Molecular and functional characterisation of the spindle position checkpoint in budding yeast

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In all eukaryotic cells surveillance mechanisms, named checkpoints, make cell cycle progression dependent upon the successful completion of previous steps. Checkpoints are of extreme importance for the maintenance and accurate segregation of the genome. In the budding yeast, Saccharomyces cerevisiae, mitotic checkpoints ensure that each daughter cell will be provided with identical sets of chromosomes. The spindle position checkpoint (SPOC) senses any failure of cytoplasmic microtubules to correctly position the nucleus between mother and daughter cells. Thus making the transition out of mitosis and subsequent cell division (cytokinesis) dependent upon the correct partitioning of one set of chromosomes into the daughter cell. In the absence of SPOC, mutant cells defective in cytoplasmic microtubules undergo an aberrant mitosis and become aneuploid, reinforcing the importance of the SPOC in maintaining genome integrity (Figure 1).

The mitotic exit network (MEN) is a signalling transduction cascade that promotes exit from mitosis and cytokinesis. The SPOC inhibits the MEN via a two component GTPase activating protein (GAP) complex, composed of Bfa1 and Bub2. When the nucleus fails to move into the daughter cell, it is the function of Bub2-Bfa1 GAP complex to keep one of the most upstream components of the MEN, the Ras-like GTPase Tem1, in its GDP-inactive form until defects are corrected. Bfa1 and Bub2 associate with the yeast centrosome, called spindle pole body (SPB). Interestingly, Bfa1 and Bub2 are only found on the SPB that enters the daughter cell during mitosis. In response to cytoplasmic microtubules defects, Bfa1 and Bub2 are regulated in two ways. First, Bfa1 and Bub2 are re-located to both SPBs, a process that likely increases the GAP activity of the complex and makes Tem1 inactivation more efficient. Second, phosphorylation of Bfa1 by the polo kinase Cdc5, which decreases the GAP activity of the complex, is inhibited. Thus, localisation and GAP activity of Bub2-Bfa1 can be modulated by cytoplasmic microtubules. In this context it is interesting to note that Bfa1 and Bub2 directly bind to core SPB proteins involved in organisation of cytoplasmic microtubules. Recently, the Kin4 kinase has been identified as a novel component of the SPOC. In case of misalignment spindles, Kin4 phosphorylates Bfa1 and thereby inhibits its subsequent phosphorylation by Cdc5. In contrast to other mitotic regulators, Kin4 localises preferentially in the mother cell body. How Kin4 and Bub2-Bfa1 complex are able to respond to changes in cytoplasmic microtubule behaviour at a molecular level is unclear. Our work is therefore focusing on the regulation of Bub2-Bfa1, and on the identification and characterisation of novel components of the SPOC. For this, we are employing a combination of genomics, proteomics and advanced microscopy approaches.

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