Kinetic analysis of tubulin exchange at microtubule ends at low vinblastine concentrations

We have investigated the effects of vinblastine at micromolar concentrations and below on the dynamics of tubulin exchange at the ends of microtubule-associated-protein-rich bovine brain microtubules. The predominant behavior of these microtubules at polymer-mass steady state under the conditions examined was tubulin flux, i.e., net addition of tubulin at one end of each microtubule, operationally defined as the assembly or A end, and balanced net loss at the opposite (disassembly or D) end. No dynamic instability behavior could be detected by video-enhanced dark-field microscopy. Addition of vinblastine to the microtubules at polymer-mass steady state resulted in an initial concentration-dependent depolymerization predominantly at the A ends, until a new steady-state plateau at an elevated critical concentration was established. Microtubules ultimately attained the same stable polymer-mass plateau when vinblastine was added prior to initiation of polymerization as when the drug was added to already polymerized microtubules. Vinblastine inhibited tubulin exchange at the ends of the microtubules at polymer-mass steady state, as determined by using microtubules differentially radiolabeled at their opposite ends. Inhibition of tubulin exchange occurred at concentrations of vinblastine that had very little effect on polymer mass. Both the initial burst of incorporation that occurs in control microtubule suspensions following a pulse of labeled GTP and the relatively slower linear incorporation of label that follows the initial burst were inhibited in a concentration-dependent manner by vinblastine. Both processes were inhibited to the same extent at all vinblastine concentrations examined. If the initial burst of label incorporation represents a low degree of dynamic instability (very short excursions of growth and shortening of the microtubules at one or both ends), then vinblastine inhibits both dynamic instability and flux to similar extents. The ability of vinblastine to inhibit tubulin exchange at microtubule ends in the micromolar concentration range appeared to be mediated by the reversible binding of vinblastine to tubulin binding sites exposed at the polymer ends. Determination by dilution analysis of the effects of vinblastine on the apparent dissociation rate constants for tubulin loss at opposite microtubule ends indicated that a principal effect of vinblastine is to decrease the dissociation rate constant at A ends (i.e., it produces a kinetic cap at A ends), whereas it has no effect on the D-end dissociation rate constant.     

Taxol stabilization of microtubules in vitro: dynamics of tubulin addition and loss at opposite microtubule ends

We have investigated the effects of taxol on steady-state tubulin flux and on the apparent molecular rate constants for tubulin addition and loss at the two ends of bovine brain microtubules in vitro. These microtubules, which consist of a mixture of 70% tubulin and 30% microtubule-associated proteins (MAPs), undergo a net addition of tubulin at one end of each microtubule (A end) and a precisely balanced net loss of tubulin at the opposite end (D end) at steady state in vitro. They do not exhibit to a detectable extent the "dynamic instability" behavior described recently for MAP-free microtubules, which would be evident as an increase in the mean microtubule length and a decrease in the number of microtubules in the suspensions [Mitchison, T., & Kirschner, M. (1984) Nature (London) 312, 237-242]. We used a double-label procedure in which microtubules were labeled with tritium and carbon-14 at A ends and carbon-14 at D ends to distinguish the two ends, combined with a microtubule collection procedure that permitted rapid and accurate analysis of retention of the two labels in the microtubules. We found that taxol slowed the flux of tubulin in a concentration-dependent manner, with 50% inhibition occurring between 5 and 7 microM drug. The effects of taxol on the apparent molecular rate constants for tubulin addition and loss at the two microtubule ends were determined by dilution analysis at an intermediate taxol concentration. The results indicated that taxol decreased the magnitudes of the dissociation rate constants at the two ends to similar extents, while exerting little effect on the association rate constants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regeneration of mirror symmetrical limbs in the axolotl

Measurements of tubulin exchange into and from bovine brain microtubules at steady state in vitro were made with 3H-GTP as a marker for tubulin addition to or loss from microtubules. Tubulin has an exchangeable GTP binding site that becomes nonexchangeable in the microtubule. We found that tubulin addition to and loss from microtubules under steady state conditions occurred at equivalent rates, that loss and gain were linear, and that exchange rates (percentage of total tubulin in microtubules lost or gained per hour) were dependent upon microtubule length. Furthermore, we found that podophyllotoxin blocked steady state assembly, but did not alter the rate of steady state tubulin loss. When the assembling microtubule end was pulsed with 3H-GTP at steady state, the label was almost completely retained during a subsequent chase. We conclude that the microtubule assembly-disassembly "equilibrium" is a steady state summation of two different reactions which occur at opposite ends of the microtubule, and that assembly and disassembly occur predominantly and perhaps exclusively at the opposite ends under steady state conditions in vitro.     

Stathmin strongly increases the minus end catastrophe frequency and induces rapid treadmilling of bovine brain microtubules at steady state in vitro

Stathmin is a ubiquitous microtubule destabilizing protein that is believed to play an important role linking cell signaling to the regulation of microtubule dynamics. Here we show that stathmin strongly destabilizes microtubule minus ends in vitro at steady state, conditions in which the soluble tubulin and microtubule levels remain constant. Stathmin increased the minus end catastrophe frequency approximately 13-fold at a stathmin:tubulin molar ratio of 1:5. Stathmin steady-state catastrophe-promoting activity was considerably stronger at the minus ends than at the plus ends. Consistent with its ability to destabilize minus ends, stathmin strongly increased the treadmilling rate of bovine brain microtubules. By immunofluorescence microscopy, we also found that stathmin binds to purified microtubules along their lengths in vitro. Co-sedimentation of purified microtubules polymerized in the presence of a 1:5 initial molar ratio of stathmin to tubulin yielded a binding stoichiometry of 1 mol of stathmin per approximately 14.7 mol of tubulin in the microtubules. The results firmly establish that stathmin can increase the steady-state catastrophe frequency by a direct action on microtubules, and furthermore, they indicate that an important regulatory action of stathmin in cells may be to destabilize microtubule minus ends.     

Mechanism of the microtubule GTPase reaction

The rate of GTP hydrolysis by microtubules has been measured at tubulin subunit concentrations where microtubules undergo net disassembly. This was made possible by using microtubules stabilized against disassembly by reaction with ethylene glycol bis-(succinimidylsuccinate) (EGS) as sites for the addition of tubulin-GTP subunits. The tubulin subunit concentration was varied from 25 to 90% of the steady state concentration, and there was no net elongation of stabilized microtubule seeds. The GTPase rate with EGS microtubules was linearly proportional to the tubulin-GTP subunit concentration when this concentration was varied by dilution and by using GDP to compete with GTP for the tubulin E-site. The linear dependence of the rate is consistent with a GTP mechanism in which hydrolysis is coupled to the tubulin-GTP subunit addition to microtubule ends. It is inconsistent with reaction schemes in which: microtubules are capped by a single tubulin-GTP subunit, which hydrolyzes GTP when a tubulin-GTP subunit adds to the end; hydrolysis occurs primarily in subunits at the interface of a tubulin-GTP cap and the tubulin-GDP microtubule core; hydrolysis is not coupled to subunit addition and occurs randomly in subunits in a tubulin-GTP cap. It was also found that GDP inhibition of the microtubule GTPase rate results from GDP competition for GTP at the tubulin subunit E-site. There is no additional effect of GDP on the GTPase rate resulting from exchange into tubulin subunits at microtubule ends.     

A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end

We have isolated a protein factor from Xenopus eggs that promotes microtubule assembly in vitro. Assembly promotion was associated with a 215-kD protein after a 1,000-3,000-fold enrichment of activity. The 215-kD protein, termed Xenopus microtubule assembly protein (XMAP), binds to microtubules with a stoichiometry of 0.06 mol/mol tubulin dimer. XMAP is immunologically distinct from the Xenopus homologues to mammalian brain microtubule-associated proteins; however, protein species immunologically related to XMAP with different molecular masses are found in Xenopus neuronal tissues and testis. XMAP is unusual in that it specifically promotes microtubule assembly at the plus-end. At a molar ratio of 0.01 mol XMAP/mol tubulin the assembly rate of the microtubule plus-end is accelerated 8-fold while the assembly rate of the minus-end is increased only 1.8-fold. Under these conditions XMAP promotes a 10-fold increase in the on-rate constant (from 1.4 s-1.microM-1 for microtubules assembled from pure tubulin to 15 s-1.microM-1), and a 10-fold decrease in off-rate constant (from 340 to 34 s-1). Given its stoichiometry in vivo, XMAP must be the major microtubule assembly factor in the Xenopus egg. XMAP is phosphorylated during M-phase of both meiotic and mitotic cycles, suggesting that its activity may be regulated during the cell cycle.     

Stability of microtubule protein over the pH range: 6.9--9.5.

The pH stability range of a microtubule protein preparation has been investigated between 6.9 and 9.5. Microtubule protein was exposed to various pH values in this range and then returned to pH 6.9. The appearance of microtubules as verified by electron microscopy and sedimentation analysis under polymerizing conditions was taken as an indication of a conformationally stable protein. Between pH 6.9 and pH 8.0 the loss in the ability to form microtubules was found to be reversible, at pH 8.2 it was partially reversible, above pH 8.2 it was irreversible. Tubulin and the microtubule-associated protein fraction were separately exposed to high pH. It was observed that tubulin exposed to high pH can still form microtubules in the presence of untreated microtubule-associated protein. On the other hand, microtubule-associated protein exposed to high pH could not initiate microtubule assembly with untreated tubulin. It was concluded from these observations that the loss in the ability of a microtubule protein preparation to assemble at high pH is due to a change in the microtubule-associated protein fraction and that tubulin is conformationally stable even after exposure to pH 9.5.     

Stathmin family protein SCG10 differentially regulates the plus and minus end dynamics of microtubules at steady state in vitro: implications for its role in neurite outgrowth

SCG10 (superior cervical ganglia neural-specific 10 protein) is a neuron specific member of the stathmin family of microtubule regulatory proteins that like stathmin can bind to soluble tubulin and depolymerize microtubules. The direct actions of SCG10 on microtubules themselves and on their dynamics have not been investigated previously. Here, we analyzed the effects of SCG10 on the dynamic instability behavior of microtubules in vitro, both at steady state and early during microtubule polymerization. In contrast to stathmin, whose major action on dynamics is to destabilize microtubules by increasing the switching frequency from growth to shortening (the catastrophe frequency) at microtubule ends, SCG10 stabilized the plus ends both at steady state and early during polymerization by increasing the rate and extent of growth. For example, early during polymerization at high initial tubulin concentrations (20 microM), a low molar ratio of SCG10 to tubulin of 1:30 increased the growth rate by approximately 50%. In contrast to its effects at plus ends, SCG10 destabilized minus ends by increasing the shortening rate, the length shortened during shortening events, and the catastrophe frequency. Consistent with its ability to modulate microtubule dynamics at steady state, SCG10 bound to purified microtubules along their lengths. The dual activity of SCG10 at opposite microtubule ends may be important for its role in regulating growth cone microtubule dynamics. SCG10's ability to promote plus end growth may facilitate microtubule extension into filopodia, and its ability to destabilize minus ends could provide soluble tubulin for net plus end elongation.     

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.     

Kinetic analysis of tubulin assembly in the presence of the microtubule-associated protein TOGp

The microtubule-associated protein TOGp, which belongs to a widely distributed protein family from yeasts to humans, is highly expressed in human tumors and brain tissue. From purified components we have determined the effect of TOGp on thermally induced tubulin association in vitro in the presence of 1 mm GTP and 3.4 m glycerol. Physicochemical parameters describing the mechanism of tubulin polymerization were deduced from the kinetic curves by application of the classical theoretical models of tubulin assembly. We have calculated from the polymerization time curves a range of parameters characteristic of nucleation, elongation, or steady state phase. In addition, the tubulin subunits turnover at microtubule ends was deduced from tubulin GTPase activity. For comparison, parallel experiments were conducted with colchicine and taxol, two drugs active on microtubules and with tau, a structural microtubule-associated protein from brain tissue. TOGp, which decreases the nucleus size and the tenth time of the reaction (the time required to produce 10% of the final amount of polymer), shortens the nucleation phase of microtubule assembly. In addition, TOGp favors microtubule formation by increasing the apparent first order rate constant of elongation. Moreover, TOGp increases the total amount of polymer by decreasing the tubulin critical concentration and by inhibiting depolymerization during the steady state of the reaction.     

Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate

Although the mechanism of microtubule dynamic instability is thought to involve the hydrolysis of tubulin-bound GTP, the mechanism of GTP hydrolysis and the basis of microtubule stability are controversial. Video microscopy of individual microtubules and dilution protocols were used to examine the size and lifetime of the stabilizing cap. Purified porcine brain tubulin (7-23 microM) was assembled at 37 degrees C onto both ends of isolated sea urchin axoneme fragments in a miniature flow cell to give a 10-fold variation in elongation rate. The tubulin concentration in the region of microtubule growth could be diluted rapidly (by 84% within 3 s of the onset of dilution). Upon perfusion with buffer containing no tubulin, microtubules experienced a catastrophe (conversion from elongation to rapid shortening) within 4-6 s on average after dilution to 16% of the initial concentration, independent of the predilution rate of elongation and length. Based on extrapolation of catastrophe frequency to zero tubulin concentration, the estimated lifetime of the stable cap after infinite dilution was less than 3-4 s for plus and minus ends, much shorter than the approximately 200 s observed at steady state (Walker, R. A., E. T. O'Brien, N. K. Pryer, M. Soboeiro, W. A. Voter, H. P. Erickson, and E. D. Salmon. 1988. J. Cell Biol. 107:1437-1448.). We conclude that during elongation, both plus and minus ends are stabilized by a short region (approximately 200 dimers or less) and that the size of the stable cap is independent of 10-fold variation in elongation rate. These results eliminate models of dynamic instability which predict extensive "build-up" stabilizing caps and support models which constrain the cap to the elongating tip. We propose that the cell may take advantage of such an assembly mechanism by using "catastrophe factors" that can promote frequent catastrophe even at high elongation rates by transiently binding to microtubule ends and briefly inhibiting GTP-tubulin association.     

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.     

Changes in the hydrodynamic properties of microtubules induced by taxol

Microtubule assembly was followed and monitored by (1) the turbidity at 350 nm, (2) the weight of the pelleted microtubules, (3) linear dichroism, LD tau, of the turbidity upon flow orientation, (4) the specific viscosity, eta spec, and (5) electron microscopy. These five methods showed the same features for normal microtubule assembly, but were different in the presence of taxol, a drug which binds to tubulin. The The apparent steady state of microtubule assembly in the presence of taxol as found by turbidity or the weight of pelleted polymer did not represent a stable state, as both LD tau and eta spec continued to change for a much longer time. Microtubules assembled in the presence of taxol from microtubule proteins as well as from purified tubulin were difficult to orient, as high flow gradients were needed and the maximal LD tau value represented only 20% of the LD tau for normal microtubules. In contrast to the slow relaxation of normal microtubules, rapid relaxation to random orientation was found in the presence of taxol. Low orientability was also indicated by electron micrographs, in which pelleted microtubules were seen to be randomly oriented in the presence of taxol. Taxol induced a very high eta spec, 4-times the steady-state value in the initial phase of assembly, which slowly declined again to a steady state, an effect which was also found for assembly of purified tubulin assembled in the absence of the microtubule-associated proteins. The presence of taxol did not change the relative amount and composition of the microtubule-associated proteins in the assembled microtubules. The results therefore suggest that taxol alters the hydrodynamic properties of the microtubules due to its interaction with tubulin and that this alteration is not an effect of the microtubule-associated proteins.     

Effects of magnesium on the dynamic instability of individual microtubules

We investigated the effect of magnesium ion (Mg) on the parameters of dynamic instability of individual porcine brain microtubules. Rates of elongation and rapid shortening were measured by using video-enhanced DIC light microscopy and evaluated by using computer-generated plots of microtubule length vs time. Increasing [Mg] from 0.25 to 6 mM increased the second-order association rate constant for elongation about 25% at each end. At plus ends, this resulted in a 1.5-2-fold increase in elongation rates over the tubulin concentrations explored. Rapid shortening rates were more dramatically affected by Mg. As [Mg] was increased from 0.25 to 6 mM, the average rate of rapid shortening increased about 3-fold at plus ends and 4-5-fold at minus ends. The ends had roughly equivalent average rates at low [Mg], of 30-45 microns/min. At any Mg concentration, rates of disassembly varied from one microtubule to another, and often an individual microtubule would exhibit more than one rate during a single shortening phase. Individual rates at 6 mM Mg varied from 12 to 250 microns/min. Over the concentration range explored, Mg affected the frequencies of transition from elongation to shortening and back only at minus ends. Minus ends were relatively stable at low [Mg], having 4 times the frequency of rescue than at high [Mg], and a lower frequency of catastrophe (particularly evident at low tubulin concentrations). Plus ends, surprisingly, were highly unstable at all Mg concentrations investigated, having about the same transition frequencies as did the least stable (high Mg) minus ends. Our results have implications for models of the GTP cap, again emphasizing that GTP caps cannot build up in proportion to elongation rate, and must be constrained to the tips of growing microtubules.     

Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle

The budding yeast protein Kip3p is a member of the conserved kinesin-8 family of microtubule motors, which are required for microtubule-cortical interactions, normal spindle assembly and kinetochore dynamics. Here, we demonstrate that Kip3p is both a plus end-directed motor and a plus end-specific depolymerase--a unique combination of activities not found in other kinesins. The ATPase activity of Kip3p was activated by both microtubules and unpolymerized tubulin. Furthermore, Kip3p in the ATP-bound state formed a complex with unpolymerized tubulin. Thus, motile kinesin-8s may depolymerize microtubules by a mechanism that is similar to that used by non-motile kinesin-13 proteins. Fluorescent speckle analysis established that, in vivo, Kip3p moved toward and accumulated on the plus ends of growing microtubules, suggesting that motor activity brings Kip3p to its site of action. Globally, and more dramatically on cortical contact, Kip3p promoted catastrophes and pausing, and inhibited microtubule growth. These findings explain the role of Kip3p in positioning the mitotic spindle in budding yeast and potentially other processes controlled by kinesin-8 family members.     

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.     

Dynamics of Antarctic fish microtubules at low temperatures

The tubulins of Antarctic fishes, purified from brain tissue and depleted of microtubule-associated proteins (MAPs), polymerized efficiently in vitro to yield microtubules at near-physiological and supraphysiological temperatures (5, 10, and 20 degrees C). The dynamics of the microtubules at these temperatures were examined through the use of labeled guanosine 5'-triphosphate (GTP) as a marker for the incorporation, retention, and loss of tubulin dimers. Following attainment of a steady state in microtubule mass at 20 degrees C, the rate of incorporation of [3H]GTP (i.e., tubulin dimers) during pulses of constant duration decreased asymptotically toward a constant, nonzero value as the interval prior to label addition to the microtubule solution increased. Concomitant with the decreasing rate of label incorporation, the average length of the microtubules increased, and the number concentration of microtubules decreased. Thus, redistribution of microtubule lengths (probably via dynamic instability and/or microtubule annealing) appears to be responsible for the time-dependent decrease in the rate of tubulin uptake. When the microtubules had attained both a steady state in mass and a constant length distribution, linear incorporation of labeled tubulin dimers over time occurred at rates of 1.45 s-1 at 5 degrees C, 0.48 s-1 at 10 degrees C, and 0.18 s-1 at 20 degrees C. Thus, the microtubules displayed greater rates of subunit flux, or treadmilling, at lower, near-physiological temperatures. At each temperature, most of the incorporated label was retained by the microtubules during a subsequent chase with excess unlabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dynamic properties of microtubules at steady state in the presence of taxol

The dynamic properties of steady-state microtubules in the presence of the antitumor drug taxol and GTP, but in the absence of microtubule-associated proteins have been studied. The molecular rate constants for the loss or gain of subunits at steady state was found to be dramatically decreased as compared with that for microtubules formed in the presence of GTP and microtubule-associated proteins but in the absence of taxol [Zeeberg, B., Reid, R., and Caplow, M. (1980) J. Biol. Chem. 255, 9891-9899]. In light of this change it was surprising to find that the degrees of directionality for subunit flux into the microtubule at steady state are nearly identical within 1.5% of each other) in the two systems. One mechanism to account for this would be for taxol to cause a nearly identical decrease in the rate constants for subunit dissociation at both ends of the microtubule, with no effect on the rate constants for subunit addition. Similar results have previously been found in studies with an endogenous effector of the microtubule steady state, a protein kinase [Jameson, J. L. and Caplow, M. (1981) Proc. Natl Acad. Sci. USA, 78, 3413-3417]. In this case it was found that phosphorylation of microtubule-associated proteins altered the molecular rate constants for tubulin subunit addition and dissociation, but had no effect on the degree of directionality for subunit flux. It will be of interest to determine whether other exogenous or endogenous effectors also act in a manner such as to leave the directionality unaltered.     

The kinesin-13 MCAK has an unconventional ATPase cycle adapted for microtubule depolymerization

Unlike other kinesins, members of the kinesin-13 subfamily do not move directionally along microtubules but, instead, depolymerize them. To understand how kinesins with structurally similar motor domains can have such dissimilar functions, we elucidated the ATP turnover cycle of the kinesin-13, MCAK. In contrast to translocating kinesins, ATP cleavage, rather than product release, is the rate-limiting step for ATP turnover by MCAK; unpolymerized tubulin and microtubules accelerate this step. Further, microtubule ends fully activate the ATPase by accelerating the exchange of ADP for ATP. This tuning of the cycle adapts MCAK for its depolymerization activity: lattice-stimulated ATP cleavage drives MCAK into a weakly bound nucleotide state that reaches microtubule ends by diffusion, and end-specific acceleration of nucleotide exchange drives MCAK into a strongly bound state that promotes depolymerization. This altered cycle accounts well for the different mechanical behaviour of this kinesin, which depolymerizes microtubules from their ends, compared to translocating kinesins that walk along microtubules. Thus, the kinesin motor domain is a nucleotide-dependent engine that can be differentially tuned for transport or depolymerization functions.     

Mechanical properties of brain tubulin and microtubules

We measured the elasticity and viscosity of brain tubulin solutions under various conditions with a cone and plate rheometer using both oscillatory and steady shearing modes. Microtubules composed of purified tubulin, purified tubulin with taxol and 3x cycled microtubule protein from pig, cow, and chicken behaved as mechanically indistinguishable viscoelastic materials. Microtubules composed of pure tubulin and heat stable microtubule-associated proteins were also similar but did not recover their mechanical properties after shearing like other samples, even after 60 min. All of the other microtubule samples were more rigid after flow orientation, suggesting that the mechanical properties of anisotropic arrays of microtubules may be substantially greater than those of randomly arranged microtubules. These experiments confirm that MAPs do not cross link microtubules. Surprisingly, under conditions where microtubule assembly is strongly inhibited (either 5 degrees or at 37 degrees C with colchicine or Ca++) tubulin was mechanically indistinguishable from microtubules at 10-20 microM concentration. By electron microscopy and ultracentrifugation these samples were devoid of microtubules or other obvious structures. However, these mechanical data are strong evidence that tubulin will spontaneously assemble into alternate structures (aggregates) in nonpolymerizing conditions. Because unpolymerized tubulin is found in significant quantities in the cytoplasm, it may contribute significantly to the viscoelastic properties of cytoplasm, especially at low deformation rates.