Regulation of the Ipl1p kinase during chromosome bi-orientation in yeast

When cells divide, chromosomes are first copied or replicated. The two identical copies remain associated but become attached to microtubules, special molecular cables that are used to pull the two copies apart during division. It is vital that each of the two daughter cells receive exactly one copy of each chromosome so that they inherit a proper complement of genetic information, and this requires that the two copies attach to microtubules from opposite ends of the cell so that they are pulled apart when the cell divides in two. Should they attach to microtubule from the same end, both copies of the chromosome will be pulled into just one of the daughter cells causing one daughter cell to gain an extra copy of the chromosome while the other daughter cell will lose its copy. Chromosomes do not automatically achieve correct attachment to microtubules and we are interested in the molecular mechanisms that are used to correct chromosome attachment errors before they have a chance to result in chromosome loss or gain events. We are using yeast as a model organism for this research because the cellular components involved are found in all higher cells from yeast to man. However, yeast constitutes a much simpler experimental system in which to investigate fundamental questions such as these. Correction of incorrect microtubule attachments in yeast cells involves an enzyme called Ipl1p that catalyses the addition of phosphate groups to proteins in the kinetochore, a complex of proteins assembled at a specific point on each chromosome and that constitutes the 'handle' onto which the microtubules can grab. When the kinetochores on both copies of a chromosome attach to microtubules from one end of the cell, Ipl1p is activated to add phosphate groups to one kinetochore, causing it to let go of its attached microtubule and allowing it to grab a different microtubule originating from the other end of the cell. This corrects the attachment error, causing the duplicated chromosomal copies to become 'bi-oriented', the state that ensures they are pulled in opposite directions when the cell divides. The objectives of our work are to understand what activates Ipl1p to perform this function, and more importantly, how its activity is then turned off when it has done its job. The most likely way that Ipl1p is regulated is through tension / the pulling force exerted on duplicated chromosomal copies when they are bi-oriented - because then the microtubules attached to them are trying to pull them apart. Before they are bi-oriented, this force would be much smaller because of their attachment to microtubules attempting to pull them both in the same (rather than opposing) directions. Ipl1p associates with two other proteins that, like Ipl1p itself also have counterparts in human cells and we will examine how tension might regulate Ipl1p through its association with these two proteins. By investigating how this mechanism works at the molecular level, we will improve our understanding of a fundamental process that ensures maintenance of genome integrity during cell division and that is relevant to human conditions such as cancer and Down's syndrome, where chromosome loss or gain events play an important role.

During cell division, newly-replicated sister chromatids don't automatically attach correctly to microtubules, and yet correct (amphitelic) attachment is vital for proper chromosome segregation in the ensuing anaphase. Amphitelic attachment (bi-orientation) is attained when sister kinetochores attach to microtubules from opposite spindle poles such that sister chromatids are pulled in opposite directions when sister chromatid cohesion is removed. The budding yeast Saccharomyces cerevisiae has proved valuable for studying chromosome bi-orientation and the conserved yeast protein kinase Ipl1p has emerged as a critical element in the process / in its absence, the majority of chromosomes fail to achieve bi-orientation and instead remain in a syntelic configuration, where both sister kinetochores are attached to microtubules from a single spindle pole. Ipl1p phosphorylates key proteins at the microtubule-kinetochore interface and is thought to promote microtubule detachment, so that the now unattached kinetochore can capture a microtubule from the opposite spindle pole. This proposal seeks to understand the molecular mechanisms that activate Ipl1p to correct syntelic attachments but then apparently prevent it continuing to destabilise kinetochore-microtubule connections once bi-orientation occurs. Two specific hypotheses will be tested, focusing on the roles of the conserved Ipl1p-associated proteins Sli15p and Bir1p: (1) That Ipl1p activity is regulated by tension applied to sister kinetochores following bi-orientation, sensed through its interactions with Bir1p and Sli15p. This will involve using bimolecular fluorescence complementation and FRET to study dynamic, tension-dependent changes in the interaction between Ipl1p, Sli15p and Bir1p and by interfering with how Sli15p responds to tension. (2) That phosphorylation of Sli15p by Ipl1p forms part of the bi-orientation mechanism. This will be investigated by phosphorylation site identification and mutagenesis.