Microtubule dynamics: an interplay of biochemistry and mechanics.

Microtubules are dynamic polymers of αß-tubulin that are essential for intracellular organization, organelle trafficking and chromosome segregation. Microtubule growth and shrinkage occur via addition and loss of αß-tubulin subunits, which are biochemical processes. Dynamic microtubules can also engage in mechanical processes, such as exerting forces by pushing or pulling against a load. Recent advances at the intersection of biochemistry and mechanics have revealed the existence of multiple conformations of αß-tubulin subunits and their central role in dictating the mechanisms of microtubule dynamics and force generation. It has become apparent that microtubule-associated proteins (MAPs) selectively target specific tubulin conformations to regulate microtubule dynamics, and mechanical forces can also influence microtubule dynamics by altering the balance of tubulin conformations. Importantly, the conformational states of tubulin dimers are likely to be coupled throughout the lattice: the conformation of one dimer can influence the conformation of its nearest neighbours, and this effect can propagate over longer distances. This coupling provides a long-range mechanism by which MAPs and forces can modulate microtubule growth and shrinkage. These findings provide evidence that the interplay between biochemistry and mechanics is essential for the cellular functions of microtubules.

CLASPs at a glance.

CLIP-associating proteins (CLASPs) form an evolutionarily conserved family of regulatory factors that control microtubule dynamics and the organization of microtubule networks. The importance of CLASP activity has been appreciated for some time, but until recently our understanding of the underlying molecular mechanisms remained basic. Over the past few years, studies of, for example, migrating cells, neuronal development, and microtubule reorganization in plants, along with in vitro reconstitutions, have provided new insights into the cellular roles and molecular basis of CLASP activity. In this Cell Science at a Glance article and the accompanying poster, we will summarize some of these recent advances, emphasizing how they impact our current understanding of CLASP-mediated microtubule regulation.

Direct observation of individual tubulin dimers binding to growing microtubules.

The biochemical basis of microtubule growth has remained elusive for over 30 years despite being fundamental for both cell division and associated chemotherapy strategies. Here, we combine interferometric scattering microscopy with recombinant tubulin to monitor individual tubulins binding to and dissociating from growing microtubule tips. We make direct, single-molecule measurements of tubulin association and dissociation rates. We detect two populations of transient dwell times and determine via binding-interface mutants that they are distinguished by the formation of one interprotofilament bond. Applying a computational model, we find that slow association kinetics with strong interactions along protofilaments best recapitulate our data and, furthermore, predicts plus-end tapering. Overall, we provide the most direct and complete experimental quantification of how microtubules grow to date.

Kinetochore-associated Stu2 promotes chromosome biorientation in vivo.

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with a self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response.

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions. Strikingly, we find that viruses encoding MT interaction-deficient A51R proteins fail to suppress a reactive oxygen species (ROS)-dependent antiviral response in macrophages that leads to a block in virion morphogenesis. Moreover, A51R-MT interactions are required for VV virulence in mice. Collectively, our data show that poxviral MAP-MT interactions overcome a cell-intrinsic antiviral ROS response in macrophages that would otherwise block virus morphogenesis and replication in animals.

Interface-acting nucleotide controls polymerization dynamics at microtubule plus- and minus-ends.

GTP-tubulin is preferentially incorporated at growing microtubule ends, but the biochemical mechanism by which the bound nucleotide regulates the strength of tubulin:tubulin interactions is debated. The 'self-acting' (cis) model posits that the nucleotide (GTP or GDP) bound to a particular tubulin dictates how strongly that tubulin interacts, whereas the 'interface-acting' (trans) model posits that the nucleotide at the interface of two tubulin dimers is the determinant. We identified a testable difference between these mechanisms using mixed nucleotide simulations of microtubule elongation: with self-acting nucleotide, plus- and minus-end growth rates decreased in the same proportion to the amount of GDP-tubulin, whereas with interface-acting nucleotide, plus-end growth rates decreased disproportionately. We then experimentally measured plus- and minus-end elongation rates in mixed nucleotides and observed a disproportionate effect of GDP-tubulin on plus-end growth rates. Simulations of microtubule growth were consistent with GDP-tubulin binding at and 'poisoning' plus-ends but not at minus-ends. Quantitative agreement between simulations and experiments required nucleotide exchange at terminal plus-end subunits to mitigate the poisoning effect of GDP-tubulin there. Our results indicate that the interfacial nucleotide determines tubulin:tubulin interaction strength, thereby settling a longstanding debate over the effect of nucleotide state on microtubule dynamics.

Catching the Conformational Wave: Measuring the Working Strokes of Protofilaments as They Curl Outward from Disassembling Microtubule Tips.

Optical traps have enabled foundational studies of how mechanoenzymes such as kinesins and dynein motors walk along microtubules, how myosins move along F-actin, and how nucleic acid enzymes move along DNA or RNA. Often the filamentous substrates serve merely as passive tracks for mechanoenzymes but microtubules and F-actin are themselves dynamic protein polymers, capable of generating movement and force independently of conventional motors. Microtubule-driven forces are particularly important during mitosis, when they align duplicated chromosomes at the metaphase plate and then pull them apart during anaphase. These vital movements depend on specialized protein assemblies called kinetochores that couple the chromosomes to the tips of dynamic microtubule filaments, thereby allowing filament shortening to produce pulling forces. Although great strides have been made toward understanding the structures and functions of many kinetochore subcomplexes, the biophysical basis for their coupling to microtubule tips remains unclear. During tip disassembly, strain energy is released when straight protofilaments in the microtubule lattice curl outward, creating a conformational wave that propagates down the microtubule. A popular viewpoint is that the protofilaments as they curl outward hook elements of the kinetochore and tug on them, transferring some of their curvature strain energy to the kinetochore. As a first step toward testing this idea, we recently developed a laser trap assay to directly measure the working strokes generated by curling protofilaments. Our "wave" assay is based on an earlier pioneering study, with improvements that allow measurement of curl-driven movements as functions of force and quantification of their conformational strain energy. In this chapter, we provide a detailed protocol for our assay and describe briefly our instrument setup and data analysis methods.

Working strokes produced by curling protofilaments at disassembling microtubule tips can be biochemically tuned and vary with species.

The disassembly of microtubules can generate force and drive intracellular motility. During mitosis, for example, chromosomes remain persistently attached via kinetochores to the tips of disassembling microtubules, which pull the sister chromatids apart. According to the conformational wave hypothesis, such force generation requires that protofilaments curl outward from the disassembling tips to exert pulling force directly on kinetochores. Rigorously testing this idea will require modifying the mechanical and energetic properties of curling protofilaments, but no way to do so has yet been described. Here, by direct measurement of working strokes generated in vitro by curling protofilaments, we show that their mechanical energy output can be increased by adding magnesium, and that yeast microtubules generate larger and more energetic working strokes than bovine microtubules. Both the magnesium and species-dependent increases in work output can be explained by lengthening the protofilament curls, without any change in their bending stiffness or intrinsic curvature. These observations demonstrate how work output from curling protofilaments can be tuned and suggest evolutionary conservation of the amount of curvature strain energy stored in the microtubule lattice.

Dividing cells duplicate their genetic information to create identical pairs of chromosomes, which then need to be equally distributed to the two future daughter cells. In preparation, each chromosome in a pair is pulled towards its final location by hollow tubes of proteins known as microtubules. To create this tugging force, the microtubule acts like a winch: the extremity attached to the chromosome gradually shortens by losing its building blocks. However, it is not clear how the microtubule can keep its grip on the chromosome while also 'falling apart' in this way. A possible explanation could stem from the way that microtubules are built, and from how they fall apart. Each tube is composed of rows of building blocks, called 'protofilaments'. As the microtubule shortens, the protofilaments first curl outwards before crumbling apart; this creates a curling action that could 'hook' the chromosome and pull on it as the microtubule shortens. This theory remains difficult to test however, in part because scientists lack ways to alter the properties of curling protofilaments in order to dissect how they work. Murray et al. aimed to fill that gap by using a technique they have previously developed, and which allows them to capture how much force curling protofilaments can apply on their environment. This approach uses an instrument known as laser tweezers to measure the pressure that microtubules exert on attached beads. With this assay, Murray et al. were able to investigate whether microtubule 'strength' is linked to protofilament length, a property that varies between species and in response to magnesium. The experiments revealed that adding magnesium not only lengthens protofilament curls but also increases the work generated from curling. In addition, they showed that yeast protofilaments create longer curls with more force compared to bovine microtubules. Together, these findings demonstrate that it is possible to fine-tune the force exerted by protofilaments on their environment by controlling their length. This knowledge could be helpful to scientists investigating the role of microtubules in cell division. Certain cancer drugs already target microtubules in order to stop rogue cells from multiplying. However, serious side-effects often emerge because these compounds also interfere with microtubule-based processes essential for healthy cells. By better understanding how protofilaments 'pull' on chromosomes, it may become possible to design targeted approaches to stop cell division but preserve the other fundamental roles that microtubules play in the body.

Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end.

Microtubule polymerization dynamics result from the biochemical interactions of αß-tubulin with the polymer end, but a quantitative understanding has been challenging to establish. We used interference reflection microscopy to make improved measurements of microtubule growth rates and growth fluctuations in the presence and absence of GTP hydrolysis. In the absence of GTP hydrolysis, microtubules grew steadily with very low fluctuations. These data were best described by a computational model implementing slow assembly kinetics, such that the rate of microtubule elongation is primarily limited by the rate of αß-tubulin associations. With GTPase present, microtubules displayed substantially larger growth fluctuations than expected based on the no GTPase measurements. Our modeling showed that these larger fluctuations occurred because exposure of GDP-tubulin on the microtubule end transiently 'poisoned' growth, yielding a wider range of growth rates compared to GTP only conditions. Our experiments and modeling point to slow association kinetics (strong longitudinal interactions), such that drugs and regulatory proteins that alter microtubule dynamics could do so by modulating either the association or dissociation rate of tubulin from the microtubule tip. By causing slower growth, exposure of GDP-tubulin at the growing microtubule end may be an important early event determining catastrophe.

Naegleria's mitotic spindles are built from unique tubulins and highlight core spindle features.

Naegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis and thus represent a unique system for studying the evolution and functional specificity of mitotic tubulins and the spindles they assemble. Previous studies show that Naegleria amoebae express a divergent α-tubulin during mitosis, and we now show that Naegleria amoebae express a second mitotic α- and two mitotic ß-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and ß-tubulins and contain residues that suggest distinct microtubule properties. These distinct residues are conserved in mitotic tubulin homologs of the "brain-eating amoeba" Naegleria fowleri, making them potential drug targets. Using quantitative light microscopy, we find that Naegleria's mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria's spindle is twisted, and its length increases during mitosis, suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers and secondary bundles function as bridging fibers.

Design, overexpression, and purification of polymerization-blocked yeast αß-tubulin mutants.

Microtubule dynamics play essential roles in intracellular organization and cell division. They result from structural and biochemical properties of αß-tubulin heterodimers and how these polymerizing subunits interact with themselves and with regulatory proteins. A broad understanding of the underlying mechanisms has been established, but fundamental questions remain unresolved. The lack of routine access to recombinant αß-tubulin represents an obstacle to deeper insight into αß-tubulin structure, biochemistry, and recognition. Indeed, the widespread reliance on animal brain αß-tubulin means that very few in vitro studies have taken advantage of powerful and ordinarily routine techniques like site-directed mutagenesis. Here we report new methods for purifying wild-type or mutant yeast αß-tubulin from inducibly overexpressing strains of Saccharomyces cerevisiae. Inducible overexpression is an improvement over existing approaches that rely on constitutive expression: it provides higher yields while also allowing otherwise lethal mutants to be purified. We also designed and purified polymerization-blocked αß-tubulin mutants. These "blocked" forms of αß-tubulin give a dominant lethal phenotype when expressed in cells; they cannot form microtubules in vitro and when present in mixtures inhibit the polymerization of wild-type αß-tubulin. The effects of blocking mutations are very specific, because purified mutants exhibit normal hydrodynamic properties, bind GTP, and interact with a tubulin-binding domain. The ability to overexpress and purify wild-type αß-tubulin, or mutants like the ones we report here, creates new opportunities for structural studies of αß-tubulin and its complexes with regulatory proteins, and for biochemical and functional studies of microtubule dynamics and its regulation.

Insights into microtubule nucleation from the crystal structure of human gamma-tubulin.

Microtubules are hollow polymers of alphabeta-tubulin that show GTP-dependent assembly dynamics and comprise a critical part of the eukaryotic cytoskeleton. Initiation of new microtubules in vivo requires gamma-tubulin, organized as an oligomer within the 2.2-MDa gamma-tubulin ring complex (gamma-TuRC) of higher eukaryotes. Structural insight is lacking regarding gamma-tubulin, its oligomerization and how it promotes microtubule assembly. Here we report the 2.7-A crystal structure of human gamma-tubulin bound to GTP-gammaS (a non-hydrolysable GTP analogue). We observe a 'curved' conformation for gamma-tubulin-GTPgammaS, similar to that seen for GDP-bound, unpolymerized alphabeta-tubulin. Tubulins are thought to represent a distinct class of GTP-binding proteins, and conformational switching in gamma-tubulin might differ from the nucleotide-dependent switching of signalling GTPases. A crystal packing interaction replicates the lateral contacts between alpha- and beta-tubulins in the microtubule, and this association probably forms the basis for gamma-tubulin oligomerization within the gamma-TuRC. Laterally associated gamma-tubulins in the gamma-TuRC might promote microtubule nucleation by providing a template that enhances the intrinsically weak lateral interaction between alphabeta-tubulin heterodimers. Because they are dimeric, alphabeta-tubulins cannot form microtubule-like lateral associations in the curved conformation. The lateral array of gamma-tubulins we observe in the crystal reveals a unique functional property of a monomeric tubulin.

The lattice as allosteric effector: structural studies of alphabeta- and gamma-tubulin clarify the role of GTP in microtubule assembly.

GTP-dependent microtubule polymerization dynamics are required for cell division and are accompanied by domain rearrangements in the polymerizing subunit, alphabeta-tubulin. Two opposing models describe the role of GTP and its relationship to conformational change in alphabeta-tubulin. The allosteric model posits that unpolymerized alphabeta-tubulin adopts a more polymerization-competent conformation upon GTP binding. The lattice model posits that conformational changes occur only upon recruitment into the growing lattice. Published data support a lattice model, but are largely indirect and so the allosteric model has prevailed. We present two independent solution probes of the conformation of alphabeta-tubulin, the 2.3 A crystal structure of gamma-tubulin bound to GDP, and kinetic simulations to interpret the functional consequences of the structural data. These results (with our previous gamma-tubulin:GTPgammaS structure) support the lattice model by demonstrating that major domain rearrangements do not occur in eukaryotic tubulins in response to GTP binding, and that the unpolymerized conformation of alphabeta-tubulin differs significantly from the polymerized one. Thus, geometric constraints of lateral self-assembly must drive alphabeta-tubulin conformational changes, whereas GTP plays a secondary role to tune the strength of longitudinal contacts within the microtubule lattice. alphabeta-Tubulin behaves like a bent spring, resisting straightening until forced to do so by GTP-mediated interactions with the growing microtubule. Kinetic simulations demonstrate that resistance to straightening opposes microtubule initiation by specifically destabilizing early assembly intermediates that are especially sensitive to the strength of lateral interactions. These data provide new insights into the molecular origins of dynamic microtubule behavior.

Microtubules form by progressively faster tubulin accretion, not by nucleation-elongation.

Microtubules are dynamic polymers that play fundamental roles in all eukaryotes. Despite their importance, how new microtubules form is poorly understood. Textbooks have focused on variations of a nucleation-elongation mechanism in which monomers rapidly equilibrate with an unstable oligomer (nucleus) that limits the rate of polymer formation; once formed, the polymer then elongates efficiently from this nucleus by monomer addition. Such models faithfully describe actin assembly, but they fail to account for how more complex polymers like hollow microtubules assemble. Here, we articulate a new model for microtubule formation that has three key features: (1) microtubules initiate via rectangular, sheet-like structures that grow faster the larger they become; (2) the dominant pathway proceeds via accretion, the stepwise addition of longitudinal or lateral layers; and (3) a "straightening penalty" to account for the energetic cost of tubulin's curved-to-straight conformational transition. This model can quantitatively fit experimental assembly data, providing new insights into biochemical determinants and assembly pathways for microtubule nucleation.

Multiple conformations of E. coli Hsp90 in solution: insights into the conformational dynamics of Hsp90.

Hsp90, an essential eukaryotic chaperone, depends upon its intrinsic ATPase activity for function. Crystal structures of the bacterial Hsp90 homolog, HtpG, and the yeast Hsp90 reveal large domain rearrangements between the nucleotide-free and the nucleotide-bound forms. We used small-angle X-ray scattering and recently developed molecular modeling methods to characterize the solution structure of HtpG and demonstrate how it differs from known Hsp90 conformations. In addition to this HtpG conformation, we demonstrate that under physiologically relevant conditions, multiple conformations coexist in equilibrium. In solution, nucleotide-free HtpG adopts a more extended conformation than observed in the crystal, and upon the addition of AMPPNP, HtpG is in equilibrium between this open state and a closed state that is in good agreement with the yeast AMPPNP crystal structure. These studies provide a unique view of Hsp90 conformational dynamics and provide a model for the role of nucleotide in effecting conformational change.

Nucleating a Microtubule: Put a γ-Tubulin Ring on It.

The γ-tubulin ring complex (γTuRC) is a microtubule nucleator that controls when and where new microtubules form. In this issue of Developmental Cell, Consolati et al. provide much-needed insight into the mechanism of γTuRC-mediated nucleation by determining the structure of human γTuRC and performing quantitative measurements of its activity.

Insights into allosteric control of microtubule dynamics from a buried ß-tubulin mutation that causes faster growth and slower shrinkage.

αß-tubulin subunits cycle through a series of different conformations in the polymer lattice during microtubule growing and shrinking. How these allosteric responses to different tubulin:tubulin contacts contribute to microtubule dynamics, and whether the contributions are evolutionarily conserved, remains poorly understood. Here, we sought to determine whether the microtubule-stabilizing effects (slower shrinking) of the ß:T238A mutation we previously observed using yeast αß-tubulin would generalize to mammalian microtubules. Using recombinant human microtubules as a model, we found that the mutation caused slow microtubule shrinking, indicating that this effect of the mutation is indeed conserved. However, unlike in yeast, ß:T238A human microtubules grew faster than wild-type and the mutation did not appear to attenuate the conformational change associated with guanosine 5'-triphosphate (GTP) hydrolysis in the lattice. We conclude that the assembly-dependent conformational change in αß-tubulin can contribute to determine the rates of microtubule growing as well as shrinking. Our results also suggest that an allosteric perturbation like the ß:T238A mutation can alter the behavior of terminal subunits without accompanying changes in the conformation of fully surrounded subunits in the body of the microtubule.

Cryo-EM structure of VASH1-SVBP bound to microtubules.

The dynamic tyrosination-detyrosination cycle of α-tubulin regulates microtubule functions. Perturbation of this cycle impairs mitosis, neural physiology, and cardiomyocyte contraction. The carboxypeptidases vasohibins 1 and 2 (VASH1 and VASH2), in complex with the small vasohibin-binding protein (SVBP), mediate α-tubulin detyrosination. These enzymes detyrosinate microtubules more efficiently than soluble αß-tubulin heterodimers. The structural basis for this substrate preference is not understood. Using cryo-electron microscopy (cryo-EM), we have determined the structure of human VASH1-SVBP bound to microtubules. The acidic C-terminal tail of α-tubulin binds to a positively charged groove near the active site of VASH1. VASH1 forms multiple additional contacts with the globular domain of α-tubulin, including contacts with a second α-tubulin in an adjacent protofilament. Simultaneous engagement of two protofilaments by VASH1 can only occur within the microtubule lattice, but not with free αß heterodimers. These lattice-specific interactions enable preferential detyrosination of microtubules by VASH1.

Long-range, through-lattice coupling improves predictions of microtubule catastrophe.

Microtubules are cylindrical polymers of αß-tubulin that play critical roles in fundamental processes such as chromosome segregation and vesicular transport. Microtubules display dynamic instability, switching stochastically between growth and rapid shrinking as a consequence of GTPase activity in the lattice. The molecular mechanisms behind microtubule catastrophe, the switch from growth to rapid shrinking, remain poorly defined. Indeed, two-state stochastic models that seek to describe microtubule dynamics purely in terms of the biochemical properties of GTP- and GDP-bound αß-tubulin predict the concentration dependence of microtubule catastrophe incorrectly. Recent studies provide evidence for three distinct conformations of αß-tubulin in the lattice that likely correspond to GTP, GDP.Pi, and GDP. The incommensurate lattices observed for these different conformations raise the possibility that in a mixed nucleotide state lattice, neighboring tubulin dimers might modulate each other's conformations and hence each other's biochemistry. We explored whether incorporating a GDP.Pi state or the likely effects of conformational accommodation can improve predictions of catastrophe. Adding a GDP.Pi intermediate did not improve the model. In contrast, adding neighbor-dependent modulation of tubulin biochemistry improved predictions of catastrophe. Because this conformational accommodation should propagate beyond nearest-neighbor contacts, our modeling suggests that long-range, through-lattice effects are important determinants of microtubule catastrophe.