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Author ORCID Identifier



Open Access Dissertation

Document Type


Degree Name

Doctor of Philosophy (PhD)

Degree Program

Molecular and Cellular Biology

Year Degree Awarded


Month Degree Awarded


First Advisor

Patricia Wadsworth

Second Advisor

Thomas Maresca

Third Advisor

Wei-Lih Lee

Fourth Advisor

Michelle Farkas

Subject Categories

Cell Biology


Developing and maintaining a multicellular organism relies on the fundamental biological process of cell division, which ensures that genetic material is equally segregated between daughter cells. During mitosis, cells completely rearrange their cytoskeleton into a bipolar spindle through the concerted efforts of microtubules, motor proteins, and microtubule-associated proteins, which cells must regulate spatially and temporally to prevent errors such as chromosomal missegregation: a major cause of cancer. Although the mitotic spindle is a validated target for chemotherapy drug resistance and redundant pathways have highlighted the need for new targets. It is therefore important to understand how proteins that help build and/or maintain the spindle are regulated. Consequently, this dissertation focuses on the regulation of two separate but somewhat redundant mitotic kinesin motor proteins, Eg5 and Kif15, in vitro and in vivo. In general, proteins are regulated several ways, including protein-protein interactions and post-translational modifications. My data show that the C-terminal 37 amino acids of the microtubule-associated protein, TPX2, which were known to regulate Eg5 localization, are also responsible for Kif15 localization. TPX2 inhibits Eg5 walking on single microtubules by acting as both a brake and a roadblock but only inhibits Kif15 as a brake. In vivo, dynamic microtubules are also involved in Kif15 behavior. These results highlight the differences in the mechanisms that govern the regulation of these motors. To further demonstrate this, I found that Eg5 activity is also regulated motor domain phosphorylation by Src kinase. Eg5 phosphomimic mutations produce monopolar spindles due to reduced Eg5 activity while non-phosphorylatable mutants result in disorganized spindles. Together, these data suggest that phosphorylation of Eg5 must be temporally regulated. Finally, using CRISPR/Cas9, I endogenously tagged Eg5 and TPX2 with EGFP in HeLa cells and quantified protein distribution. My results differed from reports using non-endogenous tags and reveal that Eg5 and TPX2 have distinct spindle localization throughout mitosis with TPX2 absent in areas where Eg5 activity is required. Additionally, I correlated fluorescence to protein concentration both locally and globally in mammalian cells, which is the first step in developing models to understand this complex biological process.


Included in

Cell Biology Commons