Investigating Mechanisms of Mitotic Spindle Positioning

The direction, or orientation, of cell division is important because it determines the fate and positions of cells within a tissue. The position of the mitotic spindle, the molecular machine that separates the chromosomes during mitosis, determines the plane of cell division. Cells sometimes use intercellular signals as spatial cues to position the mitotic spindle, but how mitotic spindles are positioned within cells in response to external cues remains unclear. To approach this question, I used the EMS cell in the early C. elegans embryo, an established model for studying cell interactions and miotic spindle orientation during development. I used contemporary genome editing strategies such as CRISPR, confocal live imaging, and classic embryological techniques, to address how proteins are deployed within cells to position mitotic spindles. The second chapter of this work is an in vivo comparison of fluorescent proteins in C. elegans. This study was a valuable technical advance and revealed which fluorescent proteins to use for in vivo live imaging. In the third chapter, using fluorescent proteins, I created tools to visualize our proteins of interest, and determined whether they were cortically enriched by cell-cell signaling mechanisms to direct mitotic spindle positioning. I found surprisingly, that APC and Dishevelled are enriched asymmetrically at the EMS cortex, but NuMA and dyenin are not. These findings have implications for better understanding how signaling pathway proteins might function as positional cues for spindle orientation, independent of the asymmetric enrichment of the canonical Gα/LGN/NuMA complexDoctor of Philosoph