The dynamic protein architecture of human kinetochores Public Deposited

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Last Modified
  • March 21, 2019
Creator
  • O'Quinn, Ryan Patrick
    • Affiliation: College of Arts and Sciences, Department of Biology
Abstract
  • Accurate segregation of genomic DNA during mitosis is one of the cell’s most vital functions. Errors in this process result in defects that are implicated in many human diseases and developmental disorders. Key to this process is the attachment of chromosomes to microtubules of the mitotic spindle, via complex protein machines at the centromere known as kinetochores. In addition to providing the spindle microtubule attachment site, kinetochores also generate and propagate a “warning system” known as the spindle assembly checkpoint (SAC) that prevents anaphase onset until all chromosomes are properly aligned at the spindle equator. Lack of resolution capability in the light microscope and lack of contrast in the electron microscope have frustrated attempts to generate a map of protein architecture within kinetochores. Here we demonstrate a new light microscopy method, which we term K-SHREC (Kinetochore Speckle High-Resolution Colocalizaton) that measures distances between fluorescent labels at an accuracy of <5 nm. We measure separation distances between 16 proteins in human kinetochores, and also reveal important mechanical properties of kinetochore protein linkages. In particular, we identify an intrakinetochore molecular switch that may function in the SAC signal. I investigate the nature of this switch further by transfecting human cancer cells with siRNAs to Spindly, a protein implicated in recruitment of dynein to kinetochores. I find that Spindly is required for timely satisfaction of this interior switch within kinetochores, and that satisfaction of this switch condition may be a key component of extinguishing the SAC “wait-anaphase” signal. These studies advance our current understanding of kinetochore substructure, and how organisms successfully complete mitosis at the molecular level.
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  • In Copyright
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Biology."
Advisor
  • Salmon, Edward D.
Degree granting institution
  • University of North Carolina at Chapel Hill
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Place of publication
  • Chapel Hill, NC
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  • Open access
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