Date of Award

1-1-2009

Language

English

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (xii, 181 pages) : illustrations (some color)

Dissertation/Thesis Chair

Conly L Rieder

Committee Members

Alexey Khodjakov, Michael P Koonce, Helder Maiato, Robert Glaser

Keywords

cyclin B, Microtubules, Mitotic checkpoint, Nocodazole, Taxol, Mitosis, Cyclin-dependent kinases, Cancer

Subject Categories

Cell Biology

Abstract

Checkpoints are regulatory pathways that control the order and timing of specific cell-cycle events. In the presence of unattached/weakly-attached kinetochores, the mitotic checkpoint (MC) arrests cells in mitosis by inhibiting the degradation of cyclin B, the regulatory subunit of Cdk1 (cyclin dependent kinase 1). Checkpoints do not arrest cells permanently, and escaping mitosis with an unsatisfied MC requires cyclin B/Cdk1 inactivation. In yeast, this occurs through an “adaptation” mechanism involving inhibitory phosphorylations and/or Cdk1-inhibitors. To determine how vertebrate cells escape mitosis when the MC cannot be satisfied I conducted live-cell imaging and immunofluorescence studies on nocodazole-treated rat kangaroo (PtK) and human (RPE1) cells dividing in the absence of microtubules (MTs). These revealed that, in contrast to yeast, vertebrate cells lack an adaptation mechanism for cyclinB/Cdk1 inactivation. Rather, an active MC cannot prevent the constant slow APC-mediated background destruction of cyclin B that ultimately allows the cells to slip out of mitosis without satisfying the checkpoint. These studies also revealed that cyclin B proteolysis was accelerated at nocodazole concentrations that allowed some MT assembly. I next determined, in live RPE1 cells, how the duration of mitosis varied in response to different spindle poisons. The duration of mitosis in drugs that prevent bipolar spindle assembly without perturbing MT dynamics (Eg5 inhibitors) was the same as in nocodazole-treated cells lacking MTs. This indicated that the mere presence of MTs does not accelerate slippage. Additionally, the data revealed that the accelerated exit from mitosis in concentrations of drugs (e.g., nocodazole, colcemid, taxol) that allow for the formation of abnormal spindles occurs because the cells ultimately satisfy the checkpoint.

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