Control of Embryonic Spindle Positioning and Gα Activity by C. elegans RIC-8

be greatly enhanced by a 50% reduction in maternal Asymmetric spindle positioning is of fundamental imgoa-1 gene dosage [3]. This indicates that goa-1 and portance for generating cell diversity during developric-8 are likely to function in the same processes, but ment. In the C. elegans 1 cell embryo, spindle positionthe phenotype of ric-8 mutants has not been directly ing has been shown to depend on heterotrimeric G compared to the phenotype of embryos depleted of protein signaling. Two Galpha subunits, GOA-1 and GOA-1 and GPA-16 by RNA interference (RNAi, [18], GPA-16 (hereafter G ), and receptor independent actigoa-1(RNAi);gpa-16(RNAi), hereafter referred to as vators of G protein signaling GPR-1 and GPR-2 (GPRG (RNAi)) and embryos depleted of GPR-1/2. To further 1/2) are required for proper regulation of spindle posiexplore the relationship between ric-8 and the G pathtioning [1]. However, it remains unclear whether G way, we reexamined the ric-8 phenotype and focused regulates spindle positioning in its GDP or GTP bound on the early events in the 1 cell embryo. In ric-8(md1909) form. Here, we investigate the role of RIC-8 in this mutant embryos, early events such as meiosis, pronupathway. RIC-8 was genetically shown to act in conclear migration, meeting, and centration are normal. cert with goa-1 to regulate centrosome movements in However, nuclear rotation is delayed in 30% of the emC. elegans [2, 3]. Interestingly, mammalian RIC-8 was bryos (n 24), the mitotic spindle fails to rock as it recently found to behave as a GEF for G subunits elongates, and it is only weakly displaced to the postein vitro [4]. We show that reduction of function of ric-8 rior, resulting in a cleavage that is more symmetric than results in a 1 cell embryo phenotype very similar to in the wild-type (see Table 1). In addition, spindle morthe phenotype of embryos depleted of G . RIC-8 is phology is symmetric with both anterior and posterior able to directly bind to GOA-1, preferentially to GOAasters having a round morphology at late anaphase in 1-GDP, consistent with a GEF role. RIC-8 is localized contrast to wild-type embryos, in which the anterior at the embryo cortex, and its activity is essential for aster is round but the posterior one has a flat appearance the asymmetric localization of GPR-1/2. We suggest (Figure 1B and [3]). None of the ric-8 mutants is a null that RIC-8 directly modulates G activity and that mutant, and depletion of RIC-8 by RNAi results in 30% G -GTP is the signaling molecule regulating spindle embryonic lethality (see below and [3]). We therefore positioning in the early embryo. tested whether depletion of RIC-8 by RNAi in a ric-8