Daniel R. Gestaut

The Ndc80 Kinetochore Complex Forms Load-Bearing Attachments to Dynamic Microtubule Tips via Biased Diffusion

Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical, and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide the combination of plasticity and strength that allows kinetochores to maintain load-bearing tip attachments during both microtubule assembly and disassembly.

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

The Dam1 complex, regulated by aurora B phosphorylation, confers a more stable microtubule association for the Ndc80 complex at kinetochores (see also related paper by Lampert et al. in this issue).

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes — too few to surround a microtubule — were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.

Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis

In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore–microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore–microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.

The Ndc80 kinetochore complex directly modulates microtubule dynamics

The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.

Direct physical study of kinetochore-microtubule interactions by reconstitution and interrogation with an optical force clamp

We detail our use of computer-controlled optical traps to study interactions between kinetochore components and dynamic microtubules. Over the last two decades optical traps have helped uncover the working principles of conventional molecular motors, such as kinesin and dynein, but only recently have they been applied to study kinetochore function. The most useful traps combine sensitive position detectors and servo-control, allowing them to be operated as force clamps that maintain constant loads on objects as they move. Our instrument, which is among the simplest designs that permits force clamping, relies on a computer-controlled piezoelectric stage and a single laser for trapping and position detection. We apply it in motility assays where beads coated with pure microtubule-binding kinetochore components are attached to the tips of individual dynamic microtubules. Like kinetochores in vivo, the beads remain tip-attached, undergoing movements coupled to filament assembly and disassembly. The force clamp provides many benefits over instruments that lack feedback control. It allows tension to be applied continuously during both assembly- and disassembly-driven movement, providing a close match to the physiological situation. It also enables tracking with high resolution, and simplifies data interpretation by eliminating artifacts due to molecular compliance. The formation of persistent, load-bearing attachments to dynamic microtubule tips is fundamental to all kinetochore activities. Our direct, physical study of kinetochore-microtubule coupling may therefore furnish insights into many vital kinetochore functions, including correction of aberrant attachments and generation of the wait-anaphase signals that delay mitosis until all kinetochores are properly attached.

Native capillary isoelectric focusing for the separation of protein complex isoforms and subcomplexes.

Here we report the use of capillary isoelectric focusing under native conditions for the separation of protein complex isoforms and subcomplexes. Using biologically relevant HIS-tag and FLAG-tag purified protein complexes, we demonstrate the separations of protein complex isoforms of the mammalian target of rapamycin complex (mTORC1 and 2) and the subcomplexes and different phosphorylation states of the Dam1 complex. The high efficiency capillary isoelectric focusing separation allowed for resolution of protein complexes and subcomplexes similar in size and biochemical composition. By performing separations with native buffers and reduced temperature (15 degrees C) we were able to maintain the complex integrity of the more thermolabile mTORC2 during isoelectric focusing and detection (<45 min). Increasing the separation temperature allowed us to monitor dissociation of the Dam1 complex into its subcomplexes (25 degrees C) and eventually its individual protein components (30 degrees C). The separation of two different phosphorylation states of the Dam1 complex, generated from an in vitro kinase assay with Mps1 kinase, was straightforward due to the large pI shift upon multiple phosphorylation events. The separation of the protein complex isoforms of mTORC, on the other hand, required the addition of a small pI range (4-6.5) of ampholytes to improve resolution and stability of the complexes. We show that native capillary isoelectric focusing is a powerful method for the difficult separations of large, similar, unstable protein complexes. This method shows potential for differentiation of protein complex isoform and subcomplex compositions, post-translational modifications, architectures, stabilities, equilibria, and relative abundances under biologically relevant conditions.

Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex

Significance During cell division, multisubunit kinetochores partition chromosomes while maintaining a grip on dynamic microtubules under tension. Previous work in Caenorhabditis elegans showed that the central kinetochore component, Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) complex, increases microtubule binding of outer kinetochore complexes, but the mechanism for this enhancement remains unknown. Here, we identify new contacts between MIND and the outer kinetochore Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complex that are essential for interaction in vitro and for cell viability. Using single-molecule microscopy, we demonstrate that a single MIND complex enhances the microtubule binding of a single Ndc80 complex. Our results suggest a molecular mechanism for enhancing kinetochore–microtubule attachment by a central kinetochore component. Multiple protein subcomplexes of the kinetochore cooperate as a cohesive molecular unit that forms load-bearing microtubule attachments that drive mitotic chromosome movements. There is intriguing evidence suggesting that central kinetochore components influence kinetochore–microtubule attachment, but the mechanism remains unclear. Here, we find that the conserved Mis12/MIND (Mtw1, Nsl1, Nnf1, Dsn1) and Ndc80 (Ndc80, Nuf2, Spc24, Spc25) complexes are connected by an extensive network of contacts, each essential for viability in cells, and collectively able to withstand substantial tensile load. Using a single-molecule approach, we demonstrate that an individual MIND complex enhances the microtubule-binding affinity of a single Ndc80 complex by fourfold. MIND itself does not bind microtubules. Instead, MIND binds Ndc80 complex far from the microtubule-binding domain and confers increased microtubule interaction of the complex. In addition, MIND activation is redundant with the effects of a mutation in Ndc80 that might alter its ability to adopt a folded conformation. Together, our results suggest a previously unidentified mechanism for regulating microtubule binding of an outer kinetochore component by a central kinetochore complex.

Reconstitution and Functional Analysis of Kinetochore Subcomplexes

Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).