Dynamic Microtubule Tips Research Articles

Three interacting regions of the Ndc80 and Dam1 complexes support microtubule tip-coupling under load.

Accurate mitosis requires kinetochores to make persistent, load-bearing attachments to dynamic microtubule tips, thereby coupling chromosome movements to tip growth and shortening. This tip-coupling behavior depends on the conserved Ndc80 complex and, in budding yeast, on the Dam1 complex, which bind each other directly via three distinct interacting regions. The functional relevance of these multiple interactions was mysterious. Here we show that interactions between two of these regions support the high rupture strengths that occur when applied force is rapidly increased and also support the stability of tip-coupling when force is held constant over longer durations. The contribution of either of these two regions to tip-coupling is reduced by phosphorylation by Aurora B kinase. The third interaction region makes no apparent contribution to rupture strength, but its phosphorylation by Aurora B kinase specifically decreases the long-term stability of tip-coupling. The specific reduction of long-term stability relative to short-term strength might have important implications for mitotic error correction.

Magnesium increases the chemo-mechanical energy released by curling protofilaments during microtubule disassembly

Disassembling microtubules provide forces that drive chromosomal movement in dividing cells. This motion can occur independently of conventional motor activity, and instead relies on the tenacious coupling of kinetochores to dynamic microtubule tips. During microtubule disassembly, kinetochores maintain load-bearing tip attachments even as thousands of tubulin subunits are lost from the tip per second. Despite recent advances in our structural and functional understanding of kinetochores and microtubules, it remains unknown how these molecular machines sustain such persistent attachments.

Regulation of microtubule dynamics, mechanics and function through the growing tip.

Microtubule dynamics and their control are essential for the normal function and division of all eukaryotic cells. This plethora of functions is, in large part, supported by dynamic microtubule tips, which can bind to various intracellular targets, generate mechanical forces and couple with actin microfilaments. Here, we review progress in the understanding of microtubule assembly and dynamics, focusing on new information about the structure of microtubule tips. First, we discuss evidence for the widely accepted GTP cap model of microtubule dynamics. Next, we address microtubule dynamic instability in the context of structural information about assembly intermediates at microtubule tips. Three currently discussed models of microtubule assembly and dynamics are reviewed. These are considered in the context of established facts and recent data, which suggest that some long-held views must be re-evaluated. Finally, we review structural observations about the tips of microtubules in cells and describe their implications for understanding the mechanisms of microtubule regulation by associated proteins, by mechanical forces and by microtubule-targeting drugs, prominently including cancer chemotherapeutics.

A CLK1-KKT2 Signaling Pathway Regulating Kinetochore Assembly in Trypanosoma brucei.

ABSTRACTDuring mitosis, eukaryotic cells must duplicate and separate their chromosomes in a precise and timely manner. The apparatus responsible for this is the kinetochore, which is a large protein structure that links chromosomal DNA and spindle microtubules to facilitate chromosome alignment and segregation. The proteins that comprise the kinetochore in the protozoan parasite Trypanosoma brucei are divergent from yeast and mammals and comprise an inner kinetochore complex composed of 24 distinct proteins (KKT1 to KKT23, KKT25) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2, and KKT3. We recently reported the identification of a specific trypanocidal inhibitor of T. brucei CLK1, an amidobenzimidazole, AB1. We now show that chemical inhibition of CLK1 with AB1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. Here, we show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 by CLK1 to be essential for KKT2 function and for kinetochore assembly. Additionally, KKT2 protein kinase activity is required for parasite proliferation but not for assembly of the inner kinetochore complex. We also show that chemical inhibition of the aurora kinase AUK1 does not affect CLK1 phosphorylation of KKT2, indicating that AUK1 and CLK1 are in separate regulatory pathways. We propose that CLK1 is part of a divergent signaling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2.

Cdk1 Phosphorylation of the Dam1 Complex Strengthens Kinetochore-Microtubule Attachments

To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1, 2, 3, 4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5, 6, 7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8, 9, 10, 11, 12, 13, 14, 15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10, 11, 12, 13, 14, 15, 18, 19, 20, 21, 22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.

Branched actin networks are assembled on microtubules by adenomatous polyposis coli for targeted membrane protrusion.

Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.

Reconstitution reveals two paths of force transmission through the kinetochore.

Partitioning duplicated chromosomes equally between daughter cells is a microtubule-mediated process essential to eukaryotic life. A multi-protein machine, the kinetochore, drives chromosome segregation by coupling the chromosomes to dynamic microtubule tips, even as the tips grow and shrink through the gain and loss of subunits. The kinetochore must harness, transmit, and sense mitotic forces, as a lack of tension signals incorrect chromosome-microtubule attachment and precipitates error correction mechanisms. But though the field has arrived at a 'parts list' of dozens of kinetochore proteins organized into subcomplexes, the path of force transmission through these components has remained unclear. Here we report reconstitution of functional Saccharomyces cerevisiae kinetochore assemblies from recombinantly expressed proteins. The reconstituted kinetochores are capable of self-assembling in vitro, coupling centromeric nucleosomes to dynamic microtubules, and withstanding mitotically relevant forces. They reveal two distinct pathways of force transmission and Ndc80c recruitment.

The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip-coupling protein.

Kinetochores are multiprotein machines that drive chromosome segregation by maintaining persistent, load-bearing linkages between chromosomes and dynamic microtubule tips. Kinetochores in commonly studied eukaryotes bind microtubules through widely conserved components like the Ndc80 complex. However, in evolutionarily divergent kinetoplastid species such as Trypanosoma brucei, which causes sleeping sickness, the kinetochores assemble from a unique set of proteins lacking homology to any known microtubule-binding domains. Here, we show that the T. brucei kinetochore protein KKT4 binds directly to microtubules and maintains load-bearing attachments to both growing and shortening microtubule tips. The protein localizes both to kinetochores and to spindle microtubules in vivo, and its depletion causes defects in chromosome segregation. We define a microtubule-binding domain within KKT4 and identify several charged residues important for its microtubule-binding activity. Thus, despite its lack of significant similarity to other known microtubule-binding proteins, KKT4 has key functions required for driving chromosome segregation. We propose that it represents a primary element of the kinetochore-microtubule interface in kinetoplastids.

Biophysics of Microtubule End Coupling at the Kinetochore.

The main physiological function of mitotic kinetochores is to provide durable attachment to spindle microtubules, which segregate chromosomes in order to partition them equally between the two daughter cells. Numerous kinetochore components that can bind directly to microtubules have been identified, including ATP-dependent motors and various microtubule-associated proteins with no motor activity. A major challenge facing the field is to explain chromosome motions based on the biochemical and structural properties of these individual kinetochore components and their assemblies. This chapter reviews the molecular mechanisms responsible for the motions associated with dynamic microtubule tips at the single-molecule level, as well as the activities of multimolecular ensembles called couplers. These couplers enable persistent kinetochore motion even under load, but their exact composition and structure remain unknown. Because no natural or artificial macro-machines function in an analogous manner to these molecular nano-devices, understanding their underlying biophysical mechanisms will require conceptual advances.

A TOG Protein Confers Tension Sensitivity to Kinetochore-Microtubule Attachments

The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of kinetochore-microtubule attachments that come under tension due to opposing pulling forces exerted on sister kinetochores by dynamic microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to kinetochore-microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 kinetochore complex that strengthens its attachment to microtubule tips. Strikingly, Stu2 can either stabilize or destabilize kinetochore attachments, depending on the level of kinetochore tension and whether the microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of microtubule dynamics. Altogether, our results demonstrate how a kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation.

A Mathematical Model of Force Generation by Flexible Kinetochore-Microtubule Attachments

Important mechanical events during mitosis are facilitated by the generation of force by chromosomal kinetochore sites that attach to dynamic microtubule tips. Several theoretical models have been proposed for how these sites generate force, and molecular diffusion of kinetochore components has been proposed as a key component that facilitates kinetochore function. However, these models do not explicitly take into account the recently observed flexibility of kinetochore components and variations in microtubule shape under load. In this paper, we develop a mathematical model for kinetochore-microtubule connections that directly incorporates these two important components, namely, flexible kinetochore binder elements, and the effects of tension load on the shape of shortening microtubule tips. We compare our results with existing biased diffusion models and explore the role of protein flexibility inforce generation at the kinetochore-microtubule junctions. Our model results suggest that kinetochore component flexibility and microtubule shape variation under load significantly diminish the need for high diffusivity (or weak specific binding) of kinetochore components; optimal kinetochore binder stiffness regimes are predicted by our model. Based on our model results, we suggest that the underlying principles of biased diffusion paradigm need to be reinterpreted.

Direct Evidence for Sister Kinetochore Fusion in Meiosis I

For decades, biologists have wondered how replicated sister chromatids, which always separate from one another during normal mitotic cell division, instead co-migrate together during phase I of meiosis, the specialized division that produces eggs and sperm. Genetic, cytological, and biochemical studies using budding yeast have suggested that sister kinetochores, which link the sister chromatids to microtubules and drive their movement, may be mechanically fused by a meiosis I-specific kinetochore-binding factor called monopolin. But thus far the evidence for monopolin-dependent sister kinetochore fusion has been indirect, and the possibility that monopolin could instead promote co-migration by binding to and inhibiting one of the two sister kinetochores has not been excluded. To test these ideas directly, we are isolating native meiotic and mitotic kinetochore particles, reconstituting their function in vitro, and applying advanced tools for manipulating and tracking individual molecules. Using laser trapping, we find that kinetochore particles from meiosis I form substantially stronger attachments to dynamic microtubule tips than those from mitosis or from meiosis II. The high strength of meiosis I particles is lost if DNA replication is blocked during pre-meiotic S-phase, demonstrating that sisters are required. High strength is also lost if monopolin is disrupted prior to kinetochore particle isolation and, conversely, the strength of mitotic kinetochores can be increased by artificially inducing monopolin expression. Quantitative fluorescence microscopy also confirms that meiosis I particles have more copies of the core microtubule-binding component, Nuf2. Together these data suggest that sister kinetochores are mechanically fused by the monopolin complex during meiosis I, in such a way that the microtubule-binding elements from the two sister kinetochores can cooperate together to form a single attachment site.

Kinetochore kinesin CENP-E is a processive bi-directional tracker of dynamic microtubule tips

During vertebrate mitosis, the centromere-associated kinesin CENP-E transports misaligned chromosomes to the plus ends of spindle microtubules. Subsequently, the kinetochores that form at the centromeres establish stable associations with microtubule ends, which assemble and disassemble dynamically. Here we provide evidence that after chromosomes have congressed and bi-oriented, the CENP-E motor continues to play an active role at kinetochores, enhancing their links with dynamic microtubule ends. Using a combination of single molecule approaches and laser trapping in vitro we demonstrate that once reaching microtubule ends, CENP-E converts from a lateral transporter into a microtubule tip-tracker which maintains association with both assembling and disassembling microtubule tips. Computational modeling of this behavior supports our proposal that CENP-E tip-tracks bi-directionally via a “tethered motor” mechanism, which relies on both the motor and tail domains of CENP-E. Our results provide a molecular framework for CENP-E's contribution to the stability of attachments between kinetochores and dynamic microtubule ends.

Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms

During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.

Phosphoregulation of the Ndc80 and Dam1 Subcomplexes Promotes Release of Kinetochores from Dynamic Microtubules via Multiple Mechanisms

During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. Aurora B kinase phosphorylates the major microtubule binding subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 versus Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Composition of the purified particles was unaffected by the phosphomimetic mutations, suggesting that phosphorylation at these sites does not disrupt kinetochore structure. Rupture forces were reduced by phosphomimetics on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Likewise, phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Together our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms - by directly weakening the kinetochore-microtubule interface, and also by destabilizing kinetochore attached microtubule tips.

Kinetochore Kinesin CENP-E Tracks the Tips of Dynamic Microtubules via the ‘Tethered Motor’ Mechanism

Member of kinesin-7 family CENP-E is a kinetochore-associated plus-end-directed motor, which is important for faithful chromosome segregation in mitosis. CENP-E assists chromosome transport to the spindle midzone, where the microtubule plus ends are located. Here we report that once reaching the dynamic microtubule ends in vitro, CENP-E converts from a lateral transporter into a microtubule tip-tracker, stably associating with the tips of both assembling and disassembling microtubules. We show that the binding between kinetochores and dynamic microtubule ends is destabilized in live cells when CENP-E function is perturbed via an inhibition or RNAi depletion, implying that CENP-E plays an important mitotic role at the kinetochore-associated microtubule tips. To determine the molecular mechanism of CENP-E tip-tracking, we characterized two purified recombinant fragments of CENP-E: one containing the motor and neck domains and the second with the dimeric C-terminal tails. The motor-containing truncated protein walked on the microtubule wall in essentially the same manner as the full length CENP-E, while the C-terminal tail exhibited rapid diffusion. Neither of these fragments showed the tip-tracking, however, this activity was recapitulated by artificially joining these two proteins by conjugating to Qdots. A computational model of CENP-E motility successfully described the tip-tracking ability by repeating the cycles of plus-end-directed walking and the tail-mediated diffusion of the microtubule wall-tethered motor. This novel “tethered motor” mechanism of tip-tracking does not rely on the specific properties of the assembling or disassembling microtubule tips, explaining why CENP-E can tip-track bi-directionally, i.e. with the growing and shortening microtubule ends. Together, these results establish the requirement for CENP-E in stably linking the kinetochores to dynamic microtubule tips, and provide a detailed molecular mechanism to explain how CENP-E can achieve this function.

Tension Directly Stabilizes Reconstituted Kinetochore-Microtubule Attachments

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct ‘bi-oriented’ kinetochore-microtubule attachments, which come under tension due to opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, via a catch bond-like mechanismthat does not require Aurora B. Based on these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

The Ndc80 Loop Region Facilitates Formation of Kinetochore Attachment to the Dynamic Microtubule Plus End

SummaryProper chromosome segregation in mitosis relies on correct kinetochore-microtubule (KT-MT) interactions. The KT initially interacts with the lateral surface of a single MT (lateral attachment) extending from a spindle pole and is subsequently anchored at the plus end of the MT (end-on attachment) [1]. The conversion from lateral to end-on attachment is crucial because end-on attachment is more robust [2–4] and thought to be necessary to sustain KT-MT attachment when tension is applied across sister KTs upon their biorientation [1]. The mechanism for this conversion is still elusive. The Ndc80 complex is an essential component of the KT-MT interface [1, 5], and here we studied a role of the Ndc80 loop region, a distinct motif looping out from the coiled-coil shaft of the complex [6], in Saccharomyces cerevisiae. With deletions or mutations of the loop region, the lateral KT-MT attachment occurred normally; however, subsequent conversion to end-on attachment was defective, leading to failure in sister KT biorientation. The Ndc80 loop region was required for Ndc80-Dam1 interaction and KT loading of the Dam1 complex, which in turn supported KT tethering to the dynamic MT plus end [3, 7]. The Ndc80 loop region, therefore, has an important role in the conversion from lateral to end-on attachment, a crucial maturation step of KT-MT interaction.

Tension directly stabilizes reconstituted kinetochore-microtubule attachments

Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B.

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.