembly rate, in a concentration-dependent manner. Evidence is also presented indicating that ATP did not bind to phosphocellulose-purified tubulin. These results further contribute to indicate that ATP and cyclic AMP, acting coordinately to control the phosphorylation extent of microtubule proteins are important factors to determine microtubule stability within the cell. Some implications of this mechanism for the regulation by cAMP of the initiation of DNA synthesis and mitosis are considered.Abbreviations MAPs microtubule-associated proteins - MAP2 microtubule-associated protein 2, Mes-4-morpholinoethanesulfonic acid - EGTA ethylene-glycol bis (-aminoethyl ether)N,N,N,N-tetraacetic acid - PMSF phenylmethylsulfonyl fluoride - PEI polyethyleneimine - PC phosphocellulose - DEAE Diethylaminoethyl - SDS-PAGE polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate     

Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell.     

Effects of ultraviolet light on the in vitro assembly of microtubules

Exposure of microtubular protein to ultraviolet light inhibits its assembly into morphologically normal microtubules. This effect appeared to result primarily from damage to the tubulin dimers. The damage consisted of a conformational change, a loss of two free sulfhydryl groups, a production of higher molecular weight cross-linked species, and the formation of aggregated amorphous material upon polymerization.     

The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules

A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer.     

The effect of ruthenium red on the assembly and disassembly of microtubules and on rapid axonal transport

The assembly of microtubules was found to decrease in proportion to the amount of added ruthenium red, indicating a high affinity of ruthenium red for the microtubule system. An equimolar amount of ruthenium red per tubulin dimer inhibited the microtubule assembly completely and disassembled existing microtubules. Binding of ruthenium red to tubulin is accompanied by a shift in the absorption maximum from 535 to 538 nm. The binding is very strong, as shown by the finding that ruthenium red could not be displaced from tubulin by gel chromatography on Sephadex, or by the addition of Ca²⁺ or Mg²⁺. The binding of ruthenium red to tubulin did not affect the single colchicine site, nor the Mg²⁺ site(s), as shown by use of Mn²⁺ as an EPR probe. Ruthenium red also interfered with microtubules in an intact cell system, as it inhibited rapid axonal transport in the frog sciatic nerve, measured by the accumulation of [³H]leucine-labelled proteins in front of a ligature.     

The novel microtubule-associated protein MAP3 contributes to the in vitro assembly of brain microtubules

MAP3 is a novel microtubule-associated protein found in brain and a variety of other tissues (Huber, G., Alaimo-Beuret, D., and Matus, A. (1985) J. Cell Biol. 100, 496-507). In this study, monoclonal antibodies were used to assess its influence on the polymerization of brain tubulin. When added to unpolymerized brain microtubules, anti-MAP3 IgG produced a dose-related inhibition of subsequent assembly. Under the same circumstances, nonimmune mouse IgG did not influence either the rate or the extent of tubulin polymerization. We also used immobilized antibodies to deplete brain MAPs selectively in either MAP3 or MAP1. MAP3-depleted MAPs showed a reproducible decrease in activity compared to control preparations that had been exposed to immobilized nonimmune IgG. MAP1-depleted MAPs did not differ significantly in performance from the nonimmune treated controls. We conclude that MAP3 contributes to the net assembly of brain microtubules observed in vitro. This may be particularly relevant in neonatal animals where brain MAP3 is more abundant than in the adult.     

cAMP inhibits the in vitro assembly of microtubules.

A new method for assaying microtubule assembly is described. The method utilizes the colchicine binding property of tubulin. This technique was used to study the effect of cyclic AMP on tubulin assembly using 100,000g supernatant and cycle-purified tubulin prepared from porcine brain. Cyclic AMP, in the presence of NaF, inhibited tubulin assembly from 100,000g supernatant but had no effect on cycle-purified tubulin.     

Effects of vanadate on the assembly and disassembly of purified tubulin

Sodium-orthovanadate (100-700 microM) added to purified pig brain microtubule protein (molar ratios 13-90 moles vanadate/mole tubulin) inhibits to a considerable extent the assembly (up to 65%) and the disassembly rates (up to 60%) of microtubules, as determined by turbidimetry. Vanadate added to preformed microtubules did not appreciably alter the turbidity level of the samples, however, the disassembly rates were decreased in the same manner as when vanadate was added prior to polymerization. Microtubule protein kept on ice for 3-6 hours became more susceptible to vanadate than freshly prepared protein. The effect of vanadate was independent of the GTP concentration at which the polymerization assays were performed (0.025 to 1 mM GTP). In the presence of taxol, which increases the rate and extent of microtubule formation, vanadate had no effect on assembly rates. Disassembly was inhibited, however, much less than in the presence of vanadate alone. Electron microscopy and polyacrylamide gel electrophoresis did not reveal differences between microtubules prepared in the presence or in the absence of vanadate. This is consistent with the notion that vanadate does not interfere with the interaction between tubulin and the high-molecular weight microtubule-associated proteins. Apparently vanadate brings about an allosteric change of the microtubule protein(s) resulting in the abnormal polymerization kinetics of tubulin found in our study. The above results may be relevant for studies where the effects of vanadate on intracellular motility are interpreted as being solely due to a specific inhibition of ATPases.     

Cell cycle-dependent assembly and disassembly of cytoplasmic microtubules in the plasmodium of the myxomycete Physarum polycephalum.

The giant syncytium of Physarum plasmodia possesses a complex cytoplasmic microtubule network except during the occurrence of the intranuclear mitosis. In early prophase stages, intranuclear spindles assemble concomitantly as the cytoplasmic microtubule network disassembles. No cytoplasmic microtubules are present in metaphase. They begin to reassemble in telophase. The complex cytoplasmic microtubule network reappears in early reconstruction stages. The assembly of cytoplasmic microtubules occurs on cytoplasmic foci, both in telophase stage and during rewarming after cold microtubule disassembly. These foci, independent of the nuclei, correspond to the foci observed in the cytoplasm during interphase, both by immunofluorescence and electron microscopy. As cytoplasmic and intranuclear microtubule-organizing centers are spatially distinct, plasmodial syncytia offer the possibility to study the effects of cell regulatory pathways on two types of microtubule-organizing centers that differ in their nucleating activity during the cell cycle.     

Turbidimetric studies of the in vitro assembly and disassembly of porcine neurotubules

Turbidity measurements have been used to study the in vitro assembly and disassembly of porcine neurotubules. All measurements were carried out with tubulin with a purity higher than 80%. Tubules formed by in vitro assembly of this protein are so long that the turbidity is insensitive to length and is a function only of the total mass of high molecular weight material. Porcine tubulin shows a critical concentration for assembly of about 0.2 mg/ml under optimal conditions, pH 6.6, 0.1m-2-(N-morpholino)ethane sulfonic acid, 26 to 37 °C. Under these conditions assembly and disassembly are essentially fully reversible in the presence of excess GTP. The kinetics of assembly show an initial lag and initial rates which are strongly temperature dependent. Our samples show a concentration dependence of no more than second order. The apparent activation enthalpy of assembly is 25 kcal/mol; the apparent reaction enthalpy of assembly for the chain propagation step is 21 kcal/mol. Disassembly kinetics show an apparent negative activation enthalpy of ?28 kcal/mol. They are independent of tubule length implying a slow activation step followed by rapid depolymerization. At 20 °C, cycles of polymerization and depolymerization show hysteresis effects in the assembly kinetics though not in disassembly rates or final states. This is most easily explained by postulating a slow reversible inactivation at 4 °C of the initiation complex for tubule assembly. Conditions are reported for producing tubulin in a state which cannot assemble in aqueous buffer unless nucleotides are added. GTP, ATP and ADP, but not GDP, are effective in promoting tubule assembly. An adenylate kinase impurity in our preparation may be the cause of this unusual effect. Whether or not it is actually associated with tubulin or tubules is unknown.     

A reassessment of the factors affecting microtubule assembly and disassembly in vitro

Current models of microtubule assembly from pure tubulin involve a nucleation phase followed by microtubule elongation at a constant polymer number. Both the rate of microtubule nucleation and elongation are thought to be tightly influenced by the free GTP-tubulin concentration, in a law of mass action-dependent manner. However, these basic hypotheses have remained largely untested due to a lack of data reporting actual measurements of the microtubule length and number concentration during microtubule assembly.Here, we performed simultaneous measurements of the polymeric tubulin concentration, of the free GTP-tubulin concentration, and of the microtubule length and number concentration in both polymerizing and depolymerizing conditions. In agreement with previous work we find that the microtubule nucleation rate is strongly dependent on the initial GTP-tubulin concentration. But we find that microtubule nucleation persists during microtubule elongation. At any given initial tubulin-GTP concentration, the microtubule nucleation rate remains constant during polymer assembly, despite the wide variation in free GTP-tubulin concentration. We also find a remarkable constancy of the rate of microtubule elongation during assembly. Apparently, the rate of microtubule elongation is intrinsic to the polymers, insensitive to large variations of the free GTP-tubulin concentration. Finally we observe that when, following assembly, microtubules depolymerize below the free GTP-tubulin critical concentration, the rate-limiting factor for disassembly is the frequency of microtubule catastrophe. At all time-points during disassembly, the microtubule catastrophe frequency is independent of the free GTP-tubulin concentration but, as the microtubule nucleation rate, is strongly dependent on the initial free GTP-tubulin concentration. We conclude that the dynamics of both microtubule assembly and disassembly depend largely on factors other than the free GTP-tubulin concentration. We propose that intrinsic structural factors and endogenous regulators, whose concentration varies with the initial conditions, are also major determinants of these dynamics.     

Human chromosomes and centrioles as nucleating sites for the in vitro assembly of microtubules from bovine brain tubulin

Treatment of HeLa cells with Colcemid at concentrations of 0.06-0.10 mug/ml leads to irreversible arrest in mitosis. Colcemid-arrested cells contained few microtubules, and many kinetochores and centrioles were free of microtubule association. When these cells were exposed to microtubule reassembly buffer containing Triton X-100 and bovine brain tubulin at 37 degrees C, numerous microtubules were reassembled at all kinetochores of metaphase chromosomes and in association with centriole pairs. When bovine brain tubulin was eliminated from the reassembly system, microtubules failed to assemble at these sites. Similarly, when EGTA was eliminated from the reassembly system, microtubules failed to polymerize. These results are consistent with other investigations of in vitro microtubule assembly and indicate that HeLa chromosomes and centrioles can serve as nucleating sites for the assembly of microtubules from brain tubulin. Both chromosomes and centrioles became displaced from their C-metaphase configurations during tubulin reassembly, indicating that their movements were a direct result of microtubule formation. Although both kinetochore- and centriole- associated microtubules were assembled and movement occurred, we did not observe direct extension of microtubules from kinetochores to centrioles. This system should prove useful for experimental studies of spindle microtubule formation and chromosome movement in mammalian cells.     

Effects of barbiturates on ultrastructure and polymerization of microtubules in vitro

Summary Barbiturates were examined for in vitro effects on ultrastructure of the frog sciatic system and polymerization of microtubules (MT) in a brain supernatant. Exposure for 5–17 h to 2.0 mM barbiturates caused a considerable loss of MT in ganglionic cell bodies and sciatic axons. This was mostly followed by a proliferation of 10 nm filaments. Under similar conditions treatment with 1 mM NaCN or 0.1 mM 2,4-DNP did not change the number or ultrastructure of MT and filaments.Eight barbiturates, varying in binding ratios to serum albumin and partition coefficients, were tested for effects on polymerization of MT using viscometry. Inhibitory effects were found which correlated with their reported ability to bind to albumin and brain fractions. Dimethylsulphoxide and ethanol were used as solvents for some of the barbiturates. These solvents at 1% had stabilizing effects on MT.The present results are discussed in relation to previous findings of inhibition of rapid axonal transport in vitro in the frog sciatic system by barbiturates.The present work was supported by grants from Statens Naturvetenskapliga Forskningsråd (No. 2535-0011), Statens Medicinska Forskningsråd (B 75-12x-2543-07), Wilhelm och Martina Lundgrens Vetenskapsfond, Magnus Bergwalls Stifteise och Göteborgs Kungl. Vetenskapsoch Vitterhetssamhälle. Thanks are due to Miss Monica Lindhé for her expert technical assistance.     

In vitro depolymerization dynamics of brain endogenous microtubules

A subcellular fraction containing fragments of endogenous microtubules stabilized in 50% glycerol was separated by diferential centrifugation of rat brain homogenates. The pellets were suspended in glycerol-deficient media, and microtubule depolymerization was monitored by measuring the decrease of sedimentable tubulin. Concomitantly, the number and size of microtubules in the suspensions were followed via electron microscopy. Depolymerization was accompanied by a proportional decrease in the number of microtubules, whereas the average size did not change significantly. After approximately 20 min, a subpopulation of microtubules became stable and did not suffer further depolymerization. These results indicate that upon dilution some microtubules completely depolymerize, whereas others remain stable in the glycerol-deficient medium. The degree of depolymerization depended on both the volume of the resuspension media and on the final glycerol concentration. The results suggest that the depolymerization of the remaining microtubules is prevented by stabilizing factors released from depolymerizing microtubules. Tubulin dimers are not one of these factors, since depolymerization was not altered by the addition of colchicine or by changing the concentration of free tubulin in the medium.     

Mechanisms of synapse assembly and disassembly

The mechanisms that govern synapse formation and elimination are fundamental to our understanding of neural development and plasticity. The wiring of neural circuitry requires that vast numbers of synapses be formed in a relatively short time. The subsequent refinement of neural circuitry involves the formation of additional synapses coincident with the disassembly of previously functional synapses. There is increasing evidence that activity-dependent plasticity also involves the formation and disassembly of synapses. While we are gaining insight into the mechanisms of both synapse assembly and disassembly, we understand very little about how these phenomena are related to each other and how they might be coordinately controlled to achieve the precise patterns of synaptic connectivity in the nervous system. Here, we review our current understanding of both synapse assembly and disassembly in an effort to unravel the relationship between these fundamental developmental processes.     

Adenovirus binds to rat brain microtubules in vitro.

We have found by negative staining electron microscopy that when similar concentrations of adenovirus and reovirus (viruses of about the same diameter, 75 to 80 nm, and density, 1.34 to 1.36 g/cm3) were incubated with a carbon support film containing microtubules, 72% of adenovirus on the grid, but only 32% (equivalent to random association) of reovirus, were associated with microtubules. Similar concentrations of both larger and smaller particles, such as polystyrene latex spheres and coliphage f2, also exhibited a low degree of interaction, viz., 17 to 37%, with microtubules. Moreover, 90% of microtubule-associated adenovirus binds to within +/- 4 nm of the edge of microtubules, but lower fractions (again equivalent to a random association) of the other particles bind to the edge of the microtubules. The mechanism behind this phenomenon, which we denote as "edge binding," is presently obscure; however, it provides us with a second, albeit empirical, method to distinguish between the microtubular association of adenovirus and other particles. We found that edge binding of adenovirus also occurred when adenovirus was initially placed on the carbon support film and then incubated with microtubules and when adenovirus and microtubules were mixed prior to placement on the support. In contrast, reovirus or the other particles prepared by similar techniques exhibited a random amount of edge binding. The binding of adenovirus appears to involve the hexon capsomers of the virion since (i) high resolution electron micrographs showed that the edge of the virus was in contact with the edge of the microtubules, and (ii) adenovirions briefly treated with formamide to remove pentons and fibers bind as efficiently as intact virions. Core structures, which were obtained by further formamide degradation of the virion, do not associate with microtubules. These observations support the hypothesis of Dales and Chardonnet (1973) that the transport of adenovirions within infected cells is mediated by interaction with microtubules.     

In vitro assembly of microtubules from tubulins of several higher plants

Tubulins were isolated by a combination of affinity (ethyl N-phenylcarbamate-Sepharose 4B) and ion exchange (DEAE-Sephacel) chromatography from several higher plants (mung bean, pea, whole pod bean, zucchini, cucumber seedlings and carrot suspension cultured cells). All these higher plant tubulins readily polymerized to microtubules in a polymerization medium containing GTP, Mg2+, EGTA, leupeptin and DMSO. Tubulins from mung bean, pea and whole pod bean showed identical behaviour on polyacrylamide gel electrophoresis but differed from carrot zucchini and cucumber tubulin. Consequently, tubulin of higher plants seems to have different molecular properties in different plant species.