In vitro polymerization of microtubules from HeLa cells

Although the purification of microtubules from brain by alternate cycles of polymerization and depolymerization in vitro has become routine, the application of this method to non-neural cultured cells has been less successful. Previous investigations have suggested that it was necessary to use substrate-grown cells and 4 M glycerol to obtain microtubules from cultured cells. We have developed a method for preparing microtubules from HeLa cells in spinner cultures without the use of glycerol. Microtubules can be readily carried through two complete cycles of polymerization at 37 degrees C and depolymerization at 4 degrees C in vitro. The microtubules obtained are morphologically similar to brain microtubules in electron micrographs, and the tubulin subunits have mobilities similar to those of brain tubulins on polyacrylamide gels. Typical yields in the second polymerization pellet are about 1 mg protein/ml of packed cells or 2.5-3.0% of the total protein in the soluble cell extract. The major nontubulin protein present after two cycles of polymerization and depolymerization has an apparent mol wt of 68,000 daltons. If glycerol is used during polymerization, this band is virtually absent.     

Microtubule and Actin Interplay Drive Intracellular c-Src Trafficking

The proto-oncogene c-Src is involved in a variety of signaling processes. Therefore, c-Src spatiotemporal localization is critical for interaction with downstream targets. However, the mechanisms regulating this localization have remained elusive. Previous studies have shown that c-Src trafficking is a microtubule-dependent process that facilitates c-Src turnover in neuronal growth cones. As such, microtubule depolymerization lead to the inhibition of c-Src recycling. Alternatively, c-Src trafficking was also shown to be regulated by RhoB-dependent actin polymerization. Our results show that c-Src vesicles primarily exhibit microtubule-dependent trafficking; however, microtubule depolymerization does not inhibit vesicle movement. Instead, vesicular movement becomes both faster and less directional. This movement was associated with actin polymerization directly at c-Src vesicle membranes. Interestingly, it has been shown previously that c-Src delivery is an actin polymerization-dependent process that relies on small GTPase RhoB at c-Src vesicles. In agreement with this finding, microtubule depolymerization induced significant activation of RhoB, together with actin comet tail formation. These effects occurred downstream of GTP-exchange factor, GEF-H1, which was released from depolymerizing MTs. Accordingly, GEF-H1 activity was necessary for actin comet tail formation at the Src vesicles. Our results indicate that regulation of c-Src trafficking requires both microtubules and actin polymerization, and that GEF-H1 coordinates c-Src trafficking, acting as a molecular switch between these two mechanisms.     

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.     

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.     

Cold adaptation of microtubule assembly and dynamics. Structural interpretation of primary sequence changes present in the alpha- and beta-tubulins of Antarctic fishes

The microtubules of Antarctic fishes, unlike those of homeotherms, assemble at very low temperatures (-1.8 degrees C). The adaptations that enhance assembly of these microtubules are intrinsic to the tubulin dimer and reduce its critical concentration for polymerization at 0 degrees C to approximately 0.9 mg/ml (Williams, R. C., Jr., Correia, J. J., and DeVries, A. L. (1985) Biochemistry 24, 2790-2798). Here we demonstrate that microtubules formed by pure brain tubulins of Antarctic fishes exhibit slow dynamics at both low (5 degrees C) and high (25 degrees C) temperatures; the rates of polymer growth and shortening and the frequencies of interconversion between these states are small relative to those observed for mammalian microtubules (37 degrees C). To investigate the contribution of tubulin primary sequence variation to the functional properties of the microtubules of Antarctic fishes, we have sequenced brain cDNAs that encode 9 alpha-tubulins and 4 beta-tubulins from the yellowbelly rockcod Notothenia coriiceps and 4 alpha-tubulins and 2 beta-tubulins from the ocellated icefish Chionodraco rastrospinosus. The tubulins of these fishes were found to contain small sets of unique or rare residue substitutions that mapped to the lateral, interprotofilament surfaces or to the interiors of the alpha- and beta-polypeptides; longitudinal interaction surfaces are not altered in the fish tubulins. Four changes (A278T and S287T in alpha; S280G and A285S in beta) were present in the S7-H9 interprotofilament "M" loops of some monomers and would be expected to increase the flexibility of these regions. A fifth lateral substitution specific to the alpha-chain (M302L or M302F) may increase the hydrophobicity of the interprotofilament interaction. Two hydrophobic substitutions (alpha:S187A in helix H5 and beta:Y202F in sheet S6) may act to stabilize the monomers in conformations favorable to polymerization. We propose that cold adaptation of microtubule assembly in Antarctic fishes has occurred in part by evolutionary restructuring of the lateral surfaces and the cores of the tubulin monomers.     

SB401, a pollen-specific protein from Solanum berthaultii, binds to and bundles microtubules and F-actin

We characterize a novel, pollen-specific, microtubule-associated protein, SB401, found in Solanum berthaultii. This protein binds to and bundles taxol-stabilized microtubules and enhances tubulin polymerization in a concentration-dependent manner, particularly at lower temperatures. Electron microscopy revealed that the protein decorates the entire length of microtubules. Cross-linking and electrophoresis studies showed that SB401 protein forms dimers, and suggest that dimerization could account for bundling. Double immunofluorescent staining of pollen tubes of S. berthaultii showed that SB401 protein co-localized with cortical microtubule bundles. SB401 protein also binds to and bundles actin filaments, and could connect actin filaments to microtubules. SB401 protein had a much higher affinity for microtubules than for actin filaments. In the presence of both cytoskeletal elements, the protein preferentially bound microtubules to form bundles. These results demonstrate that SB401 protein may have important roles in organizing the cytoskeleton in pollen tubes.     

Polymerization of Antarctic fish tubulins at low temperatures: energetic aspects

Tubulins were purified from the brain tissues of three Antarctic fishes, Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus, by ion-exchange chromatography and one cycle of temperature-dependent microtubule assembly and disassembly in vitro, and the functional properties of the protein were examined. The preparations contained the alpha- and beta-tubulins and were free of microtubule-associated proteins. At temperatures between 0 and 24 degrees C, the purified tubulins polymerized readily and reversibly to yield both microtubules and microtubule polymorphs (e.g., "hooked" microtubules and protofilament sheets). Critical concentrations for polymerization of the tubulins ranged from 0.87 mg/mL at 0 degrees C to 0.02 mg/mL at 18 degrees C. The van't Hoff plot of the apparent equilibrium constant for microtubule elongation at temperatures between 0 and 18 degrees C was linear and gave a standard enthalpy change (delta H degree) of +26.9 kcal/mol and a standard entropy change (delta S degree) of +123 eu. At 10 degrees C, tubulin from N. gibberifrons polymerized efficiently at high ionic strength; the critical concentration increased monotonically from 0.041 to 0.34 mg/mL as the concentration of NaCl added to the assembly buffer was increased from 0 to 0.4 M. Together, the results indicate that the polymerization of tubulins from the Antarctic fishes is entropically driven and suggest that an increased reliance on hydrophobic interactions underlies the energetics of microtubule formation at low temperatures. Thus, evolutionary modification to increase the proportion of hydrophobic interactions (relative to other bond types) at sites of interdimer contact may be one adaptive mechanism that enables the tubulins of cold-living poikilotherms to polymerize efficiently at low temperatures.     

Expression of cold-adapted beta-tubulins confer cold-tolerance to human cellular microtubules

Isolated microtubule proteins from the cold-adapted fish, Atlantic cod (Gadus morhua), assemble at temperatures between 8 and 30 degrees C, while avian and mammalian microtubules normally do not assemble at temperatures below 20 degrees C. Tubulin, the main component in microtubules, is expressed as many isotypes. Microtubules with different isotype composition have been shown to have different dynamic properties in vitro. Our hypothesis was that cold-tolerance of microtubules is caused by tubulin isotypes that differ in the primary sequence compared to mammalian tubulins. Here we show that transfection of human HepG2 cells with cod beta-tubulin induced cold-adaptation of the endogenous microtubules. Incorporation of one single tubulin isotype can induce cold-tolerance to cold-intolerant microtubules. Three cod beta-tubulin isotypes were tested and two of these (beta1 and beta2) transferred cold-tolerance to HepG2 microtubules, thus not all cod beta-tubulins were able to confer cold-stability.     

Stochastic computer model of cellular microtubule dynamics

A computer model of the system of microtubules has been developed to study the mechanisms of action of various factors on this system. The model describes the process of polymerization/depolymerization of microtubules as a set of chemical reactions with certain rate constants using a stochastic approach. Microtubules are visualized in the program field, which makes the model visual. The program imitates the dynamics and structure of the system of cellular microtubules with great, reliability. The parameters generated by the model correlate with the corresponding parameters of microtubules in living cells. We are going to develop this approach to modeling microtubules and similar structures to bring them into a better accord with living systems and to study the influence of various factors on these systems.     

Taxol effect on tubulin polymerization and associated guanosine 5'-triphosphate hydrolysis

Taxol has been used as a tool to investigate the relationship between microtubule assembly and guanosine 5'-triphosphate (GTP) hydrolysis. The data support the model previously proposed [Carlier, M.-F., & Pantaloni, D. (1981) Biochemistry 20, 1918] that GTP hydrolysis is not tightly coupled to the polymerization process but takes place as a monomolecular process following polymerization. The results further indicate that the energy liberated by GTP hydrolysis is not responsible for the subsequent blockage of GDP on polymerized tubulin. When tubulin is polymerized in the presence of 10-100 microM taxol, the rapid formation of a large number of very short microtubules (l less than 1 micron) is accompanied by the development of turbidity to a lesser extent than what is observed when the same weight amount of longer microtubules (l = 5 microns) is formed. A slower subsequent turbidity increase corresponds to the length redistribution of these short microtubules into 3-5-fold longer ones without any change in the weight amount of polymer. The evolution of the rate of length redistribution with the concentration of taxol suggests a model within which taxol would bind to dimeric tubulin and to tubulin present at the ends of microtubules with a somewhat 10-fold lower affinity than to polymerized tubulin embedded in the bulk of microtubules. In agreement with this model, binding of taxol to the tubulin-colchicine complex in the dimeric form could be measured from the increase in the GTPase activity of the tubulin-colchicine complex accompanying taxol binding.     

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.     

Differentiation between dynamic instability and end-to-end annealing models for length changes of steady-state microtubules

Short microtubules can be formed by shearing a sample at polymerization steady state of microtubules formed by glycerol-induced assembly of pure tubulin dimer. Such short microtubules show a rapid increase in mean length. The rate of this increase is too fast to be accounted for by statistical redistribution of subunits between microtubules. We propose that the fast length changes are a result of the end-to-end annealing of microtubules demonstrated by Rothwell et al. (Rothwell, S. W., Grasser, W. A., and Murphy, D. B. (1986) J. Cell Biol. 102, 619-627). This proposal has been tested by measuring the rate of annealing of free microtubules to Tetrahymena axonemes under conditions identical to those used for the lengthening of sheared microtubules. That free microtubules anneal to axonemal microtubules is indicated by the following observations. Axonemes elongate at both ends in the presence of steady state microtubules, as predicted for a symmetrical annealing process; under conditions where the microtubule number concentration is greater than that for axonemes, the initial rate of axoneme elongation is more rapid with a low concentration of long microtubules at steady state than with a high number concentration of short microtubules at steady state. These observations are inconsistent with the predictions of a model based on microtubule dynamic instability (Mitchison, T., and Kirschner, M. (1984) Nature 312, 237-242). The annealing rate observed with axonemes can account for the rate of elongation of sheared steady state microtubules.     

Role of tubulin-associated proteins in microtubule nucleation and elongation

Previous experiments have shown that a fraction of microtubule-associated proteins is essential for the self-assembly of microtubules in vitro. When tubulin was titrated with increasing concentrations of these non-tubulin accessory factors, both the rate and extent of polymerization increased in a sigmoidal as opposed to a stoichiometric fashion. The non-tubulin proteins promoted the nucleation of microtubules as determined from the analysis of the kinetics of tubulin selfassembly and the examination of the microtubule length distribution following polymerization. The effect of the non-tubulin factors on microtubule elongation was determined by kinetic experiments in which purified tubulin subunits were added to microtubule seeds and the initial rate of polymerization was measured under conditions where spontaneous self-assembly was below detectable levels. In addition, microtubule growth was also observed when isolated flagellar axonemes were incubated with purified tubulin subunits indicating that the non-tubulin factors were not an absolute requirement for elongation. Analysis of the data in terms of the condensation mechanism of microtubule assembly indicated that the non-tubulin proteins stimulated the growth of microtubules not by increasing the rate of polymerization but by decreasing the rate of depolyerization. The mechanism by which these accessory factors promote tubulin assembly may be summarized as follows: under the conditions employed, they are required for tubulin initiation but not for elongation; the factors affect the extent and net rate at which polymer is formed by binding to the polymer, thereby stabilizing the formed microtubules and consequently shifting the equilibrium to favor assembly.     

Microtubule flux: drivers wanted

While the metaphase spindle maintains a constant shape and size during cell division, its major component microtubules are continuously being polymerized, depolymerized and transported towards the two spindle poles in a process called microtubule poleward flux. This process has been observed in all metazoan cells. Recent studies have indicated that Kinesin-5s, which can drive the relative sliding of microtubules, and kinesin-13s, which regulate microtubule polymerization, are directly involved in microtubule poleward flux. The availability of molecular and chemical tools to perturb protein functions together with improvements in imaging and analytical methods have allowed the examination of these two kinesins' roles in poleward flux at high temporal and spatial resolution. These advances have shed some light on the molecular mechanisms that drive microtubule poleward flux.     

Biochemical and electron microscopy analysis of the endotoxin binding to microtubulesin vitro

The mechanisms involved in cellular activation and damage by bacterial endotoxins are not completely defined. In particular, there is little information about possible intracellular targets of endotoxins. Recently, the participation of a microtubule associated protein in endotoxin actions on macrophages has been suggested. In the present work, we have studied the effect ofE. coli lipopolysaccharide on the polymerization of microtubular proteinin vitro. Electrophoretic analysis of the polymerization mixtures showed that the endotoxin inhibited the polymerization when present at high concentrations. At lower concentrations, LPS selectively displaced the microtubule associated protein MAP-2 from the polymerized microtubules. Electron microscopy showed that LPS binds to microtubules of tubulin+MAPs and to microtubules of purified tubulin (without MAPs) polymerized with taxol. Gel filtration experiments confirmed the binding of LPS to tubulin, and by ligand blot assays an interaction LPS — MAP-2 was detected. The ability of LPS to interact with microtubular proteins suggests a possible participation of microtubules on the cellular effects of endotoxins.     

Microtubules and the formation of acetylcholine receptor clusters in chick embryonic muscle cells

We have used the microtubule-stabilizing drug taxol to examine the relationship between microtubules and the appearance and cell surface distribution of acetylcholine receptors (AChRs) in primary cultures of chick embryonic muscle cells. Taxol at a 5-microM concentration induced the large scale polymerization of tubulin in muscle cells that was most obvious as intermittent bundles of microtubules along the myotube. Prominent bundles of microtubules were also clearly visible in the fibroblasts. This concentration of taxol had no significant effect on the incorporation rate, increased synthesis induced by brain extract or the total cell surface number of AChRs measured over a 24-h period. Thus, excess polymerization of microtubules does not affect the movement of receptors to the cell surface. However, when cell surface AChR distribution was examined using rhodamine-conjugated alpha-bungarotoxin, taxol treatment of myotubes was shown to induce the aggregation of receptors. If receptors were labeled before taxol addition, aggregation of these prelabeled receptors was also seen, a result indicating that taxol can induce the movement of receptors already in the membrane. We believe this evidence further implicates microtubules as being involved in the movement of these cell surface receptors in the plane of the myotube membrane.     

Aminazine--highly effective inhibitor of tubulin polymerization

Aminasine was found to repress tubulin polymerization into microtubules. This process is associated with the formation of anomalous structures--aggregates which contain, except tubulin, an increased amount of microtubule-associating proteins. The effect of aminasine on microtubules is specific, since it can be observed both in vitro and in vivo. Aminasine seems to be a helpful tool in studying the functions of tubulin and microtubules.     

Linkages between the effects of taxol, colchicine, and GTP on tubulin polymerization

A comparative study has been carried out of the effects of taxol on the polymerizations into microtubules of microtubule-associated protein-free tubulin, prepared by the modified Weisenberg procedure, and of the tubulin-colchicine complex into large aggregates. Taxol enhances, to a much greater extent, the stability of microtubules than that of the tubulin-colchicine polymers so that, with highly purified tubulin, assembly into microtubules takes place at 10 degrees C, even in the absence of exogenous GTP. The polymerization of tubulin-colchicine requires both heat and GTP, and the process is reversed by cooling. These results indicate that in both systems polymerization is linked to interactions with taxol and GTP, the interplay of linkage free energies imparting the observed polymer stabilities. In the case of microtubule formation, the linkage free energy provided by taxol binding is approximately -3.0 kcal/mol of alpha-beta-tubulin dimer, whereas this quantity is reduced to approximately -0.5 kcal/mol in tubulin-colchicine, indicating the expenditure of much more binding free energy in the latter case for overcoming unfavorable factors, such as steric hindrance and geometric strain. The difference in the effect of GTP on the two polymerization processes reflects the respective abilities of the bindings of taxol to the two states of tubulin to overcome the loss of the linkage free energy of GTP binding. Analysis of the linkages leads to the conclusions that taxol need not change qualitatively the mechanism of microtubule assembly and that tubulin with the E-site unoccupied by nucleotide should have the capacity to form microtubules, the reaction being extremely weak.     

A regulatory model for spindle function during mitosis

Structural information on the mitotic spindle of Saccharomyces cerevisiae obtained from isolated whole mount preparations has shown that the spindle undergoes a two-fold increase in length whilst comprising only a single microtubule continuous between the two spindle pole bodies. Further data from immunofluorescence microscopy on the timing of anaphase B has suggested that microtubules do not directly produce the required force, but instead have a more passive role. Here a regulatory function for spindle microtubules during mitosis is explored and the existence of a non-microtubule force-generating system is postulated. Thus it is suggested that the continuous microtubules govern the velocity of anaphase B by providing a resistive force that is itself regulated by the number of microtubules and their rate of polymerization. On this basis a model for the forces acting on a spindle pole body during anaphase is proposed.     

Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.