LET-99: A novel regulator of G protein signaling and spindle positioning in Caenorhabditis elegans embryos

Asymmetric cell divisions are an essential step for normal development and tissue homeostasis in a number of organisms. The evolutionary conserved PAR proteins organize polarity for asymmetric cell divisions. Downstream of PAR proteins, G protein signaling regulates mitotic spindle positioning in many systems including C. elegans and Drosophila . In C. elegans, two partially redundant Galpha subunits, GOA-1 and GPA-16, the GoLoco protein GPR-1/2, and the coiled-coil protein LIN-5 act together to generate the cortical pulling forces that mediate asymmetric posterior spindle displacement at metaphase/anaphase of the first asymmetric division. GPR-1/2 are asymmetrically enriched at the posterior cortex, and this localization is indispensible for the posterior directed large pulling forces at this time. The DEP domain protein LET-99 is localized asymmetrically in a posterior cortical band by PAR proteins and is required for spindle positioning. LET-99 inhibits cortical localization of GPR-1/2. However, the molecular architecture of the active complex in G protein signaling was not clear. Further, it was not known at which step LET-99 inhibits G protein signaling to prevent cortical accumulation of GPR-1/2 and thus create the asymmetric distribution of cortical forces.;In this dissertation, I found that LIN-5 and GPR-1/2 are dynamically colocalized to cortical force generation domains in a par-3 and let-99 dependent manner during spindle positioning. Further, Galpha and GPR-1/2 were required for cortical LIN-5 localization, and all three proteins were necessary for normal spindle alignment. LIN-5 did not interact with Galpha in the absence of GPR-1/2. These results suggest that a conserved ternary complex of Galpha/GPR-1/2/LIN-5 is present and required for both centration/rotation and asymmetric spindle displacement. I also found that LET-99 was able to inhibit the cortical localization of GPR-1/2 to establish asymmetric localization of GPR-1/2 at the cortex when GPA-16 was the only Galpha present, but not when GPA-16 was the only Galpha present. In let-99 embryos, GOA-1 and GPB-1 were significantly accumulated at the cortex; however, cortical localization of GPA-16 is similar to wild type. Further, LET-99 bound to the GDP and GTP form of GOA-1 and GPA-16 and Gbeta. These results suggest that LET-99 utilizes two distinct mechanisms to antagonize the two Galpha subunits to regulate spindle positioning.