The precise partitioning of duplicated chromosomes to daughter cells is essential for the development and survival of all organisms. Defects in segregation lead to aneuploidy, the state where entire chromosomes are gained or lost. Aneuploidy is a hallmark of the majority of tumor cells and has been postulated to be a major factor in the evolution of cancer. Aneuploidy is also the leading cause of spontaneous miscarriages and hereditary birth defects in humans. The goal of this proposal is to elucidate the mechanisms that regulate chromosome segregation and therefore ensure genomic stability. Chromosome segregation requires forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, the highly conserved structures that assemble onto centromeric chromatin. The simplest eukaryotic kinetochore is in budding yeast where ~38 unique 'core'proteins that exist in 8 subcomplexes assemble into a structure estimated to be >5 MDa. Accurate segregation requires kinetochores to maintain load-bearing attachments to the ends of microtubules that are continually growing and shrinking. Key to proper segregation is the ability of sister kinetochores to biorient and attach to microtubules from opposite poles. Kinetochores also engage a highly conserved signal transduction system called the spindle checkpoint to halt the cell cycle when kinetochore-microtubule interactions are aberrant. To fully elucidate the mechanisms that ensure faithful chromosome segregation, it is critical to understand the diverse functions of kinetochores and their regulation. This proposal will use purified kinetochores and a combination of biochemical, biophysical and structural approaches to address a number of outstanding questions: 1) How does phosphorylation regulate the diverse functions of kinetochores? 2) How do kinetochores regulate the spindle checkpoint? and 3) What is the architecture of a yeast kinetochore? The proposal will use budding yeast for these studies because they are amenable to biochemical, genetic and cytological studies, and the yeast kinetochore is the best characterized to date. Taken together, these studies of kinetochores and their regulation in budding yeast will lead toward an understanding of the fundamental mechanisms of segregation in all eukaryotes. This work will not only elucidate important aspects about the process of segregation, but will aid in the design of better therapeutic interventions in the long-term.

Public Health Relevance

All cells must inherit the right number of chromosomes every time they divide because the wrong number of chromosomes is a hallmark of cancer and birth defects. We are therefore studying the process of chromosome partitioning to daughter cells when they divide to understand the basis for a number of human diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064386-12
Application #
8508946
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2002-02-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
12
Fiscal Year
2013
Total Cost
$353,587
Indirect Cost
$146,159
Name
Fred Hutchinson Cancer Research Center
Department
Type
DUNS #
078200995
City
Seattle
State
WA
Country
United States
Zip Code
98109
Sarangapani, Krishna K; Duro, Eris; Deng, Yi et al. (2014) Sister kinetochores are mechanically fused during meiosis I in yeast. Science 346:248-51
London, Nitobe; Biggins, Sue (2014) Signalling dynamics in the spindle checkpoint response. Nat Rev Mol Cell Biol 15:736-47
Driver, Jonathan W; Powers, Andrew F; Sarangapani, Krishna K et al. (2014) Measuring kinetochore-microtubule interaction in vitro. Methods Enzymol 540:321-37
London, Nitobe; Biggins, Sue (2014) Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint. Genes Dev 28:140-52
Umbreit, Neil T; Miller, Matthew P; Tien, Jerry F et al. (2014) Kinetochores require oligomerization of Dam1 complex to maintain microtubule attachments against tension and promote biorientation. Nat Commun 5:4951
Akiyoshi, Bungo; Nelson, Christian R; Biggins, Sue (2013) The aurora B kinase promotes inner and outer kinetochore interactions in budding yeast. Genetics 194:785-9
Biggins, Sue (2013) The composition, functions, and regulation of the budding yeast kinetochore. Genetics 194:817-46
Akiyoshi, Bungo; Nelson, Christian R; Duggan, Nicole et al. (2013) The Mub1/Ubr2 ubiquitin ligase complex regulates the conserved Dsn1 kinetochore protein. PLoS Genet 9:e1003216
Sarangapani, Krishna K; Akiyoshi, Bungo; Duggan, Nicole M et al. (2013) Phosphoregulation promotes release of kinetochores from dynamic microtubules via multiple mechanisms. Proc Natl Acad Sci U S A 110:7282-7
London, Nitobe; Ceto, Steven; Ranish, Jeffrey A et al. (2012) Phosphoregulation of Spc105 by Mps1 and PP1 regulates Bub1 localization to kinetochores. Curr Biol 22:900-6

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