Dilution-induced disassembly of microtubules: relation to dynamic instability and the GTP cap

Microtubules were assembled from purified tubulin in the buffer originally used to study dynamic instability (100 mM PIPES, 2 mM EGTA, 1 mM magnesium, 0.2 mM GTP) and then diluted in the same buffer to study the rate of disassembly. Following a 15-fold dilution, microtubule polymer decreased linearly to about 20% of the starting value in 15 sec. We determined the length distribution of microtubules before dilution, and prepared computer simulations of polymer loss for different assumed rates of disassembly. Our experimental data were consistent with a disassembly rate per microtubule of 60 microns/min. This is the total rate of depolymerization for microtubules in the rapid shortening phase, as determined by light microscopy of individual microtubules (Walker et al.: Journal of Cell Biology 107:1437-1448, 1988). We conclude, therefore, that microtubules began rapid shortening at both ends upon dilution. Moreover, since we could detect no lag between dilution and the onset of rapid disassembly, the transition from elongation to rapid shortening apparently occurred within 1 sec following dilution. Assuming that this transition (catastrophe) involves the loss of the GTP cap, and that cap loss is achieved by the sequential dissociation of GTP-tubulin subunits following dilution, we can estimate the maximum size of the cap based on the kinetic data and model interpretation of Walker et al. The cap is probably shorter than 40 and 20 subunits at the plus and minus ends, respectively.     

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.     

Phase dynamics at microtubule ends: the coexistence of microtubule length changes and treadmilling

The length dynamics both of microtubule-associated protein (MAP)-rich and MAP-depleted bovine brain microtubules were examined at polymer mass steady state. In both preparations, the microtubules exhibited length redistributions shortly after polymer mass steady state was attained. With time, however, both populations relaxed to a state in which no further changes in length distributions could be detected. Shearing the microtubules or diluting the microtubule suspensions transiently increased the extent to which microtubule length redistributions occurred, but again the microtubules relaxed to a state in which changes in the polymer length distributions were not detected. Under steady-state conditions of constant polymer mass and stable microtubule length distribution, both MAP-rich and MAP-depleted microtubules exhibited behavior consistent with treadmilling. MAPs strongly suppressed the magnitude of length redistributions and the steady-state treadmilling rates. These data indicate that the inherent tendency of microtubules in vitro is to relax to a steady state in which net changes in the microtubule length distributions are zero. If the basis of the observed length redistributions is the spontaneous loss and regain of GTP-tubulin ("GTP caps") at microtubule ends, then in order to account for stable length distributions the microtubule ends must reside in the capped state far longer than in the uncapped state, and uncapped microtubule ends must be rapidly recapped. The data suggest that microtubules in cells may have an inherent tendency to remain in the polymerized state, and that microtubule disassembly must be induced actively.     

Generation of microtubule stability subclasses by microtubule- associated proteins: implications for the microtubule "dynamic instability" model

We have developed a method to distinguish microtubule associated protein (MAP)-containing regions from MAP-free regions within a microtubule, or within microtubule sub-populations. In this method, we measure the MAP-dependent stabilization of microtubule regions to dilution-induced disassembly of the polymer. The appropriate microtubule regions are identified by assembly in the presence of [3H]GTP, and assayed by filter trapping and quantitation of microtubule regions that contain label. We find that MAPs bind very rapidly to polymer binding sites and that they do not exchange from these sites measurably once bound. Also, very low concentrations of MAPs yield measurable stabilization of local microtubule regions. Unlike the stable tubule only polypeptide (STOP) proteins, MAPs do not exhibit any sliding behavior under our assay conditions. These results predict the presence of different stability subclasses of microtubules when MAPs are present in less than saturating amounts. The data can readily account for the observed "dynamic instability" of microtubules through unequal MAP distributions. Further, we report that MAP dependent stabilization is quantitatively reversed by MAP phosphorylation, but that calmodulin, in large excess, has no specific influence on MAP protein activity when MAPs are on microtubules.     

Dynamics of a protein polymer: the assembly and disassembly pathways of the MuB transposition target complex

MuB assembles into a polymer on DNA in the presence of ATP and is directly involved in the selection of an appropriate site on the Escherichia coli chromosome for the insertion of the bacteriophage Mu genome. We have developed an assay using fluorescently tagged proteins to monitor the polymeric state of MuB via fluorescence resonance energy transfer. We show that polymer assembly is initiated by the formation of an ATP-MuB complex. MuB then self-associates into a protomer before binding to DNA. Upon binding to DNA, a dramatic increase in energy transfer is observed, suggesting a conformational change within MuB. Polymer disassembly is much slower than assembly and is greatly stimulated by the MuA transposase. Additionally, MuB is readily exchanged between polymers, and ATP hydrolysis is directly coupled to polymer disassembly. Our data support a model in which a combination of rapid polymer assembly, MuA-mediated disassembly, followed by rapid reassembly of the polymer allows MuB to sample multiple DNA targets until an appropriate site is located for the insertion of the bacteriophage genome.     

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.     

Polymer motors: pushing out the front and pulling up the back

Mechanical work in cells is performed by specialized motor proteins that operate in a continuous mechanochemical cycle. Less complex, but still efficient, 'one-shot' motors evolved based on the assembly and disassembly of polymers. We review the mechanisms of pushing and pulling by actin and microtubule filaments and the organizational principles of actin networks. We show how these polymer force generators are used for the propulsion of intracellular pathogens, protrusion of lamellipodia and mitotic movements. We discuss several examples of cellular forces generated by the assembly and disassembly of polymer gels.     

Podophyllotoxin poisoning of microtubules at steady-state: effect of substoichiometric and superstoichiometric concentrations of drug

Summary Depolymerization kinetics of microtubules assembled to steady-state by pod ophyllotoxin treatment show a dose-dependent effect of this mitotic poison on the net rate of microtubule disassembly. Pulse-chase experiments with microtubules at steady-state indicate that the depolymerization effect induced by superstoichiometric concentrations of podophyllotoxin relative to tubulin is polar and time-dependent, i.e. the rate of tubulin loss decreases along with the time of treatment in the presence of the drug. Under these conditions the rate of microtubule disassembly is much faster than one could expect from a unique effect of drug-tubulin complex on the microtubule assembly end. Podophyllotoxin-tubulin complex is not able to induce active depolymerization of microtubules, while free podophyllotoxin is. These results are consistent with the hypothesis that this drug acts on the microtubule assembly-disassembly process by two different mechanisms: 1) as a free drug, it actively promotes polar depolymerization of microtubules, and 2) as a drug-tubulin complex, it retards the addition of subunits into the microtubule ends.     

Structural plugs at microtubule ends may regulate polymer dynamics in vitro

Microtubules contain in their lumens distinct structures (plugs) that influence their dynamic behavior in vitro. As observed by electron microscopy, plugs are stain-occluding structures 10-30 nm in length that occur along the lengths and at the ends of microtubules. Plugs occur at a frequency of 20-40% at the ends of microtubules assembled from cycled microtubule protein containing MAPs. While the composition of plugs is not known, preliminary evidence suggests that they are accretions of tubulin, that they are labile, and that they are more common in preparations containing MAPs. When polymers are induced to depolymerize by endwise subunit dissociation, the frequency of plugged microtubule ends increases transiently, suggesting that plugs temporarily stabilize microtubules. The functional significance of plugs may be that they prevent the sudden complete loss of microtubules through catastrophic disassembly. It is possible that plugs, by slowing the rate of disassembly, enable a polymer to add GTP-tubulin subunits, thereby forming a stabilizing GTP-cap. These observations suggest that plugs may stabilize polymers and account for the frequent transitions from shortening to growing phases that characterize dynamic instability.     

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.     

The stabilization of microtubules in isolated spindles by tubulin-colchicine complex

We have analyzed the effect of colchicine and tubulin dimer-colchicine complex (T-C) on microtubule assembly in mitotic spindles. Cold- and calcium-labile mitotic spindles were isolated from embryos of the sea urchin Lytechinus variegatus employing EGTA/glycerol stabilization buffers. Polarization microscopy and measurements of spindle birefringent retardation (BR) were used to record the kinetics of microtubule assembly-disassembly in single spindles. When isolated spindles were perfused out of glycerol stabilizing buffer into a standard in vitro microtubule reassembly buffer (0.1 M Pipes, pH 6.8, 1 mM EGTA, 0.5 mM MgCl2, and 0.5 mM GTP) lacking glycerol, spindle BR decreased with a half-time of 120 s. Colchicine at 1 mM in this buffer had no effect on the rate of spindle microtubule disassembly. Inclusion of 20 microM tubulin or microtubule protein, purified from porcine brain, in this buffer resulted in an augmentation of spindle BR. Interestingly, in the presence of 20 microM T-C, spindle BR did not increase, but was reversibly stabilized; subsequent perfusion with reassembly buffer without T-C resulted in depolymerization. This behavior is striking in contrast to the rapid depolymerization of spindle microtubules induced by colchicine and T-C in vivo. These results support the current view that colchicine does not directly promote microtubule depolymerization. Rather, it is T-C complex that alters microtubule assembly, by reversibly binding to microtubules and inhibiting elongation. In vivo, colchicine can induce depolymerization of nonkinetochore spindle microtubules within 20 s. In vitro, colchicine blocks further microtubule assembly, but does not induce rapid disassembly.(ABSTRACT TRUNCATED AT 250 WORDS)     

The interaction of TOGp with microtubules and tubulin

TOGp is the human homolog of XMAP215, a Xenopus microtubule-associated protein that promotes rapid microtubule assembly at plus ends. These proteins are thought to be critical for microtubule assembly and/or mitotic spindle formation. To understand how TOGp interacts with the microtubule lattice, we cloned full-length TOGp and various truncations for expression in a reticulocyte lysate system. Based on microtubule co-pelleting assays, the microtubule binding domain is contained within a basic 600-amino acid region near the N terminus, with critical domains flanking a region homologous to the microtubule binding domain found in the related proteins Stu2p (S. cerevisiae) and Dis1 (S. pombe). Both full-length TOGp and the N-terminal fragment show enhanced binding to microtubule ends. Full-length TOGp also binds altered polymer lattice structures including parallel protofilament sheets, antiparallel protofilament sheets induced with zinc ions, and protofilament rings, suggesting that TOGp binds along the length of individual protofilaments. The C-terminal region of TOGp has a low affinity for microtubule polymer but binds tubulin dimer. We propose a model to explain the microtubule-stabilizing and/or assembly-promoting functions of the XMAP215/TOGp family of microtubule-associated proteins based on the binding properties we have identified.     

Dynamics of microtubules bundled by microtubule associated protein 2C (MAP2C)

MAP2C is a microtubule-associated protein abundant in immature nerve cells. We isolated a cDNA clone encoding whole mouse MAP2C of 467 amino acid residues. In fibroblasts transiently transfected with cDNA of MAP2C, interphase microtubule networks were reorganized into microtubule bundles. To reveal the dynamic properties of microtubule bundles, we analyzed the incorporation sites of exogenously introduced tubulin by microinjection of biotin-labeled tubulin and the turnover rate of microtubule bundles by photoactivation of caged fluorescein- labeled tubulin. The injected biotin-labeled tubulin was rapidly incorporated into distal ends of preexisting microtubule bundles, suggesting a concentration of the available ends of microtubules at this region. Although homogenous staining of microtubule bundles with antibiotin antibody was observed 2 h after injection, the photoactivation study indicated that turnover of microtubule bundles was extremely suppressed and < 10% of tubulin molecules would be exchanged within 1 h. Multiple photoactivation experiments provided evidence that neither catastrophic disassembly at the distal ends of bundles nor concerted disassembly due to treadmilling at the proximal ends could explain the observed rapid incorporation of exogenously introduced tubulin molecules. We conclude that microtubules bundled by MAP2C molecules are very stable while the abrupt increase of free tubulin molecules by microinjection results in rapid assembly from the distal ends within the bundles as well as free nucleation of small microtubules which are progressively associated laterally with preexisting microtubule bundles. This is the first detailed study of the function of MAPs on the dynamics of microtubules in vivo.     

Fluorescence spot tests for DNA endonuclease, ligase, and topoisomerase activities

A rapid dilution cuvette has been designed for measurement of the rates of microtubule disassembly and the factors which stabilize tubules against dilution-induced depolymerization. The device minimizes microtubule shearing by obviating the passage of microtubules through small orifices, and optical measurements of the turbidity changes during disassembly are optimized by use of a long-path-length configuration. Details on the design and application of the rapid dilution cuvette to probe microtubule disassembly are described here. In principle, the method may be applied to study any indefinite polymerization process which is characterized by critical concentration phenomena.     

A Thermodynamic Model of Microtubule Assembly and Disassembly

Microtubules are self-assembling polymers whose dynamics are essential for the normal function of cellular processes including chromosome separation and cytokinesis. Therefore understanding what factors effect microtubule growth is fundamental to our understanding of the control of microtubule based processes. An important factor that determines the status of a microtubule, whether it is growing or shrinking, is the length of the GTP tubulin microtubule cap. Here, we derive a Monte Carlo model of the assembly and disassembly of microtubules. We use thermodynamic laws to reduce the number of parameters of our model and, in particular, we take into account the contribution of water to the entropy of the system. We fit all parameters of the model from published experimental data using the GTP tubulin dimer attachment rate and the lateral and longitudinal binding energies of GTP and GDP tubulin dimers at both ends. Also we calculate and incorporate the GTP hydrolysis rate. We have applied our model and can mimic published experimental data, which formerly suggested a single layer GTP tubulin dimer microtubule cap, to show that these data demonstrate that the GTP cap can fluctuate and can be several microns long.     

Fast disassembly of microtubules induced by Mg2+ or Ca2+

The extent and rate of disassembly of microtubules induced by the addition of high concentrations of magnesium and calcium have been measured. At 25 degrees C, the rate constant for microtubule disassembly increases more than ten-fold on increasing [Mg2+] from 4.0 to 20 mM. The process is even more sensitive to [Ca2+], showing similar enhancement on increasing [Ca2+] from 0.5 to 8 mM. Electron microscopy indicates that the disassembly is an end-dependent process. Complete microtubule disassembly occurs at concentrations in excess of 10 and 2 mM for Mg2+ and Ca2+, respectively; this suggests the importance of binding to weak sites for both ions. The sensitivity to ionic composition explains the wide variations in the published values for k-, under varying conditions. The results indicate the potential range of microtubule disassembly rates which may be encountered under different conditions in vitro and in vivo. The highest values of k- (ca. 3000 s-1) would imply microtubule shortening rates in excess of 100 um per minute.     

Dynamics of microtubule depolymerization in monocytes

Human monocytes, which contain few interphase microtubules (35.+/- 7.7), were used to study the dynamics of microtubule depolymerization. Steady-state microtubule assembly was abruptly blocked with either high concentrations of nocodazole (10 micrograms/ml) or exposure to cold temperature (3 degrees C). At various times after inhibition of assembly, cells were processed for anti-tubulin immunofluorescence microscopy. Stained cells were observed with an intensified video camera attached to the fluorescence microscope. A tracing of the entire length of each individual microtubule was made from the image on the television monitor by focusing up and down through the cell. The tracings were then digitized into a computer. All microtubules were seen to originate from the centrosome, with an average length in control cells of 7.1 +/- 2.7 microns (n = 957 microtubules). During depolymerization, the total microtubule polymer and the number of microtubules per cell decreased rapidly. In contrast, there was a slow decrease in the average length of the persisting microtubules. The half-time for both the loss of total microtubule polymer and microtubule number per cell was approximately 40 s for nocodazole-treated cells. The rate-limiting step in the depolymerization process was the rate of initiation of disassembly. Once initiated, depolymerization appeared catastrophic. Further kinetic analysis revealed two classes of microtubules: 70% of the microtubule population was very labile and initiated depolymerization at a rate approximately 23 times faster than a minor population of persistent microtubules. Cold treatment yielded qualitatively similar characteristics of depolymerization, but the initiation rates were slower. In both cases there was a significant asynchrony and heterogeneity in the initiation of depolymerization among the population of microtubules.     

The mechanism of calcium-induced microtubule disassembly

We have examined the mechanism of calcium induced microtubule dissasembly by combined kinetic and steady state analysis. Our results indicate that calcium induces microtubule disassembly by binding directly to the wall of the microtubule and promoting dissociation of subunits from the ends.     

Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates

Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995- 8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.     

The effect of podophyllotoxin on microtubule dynamics

We have investigated the effects of podophyllotoxin on the dynamic properties of microtubules assembled from pure tubulin dimer. Excess podophyllotoxin causes the complete disassembly of microtubules, through formation of a tubulin-GTP-podophyllotoxin ternary complex with a dissociation rate constant of 160 s-1 at 37 degrees C, similar to that found upon extensive isothermal dilution in this buffer system. Addition of substoichiometric concentrations of podophyllotoxin causes partial disassembly of microtubules through production of an equivalent amount of the ternary complex. Microtubule length measurements and incorporation of [3H]GTP-tubulin dimer show that podophyllotoxin can suppress the dynamic instability of tubulin dimer microtubules and that it acts substoichiometrically in so doing. We interpret the action of substoichiometric podophyllotoxin on microtubule ends in terms of effects on interconversion of growing and shrinking microtubules in a dynamic system in which tubulin-GTP-podophyllotoxin is kinetically analogous to tubulin-GTP in addition and to tubulin-GDP in dissociation. The ability to suppress dynamic instability may be one way in which drugs such as podophyllotoxin, acting at relatively low concentrations, are able to arrest cell growth and development in a selective way, without necessarily affecting the integrity of the major part of the cytoskeletal microtubule network.