UC Davis

Asymmetric cell division is the process in which one cell divides to give rise to two daughter cells with different cell fates. This process is important throughout development and during stem cell maintenance. Defects in this process can lead to developmental defects and cancer. There are three important steps for a cell to divide asymmetrically. First the cell must generate a polarity axis, such as an anterior-posterior axis. Next the cell must distribute cell fate determinants, such as transcription factors, along this polarity axis. Finally, the cell must orient its spindle along this polarity axis so that when the cell divides each daughter cell receives the correct cell fate determinants. My dissertation work aimed to better understand this process. Specifically, I examined how a cell generates a polarity axis and how the spindle orients along this axis using the early C. elegans embryo as a model.In the one-cell C. elegans embryo, polarity is established by the highly conserved PAR proteins which form two mutually exclusive domains on the membrane, in the anterior and posterior. These domains are maintained by the kinase activity of PKC-3 in the anterior and PAR-1 in the posterior. Polarity establishment and maintenance at the one-cell stage have been well studied, but the mechanisms of polarity establishment in the P1 cell had not been examined. In my work, I found that there are two redundant pathways for polarity establishment. First I identified a novel early pathway in which PAR-1, and its downstream cytoplasmic factors MEX-5 and PLK-1 are required. Then through double mutant analysis, I identified a redundant pathway, similar to the P0 cell pathway, in which AIR-1 and actomyosin flow are required. The PAR polarity proteins also control cytoplasmic polarity and the orientation of the spindle to generate an asymmetric cell division. One of the downstream targets of the PAR proteins is LET-99, which localizes into a posterior-lateral band and acts as the link between cortical polarity and spindle positioning. LET-99 locally inhibits the force generating complex in its band causing asymmetric pulling forces which orient the spindle along the anterior-posterior axis. My work aimed to identify the mechanism by which LET-99 is localized to the membrane and how it is restricted from the anterior. Through a structure function analysis of LET-99, I found that the C-terminus of LET-99 is required for its cortical localization. I also found that PAR-3, PKC-3, and CDC-42 are all required for LET-99 localization. Our analysis of different LET-99 deletions is consistent with a role for PKC-3 in phosphorylating LET-99 to inhibit anterior localization. Overall, my studies contribute to our understanding of how cells generate a polarity axis and regulate spindle positioning.