Our goal is to understand how the nanoscale arrangement of kinetochore proteins shapes its functional and regulatory mechanisms. The kinetochore is a macromolecular motor that drives chromosome movement and ensures their accurate segregation during cell division. Kinetochore force generation required for chromosome movement is critical for inheritance of a complete genome by both daughter cells. Kinetochore misregulation leads to chromosomal instability, which has been linked to tumorigenesis, developmental defects, as well as age- related infertility. Therefore, definition of the biophysical mechanism of kinetochore force generation is necessary to develop a mechanistic understanding of disease relevant mutations in kinetochore proteins. Although the last decade has witnessed tremendous progress in our understanding the protein composition of the kinetochore, a mechanistic understanding of its function as a force generator remains elusive. The primary obstacle in further progress is a lack of understanding of the molecular architecture of the kinetochore. Therefore, we propose a novel 'architecture-function'approach to establish mechanistic link between kinetochore architecture and its function.
Aim 1 : Develop a new fluorescence microscopy method to reconstruct the nanoscale kinetochore architecture. We have developed a new technique to determine nanoscale distribution of proteins in live cells. Our preliminary reconstruction of kinetochore architecture suggests an integrative model of how the kinetochore generates microtubule polymerization and depolymerization coupled force. Our technique will be useful for determining the architecture of other cellular machines.
Aim 2 : Determine how the location of force generating molecules defines their function. We will subject our new model to an 'architecture-function'analysis, wherein we will study the impact of changes in kinetochore architecture on its function. This work will define the biophysical principles of force generation by the kinetochore.
Aim 3 : Define the minimal architectural specification for the kinetochore. We will use in vitro experiments and artificial kinetochore protein assemblies to determine the necessary and sufficient architectural features of a key kinetochore protein, Ndc80, for reconstituting its distribution and function observed in vivo. This work will establish a framework for building artificial kinetochores in cells.

Public Health Relevance

Our work will define the biophysical principles of force generation within the kinetochore. Complete understanding of this fundamental cellular process will provide a mechanistic insight into how kinetochore misregulation can lead to chromosome missegregation, which is a major cause of tumorigenesis, developmental defects, and age-related infertility. Our work will also develop a conceptual framework to guide our long-term efforts of designing artificial kinetochores.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM105948-01
Application #
8480061
Study Section
Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
Program Officer
Deatherage, James F
Project Start
2013-04-01
Project End
2018-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
1
Fiscal Year
2013
Total Cost
$280,635
Indirect Cost
$90,635
Name
University of Michigan Ann Arbor
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Aravamudhan, Pavithra; Felzer-Kim, Isabella; Gurunathan, Kaushik et al. (2014) Assembling the protein architecture of the budding yeast kinetochore-microtubule attachment using FRET. Curr Biol 24:1437-46