Date of Award
Doctor of Philosophy (PhD)
Cross Fred Laboratory
The cell cycle consists of a series of events, including replication and segregation of the genome, that occurs in order to ensure successful reproduction of cells. In fungi and animals, this process is carefully regulated by a set of protein complexes with alternating, oscillating activity. A well established model has been developed for animals and fungi in which the activities of various cyclin-dependent kinases (CDKs) and the anaphase promoting complex (APC) drive the events of the cell cycle at the appropriate time and in the appropriate order. While this model has been extremely useful for understanding cell division in these lineages, it is not necessarily applicable to other groups of eukaryotes. Animals and fungi belong to a relatively recently diverged group called the Opisthokonts, so their shared features do not necessarily extend to their eukaryotic cousins, including plants, a very important lineage of particular concern to humanity. Chlamydomonas reinhardtii is a unicellular member of the plant kingdom. Its simple genome (compared to land plants) and easily observed cell division cycle have facilitated the collection of a large number of conditional mutations that block the cell cycle at high temperature. Mutations in two CDKs, CDKA1 and CDKB1, and two subunits of the APC are included in this set. The phenotypes of these mutants at restrictive-temperature revealed that CDKA1, unlike its ortholog in the Opisthokonts, is not required for mitosis, and instead plays a role in cell cycle initiation. CDKB1, on the other hand, is a plant-specific CDK and is required for promoting the events of mitosis. The APC plays a similar role to its counterpart in Opisthokonts in driving the metaphase-to-anaphase transition. In this thesis, we present the results of our efforts to better understand the function and regulation of the kinases CDKA1 and CDKB1 and two Chlamydomonas cyclins, CYCA1 and CYCB1. We characterize the role of these molecules in cell division timing, DNA replication, spindle formation, and cytokinesis and explore the nature of their regulatory interactions and their control by the APC. We also describe a genetic screen to identify parallel pathways that promote cell cycle initiation alongside CDKA1 and speculate on a possible common thread among the identified mutations. A genetic screen for genes involved in cell cycle initiation uncovers many null mutations in CDKA1, showing definitively that CDKA1 is inessential for cell division in Chlamydomonas, and likely all plants. Disruption of both CDKA1 and CYCA1 results in a delay in cell division, and CYCA1 is specifically required for biochemical activity of CDKA1, suggesting they may act as a complex to promote the initiation of cell division. CYCB1 is required for timely DNA replication and mitotic spindle formation in a similar manner to CDKB1, and, consistently, is also required for biochemical activation of CDKB1. We propose that CYCB1 and CDKB1 form a complex and together constitute the primary mitotic inducer in Chlamydomonas. Both CDKA1 and CDKB1 are downregulated by the APC, and CDKA1 kinase activity is inhibited by CYCB1- CDKB1. A model is presented incorporating these and prior findings concerning the function and regulatory interaction among these cell cycle regulators. Several possible positive and negative feedback loops become apparent which may ensure switch-like activation or appropriate ordering of the activity of various complexes.
Atkins, Kenneth C., "Cell Cycle Control by Cyclin-CDKS in Chlamydomonas ReinhardtII" (2018). Student Theses and Dissertations. 424.