Asymmetric Spindle Positioning and Intracellular Trafficking in C. Elegans Embryos

Asymmetric cell division is critical for generating daughter cells that possess distinct fates during development and in stem cell lineages. Correct orientation of the mitotic spindle with respect to the polarity axis is crucial to this process. In the one-cell stage embryo of the nematode C. elegans, polarity cues define an anterior-posterior axis, and the mitotic spindle is positioned asymmetrically towards the posterior of the embryo in response to these cues, resulting in daughter cells that differ not only in fates, but also in sizes. Although the way by which force is generated has becoming increasingly well understood, the mechanisms by which polarity cues regulate the asymmetric distribution of active cortical force generators that pull on astral microtubules to position the spindle remained unclear at the onset of my thesis. During the course of our work, we found that intracellular trafficking plays a crucial role in the distribution and levels of proteins involved in asymmetric spindle positioning. Firstly, we found that the distribution of the Gβ protein GPB-1, a negative regulator of pulling forces, varies across the cell cycle, with levels at the cell membrane being lowest during mitosis. GPB-1 transits through the endosomal network in a dynamin-, clathrin- and RAB-5-dependent manner. We also found that GPB-1 trafficking is more pronounced on the anterior side and that this asymmetry is regulated by A-P polarity cues. In addition, we demonstrate that GPB-1 depletion results in the loss of GPR-1/2 asymmetry during mitosis, and thus symmetric pulling forces during mitosis. Secondly, we found that the clathrin heavy chain modulates spindle-positioning forces as well as the cortical distribution of positive (GPR-1/2 and DHC-1) and negative (GPB-1 and LET-99) regulators of pulling forces. Although the mechanisms by which it does so remain unclear, our data suggests that clathrin plays multiple roles in the one-cell stage C. elegans embryos, including in recycling GPB-1 to the plasma membrane. Overall, our results lead us to propose that the clathrin heavy chain and the modulation of Gβγ trafficking play a critical role during asymmetric division of one-cell stage C. elegans embryos.