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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Nuclear and Cytoplasmic Structure/Function and Dynamics Study Section (NCSD)
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Deatherage, James F
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University of Michigan Ann Arbor
Anatomy/Cell Biology
Schools of Medicine
Ann Arbor
United States
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Humphrey, Lauren; Felzer-Kim, Isabella; Joglekar, Ajit P (2018) Stu2 acts as a microtubule destabilizer in metaphase budding yeast spindles. Mol Biol Cell 29:247-255
Joglekar, Ajit P; Kukreja, Alexander A (2017) How Kinetochore Architecture Shapes the Mechanisms of Its Function. Curr Biol 27:R816-R824
Aravamudhan, Pavithra; Chen, Renjie; Roy, Babhrubahan et al. (2016) Dual mechanisms regulate the recruitment of spindle assembly checkpoint proteins to the budding yeast kinetochore. Mol Biol Cell 27:3405-3417
Joglekar, Ajit P; Aravamudhan, Pavithra (2016) How the kinetochore switches off the spindle assembly checkpoint. Cell Cycle 15:7-8
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Aravamudhan, Pavithra; Goldfarb, Alan A; Joglekar, Ajit P (2015) The kinetochore encodes a mechanical switch to disrupt spindle assembly checkpoint signalling. Nat Cell Biol 17:868-79
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
Aravamudhan, Pavithra; Felzer-Kim, Isabella; Joglekar, Ajit P (2013) The budding yeast point centromere associates with two Cse4 molecules during mitosis. Curr Biol 23:770-4
Joglekar, Ajit; Chen, Renjie; Lawrimore, Joshua (2013) A Sensitized Emission Based Calibration of FRET Efficiency for Probing the Architecture of Macromolecular Machines. Cell Mol Bioeng 6:369-382