Evidence that S100 proteins regulate microtubule assembly and stability in rat brain extracts

Microtubule re-assembly in rat brain extracts was inhibited by antibodies to S100 proteins. Anti-S100 antibodies caused an increase in the cold-stability of microtubules and this effect was abolished by the presence of short lengths of microtubules formed under control conditions. Anti-S100 antibodies had no effect on the stimulation of assembly or the increase in microtubule stability caused by low zinc concentrations. Addition of exogenous S100a and S100b to brain extracts had different effects on assembly; S100a caused an inhibition of assembly while S100b stimulated the early phase of assembly. The data suggest that endogenous S100b is involved in the regulation of microtubule assembly in brain extracts.     

Microtubule assembly in cytoplasmic extracts of Xenopus oocytes and eggs

We have investigated the differences in microtubule assembly in cytoplasm from Xenopus oocytes and eggs in vitro. Extracts of activated eggs could be prepared that assembled extensive microtubule networks in vitro using Tetrahymena axonemes or mammalian centrosomes as nucleation centers. Assembly occurred predominantly from the plus-end of the microtubule with a rate constant of 2 microns.min-1.microM-1 (57 s-1.microM-1). At the in vivo tubulin concentration, this corresponds to the extraordinarily high rate of 40-50 microns.min-1. Microtubule disassembly rates in these extracts were -4.5 microns.min-1 (128 s-1) at the plus-end and -6.9 microns.min-1 (196 s-1) at the minus-end. The critical concentration for plus-end microtubule assembly was 0.4 microM. These extracts also promoted the plus-end assembly of microtubules from bovine brain tubulin, suggesting the presence of an assembly promoting factor in the egg. In contrast to activated eggs, assembly was never observed in extracts prepared from oocytes, even at tubulin concentrations as high as 20 microM. Addition of oocyte extract to egg extracts or to purified brain tubulin inhibited microtubule assembly. These results suggest that there is a plus-end-specific inhibitor of microtubule assembly in the oocyte and a plus-end-specific promoter of assembly in the eggs. These factors may serve to regulate microtubule assembly during early development in Xenopus.     

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.     

Erythrocyte microtubule assembly in vitro. Determination of the effects of erythrocyte tau, tubulin isoforms, and tubulin oligomers on erythrocyte tubulin assembly, and comparison with brain microtubule assembly

Two tubulin variants, isolated from chicken brain and erythrocytes and known to have different peptide maps and electrophoretic properties, are demonstrated to exhibit different assembly properties in vitro: 1) erythrocyte tubulin assembles with greater efficiency (lower critical concentration, greater elongation rate) but exhibits a lower nucleation rate than brain tubulin, and 2) erythrocyte tubulin readily forms oligomers whose presence significantly retards the rate of elongation, suggesting that tubulin oligomers may also be important for determining the rate of assembly and the length of microtubules in erythrocytes. Erythrocyte tubulin isolated by cycles of in vitro assembly-disassembly is also demonstrated to contain a 67-kDa tau factor that greatly enhances microtubule nucleation but has little effect on elongation rates or critical concentration. Immunofluorescence microscopy with tau antibody indicates that tau is specifically associated with marginal band microtubules, suggesting that it may be important for determining microtubule function in vivo.     

Purification, characterization, and assembly properties of tubulin from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus

Tubulin was purified from unfertilized eggs of the sea urchin Strongylocentrotus purpuratus by chromatography of an egg supernatant fraction on DEAE-Sephacel or DEAE-cellulose followed by cycles of temperature-dependent microtubule assembly and disassembly in vitro. After two assembly cycles, the microtubule protein consisted of the alpha- and beta-tubulins (greater than 98% of the protein) and trace quantities of seven proteins with molecular weights less than 55 000; no associated proteins with molecular weights greater than tubulin were observed. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on urea-polyacrylamide gradient gels, the alpha- and beta-tubulins did not precisely comigrate with their counterparts from bovine brain. Two-dimensional electrophoresis revealed that urchin egg tubulin contained two major alpha-tubulins and a single major beta species. No oligomeric structures were observed in tubulin preparations maintained at 0 degrees C. Purified egg tubulin assembled efficiently into microtubules when warmed to 37 degrees C in a glycerol-free polymerization buffer containing guanosine 5'-triphosphate. The critical concentration for assembly of once- or twice-cycled egg tubulin was 0.12-0.15 mg/mL. Morphologically normal microtubules were observed by electron microscopy, and these microtubules were depolymerized by exposure to low temperature or to podophyllotoxin. Chromatography of a twice-cycled egg tubulin preparation on phosphocellulose did not alter its protein composition and did not affect its subsequent assembly into microtubules. At concentrations above 0.5-0.6 mg/mL, a concentration-dependent "overshoot" in turbidity was observed during the assembly reaction. These results suggest that egg tubulin assembles into microtubules in the absence of the ring-shaped oligomers and microtubule-associated proteins that characterize microtubule protein from vertebrate brain.     

The effect of polyamines on tubulin assembly

The assembly of cold solubilized microtubules prepared from calf brain and the polymerization of tubulin purified from this material are facilitated by polyamines at physiological concentrations. The number of free amino groups in the polyamine determines the ability of the polyamines to promote microtubule formation. Spermine with four amino groups was the most effective polyamine tested. Spermidine and N'-acetylspermine with 3 amino groups were less effective than spermine but more effective than N8-acetylspermidine and putrescine which contain two free amino groups. Microtubule formation may therefore be controlled by alterations in the nature and amounts of polyamines present.     

Cell cycle-dependent, in vitro assembly of microtubules onto pericentriolar material of HeLa cells

A centriolar complex comprising a pair of centrioles and a cloud of pericentriolar materials is located at the point of covergence of the microtubules of the mitotic apparatus. The in vitro assembly of microtubules was observed onto these complexes in the 1,400 g supernatant fraction of colcemid-blocked, mitotic HeLa cells lysed into solutions containing tubulin and Triton X-100. Dark-field microscopy provided a convenient means by which this process could be visualized directly. When this 1,400 g supernate was incubated at 30 degrees C and centrifuged into a discontinuous sucrose gradient, a band containing centriolar complexes and assembled microtubles was obtained at 50-60% sucrose interface. Ultrastructual analysis indicated that the majority of the microtubules assembled predominantly from the pericentriolar material but also onto the centrioles. When cells were synchronized by a double thymide block, the assembly of microtubules onto centriolar complexes was observed only in lysates of mitotic cells; no assembly was seen in lysed material of interphase cells. Microtubule assembly occured onto centriolar complexes in solutions of either 100,000 g brain supernate, 2 X cycled tubulin, or purified tubulin dimers. This study demonstrates that the pericentriolar material becomes competent as a microtubule-organizing center (MTOC) at the time of mitosis. With use of the techniques described, a method for the isolation of centriolar complexes may be developed.     

EFFECTS OF LIDOCAINE ON RABBIT BRAIN MICROTUBULAR PROTEIN1

Microtubule repolymerization from a crude supernatant fraction prepared from rabbit brain was followed quantitatively by viscometry and electron microscopy. Lidocaine inhibits this repolymerization in a dose-dependent fashion and the effect is completely reversible upon removal of the lidocaine by dialysis. Direct counting of microtubules by electron microscopy indicates that the local anesthetic decreases the number of tubules without significantly affecting their length. An association constant of lidocaine for tubulin is estimated at 7.5 mm . Procaine and etidocaine were also found to affect the polymerization of microtubules with results similar to those found with lidocaine.     

Enhanced microtubule binding and tubulin assembly properties of conformationally constrained paclitaxel derivatives

Microtubule binding and tubulin assembly promotion by a series of conformationally restricted paclitaxel (PTX) derivatives was investigated. In these derivatives, the C-4 acetate of the taxane is tethered to the C-3' phenyl at ortho and meta positions with different length linkers. The apparent affinity of these derivatives for GMPCPP-stabilized microtubules was assessed by a competition assay, and their influence on microtubule polymerization was evaluated by measuring the critical concentration of GDP-tubulin in the presence of the respective molecule. In general, taxane derivatives with higher apparent affinity for microtubules induced tubulin assembly more efficiently. Among the derivatives, molecules with the shortest tether display the strongest affinity for microtubules. These derivatives exhibited enhanced microtubule stabilization properties and efficiently induced GDP-tubulin assembly into microtubules at low temperature of 12 degrees C and in the absence of Mg2+ ions in 0.1 M PIPES. Based on molecular dynamics simulations, we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.     

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.     

The multiple phosphorylation of the microtubule-associated protein MAP2 controls the MAP2:tubulin interaction

Pre-phosphorylation of the microtubule-associated protein MAP2 with the co-purifying cAMP-independent protein kinase (a) decrease the affinity of MAP2 for taxol-stabilised microtubules, (b) increases the dissociation rate constant for microtubule polymerisation, each of which is dependent upon the level of phosphorylation, but (c) has no effect on the association rate constant. Microtubule assembly has no effect on the kinetics of phosphorylation, whereas phosphorylation of pre-assembled microtubules causes their immediate depolymerisation at a rate which is proportional to the initial rate of phosphorylation. The results suggest that the modulated phosphorylation of MAP2 may regulate microtubule length in vivo.     

Different assembly properties of cod, bovine, and rat brain microtubules.

Assembly properties of cod, bovine, and rat brain microtubules were compared. Estramustine phosphate, heparin, poly-L-aspartic acid, as well as NaCl, inhibited the assembly and disassembled both bovine and rat microtubules by inhibition of the binding between tubulin and MAPs. The assembly of cod brain microtubules was in contrast only marginally affected by these agents, in spite of a release of the MAPs. The results suggest that cod tubulin has a high intrinsic ability to assemble. This was confirmed by studies on phosphocellulose-purified cod tubulin, since the critical concentration for assembly was independent of the presence or absence of MAPs. The results show therefore that cod brain tubulin has, in contrast to bovine and rat brain tubulins, a high propensity to assembly under conditions which normally require the presence of MAPs. Even if cod MAPs, which have an unusual protein composition, were not needed for the assembly of cod microtubules, they were able to induce assembly of bovine brain tubulin. Both cod and bovine MAPs bound to cod microtubules, and bovine MAP1 and MAP2 bound to, and substituted at least the 400 kDa cod protein. This suggests that the tubulin-binding sites and the assembly-stimulatory ability of MAPs are common properties of MAPs from different species, independent of the tubulin assembly propensity.     

Pressure-induced depolymerization of spindle microtubules. II. Thermodynamics of in vivo spindle assembly

The present experiments were designed to test whether the simple equilibrium assembly model proposed by Inoué could predict variations in spindle microtubule assembly in response to changes in hydrostatic pressure as it does for changes in temperature. The results were also analyzed according to a model based on nucleated condensation polymerization since this recently appears to be the mechanism by which purified brain microtubules are assembled in vitro. Equilibrium birefringence (BR) of the meiotic metaphase-arrested spindle was measured in vivo as a function of hydrostatic pressure and temperature in Chaetopterus oocytes using a miniature microscope pressure chamber. Increasing pressure in steps to 3,000 psi at temperatures below 22 degrees C did produce decreases in spindle equilibrium BR predictable directly from the simple equilibrium model of spindle assembly. Thermodynamic analysis of the pressure data yielded a value of delta V congruent to 400 ml/mol of polymerizing unit. Theoretical curves based on the nucleated condensation model can also be made to fit the data, but semilog plots of the dependence of the equilibrium constant versus pressure and versus reciprocal temperature are biphasic, suggesting that either the size of the polymerizing unit changes or more than one equilibrium constant governs the assembly reaction. That the same value of delta V, 90 ml/mol, was estimated from both the majority of the spindle BR data and data for the assembly of neural microtubules in vitro supports the possibility that spindle microtubules are assembled by a nucleated condensation mechanism.     

The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae

Microtubule assembly in Saccharomyces cerevisiae is initiated from sites within spindle pole bodies (SPBs) in the nuclear envelope. Microtubule plus ends are thought to be organized distal to the SPBs, while minus ends are proximal. Several hypotheses for the function of microtubule motor proteins in force generation and regulation of microtubule assembly propose that assembly and disassembly occur at minus ends as well as at plus ends. Here we analyse microtubule assembly relative to the SPBs in haploid yeast cells expressing green fluorescent protein fused to alpha-tubulin, a microtubule subunit. Throughout the cell cycle, analysis of fluorescent speckle marks on cytoplasmic astral microtubules reveals that there is no detectable assembly or disassembly at minus ends. After laser-photobleaching, metaphase spindles recover about 63% of the bleached fluorescence, with a half-life of about 1 minute. After anaphase onset, photobleached marks in the interpolar spindle are persistent and do not move relative to the SPBs. In late anaphase, the elongated spindles disassemble at the microtubule plus ends. These results show for astral and anaphase interpolar spindle microtubules, and possibly for metaphase spindle microtubules, that microtubule assembly and disassembly occur at plus, and not minus, ends.     

Interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly

We previously reported that direct incorporation of GDP (i.e., without an initial hydrolysis of GTP) into microtubules occurs throughout an assembly cycle in a constant proportion. The exact proportion varied with reaction conditions, becoming greater under all conditions in which tubulin-GDP increased relative to tubulin-GTP (low Mg2+ and GTP concentrations, high tubulin concentrations, and in the presence of exogenous GDP). These findings led us to explore further interrelationships of tubulin-GDP and tubulin-GTP in microtubule assembly. We have now determined the minimum amount of tubulin-GTP required for the initiation of microtubule assembly and the relative efficiency with which tubulin-GDP participates in microtubule elongation. When GTP, GDP, and tubulin concentrations were varied at a constant Mg2+ concentration (0.2 mM), initiation of assembly required that 35% of the nucleotide-bearing tubulin be in the form of tubulin-GTP, and incorporation of tubulin-GDP into microtubules during elongation was only 60% as efficient as would be predicted on the basis of its proportional concentration in the reaction mixtures. Very different results were obtained when the Mg2+ concentration was varied. Even though Mg2+ enhances the binding of GTP to tubulin (the equilibrium constant for the exchange of GTP for GDP was 0.2 in the absence of exogenous Mg2+, 3 with 0.2 mM Mg2+, 5 with 0.5 mM Mg2+, and 11 with 2 and 4 mM Mg2+), as Mg2+ was increased the proportion of tubulin-GTP required for the initiation of microtubule assembly rose greatly, and the direct incorporation of tubulin-GDP into microtubules during elongation became progressively more efficient. In the absence of exogenous Mg2+, only 20% tubulin-GTP was required for initiation, and tubulin-GDP was directly incorporated into microtubules half as efficiently as would be predicted on the basis of its concentration in the reaction mixture. At the highest Mg2+ concentration examined (4 mM), 80% tubulin-GTP was required for initiation of assembly, and tubulin-GDP was incorporated into microtubules as efficiently as tubulin-GTP.     

Microtubule assembly and disassembly at alkaline pH

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.     

Estimation of the diffusion-limited rate of microtubule assembly.

Microtubule assembly is a complex process with individual microtubules alternating stochastically between extended periods of assembly and disassembly, a phenomenon known as dynamic instability. Since the discovery of dynamic instability, molecular models of assembly have generally assumed that tubulin incorporation into the microtubule lattice is primarily reaction-limited. Recently this assumption has been challenged and the importance of diffusion in microtubule assembly dynamics asserted on the basis of scaling arguments, with tubulin gradients predicted to extend over length scales exceeding a cell diameter, approximately 50 microns. To assess whether individual microtubules in vivo assemble at diffusion-limited rates and to predict the theoretical upper limit on the assembly rate, a steady-state mean-field model for the concentration of tubulin about a growing microtubule tip was developed. Using published parameter values for microtubule assembly in vivo (growth rate = 7 microns/min, diffusivity = 6 x 10(-12) m2/s, tubulin concentration = 10 microM), the model predicted that the tubulin concentration at the microtubule tip was approximately 89% of the concentration far from the tip, indicating that microtubule self-assembly is not diffusion-limited. Furthermore, the gradients extended less than approximately 50 nm (the equivalent of about two microtubule diameters) from the microtubule tip, a distance much less than a cell diameter. In addition, a general relation was developed to predict the diffusion-limited assembly rate from the diffusivity and bulk tubulin concentration. Using this relation, it was estimated that the maximum theoretical assembly rate is approximately 65 microns/min, above which tubulin can no longer diffuse rapidly enough to support faster growth.     

Monoclonal antibody refolding and assembly: Protein disulfide isomerase reaction kinetics

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (MAb) refolding and assembly which accompanies disulfide bond formation. The MAbin vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.     

Monoclonal antibody refolding and assembly: Protein disulfide isomerase reaction kinetics

The protein disulfide isomerase (PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody (MAb) refulding and assembly which accompanies disulfide bond formation. The MAbin vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hyboridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specifle MAb productivity exists.     

The effects of dimethyl sulfoxide on the kinetics of tubulin assembly

The kinetics of tubulin assembly were examined in the absence and presence of dimethyl sulfoxide at 37 degrees C. Inclusion of 1.4 M (10%) dimethyl sulfoxide lowered the critical protein concentration about 8-10-fold, from 9.4 microM in the absence of the organic solvent to 1.1 microM in its presence. This decrease was due solely to an effect on k-, the off rate constant. The on rate constant k+, was essentially unaffected. Another effect of dimethyl sulfoxide was in the nucleation process. The pseudo-first-order rate constant of elongation, kapp (k+[m]), was greatly increased by inclusion of dimethyl sulfoxide. This was due to an increase in the microtubule number concentration, [m]. The microtubules formed in the presence of dimethyl sulfoxide were much shorter than those formed in its absence, accounting for the higher number concentration. The nucleation number, n, was calculated by plots of ln kapp vs. ln c0 or ln t10% vs. ln c0, and the value appeared to be about 4 to 5, although some variability was found. It was shown that a plot of kapp vs. c0 to determine n, is not appropriate because of the inability to distinguish between linear and curved plots in the range of tubulin concentration used in assembly studies.