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Functions of Gamma-tubulin in the Spindle Assembly Checkpoint and APC/C Regulation in Aspergillus nidulans

Edgerton, Heather Dawn
http://rave.ohiolink.edu/etdc/view?acc_num=osu1374159200

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Molecular, Cellular and Developmental Biology.
Studies from our lab demonstrated that a cold-sensitive gamma-tubulin mutant allele, mipAD159, causes defects in the coordination of late mitotic events at restrictive temperatures with observed phenotypes such as abnormal chromosome segregation and inhibition of anaphase A (Prigozhina et al., 2004). These abnormalities are not a result of defects in microtubule nucleation since gamma-tubulin localized normally to the spindle pole body (SPB), microtubules were abundant and mitotic spindle formation and elongation appeared to be normal (Prigozhina et al., 2004). Nayak et al. (2010) further examined gamma-tubulin's role in mitotic regulation and determined that, at restrictive temperatures, mipAD159 caused a failure of accumulation of cyclin B, cyclin dependent kinase 1 (Cdk1) and the phosphatase, Ancdc14, in a subset of nuclei. These nuclei were removed from the cell cycle while other nuclei in the same cell accumulated these proteins and cycled normally. Extensive analysis revealed that this failure of accumulation was due to a nuclear autonomous failure of inactivation of the anaphase promoting complex/cyclosome (APC/C) sometime between late mitosis and S phase. The two projects I have focused on are directed toward further elucidating gamma-tubulin's role in cell cycle regulation. Many mitotic regulatory proteins are known to localize to the SPB, or the centrosome, its functional equivalent in higher organisms, in mitosis. Therefore, gamma-tubulin might be interacting with such proteins and such interactions might be altered in strains carrying mipAD159. I decided to focus on the spindle assembly checkpoint (SAC) proteins Mad2, Mps1, Bub3, BubR1 and Cdc20. I identified the A. nidulans homolog of each, created fluorescent protein fusions, and observed them in vivo by spinning disk confocal microscopy. I found that these proteins are physically separate from each other in interphase, keeping the SAC inactive until mitosis, when they are all at the SPB/kinetochore region. Additionally, I found that Mps1 and BubR1 fail to accumulate in a subset of mipAD159 nuclei, and such nuclei are unable to establish a proper SAC. In other organisms, APC/C binds to two activators. APC/CCdh1 is known to be active in G1, preventing premature accumulation of S-phase cyclins, and it has to be inactivated for the cell to proceed into S phase. APC/CCdc20 is active in mitosis and is required for anaphase and mitotic exit. One unanswered question is which form of the APC/C is constitutively active in mipAD159 nuclei. Since cyclin B is both an S-phase and mitotic cyclin in A. nidulans, either failure of inactivation of APC/CCdh1 at G1/S or failure of inactivation of APC/CCdc20 at the end of mitosis would lead to continuous destruction of cyclin B. I identified the A. nidulans homolog of Cdh1 (designated CdhA) created a GFP fusion, deleted the gene, and analyzed both the fusion and the deletion by time-lapse microscopy. Here, I report my data showing that gamma-tubulin has a critical role in inactivating APC/CCdh1 at the G1/S boundary.
Harold Fisk (Advisor)
Berl Oakley (Advisor)
Stephen Osmani (Committee Member)
Hay-Oak Park (Committee Member)
223 p.
Biology; Cellular Biology; Genetics; Molecular Biology
cell cycle; mitosis; G1 phase; Spindle Assembly Checkpoint; Ananphase Promoting ComplexCyclosome; gamma-tubulin; spindle pole body; Aspergillus nidulans; fungi

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Citations

  • Edgerton, H. D. (2013). Functions of Gamma-tubulin in the Spindle Assembly Checkpoint and APC/C Regulation in Aspergillus nidulans [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374159200

    APA Style (7th edition)

  • Edgerton, Heather. Functions of Gamma-tubulin in the Spindle Assembly Checkpoint and APC/C Regulation in Aspergillus nidulans. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1374159200.

    MLA Style (8th edition)

  • Edgerton, Heather. "Functions of Gamma-tubulin in the Spindle Assembly Checkpoint and APC/C Regulation in Aspergillus nidulans." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374159200

    Chicago Manual of Style (17th edition)

osu1374159200
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This open access ETD is published by The Ohio State University and OhioLINK.