Elucidating the role of Bub1 in human checkpoint signalling

The spindle assembly checkpoint (SAC) establishes a delay before anaphase, until all chromosomes are equally and stably attached to spindle microtubules via kinetochores. This acts as a surveillance mechanism to prevent chromosome segregation defects and safeguard genomic stability. The exact mechanisms regulating checkpoint activation and mitotic progression remain unclear. Previous work has shown that SAC activation requires Mps1-dependant phosphorylation of KNL1 MELT motifs. This allows recruitment of Bub1-Bub3, and subsequent localisation of BubR1-Bub3 and Mad1/Mad2 complexes onto Bub1. Localisation of Mad1/Mad2 to the kinetochore allows production of the mitotic checkpoint complex (MCC), which inhibits the APC/C to prevent initiation of anaphase. This was thought to represent a single, linear pathway for SAC activation. However, recent work in human cells has provided evidence that the Rod-Zwilch-ZW10 (RZZ) complex can offer a second receptor for Mad1/Mad2 complexes, thus allowing two separable pathways for SAC activation and maintenance in human cells. We have tested this dual-pathway model by knocking out BUB1 in human hTERT-RPE1 cells using CRISPR-Cas9 technology. These Bub11-23 cells are able to generate a functional checkpoint in the presence of unattached kinetochores, however, Bub11-23 cells were later found to express residual amounts of Bub1 protein, referred to as ‘zombie’ Bub1. We have therefore repeated all key experiments using Bub11-23 cells in the presence of Bub1 siRNA to deplete ‘zombie’ Bub1, and again shown that these cells are able to generate a functional checkpoint in the presence of unattached kinetochores, although maintenance is perturbed. Furthermore, we have found that Bub1 plays an important role in the regulation of unperturbed mitosis which is at least partially mediated through an uncharacterised premitotic function. Together, these data provide further evidence for a Bub1-independent mechanism of checkpoint activation at unattached kinetochores, and uncover a novel role for Bub1 during interphase

Age-dependent loss of cohesion protection in human oocytes

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage, and chromosomal disorders. Such aneuploidies increase greatly as women age, with defective linkages between sister chromatids (cohesion) in meiosis as a common cause. We found that loss of a specific pool of the cohesin protector protein, shugoshin 2 (SGO2), may contribute to this phenomenon. Our data indicate that SGO2 preserves sister chromatid cohesion in meiosis by protecting a ‘‘cohesin bridge’’ between sister chromatids. In human oocytes, SGO2 localizes to both sub-centromere cups and the pericentromeric bridge, which spans the sister chromatid junction. SGO2 normally colocalizes with cohesin; however, in meiosis II oocytes from older women, SGO2 is frequently lost from the pericentromeric bridge and sister chromatid cohesion is weakened. MPS1 and BUB1 kinase activities maintain SGO2 at sub-centromeres and the pericentromeric bridge. Removal of SGO2 throughout meiosis I by MPS1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, SGO2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that impaired SGO2 localization weakens cohesion integrity and may contribute to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age

The first mitotic division of human embryos is highly error prone

Human beings are made of ~50 trillion cells which arise from serial mitotic divisions of a single cell - the fertilised egg. Remarkably, the early human embryo is often chromosomally abnormal, and many are mosaic, with the karyotype differing from one cell to another. Mosaicism presumably arises from chromosome segregation errors during the early mitotic divisions, although these events have never been visualised in living human embryos. Here, we establish live cell imaging of chromosome segregation using normally fertilised embryos from an egg-share-to-research programme, as well as embryos deselected during fertility treatment. We reveal that the first mitotic division has an extended prometaphase/metaphase and exhibits phenotypes that can cause nondisjunction. These included multipolar chromosome segregations and lagging chromosomes that lead to formation of micronuclei. Analysis of nuclear number and size provides evidence of equivalent phenotypes in 2-cell human embryos that gave rise to live births. Together this shows that errors in the first mitotic division can be tolerated in human embryos and uncovers cell biological events that contribute to preimplantation mosaicism

Bub1 is not required for the checkpoint response to unattached kinetochores in diploid human cells

Error-free chromosome segregation during mitosis depends on a functional spindle assembly checkpoint (SAC). The SAC is a multi-component signaling system that is recruited to incorrectly attached kinetochores to catalyze the formation of a soluble inhibitor, known as the mitotic checkpoint complex (MCC), which binds and inhibits the anaphase promoting complex [1]. We have previously proposed that two separable pathways, composed of KNL1-Bub3-Bub1 (KBB) and Rod-Zwilch-Zw10 (RZZ), recruit Mad1-Mad2 complexes to human kinetochores to activate the SAC [2]. We refer to this as the dual pathway model. Although Bub1 is absolutely required for MCC formation in yeast (which lack RZZ), there is conflicting evidence as to whether this is also the case in human cells based on siRNA studies [2-5]. Here we report, using genome editing, that Bub1 is not strictly required for the SAC response to unattached kinetochores in human diploid hTERT-RPE1 cells, consistent with the dual pathway model

Data for The first mitotic division of human embryos is highly error prone

Aneuploidy in human embryos is surprisingly prevalent and increases drastically with maternal age, resulting in miscarriages, infertility and birth defects. Frequent errors during the meiotic divisions cause this aneuploidy, while age-independent errors during the first cleavage divisions of the embryo also contribute. However, the underlying mechanisms are poorly understood, largely because these events have never been visualised in living human embryos. Here, using cell-permeable DNA dyes, we film chromosome segregation during the first and second mitotic cleavage divisions in human embryos from women undergoing assisted reproduction following ovarian stimulation. We show that the first mitotic division takes several hours to complete and is highly variable. Timings of key mitotic events were, however, largely consistent with clinical videos of embryos that gave rise to live births. Multipolar divisions and lagging chromosomes during anaphase were frequent with no maternal age association. In contrast, the second mitosis was shorter and underwent mostly bipolar divisions with no detectable lagging chromosomes. We propose that the first mitotic division in humans is a unique and highly error-prone event, which contributes to fetal aneuploidies