Tubulin Subunits Research Articles

Chemistry and Biology of Novel Microtubule-Destabilizing Agents that Bind α-Tubulin

Bioactive compounds capable of interacting with tubulins and microtubules represent powerful weapons in cancer chemotherapy. Most of these compounds, including the well-established chemotherapeutic agents colchicine (1), podophyllotoxin (3), vinblastine (4) and vincristine (5) that interfere with the formation and growth of microtubules, or Taxol (11) and the epothilones (12-13), which promote tubulin polymerization and stabilize microtubules, share an interaction with β-tubulin, one of the two subunits of the globular heterodimeric protein tubulin. In contrast, few compounds have been identified as interacting with α-tubulin. A select group of natural compounds are pironetin (18) and the hemiasterlins, and the synthetic compounds cyanoacrylates (21-22), dinitroanilines (23-25) and tetrahydrofuranyl derivatives COBRA-0 (133) and COBRA-1 (26). In addition to their interaction with the α-tubulin subunit, all these compounds share the capacity of destabilizing microtubules. In the present review, we will discuss the most relevant aspects of the biology and chemistry of these compounds, with an emphasis on their antiproliferative properties and potential as anticancer drugs.

TXR1-mediated thrombospondin repression: a novel mechanism of resistance to taxanes?

The usefulness of chemotherapeutic agents for the clinical treatment of cancer relies on their toxic effects on dividing cells. While most anti-cancer drugs directly affect DNA synthesis or integrity, the vinca-alkaloids and taxanes act by disrupting the cellular microtubule network (depicted schematically in Fig. 1; for review, see Zhou and Giannakakou 2005). In addition to providing structural integrity as part of the cytoskeleton, microtubules function as the cell’s freeway system by providing the infrastructure along which cargo can be transported by motor proteins such as kinesin and dynein. During cell division, microtubules are critically important for the formation of the mitotic spindle, which dictates the proper segregation of chromosomes during mitosis. Microtubules are synthesized through the polymerization of and -tubulin subunits on their fast-growing, or plus, ends. At the same time, they are degraded on their slow-growing, or minus, ends by the release of / dimers into the cytosol. The combined processes of assembly and disassembly cause microtubules to be in a state of “dynamic instability,” which allows the growing or shortening of individual microtubules as required. Anti-microtubule agents exert their anti-cancer effect by disrupting normal microtubule dynamics in distinct fashions. Whereas vinca-alkaloids are able to bind to free tubulin, thereby preventing microtubule assembly and concomitant mitotic spindle formation, they cannot associate with polymerized tubulin present in microtubules. In contrast, taxanes interact with polymerized tubulin to cause microtubule stabilization. As a result, cells become overloaded with microtubules and are unable to resolve the mitotic spindle, causing a mitotic arrest or apoptosis. Taxol, the prototype chemotherapeutic of the taxane family, was isolated in the early 1970s from Taxus brevifolia. In recent years, taxol/paclitaxel and taxotere/docetaxel, a semisynthetic taxane identified in the 1980s, have become part of standard drug treatment regimens and are widely used in the clinic to treat a variety of human malignancies, including breast, ovarian, lung, gastric, and prostate cancers. The latter two are of special interest because taxanes may prove to offer some advances in the treatment of these otherwise refractory diseases.

Microtubule dynamic instability

What are microtubules? One of three types of protein polymer that make up the cellular cytoskeleton, an elaborate array of filaments that are used to establish cell shape, facilitate cell motility, organize organelles and assist with cell division. Microtubules are stiff tubes, about 25 nm in diameter. During interphase, they serve as tracks on which organelles and the nucleus are positioned by molecular motor proteins. During mitosis, microtubules form a structure called the mitotic spindle which physically segregates the chromosomes into the two daughter cells. What is microtubule dynamic instability? Microtubules have special dynamic properties. In a population of microtubules, at any point in time, a subset of microtubules are rapidly growing while others are quickly shrinking (although sometimes some microtubules also sit still in a ‘paused’ state). This intriguing property is explained by the observation that individual microtubules switch randomly between growing and shrinking states, sometimes changing back and forth several times in the course of their lifetime. This combination of growth, shrinkage and rapid transitions between the two is known as dynamic instability. Why is dynamic instability useful biologically? Speed. Dynamic instability allows the cell to rapidly reorganize the cytoskeleton when necessary. Dynamic microtubules are individually short-lived, so arrays of microtubules are continuously in the process of re-creation. Because microtubule growth and shrinkage are active processes, consuming energy, this turnover can be fast, on the order of minutes. This means that an array of microtubules can adapt quickly to changes in the environment, adopting new spatial arrangements in response to cellular needs. Examples include the reorganization of the cytoskeleton in the transition to mitosis and the extension of growth cones from neurons. How does dynamic instability work? Microtubules are made from subunits of the protein tubulin which are bound to the energy carrier guanosine triphosphate (GTP). The cell consumes energy to keep the concentration of GTP-tubulin high above the critical concentration for polymerization, far from equilibrium, so that subunits rapidly associate with microtubule ends and the microtubules grow. After subunits are incorporated into a microtubule, their GTP is hydrolyzed into GDP, releasing energy. It is believed that some of this energy goes to deform the tubulin subunit, changing it so it would be most stable in a curved state. This puts pressure on the microtubule to splay apart like a banana peeling. However, the GDP-tubulin is not free to curve outwards while it is trapped in the microtubule lattice — the curving can only begin at the ends. While the ends are stable, a microtubule will grow, but once an end begins to come apart, the splaying propagates down the microtubule (Figure 1). The energy stored in the tubulin subunits is released as the microtubule rapidly shrinks. What makes ends fall apart? We do not know what triggers ends to come apart. One possibility is that a layer of non-hydrolyzed, relatively stable GTP-tubulin remains at the microtubule end. In this case, the microtubule as a whole is stable and continues to grow. If this protective ‘cap’ is lost, the microtubule begins to splay. Alternatively, some other relatively rare ‘tearing’ event might begin microtubule shrinkage. So if dynamic instability consumes energy, can it do work? Yes! Growing microtubules can push against things, producing forces up to 4pN — comparable to the force produced by a kinesin motor, and enough to buckle the microtubules. Also, if the end of a shrinking microtubule is attached to an object, it can pull as it shrinks. In fact, microtubules attached to special sites on chromosomes are thought to use this type of force to pull the chromosomes apart during anaphase. How is dynamic instability regulated? Some microtubule-associated proteins, or MAPs, can attach along the sides of the microtubules, slowing or reversing shrinkage. These MAPs are dominant in interphase cells and neurons, which is why microtubules in these cells are not very dynamic. Other MAPs, dominant in mitosis, attach to microtubule ends, where they can stabilize or destabilize microtubules by changing the frequencies of transitions between growing and shrinking states. The activity of many of these MAPs is highly regulated in time and in space. Why is spatial regulation of dynamic instability important? Dynamic instability allows microtubules to rapidly explore space. If microtubules are preferentially stabilized or destabilized when they reach certain locations, the cell can build microtubule arrays with specific shapes. For example, during mitosis, selective stabilization of microtubules that encounter chromosomes is thought to help form the mitotic spindle.

Microtubules as mechanical force sensors

Microtubules are polymers of tubulin subunits (dimers) arranged on a hexagonal lattice. Each tubulin dimer comprises two monomers, the α-tubulin and β-tubulin, and can be found in two states. In the first state a mobile negative charge is located into the α-tubulin monomer and in the second into the β-tubulin monomer. Each tubulin dimer is modeled as an electrical dipole coupled to its neighbors by electrostatic forces. The location of the mobile charge in each dimer depends on the location of the charges in the dimer's neighborhood. Mechanical forces that act on the microtubule affect the distances between the dimers and alter the electrostatic potential. Changes in this potential affect the mobile negative charge location in each dimer and the charge distribution in the microtubule. The net effect is that mechanical forces affect the charge distribution in microtubules. We propose to exploit this effect and use microtubules as mechanical force sensors. We model each dimer as a two-state quantum system and, following the quantum computation paradigm, we use discrete quantum random walk on the hexagonal microtubule lattice to determine the charge distribution. Different forces applied on the microtubule are modeled as different coin biases leading to different probability distributions of the quantum walker location, which are directly connected to different charge distributions. Simulation results show that there is a strong indication that microtubules can be used as mechanical force sensors and that they can also detect the force directions and magnitudes.

Assembly dynamics of microtubules at molecular resolution

Microtubules are highly dynamic protein polymers that form a crucial part of the cytoskeleton in all eukaryotic cells. Although microtubules are known to self-assemble from tubulin dimers, information on the assembly dynamics of microtubules has been limited, both in vitro and in vivo, to measurements of average growth and shrinkage rates over several thousands of tubulin subunits. As a result there is a lack of information on the sequence of molecular events that leads to the growth and shrinkage of microtubule ends. Here we use optical tweezers to observe the assembly dynamics of individual microtubules at molecular resolution. We find that microtubules can increase their overall length almost instantaneously by amounts exceeding the size of individual dimers (8 nm). When the microtubule-associated protein XMAP215 (ref. 6) is added, this effect is markedly enhanced and fast increases in length of about 40-60 nm are observed. These observations suggest that small tubulin oligomers are able to add directly to growing microtubules and that XMAP215 speeds up microtubule growth by facilitating the addition of long oligomers. The achievement of molecular resolution on the microtubule assembly process opens the way to direct studies of the molecular mechanism by which the many recently discovered microtubule end-binding proteins regulate microtubule dynamics in living cells.

Antagonistic activities of Klp10A and Orbit regulate spindle length, bipolarity and function in vivo

The metaphase-spindle steady-state length occurs as spindle microtubules ;flux', incorporating new subunits at their plus ends, while simultaneously losing subunits from their minus ends. Orbit/Mast/CLASP is required for tubulin subunit addition at kinetochores, and several kinesins regulate spindle morphology and/or flux by serving as microtubule depolymerases. Here, we use RNA interference in S2 cells to examine the relationship between Orbit and the four predicted kinesin-type depolymerases encoded by the Drosophila genome (Klp10A, Klp59C, Klp59D and Klp67A). Single depletion of Orbit results in monopolar spindles, mitotic arrest and a subsequent increase in apoptotic cells. These phenotypes are rescued by co-depleting Klp10A but none of the other three depolymerases. Spindle bipolarity is restored by preventing the spindle collapse seen in cells that lack Orbit, leading to functional spindles that are similar to controls in shape and length. We conclude that Klp10A exclusively antagonises Orbit in the regulation of bipolar spindle formation and maintenance.

Microtubule Structural Dynamics Measured with Impedance Spectroscopy

Microtubules are ubiquitous cytoskeletal proteins that play an important role in a variety of cellular processes. It has been demonstrated that microtubules are essential components in cellular activities as diverse as mitotic spindle formation, dendrite outgrowth at synapses, and vesicle transport. In vivo, these proteins exist in a constant state of dynamic instability, alternating between the polymerized microtubule and depolymerized tubulin subunits. Due to this property, very little is known about the dynamic structural features of microtubules. We use impedance spectroscopy to study structural details of microtubules, probing polymerization and binding events in nearly real time without the need for optically active labels. Using this method, we have obtained data for polymerization rates and microtubule-associated protein 2 (MAP2) and tau binding that agree with previously published results. Additionally, we can distinguish the presence of multiple conduction pathways along the microtubule, which correspond to the presence of various isoforms of tubulin present in the sample. Finally, we look at single neuronal cells in an attempt to monitor microtubule behavior in vivo. We then discuss the greater applicability of this technique in the biological sciences.

The evolution of the structure of tubulin and its potential consequences for the role and function of microtubules in cells and embryos

This paper discusses the results of homology modeling and resulting calculation of key structural and physical properties for close to 300 tubulin sequences, including alpha, beta, gamma, delta and epsilon -tubulins. The basis for our calculations was the structure of the tubulin dimer published several years ago by Nogales et al. (1998), later refined to 3.5 resolution by Lowe et al. (2001). While, it appears that the alpha, beta and gamma-tubulins segregate into distinct structural families, we have found several differences in the physical properties within each group. Each of the alpha, beta and gamma- tubulin groups exhibit major differences in their net electric charge, dipole moments and dipole vector orientations. These properties could influence functional characteristics such as microtubule stability and assembly kinetics, due to their effects on the strength of protein-protein interactions. In addition to the general structural trends between tubulin isoforms, we have observed that the carboxy-termini of alpha and beta-tubulin exists in at least two stable configurations, either projecting away from the tubulin (or microtubule) surface, or collapsed onto the surface. In the latter case, the carboxy-termini form a lattice distinctly different from that of the well-known A and B lattices formed by the tubulin subunits. However, this C-terminal lattice is indistinguishable from the lattice formed when the microtubule-associated protein tau binds to the microtubule surface. Finally, we have discussed how tubulin sequence diversity arose in evolution giving rise to its particular phylogeny and how it may be used in cell- and tissue-specific expression including embryonal development.

Microtubule disassembly and inhibition of mitosis by a novel synthetic pharmacophore

Microtubule drugs, which block cell cycle progression through mitosis, have seen widespread use in cancer chemotherapies. Although microtubules are subject to regulation by signal transduction mechanisms, their pharmacological modulation has so far relied on compounds that bind to the tubulin subunit. A new microtubule pharmacophore, diphenyleneiodonium, causing disassembly of the microtubule cytoskeleton is described here. Although this synthetic compound does not affect the assembly state of purified microtubules, it profoundly suppresses microtubule assembly in vivo, causes paclitaxel-stabilized microtubules to cluster around the centrosomes, and selectively disassembles dynamic microtubules. Similar to other microtubule drugs, this new pharmacophore blocks mitotic spindle assembly and mitotic cell division.

Interaction of kinesin motors, microtubules, and MAPs

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the "conventional" motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize the evidence that kinesin binds mainly to beta tubulin with the switch-2 helix close to the intradimer interface between alpha and beta tubulin.

Docetaxel in ovarian cancer

Docetaxel, a semisynthetic taxane, is a potent inhibitor of cell replication and, similar to paclitaxel, promotes in vitro assembly of stable microtubules and, therefore, prevents the depolymerisation process. Docetaxel has a higher affinity for the tubulin subunit and is associated with a 100-fold greater phosphorylation of BCL-2 inducing apoptosis. Docetaxel in combination with carboplatin demonstrates similar activity to paclitaxel/carboplatin in the upfront management of advanced ovarian cancer with less neurological, but greater haematological toxicity. This article reviews the rationale and indications for the use of docetaxel in ovarian cancer.

Dynamic instability of microtubules: Effect of catastrophe-suppressing drugs

Microtubules are stiff filamentary proteins that constitute an important component of the cytoskeleton of cells. These are known to exhibit a dynamic instability. A steadily growing microtubule can suddenly start depolymerizing very rapidly; this phenomenon is known as a "catastrophe." However, often a shrinking microtubule is "rescued" and starts polymerizing again. Here we develop a model for the polymerization-depolymerization dynamics of microtubules in the presence of catastrophe-suppressing drugs. Solving the dynamical equations in the steady state, we derive exact analytical expressions for the length distributions of the microtubules tipped with drug-bound tubulin subunits as well as those of the microtubules, in the growing and shrinking phases, tipped with drug-free pure tubulin subunits. We also examine the stability of the steady-state solutions.

Microtubules: Mechanical Meets Chemical

Although the cylindrical shape and regular lattice of microtubules is beguilingly simple, the polymerization dynamics have proven aggravatingly complex, and the molecular basis of dynamic instability, the behavior whereby microtubules alternate between periods of slow growth and rapid shortening, has eluded accurate mechanistic description. In part this is due to insufficient experimental resolution—light microscopy is limited by the light resolution limit, approximately the length of 30 tubulin subunits (1,2)—but is also due to the lack of a comprehensive mechanistic model tying mechanical properties with molecular kinetics.

Microtubule‐dependent nuclear‐cytoplasmic shuttling of Runx2

RUNX/AML transcription factors are critical regulators of cell growth and differentiation in multiple lineages and have been linked to human cancers including acute myelogenous leukemia (RUNX1), as well as breast (RUNX2) and gastric cancers (RUNX3). RUNX proteins are targeted to gene regulatory micro-environments within the nucleus via a specific subnuclear targeting signal. However, the dynamics of RUNX distribution and compartmentalization between the cytoplasm and nucleus is minimally understood. Here we show by immunofluorescence microscopy that RUNX2 relocates from the nucleus to the cytoplasm when microtubules are stabilized by the chemotherapeutic agent taxol. The taxol-dependent cytoplasmic accumulation of RUNX2 is inhibited by leptomycin B, which blocks CRM-1 dependent nuclear export, and is not affected by the protein synthesis inhibitor cycloheximide. Using biochemical assays, we show that endogenous RUNX2 associates with stabilized microtubules in a concentration-dependent manner and that the RUNX2 amino terminus mediates the microtubule association. In soluble fractions of cells, RUNX2 co-immunoprecipitates alpha tubulin suggesting that microtubule binding involves the alpha/beta tubulin subunits. We conclude that RUNX2 associates with microtubules and shuttles between the nucleus and the cytoplasm. We propose that nuclear-cytoplasmic shuttling of RUNX2 may modulate its transcriptional activity, as well as its ability to interface with signal transduction pathways that are integrated at RUNX2 containing subnuclear sites. It is possible that taxol-induced acute depletion of the nuclear levels of RUNX2 and/or other cell growth regulatory factors may represent an alternative pathway by which taxol exerts its biological effects during cancer chemotherapies.

LIM Kinase 1 Coordinates Microtubule Stability and Actin Polymerization in Human Endothelial Cells

Microtubule (MT) destabilization promotes the formation of actin stress fibers and enhances the contractility of cells; however, the mechanism involved in the coordinated regulation of MTs and the actin cytoskeleton is poorly understood. LIM kinase 1 (LIMK1) regulates actin polymerization by phosphorylating the actin depolymerization factor, cofilin. Here we report that LIMK1 is also involved in the MT destabilization. In endothelial cells endogenous LIMK1 co-localizes with MTs and forms a complex with tubulin via the PDZ domain. MT destabilization induced by thrombin or nocodazole resulted in a decrease of LIMK1 colocalization with MTs. Overexpression of wild type LIMK1 resulted in MT destabilization, whereas the kinase-dead mutant of LIMK1 (KD) did not affect MT stability. Importantly, down-regulation of endogenous LIMK1 by small interference RNA resulted in abrogation of the thrombin-induced MTs destabilization and the inhibition of thrombin-induced actin polymerization. Expression of Rho kinase 2, which phosphorylates and activates LIMK1, dramatically decreases the interaction of LIMK1 with tubulin but increases its interaction with actin. Interestingly, expression of KD-LIMK1 or small interference RNA-LIMK1 prevents thrombin-induced microtubule destabilization and F-actin formation, suggesting that LIMK1 activity is required for thrombin-induced modulation of microtubule destabilization and actin polymerization. Our findings indicate that LIMK1 may coordinate microtubules and actin cytoskeleton.

Nitrotyrosination of plant α-tubulin: potential mechanisms of influence on cellular processes

3-Nitrotyrosine, the product of interaction between the free tyrosine with nitric oxide, is characterized by its posttranslational incorporation into carboxyl terminus of α-tubulin molecule via tubulin-tyrosine ligase, similar to unmodified tyrosine residue. However, α-tubulin C-terminal nitrotyrosination is irreversible unlike an enzymatically regulated tyrosination/detyrosination cycle. The nitrotyrosination of α-tubulin results in an appearance of some changes in animal cell morphology and their microtubule organization, a loss of some cellular functions, and in intracellular redistribution of cytoplasmic dynein. Since, nitrotyrosination in plant cell has not been investigated, the aim of our study was to reconstruct the spatial structure of nitrotyrosinated plant α-tubulin with following modeling of possible mechanisms of this posttranslational modification influence on cellular processes. The last 10–12 amino acid residues of α-tubulin C-terminal region, unlike the main part of the molecule, form a free oscillating tail that can be considered as individual structural domain. The results of reconstructing the spatial structure of detyrosinated, tyrosinated and nitrotyrosinated goosegrass (Eleusine indica (L.) Gaerth.) α-tubulin forms and studies of their behavior testify that these modifications change substantially the level of mobility of flexible α-tubulin C-terminal domain. We found that tyrosination increases substantially the mobility of C-terminus (see Figure ​Figure11). Figure 1 Modelling of different allowed conformation of C-terminal tails of α-tubulin from goosegrass: A. Detyrosinated molecule; B. Tyrosinated molecule; C. Nitrotyrosinated molecule The root mean square deviation (RMSD) in coordinates of amino acid residues composing this region grows from 0.48 A for detyrosinated form, to 0.55 A for tyrosinated α-tubulin as was calculated on the basis of molecular fluctuations trajectory in 500 ps interval. These results correlate with data about predomination of detyrosinated α-tubulin in long-time living microtubules and vice versa. The breakdown of tyrosination/detyrosination cycle due to incorporation the nitrotyrosine into C-terminus should resulted in predomination of short-time living microtubules over long-time living microtubules. It is necessary to note that mobility of nitrotyrosinated terminus is lower than the tyrosinated one, but it is higher than the detyrosinated terminus. In the last case RMSD is 0.52 A. It can lead to the appearance of microtubules with a certain intermediate lifetime and, as a consequence, modify the microtubules organization in cells. It is known that the tubulin C-terminal region is responsible for interactions with structural and motor MAPs. In combination with the above mentioned data about the redistribution of cytoplasmic dynein after nitrotyrosination of α-tubulin we can speculate that nitrotyrosination can influence processes of tubulin-MAP recognition and interaction. This assumption is confirmed by our results on modeling the interaction of α-tubulin with the MT-binding domain of dynein heavy chains. The high content of dicarboxylic amino acid residues, especially glutamate residues, is the hallmark of the flexible C-terminal region of both tubulin subunits. It has to be correlated with the necessity of presence, a surface enriched with diamidate amino acid residues in the dynein MT-binding domain structure. The surface of dynein MT-binding domain formed by residues Lys3148, Lys3151, Lys3154, Lys3155, Arg3162, Lys3287, Lys3300, Arg3327, Lys3328 is correspondent to this factor. The surface is flanked by residues Asp3144, Asp3146 and Asp3320, Asp3321 on both sides. Included in nitrotyrosine nitro-group possesses an additional negative charge, that is not typical for dynein-tubulin interaction, and locates close to flanking residues. This location results in a strong decrease in the affinity between the dynein contact surface and α-tubulin C-terminal region.

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

The dynamic behaviour of microtubules and their contributions to hyphal tip growth in Aspergillus nidulans

Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of Aspergillus nidulans expressing GFP-alpha-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT-organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the 'cytomatrix', thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.

Mechanical Stress Induced Mechanism of Microtubule Catastrophes

Microtubules assembled in vitro from pure tubulin can switch occasionally from growing to shrinking states or resume assembly, an unusual behavior termed "dynamic instability of microtubule growth". Its origin remains unclear and several models have been proposed, including occasional switching of the microtubules into energetically unfavorable configurations during assembly. In this study, we have asked whether the excess energy accumulated in these configurations would be of sufficient magnitude to destabilize the capping region that must exist at the end of growing microtubules. For this purpose, we have analyzed the frequency distribution of microtubules assembled in vitro from pure tubulin, and modeled the different mechanical constraints accumulated in their wall. We find that the maximal excess energy that the microtubule lattice can store is in the order of 11 kBT per dimer. Configurations that require distortions up to approximately 20 kBT are allowed at the expense of a slight conformational change, and larger distortions are not observed. Modeling of the different elastic deformations suggests that the excess energy is essentially induced by protofilament skewing, microtubule radial curvature change and inter-subunit shearing, distortions that must destabilize further the tubulin subunits interactions. These results are consistent with the hypothesis that unfavorable closure events may trigger the catastrophes observed at low tubulin concentration in vitro. In addition, we propose a novel type of representation that describes the stability of microtubule assembly systems, and which might be of considerable interest to study the effects of stabilizing and destabilizing factors on microtubule structure and dynamics.

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.