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Evaluating the effect dynein and related proteins exhibit on the spindle assembly checkpoint and kinetochore




Biebighauser, Tyler, author
Markus, Steven, advisor
DeLuca, Jennifer, advisor
Di Pietro, Santiago, committee member
Hoerndli, Fred, committee member

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To ensure that cell division is faithfully carried out without causing genetic errors, eukaryotic cells have evolved several conserved checkpoints during mitosis. One such checkpoint, the Spindle Assembly Checkpoint (SAC), blocks the cell from progressing through metaphase until all chromosomes have become bi-oriented by microtubules. Only once this occurs can the cell progress into anaphase to separate the sister chromatids. Errors in this checkpoint have been linked with aneuploidy, which itself is linked with oncogenesis. Naturally there are many layers of regulation within the SAC, most of which are associated with a proteinaceous structure on the sister chromatid – the kinetochore. The molecular motor dynein, and its kinetochore localized co-factors play several roles in this regulation. In one of these roles, dynein strips away kinetochore localized signal proteins upon microtubule bi-orientation, to weaken the strength of the SAC. We initially set out to test whether this process of SAC stripping has further levels of regulation, or if all dynein requires to strip these signal proteins is the presence of a microtubule. We used in-vitro motility assays to investigate whether dynein's motility along microtubules is changed depending on the length of its kinetochore localized cargo adapter, spindly. We purified truncated versions of spindly to test if it undergoes regulation analogous to other dynein cargo adapters. These in-vitro motility assays showed no difference in dynein motility past a certain length required to confer motility. Interestingly, we observed that some of the shorter spindly truncations undergo phase separation both in-vitro in the right conditions and in-vivo when transfected into HeLa cells. We postulate that this phase separation could have implications in a process called fibrous corona expansion, which occurs on a kinetochore that has spent a long time in prometaphase without attaching to a microtubule. In total these studies shed light on the nature of interactions at the kinetochore, and the complexity of regulation as it pertains to dynein mediated kinetochore stripping.


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spindle assembly checkpoint


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