Regulation of the Anaphase-Promoting Complex Examined at the Single Cell Level

<p>Cell cycle transitions are driven by oscillations of cyclin-cyclin dependent kinase (CDK) activity and associated cyclin degradation, mediated by ubiquitylation by the anaphase-promoting complex (APC). In this work, I analyzed the regulation of the APC by its activator Cdh1 in budding yeast in single cells. Inactivation of APC-Cdh1 is an important regulatory transition leading to mitotic entry. I developed and characterized a fluorescent biosensor to measure the dynamics of APC-Cdh1 activity in single cells by quantitative time-lapse microscopy. I found that APC-Cdh1 is inactivated with very reliable timing, in contrast with other cell cycle events that occur with considerable variability in timing. The activity of APCCdh1 is restrained by multisite phosphorylation by early cyclin-CDKs. Complete removal of phosphorylation control of Cdh1 results in cell cycle arrest before mitotic entry, because persistent APC-Cdh1 activity prevents mitotic cyclin gene expression and accumulation of mitotic cyclins. I show that partial phosphorylation of Cdh1 allows for partial inactivation of APC-Cdh1. Interestingly, incompletely restrained APC-Cdh1 activity causes a variable phenotype in cell cycle progression on the single cell level. This partially penetrant phenotype, caused by incomplete inactivation of APC-Cdh1, is highly complex; even though some of the cells arrest in the cell cycle, they occasionally complete later cell cycle events with delay and in incorrect order. I show that Cdh1 can be phosphorylated by multiple cyclin-CDKs, and that additional mechanisms of APCCdh1 inactivation besides phosphorylation also contribute to robust inactivation. In the last part of the thesis, I examine the global cell cycle-associated transcriptional program and its regulation by cyclin-dependent kinase activity.</p>

Name thesis:degree_name

Doctor of Philosophy (PhD)

Level thesis:degree_level

Thesis

Year

2016

Author dc:creator

• Ondracka, Andrej

Contributors dc:contributor

• Frederick R. Cross

• cell cycle
• APC-Cdh1
• phosphorylation
• cyclin-CDK
• yeast
• single-cell analysis
• Life Sciences

Repository record dc:identifier

https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/312

OAI identifier oai:identifier

oai:digitalcommons.rockefeller.edu:student_theses_and_dissertations-1318