In multicellular organisms, asymmetric cell division generates cell type diversity during both development and tissue homeostasis. For successful asymmetric division to proceed, a cellular axis of polarity, or asymmetry, must be established via any of several molecular mechanisms. This step is followed by two sequential processes requiring precise coordination of the various components of the cytoskeleton. First, the mitotic spindle must be positioned in alignment with the axis of polarity; second, cytokinesis must occur at the right position and time, as instructed by the spindle, to segregate chromosomes and bisect the axis of polarity. In this dissertation I present my contributions to two research projects related to cytoskeletal regulation during asymmetric cell division. Chapter I reviews the state of the field prior to my studies and introduces the two research projects in context. Chapter II is a manuscript in revision for Journal of Cell Science that relates our findings from the first project, which aimed to understand the role of the DEPDC1 homolog LET-99 during cytokinesis. LET-99 was previously characterized as a negative regulator of the dynein-containing force generating complex during spindle positioning, but had also been shown to play a separable role in cytokinesis. With others, I demonstrated that during the first mitotic division LET-99 and the Rac1 homolog CED-10 have antagonistic roles in regulating the balance of branched vs linear actin during cytokinesis to achieve robust and timely furrowing. These roles appear to be global rather than spatially restricted to the contractile ring. However, the findings suggest that CED-10 does not interact with LET-99 to promote spindle positioning in the first mitotic division, further confirming that LET-99’s role as a regulator of the actomyosin cytoskeleton and cytokinesis is separable from its role in spindle positioning. Chapter III is a manuscript in preparation that documents our investigations into the role of CED-10 in spindle orientation of the asymmetrically dividing EMS cell. Two signaling pathways that regulate this spindle orientation were previously identified: a Wnt/Frizzled pathway and a MES-1/SRC-1 pathway. I determined that CED-10 works upstream or at the level of SRC-1 in the MES-1/SRC-1 signaling pathway and furthermore that CED-10 contributes to cortical localization of the same dynein-containing force generating complex involved in spindle positioning at the one-cell stage. I also found that CED-10’s molecular function in this context may be to promote branched actin formation at the EMS/P2 contact, but this evidence is partially contradicted by our findings that loss of the branched-actin regulator GEX-3 does not enhance the rate of EMS spindle rotation defects in Wnt/MOM-2-depleted embryos. An additional curious finding is that CED-10 has a previously unreported role in P1 spindle rotation, as do the branched actin nucleator Arp2/ARX-2 (Arp2) and the Frizzled ortholog MOM-5. Although this research was performed using nematodes as a model organism, the results have broad implications for all animals because most of the molecular components involved are evolutionarily conserved. By understanding in detail the mechanisms of different types of asymmetric divisions, we can understand both normal development and diseases such as cancer, which involves the dysregulation of asymmetric division.

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.