The Kinetochore-Microtubule Coupling Machinery Is Repurposed in Sensory Nervous System Morphogenesis.
ABSTRACT: Dynamic coupling of microtubule ends to kinetochores, built on the centromeres of chromosomes, directs chromosome segregation during cell division. Here, we report that the evolutionarily ancient kinetochore-microtubule coupling machine, the KMN (Knl1/Mis12/Ndc80-complex) network, plays a critical role in neuronal morphogenesis. We show that the KMN network concentrates in microtubule-rich dendrites of developing sensory neurons that collectively extend in a multicellular morphogenetic event that occurs during C. elegans embryogenesis. Post-mitotic degradation of KMN components in sensory neurons disrupts dendritic extension, leading to patterning and functional defects in the sensory nervous system. Structure-guided mutations revealed that the molecular interface that couples kinetochores to spindle microtubules also functions in neuronal development. These results identify a cell-division-independent function for the chromosome-segregation machinery and define a microtubule-coupling-dependent event in sensory nervous system morphogenesis.
Project description:The kinetochore provides a vital connection between chromosomes and spindle microtubules [1, 2]. Defining the molecular architecture of the core kinetochore components is critical for understanding the mechanisms by which the kinetochore directs chromosome segregation. The KNL1/Mis12 complex/Ndc80 complex (KMN) network acts as the primary microtubule-binding interface at kinetochores  and provides a platform to recruit regulatory proteins . Recent work found that the inner kinetochore components CENP-C and CENP-T act in parallel to recruit the KMN network to kinetochores [5-8]. However, due to the presence of these dual pathways, it has not been possible to distinguish differences in the nature of kinetochore assembly downstream of CENP-C or CENP-T. Here, we separated these pathways by targeting CENP-C and CENP-T independently to an ectopic chromosomal locus in human cells. Our work reveals that the organization of the KMN network components downstream of CENP-C and CENP-T is distinct. CENP-C recruits the Ndc80 complex through its interactions with KNL1 and the Mis12 complex. In contrast, CENP-T directly interacts with Ndc80, which in turn promotes KNL1/Mis12 complex recruitment through a separate region on CENP-T, resulting in functional relationships for KMN network localization that are inverted relative to the CENP-C pathway. We also find that distinct regulatory paradigms control the assembly of these pathways, with Aurora B kinase promoting KMN network recruitment to CENP-C and cyclin-dependent kinase (CDK) regulating KMN network recruitment to CENP-T. This work reveals unexpected complexity for the architecture and regulation of the core components of the kinetochore-microtubule interface.
Project description:The spindle assembly checkpoint (SAC) arrests cells in mitosis by sensing unattached kinetochores, until all chromosomes are bi-oriented by spindle microtubules. Kinetochore accumulation of the SAC component Mad1-Mad2 is crucial for SAC activation. However, the mechanism by which Mad1-Mad2 accumulation at kinetochores is regulated is not clear. Here we find that Cep57 is localized to kinetochores in human cells, and binds to Mis12, a KMN (KNL1/Mis12 complex/Ndc80 complex) network component. Cep57 also interacts with Mad1, and depletion of Cep57 results in decreased kinetochore localization of Mad1-Mad2, reduced SAC signalling and increased chromosome segregation errors. We also show that the microtubule-binding activity of Cep57 is involved in the timely removal of Mad1 from kinetochores. Thus, these findings reveal that the KMN network-binding protein Cep57 is a mitotic kinetochore component, and demonstrate the functional connection between the KMN network and the SAC.
Project description: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.
Project description:During mitosis, the spindle checkpoint senses kinetochores not properly attached to spindle microtubules and prevents precocious sister-chromatid separation and aneuploidy. The constitutive centromere-associated network (CCAN) at inner kinetochores anchors the KMN network consisting of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C) at outer kinetochores. KMN is a critical kinetochore receptor for both microtubules and checkpoint proteins. Here, we show that nearly complete inactivation of KMN in human cells through multiple strategies produced strong checkpoint defects even when all kinetochores lacked microtubule attachment. These KMN-inactivating strategies reveal multiple KMN assembly mechanisms at human mitotic kinetochores. In one mechanism, the centromeric kinase Aurora B phosphorylates Mis12C and strengthens its binding to the CCAN subunit CENP-C. In another, CENP-T contributes to KMN attachment in a CENP-H-I-K-dependent manner. Our study provides insights into the mechanisms of mitosis-specific assembly of the checkpoint platform KMN at human kinetochores.
Project description:Chromosome segregation requires assembly of kinetochores on centromeric chromatin to mediate interactions with spindle microtubules and control cell-cycle progression. To elucidate the protein architecture of human kinetochores, we developed a two-color fluorescence light microscopy method that measures average label separation, Delta, at <5 nm accuracy. Delta analysis of 16 proteins representing core structural complexes spanning the centromeric chromatin-microtubule interface, when correlated with mechanical states of spindle-attached kinetochores, provided a nanometer-scale map of protein position and mechanical properties of protein linkages. Treatment with taxol, which suppresses microtubule dynamics and activates the spindle checkpoint, revealed a specific switch in kinetochore architecture. Cumulatively, Delta analysis revealed that compliant linkages are restricted to the proximity of chromatin, suggested a model for how the KMN (KNL1/Mis12 complex/Ndc80 complex) network provides microtubule attachment and generates pulling forces from depolymerization, and identified an intrakinetochore molecular switch that may function in controlling checkpoint activity.
Project description:Kinetochores, multisubunit protein assemblies, connect chromosomes to spindle microtubules to promote chromosome segregation. The 10-subunit KMN assembly (comprising KNL1, MIS12, and NDC80 complexes, designated KNL1C, MIS12C, and NDC80C) binds microtubules and regulates mitotic checkpoint function through NDC80C and KNL1C, respectively. MIS12C, on the other hand, connects the KMN to the chromosome-proximal domain of the kinetochore through a direct interaction with CENP-C. The structural basis for this crucial bridging function of MIS12C is unknown. Here, we report crystal structures of human MIS12C associated with a fragment of CENP-C and unveil the role of Aurora B kinase in the regulation of this interaction. The structure of MIS12:CENP-C complements previously determined high-resolution structures of functional regions of NDC80C and KNL1C and allows us to build a near-complete structural model of the KMN assembly. Our work illuminates the structural organization of essential chromosome segregation machinery that is conserved in most eukaryotes.
Project description:Kinetochores are superprotein complexes that orchestrate chromosome segregation via a dynamic interaction with spindle microtubules. A physical connection between CENP-C and the Mis12-Ndc80-Knl1 (KMN) protein network is an important pathway that is used to assemble kinetochores on CENP-A nucleosomes. Multiple outer kinetochore components are phosphorylated by Aurora B kinase to activate the spindle assembly checkpoint (SAC) and to ensure accurate chromosome segregation. However, it is unknown whether Aurora B can phosphorylate inner kinetochore components to facilitate proper mitotic chromosome segregation. Here, we reported the structure of the fission yeast Schizosaccharomyces pombe Mis12-Nnf1 complex and showed that N-terminal residues 26-50 in Cnp3 (the CENP-C homolog of S. pombe) are responsible for interacting with the Mis12 complex. Interestingly, Thr28 of Cnp3 is a substrate of Ark1 (the Aurora B homolog of S. pombe), and phosphorylation impairs the interaction between the Cnp3 and Mis12 complex. The expression of a phosphorylation-mimicking Cnp3 mutant results in defective chromosome segregation due to improper kinetochore assembly. These results establish a previously uncharacterized regulatory mechanism involved in CENP-C-Mis12-facilitated kinetochore attachment error correction to ensure accurate chromosome segregation during mitosis.
Project description:The establishment of proper kinetochore-microtubule attachments facilitates faithful chromosome segregation. Incorrect attachments activate the spindle assembly checkpoint (SAC), which blocks anaphase onset via recruitment of a cohort of SAC components (Mph1/MPS1, Mad1, Mad2, Mad3/BubR1, Bub1 and Bub3) to kinetochores. KNL1, a component of the outer kinetochore KMN network (KNL1/Mis12 complex/Ndc80 complex), acts as a platform for Bub1 and Bub3 localization upon its phosphorylation by Mph1/MPS1. The Ndc80 protein, a major microtubule-binding site, is critical for MPS1 localization to the kinetochores in mammalian cells. Here we characterized the newly isolated mutant ndc80-AK01 in fission yeast, which contains a single point mutation within the hairpin region. This hairpin connects the preceding calponin-homology domain with the coiled-coil region. ndc80-AK01 was hypersensitive to microtubule depolymerizing reagents with no apparent growth defects without drugs. Subsequent analyses indicated that ndc80-AK01 is defective in SAC signaling, as mutant cells proceeded into lethal cell division in the absence of microtubules. Under mitotic arrest conditions, all SAC components (Ark1/Aurora B, Mph1, Bub1, Bub3, Mad3, Mad2 and Mad1) did not localize to the kinetochore. Further genetic analyses indicated that the Ndc80 hairpin region might act as a platform for the kinetochore recruitment of Mph1, which is one of the most upstream SAC components in the hierarchy. Intriguingly, artificial tethering of Mph1 to the kinetochore fully restored checkpoint signaling in ndc80-AK01 cells, further substantiating the notion that Ndc80 is a kinetochore platform for Mph1. The hairpin region of Ndc80, therefore, plays a critical role in kinetochore recruitment of Mph1.
Project description:Kinetochores attach the replicated chromosomes to the mitotic spindle and orchestrate their transmission to the daughter cells. Kinetochore-spindle binding and chromosome segregation are mediated by the multi-copy KNL1(Spc105), MIS12(Mtw1) and NDC80(Ndc80) complexes that form the so-called KMN network. KMN-spindle attachment is regulated by the Aurora B(Ipl1) and MPS1(Mps1) kinases. It is unclear whether other mechanisms exist that support KMN activity during the cell cycle. Using budding yeast, we show that kinetochore protein Cnn1 localizes to the base of the Ndc80 complex and promotes a functionally competent configuration of the KMN network. Cnn1 regulates KMN activity in a spatiotemporal manner by inhibiting the interaction between its complexes. Cnn1 activity peaks in anaphase and is driven by the Cdc28, Mps1 and Ipl1 kinases.
Project description:Accurate chromosome segregation during mitosis and meiosis is crucial for cellular and organismal viability. Kinetochores connect chromosomes with spindle microtubules and are essential for chromosome segregation. These large protein scaffolds emerge from the centromere, a specialized region of the chromosome enriched with the histone H3 variant CENP-A. In most eukaryotes, the kinetochore core consists of the centromere-proximal constitutive centromere-associated network (CCAN), which binds CENP-A and contains 16 subunits, and of the centromere-distal Knl1 complex, Mis12 complex, Ndc80 complex (KMN) network, which binds microtubules and contains 10 subunits. In the fruitfly, Drosophila melanogaster, the kinetochore underwent remarkable simplifications. All CCAN subunits, with the exception of centromeric protein C (CENP-C), and two KMN subunits, Dsn1 and Zwint, cannot be identified in this organism. In addition, two paralogues of the KMN subunit Nnf1 (Nnf1a and Nnf1b) are present. Finally, the Spc105R subunit, homologous to human Knl1/CASC5, underwent considerable sequence changes in comparison with other organisms. We combined biochemical reconstitution with biophysical and structural methods to investigate how these changes reflect on the organization of the Drosophila KMN network. We demonstrate that the Nnf1a and Nnf1b paralogues are subunits of distinct complexes, both of which interact directly with Spc105R and with CENP-C, for the latter of which we identify a binding site on the Mis12 subunit. Our studies shed light on the structural and functional organization of a highly divergent kinetochore particle.