Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules

Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.     

Microtubule structure at improved resolution.

Microtubule architecture can vary with eukaryotic species, with different cell types, and with the presence of stabilizing agents. For in vitro assembled microtubules, the average number of protofilaments is reduced by the presence of sarcodictyin A, epothilone B, and eleutherobin (similarly to taxol) but increased by taxotere. Assembly with a slowly hydrolyzable GTP analogue GMPCPP is known to give 96% 14 protofilament microtubules. We have used electron cryomicroscopy and helical reconstruction techniques to obtain three-dimensional maps of taxotere and GMPCPP microtubules incorporating data to 14 A resolution. The dimer packing within the microtubule wall is examined by docking the tubulin crystal structure into these improved microtubule maps. The docked tubulin and simulated images calculated from "atomic resolution" microtubule models show tubulin heterodimers are aligned head to tail along the protofilaments with the beta subunit capping the microtubule plus end. The relative positions of tubulin dimers in neighboring protofilaments are the same for both types of microtubule, confirming that conserved lateral interactions between tubulin subunits are responsible for the surface lattice accommodation observed for different microtubule architectures. Microtubules with unconventional protofilament numbers that exist in vivo are likely to have the same surface lattice organizations found in vitro. A curved "GDP" tubulin conformation induced by stathmin-like proteins appears to weaken lateral contacts between tubulin subunits and could block microtubule assembly or favor disassembly. We conclude that lateral contacts between tubulin subunits in neighboring protofilaments have a decisive role for microtubule stability, rigidity, and architecture.     

Microtubules and tubulin sheet polymers elongate from isolated axonemes in vitro as observed by negative-stain electron microscopy

Microtubules are an important cytoskeletal component involved in cell motility and morphogenesis. They are unique polymers because they are highly dynamic in vivo and in vitro, displaying spontaneous transitions between phases of elongation and rapid shortening. This property has been termed microtubule dynamic instability. Here we describe the application of negative-stain electron microscopy to examine the morphology of microtubules. The purpose was to provide insight into the structural basis of dynamic instability. Highly purified porcine brain tubulin was seeded from isolated axoneme seeds and the morphologies of the tubulin polymers were examined. As previously reported, tubulin polymer sheets in addition to intact MTs were observed during elongation. Cross-sections of identical preparations displayed intact circles (MTs) and c-shaped polymers. The fraction of sheets (28%) observed by negative staining was identical to the fraction of c-shaped polymers in cross-sections. These results suggest that tubulin polymer elongation is not strictly helical due to the lack of helical symmetry of tubulin sheets. Finally, we document the novel effect of glutaraldehyde fixation on MTs. Fixation of MTs in 1% glutaraldehyde for longer than 2 min severely disrupted MT protofilament structure and induced MT curvature at a gross level. Even at 1 min, thin, thread-like structures were observed that were only present in glutaraldehyde-fixed samples. It is therefore an advantage to minimize the extent of glutaraldehyde fixation.     

Giardia lamblia EB1 is a functional homolog of yeast Bim1p that binds to microtubules

Giardia lamblia, with two nuclei and a distinct polarized morphology, is an interesting organism for investigating how distribution of its microtubule (MT) is controlled during its cell cycle. In this study, we identified the end-binding protein 1 (EB1) of G. lamblia, a well-known microtubule-associated protein that organizes MTs in eukaryotes. Immunofluorescence assays using recombinant EB1 (rEB1)-specific antibodies demonstrated EB1 localization in nuclear membrane as well as in some cytoskeletal structures such as axomenes and median bodies of trophozoites of G. lamblia. Complementation experiments using the BIM1 knock-out mutant of yeast, the yeast homolog of mammalian EB1, showed that giardial EB1 was able to carry out a homologous function in controlling MT dynamics. In addition, rEB1 of G. lamblia co-precipitated with MTs by an in vitro binding assay, thereby demonstrating that G. lamblia EB1 is a MT-associated protein. These results, taken together, suggest that G. lamblia EB1 is a functional homolog of eukaryotic EB1 and is likely to be a determinant for MT distribution.     

New insights into microtubule structure and function from the atomic model of tubulin

The structure of tubulin has recently been solved by electron crystallography of zinc-induced tubulin sheets. Because tubulin was studied in a polymerized state, the model contains information on the interactions between monomers that give rise to the αβ dimer as well as contacts between adjacent dimers that result in the structure of the protofilament. The model includes the binding site of taxol, an anti-cancer agent that acts by stabilizing microtubules. The present tubulin model gives the first structural framework for understanding microtubule polymerization and its regulation by nucleotides and anti-mitotic drugs at the molecular level. Received: 15 December 1997 / Revised version: 25 January 1998 / Accepted: 2 February 1998     

Adenomatous polyposis coli on microtubule plus ends in cell extensions can promote microtubule net growth with or without EB1

In interphase cells, the adenomatous polyposis coli (APC) protein accumulates on a small subset of microtubules (MTs) in cell protrusions, suggesting that APC may regulate the dynamics of these MTs. We comicroinjected a nonperturbing fluorescently labeled monoclonal antibody and labeled tubulin to simultaneously visualize dynamics of endogenous APC and MTs in living cells. MTs decorated with APC spent more time growing and had a decreased catastrophe frequency compared with non-APC-decorated MTs. Endogenous APC associated briefly with shortening MTs. To determine the relationship between APC and its binding partner EB1, we monitored EB1-green fluorescent protein and endogenous APC concomitantly in living cells. Only a small fraction of EB1 colocalized with APC at any one time. APC-deficient cells and EB1 small interfering RNA showed that EB1 and APC localized at MT ends independently. Depletion of EB1 did not change the growth-stabilizing effects of APC on MT plus ends. In addition, APC remained bound to MTs stabilized with low nocodazole, whereas EB1 did not. Thus, we demonstrate that the association of endogenous APC with MT ends correlates directly with their increased growth stability, that this can occur independently of its association with EB1, and that APC and EB1 can associate with MT plus ends by distinct mechanisms.     

Structural rearrangements in tubulin following microtubule formation

Microtubules are essential cytoskeletal structures that mediate several dynamic processes in a cell. To shed light on the structural processes relating to microtubule formation and dynamic instability, we investigated microtubules composed of 15 protofilaments using cryo-electron microscopy, helical image reconstruction and computational modelling. Analysis of the configuration of the alpha beta-tubulin heterodimer shows distinct structural differences in both subunits, and illustrates that the tubulin subunits have different roles in the microtubule lattice. Our modelling data suggest that after GTP hydrolysis microtubules, adopt a conformational state somewhere between a straight protofilament conformation--as found in zinc-induced tubulin sheets--and an outward curved conformation--as found in tubulin-stathmin complexes. The tendency towards a curved conformation seems to be mediated mostly by beta-tubulin, whereas alpha-tubulin resembles a state more related to the straight structure. Our data suggest a possible explanation of dynamic instability of microtubules, and for nucleotide-sensitive microtubule-binding properties of microtubule-associated proteins and molecular motors.     

Structural basis of microtubule plus end tracking by XMAP215, CLIP-170, and EB1

Microtubule plus end binding proteins (+TIPs) localize to the dynamic plus ends of microtubules, where they stimulate microtubule growth and recruit signaling molecules. Three main +TIP classes have been identified (XMAP215, EB1, and CLIP-170), but whether they act upon microtubule plus ends through a similar mechanism has not been resolved. Here, we report crystal structures of the tubulin binding domains of XMAP215 (yeast Stu2p and Drosophila Msps), EB1 (yeast Bim1p and human EB1), and CLIP-170 (human), which reveal diverse tubulin binding interfaces. Functional studies, however, reveal a common property that native or artificial dimerization of tubulin binding domains (including chemically induced heterodimers of EB1 and CLIP-170) induces tubulin nucleation/assembly in vitro and, in most cases, plus end tracking in living cells. We propose that +TIPs, although diverse in structure, share a common property of multimerizing tubulin, thus acting as polymerization chaperones that aid in subunit addition to the microtubule plus end.     

The C-termini of tubulin and the specific geometry of tubulin substrates influence the depolymerization activity of MCAK

MCAK is a Kinesin-13 that depolymerizes microtubules (MTs) and regulates MT dynamics. We used subtilisin-treated MTs (MTs lacking the C-termini of α- and β-tubulin) and alternative tubulin substrates to study which structural and geometrical features of the MT are critical for MCAK activity. We found that removal of the C-termini significantly decreased the efficiency of MCAK-induced depolymerization, which was not due to a reduction of end-specific binding. We also found that depolymerization of SMTs led to an increase in the stabilization of curved oligomeric tubulin products. Using alternative tubulin substrates with different geometries, we found that MCAK depolymerized parallel and anti-parallel tubulin sheets. However, MCAK did not depolymerize tubulin rings regardless of the presence or absence of the tubulin C-termini. We propose that localization of MCAK to the ends of MTs is independent of tubulin C-termini, that MCAK stabilizes a curved conformation at the end of the MT, and that efficient release of this complex is dependent on the presence of the C-termini of tubulin.αβ     

Effect of microtubule-associated proteins on the protofilament number of microtubules assembled in vitro

In order to demonstrate the effect of microtubule-associated proteins on the protofilament number of microtubules, we used different systems of microtubule formation in vitro in which these proteins are either functionally eliminated (by DNA or glycerol) or absent (purified tubulin). The results obtained by electron microscopy of ultrathin-sectioned material indicate that under standard conditions in the presence of microtubule-associated proteins microtubules are formed consisting predominantly of 14 protofilaments. In cases of deficiency of microtubule-associated proteins, the mean value of the protofilament number is lower, and the protofilament number within the microtubule population varies remarkably. On the other hand, the action of microtubule-associated proteins is enhanced by histones resulting in increased protofilament numbers. A model is proposed illustrating that the quality and the quantity of microtubule-associated proteins bound to microtubules determine the curvature between the protofilaments and restrict the variety of their binding angles. In this way the microtubule-associated proteins may be regarded as an important factor in determining the structural fidelity of microtubules.     

Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin

Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215^msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215^msps polymerase and the EB1–Sentin duo.     

Assembly and three-dimensional image reconstruction of tubulin hoops

The three-dimensional structure of tubulin hoops has been determined by image reconstruction. The surface lattice of hoops is similar to that of microtubules, but in addition hoops possess a superstructure of protofilament triplets. The protofilaments differ mainly in their apparent volumes and lateral spacings. The volumes depend strongly on the orientation on the carbon support, while the spacings do not. The differences of appearance do not reflect changes of intrinsic subunit structure. They are explained by differential staining related to the orientation and packing of protofilament. Microtubule-associated proteins do not contribute to the average subunit structure. All apparent protofilament structures differ from that expected from X-ray patterns of microtubules in terms of subunit tilt and distribution of contrast. It is concluded that the negatively stained structure is a reliable representation of the arrangement of protein subunits, but not of their shape. Tubulin hoops occur in conditions of microtubule assembly near the critical concentration in a stabilizing buffer. Their formation depends on microtubule-associated proteins and on the initial presence of tubulin oligomers, which may associate into short protofilament triplets. If their elongation is rapid compared to lateral aggregation, they form closed hoops. The growth phase is followed by a redistribution phase, during which hoops disappear in favour of microtubules. This behaviour is explained by kinetic overshoot assembly. Each triplet resembles an incomplete microtubule wall so that the junction between two triplets may be compared to a junction between microtubule walls. Such junctions are formed by a closely spaced pair of protofilaments. They are analogous to junctions between microtubules and incomplete microtubule walls, and they have the same clockwise curvature when viewed at the growing end.     

EB1 Recognizes the Nucleotide State of Tubulin in the Microtubule Lattice

Plus-end-tracking proteins (+TIPs) are localized at the fast-growing, or plus end, of microtubules, and link microtubule ends to cellular structures. One of the best studied +TIPs is EB1, which forms comet-like structures at the tips of growing microtubules. The molecular mechanisms by which EB1 recognizes and tracks growing microtubule ends are largely unknown. However, one clue is that EB1 can bind directly to a microtubule end in the absence of other proteins. Here we use an in vitro assay for dynamic microtubule growth with two-color total-internal-reflection-fluorescence imaging to investigate binding of mammalian EB1 to both stabilized and dynamic microtubules. We find that under conditions of microtubule growth, EB1 not only tip tracks, as previously shown, but also preferentially recognizes the GMPCPP microtubule lattice as opposed to the GDP lattice. The interaction of EB1 with the GMPCPP microtubule lattice depends on the E-hook of tubulin, as well as the amount of salt in solution. The ability to distinguish different nucleotide states of tubulin in microtubule lattice may contribute to the end-tracking mechanism of EB1.     

Microtubule Assembly from Single Flared Protofilaments—Forget the Cozy Corner?

A paradigm shift for models of MT assembly is suggested by a recent cryo-electron microscopy study of microtubules (MTs). Previous assembly models have been based on the two-dimensional lattice of the MT wall, where incoming subunits can add with longitudinal and lateral bonds. The new study of McIntosh et al. concludes that the growing ends of MTs separate into flared single protofilaments. This means that incoming subunits must add onto the end of single protofilaments, forming only a longitudinal bond. How can growth of single-stranded protofilaments exhibit cooperative assembly with a critical concentration? An answer is suggested by FtsZ, the bacterial tubulin homolog, which assembles into single-stranded protofilaments. Cooperative assembly of FtsZ is thought to be based on conformational changes that switch the longitudinal bond from low to high affinity when the subunit is incorporated in a protofilament. This novel mechanism may also apply to tubulin assembly and could be the primary mechanism for assembly onto single flared protofilaments.     

Formation of double-walled microtubules and multilayered tubulin sheets by basic proteins

Some basic proteins enable microtubule protein to form special assembly products in vitro, known as double-walled microtubules. Using histones (H1, core histones) as well as the human encephalitogenic protein to induce the formation of double-walled microtubules, we made the following electron microscopic observations: (1) Double-walled microtubules consist of an "inner" microtubule which is covered by electron-dense material, apparently formed from the basic protein, and by a second tubulin wall. (2) The tubulin of the second wall seems to be arranged as protofilaments, surrounding the inner microtubule in a helical or ring-like manner. (3) The surface of double-walled microtubules lacks the projections of microtubule-associated proteins, usually found on microtubules. (4) In the case of protofilament ribbons (incomplete microtubules), H1 binds exclusively to their convex sides that correspond to the surface of microtubules. Zn2+-induced tubulin sheets, consisting in contrast to microtubules of alternately arranged protofilaments, are covered by H1 on both surfaces. Furthermore, multilayered sheet aggregates appeared. The results indicate that the basic proteins used interact only with that protofilament side which represents the microtubule surface. In accordance with this general principle, models on the structure of double-walled microtubules and multilayered tubulin sheets were derived.     

Reversible unfolding of FtsZ cell division proteins from archaea and bacteria. Comparison with eukaryotic tubulin folding and assembly

The stability, refolding, and assembly properties of FtsZ cell division proteins from Methanococcus jannaschii and Escherichia coli have been investigated. Their guanidinium chloride unfolding has been studied by circular dichroism spectroscopy. FtsZ from E. coli and tubulin released the bound guanine nucleotide, coinciding with an initial unfolding stage at low denaturant concentrations, followed by unfolding of the apoprotein. FtsZ from M. jannaschii released its nucleotide without any detectable secondary structural change. It unfolded in an apparently two-state transition at larger denaturant concentrations. Isolated FtsZ polypeptide chains were capable of spontaneous refolding and GTP-dependent assembly. The homologous eukaryotic tubulin monomers misfold in solution, but fold within the cytosolic chaperonin CCT. Analysis of the extensive tubulin loop insertions in the FtsZ/tubulin common core and of the intermolecular contacts in model microtubules and tubulin-CCT complexes shows a loop insertion present at every element of lateral protofilament contact and at every contact of tubulin with CCT (except at loop T7). The polymers formed by purified FtsZ have a distinct limited protofilament association in comparison with microtubules. We propose that the loop insertions of tubulin and its CCT-assisted folding coevolved with the lateral association interfaces responsible for extended two-dimensional polymerization into microtubule polymers.     

Effects of tubulin acetylation and tubulin acetyltransferase binding on microtubule structure

Tubulin undergoes posttranslational modifications proposed to specify microtubule subpopulations for particular functions. Most of these modifications occur on the C-termini of tubulin and may directly affect the binding of microtubule-associated proteins (MAPs) or motors. Acetylation of Lys-40 on α-tubulin is unique in that it is located on the luminal surface of microtubules, away from the interaction sites of most MAPs and motors. We investigate whether acetylation alters the architecture of microtubules or the conformation of tubulin, using cryo–electron microscopy (cryo-EM). No significant changes are observed based on protofilament distributions or microtubule helical lattice parameters. Furthermore, no clear differences in tubulin structure are detected between cryo-EM reconstructions of maximally deacetylated or acetylated microtubules. Our results indicate that the effect of acetylation must be highly localized and affect interaction with proteins that bind directly to the lumen of the microtubule. We also investigate the interaction of the tubulin acetyltransferase, αTAT1, with microtubules and find that αTAT1 is able to interact with the outside of the microtubule, at least partly through the tubulin C-termini. Binding to the outside surface of the microtubule could facilitate access of αTAT1 to its luminal site of action if microtubules undergo lateral opening between protofilaments.     

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.     

Template-free 13-protofilament microtubule-MAP assembly visualized at 8 A resolution

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.     

Cryo-EM Structure (4.5-Å) of Yeast Kinesin-5–Microtubule Complex Reveals a Distinct Binding Footprint and Mechanism of Drug Resistance

Kinesin-5s are microtubule-dependent motors that drive spindle pole separation during mitosis. We used cryo-electron microscopy to determine the 4.5-Å resolution structure of the motor domain of the fission yeast kinesin-5 Cut7 bound to fission yeast microtubules and explored the topology of the motor–microtubule interface and the susceptibility of the complex to drug binding. Despite their non-canonical architecture and mechanochemistry, Schizosaccharomyces pombe microtubules were stabilized by epothilone at the taxane binding pocket. The overall Cut7 footprint on the S. pombe microtubule surface is altered compared to mammalian tubulin microtubules because of their different polymer architectures. However, the core motor–microtubule interaction is tightly conserved, reflected in similar Cut7 ATPase activities on each microtubule type. AMPPNP-bound Cut7 adopts a kinesin-conserved ATP-like conformation including cover neck bundle formation. However, the Cut7 ATPase is not blocked by a mammalian-specific kinesin-5 inhibitor, consistent with the non-conserved sequence and structure of its loop5 insertion.