Asymmetric positioning of the mitotic spindle is key generation of cells with different sizes and fates during development. In one cell stage C.elegans embryos, asymmetric spindle positioning is under the control of polarity cues, which are translated into the generation of differential pulling forces acting on spindle poles with a stronger net force acting on the posterior spindle pole. In addition, simultaneous inactivation of two Galpha subunits,
goa-1 and
gpa-16, or o f the interacting Goloco proteins GPR-1/2 results in extreme reduction of pulling forces and a symmetric division. We investigated the contribution of
ric-8 to spindle positioning in one cell stage C. elegans embryos.
ric-8 is re q uired for Gqalpha signaling in the nervous system, and has been postulated to act in concert with
goa-1 in early embryo. We established that RIC-8 is required for generation of pulling forces on spindle poles, and that it physically interacts w it h both GOA-1 and GPA-16 in vitro and in vivo. We found that RIC-8 is mainly cytoplasmic but enriched slightly at the cell cortex, and investigated interdependency of localization of RIC-8 and Galpha proteins. Our biochemical assays sh ow th at RIC-8 binds preferentially to GOA-1.GDP and that it promotes nucleotide exchange, just like it has been reported for its mammalian counterpart. Therefore, RIC-8 functions as a GEF. Taken together, this and previous studies to date supports a wo r ki ng m odel of asymmetric spindle positioning in which GPR-1/2 and RIC-8 act sequentially to ensure proper Galpha signaling and the generation pulling forces on the spindle poles.