Sequential multisite phospho-regulation of KNL1-BUB3 interfaces at mitotic kinetochores.

Regulated recruitment of the kinase-adaptor complex BUB1/BUB3 to kinetochores is crucial for correcting faulty chromosome-spindle attachments and for spindle assembly checkpoint (SAC) signaling. BUB1/BUB3 localizes to kinetochores by binding phosphorylated MELT motifs (MELpT) in the kinetochore scaffold KNL1. Human KNL1 has 19 repeats that contain a MELT-like sequence. The repeats are, however, larger than MELT, and repeat sequences can vary significantly. Using systematic screening, we show that only a limited number of repeats is "active." Repeat activity correlates with the presence of a vertebrate-specific SHT motif C-terminal to the MELT sequence. SHT motifs are phosphorylated by MPS1 in a manner that requires prior phosphorylation of MELT. Phospho-SHT (SHpT) synergizes with MELpT in BUB3/BUB1 binding in vitro and in cells, and human BUB3 mutated in a predicted SHpT-binding surface cannot localize to kinetochores. Our data show sequential multisite regulation of the KNL1-BUB1/BUB3 interaction and provide mechanistic insight into evolution of the KNL1-BUB3 interface.

Guided by Light: Optical Control of Microtubule Gliding Assays.

Force generation by molecular motors drives biological processes such as asymmetric cell division and cell migration. Microtubule gliding assays in which surface-immobilized motor proteins drive microtubule propulsion are widely used to study basic motor properties as well as the collective behavior of active self-organized systems. Additionally, these assays can be employed for nanotechnological applications such as analyte detection, biocomputation, and mechanical sensing. While such assays allow tight control over the experimental conditions, spatiotemporal control of force generation has remained underdeveloped. Here we use light-inducible protein-protein interactions to recruit molecular motors to the surface to control microtubule gliding activity in vitro. We show that using these light-inducible interactions, proteins can be recruited to the surface in patterns, reaching a ∼5-fold enrichment within 6 s upon illumination. Subsequently, proteins are released with a half-life of 13 s when the illumination is stopped. We furthermore demonstrate that light-controlled kinesin recruitment results in reversible activation of microtubule gliding along the surface, enabling efficient control over local microtubule motility. Our approach to locally control force generation offers a way to study the effects of nonuniform pulling forces on different microtubule arrays and also provides novel strategies for local control in nanotechnological applications.

Aurora B phosphorylates spatially distinct targets to differentially regulate the kinetochore-microtubule interface.

Accurate chromosome segregation requires carefully regulated interactions between kinetochores and microtubules, but how plasticity is achieved to correct diverse attachment defects remains unclear. Here we demonstrate that Aurora B kinase phosphorylates three spatially distinct targets within the conserved outer kinetochore KNL1/Mis12 complex/Ndc80 complex (KMN) network, the key player in kinetochore-microtubule attachments. The combinatorial phosphorylation of the KMN network generates graded levels of microtubule-binding activity, with full phosphorylation severely compromising microtubule binding. Altering the phosphorylation state of each protein causes corresponding chromosome segregation defects. Importantly, the spatial distribution of these targets along the kinetochore axis leads to their differential phosphorylation in response to changes in tension and attachment state. In total, rather than generating exclusively binary changes in microtubule binding, our results suggest a mechanism for the tension-dependent fine-tuning of kinetochore-microtubule interactions.

Dissecting the roles of human BUB1 in the spindle assembly checkpoint.

Mitotic chromosome segregation is initiated by the anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 (forming APC/C(CDC20)). APC/C(CDC20) is inhibited by the spindle assembly checkpoint (SAC) when chromosomes have not attached to spindle microtubules. Unattached kinetochores catalyze the formation of a diffusible APC/C(CDC20) inhibitor that comprises BUBR1 (also known as BUB1B), BUB3, MAD2 (also known as MAD2L1) and a second molecule of CDC20. Recruitment of these proteins to the kinetochore, as well as SAC activation, rely on the mitotic kinase BUB1, but the molecular mechanism by which BUB1 accomplishes this in human cells is unknown. We show that kinetochore recruitment of BUBR1 and BUB3 by BUB1 is dispensable for SAC activation. Unlike its yeast and nematode orthologs, human BUB1 does not associate stably with the MAD2 activator MAD1 (also known as MAD1L1) and, although required for accelerating the loading of MAD1 onto kinetochores, BUB1 is dispensable for the maintenance of steady-state levels of MAD1 there. Instead, we identify a 50-amino-acid segment that harbors the recently reported ABBA motif close to a KEN box as being crucial for the role of BUB1 in SAC signaling. The presence of this segment correlates with SAC activity and efficient binding of CDC20 but not of MAD1 to kinetochores.

Mps1 promotes rapid centromere accumulation of Aurora B.

Aurora B localization to mitotic centromeres, which is required for proper chromosome alignment during mitosis, relies on Haspin-dependent histone H3 phosphorylation and on Bub1-dependent histone H2A phosphorylation--which interacts with Borealin through a Shugoshin (Sgo) intermediate. We demonstrate that Mps1 stimulates the latter recruitment axis. Mps1 activity enhances H2A-T120ph and is critical for Sgo1 recruitment to centromeres, thereby promoting Aurora B centromere recruitment in early mitosis. Importantly, chromosome biorientation defects caused by Mps1 inhibition are improved by restoring Aurora B centromere recruitment. As Mps1 kinetochore localization reciprocally depends on Aurora B, we propose that this Aurora B-Mps1 recruitment circuitry cooperates with the Aurora B-Haspin feedback loop to ensure rapid centromere accumulation of Aurora B at the onset of mitosis.

A unique residue in rab3c determines the interaction with novel binding protein Zwint-1.

Exocytic events are tightly regulated cellular processes in which rab GTPases and their interacting proteins perform an important function. We set out to identify new binding partners of rab3, which mediates regulated secretion events in specialized cells. We discovered Zwint-1 as a rab3 specific binding protein that bound preferentially to rab3c. The interaction depends on a critical residue in rab3c that determines the binding efficiency of Zwint-1, which is immaterial for interaction with rabphilin3a. Rab3c and Zwint-1 are expressed highly in brain and colocalized extensively in primary hippocampal neurons. We also found that SNAP25 bound to the same region in Zwint-1 as rab3c, suggesting a new role for the kinetochore protein Zwint-1 in presynaptic events that are regulated by rab3 and SNAP25.

Inner centromere localization of the CPC maintains centromere cohesion and allows mitotic checkpoint silencing.

Faithful chromosome segregation during mitosis requires that the kinetochores of all sister chromatids become stably connected to microtubules derived from opposite spindle poles. How stable chromosome bi-orientation is accomplished and coordinated with anaphase onset remains incompletely understood. Here we show that stable chromosome bi-orientation requires inner centromere localization of the non-enzymatic subunits of the chromosomal passenger complex (CPC) to maintain centromeric cohesion. Precise inner centromere localization of the CPC appears less relevant for Aurora B-dependent resolution of erroneous kinetochore-microtubule (KT-MT) attachments and for the stabilization of bi-oriented KT-MT attachments once sister chromatid cohesion is preserved via knock-down of WAPL. However, Aurora B inner centromere localization is essential for mitotic checkpoint silencing to allow spatial separation from its kinetochore substrate KNL1. Our data infer that the CPC is localized at the inner centromere to sustain centromere cohesion on bi-oriented chromosomes and to coordinate mitotic checkpoint silencing with chromosome bi-orientation.

Understanding force-generating microtubule systems through in vitro reconstitution.

Microtubules switch between growing and shrinking states, a feature known as dynamic instability. The biochemical parameters underlying dynamic instability are modulated by a wide variety of microtubule-associated proteins that enable the strict control of microtubule dynamics in cells. The forces generated by controlled growth and shrinkage of microtubules drive a large range of processes, including organelle positioning, mitotic spindle assembly, and chromosome segregation. In the past decade, our understanding of microtubule dynamics and microtubule force generation has progressed significantly. Here, we review the microtubule-intrinsic process of dynamic instability, the effect of external factors on this process, and how the resulting forces act on various biological systems. Recently, reconstitution-based approaches have strongly benefited from extensive biochemical and biophysical characterization of individual components that are involved in regulating or transmitting microtubule-driven forces. We will focus on the current state of reconstituting increasingly complex biological systems and provide new directions for future developments.

Reconstitution of Basic Mitotic Spindles in Spherical Emulsion Droplets.

Mitotic spindle assembly, positioning and orientation depend on the combined forces generated by microtubule dynamics, microtubule motor proteins and cross-linkers. Growing microtubules can generate pushing forces, while depolymerizing microtubules can convert the energy from microtubule shrinkage into pulling forces, when attached, for example, to cortical dynein or chromosomes. In addition, motor proteins and diffusible cross-linkers within the spindle contribute to spindle architecture by connecting and sliding anti-parallel microtubules. In vivo, it has proven difficult to unravel the relative contribution of individual players to the overall balance of forces. Here we present the methods that we recently developed in our efforts to reconstitute basic mitotic spindles bottom-up in vitro. Using microfluidic techniques, centrosomes and tubulin are encapsulated in water-in-oil emulsion droplets, leading to the formation of geometrically confined (double) microtubule asters. By additionally introducing cortically anchored dynein, plus-end directed microtubule motors and diffusible cross-linkers, this system is used to reconstitute spindle-like structures. The methods presented here provide a starting point for reconstitution of more complete mitotic spindles, allowing for a detailed study of the contribution of each individual component, and for obtaining an integrated quantitative view of the force-balance within the mitotic spindle.

A molecular basis for the differential roles of Bub1 and BubR1 in the spindle assembly checkpoint.

The spindle assembly checkpoint (SAC) monitors and promotes kinetochore-microtubule attachment during mitosis. Bub1 and BubR1, SAC components, originated from duplication of an ancestor gene. Subsequent sub-functionalization established subordination: Bub1, recruited first to kinetochores, promotes successive BubR1 recruitment. Because both Bub1 and BubR1 hetero-dimerize with Bub3, a targeting adaptor for phosphorylated kinetochores, the molecular basis for such sub-functionalization is unclear. We demonstrate that Bub1, but not BubR1, enhances binding of Bub3 to phosphorylated kinetochores. Grafting a short motif of Bub1 onto BubR1 promotes Bub1-independent kinetochore recruitment of BubR1. This gain-of-function BubR1 mutant cannot sustain a functional checkpoint. We demonstrate that kinetochore localization of BubR1 relies on direct hetero-dimerization with Bub1 at a pseudo-symmetric interface. This pseudo-symmetric interaction underpins a template-copy relationship crucial for kinetochore-microtubule attachment and SAC signaling. Our results illustrate how gene duplication and sub-functionalization shape the workings of an essential molecular network.

Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation.

Fidelity of chromosome segregation relies on coordination of chromosome biorientation and the spindle checkpoint. Central to this is the kinetochore scaffold KNL1 that integrates the functions of various mitotic regulators including BUB1 and BUBR1. We show that KNL1 contains an extensive array of short linear sequence modules that encompass TxxΩ and MELT motifs and that can independently localize BUB1. Engineered KNL1 variants with few modules recruit low levels of BUB1 to kinetochores but support a robust checkpoint. Increasing numbers of modules concomitantly increase kinetochore BUB1 levels and progressively enhance efficiency of chromosome biorientation. Remarkably, normal KNL1 function is maintained by replacing all modules with a short array of naturally occurring or identical, artificially designed ones. A minimal array of generic BUB recruitment modules in KNL1 thus suffices for accurate chromosome segregation. Widespread divergence in the amount and sequence of these modules in KNL1 homologues may represent flexibility in adapting regulation of mitotic processes to altered requirements for chromosome segregation during evolution.

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B.

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization.

Integration of kinase and phosphatase activities by BUBR1 ensures formation of stable kinetochore-microtubule attachments.

Maintenance of chromosomal stability depends on error-free chromosome segregation. The pseudokinase BUBR1 is essential for this, because it is a core component of the mitotic checkpoint and is required for formation of stable kinetochore-microtubule attachments. We have identified a conserved and highly phosphorylated domain (KARD) in BUBR1 that is crucial for formation of kinetochore-microtubule attachments. Deletion of this domain or prevention of its phosphorylation abolishes formation of kinetochore microtubules, which can be reverted by inhibiting Aurora B activity. Phosphorylation of KARD by PLK1 promotes direct interaction of BUBR1 with the PP2A-B56α phosphatase that counters excessive Aurora B activity at kinetochores. As a result, removal of BUBR1 from mitotic cells or inhibition of PLK1 reduces PP2A-B56α kinetochore binding and elevates phosphorylation of Aurora B substrates on the outer kinetochore. We propose that PLK1 and BUBR1 cooperate to stabilize kinetochore-microtubule interactions by regulating PP2A-B56α-mediated dephosphorylation of Aurora B substrates at the kinetochore-microtubule interface.

Evolution and function of the mitotic checkpoint.

The mitotic checkpoint evolved to prevent cell division when chromosomes have not established connections with the chromosome segregation machinery. Many of the fundamental molecular principles that underlie the checkpoint, its spatiotemporal activation, and its timely inactivation have been uncovered. Most of these are conserved in eukaryotes, but important differences between species exist. Here we review current concepts of mitotic checkpoint activation and silencing. Guided by studies in model organisms and our phylogenomics analysis of checkpoint constituents and their functional domains and motifs, we highlight ancient and taxa-specific aspects of the core checkpoint modules in the context of mitotic checkpoint function.

The vertebrate mitotic checkpoint protein BUBR1 is an unusual pseudokinase.

Chromosomal stability is safeguarded by a mitotic checkpoint, of which BUB1 and Mad3/BUBR1 are core components. These paralogs have similar, but not identical, domain organization. We show that Mad3/BUBR1 and BUB1 paralogous pairs arose by nine independent gene duplications throughout evolution, followed by parallel subfunctionalization in which preservation of the ancestral, amino-terminal KEN box or kinase domain was mutually exclusive. In one exception, vertebrate BUBR1-defined by the KEN box-preserved the kinase domain but allowed nonconserved degeneration of catalytic motifs. Although BUBR1 evolved to a typical pseudokinase in some vertebrates, it retained the catalytic triad in humans. However, we show that putative catalysis by human BUBR1 is dispensable for error-free chromosome segregation. Instead, residues that interact with ATP in conventional kinases are essential for conformational stability in BUBR1. We propose that parallel evolution of BUBR1 orthologs rendered its kinase function dispensable in vertebrates, producing an unusual, triad-containing pseudokinase.

Regulated targeting of protein phosphatase 1 to the outer kinetochore by KNL1 opposes Aurora B kinase.

Regulated interactions between kinetochores and spindle microtubules are essential to maintain genomic stability during chromosome segregation. The Aurora B kinase phosphorylates kinetochore substrates to destabilize kinetochore-microtubule interactions and eliminate incorrect attachments. These substrates must be dephosphorylated to stabilize correct attachments, but how opposing kinase and phosphatase activities are coordinated at the kinetochore is unknown. Here, we demonstrate that a conserved motif in the kinetochore protein KNL1 directly interacts with and targets protein phosphatase 1 (PP1) to the outer kinetochore. PP1 recruitment by KNL1 is required to dephosphorylate Aurora B substrates at kinetochores and stabilize microtubule attachments. PP1 levels at kinetochores are regulated and inversely proportional to local Aurora B activity. Indeed, we demonstrate that phosphorylation of KNL1 by Aurora B disrupts the KNL1-PP1 interaction. In total, our results support a positive feedback mechanism by which Aurora B activity at kinetochores not only targets substrates directly, but also prevents localization of the opposing phosphatase.