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The role of Ipl1-dependent phosphorylation of Mad3p in the spindle checkpoint mechanism that restrains anaphase when sister kinetochores lack tension

Reference	BB/F009453/1
Principal Investigator / Supervisor	Professor Michael Stark
Co-Investigators / Co-Supervisors
Institution	University of Dundee
Department	College of Life Sciences
Funding type	Research
Value (£)	356,588
Status	Completed
Type	Research Grant
Start date	19/06/2008
End date	18/06/2011
Duration	36 months

Abstract

When cells enter mitosis with chromosomes incorrectly attached to the mitotic spindle, the spindle checkpoint delays anaphase. The checkpoint components are conserved and have been studied extensively in the yeast Saccharomyces cerevisiae. After replication, many sister chromatids pairs become attached to microtubules from one spindle pole (syntelic attachment) and Ipl1p (Aurora B) kinase is required to promote their biorientation so that they can be correctly segregated. We have found that Ipl1p kinase also activates the spindle checkpoint through phosphorylation of the checkpoint protein Mad3p. This novel pathway appears to respond to sister kinetochores that are not under tension from the mitoic spindle rather than unattached kinetochores, so it could be important for delaying anaphase in response to syntelic chromosomes. We propose to investigate how Ipl1p-dependent Mad3p phosphorylation delays anaphase. Firstly, we will use phosphospecific antibodies follow the kinetics of Mad3p phosphorylation by western blot analysis of synchronous yeast cell extracts and test its dependence on the other known checkpoint proteins. Secondly, we will assess the role of the Ipl1p-Mad3p pathway under additional conditions to test more rigorously its generality and its specificity for sensing lack of sister kinetochore tension as opposed to unattached kinetochores. Thirdly, we will make phosphomimetic mutants of Mad3p that should be constitutively activated and use these to probe the mechanisms through which Mad3p phosphorylation functions. Finally, we will examine how Mad3p phosphorylation affects the stability and protein-protein interactions of checkpoint components. We will address the latter question both by co-immune precipitation and using a recently developed method (SILAC), which involves heavy isotopic labelling and mass spectrometry analysis so that the relative abundance of proteins interacting with phosphorylated and non-phosphorylated Mad3p can be compared.

Summary

When cells divide, each chromosome is precisely copied and then one copy is distributed to each of the two daughter cells. The accuracy of this distribution process, termed chromosome segregation, is clearly vital since mistakes will result in chromosome loss in one daughter cell and gain chromosome gain in the other. Cells therefore have evolved mechanisms to ensure the accuracy of this process. The chromosomes are segregated to the two daughter cells by becoming attached to molecular cables, termed microtubules, that eventually pull them in opposite directions, but if this attachment is incorrect or fails to happen then segregation during division will fail. Cells therefore have mechanisms both to correct the attachment of chromosomes to microtubules when there is a problem, and to delay the cell from trying to divide until all chromosomes are correctly attached. The mechanism that delays cell division in response to incorrectly attached chromosomes it is present in all higher organisms and much of the work leading to our current understanding of the checkpoint mechanism has been carried out in yeast, a model organism that is used for investigating fundamental questions concerning how cells function. We have been using yeast to investigate the function of a regulatory protein involved in correcting the attachment of chromosomes to microtubules, and discovered that in addition to this function it also provides the signal that delays division while the correction process occurs. However, whereas until now the main factor demonstrated to delay division is the presence of chromosomes that are not attached to microtubules, the mechanism we have discovered appears to respond to chromosomes that are attached to microtubules but not subject to pulling forces exerted by them. This is the situation that would prevail if both copies of a chromosome became incorrectly attached to microtubules such that they would subsequently pull them into the same daughter cell. The objectives of our proposed work are therefore to understand how this new mechanism functions, since it may be the major pathway by which cells allow time to correct an important category of incorrectly attached chromosomes. By investigating how this mechanism operates at the molecular level, we will improve our understanding of a fundamental process that ensures the 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.

Committee	Closed Committee - Biochemistry & Cell Biology (BCB)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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