Signaling protein abundance modulates the strength of the spindle assembly checkpoint.

During mitosis, unattached kinetochores in a dividing cell signal to the spindle assembly checkpoint (SAC) to delay anaphase onset and prevent chromosome missegregation.1,2,3,4 The signaling activity of these kinetochores and the likelihood of chromosome missegregation depend on the amount of SAC signaling proteins each kinetochore recruits.5,6,7,8 Therefore, factors that control SAC protein recruitment must be thoroughly understood. Phosphoregulation of kinetochore and SAC signaling proteins due to the concerted action of many kinases and phosphatases is a significant determinant of the SAC protein recruitment to signaling kinetochores.9 Whether the abundance of SAC proteins also influences the recruitment and signaling activity of human kinetochores has not been studied.8,10 Here, we reveal that the low cellular abundance of the SAC signaling protein Bub1 limits its own recruitment and that of BubR1 and restricts the SAC signaling activity of the kinetochore. Conversely, Bub1 overexpression results in higher recruitment of SAC proteins, producing longer delays in anaphase onset. We also find that the number of SAC proteins recruited by a signaling kinetochore is inversely correlated with the total number of signaling kinetochores in the cell. This correlation likely arises from the competition among the signaling kinetochores to recruit from a limited pool of signaling proteins, including Bub1. The inverse correlation may allow the dividing cell to prevent a large number of signaling kinetochores in early prophase from generating an overly large signal while enabling the last unaligned kinetochore in late prometaphase to signal at the maximum strength.

The structural flexibility of MAD1 facilitates the assembly of the Mitotic Checkpoint Complex.

The spindle assembly checkpoint (SAC) safeguards the genome during cell division by generating an effector molecule known as the Mitotic Checkpoint Complex (MCC). The MCC comprises two subcomplexes: BUBR1:BUB3 and CDC20:MAD2, and the formation of CDC20:MAD2 is the rate-limiting step during MCC assembly. Recent studies show that the rate of CDC20:MAD2 formation is significantly accelerated by the cooperative binding of CDC20 to the SAC proteins MAD1 and BUB1. However, the molecular basis for this acceleration is not fully understood. Here, we demonstrate that the structural flexibility of MAD1 at a conserved hinge near the C-terminus is essential for catalytic MCC assembly. This MAD1 hinge enables the MAD1:MAD2 complex to assume a folded conformation in vivo. Importantly, truncating the hinge reduces the rate of MCC assembly in vitro and SAC signaling in vivo. Conversely, mutations that preserve hinge flexibility retain SAC signaling, indicating that the structural flexibility of the hinge, rather than a specific amino acid sequence, is important for SAC signaling. We summarize these observations as the 'knitting model' that explains how the folded conformation of MAD1:MAD2 promotes CDC20:MAD2 assembly.

BubR1 recruitment to the kinetochore via Bub1 enhances spindle assembly checkpoint signaling.

During mitosis, unattached kinetochores in a dividing cell activate the spindle assembly checkpoint (SAC) and delay anaphase onset by generating the anaphase-inhibitory mitotic checkpoint complex (MCC). These kinetochores generate the MCC by recruiting its constituent proteins, including BubR1. In principle, BubR1 recruitment to signaling kinetochores should increase its local concentration and promote MCC formation. However, in human cells BubR1 is mainly thought to sensitize the SAC to silencing. Whether BubR1 localization to signaling kinetochores by itself enhances SAC signaling remains unknown. Therefore, we used ectopic SAC activation (eSAC) systems to isolate two molecules that recruit BubR1 to the kinetochore, the checkpoint protein Bub1 and the KI and MELT motifs in the kinetochore protein KNL1, and observed their contribution to eSAC signaling. Our quantitative analyses and mathematical modeling show that Bub1-mediated BubR1 recruitment to the human kinetochore promotes SAC signaling and highlight BubR1's dual role of strengthening the SAC directly and silencing it indirectly.

Kre28-Spc105 interaction is essential for Spc105 loading at the kinetochore.

Kinetochore (KTs) are macromolecular protein assemblies that attach sister chromatids to spindle microtubules (MTs) and mediate accurate chromosome segregation during mitosis. The outer KT consists of the KMN network, a protein super-complex comprising Knl1 (yeast Spc105), Mis12 (yeast Mtw1), and Ndc80 (yeast Ndc80), which harbours sites for MT binding. Within the KMN network, Spc105 acts as an interaction hub of components involved in spindle assembly checkpoint (SAC) signalling. It is known that Spc105 forms a complex with KT component Kre28. However, where Kre28 physically localizes in the budding yeast KT is not clear. The exact function of Kre28 at the KT is also unknown. Here, we investigate how Spc105 and Kre28 interact and how they are organized within bioriented yeast KTs using genetics and cell biological experiments. Our microscopy data show that Spc105 and Kre28 localize at the KT with a 1 : 1 stoichiometry. We also show that the Kre28-Spc105 interaction is important for Spc105 protein turn-over and essential for their mutual recruitment at the KTs. We created several truncation mutants of kre28 that affect Spc105 loading at the KTs. When over-expressed, these mutants sustain the cell viability, but SAC signalling and KT biorientation are impaired. Therefore, we conclude that Kre28 contributes to chromosome biorientation and high-fidelity segregation at least indirectly by regulating Spc105 localization at the KTs.

Aurora B phosphorylates Bub1 to promote spindle assembly checkpoint signaling.

Accurate chromosome segregation during cell division requires amphitelic chromosome attachment to the spindle apparatus. It is ensured by the combined activity of the spindle assembly checkpoint (SAC),1 a signaling mechanism that delays anaphase onset in response to unattached chromosomes, and an error correction mechanism that eliminates syntelic attachments.2 The SAC becomes active when Mps1 kinase sequentially phosphorylates the kinetochore protein Spc105/KNL1 and the signaling proteins that Spc105/KNL1 recruits to facilitate the production of the mitotic checkpoint complex (MCC).3-8 The error correction mechanism is regulated by the Aurora B kinase, but Aurora B also promotes SAC signaling via indirect mechanisms.9-12 Here we present evidence that Aurora B kinase activity directly promotes MCC production by working downstream of Mps1 in budding yeast and human cells. Using the ectopic SAC activation (eSAC) system, we find that the conditional dimerization of Aurora B in budding yeast and an Aurora B recruitment domain in HeLa cells with either Bub1 or Mad1, but not the phosphodomain of Spc105/KNL1, leads to ectopic MCC production and mitotic arrest.13-16 Importantly, Bub1 must recruit both Mad1 and Cdc20 for this ectopic signaling activity. These and other data show that Aurora B cooperates with Bub1 to promote MCC production, but only after Mps1 licenses Bub1 recruitment to the kinetochore. This direct involvement of Aurora B in SAC signaling may maintain SAC signaling even after Mps1 activity in the kinetochore is lowered.

Microtubule Attachment and Centromeric Tension Shape the Protein Architecture of the Human Kinetochore.

The nanoscale protein architecture of the kinetochore plays an integral role in specifying the mechanisms underlying its functions in chromosome segregation. However, defining this architecture in human cells remains challenging because of the large size and compositional complexity of the kinetochore. Here, we use Förster resonance energy transfer to reveal the architecture of individual kinetochore-microtubule attachments in human cells. We find that the microtubule-binding domains of the Ndc80 complex cluster at the microtubule plus end. This clustering occurs only after microtubule attachment, and it increases proportionally with centromeric tension. Surprisingly, Ndc80 complex clustering is independent of the organization and number of its centromeric receptors. Moreover, this clustering is similar in yeast and human kinetochores despite significant differences in their centromeric organizations. These and other data suggest that the microtubule-binding interface of the human kinetochore behaves like a flexible "lawn" despite being nucleated by repeating biochemical subunits.

The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness of the spindle assembly checkpoint.

During mitosis, the Spindle Assembly Checkpoint (SAC) maintains genome stability while also ensuring timely anaphase onset. To maintain genome stability, the SAC must be strong to delay anaphase even if just one chromosome is unattached, but for timely anaphase onset, it must promptly respond to silencing mechanisms. How the SAC meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of 'MELT' motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Many strong MELT motifs per Spc105/KNL1 minimize chromosome missegregation, but too many delay anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for much more accurate chromosome segregation. We propose that the necessity of balancing SAC strength and responsiveness drives the dual evolutionary trend of the amplification of MELT motif number, but degeneration of their functionally optimal amino acid sequence.

Delineating the contribution of Spc105-bound PP1 to spindle checkpoint silencing and kinetochore microtubule attachment regulation.

Accurate chromosome segregation during cell division requires the spindle assembly checkpoint (SAC), which detects unattached kinetochores, and an error correction mechanism that destabilizes incorrect kinetochore-microtubule attachments. While the SAC and error correction are both regulated by protein phosphatase 1 (PP1), which silences the SAC and stabilizes kinetochore-microtubule attachments, how these distinct PP1 functions are coordinated remains unclear. Here, we investigate the contribution of PP1, docked on its conserved kinetochore receptor Spc105/Knl1, to SAC silencing and attachment regulation. We find that Spc105-bound PP1 is critical for SAC silencing but dispensable for error correction; in fact, reduced PP1 docking on Spc105 improved chromosome segregation and viability of mutant/stressed states. We additionally show that artificially recruiting PP1 to Spc105/Knl1 before, but not after, chromosome biorientation interfered with error correction. These observations lead us to propose that recruitment of PP1 to Spc105/Knl1 is carefully regulated to ensure that chromosome biorientation precedes SAC silencing, thereby ensuring accurate chromosome segregation.

Ectopic Activation of the Spindle Assembly Checkpoint Signaling Cascade Reveals Its Biochemical Design.

Switch-like activation of the spindle assembly checkpoint (SAC) is critical for accurate chromosome segregation and for cell division in a timely manner. To determine the mechanisms that achieve this, we engineered an ectopic, kinetochore-independent SAC activator: the "eSAC." The eSAC stimulates SAC signaling by artificially dimerizing Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the kinetochore signaling scaffold. By exploiting variable eSAC expression in a cell population, we defined the dependence of the eSAC-induced mitotic delay on eSAC concentration in a cell to reveal the dose-response behavior of the core signaling cascade of the SAC. These quantitative analyses and subsequent mathematical modeling of the dose-response data uncover two crucial properties of the core SAC signaling cascade: (1) a cellular limit on the maximum anaphase-inhibitory signal that the cascade can generate due to the limited supply of SAC proteins and (2) the ability of the KNL1 phosphodomain to produce the anaphase-inhibitory signal synergistically, when it recruits multiple SAC proteins simultaneously. We propose that these properties together achieve inverse, non-linear scaling between the signal output per kinetochore and the number of signaling kinetochores. When the number of kinetochores is low, synergistic signaling by KNL1 enables each kinetochore to produce a disproportionately strong signal output. However, when many kinetochores signal concurrently, they compete for a limited supply of SAC proteins. This frustrates synergistic signaling and lowers their signal output. Thus, the signaling activity of unattached kinetochores will adapt to the changing number of signaling kinetochores to enable the SAC to approximate switch-like behavior.

Stu2 acts as a microtubule destabilizer in metaphase budding yeast spindles.

The microtubule-associated protein Stu2 (XMAP215) has the remarkable ability to act either as a polymerase or as a destabilizer of the microtubule plus end. In budding yeast, it is required for the dynamicity of spindle microtubules and also for kinetochore force generation. To understand how Stu2 contributes to these distinct activities, we analyzed the contributions of its functional domains to its localization and function. We find that Stu2 colocalizes with kinetochores using its TOG domains, which bind GTP-tubulin, a coiled-coil homodimerization domain, and a domain that interacts with plus-end interacting proteins. Stu2 localization is also promoted by phosphorylation at a putative CDK1 phosphorylation site located within its microtubule-binding basic patch. Surprisingly, however, we find that kinetochore force generation is uncorrelated with the amount of kinetochore-colocalized Stu2. These and other data imply that Stu2 colocalizes with kinetochores by recognizing growing microtubule plus ends within yeast kinetochores. We propose that Stu2 destabilizes these plus ends to indirectly contribute to the "catch-bond" activity of the kinetochores.

How Kinetochore Architecture Shapes the Mechanisms of Its Function.

The eukaryotic kinetochore is a sophisticated multi-protein machine that segregates chromosomes during cell division. To ensure accurate chromosome segregation, it performs three major functions using disparate molecular mechanisms. It operates a mechanosensitive signaling cascade known as the spindle assembly checkpoint (SAC) to detect and signal the lack of attachment to spindle microtubules, and delay anaphase onset in response. In addition, after attaching to spindle microtubules, the kinetochore generates the force necessary to move chromosomes. Finally, if the two sister kinetochores on a chromosome are both attached to microtubules emanating from the same spindle pole, they activate another mechanosensitive mechanism to correct the monopolar attachments. All three of these functions maintain genome stability during cell division. The outlines of the biochemical activities responsible for these functions are now available. How the kinetochore integrates the underlying molecular mechanisms is still being elucidated. In this Review, we discuss how the nanoscale protein organization in the kinetochore, which we refer to as kinetochore 'architecture', organizes its biochemical activities to facilitate the realization and integration of emergent mechanisms underlying its three major functions. For this discussion, we will use the relatively simple budding yeast kinetochore as a model, and extrapolate insights gained from this model to elucidate functional roles of the architecture of the much more complex human kinetochore.

A Cell Biological Perspective on Past, Present and Future Investigations of the Spindle Assembly Checkpoint.

The spindle assembly checkpoint (SAC) is a quality control mechanism that ensures accurate chromosome segregation during cell division. It consists of a mechanochemical signal transduction mechanism that senses the attachment of chromosomes to the spindle, and a signaling cascade that inhibits cell division if one or more chromosomes are not attached. Extensive investigations of both these component systems of the SAC have synthesized a comprehensive understanding of the underlying molecular mechanisms. This review recounts the milestone results that elucidated the SAC, compiles a simple model of the complex molecular machinery underlying the SAC, and highlights poorly understood facets of the biochemical design and cell biological operation of the SAC that will drive research forward in the near future.

Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore.

Recruitment of spindle assembly checkpoint (SAC) proteins by an unattached kinetochore leads to SAC activation. This recruitment is licensed by the Mps1 kinase, which phosphorylates the kinetochore protein Spc105 at one or more of its six MELT repeats. Spc105 then recruits the Bub3-Bub1 and Mad1-Mad2 complexes, which produce the inhibitory signal that arrests cell division. The strength of this signal depends, in part, on the number of Bub3-Bub1 and Mad1-Mad2 molecules that Spc105 recruits. Therefore regulation of this recruitment will influence SAC signaling. To understand this regulation, we established the physiological binding curves that describe the binding of Bub3-Bub1 and Mad1-Mad2 to the budding yeast kinetochore. We find that the binding of both follows the mass action law. Mps1 likely phosphorylates all six MELT repeats of Spc105. However, two mechanisms prevent Spc105 from recruiting six Bub3-Bub1 molecules: low Bub1 abundance and hindrance in the binding of more than one Bub3-Bub1 molecule to the same Spc105. Surprisingly, the kinetochore recruits two Mad1-Mad2 heterotetramers for every Bub3-Bub1 molecule. Finally, at least three MELT repeats per Spc105 are needed for accurate chromosome segregation. These data reveal that kinetochore-intrinsic and -extrinsic mechanisms influence the physiological operation of SAC signaling, potentially to maximize chromosome segregation accuracy.

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds.

DNA origami provides a versatile platform for conducting 'architecture-function' analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.

The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling.

The spindle assembly checkpoint (SAC) is a unique signalling mechanism that responds to the state of attachment of the kinetochore to spindle microtubules. SAC signalling is activated by unattached kinetochores, and it is silenced after these kinetochores form end-on microtubule attachments. Although the biochemical cascade of SAC signalling is well understood, how kinetochore-microtubule attachment disrupts it remained unknown. Here we show that, in budding yeast, end-on microtubule attachment to the kinetochore physically separates the Mps1 kinase, which probably binds to the calponin homology domain of Ndc80, from the kinetochore substrate of Mps1, Spc105 (KNL1 orthologue). This attachment-mediated separation disrupts the phosphorylation of Spc105, and enables SAC silencing. Additionally, the Dam1 complex may act as a barrier that shields Spc105 from Mps1. Together these data suggest that the protein architecture of the kinetochore encodes a mechanical switch. End-on microtubule attachment to the kinetochore turns this switch off to silence the SAC.

Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET.

BACKGROUND: The kinetochore is a multiprotein machine that couples chromosome movement to microtubule (MT) polymerization and depolymerization. It uses numerous copies of at least three MT-binding proteins to generate bidirectional movement. The nanoscale organization of these proteins within the kinetochore plays an important role in shaping the mechanisms that drive persistent, bidirectional movement of the kinetochore. RESULTS: We used fluorescence resonance energy transfer (FRET) between genetically encoded fluorescent proteins fused to kinetochore subunits to reconstruct the nanoscale organization of the budding yeast kinetochore. We performed >60 FRET and high-resolution colocalization measurements involving the essential MT-binding kinetochore components: Ndc80, Dam1, Spc105, and Stu2. These measurements reveal that neighboring Ndc80 complexes within the kinetochore are narrowly distributed along the length of the MT. Dam1 complex molecules are concentrated near the MT-binding domains of Ndc80. Stu2 localizes in high abundance within a narrowly defined territory within the kinetochore centered ∼20 nm on the centromeric side of the Dam1 complex. CONCLUSIONS: Our data show that the MT attachment site of the budding yeast kinetochore is well organized. Ndc80, Dam1, and Stu2 are all narrowly distributed about their average positions along the kinetochore-MT axis. The relative organization of these components, their narrow distributions, and their known MT-binding properties together elucidate how their combined actions generate persistent, bidirectional kinetochore movement coupled to MT polymerization and depolymerization.

A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines.

Macromolecular machines participate in almost every cell biological function. These machines can take the form of well-defined protein structures such as the kinetochore, or more loosely organized protein assemblies like the endocytic coat. The protein architecture of these machines-the arrangement of multiple copies of protein subunits at the nanoscale, is necessary for understanding their cell biological function and biophysical mechanism. Defining this architecture in vivo presents a major challenge. High density of protein molecules within macromolecular machines severely limits the effectiveness of super-resolution microscopy. However, this density is ideal for Forster Resonance Energy Transfer (FRET), which can determine the proximity between neighboring molecules. Here, we present a simple FRET quantitation scheme that calibrates a standard epifluorescence microscope for measuring donor-acceptor separations. This calibration can be used to deduce FRET efficiency fluorescence intensity measurements. This method will allow accurate determination of FRET efficiency over a wide range of values and FRET pair number. It will also allow dynamic FRET measurements with high spatiotemporal resolution under cell biological conditions. Although the poor maturation efficiency of genetically encoded fluorescent proteins presents a challenge, we show that its effects can be alleviated. To demonstrate this methodology, we probe the in vivo architecture of the γ-Tubulin Ring. Our technique can be applied to study the architecture and dynamics of a wide range of macromolecular machines.

Plasticity and epigenetic inheritance of centromere-specific histone H3 (CENP-A)-containing nucleosome positioning in the fission yeast.

Nucleosomes containing the specific histone H3 variant CENP-A mark the centromere locus on each chromatin and initiate kinetochore assembly. For the common type of regional centromeres, little is known in molecular detail of centromeric chromatin organization, its propagation through cell division, and how distinct organization patterns may facilitate kinetochore assembly. Here, we show that in the fission yeast S. pombe, a relatively small number of CENP-A/Cnp1 nucleosomes are found within the centromeric core and that their positioning relative to underlying DNA varies among genetically homogenous cells. Consistent with the flexible positioning of Cnp1 nucleosomes, a large portion of the endogenous centromere is dispensable for its essential activity in mediating chromosome segregation. We present biochemical evidence that Cnp1 occupancy directly correlates with silencing of the underlying reporter genes. Furthermore, using a newly developed pedigree analysis assay, we demonstrated the epigenetic inheritance of Cnp1 positioning and quantified the rate of occasional repositioning of Cnp1 nucleosomes throughout cell generations. Together, our results reveal the plasticity and the epigenetically inheritable nature of centromeric chromatin organization.

The budding yeast point centromere associates with two Cse4 molecules during mitosis.

The centromere is defined by the incorporation of the centromere-specific histone H3 variant centromere protein A (CENP-A). Like histone H3, CENP-A can form CENP-A-H4 heterotetramers in vitro. However, the in vivo conformation of CENP-A chromatin has been proposed by different studies as hemisomes, canonical, or heterotypic nucleosomes. A clear understanding of the in vivo architecture of CENP-A chromatin is important, because it influences the molecular mechanisms of the assembly and maintenance of the centromere and its function in kinetochore nucleation. A key determinant of this architecture is the number of CENP-A molecules bound to the centromere. Accurate measurement of this number can limit possible centromere architectures. The genetically defined point centromere in the budding yeast Saccharomyces cerevisiae provides a unique opportunity to define this number accurately, as this 120-bp-long centromere can at the most form one nucleosome or hemisome. Using novel live-cell fluorescence microscopy assays, we demonstrate that the budding yeast centromere recruits two Cse4 (ScCENP-A) molecules. These molecules are deposited during S phase and they remain stably bound through late anaphase. Our studies suggest that the budding yeast centromere incorporates a Cse4-H4 tetramer.