E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G 2 -M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/ anaphase checkpoint. [Mol Cancer Ther 2005;4(7): 1086 -95]

The growing and shortening dynamics of individual bovine brain microtubules at their plus ends at steady state in vitro, assembled from isotypically pure a4u, abim, or acsv tubulin dimers, were determined by differential interference contrast video microscopy. Microtubules assembled from the purified afm isotype were considerably more dynamic than microtubules made from the apu or a4iv isotypes or from unfractionated phosphocellulose-purified tubulin. Furthermore, increasing the proportion of the aoil isotype in a mixture of the alI and atom isotypes suppressed microtubule dynamics, demonstrating that microtubule dynamics can be influenced by the tubulin isotype composition.The data support the hypothesis that cells might determine the dynamic properties and functions of its microtubules in part by altering the relative amounts of the different tubulin isotypes.Microtubules (1) are required for structural organization and for many kinds of movements within the eukaryotic cell cytoplasm. They are especially prominent in the central nervous system where they appear to be essential for the organization and function of axonal and dendritic processes of neurons. Although it is unclear how microtubule functions in cells are controlled, a large body of evidence indicates that the dynamic properties of the microtubules play an important role (for reviews, see refs. 2 and 3). For example, microtubule dynamics increase dramatically when cells progress from interphase to mitosis (4-6), and the rapid dynamics of spindle microtubules appear to be important for establishing spindle microtubule organization, for facilitating the linkage of chromosomes to the spindles, and for chromosome movement (2-7).Little is known about how microtubule dynamics are regulated in cells. In vitro and in cells, microtubule ends switch between states of growing and shortening, a process known as "dynamic instability" (8-13), apparently due to the gain and loss ofa stabilizing GTP-or GDP-P1-liganded tubulin cap at the microtubule ends. Also, in vitro and in cells, net growing of microtubules can occur at one microtubule end and net shortening can occur at the opposite end, a process termed "treadmilling" or "flux" (9, 14-16). Both dynamic instability and treadmilling are logical targets for control. Biochemical studies in vitro have indicated that microtubule dynamics in cells could be regulated at several levels. One possibility is that control of microtubule dynamics involves regulation of the gain and loss of the stabilizing cap. A second possibility is that control of microtubule dynamics occurs through interactions of microtubule-associated proteins (MAPs) with microtubule surfaces and ends.Another possible mechanism for control of microtubule polymerization dynamics could involve the isotypic composition of the tubulin itself. Tubulin is composed oftwo 50-kDa polypeptide subunits, a and (3, which exist in multiple forms called isotypes. For example, mammalian brain tubulin consists of at least five a-and five j-tubulin isotypes (17,...

Tau is a neuronal microtubule-associated protein that promotes microtubule assembly, stability, and bundling in axons. Two distinct regions of tau are important for the tau-microtubule interaction, a relatively well-characterized "repeat region" in the carboxyl terminus (containing either three or four imperfect 18-amino acid repeats separated by 13- or 14-amino acid long inter-repeats) and a more centrally located, relatively poorly characterized proline-rich region. By using amino-terminal truncation analyses of tau, we have localized the microtubule binding activity of the proline-rich region to Lys215-Asn246 and identified a small sequence within this region, 215KKVAVVR221, that exerts a strong influence on microtubule binding and assembly in both three- and four-repeat tau isoforms. Site-directed mutagenesis experiments indicate that these capabilities are derived largely from Lys215/Lys216 and Arg221. In marked contrast to synthetic peptides corresponding to the repeat region, peptides corresponding to Lys215-Asn246 and Lys215-Thr222 alone possess little or no ability to promote microtubule assembly, and the peptide Lys215-Thr222 does not effectively suppress in vitro microtubule dynamics. However, combining the proline-rich region sequences (Lys215-Asn246) with their adjacent repeat region sequences within a single peptide (Lys215-Lys272) enhances microtubule assembly by 10-fold, suggesting intramolecular interactions between the proline-rich and repeat regions. Structural complexity in this region of tau also is suggested by sequential amino-terminal deletions through the proline-rich and repeat regions, which reveal an unusual pattern of loss and gain of function. Thus, these data lead to a model in which efficient microtubule binding and assembly activities by tau require intramolecular interactions between its repeat and proline-rich regions. This model, invoking structural complexity for the microtubule-bound conformation of tau, is fundamentally different from previous models of tau structure and function, which viewed tau as a simple linear array of independently acting tubulin-binding sites.

Cellular factors tightly regulate the architecture of bundles of filamentous cytoskeletal proteins, giving rise to assemblies with distinct morphologies and physical properties, and a similar control of the supramolecular organization of nanotubes and nanorods in synthetic materials is highly desirable. However, it is unknown what principles determine how macromolecular interactions lead to assemblies with defined morphologies. In particular, electrostatic interactions between highly charged polyelectrolytes, which are ubiquitous in biological and synthetic self-assembled structures, are poorly understood. We have used a model system consisting of microtubules (MTs) and multivalent cations to examine how microscopic interactions can give rise to distinct bundle phases in biological polyelectrolytes. The structure of these supramolecular assemblies was elucidated on length scales from subnanometer to micrometer with synchrotron x-ray diffraction, transmission electron microscopy, and differential interference contrast microscopy. Tightly packed hexagonal bundles with controllable diameters were observed for large trivalent, tetravalent, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of 2D dynamic assemblies of MTs with linear, branched, and loop topologies. This new bundle phase is an experimental example of nematic membranes. The morphologically distinct MT assemblies give insight into general features of bundle formation and may be used as templates for miniaturized materials with applications in nanotechnology and biotechnology.cation ͉ like-charge attraction ͉ x-ray I n this article, we present our findings on the assembly behavior of microtubules (MTs), a model nanoscale tubule. MTs are hollow, cylindrical protein polymers, with inner and outer diameters of Ϸ15 and 25 nm, respectively, involved in a variety of cellular functions, including cell division, intracellular transport, and cell morphology. MTs often assemble into arrays and bundles as in axostyles in protozoa, the cortical array in plants, the mitotic spindle, and neuronal processes (1). MT-associated proteins (MAPs) regulate the interactions between MTs, giving rise to MT bundles with various degrees of order and different MT-MT spacings, both in native biological structures such as axons and dentrites and in in vivo overexpression experiments (2). In vitro experiments show that subtle mutations in MAPs can lead to bundles with radically different structures, converting hexagonally packed bundles into linear chains of MTs (3). However, it is unclear how MAP-controlled, MT-MT interactions lead to bundles with the observed architectures.Understanding the fundamental mechanisms underlying the nature of the self-assembly of nanometer-scale tubules and rods is also important from a technological perspective. Nanotubes are currently being developed as miniaturized materials with applications as circuitry components, templates for nanosized wires and optic...

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Intermolecular interactions between charged membranes and biological polyelectrolytes, tuned by physical parameters, which include the membrane charge density and bending rigidity, the membrane spontaneous curvature, the biopolymer curvature, and the overall charge of the complex, lead to distinct structures and morphologies. The self-assembly of cationic liposome-microtubule (MT) complexes was studied, using synchrotron x-ray scattering and electron microscopy. Vesicles were found to either adsorb onto MTs, forming a ''beads on a rod'' structure, or undergo a wetting transition and coating the MT. Tubulin oligomers then coat the external lipid layer, forming a tunable lipid-protein nanotube. The beads on a rod structure is a kinetically trapped state. The energy barrier between the states depends on the membrane bending rigidity and charge density. By controlling the cationic lipid͞tubulin stoichiometry it is possible to switch between two states of nanotubes with either open ends or closed ends with lipid caps, a process that forms the basis for controlled chemical and drug encapsulation and release.polyelectrolyte lipid complexes ͉ small angle x-ray scattering ͉ nanotubebased drug delivery ͉ membrane ͉ tubulin

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.

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 ϳ13-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 ϳ14.7 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.