The microtubule-destabilizing kinesin XKCM1 is required for chromosome positioning during spindle assembly

Xenopus kinesin catastrophe modulator-1 (XKCM1) is a Kin I kinesin family member that uses the energy of ATP hydrolysis to depolymerize microtubules. We demonstrated previously that XKCM1 is essential for mitotic-spindle assembly in vitro and acts by regulating microtubule dynamics as a pure protein, in extracts and in cells. A portion of the XKCM1 pool is specifically localized to centromeres during mitosis and may be important in chromosome movement. To selectively analyze the function of centromere-bound XKCM1, we generated glutathione-S-transferase (GST) fusion proteins containing the N-terminal globular domain (GST-NT), the centrally located catalytic domain (GST-CD), and the C-terminal alpha-helical tail (GST-CT) of XKCM1. The GST-NT protein targeted to centromeres during spindle assembly, suggesting that the N-terminal domain of XKCM1 is sufficient for centromere localization. Addition of GST-NT prior to or after spindle assembly replaced endogenous XKCM1, indicating that centromere targeting is a dynamic process. Loss of endogenous XKCM1 from centromeres caused a misalignment of chromosomes on the metaphase plate without affecting global spindle structure. These results suggest that centromere bound XKCM1 has an important role in chromosome positioning on the spindle.     

The Dam1 ring binds to the E-hook of tubulin and diffuses along the microtubule

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.     

A comparison of the ability of XMAP215 and tau to inhibit the microtubule destabilizing activity of XKCM1

During mitosis, microtubules not only grow fast, but also have a high rate of catastrophe. This is achieved in part by the activity of the MAP, XMAP215, which can stimulate the growth rate of microtubules without fully inhibiting the function of the catastrophe-kinesin XKCM1. We do not know whether this activity is particular to XMAP215, or is a general property of all MAPs. Here, we compare the activities of XMAP215 with the neuronal MAP tau, in opposing the destabilizing activity of the non-conventional kinesin XKCM1. We show that tau is a much more potent inhibitor of XKCM1 than XMAP215. Because tau completely suppresses XKCM1 activity, even at low concentrations, the combination of tau and XKCM1 is unable to generate mitotic microtubule dynamics.     

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.     

In vitro depolymerization dynamics of brain endogenous microtubules

A subcellular fraction containing fragments of endogenous microtubules stabilized in 50% glycerol was separated by diferential centrifugation of rat brain homogenates. The pellets were suspended in glycerol-deficient media, and microtubule depolymerization was monitored by measuring the decrease of sedimentable tubulin. Concomitantly, the number and size of microtubules in the suspensions were followed via electron microscopy. Depolymerization was accompanied by a proportional decrease in the number of microtubules, whereas the average size did not change significantly. After approximately 20 min, a subpopulation of microtubules became stable and did not suffer further depolymerization. These results indicate that upon dilution some microtubules completely depolymerize, whereas others remain stable in the glycerol-deficient medium. The degree of depolymerization depended on both the volume of the resuspension media and on the final glycerol concentration. The results suggest that the depolymerization of the remaining microtubules is prevented by stabilizing factors released from depolymerizing microtubules. Tubulin dimers are not one of these factors, since depolymerization was not altered by the addition of colchicine or by changing the concentration of free tubulin in the medium.     

Bundling of microtubules by synapsin 1. Characterization of bundling and interaction of distinct sites in synapsin 1 head and tail domains with different sites in tubulin.

Synapsin 1 is a nerve terminal phosphoprotein whose role seems to encompass the linking of small synaptic vesicles to the cytoskeleton. Synapsin 1 can join small synaptic vesicles to neuronal spectrin, microfilaments and microtubules; it can also bundle microtubules and microfilaments. In this paper, the mode of interaction between synapsin 1 and microtubules has been investigated. Bundling is shown to be highly cooperative: the apparent Hill coefficient is 3.06 +/- 0.3, and bundling is half-maximal at 0.63 +/- 0.02 microM. Bundling occurs either when whole synapsin 1 preparations (containing monomers and oligomers) or when monomeric synapsin 1 is added to microtubules. However, it is not clear that synapsin 1 remains monomeric in the presence of microtubules. Synapsin 1-microtubule mixtures contain two types of filament. One type is characterised by microtubules often with synapsin 1 bound to their surface. The other type is composed of filaments of diameter 15 +/- 5 nm. This filament type is granular and made up in part of 14-nm-diameter particles. These dimensions are consistent with their being made up of polymerised synapsin 1. It is possible that microtubules induce the polymerisation of synapsin 1. Synapsin 1 had independent tubulin binding sites in the N-terminal head domain and in the C-terminal tail domain. Whole synapsin 1 can interact with tubulin after it has been digested to remove the tubulin C terminus (des-C-terminal tubulin). The interaction of des-C-terminal tubulin with synapsin 1 appears to be via the head domain, since 125I-des-C-terminal tubulin only shows specific binding to the head domain on gel blots. By contrast intact tubulin binds to both head and tail domains. Binding to the tail domain can be inhibited by a synthetic peptide representing the microtubule-associated protein 2 (MAP2) binding site of class II beta tubulin. These results suggest a model for microtubule bundling by synapsin 1 in which independent sites in the head and tail domains of synapsin 1 cross-link microtubules by interactions with two distinct sites in tubulin.     

A microtubule-binding Rho-GEF controls cell morphology during convergent extension of Xenopus laevis

During Xenopus development, convergent extension movements mediated by cell intercalation drive axial elongation. While many genes required for convergent extension have been identified, little is known of regulation of the cytoskeleton during these cell movements. Although microtubules are required for convergent extension, this applies only to initial stages of gastrulation, between stages 10 and 10.5. To examine the cytoskeleton more directly during convergent extension, we visualized actin and microtubules simultaneously in live explants using spinning disk confocal fluorescence microscopy. Microtubule depolymerization by nocodazole inhibits lamellipodial protrusions and cell-cell contact, thereby inhibiting convergent extension. However, neither taxol nor vinblastine, both of which block microtubule dynamics while stabilizing a polymer form of tubulin, inhibits lamellipodia or convergent extension. This suggests an unusual explanation: the mass of polymerized tubulin, not dynamics of the microtubule cytoskeleton, is crucial for convergent extension. Because microtubule depolymerization elicits striking effects on actin-based protrusions, the role of Rho-family GTPases was tested. The effects of nocodazole are partially rescued using dominant negative Rho, Rho-kinase inhibitor, or constitutively active Rac, suggesting that microtubules regulate small GTPases, possibly via a guanine-nucleotide exchange factor. We cloned full-length XLfc, a microtubule-binding Rho-GEF. Nucleotide exchange activity of XLfc is required for nocodazole-mediated inhibition of convergent extension; constitutively active XLfc recapitulates the effects of microtubule depolymerization. Morpholino knockdown of XLfc abrogates the ability of nocodazole to inhibit convergent extension. Therefore, we believe that XLfc is a crucial regulator of cell morphology during convergent extension, and microtubules limit its activity through binding to the lattice.     

Microtubule dynamics and tubulin interacting proteins

Microtubule dynamics are crucial in generation of the mitotic spindle. During the transition from interphase to mitosis, there is an increase in the frequency of microtubule catastrophes. Recent work has identified two proteins, Op 18/stathmin and XKCM1, which can promote microtubule catastrophes in vitro and in cells or extracts. Although both of these proteins share the ability to bind tubulin dimers, their mechanisms of action in destabilizing microtubules are distinct.     

Recognition of C-terminal amino acids in tubulin by pore loops in Spastin is important for microtubule severing

Spastin, an AAA ATPase mutated in the neurodegenerative disease hereditary spastic paraplegia, severs microtubules. Many other AAA proteins form ring-shaped hexamers and contain pore loops, which project into the ring's central cavity and act as ratchets that pull on target proteins, leading, in some cases, to conformational changes. We show that Spastin assembles into a hexamer and that loops within the central pore recognize C-terminal amino acids of tubulin. Key pore loop amino acids are required for severing, including one altered by a disease-associated mutation. We also show that Spastin contains a second microtubule binding domain that makes a distinct interaction with microtubules and is required for severing. Given that Spastin engages the MT in two places and that both interactions are required for severing, we propose that severing occurs by forces exerted on the C-terminal tail of tubulin, which results in a conformational change in tubulin, which releases it from the polymer.     

Identification of a distinct class of vinblastine binding sites on microtubules

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.     

The acetylation of alpha-tubulin and its relationship to the assembly and disassembly of microtubules

A tight association between Chlamydomonas alpha-tubulin acetyltransferase (TAT) and flagellar axonemes, and the cytoplasmic localization of both tubulin deacetylase (TDA) and an inhibitor of tubulin acetylation have been demonstrated by the use of calf brain tubulin as substrate for these enzymes. A major axonemal TAT of 130 kD has been solubilized by high salt treatment, purified, and characterized. Using the Chlamydomonas TAT with brain tubulin as substrate, we have studied the effects of acetylation on the assembly and disassembly of microtubules in vitro. We also determined the relative rates of acetylation of tubulin dimers and polymers. The acetylation does not significantly affect the temperature-dependent polymerization or depolymerization of tubulin in vitro. Furthermore, polymerization of tubulin is not a prerequisite for the acetylation, although the polymer is a better substrate for TAT than the dimer. The acetylation is sensitive to calcium ions which completely inhibit the acetylation of both dimers and polymers of tubulin. Acetylation of the dimer is not inhibited by colchicine; the effect of colchicine on acetylation of the polymer can be explained by its depolymerizing effect on the polymer.     

Possible treadmilling in zinc(II)-induced sheet polymers of bovine brain microtubule protein

We have examined the ability of zinc(II)-induced sheet polymers, formed from thrice-cycled bovine brain microtubule protein prepared in the absence of glycerol, to exchange with tubulin subunits at steady state. By a rapid filtration assay in which labeled GTP was used as a marker for tubulin addition and loss, we found that steady-state sheet polymers, formed in 0.5 mM-ZnCl2, 1 mM-dithiothreitol, and 100 mM-2-(N-morpholino)ethanesulfonic acid (pH 6.75) in the presence of a GTP-regenerating system at 37 degrees C, incorporated the label in a time-dependent manner to a maximum level. The steady-state uptake of label was inhibited by colchicine, podophyllotoxin and vinblastine. In pulse-chase experiments, we observed that label added onto sheet polymers in a short pulse was retained for a period equal to that required by the polymers to become fully labeled in a continuous pulse; thereafter, the label was lost gradually to a baseline level. An average of 82% of the label was retained in the sheet polymers after a "cold" chase of equal duration to the time of the pulse. Sheet polymers assembled from microtubule protein prepared in the presence of glycerol gave similar results. Using a double-labeling procedure to analyze tubulin addition and loss simultaneously, we found that the rates of steady-state addition and loss were similar. Sheet polymers retained their structural integrity throughout these experiments, as determined by electron microscopy. We believe that the data are consistent with a "treadmilling" mechanism of polymerization and depolymerization, analogous to that documented to occur in steady-state microtubules in vitro. Such a mechanism is discussed in the context of recent findings from structural studies, and a model consistent with established structural data is offered.     

Thermal hysteresis in microtubule assembly/disassembly dynamics: The aging-induced degradation of tubulin dimers

The assembly/disassembly of biological macromolecules plays an important role in their biological functionalities. Although the dynamics of tubulin polymers and their super-assembly into microtubule structures is critical for many cellular processes, details of their cyclical polymerization/depolymerization are not fully understood. Here, we use a specially designed light scattering technique to continuously examine the effects of temperature cycling on the process of microtubule assembly/disassembly. We observe a thermal hysteresis loop during tubulin assembly/disassembly, consistently with earlier reports on the coexistence of tubulin and microtubules as a phase transition. In a cyclical process, the structural hysteresis has a kinetic component that depends on the rate of temperature change but also an intrinsic thermodynamic component that depends on the protein topology, possibly related to irreversible processes. Analyzing the evolution of such thermal hysteresis loops over successive cycles, we found that the assembly/disassembly ceases after some time, which is indicative of protein aging leading to its inability to self-assemble after a finite number of temperature cycles. The emergence of assembly-incompetent tubulin could have major consequences for human pathologies related to microtubules, including aging, neurodegenerative diseases and cancer.     

The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B

The kinetics of microtubule assembly were investigated by monitoring changes in turbidity which result from the scattering of incident light by the polymer. These studies indicated that assembly occurred by a pathway involving a nucleation phase, followed by an elongation phase as evidenced by a lag in the polymerization kinetics, followed by a psuedo-first-order exponential increase in turbidity. Analytical ultracentrifugation of solutions polymerized to equilibrium showed that 6 S tubulin was the only species detectable in equilibrium with microtubules. Investigation of the elongation reaction in mixtures of 6 S tubulin and microtubule fragments demonstrated that: (1) the net rate of assembly was the sum of the rates of polymerization and depolymerization; (2) the rate of polymerization was proportional to the product of the microtubule number concentration and the 6 S tubulin concentration; and (3) the rate of depolymerization was proportional to the number concentration of microtubules. These results demonstrate that microtubule assembly occurs by a condensation polymerization mechanism consisting of distinct nucleation and elongation steps. Microtubules are initiated in a series of protein association reactions in a pathway that has not been fully elucidated. Elongation proceeds by the consecutive association of 6 S tubulin subunits onto the ends of existing microtubules. Similarly, depolymerization occurs by dissociation of 6 S subunits from the ends of microtubules. The rate constants measured for polymerization and depolymerization at 30 °C were 4 × 10⁶m^?1 s^?1 and 7 s^?1, respectively.     

MCAK-independent functions of ch-Tog/XMAP215 in microtubule plus-end dynamics

The formation of a functional bipolar mitotic spindle is essential for genetic integrity. In human cells, the microtubule polymerase XMAP215/ch-Tog ensures spindle bipolarity by counteracting the activity of the microtubule-depolymerizing kinesin XKCM1/MCAK. Their antagonistic effects on microtubule polymerization confer dynamic instability on microtubules assembled in cell-free systems. It is, however, unclear if a similar interplay governs microtubule behavior in mammalian cells in vivo. Using real-time analysis of spindle assembly, we found that ch-Tog is required to produce or maintain long centrosomal microtubules after nuclear-envelope breakdown. In the absence of ch-Tog, microtubule assembly at centrosomes was impaired and microtubules were nondynamic. Interkinetochore distances and the lengths of kinetochore fibers were also reduced in these cells. Codepleting MCAK with ch-Tog improved kinetochore fiber length and interkinetochore separation but, surprisingly, did not rescue centrosomal microtubule assembly and microtubule dynamics. Our data therefore suggest that ch-Tog has at least two distinct roles in spindle formation. First, it protects kinetochore microtubules from depolymerization by MCAK. Second, ch-Tog plays an essential role in centrosomal microtubule assembly, a function independent of MCAK activity. Thus, the notion that the antagonistic activities of MCAK and ch-Tog determine overall microtubule stability is too simplistic to apply to human cells.     

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.     

MAP4 counteracts microtubule catastrophe promotion but not tubulin-sequestering activity in intact cells

Microtubules are polar polymers that continually switch between phases of elongation and shortening, a property referred to as dynamic instability. The ubiquitous microtubule associated protein 4 (MAP4) shows rescue-promoting activity during in vitro assembly of microtubules (i.e., promotes transitions from shortening to elongation), but its regulatory role in intact cells is poorly defined. Here, we demonstrate that ectopic MAP4 promotes outgrowth of extended MTs during beta1-integrin-induced cell spreading. An inducible cotransfection protocol was employed to further analyze the regulatory role of MAP4 in human leukemia cells with microtubules partially destabilized by either ectopic tubulin-sequestering proteins or proteins that promote catastrophes (i.e., transitions from elongation to shortening). Coexpression of proteins that sequester free tubulin heterodimers with different efficiencies was found to abolish microtubule stabilization by MAP4. In contrast, however, the microtubule-stabilizing activity of MAP4 was found to suppress the activities of two distinct and specific catastrophe promoters, namely, XKCM1 and a nonsequestering truncation derivative of Op18/stathmin. These observations reveal specificity in the microtubule-stabilizing activity of MAP4 that differentiates between two mechanistically distinct types of MT destabilization.     

Effects of Taxol on the Development of the Cell Plate and of the Phragmoplast in Tobacco BY-2 Cells

To ascertain whether accumulation of vesicles at the site ofcell-plate formation in the phragmoplast is caused by the translocationof vesicles along phragmoplast microtubules or by the translocationof vesicles that is mediated by depolymerization of phragmoplastmicrotubules at the equatorial plane, we examined the effectsof taxol, an inhibitor of the depolymerization of microtubules,on the accumulation of vesicles at the equatorial region ofthe phragmoplast in tobacco BY-2 cells. Taxol caused an increase in the accumulation of vesicles atthe equatorial plane of the phragmoplast while simultaneouslyinhibiting the centrifugal growth of the phragmoplast. The depolymerization of microtubules does not seem to be involvedin the accumulation of vesicles at the site of cell-plate formation,but this process appears to be required for the centrifugalgrowth of the phragmoplast. Our results suggest that the translocationof vesicles is mediated by some kind of translocator, whichmoves along phragmoplast microtubules, and that the polymerizationof microtubules at the growing edges of the phragmoplast requiresa supply of free tubulin from preexisting microtubules. (Received May 14, 1992; Accepted October 12, 1992)