Reconstituting the spindle assembly checkpoint and the signalling roles of Mad1

Cell division allows the passage of genetic information to a new cell. During this process, maintaining chromosome transmission fidelity is important in preventing diseases such as cancer and Down’s syndrome. To ensure accurate chromosome segregation, eukaryotes have developed a cell cycle control mechanism that monitors kinetochore-microtubule attachments, known as the spindle assembly checkpoint (SAC). The SAC is active in metaphase and is able to sense a lack of tension and incorrect attachments between kinetochores and microtubules. This leads to a metaphase arrest, allowing time for error correction to take place before anaphase onset. The Mad and Bub proteins, along with Mps1 kinase are central to this signalling pathway which leads to the formation of the mitotic checkpoint complex (MCC) — the key inhibitor of the anaphase promoting complex/cyclosome (APC/C). APC/C inhibition prevents proteolytic degradation of Securin and Cyclin B, blocking cells in metaphase. Although we are familiar with the components of the SAC pathway, the mechanism by which they interact to form the MCC remains unclear. It is well established that SAC signalling is initiated at kinetochores. These are complex structures that are involved in key mitotic functions such as microtubule attachment and biorientation of sister chromatids. To study the checkpoint without interfering with kinetochore function, we have devised a minimalist approach. This study describes an ectopic reconstitution of SAC activation and silencing in S. pombe. Using abscisic acid induced dimerisation, we are able to control the co-recruitment of the checkpoint proteins KNL1 and Mps1 to recapitulate robust SAC arrest and silencing. Additionally, we provide insight into how S. pombe and HeLa cells respond to a prolonged ectopic arrest. It is widely accepted that Mad1 recruits Mad2 to unattached kinetochores, enabling MCC formation. However, recent findings point towards a more active role of Mad1 in checkpoint activation. This study shows that Mad1 interacts with Bub1 in S. pombe to form a scaffold complex that is essential for SAC function. We also investigate Mad1 C-terminal mutants to further dissect the roles of Mad1 and find that it forms a complex with the APC/C coactivator Cdc20. As a result, this study provides evidence in support of the hypothesis that the C-terminus of Mad1 has additional roles in SAC signalling aside from Mad2 kinetochore recruitment

‘SynCheck’: new tools for dissecting Bub1 checkpoint functions

The accurate segregation of DNA during cell division is essential for the viability of future cellular generations. Genetic material is packaged in the form of chromosomes during cell division, and chromosomes are segregated equally into two daughter cells. Chromosome mis-distribution leads to genetic disorders (e.g. Down’s syndrome), aneuploidy and cancer. The spindle checkpoint ensures proper chromosome segregation by monitoring kinetochore-microtubule interactions. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins that combine to form a diffusible inhibitor (the Mitotic Checkpoint Complex-MCC). The MCC delays anaphase, thus giving cells time to fix attachment errors. Although the major checkpoint proteins were identified several years ago, we have only just begun to understand how they assemble at unattached kinetochores to generate the checkpoint signal. Yeast genetics and proteomics have revealed that kinetochores are highly complex molecular machines with almost 50 kinetochore components and ~10 components of the spindle checkpoint machinery. Such complexity makes the separation of error correction, kinetochore bi-orientation and microtubule attachment functions very challenging. To circumvent this complexity, a synthetic version of the spindle checkpoint (SynCheck), based on tetO array was engineered at an ectopic location on a chromosome arm away from kinetochores in S. pombe. This work describes that combined targeting, initially of KNL1Spc7 with Mps1Mph1 and later of Bub1 (but not Mad1) with Mps1Mph1 fragments, was able to activate the spindle checkpoint and generate a robust arrest. The system is based on, soluble complexes, which were formed between KNL1Spc7 or Bub1 with Mps1Mph1. The synthetic checkpoint or ‘Syncheck’ is independent of localisation of the checkpoint components to the kinetochores, to spindle pole bodies (SPBs) and to nuclear pores. By using the synthetic tethering system a Mad1-Bub1 complex was identified for the first time in S.pombe. Bub1- Mad1 complex formation is crucial for checkpoint activation. Bub1-Mad1 gets phosphorylated itself and is thought to act as an assembly platform for MCC production and thereby generation of the “wait anaphase” signal. The ectopic tetO array is an important tool, not only for generating MCC formation and activating the spindle checkpoint, but also for providing a nice system for analysing in vivo protein-protein interactions. The ectopic array is capable of not only recruiting checkpoint components, but also recruiting them in a physiological manner (similar to the unattached kinetochores). For this reason it was decided to adopt this system to examine the role of the conserved Bub1TPR domain in the recruitment of other spindle checkpoint proteins. This work represents two novel functions for the S. pombe Bub1TPR domain. For the first time in S. pombe, both in vivo tethering and in vitro experiments with purified, recombinant proteins showed that the Bub1 has the ability to homodimerise and to form a complex with Mad3BubR1 through its TPR domain. These results revealed that complex formation of Bub1 with Mad3BubR1 is important for checkpoint signalling and that the highly conserved TPR domains in BubR1Mad3 and Bub1 have key roles to play in their interactions

Tension-Induced Error Correction and Not Kinetochore Attachment Status Activates the SAC in an Aurora-B/C-Dependent Manner in Oocytes

International audienceCell division with partitioning of the genetic material should take place only when paired chromosomes named bivalents (meiosis I) or sister chromatids (mitosis and meiosis II) are correctly attached to the bipolar spindle in a tension-generating manner. For this to happen, the spindle assembly checkpoint (SAC) checks whether unattached kinetochores are present, in which case anaphase onset is delayed to permit further establishment of attachments. Additionally, microtubules are stabilized when they are attached and under tension. In mitosis, attachments not under tension activate the so-named error correction pathway depending on Aurora B kinase substrate phosphorylation. This leads to microtubule detachments, which in turn activates the SAC [1, 2, 3]. Meiotic divisions in mammalian oocytes are highly error prone, with severe consequences for fertility and health of the offspring [4, 5]. Correct attachment of chromosomes in meiosis I leads to the generation of stretched bivalents, but—unlike mitosis—not to tension between sister kinetochores, which co-orient. Here, we set out to address whether reduction of tension applied by the spindle on bioriented bivalents activates error correction and, as a consequence, the SAC. Treatment of oocytes in late prometaphase I with Eg5 kinesin inhibitor affects spindle tension, but not attachments, as we show here using an optimized protocol for confocal imaging. After Eg5 inhibition, bivalents are correctly aligned but less stretched, and as a result, Aurora-B/C-dependent error correction with microtubule detachment takes place. This loss of attachments leads to SAC activation. Crucially, SAC activation itself does not require Aurora B/C kinase activity in oocytes

Deaths due to miscellaneous infections by year in Greece during 2003–2010.

<p>Deaths due to miscellaneous infections by year in Greece during 2003–2010.</p

Deaths due to septicemia by year in Greece during 2003–2010.

<p>Deaths due to septicemia by year in Greece during 2003–2010.</p

Deaths caused by infectious diseases in Greece from 2003 to 2010 stratified by site of infection.

*<p>The percentage is calculated using the total infections of each year. ** The percentage is calculated using the total infections of each site during the whole study period. <b>Abbreviations:</b> TB: tuberculosis, RTI: respiratory tract infection, IAI: intra-abdominal infection, UTI: urinary tract infection, CNS: central nervous system.</p

Deaths due to pneumonia and pulmonary tuberculosis by year in Greece during 2003–2010.

<p>Deaths due to pneumonia and pulmonary tuberculosis by year in Greece during 2003–2010.</p