Counteraction between Astrin-PP1 and Cyclin-B-CDK1 pathways protects chromosome-microtubule attachments independent of biorientation

Defects in chromosome-microtubule attachment can cause chromosomal instability (CIN), frequently associated with infertility and aggressive cancers. Chromosome-microtubule attachment is mediated by a large macromolecular structure, the kinetochore. Sister kinetochores of each chromosome are pulled by microtubules from opposing spindle-poles, a state called biorientation which prevents chromosome missegregation. Kinetochore-microtubule attachments that lack the opposing-pull are detached by Aurora-B/Ipl1. It is unclear how mono-oriented attachments that precede biorientation are spared despite the lack of opposing-pull. Using an RNAi-screen, we uncover a unique role for the Astrin-SKAP complex in protecting mono-oriented attachments. We provide evidence of domains in the microtubule-end associated protein that sense changes specific to end-on kinetochore-microtubule attachments and assemble an outer-kinetochore crescent to stabilise attachments. We find that Astrin-PP1 and Cyclin-B-CDK1 pathways counteract each other to preserve mono-oriented attachments. Thus, CIN prevention pathways are not only surveying attachment defects but also actively recognising and stabilising mature attachments independent of biorientation.

Mitotic chromosome condensation requires phosphorylation of the centromeric protein KNL-2 in C. elegans

Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1 in vitro, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans.

MKLP2 functions in early mitosis to ensure proper chromosome congression

Mitotic kinesin-like protein 2 (MKLP2) is a motor protein with a well-established function in promoting cytokinesis. However, our results with siRNAs targeting MKLP2 and small molecule inhibitors of MKLP2 (MKLP2i) along with the observations of others suggested a function earlier in mitosis, prior to anaphase. In this study we provide direct evidence that MKLP2 facilitates chromosome congression in prometaphase. We employed live imaging to observe HeLa cells with fluorescently tagged histones treated with MKLP2i and discovered a pronounced chromosome congression defect. We show that MKLP2 inhibited cells had a significant increase in unstable kinetochore-microtubule attachments due to impaired error correction of syntelic attachments. We propose that MKLP2 mediates kinetochore microtubule attachment stability by regulating Aurora Kinase and a downstream target, pHEC1 (Ser 55). Lastly, we show that MKLP2 inhibition results in aneuploidy, confirming that MKLP2 safeguards cells against chromosomal instability.

Bayesian inference of multi-point macromolecular architecture mixtures at nanometre resolution

Gaussian spot fitting methods have significantly extended the spatial range where fluorescent microscopy can be used, with recent techniques approaching nanometre (nm) resolutions. However, small inter-fluorophore distances are systematically over-estimated for typical molecular scales (≲ 50nm). This bias can be corrected computationally, but current algorithms are limited to correcting distances between pairs of fluorophores. Here we present a flexible Bayesian computational approach that infers the distances and angles between multiple markers and has several advantages over these previous methods. Specifically it improves confidence intervals for small lengths, estimates measurement errors of each fluorescent marker individually and infers the correlations between polygon lengths. The latter is essential for determining the full multi-fluorophore 3D architecture. We further developed the algorithm to infer the mixture composition of a heterogeneous population of multiple polygon states. We use our algorithm to analyse the 3D architecture of the human kinetochore, a macro-molecular complex that is essential for high fidelity cell division. We examine the conformational change induced by microtubule attachment using triple fluorophore marked data and demonstrate for the first time that in metaphase kinetochore conformation is heterogeneous.

Mitotic chromosome condensation requires phosphorylation of the centromeric protein KNL-2 in C. elegans

Centromeres are chromosomal regions that serve as sites for kinetochore formation and microtubule attachment, processes that are essential for chromosome segregation during mitosis. Centromeres are almost universally defined by the histone variant CENP-A. In the holocentric nematode C. elegans, CENP-A deposition depends on the loading factor KNL-2. Depletion of either CENP-A or KNL-2 results in defects in centromere maintenance, chromosome condensation and kinetochore formation, leading to chromosome segregation failure. Here, we show that KNL-2 is phosphorylated by CDK-1, and that mutation of three C-terminal phosphorylation sites causes chromosome segregation defects and an increase in embryonic lethality. In strains expressing phosphodeficient KNL-2, CENP-A and kinetochore proteins are properly localised, indicating that the role of KNL-2 in centromere maintenance is not affected. Instead, the mutant embryos exhibit reduced mitotic levels of condensin II on chromosomes and significant chromosome condensation impairment. Our findings separate the functions of KNL-2 in CENP-A loading and chromosome condensation and demonstrate that KNL-2 phosphorylation regulates the cooperation between centromeric regions and the condensation machinery in C. elegans.

Knl1 participates in spindle assembly checkpoint signaling in maize

The Knl1-Mis12-Ndc80 (KMN) network is an essential component of the kinetochore–microtubule attachment interface, which is required for genomic stability in eukaryotes. However, little is known about plant Knl1 proteins because of their complex evolutionary history. Here, we cloned the Knl1 homolog from maize (Zea mays) and confirmed it as a constitutive central kinetochore component. Functional assays demonstrated their conserved role in chromosomal congression and segregation during nuclear division, thus causing defective cell division during kernel development when Knl1 transcript was depleted. A 145 aa region in the middle of maize Knl1, that did not involve the MELT repeats, was associated with the interaction of spindle assembly checkpoint (SAC) components Bub1/Mad3 family proteins 1 and 2 (Bmf1/2) but not with the Bmf3 protein. They may form a helical conformation with a hydrophobic interface with the TPR domain of Bmf1/2, which is similar to that of vertebrates. However, this region detected in monocots shows extensive divergence in eudicots, suggesting that distinct modes of the SAC to kinetochore connection are present within plant lineages. These findings elucidate the conserved role of the KMN network in cell division and a striking dynamic of evolutionary patterns in the SAC signaling and kinetochore network.

Mps1 promotes poleward chromosome movements in meiotic prometaphase

Mps1 is a kinase that regulates several steps in mitosis and meiosis. Mps1 is essential for the spindle checkpoint and helps stabilize attachment of kinetochores to microtubules. Here we show that following microtubule attachment, Mps1 promotes microtubule depolymerization to trigger migration of the chromosome toward the spindle pole.

Physiological relevance of post-translational regulation of the spindle assembly checkpoint protein BubR1

BubR1 is an essential component of the spindle assembly checkpoint (SAC) during mitosis where it functions to prevent anaphase onset to ensure proper chromosome alignment and kinetochore-microtubule attachment. Loss or mutation of BubR1 results in aneuploidy that precedes various potential pathologies, including cancer and mosaic variegated aneuploidy (MVA). BubR1 is also progressively downregulated with age and has been shown to be directly involved in the aging process through suppression of cellular senescence. Post-translational modifications, including but not limited to phosphorylation, acetylation, and ubiquitination, play a critical role in the temporal and spatial regulation of BubR1 function. In this review, we discuss the currently characterized post-translational modifications to BubR1, the enzymes involved, and the biological consequences to BubR1 functionality and implications in diseases associated with BubR1. Understanding the molecular mechanisms promoting these modifications and their roles in regulating BubR1 is important for our current understanding and future studies of BubR1 in maintaining genomic integrity as well as in aging and cancer.