ABSTRACT
Faithful chromosome segregation requires that sister chromatids establish bi-oriented kinetochore-microtubule attachments. The spindle assembly checkpoint (SAC) prevents premature anaphase onset with incomplete attachments. However, how microtubule attachment and checkpoint signaling are coordinated remains unclear. The conserved kinase Mps1 initiates SAC signaling by localizing transiently to kinetochores in prometaphase and is released upon bi-orientation. Using biochemistry, structure predictions, and cellular assays, we shed light on this dynamic behavior in Saccharomyces cerevisiae. A conserved N-terminal segment of Mps1 binds the neck region of Ndc80:Nuf2, the main microtubule receptor of kinetochores. Mutational disruption of this interface, located at the backside of the paired CH domains and opposite the microtubule-binding site, prevents Mps1 localization, eliminates SAC signaling, and impairs growth. The same interface of Ndc80:Nuf2 binds the microtubule-associated Dam1 complex. We demonstrate that the error correction kinase Ipl1/Aurora B controls the competition between Dam1 and Mps1 for the same binding site. Thus, binding of the Dam1 complex to Ndc80:Nuf2 may release Mps1 from the kinetochore to promote anaphase onset.
Subject(s)
Cell Cycle Proteins , Kinetochores , Microtubules , Protein Serine-Threonine Kinases , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Kinetochores/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , Microtubules/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubule-Associated Proteins/genetics , Nuclear ProteinsABSTRACT
Accurate chromosome segregation in mitosis relies on sister kinetochores forming stable attachments to microtubules (MTs) extending from opposite spindle poles and establishing biorientation. To achieve this, erroneous kinetochore-MT interactions must be resolved through a process called error correction, which dissolves improper kinetochore-MT attachment and allows new interactions until biorientation is achieved. The Aurora B kinase plays key roles in driving error correction by phosphorylating Dam1 and Ndc80 complexes, while Mps1 kinase, Stu2 MT polymerase and phosphatases also regulate this process. Once biorientation is formed, tension is applied to kinetochore-MT interaction, stabilizing it. In this review article, we discuss the mechanisms of kinetochore-MT interaction, error correction and biorientation. We focus mainly on recent insights from budding yeast, where the attachment of a single MT to a single kinetochore during biorientation simplifies the analysis of error correction mechanisms.
Subject(s)
Saccharomyces cerevisiae Proteins , Saccharomycetales , Saccharomyces cerevisiae/genetics , Kinetochores , Microtubules/genetics , Mitosis , Chromosome Segregation , Saccharomyces cerevisiae Proteins/geneticsABSTRACT
For correct chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (biorientation). For this, aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase. Once biorientation is formed, tension is applied on kinetochore-microtubule interaction, stabilizing this interaction. The mechanism for this tension-dependent process has been debated. Here, we study how Aurora B localizations at different kinetochore sites affect the biorientation establishment and maintenance in budding yeast. Without the physiological Aurora B-INCENP recruitment mechanisms, engineered recruitment of Aurora B-INCENP to the inner kinetochore, but not to the outer kinetochore, prior to biorientation supports the subsequent biorientation establishment. Moreover, when the physiological Aurora B-INCENP recruitment mechanisms are present, an engineered Aurora B-INCENP recruitment to the outer kinetochore, but not to the inner kinetochore, during metaphase (after biorientation establishment) disrupts biorientation, which is dependent on the Aurora B kinase activity. These results suggest that the spatial separation of Aurora B from its outer kinetochore substrates is required to stabilize kinetochore-microtubule interaction when biorientation is formed and tension is applied on this interaction. Meanwhile, Aurora B exhibits dynamic turnover on the centromere/kinetochore during early mitosis, a process thought to be crucial for error correction and biorientation. However, using the engineered Aurora B-INCENP recruitment to the inner kinetochore, we demonstrate that, even without such a turnover, Aurora B-INCENP can efficiently support biorientation. Our study provides important insights into how Aurora B promotes error correction for biorientation in a tension-dependent manner.
Subject(s)
Chromosome Segregation , Kinetochores , Aurora Kinase B/genetics , Centromere , Microtubules , MitosisABSTRACT
Cell division events require regulatory systems to ensure that events happen in a distinct order. The classic view of temporal control of the cell cycle posits that cells order events by linking them to changes in Cyclin Dependent Kinase (CDK) activities. However, a new paradigm is emerging from studies of anaphase where chromatids separate at the central metaphase plate and then move to opposite poles of the cell. These studies suggest that distinct events are ordered depending upon the location of each chromosome along its journey from the central metaphase plate to the elongated spindle poles. This system is dependent upon a gradient of Aurora B kinase activity that emerges during anaphase and acts as a spatial beacon to control numerous anaphase/telophase events and cytokinesis. Recent studies also suggest that Aurora A kinase activity specifies proximity of chromosomes or proteins to spindle poles during prometaphase. Together these studies argue that a key role for Aurora kinases is to provide spatial information that controls events depending upon the location of chromosomes or proteins along the mitotic spindle.
ABSTRACT
For correct chromosome segregation in mitosis, eukaryotic cells must establish chromosome biorientation where sister kinetochores attach to microtubules extending from opposite spindle poles. To establish biorientation, any aberrant kinetochore-microtubule interactions must be resolved in the process called error correction. For resolution of the aberrant interactions in error correction, kinetochore-microtubule interactions must be exchanged until biorientation is formed (the SWAP process). At initiation of biorientation, the state of weak kinetochore-microtubule interactions should be converted to the state of stable interactions (the SWITCH process)-the conundrum of this conversion is called the initiation problem of biorientation. Once biorientation is established, tension is applied on kinetochore-microtubule interactions, which stabilizes the interactions (the STABILIZE process). Aurora B kinase plays central roles in promoting error correction, and Mps1 kinase and Stu2 microtubule polymerase also play important roles. In this article, we review mechanisms of error correction by considering the SWAP, SWITCH, and STABILIZE processes. We mainly focus on mechanisms found in budding yeast, where only one microtubule attaches to a single kinetochore at biorientation, making the error correction mechanisms relatively simpler.
Subject(s)
Kinetochores , Microtubules , Aurora Kinase B/genetics , Chromosome Segregation , MitosisABSTRACT
Correct chromosome segregation in mitosis relies on chromosome biorientation, in which sister kinetochores attach to microtubules from opposite spindle poles prior to segregation. To establish biorientation, aberrant kinetochore-microtubule interactions must be resolved through the error correction process. During error correction, kinetochore-microtubule interactions are exchanged (swapped) if aberrant, but the exchange must stop when biorientation is established. In this article, we discuss recent findings in budding yeast, which have revealed fundamental molecular mechanisms promoting this "swap and stop" process for error correction. Where relevant, we also compare the findings in budding yeast with mechanisms in higher eukaryotes. Evidence suggests that Aurora B kinase differentially regulates kinetochore attachments to the microtubule end and its lateral side and switches relative strength of the two kinetochore-microtubule attachment modes, which drives the exchange of kinetochore-microtubule interactions to resolve aberrant interactions. However, Aurora B kinase, recruited to centromeres and inner kinetochores, cannot reach its targets at kinetochore-microtubule interface when tension causes kinetochore stretching, which stops the kinetochore-microtubule exchange once biorientation is established.
Subject(s)
Kinetochores , Saccharomycetales , Aurora Kinase B/genetics , Chromosome Segregation , Microtubules/physiology , MitosisABSTRACT
Mitotic spindle is a self-assembling macromolecular machine responsible for the faithful segregation of chromosomes during cell division. Assembly of the spindle is believed to be governed by the 'Search & Capture' (S&C) principle in which dynamic microtubules explore space in search of kinetochores while the latter capture microtubules and thus connect chromosomes to the spindle. Due to the stochastic nature of the encounters between kinetochores and microtubules, the time required for incorporating all chromosomes into the spindle is profoundly affected by geometric constraints, such as the size and shape of kinetochores as well as their distribution in space at the onset of spindle assembly. In recent years, several molecular mechanisms that control these parameters have been discovered. It is now clear that stochastic S&C takes place in structured space, where components are optimally distributed and oriented to minimize steric hindrances. Nucleation of numerous non-centrosomal microtubules near kinetochores accelerates capture, while changes in the kinetochore architecture at various stages of spindle assembly promote proper connection of sister kinetochores to the opposite spindle poles. Here we discuss how the concerted action of multiple facilitating mechanisms ensure that the spindle assembles rapidly yet with a minimal number of errors.
Subject(s)
Kinetochores/metabolism , Morphogenesis/physiology , Spindle Apparatus/metabolism , HumansABSTRACT
During mitosis, the identical sister chromatids of each chromosome must attach through their kinetochores to microtubules emanating from opposite spindle poles. This process, referred to as chromosome biorientation, is essential for equal partitioning of the genetic information to the two daughter cells. Defects in chromosome biorientation can give rise to aneuploidy, a hallmark of cancer and genetic diseases. A conserved surveillance mechanism called spindle assembly checkpoint (SAC) prevents the onset of anaphase until biorientation is attained. Key to chromosome biorientation is an error correction mechanism that allows kinetochores to establish proper bipolar attachments by disengaging faulty kinetochore-microtubule connections. Error correction relies on the Aurora B and Mps1 kinases that also promote SAC signaling, raising the possibility that they are part of a single sensory device responding to improper attachments and concomitantly controlling both their disengagement and a temporary mitotic arrest. In budding yeast, Aurora B and Mps1 promote error correction independently from one another, but while the substrates of Aurora B in this process are at least partially known, the mechanism underlying the involvement of Mps1 in the error correction pathway is unknown. Through the characterization of a novel mps1 mutant and an unbiased genetic screen for extragenic suppressors, we recently gained evidence that a common mechanism based on Mps1-dependent phosphorylation of the Knl1/Spc105 kinetochore scaffold and subsequent recruitment of the Bub1 kinase is critical for the function of Mps1 in chromosome biorientation as well as for SAC activation (Benzi et al. EMBO Rep, 2020).
Subject(s)
Aurora Kinase B/genetics , Chromosome Segregation/genetics , M Phase Cell Cycle Checkpoints/genetics , Microtubule-Associated Proteins/genetics , Protein Serine-Threonine Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Humans , Kinetochores/metabolism , Phosphorylation/genetics , Saccharomycetales/geneticsABSTRACT
The Mps1 kinase corrects improper kinetochore-microtubule attachments, thereby ensuring chromosome biorientation. Yet, its critical phosphorylation targets in this process remain largely elusive. Mps1 also controls the spindle assembly checkpoint (SAC), which halts chromosome segregation until biorientation is attained. Its role in SAC activation is antagonised by the PP1 phosphatase and involves phosphorylation of the kinetochore scaffold Knl1/Spc105, which in turn recruits the Bub1 kinase to promote assembly of SAC effector complexes. A crucial question is whether error correction and SAC activation are part of a single or separable pathways. Here, we isolate and characterise a new yeast mutant, mps1-3, that is severely defective in chromosome biorientation and SAC signalling. Through an unbiased screen for extragenic suppressors, we found that mutations lowering PP1 levels at Spc105 or forced association of Bub1 with Spc105 reinstate both chromosome biorientation and SAC signalling in mps1-3 cells. Our data argue that a common mechanism based on Knl1/Spc105 phosphorylation is critical for Mps1 function in error correction and SAC signalling, thus supporting the idea that a single sensory apparatus simultaneously elicits both pathways.
Subject(s)
Chromosome Segregation , Saccharomyces cerevisiae Proteins , Cell Cycle Proteins/genetics , Kinetochores , M Phase Cell Cycle Checkpoints/genetics , Saccharomyces cerevisiae Proteins/genetics , Spindle Apparatus/geneticsABSTRACT
Polo-like kinase 1 (PLK1) plays a fundamental role in the spatiotemporal control of mitosis. Cells lacking PLK1 activity exhibit characteristic chromosome misalignment due to defects in microtubule-kinetochore organization and attachment. In our recently published paper, we uncover a new role for PLK1 in the preservation and maintenance of centromere integrity.
ABSTRACT
For proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3-6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.
Subject(s)
Aurora Kinases/genetics , Centromere/metabolism , Kinetochores/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Aurora Kinases/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
The chromosomal passenger complex (CPC) is a key regulator of eukaryotic cell division, consisting of the protein kinase Aurora B/Ipl1 in association with its activator (INCENP/Sli15) and two additional proteins (Survivin/Bir1 and Borealin/Nbl1). Here, we report a genome-wide genetic interaction screen in Saccharomyces cerevisiae using the bir1-17 mutant, identifying through quantitative fitness analysis deletion mutations that act as enhancers and suppressors. Gene knockouts affecting the Ctf19 kinetochore complex were identified as the strongest enhancers of bir1-17, while mutations affecting the large ribosomal subunit or the mRNA nonsense-mediated decay pathway caused strong phenotypic suppression. Thus, cells lacking a functional Ctf19 complex become highly dependent on Bir1 function and vice versa. The negative genetic interaction profiles of bir1-17 and the cohesin mutant mcd1-1 showed considerable overlap, underlining the strong functional connection between sister chromatid cohesion and chromosome biorientation. Loss of some Ctf19 components, such as Iml3 or Chl4, impacted differentially on bir1-17 compared with mutations affecting other CPC components: despite the synthetic lethality shown by either iml3∆ or chl4∆ in combination with bir1-17, neither gene knockout showed any genetic interaction with either ipl1-321 or sli15-3 Our data therefore imply a specific functional connection between the Ctf19 complex and Bir1 that is not shared with Ipl1.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Genetic Fitness , Kinetochores/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/physiology , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/metabolism , Epistasis, Genetic , Gene Deletion , Gene Expression , Genetic Association Studies , Microbial Viability/genetics , Mutation , Phenotype , Protein Binding , CohesinsABSTRACT
The segregation of chromosomes is coordinated at multiple levels to prevent chromosome loss, a phenotype frequently observed in cancers. We recently described an essential role for telomeres in the physical separation of chromosomes and identified Aurora B kinase as a double agent involved in the separation of centromeric and telomeric heterochromatin.