Microtubule dynamics and microtubule caps: a time-resolved cryo- electron microscopy study

Microtubules display the unique property of dynamic instability characterized by phase changes between growth and shrinkage, even in constant environmental conditions. The phases can be synchronized, leading to bulk oscillations of microtubules. To study the structural basis of dynamic instability we have examined growing, shrinking, and oscillating microtubules by time-resolved cryo-EM. In particular we have addressed three questions which are currently a matter of debate: (a) What is the relationship between microtubules, tubulin subunits, and tubulin oligomers in microtubule dynamics?; (b) How do microtubules shrink? By release of subunits or via oligomers?; and (c) Is there a conformational change at microtubule ends during the transitions from growth to shrinkage and vice versa? The results show that (a) oscillating microtubules coexist with a substantial fraction of oligomers, even at a maximum of microtubule assembly; (b) microtubules disassemble primarily into oligomers; and (c) the ends of growing microtubules have straight protofilaments, shrinking microtubules have protofilaments coiled inside out. This is interpreted as a transition from a tense to a relaxed conformation which could be used to perform work, as suggested by some models of poleward chromosome movement during anaphase.     

Microtubule oscillations. Role of nucleation and microtubule number concentration

Microtubules are capable of performing synchronized oscillations of assembly and disassembly which has been explained by reaction mechanisms involving tubulin subunits, oligomers, microtubules, and GTP. Here we address the question of how microtubule nucleation or their number concentration affects the oscillations. Assembly itself requires a critical protein concentration (Cc), but oscillations require in addition a critical microtubule number concentration (CMT). In spontaneous assembly this can be achieved with protein concentrations Cos well above the critical concentration Cc because this enhances the efficiency of nucleation. Seeding with microtubules can either generate oscillations or suppress them, depending on how the seeds alter the effective microtubule number concentration. The relative influence of microtubule number and total protein concentrations can be varied by the rate at which assembly conditions are induced (e.g. by a temperature rise): Fast T-jumps induce oscillations because of efficient nucleation, slow ones do not. Oscillations become damped for several reasons. One is the consumption of GTP, the second is a decrease in microtubule number, and the third is that the ratio of microtubules in the two phases (growth-competent and shrinkage-competent) approach a steady state value. This ratio can be perturbed, and the oscillations restarted, by a cold shock, addition of seeds, addition of GTP, or fragmentation. Each of these is equivalent to a change in the effective microtubule number concentration.     

Dynamics of the microtubule oscillator: role of nucleotides and tubulin-MAP interactions.

Microtubules can be induced to perform synchronous and periodic cycles of assembly and disassembly at constant temperature. The process depends on GTP hydrolysis. Time-resolved X-ray scattering using synchrotron radiation shows a cyclic interconversion of tubulin subunits, microtubules and oligomers (= short protofilament fragments). Oscillations are correlated with conditions that stabilize polymers and destabilize oligomers, and others of opposite effect. Microtubule stabilizers include GTP, Mg2+ or microtubule-associated proteins (MAPs), destabilizers include GDP or elevated ionic strength. K+ at intracellular concentrations noticeably increases the stability of tubulin-MAP oligomers, in contrast to Na+. ATP and the non-hydrolyzable analogue AMP-PNP enhance oscillations by mechanisms that are not directly linked to the role of nucleotide hydrolysis in assembly. We propose a mechanism of oscillations that include oligomers as microtubule disassembly products which transiently lock the protein in an unpolymerizable state; this may point to a role of oligomers in controlling microtubule assembly cycles in cells.     

Oscillations in microtubule polymerization: the rate of GTP regeneration on tubulin controls the period.

The essential reactions involved in the oscillatory kinetics of microtubule polymerization have been investigated. The rate of GDP dissociation from tubulin decreased cooperatively upon increasing tubulin concentration above 20 microM, consistent with the formation of GDP oligomers whose dissociation is rate limiting in nucleotide exchange. The apparent rate constant for nucleotide exchange at high tubulin concentration was 0.02 s-1 at 37 degrees C, which is the exact value needed in previous theoretical simulations to obtain oscillations with the real period of 70-80 s. A glass filter assay separating microtubules from oligomers and tubulin allowed nucleotide bound to non-microtubular tubulin during the oscillations to be monitored. In agreement with nucleotide exchange data, tubulin-bound GDP was found to oscillate in antiphase with microtubules. By varying the concentration of an enzymatic GTP-regenerating system, we could demonstrate that the period of the oscillations is directly controlled by the rate at which GTP is regenerated on tubulin, and oscillations can be observed under conditions where the dissociation of oligomers is no longer rate limiting. The possible physiological significance of GTP-regenerating systems in establishing synchrony in a microtubule population is evoked. The present data confirm and extend the model that we previously proposed to account for the oscillations.     

Tubulin oligomers and microtubule assembly studied by time-resolved X-ray scattering: separation of prenucleation and nucleation events

This paper describes a time-resolved X-ray scattering study of microtubule assembly by synchrotron radiation. The method is complementary to light scattering but allows a better distinction between oligomeric and polymeric assembly states. With an improved rapid temperature jump device, it is shown that temperature-induced microtubule assembly is preceded by prenucleation and nucleation events involving oligomers of tubulin, in analogy with earlier results from near-equilibrium temperature scans. In general, the two phases closely overlap, but in certain conditions they can be observed separately. The prenucleation events seen by X-rays can be described as a rapid temperature-dependent equilibrium, with ring oligomers dissociating into smaller oligomers and subunits at elevated temperature. Different solution conditions affect mainly the time lag between the prenucleation and nucleation phases; this in turn determines the apparent magnitude of the prenucleation steps. By contrast, the temperature dependence of the equilibrium between the prenucleation oligomers shows little influence on solution conditions. The results suggest that the ring-forming and tubule-forming assembly modes of tubulin are governed by different interactions between subunits, although they may be based on a pool of similar intermediates.     

Monotonic versus oscillating microtubule assembly: a cryo-electron microscope study

Depending on the free GTP concentration, microtubules can assemble following either a monotonic or an oscillatory mode. We have used cryoelectron microscopy to compare the tubulin assemblies characteristic of each polymerization pathway. We focus on the first assembly peak. At this particular time point, despite their strikingly different subsequent evolution, both systems are similar with regard to the extent of tubulin polymerization and to the microtubule length distribution. The present study shows that whilst the observed microtubule structures are the same in both systems, the oscillatory system shows quantities of closed ring-like tubulin oligomers, far in excess of those observed in the monotonic system. Furthermore, the conversion of the oscillating system to a monotonic one by GTP addition during the first oscillation is accompanied by a marked decrease in the number of rings. Based on these results we propose that the GTP dependent step which governs microtubule oscillations is the opening of inactive tubulin oligomers.     

Mechanism for oscillatory assembly of microtubules

Dampened oscillations of microtubule assembly can accompany polymerization at high tubulin subunit concentrations. This presumably results from a synchronization of dynamic instability behavior, which generates a large population of rapidly disassembling microtubules, that liberate tubulin-GDP oligomers. Subunits in oligomers cannot assemble until they dissociate, to allow GDP-GTP exchange. To determine whether rapidly disassembling microtubules generate oligomers directly, we measured the rate of dilution-induced disassembly of tubulin-GDP microtubules and the rate of dissociation of GDP from the so-formed tubulin-GDP subunits. The rate of GDP dissociation from liberated subunits was found to correspond to that of tubulin-GDP subunits (t1/2 = 5 s), rather than tubulin-GDP oligomers. This indicates that tubulin-GDP subunits are released from microtubules undergoing rapid disassembly. Oligomers apparently form in a side reaction from the high concentration of tubulin-GDP subunits liberated from the synchronously disassembling microtubule population. The rate of subunit dissociation is 0.11 s-1 with oligomers formed by concentrating tubulin-GDP subunits and 0.045 s-1 with oligomers formed by cold-induced microtubule disassembly. This difference provides evidence that the conformation of tubulin-GDP subunits released from rapidly disassembling microtubules differs from tubulin-GDP subunits that were not recently in the microtubule lattice.     

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.     

CLIP-170/tubulin-curved oligomers coassemble at microtubule ends and promote rescues

BACKGROUND: CLIP-170 is a microtubule binding protein specifically located at microtubule plus ends, where it modulates their dynamic properties and their interactions with intracellular organelles. The mechanism by which CLIP-170 is targeted to microtubule ends remains unclear today, as well as its precise effect on microtubule dynamics. RESULTS: We used the N-terminal part of CLIP-170 (named H2), which contains the microtubule binding domains, to investigate how it modulates in vitro microtubule dynamics and structure. We found that H2 primarily promoted rescues (transitions from shrinkage to growth) of microtubules nucleated from pure tubulin and isolated centrosomes, and stimulated microtubule nucleation. Electron cryomicroscopy revealed that H2 induced the formation of tubulin rings in solution and curved oligomers at the extremities of microtubules in assembly conditions. CONCLUSIONS: These results suggest that CLIP-170 targets specifically at microtubule plus ends by copolymerizing with tubulin and modulates microtubule nucleation, polymerization, and rescues by the same basic mechanism with tubulin oligomers as intermediates.     

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.     

Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanism

The relationship between GTP hydrolysis and microtubule assembly has been investigated by using a rapid filtration method. Microtubules assembled from phosphocellulose-purified tubulin, double-labeled with [gamma-32P]- and [3H]GTP, were trapped and washed free of unbound nucleotide on glass fiber filters. The transient accumulation of microtubule-bound GTP predicted by uncoupled GTP hydrolysis models [Carlier & Pantaloni (1981) Biochemistry 20, 1918-1924; Carlier et al. (1987) Biochemistry 26, 4428-4437] during the rapid assembly of microtubules was not detectable under our experimental conditions. By calculating hypothetical time courses for the transient accumulation of microtubule-bound GTP, we demonstrate that microtubule-bound GTP would have been detectable even if the first-order rate constant for GTP hydrolysis were 4-5 times greater than the pseudo-first-order rate constant for tubulin subunit addition to microtubules. In a similar manner, we demonstrate that if GTP hydrolysis were uncoupled from microtubule assembly but were limited to the interface between GTP subunits and GDP subunits (uncoupled vectorial hydrolysis), then microtubule-bound GTP would have been detectable if GTP hydrolysis became uncoupled from microtubule assembly at less than 50 microM free tubulin, 5 times the steady-state tubulin concentration of our experimental conditions. In addition, during rapid microtubule assembly, we have not detected any microtubule-bound Pi, which has been proposed to form a stabilizing cap at the ends of microtubules [Carlier et al. (1988) Biochemistry 27, 3555-3559]. Also, several conditions that could be expected to increase the degree of potential uncoupling between GTP hydrolysis and microtubule assembly were examined, and no evidence of uncoupling was found. Our results are consistent with models that propose cooperative mechanisms that limit GTP hydrolysis to the terminal ring of tubulin subunits [e.g., O'Brien et al. (1987) Biochemistry 26, 4148-4156]. The results are also consistent with the hypothesis that a slow conformational change in tubulin subunits after GTP hydrolysis and Pi release occurs that results in destabilized microtubule ends when such subunits become exposed at the ends.     

Isolation of microtubule protein from chicken erythrocytes and determination of the critical concentration for tubulin polymerization in vitro and in vivo

Microtubule protein isolated from nucleated chicken erythrocytes was examined with respect to composition and assembly properties to determine its significance in a microtubule bundle called the marginal band. 1) The protein contains greater than 95% tubulin with small amounts of tau polypeptides and no high molecular weight polypeptides. 2) Microtubule assembly in vitro at 37 degrees C is characterized by low levels of nucleation, despite an abundance of ring oligomers at 5 degrees C, as indicated by long lag times, slow assembly rates, and microtubules that are twice as long as brain microtubules assembled under the same conditions. 3) By radioimmunoassay and sodium dodecyl sulfate gel analysis we determined that 0.6% of erythrocyte protein is tubulin of which three-quarters is in a nonextractable form and is associated with the microtubule bundle and the cell cortex. From these values the in vivo concentrations of total tubulin and tubulin dimer subunits are 2.4 and 0.7 mg/ml, respectively. The value of 0.7 mg/ml is close to the range of values of 0.1-0.6 mg/ml for the critical concentration of erythrocyte microtubule protein in vitro, suggesting that the assembly properties of tubulin in vitro and in vivo are similar.     

Prion protein inhibits microtubule assembly by inducing tubulin oligomerization

A growing body of evidence points to an association of prion protein (PrP) with microtubular cytoskeleton. Recently, direct binding of PrP to tubulin has also been found. In this work, using standard light scattering measurements, sedimentation experiments, and electron microscopy, we show for the first time the effect of a direct interaction between these proteins on tubulin polymerization. We demonstrate that full-length recombinant PrP induces a rapid increase in the turbidity of tubulin diluted below the critical concentration for microtubule assembly. This effect requires magnesium ions and is weakened by NaCl. Moreover, the PrP-induced light scattering structures of tubulin are cold-stable. In preparations of diluted tubulin incubated with PrP, electron microscopy revealed the presence of approximately 50 nm disc-shaped structures not reported so far. These unique tubulin oligomers may form large aggregates. The effect of PrP is more pronounced under the conditions promoting microtubule formation. In these tubulin samples, PrP induces formation of the above oligomers associated with short protofilaments and sheets of protofilaments into aggregates. Noticeably, this is accompanied by a significant reduction of the number and length of microtubules. Hence, we postulate that prion protein may act as an inhibitor of microtubule assembly by inducing formation of stable tubulin oligomers.     

Tubulin subunit sorting: Analysis of the mechanisms involved in the segregation of unique tubulin isotypes within microtubule copolymers

Summary Vertebrate cells contain biochemical and genetic isotypes of tubulin which are expressed in unique combinations in different tissues and cell types. To determine if mixtures of tubulin isotypes assemblein vitro to form different classes of microtubules, we analyzed the composition of microtubule copolymers assembled from mixtures of chicken brain and erythrocyte tubulin. During microtubule elongation brain tubulin assembled onto the ends of microtubules faster than erythrocyte tubulin, resulting in copolymers with continually changing ratios of isotypes along their lengths. Unlike examples of microtubule assembly where the rate of polymerization depends on the association rate constant (k⁺) and the subunit concentration, the rate and extent of sorting in copolymers appear to depend on the dissociation rate constant (k^–), which governs the rate at which subunits are released from tubulin oligomers and microtubules and thereby made available for reassembly into copolymers. The type of microtubule seed used to initiate elongation was also found to influence the composition of copolymers, indicating that polymerization favors association of subunits of the same isotype.     

Mechanical stress induced mechanism of microtubule catastrophes

Microtubules assembled in vitro from pure tubulin can switch occasionally from growing to shrinking states or resume assembly, an unusual behavior termed "dynamic instability of microtubule growth". Its origin remains unclear and several models have been proposed, including occasional switching of the microtubules into energetically unfavorable configurations during assembly. In this study, we have asked whether the excess energy accumulated in these configurations would be of sufficient magnitude to destabilize the capping region that must exist at the end of growing microtubules. For this purpose, we have analyzed the frequency distribution of microtubules assembled in vitro from pure tubulin, and modeled the different mechanical constraints accumulated in their wall. We find that the maximal excess energy that the microtubule lattice can store is in the order of 11 kBT per dimer. Configurations that require distortions up to approximately 20 kBT are allowed at the expense of a slight conformational change, and larger distortions are not observed. Modeling of the different elastic deformations suggests that the excess energy is essentially induced by protofilament skewing, microtubule radial curvature change and inter-subunit shearing, distortions that must destabilize further the tubulin subunits interactions. These results are consistent with the hypothesis that unfavorable closure events may trigger the catastrophes observed at low tubulin concentration in vitro. In addition, we propose a novel type of representation that describes the stability of microtubule assembly systems, and which might be of considerable interest to study the effects of stabilizing and destabilizing factors on microtubule structure and dynamics.     

Structural intermediates in the assembly of taxoid-induced microtubules and GDP-tubulin double rings: time-resolved X-ray scattering.

We have studied the self-association reactions of purified GDP-liganded tubulin into double rings and taxoid-induced microtubules, employing synchrotron time-resolved x-ray solution scattering. The experimental scattering profiles have been interpreted by reference to the known scattering profiles to 3 nm resolution and to the low-resolution structures of the tubulin dimer, tubulin double rings, and microtubules, and by comparison with oligomer models and model mixtures. The time courses of the scattering bands corresponding to the different structural features were monitored during the assembly reactions under varying biochemical conditions. GDP-tubulin essentially stays as a dimer at low Mg(2+) ion activity, in either the absence or presence of taxoid. Upon addition of the divalent cations, it associates into either double-ring aggregates or taxoid-induced microtubules by different pathways. Both processes have the formation of small linear (short protofilament-like) tubulin oligomers in common. Tubulin double-ring aggregate formation, which is shown by x-ray scattering to be favored in the GDP- versus the GTP-liganded protein, can actually block microtubule assembly. The tubulin self-association leading to double rings, as determined by sedimentation velocity, is endothermic. The formation of the double-ring aggregates from oligomers, which involves additional intermolecular contacts, is exothermic, as shown by x-ray and light scattering. Microtubule assembly can be initiated from GDP-tubulin dimers or oligomers. Under fast polymerization conditions, after a short lag time, open taxoid-induced microtubular sheets have been clearly detected (monitored by the central scattering and the maximum corresponding to the J(n) Bessel function), which slowly close into microtubules (monitored by the appearance of their characteristic J(0), J(3), and J (n) - (3) Bessel function maxima). This provides direct evidence for the bidimensional assembly of taxoid-induced microtubule polymers in solution and argues against helical growth. The rate of microtubule formation was increased by the same factors known to enhance taxoid-induced microtubule stability. The results suggest that taxoids induce the accretion of the existing Mg(2+)-induced GDP-tubulin oligomers, thus forming small bidimensional polymers that are necessary to nucleate the microtubular sheets, possibly by binding to or modifying the lateral interaction sites between tubulin dimers.     

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.     

Physical and chemical properties of purified tau factor and the role of tau in microtubule assembly.

This paper describes the physical and chemical properties of purified tau, a protein which is associated with brain microtubules and which induces assembly of microtubules from tubulin. Purified tau is composed of four polypeptides which migrate at positions equivalent to molecular weights between 55,000 and 62,000 during electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. These polypeptides are shown to be closely related by peptide mapping and by amnio acid analysis. A comparison by various techniques of the high molecular weight microtubule-associated proteins with the tau polypeptides indicates no apparent relationship. Tau is found by analytical ultracentrifugation and by sedimentation equilibrium to have a sedimentation coefficient of 2.6 S and a native molecular weight of 57,000. Tau, therefore, must be highly asymmetric (an axial ratio of 20:1 using a prolate ellipsoid model), and yet possess little α-helical structure as indicated by circular dichroism. Isoelectric focusing shows tau to be a neutral or slightly basic protein. Tau is also seen to be phosphorylated by a protein kinase which copurifies with microtubules.In the assembly process, tau apparently regulates the formation of longitudinal oligomers from tubulin dimers, and hence promotes ring formation under depolymerizing conditions and microtubule formation under polymerizing conditions. The known asymmetry of the tau molecule suggests that tau induces assembly by binding to several tubulin molecules per tau molecule, thereby effectively increasing the local concentration of tubulin and inducing the formation of longitudinal filaments. The role of tau is discussed in light of reports of polymerization induced by particular non-physiological conditions and by various polycations. The formation of normal microtubules over a wide range of tubulin and tau concentrations under mild buffer conditions suggests that tau and tubulin define a complete in vitro assembly system under conditions which approach physiological.     

Direct observation of steady-state microtubule dynamics

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.     

Hydrolysis of GTP associated with the formation of tubulin oligomers is involved in microtubule nucleation.

Hydrolysis of GTP is known to accompany microtubule assembly. Here we show that hydrolysis of GTP is also associated with the formation of linear oligomers of tubulin, which are precursors (prenuclei) in microtubule assembly. The hydrolysis of GTP on these linear oligomers inhibits the lateral association of GTP-tubulin that leads to the formation of a bidimensional lattice. Therefore GTP hydrolysis interferes with the nucleation of microtubules. Linear oligomers are also formed in mixtures of GTP-tubulin and GDP-tubulin. The hydrolysis of GTP associated with heterologous interactions between GTP-tubulin and GDP-tubulin in the cooligomer takes place at a threefold faster rate than upon homologous interactions between GTP-tubulins. The implication of these results in a model of vectorial GTP hydrolysis in microtubule assembly is discussed.