ABSTRACT
The approximately thirty core subunits of kinetochores assemble on centromeric chromatin containing the histone H3 variant CENP-A and connect chromosomes with spindle microtubules. The chromatin proximal 16-subunit CCAN (constitutive centromere associated network) creates a mechanically stable bridge between CENP-A and the kinetochore's microtubule-binding machinery, the 10-subunit KMN assembly. Here, we reconstituted a stoichiometric 11-subunit human CCAN core that forms when the CENP-OPQUR complex binds to a joint interface on the CENP-HIKM and CENP-LN complexes. The resulting CCAN particle is globular and connects KMN and CENP-A in a 26-subunit recombinant particle. The disordered, basic N-terminal tail of CENP-Q binds microtubules and promotes accurate chromosome alignment, cooperating with KMN in microtubule binding. The N-terminal basic tail of the NDC80 complex, the microtubule-binding subunit of KMN, can functionally replace the CENP-Q tail. Our work dissects the connectivity and architecture of CCAN and reveals unexpected functional similarities between CENP-OPQUR and the NDC80 complex.
Subject(s)
Chromosomal Proteins, Non-Histone/ultrastructure , Kinetochores/physiology , Kinetochores/ultrastructure , Centromere/physiology , Centromere Protein A/metabolism , Centromere Protein A/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Cytoskeletal Proteins , HeLa Cells , Humans , Kinetochores/metabolism , Microtubules/metabolism , Microtubules/physiology , Nuclear Proteins/metabolismABSTRACT
Mitotic cell division requires that kinetochores form microtubule attachments that can segregate chromosomes and control mitotic progression via the spindle assembly checkpoint. During prometaphase, kinetochores shed a domain called the fibrous corona as microtubule attachments form. This shedding is mediated, in part, by the minus-end directed motor dynein, which 'strips' cargoes along K-fibre microtubules. Despite its essentiality, little is known about how dynein stripping is regulated and how it responds to attachment maturation. Lis1 (also known as PAFAH1B1) is a conserved dynein regulator that is mutated in the neurodevelopmental disease lissencephaly. Here, we have combined loss-of-function studies, high-resolution imaging and separation-of-function mutants to define how Lis1 contributes to dynein-mediated corona stripping in HeLa cells. Cells depleted of Lis1 fail to disassemble the corona and show a delay in metaphase as a result of persistent checkpoint activation. Furthermore, we find that although kinetochore-tethered Lis1-dynein is required for error-free microtubule attachment, the contribution of Lis1 to corona disassembly can be mediated by a cytoplasmic pool. These findings support the idea that Lis1 drives dynein function at kinetochores to ensure corona disassembly and prevent chromosome mis-segregation.
Subject(s)
1-Alkyl-2-acetylglycerophosphocholine Esterase , Dyneins , Kinetochores , Microtubule-Associated Proteins , Humans , Dyneins/metabolism , HeLa Cells , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints , Microtubule-Associated Proteins/genetics , Microtubule-Associated Proteins/metabolism , Microtubules/metabolism , Mitosis , 1-Alkyl-2-acetylglycerophosphocholine Esterase/genetics , 1-Alkyl-2-acetylglycerophosphocholine Esterase/metabolismABSTRACT
A universal feature of mitosis is that all chromosomes become aligned at the spindle equator--the halfway point between the two spindle poles--prior to anaphase onset. This migratory event is called congression, and is powered by centromere-bound protein machines called kinetochores. This Commentary aims to document recent advances concerning the two kinetochore-based force-generating mechanisms that drive mitotic chromosome congression in vertebrate cells: depolymerisation-coupled pulling (DCP) and lateral sliding. We aim to explore how kinetochores can 'read-out' their spatial position within the spindle, and adjust these force-generating mechanisms to ensure chromosomes reach, and then remain, at the equator. Finally, we will describe the 'life history' of a chromosome, and provide a working model for how individual mechanisms are integrated to ensure efficient and successful congression.
Subject(s)
Chromosome Pairing , Chromosome Segregation , Kinetochores/physiology , Models, Biological , Animals , Centromere , Chromosomal Puffs , HeLa Cells , Humans , Spindle Apparatus , VertebratesABSTRACT
A key step of mitosis is the congression of chromosomes to the spindle equator. Congression is driven by at least two distinct mechanisms: (1) kinetochores slide along the microtubule lattice using the plus-end directed CENP-E motor, and (2) kinetochores biorientating near the pole move to the equator through microtubule depolymerisation-coupled pulling. Here, we show that CENP-Q - a subunit of the CENP-O complex (comprising CENP-O, CENP-P, CENP-Q and CENP-U) that targets polo-like kinase (Plk1) to kinetochores - is also required for the recruitment of CENP-E to kinetochores. We further reveal a CENP-E recruitment-independent role for CENP-Q in depolymerisation-coupled pulling. Both of these functions are abolished by a single point mutation in CENP-Q (S50A) - a residue that is phosphorylated in vivo. Importantly, the S50A mutant does not affect the loading of Plk1 onto kinetochores and leaves the CENP-O complex intact. Thus, the functions of CENP-Q in CENP-E loading and depolymerisation-coupled pulling are independent from its role in Plk1 recruitment and CENP-O complex stabilisation. Taken together, our data provide evidence that phosphoregulation of CENP-Q plays a central function in coordinating chromosome congression mechanisms.
Subject(s)
Cell Cycle/physiology , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Human/metabolism , Kinetochores/metabolism , Multiprotein Complexes/metabolism , Amino Acid Substitution , Cell Cycle Proteins , Chromosomal Proteins, Non-Histone/genetics , Chromosomes, Human/genetics , HeLa Cells , Humans , Multiprotein Complexes/genetics , Mutation, Missense , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins , Polo-Like Kinase 1ABSTRACT
Accurate chromosome segregation demands efficient capture of microtubules by kinetochores and their conversion to stable bioriented attachments that can congress and then segregate chromosomes. An early event is the shedding of the outermost fibrous corona layer of the kinetochore following microtubule attachment. Centromere protein F (CENP-F) is part of the corona, contains two microtubule-binding domains, and physically associates with dynein motor regulators. Here, we have combined CRISPR gene editing and engineered separation-of-function mutants to define how CENP-F contributes to kinetochore function. We show that the two microtubule-binding domains make distinct contributions to attachment stability and force transduction but are dispensable for chromosome congression. We further identify a specialized domain that functions to limit the dynein-mediated stripping of corona cargoes through a direct interaction with Nde1. This antagonistic activity is crucial for maintaining the required corona composition and ensuring efficient kinetochore biorientation.
Subject(s)
Chromosomal Proteins, Non-Histone/genetics , Chromosome Segregation/genetics , Kinetochores , Microfilament Proteins/genetics , Microtubule-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , Chromosomes/genetics , Dyneins/genetics , HeLa Cells , Humans , Microtubules/genetics , Mutant Proteins/genetics , Protein Binding/genetics , Spindle Apparatus/geneticsABSTRACT
Kinetochores mediate chromosome congression by either sliding along the lattice of spindle microtubules or forming end-on attachments to their depolymerizing plus-ends. By following the fates of individual kinetochores as they congress in live cells, we reveal that the Ska complex is required for a distinct substep of the depolymerization-coupled pulling mechanism. Ska depletion increases the frequency of naturally occurring, force-dependent P kinetochore detachment events, while being dispensable for the initial biorientation and movement of chromosomes. In unperturbed cells, these release events are followed by reattachment and successful congression, whereas in Ska-depleted cells, detached kinetochores remain in a futile reattachment/detachment cycle that prevents congression. We further find that Ska is progressively loaded onto bioriented kinetochore pairs as they congress. We thus propose a model in which kinetochores mature through Ska complex recruitment and that this is required for improved load-bearing capacity and silencing of the spindle assembly checkpoint.