Date

5-24-2011

UMMS Affiliation

Graduate School of Biomedical Sciences, Interdisciplinary Graduate Program

Document Type

Dissertation, Doctoral

Subjects

Dissertations, UMMS; Centrosome; Cell Cycle; Cyclin-Dependent Kinase 2; Cyclin E

Disciplines

Cell Biology | Life Sciences | Medicine and Health Sciences

Abstract

This thesis comprises three separate studies that investigate the consequences of supernumary centrosomes, the effect of centrosome loss, and a control mechanism for regulating CDK2/cyclin E activity in centrosome duplication.

The centrosome is the major microtubule-organizing center of the cell. When the cell enters mitosis, it is of critical importance that the cell has exactly two centrosomes in order to properly segregate the chromosomes to two daughter cells. Supernumary centrosomes are a problem for the cell in that they increase the incidence of chromosomal instability. Aberrant centrosome numbers are seen in a number of cancers, and there has been a proposed connection between the loss of function of p53 and multiple centrosomes. We investigated the consequences of multiple centrosomes in p53-null mouse embryonic fibroblasts (MEFs) to determine how cells with multiple centrosomes can continue to propagate and become cancer. We found that even in the face of extra centrosomes, p53-null MEFs are able to divide in a bipolar fashion by bundling extra centrosomes into two spindle poles.

The centrosome has also been proposed to play a role in cell cycle control. We followed up on a previous study, which had suggested that centrosome loss causes a G1 arrest. We found that cells did not arrest in G1 due to centrosome removal as previously reported, but instead the arrest was viii dependent on additional stressors, namely the incident light used for our long-term live-cell observations. Our study showed that centrosome loss is a detectable stress that, in conjunction with additional stresses, can contribute to cell cycle arrest.

It is known that CDK2/cyclin E activity is required to promote centrosome duplication. But with the discovery of a centrosomal localization sequence (CLS) in cyclin E, we wanted to know if centrosome duplication required a specific sub-cellular localization of CDK2 kinase activity. We found that centrosome duplication in Xenopus extract was dependent on CLS-mediated centrosomal localization of cyclin E, in complex with CDK2. Our results point to a mechanism for regulating centrosome duplication in the face of high cytoplasmic CDK2/cyclin E kinase activity.